Constructs, compositions, and methods for promoting thromboangioplasticity

Abstract

Described herein are a composition or a construct including a first polypeptide and a second polypeptide. The first polypeptide includes a growth arrest-specific 6 (GAS6) polypeptide or a protein S polypeptide, and the second polypeptide includes an annexin V polypeptide. Also described is a method of treating, ameliorating and / or preventing a disease or disorder caused by or involves a vascular occlusion or damage.

Description

[0001] Attorney Docket No. 047162-7481WO 1(02817)

[0002] CONSTRUCTS, COMPOSITIONS, AND METHODS FOR PROMOTING THROMBOANGIOPLASTICITY AND / OR TREATING, AMELIORATING, AND / OR PREVENTING DISEASES OR DISORDERS ASSOCIATED WITH TISSUE DAMAGE AND / OR THROMBOTIC OCCLUSION

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] The present application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Applications No. 63 / 805,618, filed May 14, 2025, and U.S. Provisional Patent Applications No. 63 / 728,449, filed December 05, 2024, both of which are incorporated herein by reference in their entireties.

[0005] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0006] This invention was made with government support under 5R01NS111961-05 awarded by National Institutes of Health. The government has certain rights in the invention.

[0007] SEQUENCE LISTING

[0008] The XML file named "047162-7481WOl(02817)_Sequence Listing.xml" created on December 4, 2025, comprising 7,410 bytes, is hereby incorporated by reference in its entirety.

[0009] BACKGROUND

[0010] The health and survival of organisms, including humans, are largely reliant on efficient tissue repair, in view of the continuous damage, microtrauma, and regular wear and tear of tissues throughout the lifespan. When an injury occurs, a complex series of events takes place to stop bleeding, repair blood vessels, and remodel tissues, ultimately restonng normal function. Vascular occlusion or damage is also implicated in various diseases and disorders such as ischemic conditions (for example, stroke and myocardial infarction). Both tissue repair from wear and tear or physical damages and the recovery’ from diseases associated with vascular occlusion involve the thromboangioplasticity process, i.e., the removal of vascular occlusion from the blood vessel and the restoration of normal blood flow.

[0011] The thromboangioplasticity process spans the full continuum from the earliest instances of tissue injury, hemorrhage, and thrombosis, all the way through to the final stages of recanalization and repair. Existing treatments primarily act on the early phases of this

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[0013] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817) process by preventing thrombosis, limiting thrombus grow th, or dissolving the thrombus. However, there is a lack of medications that facilitate the later vascular phase of revascularization. While the existing interventions are somewhat effective in the early stages of thrombosis, they enhance the risk of hemorrhage and thus are not suitable in numerous clinical scenarios or during later stages of tissue injury'.

[0014] Therefore, there is a need for novel compounds, compositions, and methods that can speed up the thromboangioplasticity process without causing the undesirable side effects of the existing treatments. The present invention addresses this need.

[0015] SUMMARY

[0016] In some aspects, the present invention is directed to the following non-limiting embodiments:

[0017] Composition

[0018] In some embodiments, the present invention is directed to a composition.

[0019] In some embodiments, the composition comprises a first polypeptide (or a first nucleic acid encoding the first polypeptide) and / or a second polypeptide (or a second nucleic acid encoding the second polypeptide).

[0020] In some embodiments, the first polypeptide comprises a growth arrest-specific 6 (GAS6) polypeptide and / or a protein S polypeptide.

[0021] In some embodiments, the second polypeptide comprising an annexin V polypeptide.

[0022] In some embodiments, the first polypeptide and the second polypeptide are not covalently linked to each other.

[0023] In some embodiments, the first polypeptide and the second polypeptide are covalently linked to each other.

[0024] In some embodiments, both the first polypeptide and the second polypeptide are parts of a fusion protein.

[0025] In some embodiments, the further comprising a pharmaceutically acceptable carrier.

[0026] In some embodiments, the composition is formulated for parenteral administration. In some embodiments, the parenteral administration compnses intradermal administration, subcutaneous administration, intramuscular administration, or intravenous administration.

[0027] In some embodiments, the composition accelerates thromboangioplasticity process, clot removal from a blood vessel, vessel recanalization, reestablishment of blood flow, and / or tissue repair in a subject.

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[0029] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817)

[0030] In some embodiments, the composition reduces risk of hemorrhage after injury and / or risk of hemorrhagic conversion due to stroke and / or organ trauma.

[0031] In some embodiments, the composition treats, ameliorates, and / or prevents a tissue ischemia, a physical injury, a stroke, and / or a myocardial infarction in a subject in need thereof.

[0032] In some embodiments, the composition treats, ameliorates, and / or prevents a condition associated with anti-annexin V autoimmune antibodies, optionally a pregnancy loss or an antiphospholipid syndrome.

[0033] In some embodiments, the composition treats, ameliorates, and / or prevents thrombo- hemorrhage in subjects carrying a mutant annexin V.

[0034] Non-natural construct or nucleic acid encoding the same

[0035] In some aspects, the present invention is directed to a non-natural construct or a nucleic acid encoding the non-natural construct.

[0036] In some embodiments, the construct comprises a first polypeptide comprising a growth arrest-specific 6 (GAS6) polypeptide and / or a protein S polypeptide; and / or a second polypeptide comprising an annexin V polypeptide.

[0037] In some embodiments, the construct is a fusion protein comprising the first polypeptide and the second polypeptide.

[0038] In some embodiments, the construct or the nucleic acid accelerates clot removal from a blood vessel, vessel recanalization, reestablishment of blood flow, and / or tissue repair in a subject.

[0039] In some embodiments, the construct or the nucleic acid reduces a risk of hemorrhage after injury and / or the risk of hemorrhagic conversion due to stroke and / or organ trauma.

[0040] In some embodiments, the construct or the nucleic acid treats, ameliorates, and / or prevents tissue ischemia, physical injury, stroke, and / or myocardial infarction in a subject in need thereof.

[0041] In some embodiments, the construct or the nucleic acid treats, ameliorates, and / or prevents a condition associated with anti-annexin V autoimmune antibodies, optionally a pregnancy loss and / or an antiphospholipid syndrome.

[0042] In some embodiments, the construct or the nucleic acid treats, ameliorates, and / or prevents thrombo-hemorrhage in a subject carrying a mutant annexin V.

[0043] Method of treating, ameliorating, and / or preventing disease or disorder

[0044] In some aspects, the present invention is directed to a method of treating, ameliorating, and / or preventing a disease or disorder in a subject in need thereof.

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[0047] In some embodiments, the disease or disorder is caused by or involves blood vessel thromboembolic occlusion and / or embolic occlusion.

[0048] In some embodiments, the method comprises: administering to the subject an effective amount of the composition, the construct, or the nucleic acid herein.

[0049] In some embodiments, the blood vessel thromboembolic occlusion is a thromboembolic occlusion or an embolic occlusion in a microvasculature blood vessel.

[0050] In some embodiments, the disease or disorder compnses tissue ischemia, physical injury, stroke, or myocardial infarction.

[0051] In some embodiments, the disease or disorder comprises a condition associated with anti-annexin V autoimmune antibodies, optionally a pregnancy loss or an antiphospholipid syndrome.

[0052] In some embodiments, the disease or disorder comprises a thrombo-hemorrhage in a subject carrying a mutant annexin V.

[0053] In some embodiments, the composition or the construct is administered parenterally.

[0054] In some embodiments, the administration of the composition or the construct accelerates thromboangioplasticity process, clot removal from a blood vessel, vessel recanalization, reestablishment of blood flow, and / or tissue repair in a subject.

[0055] In some embodiments, the administration of the composition or the construct reduces a risk of hemorrhage after injury and / or the risk of hemorrhagic conversion due to stroke or organ trauma.

[0056] In some embodiments, the subject is a mammal, optionally a human. Method of accelerating thromboangioplasticity’ process

[0057] In some aspects, the present invention is directed to a method of accelerating thromboangioplasticity process, such as in a blood vessel, such as in a blood vessel having a thrombotic occlusion and / or an embolic occlusion.

[0058] In some embodiments, the method comprises contacting the blood vessel with the composition or the construct herein.

[0059] In some embodiments, the method comprises delivering to a cell in. within, or in proximity to the blood vessel the composition or the nucleic acid herein.

[0060] In some embodiments, the blood vessel is a microvasculature blood vessel.

[0061] BRIEF DESCRIPTION OF THE DRAWINGS

[0062] The following detailed description of exemplary embodiments will be better understood when read in conjunction with the appended drawings. For the purpose of

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[0064] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817) illustrating, non-limiting embodiments are shown in the drawings. It should be understood, however, that the instant specification is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0065] Fig. 1 illustrates certain aspects of the thromboangioplasticity process in a blood vessel, in accordance with some embodiments.

[0066] Figs. 2A-2M: Intravital imaging of endothelial plasticity following thrombosis, in accordance with some embodiments. Fig. 2A: Diagram of a procedure for generation of endogenous thrombi by focal topical application of Ferric Chloride (FeCh) to the surface of a primary branch of the middle cerebral artery’ (MCA) through a craniotomy opening. Thrombi are dislodged by gentle massaging of the surface of the vessel with forceps until dislodged thrombi occlude downstream branches located greater than 0.5 mm away from the FeCh application site. Fig. 2B: Platelet-rich thrombi can be readily visualized by imaging a transgenic reporter mouse with GFP-labeled platelets (Pf4-GFP, Green) and i.v. dye (magenta) which highlights the vessel wall at 4 days. Fig. 2C: Transmission electron microscopy (TEM) of a relocated vessel shows platelet-rich thrombus (magenta), adjacent to leukocytes (blue) and endothelium (green). Fig. 2D: Formation of early endothelial lamellipodia projections (white arrowheads) towards thrombi (i.v. dye, magenta). Magenta i.v. dye is retained within the thrombus at day 1 allowing visualization of the thrombus in a transgenic reporter mouse with GFP-labeled endothelium (Tie2-GFP). Fig. 2E: Quantification of endothelial lamellipodia projections at different time points following thrombosis (Ih n=7 mice, 25 clots; 8h n=6 mice, 21 clots; 12h n=4 mice, 9 clots; Kruskal- Wallis test with Dunn’s multiple comparisons correction). Fig. 2F: Quantification of the correlation between lamellipodia projections and propensity' for thrombus washout (n=9 mice. 18 clots; Spearman Correlation Analysis r = -0.7113, p = 0.0371). Fig. 2G: Correlated in vivo imaging, (Left Panel) TEM of a thrombus at day 4, (Right Panel) Multiple endothelial cells are involved in the envelopment of thrombi as evidenced by the presence of multiple nuclei (magenta asterisks) in the surrounding endothelium in the right panel. Fig. 2H: Time lapse imaging demonstrates the progressive envelopment of a thrombus (magenta) with endothelial projections (green) that infiltrate and compartmentalize the thrombus (white asterisks). Fig. 21: Quantification of the degree of endothelial envelopment of thrombi over time (n=44 mice, 91 clots). A linear mixed effects model assessed change in slope over time and accounted for repeated measures within mice. The fixed effect slope for time was 21.55, p <0.0001. Fig. J: High-resolution time-lapse imaging reveals the gradual formation of large tunnels in the original abluminal endothelium (white arrowhead) through which thrombi will

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[0068] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817) be extruded gradually. Notice the lack of i.v. dye (magenta) penetration into the thrombus cavity and extravascular leakage despite the presence of tunnels. Fig. 2K: Quantification of the enlargement of abluminal endothelial tunnels over time (n=59 clots in 32 mice). Pore opening increased significantly over time (Data was analyzed using a linear mixed model with time as a fixed effect. The fixed effect slope for time was 16.58, p <0.0001). Fig. 2L: Time lapse imaging showing a thrombus that is anchored to the vessel wall by endothelial projections followed by gradual extraluminal extrusion of the thrombus. Fig. 2M: Schematic of the endothelial plasticity process. Data ± SEM. Scale bar = 20pm (Figs. 2B, 2D, 2G, 2H, 2J, and 21); 10 pm (Fig. 2C and left panel of Fig. 2G); 6 pm (right panel of Fig. 2G).

[0069] Figs. 3A-3J demonstrate that SMCs invade the intraluminal thrombus cavity, leading to thrombus engulfment and extrusion, in accordance with some embodiments. Fig. 3A: Intravital imaging in SMA-mCherry:Tie2-GFP mice following thrombosis. SMC lamellipodia (red) penetrate through a large abluminal endothelial tunnel opening, migrate alongside the endothelium and invade the thrombus cavity (white arrowheads). Notice that the intravascular dye (blue) is mostly absent from the thrombus cavity, suggesting the preservation of a tight blood brain barrier during this plasticity process. Fig. 3B: Quantification of this process over time shows a close association betw een the degree of SMC invasion and the gradual enlargement of the endothelial tunnels (n= 13-31 mice, 23-55 clots; mixed effects model with Sidak's multiple comparisons test, no significant difference found at any timepoint). Fig. 3C: SMC lamellipodia can invade the thrombus cavity through multiple EC tunnel entry points (white arrowheads). Fig. 3D: Correlative in vzvo-TEM imaging of SMC invasion by intravital fluorescence microscopy (left panel, white dotted box) and (right panel) TEM imaging. TEM shows the tight interaction between EC (green) and highly invasive SMC projections (red) intermixed with remnants of undegraded thrombi. Fig. 3E: The elastin layer (white) around vessels is visualized in vivo through labeling with hydrazide 633. Notice the gaps in the labeling (white arrowheads) through which smooth muscle cell lamellipodia (red) penetrate the cavity. Fig. 3F: Quantification of SMC envelopment of the thrombus over time (n=24 clots in n=14 mice). A linear mixed effects model assessed change in slope over time and accounted for repeated measures within mice. The fixed effect slope for time was 13.54, p <0.0001. Fig. 3G: SMC lamellipodia become highly invasive, forming a dense network (red) where thrombi (white, prelabeled by i.v. anti- CD41-647 antibody administration) become fragmented and appear to be gradually degraded. Fig. 3H: Time lapse imaging demonstrates the gradual invasion and retraction of SMCs, associated with the reopening of the vessel lumen (white asterisk). Fig. 31 Quantification of

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[0071] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817) the degree of vascular recanalization overtime (recanalization index is obtained by dividing the various time points by the baseline value at day 1) (n=27 clots in 16 mice). Data was analyzed using a linear mixed model with time as a fixed effect. The fixed effect slope for time was 0.34, p <0.0001. Fig. 3J: Schematic depicting the invasion of SMCs through endothelial openings and the degradation of thrombi within the dense netw ork of SMC processes. Data ± SEM. Scale bars= 20pm (Figs. 3A, 3E, 3G. and 3H); 10 pm (Figs. 3C and the left panel of Fig. 3D); 2 pm (right panel of Fig. 3D).

