Fusion protein compositions and methods relating to hemostatic therapy

Abstract

Described herein are compositions and polypeptides comprising a collagen-binding peptide (CBP) and a von Willebrand factor binding peptide (VBP), as well as methods of use thereof.

Description

FUSION PROTEIN COMPOSITIONS AND METHODS RELATING TO HEMOSTATIC THERAPYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119 of U.S. Provisional Application No. 63 / 678,634 filed August 2, 2024, the contents of which are incorporated herein by reference in their entirety.GOVERNMENT SUPPORT

[0002] This invention was made with government support under W81XWH-22-1-0113 awarded by Defense Health Agency, Medical Research and Development Branch (DHA / MRDB). The government has certain rights in this invention.SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on July 14, 2025, is named 002806-000144WOPT_SL.txt and is 32,608 bytes in size.TECHNICAL FIELD

[0004] The technology described herein relates to methods and compositions for treatment of a hemostatic disorder or injury.BACKGROUND

[0005] A subject suffering from high volume bleeding requires extremely quick-acting therapies. For example, for trauma patients, it is well known that intervention during the “golden hour” after the trauma is critical to obtain favorable outcomes. Therapies to address uncontrolled or high volume bleeding need to be immediately deployable, often in the field, and provide rapid hemostatic effects.SUMMARY

[0006] The inventors have found that the polypeptides described herein can provide rapid decreases in bleeding. These treatments are relevant to hemostatic treatment for, e.g, patients bleeding from trauma or other serious wounds, hemorrhage, internal wounds, surgery-associated hermorrhage, oncologic clotting disorders, chemo / radiotherapy-induced myelosuppression, and hemophilia.

[0007] In one aspect of any of the embodiments, described herein is a polypeptide comprising: a collagen-binding peptide (CBP); and a von Willebrand factor binding peptide (VBP).

[0008] In some embodiments of any of the aspects, the polypeptide further comprises a linker located between the CBP and the VBP. In some embodiments of any of the aspects, the polypeptide comprises, from N-terminus to C-terminus: a) a CBP; b) a linker; andc) a VBP.In some embodiments of any of the aspects, the polypeptide comprises, from N-terminus to C- terminus: a) a VBP; b) a linker; and c) a CBP.

[0009] In some embodiments of any of the aspects, the CBP comprises, consists of, or consists essentially of the sequence of SEQ ID NO: 1 (GP{Hyp}GP{Hyp}GP{Hyp}GP{Hyp}GP{Hyp}GP{Hyp}GP{Hyp}). In some embodiments of any of the aspects, the VBP comprises, consists of, or consists essentially of the sequence of SEQ ID NO:2 (TRYLRIHPQSWVHQI) or SEQ ID NO: 7 (TRYLRIHPQSQVHQI). In some embodiments of any of the aspects, the linker comprises, consists of, or consists essentially of the sequence of SEQ ID NO:3 (GGGSGGGS). In some embodiments of any of the aspects, the polypeptide comprises, consists of, or consists essentially of the sequence of SEQ ID NO: 4 (GP{Hyp}GP{Hyp}GP{Hyp}GP{Hyp}GP{Hyp}GP{Hyp}GP{Hyp}GGGSGGGSTRYLRIHPQSW VHQI) or GP{Hyp}GP{Hyp}GP{Hyp}GP{Hyp}GP{Hyp}GP{Hyp}GP{Hyp}GGGSGGGSTRYLRIHPQSQV HQI (SEQ ID NO: 9).

[0010] In some embodiments of any of the aspects, the polypeptide is not conjugated or attached to a substrate. In some embodiments of any of the aspects, the polypeptide is not conjugated or attached to a polymer. In some embodiments of any of the aspects, the polypeptide is not conjugated or attached to a non-proteinaceous polymer.

[0011] In one aspect of any of the embodiments, described herein is a pharmaceutical composition comprising a polypeptide as described herein. In some embodiments of any of the aspects, the composition does not comprise a substrate, polymer, or protein-polymer bilayer.

[0012] In some embodiments of any of the aspects, the polymer comprises hyaluronic acid, polyvinyl alcohol, or polylactic-co-glycolic acid. In some embodiments of any of the aspects, the the polymer is hyaluronic acid.