[0072] Figs. 4A-4O demonstrate that MerTK signaling triggers EC-mediated thrombus stabilization which impacts hemorrhage and tissue injury outcomes, in accordance with some embodiments. Fig. 4A: Intravital imaging of a platelet-rich thrombus in a transgenic reporter mouse (Pf4-mTmG) with GFP-labeled platelets (green). ECs and SMCs are Tomato-positive (red) in these mice. Intravenous administration of Annexin A5-633 (white) shows robust colocalization with platelets. Fig. 4B: Intravital imaging in Tie2-GFP mice highlighting early endothelial lamellipodia protrusions (green) around thrombi (magenta, labeled by accumulation of i.v. dye). In MerTK ' mice these lamellipodia are markedly reduced at day 1. Fig. 4C: Statistical analysis showing reduced lamellipodia projections in MerTK' ' mice at various time points post thrombosis (Control n=7-9 mice; MerTK'7' n=4-6 mice). Mixed- effects model with the Geisser-Greenhouse correction with Sidak's multiple comparison test. Fig. 4D: Time lapse imaging in MerTK' ' mice shows extensive thrombus (magenta) washout at day 1. Fig. 4E: Statistical representation of the degree of thrombus washout as a function of the number of EC projections towards thrombi in both wildtype and MerTK' ' mice (control n=9 mice with 18 clots; MerTK" n=6 mice with 15 clots). Values were computed using Hotelling’s T2 test (T2 = 69.62, F=32.13, P<0.0001). Fig. 4F: Time lapse imaging shows markedly reduced endothelial thrombus envelopment in MerTK' ' mice at day 1. At day 3 there is a significant rebound leading to robust EC envelopment. Fig. 4G: Quantitative analysis of the degree of EC thrombus envelopment at various time points in wildtype controls, MerTk' ' and Gas6 inhibitor (RU301)-treated mice (Control n=91 clots in 44 mice; MerTK' ' n=33 clots in 16 mice; RU301 n=12 clots in 4 mice). Data was transformed and linear mixed-effects models were used to account for repeated measures within individual mice. Type III ANOVA with Satterthwaite approximation assessed significance, followed by Tukey’s HSD adjusted t-tests for multiple comparisons. P values for control vs MerTK" (magenta) and control vs RU301 (green). Fig. 4H: Schematic depicting the method for infusion of exogenous fibrin emboli into the common carotid artery for subsequent tissue quantifications. Fig. 41: In vivo imaging of a fibrin embolus (blue) and extensive platelet

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[0074] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817) aggregation around it (green), in Pf4-mTmG mice (EC / SMCs in red). Fig. 4J: Brain slices (blue) of mice infused with fibrin emboli (green / yellow), showing reduced fibrin emboli retention in MerTk inhibitor (UNC2881)-treated- mice 6 hrs. post emboli injection. Fig. 4K: Quantification of fibrin emboli retained in brain slices comparing controls and UNC2881- treated mice at 0.5 and 6 hrs post injection (Control 0.5h n=4 mice, UNC2881 0.5h n=5 mice, Control 6h n=l 1 mice, UNC2881 6h n=9 mice; average clot number per slice in 9 slices per mouse. Two-way ANOVA with Sidak's multiple comparison test). Fig. 4L: Kaplan-Meier survival graph showing markedly decreased mouse survival in fibrin emboli-injected, UNC2881-treated mice over the first 48hrs (Control n=12 and UNC2881 n=15 mice). Survival analyses were performed using log-rank (Mantel-Cox) tests. Fig. 4M: Hemorrhagic transformation visualized in coronal sections of H&E-stained brain slices. Fig. 4N: Quantification of the number of hemorrhages visualized in brain slices per animal (Unpaired t-test). Fig. 40: Schematic depicting reduced EC envelopment in MerT which is associated with increased thrombus washout and disruption of blood vessels with increased risk of hemorrhagic transformation and ultimately a reduction in survival. Data ± SEM. Scale bar= 20pm (Figs. 4A, 4B, and 41); 50 pm (Figs. 4D and 4F); 1 mm (Figs. 4J and 4M).

[0075] Figs. 5A-5G demonstrate that Axl signaling mediates SMC plasticity and controls the efficiency of vascular recanalization, in accordance with some embodiments. Intravital imaging of thrombi (unlabeled) in SMA-mCherry:Tie2-GFP mice in controls, MerTK or Axl knockout mice. Fig. 5 A, left panel: Abluminal EC retraction forms a large EC tunnel (white arrowheads) through which the adjacent smooth muscle (white arrows) invades the thrombus cavity. Fig. 5 A, right panel: Quantification of the degree of EC tunnel formation and concurrent SMC invasion of the thrombus cavity' over time in control mice (n=23 clots in 13 mice). Mixed effects model with Sidak's multiple comparisons test, no significant difference found at any timepoint. Fig. 5B, left panel: Images in Axl' ' mice show normal EC tunnel formation (white arrowheads) but a lack of SMCs invasion of the thrombus cavity (white arrows) at day 6. Fig. 5B, right panel: Quantification of the degree of EC tunnel formation and concurrent SMC invasion of the thrombus cavity over time in Axl' ' mice shows a marked delay in SMC invasion, which is especially pronounced between days 2 and 5 (n=33-43 clots in 27 mice). Mixed effects model with Sidak's multiple comparisons test. At days 3 and 4, p<0.0001. Fig. 5C, left panel: Imaging in MerTK " mice shows normal EC tunnel formation (white arrowheads) and SMC thrombus cavity invasion (white arrows). Fig. 5C, right panel: Quantitative analysis of the time course of EC openings relative to SMC thrombus cavity’ invasion in MerTK" mice (n=l l-20 clots in 5-9 mice) Mixed effects model with Sidak's

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[0077] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817) multiple comparisons test, no significant difference found at any timepoint. Fig. 5D: Quantification of the degree of SMC thrombus envelopment over time in controls, Axl and MerTK knockout mice (Control n=24 clots in 14 mice; MerTK" n=l l clots in 5 mice; Axl' ' n=37 clots in 21 mice). Data was transformed and linear mixed-effects models were used to account for repeated measures within individual mice. Type III ANOVA with Satterthwaite approximation assessed significance, followed by Tukey's HSD adjusted t-tests for multiple comparisons. P values for control vs Axl' ' (grey) and MerTK'7' vs Axl' ' (magenta). Fig. 5E: Quantification of lumen recanalization as fold-change of baseline (recanalization index), measured over time in controls, Axl and MerTK knockout mice (Control n=27 clots in 16 mice: MerTK' ' n=25 clots inlO mice; Axl'7' n=54 clots in 27 mice). Data was transformed and linear mixed-effects models were used to account for repeated measures within individual mice. Type III ANOVA with Satterthwaite approximation assessed significance, followed by Tukey’s HSD adjusted t-tests for multiple comparisons. P values for control vs Axl'7' (grey) and MerTK'7' vs Axl'7' (magenta). Fig. 5F: Quantification of long-term thrombus persistence (>10 days) in Control (n=66 clots), Axl'7' (n=54 clots) and MerTK' (n=22 clots) mice (Fisher’s exact test with Bonferroni correction was used to compare the proportion of persistent thrombi after 10 days in all groups). Fig. 5G: Time lapse over an interval up to 60 days in an Axl knockout mouse showing a case in which the thrombus (labeled at time point 1 with anti-CD41-647, white) has persisted intraluminally for 60 days and remains infiltrated by the SMCs, partially obstructing blood flow (i.v dye, blue). Data ± SEM. Scale bar = 20pm (Figs. 5A-5C and 5G).

[0078] Figs. 6A-6L demonstrate that Annexin A5 administration accelerates thromboangioplasticity but the effect is delayed in MerTK'7' mice. Fig. 6A: In vivo imaging showing fluorescently conjugated Annexin A5-633 (white) binding with high specificity to an intravascular thrombus. Fig. 6B: In vivo images showing robust EC lamellipodia projections towards thrombi (white arrowheads) in Annexin A5 treated mice that appear to be anchoring the thrombi to the vessel wall. Notice the long anchoring lamellipodia at 8hrs, right panel. Fig. 6C: Quantification of the number of initial lamellipodia projections towards thrombi in Vehicle (n=6 mice with 13 clots) and Annexin A5 (n=6 mice with 16 clots)- treated mice (Unpaired t-test). Fig. 6D: In vivo images showing various degrees of endothelial envelopment of thrombi in Vehicle, Annexin A2- and Annexin A5-treated mice on day 1. Fig. 6E: Quantification of the degree of EC thrombus envelopment in Vehicle-, Annexin A2- and Annexin A5-treated mice at day 1 (Vehicle n=42 mice with 86 clots; Annexin A2 n=9 mice with 17 clots; Annexin A5 n=34 mice with 72 clots. One-way

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[0081] ANOVA with Tukey’s multiple comparison correction). Fig. 6F: Time lapse imaging of the entire sequence of thromboangioplasticity in an Annexin A5-treated mouse, showing accelerated thrombus envelopment and lumen recanalization. Notice persistent binding of Annexin A5-633 (white) to the thrombus even after extravasation at day 4. Fig. 6G: Quantification of the degree of vessel lumen recanalization over time in Vehicle (n=27 clots in 16 mice), Annexin A2 (n=17 clots in 9 mice) and Annexin A5 (n=72 clots in 34 mice)- treated mice. Data was transformed and linear mixed-effects models were used to account for repeated measures within individual mice. Type III ANOVA with Satterthwaite approximation assessed significance, followed by Tukey’s HSD adjusted t-tests for multiple comparisons. P values for control vs Axl’ ’ pairwise comparisons are shown in orange. Fig. 6H: Time lapse imaging of MerTK’ ’ mice treated with Annexin A5 showing a delay in EC envelopment at day 1. Notice that there is a rebound leading to robust envelopment of the Annexin A5-633 thrombus by days 2 to 4. Fig. 61: Quantification of the degree of EC envelopment in Vehicle (n=86 clots in 42 mice), MerTK" ’ (n=33 clots in 16 mice), Annexin A5 (n=72 clots in 34 mice) and Annexin A5 treated MertK’ ’ mice (n=12 clots in 5 mice). Notice that Annexin A5 treated MerTK ” mice had a marked reduction in EC envelopment at day 1 that rapidly rebounded by day 2. Pairwise comparisons - on day 1, control differed from Annexin A5 (p < 0.0001), Annexin A5+MerTK KO (p < 0.0043), and MerTK KO (p = 0.0029), and Annexin A5 differed from both Annexin A5+MerTK KO and MerTK KO (p < 0.0001), with no differences between Annexin A5+MerTK KO vs MerTK KO (p = 0.7783). On day 2, control vs. MerTK KO (p = 0.0042); MerTK KO vs. Annexin A5+MerTK KO (p = 0.0089) and MerTK KO vs Annexin A5 (p < 0.0001) reached significance. On day 3, there were no significant differences. Data was transformed and linear mixed-effects models w ere used. Type III ANOVA with Satterthwaite approximation assessed significance, followed by Tukey’s HSD adjusted t-tests for multiple comparisons. Fig. 6J: Quantification of the percentage of lumen recanalization in Vehicle (n=27 clots in 16 mice), MerTK’ ’ (n=25 clots in 16 mice), Annexin A5 (n=71 clots in 34 mice) and Annexin A5 treated MertK’ ’ mice (n=12 clots in 5 mice). Mixed effects analysis was performed with Tukey’s HSD. Pairwise comparisons - on days 1 to 5, Annexin A5 differed significantly from vehicle (****p<0.0001 to **p=0.0085) but not on day 6 (ns, p=0.2941). Annexin A5 vs. Annexin A5+MerTK’ ’ reached significance across all timepoints (***p<0.0001 to **p=0.0076). Annexin A5 vs. MerTK” comparisons were significant across all timepoints (**p<0.0001 to *p=0.0444). MerTK’ ’ vs. Annexin A5+MerTK’ ’ showed no significant differences at any timepoint (ns, p>0.05). Vehicle vs. Annexin A5+MerTK’ ’ was significant only at timepoint 6 (*p=0.0456).

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[0084] Fig. 6K: Quantification of thrombus washout in the first 24 hrs in Vehicle (n=24 clots in 10 mice), MerTK " (n=16 clots in 6 mice), Annexin A5 (n=18 clots in 9 mice) and Annexin A5 treated MerTK' " mice (n=18 clots in 5 mice). The present study observed a marked increase in thrombus washout in Annexin A5 treated MerTK" mice consistent with the reduced EC envelopment observed at day 1. A linear mixed-effects model was used to account for multiple clots per mouse. A type III ANOVA was used to assess the overall significance and multiple comparison were controlled for using Tukey’s HSD. Fig. 6L: Schematic depicting the strong affinity of Annexin A5 for thrombi and its positive impact on EC envelopment and vascular recanalization during thromboangioplasticity. Data ± SEM. Scale bar = 20pm (Figs. 6A-6H).

[0085] Figs. 7A-7J demonstrate that the administration of Annexin A5 in a cerebral microembolic model reduces tissue injury and improves behavioral outcomes, in accordance with some embodiments. Fig. 7A: Timeline of experimental model, pharmacological treatment and outcome measurements. Fig. 7B: Annexin A5 immunofluorescence staining demonstrates a gradual increase in endogenous Annexin A5 levels in areas of ischemia (cyan), predominantly at the microvascular walls in occluded vessels (red) (right panel). Fig. 7C: Quantification of endogenous Annexin A5 immunofluorescence comparing areas of ischemia with non-ischemic areas at 6 and 72h (n=3 mice per timepoint, pixel intensity- averaged over 2 regions per mouse; tw o-w ay ANOVA with Sidak's multiple comparisons test). Measurement of fluorescence standard deviation was used to better highlight the vessel upregulation rather than using mean tissue fluorescence intensity. Fig. 7D: Immunoglobulin G immunofluorescence labeling (green) demonstrates extensive IgG leakage due to blood brain barrier breakdown in vehicle treatment. This was markedly reduced in Annexin A5- treated mice. Fig. 7E: Quantification of BBB breakdown comparing vehicle (n=12) and Annexin A5-treated mice (n=l 1) (Non-parametric, Mann-Whitney U test). Fig. 7F: Nissl stain at day 3 following occlusion reveals both infarct areas and microbleeds. Fig. 7G: Quantification of number of microbleeds, comparing vehicle and Annexin A5-treated mice (n=l 1-12 mice per treatment group, averaging 6 brain sections per mouse, non-parametric Mann- Whitney U test). Fig. 7H: Quantification of infarct area comparing vehicle and Annexin A5 treated mice (n=l 1-12 mice per treatment group, non-parametric Mann-Whitney U test). Fig. 71: Quantification of motor score (two-way ANOVA with Tukey’s multiple comparison test) and Fig. 7J, Cylinder test, show improved outcomes in Annexin A5 (i.v.) treated mice compared to controls (unpaired t-test; n=l 1-12 mice per treatment group). Data ± SEM. Scale bar = 0.5mm (Fig. 7B), 2mm (Fig. 7D), 1mm (Fig. 7F).

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[0088] Figs. 8A-8K demonstrate that vascular regression is triggered by anti-Annexin A5 antibodies in areas of thrombosis, in accordance with some embodiments. Fig. 8A: In vivo imaging at day 1 following administration of anti-Annexin A5 antibodies demonstrate a reduction in EC spreading around the thrombus. Fig. 8B: Quantification of EC plasticity following i.v. administration of Anti-Annexin A5 and A2 antibodies show a modest reduction in EC spreading for anti-Annexin A5 mAb at day 1 post-occlusion (Control IgG n= 44 mice with 92 clots, anti-Annexin A2 mAb n=8 mice with 20 clots, anti-Annexin A5 mAb n=23 mice with 61 clots; One-way ANOVA, Dunnetfs multiple comparisons test). Fig. 8C: in vivo images comparing the degree of vessel recanalization in control IgG versus anti-Annexin A5- treated mice. White arrowheads (low er panel) show persistent endothelial envelopment of the intravascular thrombus compared to the patent lumen (dim magenta) and extravasated thrombus (asterisk) in control mice (upper panel). The perivascular basal lamina is highlighted (bright magenta) due to accumulation of i.v. dye after multiple daily injections by day 6. Fig. 8D: Quantification of lumen recanalization at day 6 in IgG control and anti- Annexin A5 antibody -treated mice (Control n=6 mice with 13 clots, anti-Annexin A2 mAb n=6 mice with 16 clots, anti-Annexin A5 mAb n=9 mice with 18 clots; Nonparametric Kruskal-Wallis test, Dunn’s multiple comparison test). Fig. 8E: Time lapse imaging over 6 days in an anti-Annexin A5 antibody-treated mouse shows an enveloped intravascular thrombus at day 1 (asterisk). The vessel appears to undergo constriction in the vicinity of the thrombus at day 3 (white arrowhead) and is completely pruned by day 6, while the parent vessel remains intact (green). Fig. 8F: Time lapse imaging of a vessel with an occlusive thrombus (bright magenta) at day 1 in anti-Annexin A5 antibody-treated mice, shows gradual changes in EC architecture and vessel patency. Briefly, at day 2 the present study observed a pattern of EC remodeling with small EC protrusions forming (white arrowheads). By day 4, a region of vascular constriction in the occluded vessel is observed (white dotted box, zoomed below). By day 6 there is clear evidence of vascular regression (white dotted box, zoomed below ), concomitantly with the formation of a reticular pattern formed within the vessel lumen (white asterisk) and detachment of the vessel from the interconnected branch point (white arrowhead). Fig. 8G: Kaplan-Meier curve showing the degree of vessel survival (regression) in IgG controls, anti-Annexin A2 antibody, anti-Annexin A5 antibody and anti- Annexin A5 antibody-treated mice without thrombosis (Control IgG n=16 vessels; anti- Annexin A5 mAb n=31 vessels; anti-Annexin A2 mAb n=20 vessels; anti-Annexin A5 mAb / no thrombosis n=19 vessels; Log rank (Mantel-Cox) test, X2 = 32.31, PO.OOOl). Fig. 8H: Quantitative analysis of the different patterns of vessel outcomes following thrombosis in

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[0090] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817) primary thrombosed vessels versus immediately adjacent vessels in control IgG (n=17 vessels), anti-Annexin A2 (n=20 vessels) or anti-Annexin A5 mAb (n=40 vesselsj-treated mice. A chi - square test demonstrated significant differences in the distribution of vessel outcomes among the three groups ( x2= 18.82, p < 0.001). Pairwise chi - square comparisons were performed with Bonferroni correction (Control IgG vs AnxA2 P=1.00, X2= 1.37; Control IgG vs AnxA5 P=0.015, X2= 10.32; AnxA2 vs AnxA5 P=0.006, X2= 12.6). On further inspection in the Annexin A5 mAb-treated mice the present study noticed that regression occurred even more frequently in vessels that were adjacent to the thrombus but un-occluded (59%) compared to occluded vessels (41%). Fig. 81: Time-lapse imaging showing gradual SMC constriction (day 4) and remodeling and collapse (day 5) of a previously unoccluded vessel branch (white arrowhead), which is adjacent to a partially occluded parent vessel (asterisk). Fig. 8J: Intravital imaging of vessel pruning in SMC / EC fluorescent reporter mouse shows EC pruning. Notice SMC constriction (white arrowhead) and persistence of an SMC empty sleeve (white asterisk). Fig. 8K: Schematic summarizing coordinated multicellular processes during vascular pruning in anti-Annexin A5 treated mice. Data ± SEM. Scale bar = 20pm (Figs. 8A-8J).

[0091] Fig. 9A: Microphotography of the cranial window area showing multiple pial branches of the middle cerebral artery. Depicted is the initial location of FeCh application (black arrowhead) and the site of thrombus migration and final lodgment (white arrow). Fig. 9B: intravital imaging at day 4 in a transgenic reporter mouse with GFP-labeled platelets (Pf4-mTmG), showing platelet rich thrombi (green) and vascular remodeling around the thrombi (right panel, white arrowheads). Fig. 9C: intravenous administration of a fluorescent anti-CD41 -647 antibody (grey) highlights platelets within the intravascular thrombus in a Tie2-GFP mouse (EC, green). Fig. 9D: Injection of an intravascular dye (blue) intercalates with and highlights the thrombus as evidenced by strong colocalization with platelets labeled by anti-CD41-647 antibody (grey). Scale bars: 20pm (Figs. 9B-9D).

[0092] Figs. 10A-10D: Intravital imaging showing: (Fig. 10A) early stage endothelial lamellipodia projections at 8hrs (green, white arrowheads) that appear to be anchoring the thrombus (i.v. dye, magenta). (Figs. 10B-10C) Between days 1 and 2, the lamellipodia spread across the thrombus surface forming more robust stabilizing points of contact (white arrowheads). (Fig. 10D) Initially the endothelium makes contacts through lamellipodia that emerge from multiple sites throughout the vessel in the vicinity of the thrombus (white arrowheads). However, by day 2 the thrombus is mainly wrapped by lamellipodia polarized

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[0094] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817) to one side of the vessel, allowing the reformation of a vessel lumen (white asterisk). Scale bar: 20pm.

[0095] Fig. 1 1A: Schematic showing the pipeline for correlated in vivo to ex vivo tissue imaging of a site of thromboangioplasticity with high resolution confocal and transmission electron microscopy (TEM). Fig. 11B: Intravital imaging of a thrombus (dimly labeled w ith i.v. dye, magenta) and robustly enveloped by EC processes at day 3 (green). Fig. 11C: A tangential section of the brain that included the previously imaged cranial window, in Fig. 1 IB, was imaged with confocal microscopy, demonstrating the ability to relocate the same vessels ex vivo at a later timepoint. Fig. 1 ID: Immunofluorescence imaging following multiple injections of BrdU after thrombosis, indicates that while there is BrdU uptake in perivascular cells (red. white arrowheads), there is no colocalization with endothelial cells (green), indicating that endothelial envelopment of thrombi is likely to be mediated by EC migration rather than proliferation. See also correlated in vivo to TEM imaging, in Figs. 12A- 12E. Scale bar: 20p.m.

[0096] Fig. 12A: Top view of the surface of a vessel undergoing thromboangioplasticity shows the formation of small openings (white arrowheads) in the abluminal endothelial wall that appear to gradually coalesce and enlarge into a trans -endothelial tunnel (while dotted circle). Fig. 12B: Cross-sectional view of a vessel showing that tunnel coalescence can lead to large openings up to tens of microns in size (white arrowheads). Figs. 12C-12D: Large thrombi can undergo compartmentalization (day 1, white asterisks). Each compartment has a different spatio-temporal sequence of endothelial tunnel opening. Throughout the process there is negligible leakage of intravascular dye (magenta), despite extensive vascular remodeling and the presence of large EC tunnels. Notably, in larger thrombi, tunnel formation can happen at multiple sites (white arrowheads). Fig. 12E: Correlated light (see Fig. 2G) and TEM allowed relocation of a thrombus site for ultrastructural imaging. This reveals that despite extensive endothelial remodeling around thrombi, tight junctions are preserved (dotted box) in the vicinity of thrombi. EC (green), SMCs (red), Thrombus (light blue), Lumen (L). Scale bars: (A,B,C)=20pm; left panel of Fig. 12E= 1pm, right panel of Fig. 12E= 0.5 pm.

[0097] Fig. 13 A: Intravital imaging shows the normal formation of EC tunnels at day 3 in a Caveolin-1 knockout mouse (arrowhead). Fig. 13B: Quantification of the timing of EC tunnel formation comparing Caveolin-1 knockout with wildtype mice (WT Control n=26 mice with 64 clots, Caveolin-1 knockout n=3 mice with 6 clots) Unpaired t-test. Fig. 13C: In vivo thrombus imaging allows staining with an antibody against the Plasmalemma Vesicle

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[0100] Associated Protein (anti PV-1-647) (grey) typically present in fenestrated vessels. This data shows that there is no colocalization of PV-1 with areas of EC thrombus envelopment (red arrowheads). Interestingly, PV-1 was observed sometimes in areas distant from thrombi (white dotted rectangles and lower panels, grey) preferential binding to what appear to be nascent EC projections (Fig. 13D) but not thrombus envelopment areas where tunnels are expected to develop Scale Bars (Figs. 13A, 13C, and left panel of Fig. 13D)=20pm, (Right panels of Fig. 13D)=5pm.

[0101] Fig. 14A: Intravital imaging showing invasion of SMC lamellipodia (white arrowheads) at sites of EC tunnel openings (white arrow). Fig. 14B: Intravital imaging showing SMC projections crossing through sites with interruptions in the elastin / basal lamina layer (labeled with hydrazide-633 dye) (white arrowheads). Fig. 14C: large thrombi are fragmented into multiple endothelial compartments that are invaded by SMC projections through EC tunnels at different times (see parallel data in Figs. 12C-12D). Fig. 14D: Intravital imaging at day 5 showing a fully recanalized vessel (asterisk) with residual thrombus material labeled with anti-CD41-647 (white) encapsulated by perivascular SMC processes (red). Fig. 14E: Relocation of a thrombus for subsequent TEM of a site showing the formation of compartments containing fragments of thrombi within SMC processes (light red), and directly adjacent to endothelial processes (light green). Fig. 14F: Correlated in vivo- ex vivo immunofluorescence imaging demonstrates the presence of Ibal positive macrophages (white) within the thrombus cavity’ (red). Scale bars: (A,B,C,D.E,F)= 20pm and E= 0.5 pm.

[0102] Fig. 15 A: Distribution of thrombus sizes shows that there is no impairment in thrombus formation at 1 hour in MerTK" mice. (WT control n=10 mice with 31 clots; MerTK'7' n=6 mice with 18 clots; Mann Whitney U t-test). Fig. 15B: Comparison of tail bleeding times shows no statistical difference between different genotypes (Tie2GFP n=5 mice; Tie2GFP: MerTK' ' n=5 mice; MerTK'7' n=6 mice; One-way ANOVA with Tukey’s multiple comparisons test). Fig. 15C: Quantification of thrombus washout from 1 to 24hrs in control and MerTK' ' mice. (WT control n=9 mice with 23 clots; MerTK " n=6 mice with 16 clots) Mann Whitney U test. Data ± SEM.

[0103] Fig. 16A: Intravital imaging comparing EC plasticity at 24 hrs in control, MerTK'7' and Axl'7' mice. Fig. 16B: Quantification of EC envelopment of thrombi at 24hrs in control, MerTK'7' and Axl'7' mice. (WT control n=42 mice with 79 clots; MerTK'7' n=16 mice with 33 clots; Axl'7' n=22 mice with 43 clots) Kruskal-Wallis with Dunn’s multiple comparisons test. Data ± SEM. Scale bar: (Fig. 16A) 20pm.

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[0106] Fig. 17A: Quantification of endothelial envelopment at 24 hrs in mice pretreated with Cilengitide (oc5 / |33 and a5 / ]35 integrin inhibitor; Vehicle n=42 mice with 86 clots; Cilengitide n=6 mice with 15 clots; Unpaired t-test); or Fig. 17B: DC101 (VEGFR2 blocking antibody. Vehicle n=42 mice with 86 clots; DC101 n=6 mice with 15 clots; Unpaired t-test). Fig. 17C: Immunofluorescence imaging showing mild VEGF staining (grey) within platelet-rich thrombi (CD41, red). Data ± SEM. Scale bar: (Fig. 17C) 20pm.

[0107] Fig. 18A: Anti-Annexin A2 immunofluorescence staining at 3 days post fibrin microemboli injection shows strong vascular binding at sites of occlusion demonstrating a robust endogenous upregulation of Annexin A2. Fig. 18B; High resolution image of Annexin A2 vascular upregulation (anti- Annexin A2 mAh, white) at a site of embolus occlusions (red). Fig. 18C: Quantification of anti-Annexin 2 immunofluorescence at different time points using a measure of standard deviation to account for the vessel upregulation rather than the overall mean tissue fluorescence intensity (non-infarcted tissue n=3 mice; infarcted tissue n=3 mice; 2-way ANOVA with Sidak's multiple comparisons test). Data ± SEM. Scale bar: (Fig. 18A) 1mm.

[0108] Fig. 19A: Quantification of EC lamellipodia formed at 8hrs post thrombus occlusion in mice treated with neutralizing antibodies against Annexin A2 and A5 compared to isotype IgG control (Control IgG n=l 1 clots, Annexin A2 n=8 clots, Annexin A5 n=20 clots; One way ANOVA with Tukey's HSD). Fig. 19B: Time lapse imaging demonstrates that intravenous anti-Annexin A5 administration is associated with vessel regression in primary occluded vessels undergoing thromboangioplasticity (see also Figs. 8A-8K for regression in adjacent un-occluded vessels). Fig. 19C: anti-Annexin A5 administration leads to EC regression (green, white arrowhead), which is accompanied by a SMC remodeling (red) around regressing vessels. Data ± SEM. Scale bar: (B,C) 20pm.

[0109] Fig. 20 illustrates certain aspects of the thromboangioplasticity, in accordance with some embodiments. Thromboangioplasticity is a coordinated mechanism in the microvasculature involving endothelial and smooth muscle cell plasticity, bridging hemostasis and tissue repair. This schematic highlights a newly uncovered mechanism in microvessels ( 70 pm in diameter, which constitutes over 90% of all blood vessels in various organs), demonstrating how classical platelet-fibrin hemostasis is intimately linked with cellular remodeling by endothelial cells (ECs) and smooth muscle cells (SMCs). While traditional models of thrombosis focus on platelet aggregation and fibrin mesh formation, they do not fully account for how clots are stabilized, reorganized, and ultimately cleared

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[0111] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817) from the cerebrovasculature. The findings reveal that thromboangioplasticity is critical for bridging these events, ensuring stable clot retention (to prevent hemorrhage) and coordinated vessel repair (to re-establish blood flow). Phase 1 (Day 1-3): Hematogenous Stage Upon injury, platelets (red) adhere via von Willebrand Factor (vWF; dark blue) and integrins (orange / red), while tissue factor (TF; green) sparks a coagulation cascade generating thrombin, which converts fibrinogen into fibrin (gray), further stabilized by Factor XIII. Endogenous tPA predominantly degrades fibrin in the first ~6-8 hours, before the clot is encapsulated by ECs. Concurrently, phosphatidylserine (PtSer) (blue) is externalized on activated platelets and binds to Annexin V (hexagons; yellow). Gas6 (magenta), complexed with PtSer, then activates MerTK receptors (purple “Y’’) on ECs (pink). This induces EC lamellipodia (green arcs) that envelop the clot, anchoring it against the vessel wall and preventing premature washout or hemorrhage. Phase 2 (Day 3-6): Vascular Remodeling Once anchored, EC remodeling proceeds over time, likely through cell migration, rather than cell division. This leads to full envelopment of the thrombus. Concurrently, there is formation of large transcellular tunnels on the original abluminal endothelium (green). Despite tunnel formation, EC remodeling involves tight junction formation, preventing plasma leakage. Through these tunnels, SMCs (light-pink) penetrate the thrombus cavity, fully enveloping the thrombi, compacting and fragmenting the thrombus, leading to ultimate extrusion from the lumen. This process is mediated through Axl receptors located on SMC’s (yellow “Y’") which sense PtSer-Gas6 signals. Phase 3 (Day >5) : Resolution and Recanalization SMCs progressively extrude the compacted thrombus to recanalizes the vessel and restores blood flow. In so doing, thromboangioplasticity provides a coordinated, multicellular response to microvascular obstructions and plays a critical role in bridging hemostatic responses to injury, sparing blood vessels and allowing tissue repair.

[0112] DETAILED DESCRIPTION

[0113] The following disclosure provides many different embodiments, or examples, for implementing different features of the provided subject matter. Specific examples of components and arrangements are described below to simplify the present disclosure. These are, of course, merely examples and are not intended to be limiting. For example, the formation of a first feature over or on a second feature in the description that follows may include embodiments in which the first and second features are formed in direct contact, and may also include embodiments in which additional features may be formed between the first and second features, such that the first and second features may not be in direct contact. In

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[0115] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817) addition, the present disclosure may repeat reference numerals and / or letters in the various examples. This repetition is for the purpose of simplicity and clarity and does not in itself dictate a relationship between the various embodiments and / or configurations discussed.

[0116] While hematogenous factors have been extensively studied in relation to thrombus formation and clotting, the coordinated multicellular mechanisms at the interface between hemostasis and tissue repair in the thromboangioplasticity process are not well understood. This knowledge gap is mainly due to the classical biochemical in vitro studies of hemostasis, which have not explored the continuum between hemostasis and tissue repair at the multicellular level in living organisms.

[0117] To address this gap, novel methodologies were developed for observing endogenous thrombi and vascular cellular structures with high spatio-temporal resolution in live mice. This approach allowed the capture of the entire sequence of events, starting from the formation of endogenous thrombi to the restoration of blood flow in individual vessels. By employing this methodology, previously unknown cellular and molecular mechanisms in the thromboangioplasticity process that regulate thrombosis and vascular recanalization, which have crucial implications for tissue viability and the understanding of the pathophysiology of various disorders, were discovered.

[0118] Referring to Fig. 1, the thromboangioplasticity process can be divided into four phases. In the first phase, a thrombus is formed in the lumen of a blood vessel and the endothelial cells (ECs) lining the interior wall of the blood vessel sense the presence of the thrombus. In the second phase, the endothelial cells migrate toward and and envelop the thrombus. In the third phase, abluminal endothelial cells near the thrombus retract to allow underlining smooth muscle cells (SMCs) to penetrate into the lumen and surround the thrombus. In the fourth phase, the thrombus engulfed by the SMCs is retracted from the blood vessel and subsequently degraded, which allows the blood vessel to be recanalized.

[0119] In the study described herein (“the present study”), it was discovered that the inhibition of MerTK or Axl TAM receptors was detrimental to the thromboangioplasticity process. Specifically, the inhibition of MerTK or Axl TAM receptors prevents formation of initial endothelial lamellipodia (which is required for the endothelial cell to move and thus engulf the thrombus), which in turn led to premature thrombus washout and increased bleeding. This observation led to the hypothesis that a beneficial effect could be achieved by accelerating the initial endothelial process via the agonistic action on the MerTK receptor. MerTK or Axl TAM receptors are activated by proteins such as Growth Arrest-Specific 6 (GAS6) and protein S.

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[0121] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817)

[0122] It was further discovered that the administration of annexin V accelerated or enhanced the process downstream of the initial endothelial lamellipodia formation, such as the full envelopment of the thrombus by endothelial cells, the opening of EC fenestrations (small openings) which allows adjacent smooth muscle cells (SMCs) to infiltrate and compact the thrombus, and the final extrusion of the thrombus out of the vessel lumen.