[0013] In one aspect of any of the embodiments, described herein is a method comprising administering the polypeptide of composition of any one of the preceding claims to a subject in need of hemostatic treatment. In one aspect of any of the embodiments, described herein is a polypeptide described herein, for use in treating a subject in need of hemostatic treatment. In some embodiments of any of the aspects, the subject is a subject having or diagnosed as having hemophilia, thrombocytopenia, or Glanzmann thrombasthenia.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Fig. 1 presents exemplary peptide sequence and properties used in Example 1 and Figs. 2- 7C. Figure discloses SEQ ID NOS 4 and 4, respectively, in order of appearance.

[0015] Fig. 2 presents structural characterization using transmission electron microscopy.

[0016] Fig. 3A depicts structural characterization of an exemplary polypeptide using cryoscanning electron microscopy. The peptide hemostat exhibits highly ordered fibrils as seen in SEM and TEM. Fig. 3B depicts AFM of CBP and VBP conjugated to hyaluronic acid. This composition exhibits condensed globular forms and extended coil structures.

[0017] Figs. 4A-4B depict in vivo results using healthy mice in a tail vein laceration model.

[0018] Figs. 5A-5B depict in vivo results using thrombocytopenic mice (low levels of platelet depletion) in a tail vein laceration model.

[0019] Fig. 6 depicts in vivo results using thrombocytopenic mice (low levels of platelet depletion) in a tail vein laceration model.

[0020] Figs. 7A-7C depict in vivo results using hemophilia mice in a tail vein laceration model.

[0021] Fig. 8 depicts the characterization of covodutide.

[0022] Fig. 9 demonstrates that covodutide colocalizes with platelets on injury-relevant surfaces.

[0023] Fig. 10 demonstrates that covodutide exhibits higher binding to activated than resting platelets.

[0024] Figs. 11A-1 IB demonstrate that covodutide enhances platelet aggregation and stabilizes platelet plugs. Fig. 11A depicts a schematic of the experimental protocol. Fig. 1 IB depicts graphs of aggregtation as measured by light transmission.

[0025] Fig. 12 demonstrates that covodutide does not activate resting platelets.

[0026] Figs. 13A-13B demonstrate that covodutide exhibits favorable pharmacokinetics and biodistribution. Fig. 13 depicts a schematic of the experiment and the half-life of covodutide after injections. Fig. 13B depicts biodistribution.

[0027] Figs. 14A-14B demonstrate that covodutide does not cause toxicity. Fig. 14A depicts measurements of liver and kidney toxicity markers. Fig. 14B depicts histological analysis.

[0028] Fig. 15 depicts a summary of the foregoing mechanism of action and safety studies.

[0029] Fig. 16 demonstrates that covodutide is an effective hemostat in healthy mice using a tail vein laceration model. Total blood loss is shown.

[0030] Figs. 17A-17B demonstrate that covodutide is an effective hemostat in thrombocytopenic mice using a tail vein laceration model. Fig. 17A depicts a schematic of the experiment and the platelet count. Fig. 17B depicts total blood loss and blood loss overtime.

[0031] Figs. 18A-18B demonstrate that covodutide is an effective hemostat in hemophilic mice using a tail vein laceration model. Fig. 18A depicts a schematic of the experiment. Fig. 18B depicts total blood loss and blood loss over time.

[0032] Fig. 19 demonstrates that covodutide reduces non-compressible hemorrhage in rats using a femoral artery incision model. The figure depicts a schematic of the experimental protocol.

[0033] Fig. 20 demonstrates that covodutide reduces non-compressible hemorrhage in rats using a femoral artery incision model. Total blood loss and blood loss over time are shown.

[0034] Fig. 21 depicts a schematic of an approach to translation of covodutide.

[0035] Fig. 22 demonstrates that covodutide is a prophylactic hemostat.DETAILED DESCRIPTION

[0036] Prior art approaches for administering pro-hemostatic peptide therapies suffered from a lack of uniformity. The precise dosage or peptides were difficult to control, and the total dosage was often comparatively high due to the presence of large scaffolding materials. The inventors sought to find more effective formulations that would eliminate this disadvantage. Specifically, the inventors have shown that a single polypeptide comprising both CBP and VBP is an effective hemostatic agent with superior performance as compared to, e.g, CBP and VBP with a HA linker.