[0123] Based on these discoveries, it was hypothesized that the administration of both an agonist of the MerTK / Axl TAM receptors and annexin V would be able to achieve at least two outcomes at the same time: 1) accelerating the initial phase of endothelial projections that entrap the thrombus, which diminishes the likelihood of vessel damage and bleeding following ischemia, and 2) accelerate the subsequent stages of the vascular remodeling (e.g., EC and SMC plasticity), leading to full vessel recanalization and reestablishment of blood flow. It was hypothesized that the combination of these factors would allow the rapid reestablishment of normal tissue physiology and improves the overall outcome following tissue ischemia or injury.

[0124] To test this hypothesis, the present study generated a fusion protein including the full- length annexin V / GAS6. Preliminary results demonstrated that this fusion protein was indeed able to expedite both the initial phase and the subsequent phases of the thromboangioplasticity process. Specifically, the synergistic effect of the fusion protein hastened the initial process of endothelial engulfment, which in turn diminishes the occurrence of premature thrombus washout and secondary hemorrhaging. Additionally, the fusion protein enhanced the subsequent stage of plasticity of both endothelial and smooth muscle cells, which is crucial for the complete expulsion of a thrombus from the vessel and for the comprehensive repair of the vessel and ultimately leads to the reestablishment of normal blood flow. In other words, the novel fusion protein offers a solution that works across the full continuum of the thromboangioplasticity process, from the earliest instances of tissue injury, hemorrhage, and thrombosis, through to the final stages of recanalization and repair.

[0125] Thromboembolic occlusions of the microvasculature are implicated in various diseases and disorders. Such diseases and disorders include, for example, acute ischemic conditions including stroke and myocardial infarction, tissue injuries in various organs, and micro-occlusive diseases / disorders.

[0126] Thus, the approach herein is a novel therapeutic strategy for, among others, microocclusive disorders in multiple organs. This strategy’ also has the potential to reverse the deleterious effects of anti-annexin V antibodies present in autoimmune disorders.

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[0129] Accordingly, in some aspects, the present invention is directed to constructs and compositions for revascularizing a blood vessel having a thromboembolic occlusion, or for treating, ameliorating and / or preventing a disease caused by or involving a blood vessel restricted by thromboembolic occlusion.

[0130] Definitions

[0131] As used herein, each of the following terms has the meaning associated with it in this section. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Generally, the nomenclature used herein and the laboratory' procedures in animal pharmacology, pharmaceutical science, peptide chemistry’, and organic chemistry are those w ell-known and commonly employed in the art. It should be understood that the order of steps or order for performing certain actions is immaterial, so long as the present teachings remain operable. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety', as though individually incorporated by reference.

[0132] In the application, where an element or component is said to be included in and / or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components and can be selected from a group consisting of tw o or more of the recited elements or components.

[0133] In the methods described herein, the acts can be carried out in any order, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0134] In this document, the terms "a," "an," or "the" are used to include one or more than one unless the context clearly dictates otherwise. The term "or" is used to refer to a nonexclusive "or" unless otherwise indicated. The statement "at least one of A and B" or "at least one of A or B" has the same meaning as "A, B, or A and B."

[0135] "About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, in

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[0137] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817) certain embodiments ±5%, in certain embodiments ±1%, in certain embodiments ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0138] Composition

[0139] In some aspects, the present study is directed to compositions.

[0140] In some embodiments, the composition speeds up a thromboangioplasticity process, clot removal from a blood vessel, vessel recanalization, reestablishment of blood flow, and / or tissue repair in a subject.

[0141] In some embodiments, the composition treats, ameliorates, and / or prevents a disease or a disorder caused by or involving damage of a blood vessel or thrombotic occlusion in a blood vessel in a subject in need thereof. In some embodiments, the disease or disorder includes a tissue ischemia, a physical injury, a stroke, and / or a myocardial infarction.

[0142] In some embodiments, the composition reduces the risk of hemorrhage after injury and / or the risk of hemorrhagic conversion due to stroke or organ trauma.

[0143] In some embodiments, the composition treats, ameliorates, and / or prevents a condition associated with anti-annexin V autoimmune antibodies. In some embodiments, the condition associated with anti-annexin V autoimmune antibodies includes miscarriage and fetal loss during pregnancy (e.g., pregnancy loss, recurrent 3) and / or antiphospholipid syndrome.

[0144] In some embodiments, the composition treats, ameliorates, and / or prevents thrombo- hemorrhage in subjects carrying a mutant annexin V.

[0145] In some embodiments, the composition includes a first polypeptide including a growth arrest-specific 6 (GAS6) polypeptide and / or a protein S polypeptide; and / or a second polypeptide including an annexin V polypeptide.

[0146] In some embodiments, the GAS6 polypeptide includes the following amino acid sequence:

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[0148] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817)

[0149] In some embodiments, the GAS6 polypeptide is a protein product of the human gene occupying the same allele of the gene that produces SEQ ID NO:1. In some embodiments, the GAS6 polypeptide is an ortholog protein of SEQ ID NO: 1 in a non-human species (such as a non-human mammalian species). In some embodiments, the GAS6 polypeptide is a recombinant version of a human or non-human GAS6 protein. In some embodiments, the GAS6 polypeptide is a functionally active fragment of a human or non-human GAS6 protein, such as a fragment that is able to activate the MerTK or the Axl TAM receptors.

[0150] In some embodiments, the protein S polypeptide includes the following amino acid sequence:

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[0153] In some embodiments, the protein S polypeptide is a protein product of the human gene occupying the same allele of the gene that produces SEQ ID NO:2. In some embodiments, the protein S polypeptide is an ortholog protein of SEQ ID NO:2 in a nonhuman species (such as a non-human mammalian species). In some embodiments, the protein S polypeptide is a recombinant version of a human or non-human protein S protein. In some embodiments, the protein S polypeptide is a functionally active fragment of a human or non- human protein S protein, such as a fragment that is able to activate the MerTK and / or the Axl TAM receptor(s).

[0154] In some embodiments, the annexin V polypeptide includes the following amino acid sequence:

[0155] In some embodiments, the annexin V polypeptide is a protein product of the human gene occupying the same allele of the gene that produces SEQ ID NOG. In some embodiments, the annexin V polypeptide is an ortholog protein of SEQ ID NOG in a non- human species (such as a non-human mammalian species). In some embodiments, the annexin V polypeptide is a recombinant version of a human or non-human annexin V protein. In some embodiments, the annexin V polypeptide is a functionally active fragment of a human or non-human annexin V protein, such as a functional active fragment that is able to accelerate or enhance the process dow nstream of the initial endothelial lamellipodia formation.

[0156] In some embodiments, the composition, instead of including the first polypeptide and the second polypeptide, includes a first nucleic acid encoding the first polypeptide; and a second nucleic acid encoding the second polypeptide. In some embodiments, the first nucleic acid and / or the second nucleic acid are part of an expression vector.

[0157] In some embodiments, the first polypeptide and the second polypeptide are two separate polypeptide molecules.

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[0159] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817)

[0160] In some embodiments, the first polypeptide and the second polypeptide are not covalently linked to each other.

[0161] In some embodiments, the first polypeptide and the second polypeptide are covalently linked to each other, such as directly or through a linker. In some embodiments, the N-terminus of the first polypeptide is covalently linked to the C-terminus of the second polypeptide.

[0162] In some embodiments, the C-terminus of the first polypeptide is covalently linked to the N-terminus of the second polypeptide.

[0163] In some embodiments, both the first polypeptide and the second polypeptide are parts of the same polypeptide, such as a fusion protein.

[0164] In some embodiments, the fusion protein has a sequence identity of about 90% or more, such as about 95% or more, 98% or more, or 100% with the polypeptide set forth in SEQ ID NON.

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[0166] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817)

[0167] In some embodiments, the composition further includes a pharmaceutically acceptable carrier.

[0168] In some embodiments, the composition is formulated for a parenteral administration. Non-limiting examples of parenteral administration include intradermal administration, subcutaneous administration, intramuscular administration, intravenous administration, and the like.

[0169] Construct and Nucleic Acid Encoding the Same

[0170] In some aspects, the present invention is directed to non-natural constructs.

[0171] In some embodiments, the construct speeds up a thromboangioplasticity process, clot removal from a blood vessel, vessel recanalization, reestablishment of blood flow, and / or tissue repair in a subject.

[0172] In some embodiments, the construct treats, ameliorates, and / or prevents a disease or a disorder caused by or involving damage of a blood vessel or thrombotic occlusion in a blood vessel in a subject in need thereof. In some embodiments, the disease or disorder includes a tissue ischemia, a physical injury, a stroke, and / or a myocardial infarction.

[0173] In some embodiments, the construct reduces the risk of hemorrhage after injury and / or the risk of hemorrhagic conversion due to stroke or organ trauma.

[0174] In some embodiments, the construct treats, ameliorates, and / or prevents a condition associated with anti-annexin V autoimmune antibodies. In some embodiments, the condition associated with anti-annexin V autoimmune antibodies includes miscarriage and fetal loss during pregnancy (e.g., Pregnancy Loss, Recurrent 3) and / or antiphospholipid syndrome.

[0175] In some embodiments, the construct treats, ameliorates, and / or prevents thrombo- hemorrhage in subjects earn ing a mutant annexin V.

[0176] In some embodiments, the construct includes a first polypeptide comprising a grow th arrest-specific 6 (GAS6) polypeptide and / or a protein S polypeptide; and / or a second polypeptide comprising an annexin V polypeptide.

[0177] In some embodiments, the first polypeptide and the second polypeptide are the same as or similar to those described elsewhere herein, such as in the “Composition” section.

[0178] In some embodiments, the construct is a fusion protein including the first polypeptide and the second polypeptide.

[0179] In some aspects, the present invention is directed to a nucleic acid encoding the nonnatural construct.

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[0182] In some embodiments, the nucleic acid is part of an expression vector.

[0183] Method of Treating, Ameliorating and / or Preventing Disease or Disorder Associated with Blood Vessel Thromboembolic or Embolic Occlusion

[0184] In some aspects, the present invention is directed to a method of treating, ameliorating, and / or preventing a disease or disorder caused by or involving a blood vessel thromboembolic occlusion or embolic occlusion in a subject in need thereof.

[0185] In some embodiments, the blood vessel thromboembolic occlusion is a thromboembolic occlusion in a microvasculature blood vessel.

[0186] In some embodiments, the disease or disorder includes a tissue ischemia, a physical injury, a stroke, or a myocardial infarction.

[0187] In some embodiments, the method includes administering to the subject an effective amount of the composition, construct or nucleic acid herein. The composition, construct or nucleic acid are described elsewhere herein, such as in the ‘"Composition” and the “Construct and Nucleic Acid Encoding the Same” section.

[0188] In some embodiments, the composition or the construct is administered parenterally.

[0189] In some embodiments, the administration of the composition or the construct speeds up of a thromboangioplasticity process, a clot removal from a blood vessel, a vessel recanalization, a reestablishment of blood flow, and / or a tissue repair in the subject.

[0190] In some embodiments, the subject is a mammal.

[0191] In some embodiments, the subject is a human.

[0192] Method of Speeding Up Thromboangioplasticity process in Blood Vessel Having Thrombotic or Embolic Occlusion

[0193] In some aspects, the present invention is directed to a method of speeding up a thromboangioplasticity process in a blood vessel having a thrombotic occlusion or an embolic occlusion.

[0194] In some embodiments, the method includes contacting the blood vessel with the composition or construct herein, or delivering in a cell in, within or in proximity to the blood vessel the nucleic acid herein. In some embodiments, in the case that the nucleic acid is delivered in the cells in, within or in proximity to the blood vessel, the nucleic acid is transcribed and / or translated to produce the GAS6, protein S and / or annexin V polypeptide, which then function to speed up the thromboangioplasticity process.

[0195] In some embodiments, the blood vessel is a microvasculature blood vessel.

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[0197] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817)

[0198] Vectors

[0199] Vectors can increase the stability7of the nucleic acids, make the delivery easier, or allow the expression of the nucleic acids or protein products thereof in the cells. Accordingly, in some embodiments, the nucleic acids herein, such as those encoding the first polypeptide, the second peptide and / or the fusion protein, are incorporated into a vector.

[0200] In some embodiments, the instant specification relates to a vector, including the nucleic acid sequence of the instant specification or the construct of the instant specification. The choice of the vector will depend on the host cell in which it is to be subsequently introduced. In certain embodiments, the vector of the instant specification is an expression vector. Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. In certain embodiments, the expression vector is selected from the group consisting of a viral vector, a bacterial vector and a mammalian cell vector. Prokaryote- and / or eukaryote-vector based systems can be employed for use with the instant specification to produce polynucleotide, or their cognate polypeptides. Many such systems are commercially and widely available.

[0201] In some embodiments, the vector is a viral vector. Viral vector technology is w ell known in the art and is described, for example, in virology and molecular biology7manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. (See, e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193.

[0202] In some embodiments, the viral vector is a suitable adeno-associated virus (AAV), such as the AAV1-AAV8 family of adeno-associated viruses. In some embodiments, the viral vector is a viral vector that can infect a human. The desired nucleic acid sequence, such as the nucleic acids that encoding the polypeptides or fusion proteins herein, can be inserted between the inverted terminal repeats (ITRs) in the AAV. In various embodiments, the viral vector is an AAV2 or an AAV8. The promoter can be a thyroxine binding globulin (TBG) promoter. In various embodiments, the promoter is a human promoter sequence that enables the desired nucleic acid expression in the brain. In some embodiments, the promoter is an endothelial cell promoter, or a blood cell-specific promotor. The AAV can be a recombinant AAV, in which the capsid comes from one AAV serotype and the ITRs come from another AAV serotype. In various embodiments, the AAV capsid is selected from the group

[0203] 27

[0204] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817) consisting of a AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and a AAV8 capsid. In various embodiments, the ITR in the AAV is at least one ITR selected from the group consisting of a AAV 1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and an AAV8 ITR. In various embodiments, the instant specification contemplates an AAV8 viral vector (recombinant or non-recombinant) containing a desired nucleic acid expression sequence and at least one promoter sequence that, when administered to a subject, causes elevated systemic expression of the desired nucleic acid. In some embodiments, the viral vector is a recombinant or non-recombinant AAV2 or AAV5 containing any of the desired nucleic acid expression sequences described herein. In some embodiments, the AAV is an engineered AAVs for delivering nucleic acid across the blood brain barrier to the central and peripheral nervous systems to deliver the polypeptide and / or fusion proteins into these system. Such AAVs are described in, e.g., Chan et al., Nat Neurosci. 2017 Aug; 20(8): 1172-1 179. The entirety of this reference is incorporated herein by reference.

[0205] In some embodiments, the vector in which the nucleic acid sequence is introduced is a plasmid that is or is not integrated in the genome of a host cell when it is introduced in the cell. Illustrative, non-limiting examples of vectors in which the nucleotide sequence of the instant specification or the gene construct of the instant specification can be inserted include a tet-on inducible vector for expression in eukary ote cells.

[0206] The vector may be obtained by conventional methods known by persons skilled in the art (Sambrook et al., 2012). In certain embodiments, the vector is a vector useful for transforming animal cells.

[0207] In certain embodiments, the recombinant expression vectors may also contain nucleic acid molecules which encode a peptide or peptidomimetic inhibitor of the instant specification, described elsewhere herein.

[0208] A promoter may be one naturally associated with a gene or polynucleotide sequence, as may be obtained by isolating the 5’ non-coding sequences located upstream of the coding segment and / or exon. Such a promoter can be referred to as "endogenous." Similarly, an enhancer may be one naturally associated with a polynucleotide sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a polynucleotide sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of

[0209] 28

[0210] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817) other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not "naturally occurring," i.e., containing different elements of different transcriptional regulator}7regions, and / or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and / or nucleic acid amplification technology, including PCR™. in connection with the compositions disclosed herein (U.S. Patent 4,683,202, U.S. Patent 5,928,906). Furthermore, it is contemplated the control sequences that direct transcription and / or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.

[0211] It will be important to employ a promoter and / or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism chosen for expression. Those of skill in the art of molecular biology generally know how to use promoters, enhancers, and cell type combinations for protein expression. The promoters employed may be constitutive, tissue-specific, inducible, and / or useful under the appropriate conditions to direct high-level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

[0212] The recombinant expression vectors may also contain a selectable marker gene which facilitates the selection of transformed or transfected host cells. Suitable selectable marker genes are genes encoding proteins such as G418 and hygromycin which confer resistance to certain drugs, P-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or an immunoglobulin or portion thereof such as the Fc portion of an immunoglobulin preferably IgG. The selectable markers may be introduced on a separate vector from the nucleic acid of interest.

[0213] Combination Therapies

[0214] In some embodiments, in addition to the composition, construct and / or nucleic acid herein, the subject is further administered at least one additional agent that treats, ameliorates, and / or prevents a disease and / or disorder contemplated herein. In other embodiments, the composition, construct and / or nucleic acid and the at least one additional agent are coadministered to the subject. In yet other embodiments, the compound and the at least one additional agent are co-formulated.

[0215] Non-limiting examples of compounds that can be combined with the composition, construct and / or nucleic acid herein include thrombolytic agents (such as Streptokinase,

[0216] 29

[0217] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817)

[0218] Alteplase, Reteplase, Tenecteplase, Urokinase, Prourokinase, Anistreplase(APSAC), and the like), anticoagulant drugs (such as apixaban, dabigatran, edoxaban, rivaroxaban, warfarin, and the like), and the like.

[0219] The compounds contemplated within the disclosure are intended to be useful in combination with the composition, construct and / or nucleic acid herein. These additional compounds may comprise the composition, construct and / or nucleic acid of the present disclosure and / or at least one additional agent for treating the disease / disorder herein.

[0220] A synergistic effect may be calculated, for example, using suitable methods such as, for example, the Sigmoid-Emax equation (Holford & Scheiner, 1981, Clin. Pharmacokinet. 6:429-453), the equation of Loewe additivity (Loew e & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114:313-326) and the median-effect equation (Chou & Talalay, 1984, Adv. Enzyme Regul. 22:27-55). Each equation referred to above may be applied to experimental data to generate a corresponding graph to aid in assessing the effects of the drug combination. The corresponding graphs associated with the equations referred to above are the concentration-effect curve, isobologram curve and combination index curve, respectively.

[0221] Administration / Dosage / Formulations

[0222] The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations contemplated within the disclosure may be administered to the subject either prior to or after the onset of a disease and / or disorder contemplated herein. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations contemplated within the disclosure may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

[0223] Administration of the compositions contemplated within the disclosure to a patient, preferably a mammal, more preferably a human, may be carried out using know n procedures, at dosages and for periods of time effective to treat a disease and / or disorder contemplated herein in the patient. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the state of the disease or disorder in the patient; the age, sex, and w eight of the patient; and the ability of the therapeutic compound contemplated within the disclosure to treat a disease and / or disorder contemplated herein in the patient. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be

[0224] 30

[0225] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817) proportionally reduced as indicated by the exigencies of the therapeutic situation. A nonlimiting example of an effective dose range for a therapeutic compound contemplated within the disclosure is from about 1 and 5,000 mg / kg of body weight / per day. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.

[0226] Actual dosage levels of the active ingredients in the pharmaceutical compositions contemplated within the disclosure may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0227] In particular, the selected dosage level depends upon a variety of factors including the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds or materials used in combination with the compound, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well, know n in the medical arts.

[0228] A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds contemplated within the disclosure employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0229] In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity' of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary’ dosages for the patients to be treated; each unit containing a predetermined quantity7of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms contemplated w ithin the disclosure are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding / formulating such a therapeutic compound for the treatment of a disease and / or disorder contemplated herein.

[0230] In certain embodiments, the compositions of the disclosure are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the

[0231] 31

[0232] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817) pharmaceutical compositions of the disclosure comprise a therapeutically effective amount of a compound of the disclosure and a pharmaceutically acceptable carrier.

[0233] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

[0234] In certain embodiments, the compositions of the disclosure are administered to the patient in dosages that range from one to five times per day or more. In another embodiment, the compositions of the disclosure are administered to the patient in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every' two weeks. It is readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the disclosure varies from individual to individual depending on many factors including, but not limited to, age. disease or disorder to be treated, gender, overall health, and other factors. Thus, the disclosure should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient is determined by the attending physical taking all other factors about the patient into account.

[0235] Compounds of the disclosure for administration may be in the range of from about 1 pg to about 10,000 mg, about 20 pg to about 9,500 mg, about 40 pg to about 9,000 mg, about 75 pg to about 8,500 mg, about 150 pg to about 7,500 mg, about 200 pg to about 7,000 mg, about 3050 pg to about 6,000 mg, about 500 pg to about 5,000 mg, about 750 pg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg to about 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800 mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80 mg to about 500 mg, and any and all whole or partial increments therebetween.

[0236] 32

[0237] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817)

[0238] In some embodiments, the dose of a compound of the disclosure is from about 1 mg and about 2,500 mg. In some embodiments, a dose of a compound of the disclosure used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg. or less than about 10 mg, or less than about 5 mg. or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.

[0239] In certain embodiments, the present disclosure is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the disclosure, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of the disease / disorder of the present disclosure in a patient.

[0240] Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for intracranially, intrathecal , oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, know n to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and / or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents.

[0241] Routes of administration of any of the compositions of the disclosure include oral, nasal, rectal, intravaginal, parenteral, buccal, sublingual or topical. The compounds for use in the disclosure may be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical, intrapulmonary. intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.

[0242] 33

[0243] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817)

[0244] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present disclosure are not limited to the particular formulations and compositions that are described herein.

[0245] Oral Administration

[0246] For oral application, particularly suitable are tablets, dragees, liquids, drops, suppositories, or capsules, caplets and gelcaps. The compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutically excipients that are suitable for the manufacture of tablets. Such excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate. The tablets may be uncoated or they may be coated by known techniques for elegance or to delay the release of the active ingredients. Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert diluent.

[0247] For oral administration, the compounds of the disclosure may be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., polyvinylpyrrolidone, hydroxypropylcellulose or hydroxypropylmethylcellulose); fillers (e.g., cornstarch, lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrates (e.g., sodium starch gly collate); or wetting agents (e.g., sodium lauryl sulphate). If desired, the tablets may be coated using suitable methods and coating materials such as OPADRY™ film coating systems available from Colorcon, West Point. Pa. (e.g., OPADRY™ OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type and OPADRY™ White, 32K18400). Liquid preparation for oral administration may be in the form of solutions, syrups or suspensions. The liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g.,

[0248] 34

[0249] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817) lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); and preservatives (e.g.. methyl or propyl p-hydroxy benzoates or sorbic acid).

[0250] The present disclosure also includes a multi-layer tablet comprising a layer providing for the delayed release of one or more compounds of the disclosure, and a further layer providing for the immediate release of another medication. Using a wax / pH-sensitive polymer mix, a gastric insoluble composition may be obtained in which the active ingredient is entrapped, ensuring its delayed release.

[0251] Parenteral Administration

[0252] For parenteral administration, the compounds of the disclosure may be formulated for injection or infusion, for example, intravenous, intramuscular or subcutaneous injection or infusion, or for administration in a bolus dose and / or continuous infusion. Suspensions, solutions or emulsions in an oily or aqueous vehicle, optionally containing other lormulatory agents such as suspending, stabilizing and / or dispersing agents may be used.

[0253] Additional Administration Forms

[0254] Additional dosage forms of this disclosure include dosage forms as described in U.S. Patents Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389; 5,582,837; and 5,007,790. Additional dosage forms of this disclosure also include dosage forms as described in U.S. Patent Applications Nos. 20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and 20020051820. Additional dosage forms of this disclosure also include dosage forms as described in PCT Applications Nos. WO 03 / 35041; WO 03 / 35040; WO 03 / 35029; WO 03 / 35177; WO 03 / 35039; WO 02 / 96404; WO 02 / 32416; WO 01 / 97783; WO 01 / 56544; WO 01 / 32217; WO 98 / 55107; WO 98 / 11879; WO 97 / 47285; WO 93 / 18755; and WO 90 / 11757.

[0255] Controlled Release Formulations and Drug Delivery Systems

[0256] In certain embodiments, the formulations of the present disclosure may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.

[0257] The term sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may. although not necessarily, result in substantially constant blood levels of a drug over

[0258] 35

[0259] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817) an extended time period. The period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form.

[0260] For sustained release, the compounds may be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the compounds. As such, the compounds for use the method of the disclosure may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.

[0261] In certain embodiments of the disclosure, the compounds of the disclosure are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.

[0262] The term delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that mat, although not necessarily, includes a delay of from about 10 minutes up to about 12 hours.

[0263] The term pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration.

[0264] The term immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration.

[0265] As used herein, short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration.

[0266] As used herein, rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration.

[0267] Dosing

[0268] The therapeutically effective amount or dose of a compound of the present disclosure depends on the age, sex and weight of the patient, the current medical condition of the patient and the progression of the disease / disorder herein in the patient being treated. The skilled artisan is able to determine appropriate dosages depending on these and other factors.

[0269] A suitable dose of a compound of the present disclosure may be in the range of from about 0.01 mg to about 5,000 mg per day, such as from about 0. 1 mg to about 1,000 mg, for

[0270] 36

[0271] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817) example, from about 1 mg to about 500 mg, such as about 5 mg to about 250 mg per day. The dose may be administered in a single dosage or in multiple dosages, for example from 1 to 4 or more times per day. When multiple dosages are used, the amount of each dosage may be the same or different. For example, a dose of 1 mg per day may be administered as two 0.5 mg doses, with about a 12-hour interval between doses.

[0272] It is understood that the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.

[0273] In the case wherein the patient's status does improve, upon the doctor's discretion the administration of the modulator of the disclosure is optionally given continuously; alternatively, the dose of drug being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., a "drug holiday"). The length of the drug holiday optionally varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. The dose reduction during a drug holiday includes from 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%. 50%. 55%. 60%. 65%. 70%. 75%. 80%. 85%. 90%. 95%. or 100%.

[0274] Once improvement of the patient's conditions has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, is reduced, as a function of the patient's condition, to a level at which the improved disease is retained. In certain embodiments, patients require intermittent treatment on a longterm basis upon any recurrence of symptoms and / or infection.

[0275] The compounds for use in the method of the disclosure may be formulated in unit dosage form. The term "unit dosage form" refers to physically discrete units suitable as unitary dosage for patients undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.

[0276] 37

[0277] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817)

[0278] Toxicity and therapeutic efficacy of such therapeutic regimens are optionally determined in cell cultures or experimental animals, including, but not limited to, the determination of the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD50 and ED50. Capsid assembly modulators exhibiting high therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are optionally used in formulating a range of dosage for use in human. The dosage of such capsid assembly modulators lies preferably within a range of circulating concentrations that include the ED50 w ith minimal toxicity. The dosage optionally varies within this range depending upon the dosage form employed and the route of administration utilized.

[0279] Those skilled in the art recognizes, or is able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents were considered to be w ithin the scope of this disclosure and covered by the claims appended hereto. For example, it should be understood, that modifications in assay and / or reaction conditions, with art-recognized alternatives and using no more than routine experimentation, are within the scope of the present application.

[0280] It is to be understood that wherever values and ranges are provided herein, all values and ranges encompassed by these values and ranges, are meant to be encompassed within the scope of the present disclosure. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application.

[0281] EXAMPLES

[0282] The instant specification further describes in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless so specified. Thus, the instant specification should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0283] Example 1-1

[0284] While hematogenous factors have been extensively studied in relation to thrombus formation and clotting, the coordinated multicellular mechanisms at the interface between

[0285] 38

[0286] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817) hemostasis and tissue repair are not well understood. This knowledge gap is mainly due to the classical biochemical in vitro studies of hemostasis, which have not explored the continuum between hemostasis and tissue repair at the multicellular level in living organisms.

[0287] To address this gap, novel methodologies were developed in the study described herein (“the present study”) for observing endogenous thrombi and vascular cellular structures with high spatio-temporal resolution in live mice. This innovative approach allowed the capture of the entire sequence of events, starting from the formation of endogenous thrombi to the restoration of blood flow in individual vessels. By employing this methodology, the present study discovered previously unknown cellular and molecular mechanisms that regulate thrombosis and vascular recanalization, which have crucial implications for tissue viability and the understanding of the pathophysiology of various disorders. Therefore, the present study provides a paradigm shift in the understanding of mechanisms at the interface between hemostasis and tissue repair with broad mechanistic and translational implications.

[0288] Specifically, in the present study, methods for real-time long-term intravital imaging were developed which allowed tracking of endogenous thrombi alongside endothelial (EC) cells, smooth muscle cells (SMCs), and the vascular basal lamina in live mice. This was combined with pharmacological and genetic manipulations that uncovered previously unknown mechanisms of vascular plasticity during thrombosis and repair. Using these methodologies, the present study discovered an unappreciated vascular phase of hemostasis critical for thrombus stabilization and subsequent restoration of vessel patency and blood flow.

[0289] Referring Fig. 1, following thrombosis, a coordinated mechanism of EC and SMC plasticity is triggered. Within hours, EC lamellipodia project towards occlusive thrombi, anchoring them to the vessel wall. This provides a robust vascular mechanism of thrombus stabilization that follows the initial platelet adhesion and stabilization mechanisms, including fibrinogen polymerization.

[0290] Following this initial stabilization phase, thrombi become completely enveloped by ECs within days. This is followed by formation of fenestrations at the abluminal endothelium, through which projections from adjacent SMCs penetrate and engulf thrombi. SMC engulfment is critical for additional thrombus stabilization as well as the subsequent thrombus extraluminal extrusion, degradation and vessel lumen recanalization.

[0291] Pharmacological and genetic manipulations in live mice revealed that this orchestrated multicellular process was dependent on the platelet-enriched lipid

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[0293] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817) phosphatidylserine (PTSer) as a critical signal mediating interactions between thrombi, ECs and SMCs.

[0294] PTSer-recognition receptors MerTK and Axl played distinct, sequential roles in triggering EC and SMC remodeling, respectively. Modulation of these receptors had critical effects on the initiation of thrombus engulfment by ECs and SMCs with important consequences on hemorrhage cessation, vessel recanalization and tissue viability.

[0295] Annexin II and V which are PTSer binding proteins, had marked effects on the overall efficiency of the engulfment and recanalization process. Importantly, administration of annexin V markedly accelerated vascular plasticity leading to more effective recanalization and vessel repair after thrombosis, resulting in improved outcome in a preclinical model of stroke.

[0296] Administration of anti-annexin V antibodies, recapitulating what was observed in various autoimmune disorders, led to delayed vascular recanalization and marked vessel regression in the presence of thrombosis. This occurred through a coordinated mechanism resembling developmental vessel pruning. This result provides a novel understanding of the role of circulating anti-annexin V antibodies in pathology with wide implications for conditions of chronic microvascular thrombotic and hemorrhagic disorders as well as the treatment of antiphospholipid syndrome and for patients with mutations in the annexin V gene.

[0297] The present study thus proposed a continuum between classical intravascular hemostasis / coagulation and thromboangioplasticity (“vascular phase of hemostasis”), which is critical for thrombus stabilization, lumen recanalization and tissue repair, essential for maintaining tissue homeostasis throughout life.

[0298] Thromboangioplasticity could constitute a novel target for the treatment of a variety of disorders including organ ischemia and disorders involving systemic microvascular occlusions such as in autoimmune disease, sepsis, COVID, sickle cell disease and others

[0299] Example 1-2

[0300] The administration of anti-annexin V antibodies, mirroring conditions in various autoimmune disorders (i.e. antiphospholipid syndrome), resulted in delayed vascular recanalization and induced vascular regression. Interestingly, this regression was not limited to fully occluded vessels but also affected vessels that were previously occluded or in the vicinity of occluded vessels. This suggests that the regression was not a result of ongoing vascular hypoxia, as the vessels' GFP signal remained strong, indicating that endothelial cells

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[0303] (ECs) were not actively dying. Rather, the process resembled developmental vessel pruning or so-called anastomosis in reverse. As in developmental vessel pruning, the present study also observed endothelial stenosis, retraction, and ultimate resolution. Interestingly, because in the present study both endothelial and smooth muscle cells were imaged, the present study observed hypertrophy of surrounding smooth muscle cells (SMCs) in areas of ongoing endothelial stenosis and retraction. This suggests a potential role of SMCs in regulating the stenosis and resolution stages of vessel pruning. It would be interesting to determine if similar roles of SMCs exist during developmental vessel pruning. The finding that antiannexin V increases the prevalence of vascular pruning in areas with even modest changes in blood flow underscores the potential for annexin V to play a key signaling role in vessel pruning execution, which would be interesting to explore during developmental pruning.

[0304] Anti-annexin V autoantibodies context antiphospholipid syndrome and mutations in the annexin V gene causes propensity to thrombosis. The novel observation of a propensity to vascular pruning mediated by anti-annexin V antibodies could be a previous unappreciated mechanism of chronic disease. Excessive vascular pruning could also play a role in the placental dysfunction and prevalence of miscarriage in individuals with antiphospholipid syndrome. These findings offer a new perspective on the role of circulating anti-annexin V antibodies in pathology7, with broad implications for conditions involving chronic microvascular thrombotic and hemorrhagic disorders. This also has implications for the treatment of antiphospholipid syndrome and post infectious thrombotic microangiopathy (i.e. COVID- 19) and for patients carrying mutations in the annexin V gene)

[0305] Example 1-3

[0306] To exert agonistic effect on the MerTK receptor, the present study chose the protein GAS6 (Growth Arrest-Specific 6), as well as Protein S, a homologous protein of GAS6.

[0307] GAS6 bind to both MerTK and Axl receptors, which are normally involved in the phagocytosis of apoptotic cells. When GAS6 binds to MerTK, it serves as a bridge between phosphatidylserine (PSer) on apoptotic cells and phagocytes. Accordingly, the present study hypothesized that GAS6 could potentially bridge PSer, which is abundant in activated platelets within the thrombus, and MerTK or Axl on endothelial cells (ECs). This could accelerate signaling through these receptors, thereby speeding up endothelial plasticity.

[0308] The second part of the strategy herein stemmed from the discovery that administering annexin V accelerated the subsequent stages of the thromboangioplasticity process. Particularly, the administration of annexin V hastened the full envelopment of the thrombus

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[0310] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817) by endothelial cells (ECs), the opening of EC fenestrations - or small openings - through which adjacent smooth muscle cells (SMCs) could infiltrate and compact the thrombus, and the final extrusion of the thrombus out of the vessel lumen, culminating in full repair of the vessel wall.

[0311] The present study hypothesized that a fusion protein including both annexin V and GAS 6 would simultaneously achieve two goals: accelerating both the initial engulfment and the subsequent recanalization and repair of blood vessels, which would improve the outcomes in a broad range of microvascular conditions.

[0312] To test this hypothesis, a fusion protein including the full-length annexin V / GAS 6 was generated. The preliminary results indeed show that this fusion protein was able to expedite both processes. This approach could constitute a novel therapeutic strategy for microoclussive disorders in multiple organs.

[0313] Example 2: Phosphatidylserine signaling modulates thromboangioplasticity and promotes hemostasis and tissue repair

[0314] The health and survival of organisms are reliant on efficient tissue repair. After injury, the immediate priority is hemorrhage control, followed by cellular adaptations to reestablish blood flow, enabling tissue repair. Using intravital imaging, the present study uncovered thromboangioplasticity, a coordinated multicellular mechanism at the intersection of hemostasis and repair. Within hours of vascular damage, platelet-rich thrombi are enveloped by endothelial cells (ECs), anchoring them to the vessel wall, preventing premature reflow. Subsequently, transcellular tunnels in ECs enable smooth-muscle cell (SMC) projections to infiltrate and extrude thrombi, leading to lumen recanalization. Phosphatidylserine-mediated signaling through MerTK, Axl and Annexin A5, played cell- and stage-specific roles in modulating thromboangioplasticity. Notably, Annexin A5 administration accelerated the process, improving hemorrhage control and tissue repair, while anti-Annexin A5 antibodies, as observed in autoimmune disorders, induced vessel regression. Therefore, thromboangioplasticity profoundly impacts bleeding, vessel fate and organ viability, underscoring its central role in bridging hemostasis and tissue repair.

[0315] Example 2-1:

[0316] Organisms are continually exposed to microtrauma, regular wear and tear, and tissue damage throughout life. When an injury occurs, the immediate priority is to prevent uncontrolled hemorrhage, followed by a series of multicellular changes aimed at restoring

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[0318] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817) tissue blood flow, promoting tissue repair, and regeneration to reestablish normal organ function.

[0319] While hematogenous factors have been extensively studied in relation to thrombus formation and clotting following injury, there is a notable gap in the understanding of the transitional stages betw een hemostasis and tissue repair. This process requires precise spatiotemporal coordination to prevent premature reflow, which could exacerbate ongoing hemorrhage, while ensuring timely vessel recanalization to facilitate efficient tissue repair.

[0320] However, the multicellular in vivo dynamics and molecular mechanisms during this transitional phase are poorly understood. This knowledge gap primarily stems from the classical biochemical in vitro studies which have not focused on the continuum between hemostasis and tissue repair. Furthermore, there is a scarcity of studies examining multicellular interactions during tissue injury in the intact mammalian system.

[0321] To address this gap, the present study developed an in vivo mouse model that allows real-time visualization of the transition between vascular injury, thrombosis, and tissue repair. Extravasation of embolic clots may contribute to vascular recanalization. The present study uncovers a highly coordinated microvascular process, which termed thromboangioplasticity herein, involving endothelial cells (ECs) and smooth muscle cells (SMCs) that is rapidly triggered after the formation of a platelet-rich thrombus. Within hours of vessel injury7and thrombosis, ECs interact w ith platelets in a plasticity process that anchors thrombi to the vessel wall, preventing dislodgement and limiting hemorrhage. At the same time, ECs and SMCs initiate a sequential remodeling program that compacts and degrades the thrombi, ultimately removing them from the microvascular lumen and restoring normal blood flow7within days.

[0322] The present study uncovered a critical role of phosphatidylserine (PtSer)-mediated signaling in the thromboangioplasticity process, which involves the TAM receptors MerTK and Axl, as well as the PtSer-binding proteins Annexin A2 and A5. Disruption of this signaling had a major impact on bleeding, vessel fate, tissue repair, and viability7. Notably, Annexin A2 and A5 had marked effects on the overall efficiency of the engulfment and recanalization process. Administration of Annexin A5 accelerated vascular plasticity, leading to more effective recanalization and vessel repair after thrombosis. This resulted in improved outcomes in a preclinical model of brain microvascular occlusion. Strikingly, anti-Annexin A5 antibodies, as seen in certain autoimmune disorders, led to delayed vascular recanalization and vessel regression during thrombosis, reminiscent of developmental vessel pruning. These findings offer a new perspective on the pathological role of circulating anti-

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[0325] Annexin A5 antibodies and have implications for the treatment of thrombosis in autoimmune antiphospholipid syndrome and for patients carrying mutations in the Annexin A5 gene.

[0326] The present study thus propose that there exists a continuum between the classical intravascular hemostasis process and thromboangioplasticity. This continuum plays a pivotal role in thrombus stabilization, lumen recanalization, and tissue repair — all of which are indispensable for preserving tissue homeostasis throughout life. Therefore, thromboangioplasticity could constitute a novel therapeutic target for a range of conditions involving tissue injury, hemorrhage, aberrant thrombosis, microvascular pathology and autoimmunity.

[0327] Example 2-2: Intravital imaging for real-time visualization of thrombo-vascular interactions

[0328] The present study implemented a realistic model of microvascular thrombosis that allowed high-resolution multicellular imaging over time to investigate the precise interactions between vascular cells and endogenous thrombi in vivo. This model enabled us to visualize ECs, SMCs, and platelets, while simultaneously monitoring blood flow, thrombus evolution, and ultimate vessel recanalization. Importantly, this model also permitted genetic and pharmacological manipulations to address mechanistic questions.

[0329] The method for inducing endogenous thrombi formation in this model relies on the focal topical application of ferric chloride (FeCh) to the surface of a main branch of the middle cerebral artery (MCA). This induces endothelial injury through reactive oxygen species formation, leading to the rapid generation of a platelet-rich intravascular thrombus (Figs. 2A-2C and 9B-9D). Over the proceeding minutes, these larger thrombi break down and embolize smaller downstream branches of the MCA. For intravital imaging purposes, the present study focused on branches that were located >0.5 mm from the site of FeCh -induced thrombus, to ensure that there was no direct effect of FeCh on the local cellular environment.

[0330] Example 2-3: Newly developed thrombi are anchored intravascularly by rapidly forming endothelial lamellipodia

[0331] Using intravital confocal imaging through a cranial window7, the present study monitored the endothelial responses to endogenous thrombi in reporter mice that express green fluorescent protein in endothelial cells (Tie2-GFP). Within hours after thrombus lodgment, multiple local ECs initiated a rapid process of plasticity, forming bulb-like structures and lamellipodia that projected towards the occlusive thrombi (Figs. 2D-2E and

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[0334] 10 A). These bulbs matured into distinct projections that gradually spread across the thrombus surface, forming multiple anchoring sites at points of contact (Fig. 10B), that seemed to stabilize the thrombi within the vascular lumen, as evidenced by the fact that thrombi less enveloped by EC lamellipodia were more prone to washout (Fig. 2F). Once robust anchoring sites were established, the propensity' for thrombus washout decreased over time underscoring the potential importance of these EC structures for thrombus stabilization (7 / 29 clots dislodged between l-24h versus 0 / 12 clots dislodged from 24-48h).

[0335] While the initial projections emerged from ECs located anywhere near the thrombus, there seemed to be a selection process after which one side of the vessel became more actively involved in the engulfment process (Fig. 10D), resulting in complete envelopment of the thrombus by days 2 to 4 (Figs. 2G-2I). The present study also employed an in vivo imaging correlative light, immunofluorescence and electron microscopy approach to colocalize ultrastructural images and protein markers at specific stages of the vascular plasticity process (Figs. 2G and 11 A). Intriguingly, this revealed multiple nuclei within the EC enveloped thrombus (Fig. 2G, right panel), suggesting this plasticity process could involve either EC cell division or migration from the parent vessel. Through this correlated approach, the present study was able to relocate the occluded microvessel ex vivo after administration of bromodeoxyuridine (BrdU) and measure the extent of EC proliferation. However, the present study did not observe significant endothelial BrdU labeling, suggesting that endothelial migration rather than proliferation is the chief mechanism for EC remodeling around thrombi (Figs. 1 IB- 1 ID).

[0336] Example 2-4: Extrusion of thrombi through EC transcellular tunnels allows for vessel recanalization

[0337] Around day three post-thrombus occlusion, the present study noted the emergence of openings in the original abluminal endothelium (Figs. 2J-2K). Time-lapse imaging revealed that these EC openings appeared to originate from the coalescence of smaller fenestrationlike structures, leading to the formation of a larger EC tunnel (Fig. 12A). These fenestrations continued to enlarge, allowing for the gradual extrusion of the thrombus from the lumen and the re-establishment of vessel patency (Figs. 2J-2L). Interestingly, larger thrombi underwent compartmentalization, with distinct EC compartments forming abluminal openings at different intervals (Figs. 12C-12D). Notably, even as these tunnels enlarged to tens of microns in diameter (Fig. 12B), the present study did not detect significant leakage of intravascular dye into the adjacent perivascular space (Fig. 12D). This suggests that the

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[0339] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817) newly formed luminal endothelium around the thrombi maintains normal blood brain barrier (BBB) properties, likely due to the establishment of tight junctions (Fig. 12E).

[0340] The precise molecular mechanisms driving the formation of these EC tunnels remain unclear. Previous studies have shown that Caveolin-1 plays an important role in the formation of classical fenestrations in various vascular beds. However, when the present study examined the formation of EC tunnels in Caveolin- 1 knockout mice the present study did not observe any differences compared with controls (Figs. 13A-13B). Furthermore, plasmalemma vesicle associated protein (PLVAP), a marker of fenestrated endothelium did not co-localize with areas associated with the formation of EC tunnels (Fig. 13C). Interestingly, the present study observed that PLVAP appeared to be highly concentrated in some emerging lamellipodia in the vicinity of occlusive thrombi (Fig. 13D), though its role in this process is unclear. Without wishing to be limited by any theory, altogether this indicates that the mechanism of formation of large EC tunnels is not related to fenestrations as seen in some vascular beds.

[0341] Example 2-5: SMC invasion through EC transcellular tunnels mediates thrombus extrusion and degradation

[0342] Having shown that thrombi are gradually removed from the lumen by the formation of abluminal EC tunnels, the present study investigated the concurrent changes taking place in the surrounding SMCs. To achieve this, the present study crossbred endothelial with smooth muscle reporter mice (SMA-mCherry:Tie2-GFP mice), which enabled us to examine the dynamic interplay between ECs and SMCs in the vicinity of thrombi. Immediately after the formation of the EC tunnels, the present study observed that SMC processes began to invade the thrombus cavity (Fig. 3A-3C and Fig. 14A). The intruding SMC processes appeared to initially engage closely with adjacent ECs, extending branches in tight alignment with EC processes (Figs. 3A-3C) and ultimately invading the thrombus cavity as seen by correlated in vivo confocal-EM imaging (Fig. 3D). Furthermore, the present study was able to observe precise sites of breaks in the elastin layer between SMCs and ECs, through which SMC processes penetrated (Fig. 3E and 14B). As time progressed, SMC invasion advanced (Fig. 3F) and underwent further remodeling forming an intricate reticular network surrounding and compartmentalizing thrombi (Figs. 3G-3I). Interestingly, large thrombi were engulfed and fragmented into multiple compartments to facilitate SMC invasion and degradation (Fig. 14C-14D).

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[0345] The SMC infiltration correlates temporally with the apparent compaction of the thrombus (Figs. 3H-3J), possibly driven by SMC contractility. Moreover, infiltrating SMC processes appear to engulf thrombus fragments, potentially aiding in thrombus breakdow n (Figs. 3G-3H and 14C-14E). During the initial stages of thromboangioplasticity the present study did not observe the presence of macrophages in the thrombus vicinity; however, beginning at day 2. when EC tunnels develop and SMCs begin to invade (Fig. 3B). macrophages were also observed within the thrombus cavity (Fig. 14F), which likely aid in the thrombus degradation process. Ultimately, the coordinated SMC invasion leading to thrombus compaction, extrusion and degradation results in lumen recanalization and restoration of blood flow (Figs. 3H-3J).

[0346] Example 2-6: MerTK interaction with platelet PtSer triggers endothelial lamellipodia projections towards thrombi

[0347] The present study aimed to investigate the mechanisms that trigger vascular-specific responses to the presence of an intravascular thrombus. As in naturally occurring arterial thrombi, the platelet-rich thrombi formed in the model demonstrated strong binding to Annexin A5, a protein with affinity for membrane-exposed phosphatidylserine (PtSer) (Fig. 4A). PtSer is known to translocate to the outer membrane of cells during apoptosis, and serves as a signal for detection by phagocytes during efferocytosis. A similar PtSer translocation occurs in platelets follow ing their activation during thrombosis, whereby PtSer acts as a binding site for coagulation factors. This promotes local thrombin generation, which converts fibrinogen to fibrin, thereby culminating in the formation of a fibrin mesh that stabilizes the thrombi. In addition to its role in coagulation, it was hypothesized that interactions between ECs and PtSer on thrombi might mediate the observed EC plasticity’ following vascular occlusion.

[0348] Through mining single-cell RNA sequencing databases, it was noted that MerTK and Axl, which are PtSer-binding receptors traditionally associated with signaling during macrophage efferocytosis of apoptotic cells, were enriched in ECs and SMCs. The present study thus hypothesized that PtSer signaling through these receptors could play a role in thrombo-vascular interactions and plasticity. To explore this, the present study crossed-bred mice lacking the MerTK receptor (MerTK") with endothelial reporter mice (Tie2-GFP) to visualize the dynamics of EC plasticity- following thrombosis. In these mice, the present study did not observe significant differences in the initial thrombus formation or in tail bleeding times (Figs. 15A-15B). Interestingly, despite these observations, the present study observed a

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[0350] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817) marked increase in thrombus washout within the first 24hrs following occlusion in erTK" " mice (Figs. 4D-4E and 15C). This suggests that while MerTK may not play a significant role in the acute phase of thrombus formation it is critical for the subsequent thrombus stabilization within the vasculature.

[0351] Importantly, MerTK’ " mice exhibited a marked reduction in the formation of early EC lamellipodia projecting towards thrombi within the first 24hrs following occlusion (Figs. 4B- 4C). It was hypothesized that these early projections played a pivotal role in anchoring and stabilizing thrombi. To test this, the present study quantified thrombus washout in relation to lamellipodia formation. Strikingly, the present study found that there was an inverse correlation, such that thrombi not surrounded by EC projections showed increased susceptibility to washout compared to those interacting more robustly with EC lamellipodia. which was exacerbated by MerTK deletion (Fig. 4E). Interestingly, while MerTK"7" mice exhibited a delay in EC envelopment at 24h, EC plasticity rebounded by day 3 (Figs. 4F-4G). Similarly, using RU301 for pharmacological inhibition of the protein Growth Arrest Specific 6 (GAS-6), a PtSer-binding protein that is critical for MerTK signaling, also resulted in a delay in the EC-plasticity in response to thrombi (Fig. 4G). This data demonstrate that MerTK is an essential receptor for the initial detection of platelet PtSer by ECs, leading to the formation of projections that anchor thrombi to the vascular wall, thus preventing premature washout.

[0352] Example 2-7: MerTK signaling is critical for thrombus stabilization with major impact during tissue injury

[0353] To better understand the repercussion of delayed EC plasticity and increased washout, the present study employed a global microvascular occlusion model through injection of fibrin microemboli into the carotid artery to measure the degree of retention of emboli within the brain parenchyma (Fig. 4H). Through intravital imaging of emboli the present study w as able to show that platelets rapidly adhere and aggregate around fibrin clots (Fig. 41), therefore providing a potential source of PtSer for MerTK signaling. Using this fibrin embolization model, the present study observed that similar to endogenous thrombi, selective pharmacological inhibition of MerTK with UNC2881 led to a marked increase in embolus washout (Figs. 4J-4K). Surprisingly, despite the greater degree of w ashout, the present study noted an increase in overall mortality (Fig. 4L) and brain hemorrhages (Figs. 4M-4N). Altogether, these data demonstrate that MerTK signaling during tissue injury is critical for

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[0355] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817) thrombo-vascular interactions aimed at preventing premature washout. This has major implications on hemorrhage control, vessel integrity and overall survival (Fig. 40).

[0356] Example 2-8: AXL signaling modulates smooth muscle invasion and thrombus extrusion

[0357] The observations indicate that SMC invasion through transcellular tunnels on the abluminal ECs is cntical for the compaction and extrusion of thrombi from the vessel lumen (Figs. 3A-3J and 5A). Having shown that PtSer interaction with MerTK was a key signal triggering EC plasticity, the present study investigated whether the subsequent SMC invasion and remodeling was also mediated through PTSer receptors. Notably vascular transcriptomic data demonstrate that Axl, but not MerTK, is highly enriched in SMCs in both mice and humans. To investigate the role of Axl, the present study used SMA-mCherry:Tie2-GFP reporter mice crossed with either Axl"" or MerTK"" mice to examine the coordinated responses by both ECs and SMCs in the vicinity of thrombi. In vivo imaging revealed that in Axl"7" mice the reactivity and speed of invasion of adjacent SMCs through EC tunnels was markedly delayed and the degree of SMC invasion of the thrombus cavity and degree of lumen recanalization was reduced (Figs. 5B, 5D and 5E). This contrasted with MerTK' " mice which displayed no abnormalities in the timing and robustness of SMC invasion of thrombi (Fig. 5C). Importantly, in Axl" " mice the abnormal SMC plasticity was associated with a delay in SMC invasion (Fig. 5D) and subsequent thrombus extrusion and vessel recanalization (Fig. 5E), causing vessels to remain partially occluded for extended periods of time, in some cases persisting for weeks to months (Figs. 5F-5G). Despite the disruption in SMC responses, EC plasticity remained unaffected in Axl"7" mice (Figs. 16A-16B). Altogether, these data highlight distinct signaling mechanisms at ECs and SMCs with precise spatio-temporal patterns and demonstrates a critical role of SMCs in the dynamics of thrombus extrusion and the re-establishment of vascular patency.

[0358] Example 2-9: Annexin A5 accelerates endothelial thrombus envelopment and vascular recanalization

[0359] Having shown that PtSer sensing through Axl and MerTK receptors is critical for detecting the presence of platelet-rich thrombi and triggering ECs and SMCs plasticity, the present study investigated the potential roles of PtSer binding proteins that might modulate vascular plasticity. Annexins A2 and A5 are produced by various cell types and can bind platelet PtSer with high affinity (Fig. 6A) providing scaffolding for the assembly of clotting

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[0361] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817) factors near activated platelets. Importantly, Annexin A2 and A5 mutations and autoantibodies have been implicated in thrombo-occlusive disorders, although the mechanisms are not well understood.

[0362] To examine the potential modulatory role of Annexins in thromboangioplasticity. the present study injected either Annexin A2 or A5 intravenously following thrombus occlusion and quantified EC remodeling and rates of recanalization in vivo. Surprisingly, Annexin A5 administration led to an increase in the number of early lamelhpodia forming after occlusion (Figs. 6B-6C). Subsequently there was a marked increase in EC spreading and envelopment of thrombi in mice treated with Annexin A5 (Figs. 6D-6E). In the following days, the present study also observed an acceleration of thrombus extrusion and vessel recanalization in Annexin A5 treated mice (Figs. 6F-6G). These data indicate that Annexin A5 can play a critical role in modulating the robustness of EC plasticity during thrombosis. The precise mechanisms of how Annexin binding to thrombi promote EC plasticity is unclear. Annexin A5 has been shown to interact with both VEGF and P5 integrin receptor subunit, which could potentially promote angiogenesis. However, when the present study treated mice with a VEGFR2 blocking antibody (DC 101) or with an Integrin inhibitor (Cilengitide), the present study did not observe a significant effect on EC plasticity or vessel recanalization (Figs. 17A- 17C).

[0363] Example 2-10: The effect of recombinant Annexin A5 on thromboangioplasticity is blunted in MerTK knockout mice

[0364] Having demonstrated that MerTK plays a critical role in initiating EC plasticity7towards thrombi, the present study asked whether the effect of Annexin A5 administration on EC envelopment and recanalization was independent of, or linked to, MerTK function. The present study thus treated MerTK ' / ' mice with recombinant Annexin A5 during thrombus formation. Consistent with the earlier experiments in MerTK ‘ / _mice, the present study noted a reduction in the initial EC lamellipodia envelopment at day 1 (Figs. 6H-6I), and an increase in thrombus washout (Fig. 6K). However, treatment with Annexin A5 in MerTK ' ’ mice was unable to rescue the delay in early EC lamelhpodia envelopment (Fig. 61), vessel recanalization (Fig. 6 J) and failed to improve thrombus stabilization (Fig. 6K).

[0365] Altogether, these data indicate that the initial MerTK recognition of platelet PTSer is essential for initiating EC plasticity7and that Annexin A5 cannot directly promote EC plasticity in the absence of this initial signal. However, with intact MerTK signaling. Annexin A5 can enhance the efficiency of EC envelopment and vascular recanalization (Figs. 6A-6G).

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[0368] Together, these data indicate that an effective thromboangioplasticity response is reliant on a coordinated sequence of interdependent signaling mechanisms. Annexin A5 markedly accelerates the thromboangioplasticity response, but its effect is dependent on the initial triggering of EC plasticity through MerTK signaling (Fig. 6L).

[0369] Example 2-11: Annexin A5 administration improves recovery after tissue injury

[0370] During various types of tissue injury, it is well-established that thrombi form in the microvasculature to prevent uncontrolled hemorrhage. Interestingly, following carotid infusion of microemboli, the present study observed a local upregulation of Annexin A2 and A5 adjacent to areas of occlusion (Figs. 7A-7C and 18A-18C), which led us to investigate the potential role of Annexins in thromboangioplasticity.

[0371] Given the observation that there is a marked upregulation of Annexin A5 in the endothelium in areas of tissue injury (Fig. 7B) and that the administration of exogenous Annexin A5 led to the acceleration of thrombus envelopment and extrusion from the lumen, the present study examined if this translated into improved injury outcomes. To better understand the impact of Annexin A5 on tissue recovery after injury, the present study employed a global microvascular occlusion model through the injection of fibrin microemboli into the common carotid artery', followed by treatment with intravenous administration of Annexin A5 (4 and 24hrs after embolization, Fig. 7A). This treatment consistently improved various injury and behavioral parameters, including BBB breakdown, infarct area, incidence of microhemorrhages and motor scores (Figs. 7D-7J).

[0372] These data demonstrate that Annexin A5 has a beneficial effect during tissue injury- associated microvascular occlusion, likely by enhancing EC envelopment and thrombus stabilization and by accelerating the subsequent process of vessel recanalization (Figs. 6F- 6G). Combined with the findings showing that deletion of MerTK increases acute thrombus washout and worsens tissue injury (Figs. 4A-4O), this data indicate that the coordinated thromboangioplasticity' process is critical for tissue recovery' by preventing premature reflow and promoting vascular recanalization.

[0373] Example 2-12: Anti- Annexin A5 antibodies induce a stereotyped process of vascular regression

[0374] Having shown that administration of Annexin A2 and A5 accelerated the process of thromboangioplasticity and vessel recanalization, the present study asked whether reduction in Annexins would have an opposite and detrimental effect. The present study thus

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[0376] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817) administered either Annexin A2 or A5 neutralizing antibodies intravenously 0.5 and 24 hours after thrombus formation. The present study observed that in stark contrast with MerTK deletion, this treatment had no effect on the initial extension of EC anchoring lamellipodia, which appeared largely normal (Fig. 19A). However, by day 1 the present study noted a reduction in EC envelopment and the presence of stubby EC lamellipodia in the anti- Annexin A5 antibody treatment groups (Figs. 8A-8B). The present study also observed a striking reduction in the overall efficiency of lumen recanalization (Figs. 8C-8D). This reduction in EC plasticity by Annexin neutralization further supports the concept that Annexins have an important modulatory role during thromboangioplasticity.

[0377] Remarkably, in anti-Annexin A5 treated mice, the present study observed that a substantial proportion of vessels underwent a coordinated process of regression following thrombosis (Fig. 8E). This regression was not observed in anti-Annexin A5 antibody treated mice that did not undergo thrombosis (Fig. 8G). Importantly, high resolution images revealed a unique process of EC remodeling that included vessel stenosis and the formation of an intraluminal reticular EC pattern (Fig. 8F) reminiscent of developmental vessel pruning. In addition, the present study observed thickening of the SMCs at the same time as vessel collapse (Fig. 81), indicating a potential role of SMCs in modulating vascular regression. Interestingly, during this process the endothelial GFP signal was maintained throughout (Fig. 81), indicating that the process was not associated with concurrent EC cell death. Regression occurred in both the primary occluded vessels undergoing thromboangioplasticity (Fig. 19B) as well as in adjacent vessels that only had modest blood flow alterations (Figs. 8H-8J). The precise reason for this is unclear but, without wishing to be limited by any theory', it is possible that these vessels were transiently occluded at early stages, which may have triggered an irreversible process of regression, despite relative preservation of blood flow.

[0378] Altogether the findings with anti-Annexin A5 neutralizing antibodies, including the striking regression phenotype of anti-Annexin A5 antibodies, further support the important role of Annexins during the process of thromboangioplasticity' which could have major implications in recovery after tissue injury (Fig. 8K). Importantly, these findings can be critical for the understanding of vascular and thrombotic phenotypes in individuals with antiphospholipid syndrome carrying circulating anti-Annexin A5 antibodies as well as those with mutations in the Annexin A5 gene.

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[0380] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817)

[0381] Example 2-13:

[0382] Efficient tissue repair is essential for organismal health and survival due to continuous exposure to injury throughout life. Upon injury, a complex series of events takes place to stop bleeding, repair blood vessels, and remodel tissues, ultimately restoring normal function. Using innovative intravital cerebrovascular imaging of endogenous thrombi, the present study uncovered thromboangioplasticity, a previously unknown multicellular mechanism critical for thrombus stabilization, hemorrhage control and vascular recanalization. Thromboangioplasticity operates in small blood vessels, which constitute the majority of the overall vasculature in humans. This mechanism, bridging hemostasis and tissue repair, has significant implications for tissue maintenance and organ viabi 1 i ty. It represents a novel therapeutic target for conditions involving ischemia, hemorrhage, and aberrant thrombosis in autoimmunity, such as antiphospholipid syndrome.

[0383] Thromboangioplasticity depends on the direct interactions between aggregated platelets and immediately adjacent vascular cells (ECs and SMCs) resulting in the rapid remodeling of these structures around the thrombus, which leads to its compaction and extrusion from the lumen. The electron microscopy data showed that the envelopment of thrombi was formed by multiple EC cell bodies, however, the present study found no evidence of cell division, as demonstrated by a lack of BrdU labeling. This supports a mechanism of EC migration from adjacent parent vascular branches rather than one of local EC proliferation as seen during developmental angiogenesis.

[0384] The present study has demonstrated the critical role of the platelet-enriched lipid, PtSer, in initiating and regulating thromboangioplasticity. Specifically, the present study showed that PtSer is essential for the orchestrated multicellular interactions among thrombi, ECs and SMCs. The present study found that the receptors MerTK and Axl, which interact with PtSer, play distinct and sequential roles in this process. Signaling through MerTK is crucial for triggering the initial EC envelopment of thrombi while Axl is key for modulating SMC remodeling, with important consequences on hemorrhage cessation, vessel recanalization and tissue viability. MerTK is well-recognized as a PtSer receptor, essential for the detection of apoptotic cells during macrophage polarization in efferocytosis. Beyond macrophages, MerTK is also expressed in other cell types, including ECs and platelets, though its roles in these cells are less defined. Surface PtSer in platelets interacts with circulating procoagulant and fibrinolytic factors, thus influencing the development of a mature thrombus. In studies using MerTK-deficient mice or pharmacological inhibitors, MerTK has been suggested to modulate thrombosis by promoting platelet activation,

[0385] 53

[0386] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817) although bleeding times in MerTK deficient mice are not significantly affected. Furthermore, the MerTK ligand, GAS-6, has been implicated in platelet activation and thrombosis through allb 03 integrin. Gas6 knockout mice are protected against venous and arterial thrombosis, while pharmacological inhibition of GAS-6 reduces thrombus stabilization without overt changes in bleeding times. These observations indicate that in addition to potential effects on platelet function, the effect on thrombus stabilization can be mediated by additional factors. The data reveals that the MerTK / GAS-6 signaling pathway in ECs is essential for detecting activated, aggregated platelets, which are known to externalize PtSer. Consequently, the present study proposes a novel function of platelet PtSer interactions with MerTK in ECs, which is to trigger the formation of EC lamellipodia, which anchor and stabilize newly formed thrombi to complement the early stabilization by the fibrin mesh (Fig. 20).

[0387] Annexins A2 and A5, which are known PtSer binding proteins, had marked effects on the overall efficiency of the engulfment and recanalization process. Administration of exogenous Annexin A2 and A5 markedly accelerated vascular plasticity7leading to a more robust thrombus envelopment and subsequently to a more effective recanalization and vessel repair. Importantly, in MerTK knockout mice the effect of Annexin A5 was markedly reduced (Figs. 6A-6L). This indicates that for Annexin A5 to promote spreading of EC lamellipodia, it is essential for the vasculature to initially recognize the presence of platelet PtSer through MerTK signaling in ECs. Circulating or locally produced Annexin A5 may therefore play a role in facilitating the process of EC cell spreading and migration. However, the precise mechanisms by which the presence of Annexin A5 accelerates EC envelopment, and the subsequent vascular recanalization are yet to be elucidated. Annexin A5 has been shown to potentially function as a signaling protein through VEGFR-2 and through interactions with 05 integrin, which could promote EC spreading. The Immunofluorescence imaging revealed colocalization of VEGF with thrombi (Figs. 17A-17C), suggesting either binding of circulating VEGF or its local production from ECs or activated platelets. However, the present study showed that pharmacological inhibition of either VEGF or integrins did not alter the robustness of EC envelopment (Figs. 17A-17C). Additionally, caveolin-1 interacts with both VEGF receptors and integrins within caveolae to modulate their downstream signaling pathways critical for angiogenesis, cell migration, and endothelial function. However, the present study found that the extensive EC remodeling observed during thromboangioplasticity was independent of Caveolin-1, as Cav 1 KO mice showed no defects in thrombus envelopment and EC tunnel formation. Taken together, the lack of effects from inhibiting VEGF or integrins, or deleting Caveolin 1, along with the absence of endothelial

[0388] 54

[0389] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817) cell proliferation, suggests that thromboangioplasticity operates through a distinct mechanism from angiogenesis. Instead, the data indicate that it relies on endothelial cell migration and cellular remodeling to enable the envelopment of thrombi and recanalization.

[0390] With regards to the pathophysiological impact of thromboangioplasticity, it has been suggested that inhibition of MerTK in platelets can have an anti-aggregating effect without a significant increase in bleeding. However, in the context of disease, the present study found that MerTK in the vasculature plays a critical role in thrombus stabilization. Inhibition of MerTK prevents early EC lamellipodia formation, leading to thrombus washout, with increased microhemorrhages and worsening clinical outcomes in an embolic model of stroke. Conversely, accelerating the thromboangioplasticity process through the administration of exogenous Annexin A5 had opposite effect, reducing stroke severity and ameliorating microhemorrhagic conversion. Altogether, these findings suggest that thrombus washout during the initial hours after acute ischemia may not be beneficial. On the other hand, the data shows that the orderly process of recanalization through thromboangioplasticity that occurs over several days following occlusion, is critical for maintaining vascular integrity and preventing hemorrhage.

[0391] Regarding the mechanism of thrombus removal and vessel recanalization, the present study found that SMC infiltration into the thrombus cavity was crucial for the extrusion process. Indeed, in instances where the present study did not observe the formation of an abluminal EC tunnel opening and the subsequent infiltration of SMC processes, thrombi tended to persist within the vascular lumen for more extended intervals (Figs. 5F-5G). The role of SMCs in thrombus extrusion was further underscored by the finding that deletion of Axl, which is a PtSer receptor highly expressed in SMCs, led to a marked delay in SMC infiltration and subsequent thrombus extrusion (Figs. 5D-5E). Thus, Axl may sense PtSer in platelets, triggering SMC plasticity as in response to GAS6-Axl interactions in vitro. It is speculated that these defects in thromboangioplasticity that lead to the prolonged presence of thrombi in the vascular lumen, could contribute to focal sites of inflammatory' cell recruitment and might serve as a nidus for the potential development of atherosclerotic lesions.

[0392] Notably, while SMC infiltration w as delayed in Axl knockout mice, the formation of the EC abluminal tunnels occurred at a normal pace (Fig. 5B). This indicates that Axl does not influence EC tunnel formation, suggesting that this process occurs independently of SMC remodeling. Importantly, the formation of the abluminal EC tunnels only occurred once the thrombus was completely enveloped. This envelopment created an impermeable barrier, as

[0393] 55

[0394] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817) evidenced by the preservation of the BBB with normal tight junctions as well as the absence of small molecule leakage (Figs. 2A-2M and 12C-12E). The abluminal endothelium gradually retracted by the coalescence of smaller openings reminiscent of EC fenestrae or tunnels, which eventually formed openings that are up to tens of microns in diameter. This was associated with the infiltration of projections from adjacent SMCs that fully invaded and engulfed the thrombus. The exact mechanism of endothelial tunnel formation remains uncertain, especially since the present study found that Caveolin 1 deficient mice displayed normal tunnel formation (Figs. 13A-13D). Furthermore, PLVP1 a protein associated with EC fenestrae, was not enriched in the area of tunnel opening (Figs. 13A-13D), although the present study sometimes saw bright PLVP1 staining in early lamellipodia near but not enveloping thrombi.

[0395] The administration of anti-Annexin A5 antibodies, mirroring the autoimmune antiphospholipid syndrome, resulted in delayed vascular recanalization. This defective recanalization might be a previously underappreciated aspect in this syndrome potentially having important clinical implications. A notable finding in the study was the marked increase in vascular regression after thrombosis in mice treated with anti-Annexin A5. This regression frequently affected not only fully occluded vessels but also those previously occluded or located near occluded vessels (Figs. 8A-8K). This pattern suggests that the regression was not due to persistent vascular hypoxia, as the GFP signal in the vessels indicated that ECs remained alive, suggesting that regression is an active process that requires a metabolically viable vessel. Overall, the process resembled developmental vessel pruning or so-called anastomosis in reverse, which has been reported to have distinct phases including endothelial stenosis and vessel retraction. Interestingly, because the present study was able to image both ECs and SMCs simultaneously, the present study observed hypertrophy of surrounding SMCs in areas of ongoing vascular stenosis and retraction (Figs. 8A-8K). This indicates an active role of SMCs in regulating the stenosis and retraction stages of vessel pruning, a phenomenon that would be interesting to explore in the context of developmental vessel pruning. The discovery that anti-Annexin A5 antibodies affect vascular pruning even in areas with modest changes in blood flow, underscores the potential of Annexin A5 as a key signaling molecule during vessel pruning.

[0396] The novel observation of impaired thromboangioplasticity and increased vascular pruning in the presence of anti-Annexin A5 antibodies shows a previously unrecognized mechanism that could contribute to chronic diseases. Excessive vascular pruning has implications for the pathophysiology and treatment of autoimmune disorders,

[0397] 56

[0398] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817) antiphospholipid syndrome and post infectious thrombotic microangiopathy and for patients carrying mutations in the Annexin A5 gene. Furthermore, excessive vascular pruning also plays a role in the placental dysfunction and miscarriage observed in individuals with antiAnnexin A5 antibodies in the autoimmune antiphospholipid syndrome. Ultimately, impaired thromboangioplasticity is significant in physiological processes like menstruation, where recurrent tissue remodeling and hemorrhage occur in a controlled fashion. Its role in these processes can have critical evolutionary implications, as even minor inefficiencies in thromboangioplasticity might lead to adverse reproductive outcomes.

[0399] Altogether, the research herein indicates that thromboangioplasticity likely evolved as a robust mechanism to prevent the washout of thrombi, thereby retaining them in the vasculature, and averting early reflow during tissue injury. This process also ensures the orderly re-establishment of vascular patency and blood flow over the subsequent days (Fig. 20). The present study thus shows that thromboangioplasticity and the classical intravascular hemostasis / coagulation mechanisms exist on a continuum. This continuum is crucial for thrombus stabilization, lumen recanalization, and tissue repair, all of which are essential for maintaining tissue homeostasis throughout life.

[0400] Enumerated Embodiments

[0401] In some aspects, the present invention is directed to the following non-limiting embodiments:

[0402] Embodiment 1 : A composition, comprising: a first polypeptide comprising a grow th arrest-specific 6 (GAS6) polypeptide and / or a protein S polypeptide, or a first nucleic acid encoding the first polypeptide; and / or a second polypeptide comprising an annexin V polypeptide, or a second nucleic acid encoding the second polypeptide.

[0403] Embodiment 2: The composition of Embodiment 1, w herein the first polypeptide and the second polypeptide are not covalently linked to each other.

[0404] Embodiment 3: The composition of Embodiment 1, wherein the first polypeptide and the second polypeptide are covalently linked to each other.

[0405] Embodiment 4: The composition of Embodiment 3, wherein both the first polypeptide and the second polypeptide are parts of a fusion protein.

[0406] Embodiment 5: The composition of any one of Embodiments 1-4, further comprising a pharmaceutically acceptable carrier.

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[0408] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817)

[0409] Embodiment 6: The composition of any one of Embodiments 1-5, wherein the composition is formulated for parenteral administration, optionally wherein the parenteral administration comprises intradermal administration, subcutaneous administration, intramuscular administration, or intravenous administration.

[0410] Embodiment 7: The composition of any one of Embodiments 1-6, which

[0411] (a) accelerates thromboangioplasticity process, clot removal from a blood vessel, vessel recanalization, reestablishment of blood flow, and / or tissue repair in a subject, and / or

[0412] (b) reduces a risk of hemorrhage after injury and / or the risk of hemorrhagic conversion due to stroke and / or organ trauma.

[0413] Embodiment 8: The composition of any one of Embodiments 1-7, which

[0414] (a) treats, ameliorates, and / or prevents a tissue ischemia, a physical injury, a stroke, and / or a myocardial infarction in a subject in need thereof,

[0415] (b) treats, ameliorates, and / or prevents a condition associated with anti-annexin V autoimmune antibodies, optionally a pregnancy loss or an antiphospholipid syndrome, and / or

[0416] (c) treats, ameliorates, and / or prevents thrombo-hemorrhage in subjects carrying a mutant annexin V.

[0417] Embodiment 9: A non-natural construct or a nucleic acid encoding the non-natural construct, the construct comprising: a first polypeptide comprising a growth arrest-specific 6 (GAS6) polypeptide and / or a protein S polypeptide; and / or a second polypeptide comprising an annexin V polypeptide.

[0418] Embodiment 10: The construct or nucleic acid of Embodiment 9, which is a fusion protein comprising the first polypeptide and the second polypeptide.

[0419] Embodiment 11 : The construct or nucleic acid of any one of Embodiments 9-10, which

[0420] (a) accelerates clot removal from a blood vessel, vessel recanalization, reestablishment of blood flow, and / or tissue repair in a subject, and / or

[0421] (b) reduces a risk of hemorrhage after injury and / or the risk of hemorrhagic conversion due to stroke and / or organ trauma.

[0422] Embodiment 12: The construct or nucleic acid of any one of Embodiments 9-11, which

[0423] (a) treats, ameliorates, and / or prevents tissue ischemia, physical injury, stroke, and / or myocardial infarction in a subject in need thereof.

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[0425] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817)

[0426] (b) treats, ameliorates, and / or prevents a condition associated with anti-annexin V autoimmune antibodies, optionally a pregnancy loss and / or an antiphospholipid syndrome, and / or

[0427] (c) treats, ameliorates, and / or prevents thrombo-hemorrhage in a subject carrying a mutant annexin V.

[0428] Embodiment 13: A method of treating, ameliorating, and / or preventing a disease or disorder caused by or involving blood vessel thromboembolic occlusion and / or embolic occlusion in a subject in need thereof, the method comprising: administering to the subject an effective amount of the composition of any one of Embodiments 1-8 or the construct or nucleic acid of any one of Embodiments 9-12.

[0429] Embodiment 14: The method of Embodiment 13, wherein the blood vessel thromboembolic occlusion is a thromboembolic occlusion or an embolic occlusion in a microvasculature blood vessel.

[0430] Embodiment 15: The method of any one of Embodiments 13-14, wherein the disease or disorder comprises:

[0431] (a) tissue ischemia, physical injury, stroke, or myocardial infarction,

[0432] (b) a condition associated with anti-annexin V autoimmune antibodies, optionally a pregnancy loss or an antiphospholipid syndrome, and / or

[0433] (c) a thrombo-hemorrhage in a subject carrying a mutant annexin V.

[0434] Embodiment 16: The method of any one of Embodiments 13-15, wherein the composition or the construct is administered parenterally.

[0435] Embodiment 17: The method of any one of Embodiments 13-16, wherein the administration of the composition or the construct

[0436] (a) accelerates thromboangioplasticity process, clot removal from a blood vessel, vessel recanalization, reestablishment of blood flow, and / or tissue repair in a subject, and / or

[0437] (b) reduces a risk of hemorrhage after injury and / or the risk of hemorrhagic conversion due to stroke or organ trauma.

[0438] Embodiment 18: The method of any one of Embodiments 13-17, wherein the subject is a mammal, optionally a human.

[0439] Embodiment 19: A method of accelerating thromboangioplasticity process in a blood vessel having a thrombotic occlusion and / or an embolic occlusion, the method comprising: contacting the blood vessel with the composition of any one of Embodiments 1-8 or the construct of any one of Embodiments 9-12, or

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[0441] 56873617 2 Attorney Docket No. 047162-7481WO 1(02817) delivering to a cell in, within, or in proximity' to the blood vessel the composition of any one of Embodiments 1-8 or the nucleic acid of any one of Embodiments 9-12.

[0442] Embodiment 20: The method of Embodiment 19, wherein the blood vessel is a microvasculature blood vessel. The foregoing outlines features of several embodiments so that those skilled in the art may better understand the aspects of the present disclosure. Those skilled in the art should appreciate that they may readily use the present disclosure as a basis for designing or modifying other processes and structures for carry ing out the same purposes and / or achieving the same advantages of the embodiments introduced herein. Those skilled in the art should also realize that such equivalent constructions do not depart from the spirit and scope of the present disclosure, and that they may make various changes, substitutions, and alterations herein without departing from the spirit and scope of the present disclosure.

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Claims

Attorney Docket No. 047162-7481WO 1(02817)CLAIMSWHAT IS CLAIMED IS:

1. A composition, comprising: a first polypeptide comprising a grow th arrest-specific 6 (GAS6) polypeptide and / or a protein S polypeptide, or a first nucleic acid encoding the first polypeptide; and / or a second polypeptide comprising an annexin V polypeptide, or a second nucleic acid encoding the second polypeptide.

2. The composition of claim 1, wherein the first polypeptide and the second polypeptide are not covalently linked to each other.

3. The composition of claim 1, wherein the first polypeptide and the second polypeptide are covalently linked to each other.

4. The composition of claim 3, wherein both the first polypeptide and the second polypeptide are parts of a fusion protein.

5. The composition of any one of claims 1-4, further comprising a pharmaceutically acceptable carrier.

6. The composition of any one of claims 1-5, wherein the composition is formulated for parenteral administration, optionally wherein the parenteral administration comprises intradermal administration, subcutaneous administration, intramuscular administration, or intravenous administration.

7. The composition of any one of claims 1-6, w hich(a) accelerates thromboangioplasticity process, clot removal from a blood vessel, vessel recanalization, reestablishment of blood flow, and / or tissue repair in a subject, and / or(b) reduces a risk of hemorrhage after injury and / or the risk of hemorrhagic conversion due to stroke and / or organ trauma.

8. The composition of any one of claims 1-7. which6156873617 2Attorney Docket No. 047162-7481WO 1(02817)(a) treats, ameliorates, and / or prevents a tissue ischemia, a physical injury, a stroke, and / or a myocardial infarction in a subject in need thereof,(b) treats, ameliorates, and / or prevents a condition associated with anti-annexin V autoimmune antibodies, optionally a pregnancy loss or an antiphospholipid syndrome, and / or(c) treats, ameliorates, and / or prevents thrombo-hemorrhage in subjects carrying a mutant annexin V.

9. A non-natural construct or a nucleic acid encoding the non-natural construct, the construct comprising: a first polypeptide comprising a growth arrest-specific 6 (GAS6) polypeptide and / or a protein S polypeptide; and / or a second polypeptide comprising an annexin V polypeptide.

10. The construct or nucleic acid of claim 9, which is a fusion protein comprising the first polypeptide and the second polypeptide.

11. The construct or nucleic acid of any one of claims 9-10, w hich(a) accelerates clot removal from a blood vessel, vessel recanalization, reestablishment of blood flow, and / or tissue repair in a subject, and / or(b) reduces a risk of hemorrhage after injury and / or the risk of hemorrhagic conversion due to stroke and / or organ trauma.

12. The construct or nucleic acid of any one of claims 9-11, w hich(a) treats, ameliorates, and / or prevents tissue ischemia, physical injury, stroke, and / or myocardial infarction in a subject in need thereof,(b) treats, ameliorates, and / or prevents a condition associated with anti-annexin V autoimmune antibodies, optionally a pregnancy loss and / or an antiphospholipid syndrome, and / or(c) treats, ameliorates, and / or prevents thrombo-hemorrhage in a subject carrying a mutant annexin V.

13. A method of treating, ameliorating, and / or preventing a disease or disorder caused by or involving blood vessel thromboembolic occlusion and / or embolic occlusion in a subject in need thereof, the method comprising:6256873617 2Attorney Docket No. 047162-7481WO 1(02817) administering to the subject an effective amount of the composition of any one of claims 1-8 or the construct or nucleic acid of any one of claims 9-12.

14. The method of claim 13, wherein the blood vessel thromboembolic occlusion is a thromboembolic occlusion or an embolic occlusion in a microvasculature blood vessel.

15. The method of any one of claims 13-14, wherein the disease or disorder comprises:(a) tissue ischemia, physical injury, stroke, or myocardial infarction,(b) a condition associated with anti-annexin V autoimmune antibodies, optionally a pregnancy loss or an antiphospholipid syndrome, and / or(c) a thrombo-hemorrhage in a subject carrying a mutant annexin V.

16. The method of any one of claims 13-15, wherein the composition or the construct is administered parenterally.

17. The method of any one of claims 13-16, wherein the administration of the composition or the construct(a) accelerates thromboangioplasticity process, clot removal from a blood vessel, vessel recanalization, reestablishment of blood flow, and / or tissue repair in a subject, and / or(b) reduces a risk of hemorrhage after injury and / or the risk of hemorrhagic conversion due to stroke or organ trauma.

18. The method of any one of claims 13-17, wherein the subject is a mammal, optionally a human.

19. A method of accelerating thromboangioplasticity process in a blood vessel having a thrombotic occlusion and / or an embolic occlusion, the method comprising: contacting the blood vessel with the composition of any one of claims 1-8 or the construct of any one of claims 9-12, or delivering to a cell in, within, or in proximity to the blood vessel the composition of any one of claims 1-8 or the nucleic acid of any one of claims 9-12.

20. The method of claim 19, wherein the blood vessel is a microvasculature blood vessel.6356873617 2

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