[0037] The effectiveness of this approach is surprising because the prior art formulations utilized extremely large macromolecules (e.g,. 250 kDa per molecule) to deliver peptides. The instant compositions relate to very small individual polypeptides (e.g., 4.3 kDa per peptide), and a large number of these polypeptides must necessarily act in concert to exhibit detectable hemostatic activity. It was not predictable that this would be possible. Additionally, CBP and VBP have, when administered as independent peptides, CBP and VBP individually or together as a mix of 2 separate peptides do not exert a functional hemostatic effect, as shown previously (see Gao et al. (A polymer- based systemic hemostatic agent.Sci. Adv, 2020). Furthermore, the instant compositions show a surprising and expected ability to self-organize into fibrils, forming a matrix that promotes secure binding of platelets and endothelial cells. Such matrix organization was not observed for prior art formulations, e.g., CBP and VBP with a HA linker (see Figs. 3A and 3B).

[0038] Accordingly, described herein is a polypeptide comprising A polypeptide comprising a collagen-binding peptide (CBP); and a von Willebrand factor binding peptide (VBP).

[0039] As used herein, “collagen-binding peptide” or “CBP” refers to a peptide that binds to collagen. In some embodiments, the CBP comprises a repeating triplet that can form a triple helix when three CBPs are present. CBPs are described in the art, e.g., in Kehrel et al. Blood 1998; 91: 491-9; and Famdale et al., J Thromb Haemost 2004; 2: 561-73; and Munnix et al., Journal of Thrombosis and Haemostasis, 2008 6: 2132-2142; each of which is incorporated by reference herein in its entirety. Exemplary CBPs include GP{Hyp}GP{Hyp}GP{Hyp}GP{Hyp}GP{Hyp}GP{Hyp}GP{Hyp} (SEQ ID NO: 1), (GXX)y, where each X is proline or hydroxyproline and y is an integer from 6 to 10 (SEQ ID NO: 5), and(GXX)y, where at least half of the X’s are proline or hydroxyproline and y is an integer from 6 to 10 (SEQ ID NO: 6).

[0040] In some embodiments of any of the aspects, the CBP comprises a sequence with at least 70% sequence identity to SEQ ID NO: 1. In some embodiments of any of the aspects, the CBP comprises a sequence with at least 80% sequence identity to SEQ ID NO: 1. In some embodiments of any of the aspects, the CBP comprises a sequence with at least 85% sequence identity to SEQ ID NO: 1. In some embodiments of any of the aspects, the CBP comprises a sequence with at least 90% sequence identity to SEQ ID NO: 1. In some embodiments of any of the aspects, the CBP comprises a sequence with at least 95% sequence identity to SEQ ID NO: 1. In some embodiments of any of the aspects, the CBP comprises a sequence with at least 96% sequence identity to SEQ ID NO: 1. In some embodiments of any of the aspects, the CBP comprises a sequence with at least 97% sequence identity to SEQ ID NO: 1. In some embodiments of any of the aspects, the CBP comprises a sequence with at least 98% sequence identity to SEQ ID NO: 1. In some embodiments of any of the aspects, the CBP comprises a sequence with at least 99% sequence identity to SEQ ID NO: 1. In some embodiments of any of the aspects, the CBP comprises the sequence of SEQ ID NO: 1.

[0041] In some embodiments of any of the aspects, the CBP consists essentially of a sequence with at least 70% sequence identity to SEQ ID NO: 1. In some embodiments of any of the aspects, the CBP consists essentially of a sequence with at least 80% sequence identity to SEQ ID NO: 1. In some embodiments of any of the aspects, the CBP consists essentially of a sequence with at least 85% sequence identity to SEQ ID NO: 1. In some embodiments of any of the aspects, the CBP consists essentially of a sequence with at least 90% sequence identity to SEQ ID NO: 1. In some embodiments of any of the aspects, the CBP consists essentially of a sequence with at least 95% sequence identity to SEQ ID NO: 1. In some embodiments of any of the aspects, the CBP consists essentially of a sequence with at least 96% sequence identity to SEQ ID NO: 1. In some embodiments of any of the aspects, the CBP consists essentially of a sequence with at least 97% sequence identity to SEQ ID NO: 1. In some embodiments of any of the aspects, the CBP consists essentially of a sequence with at least 98% sequence identity to SEQ ID NO: 1. In some embodiments of any of the aspects, the CBP consists essentially of a sequence with at least 99% sequence identity to SEQ ID NO: 1. In some embodiments of any of the aspects, the CBP consists essentially of the sequence of SEQ ID NO: 1.

[0042] In some embodiments of any of the aspects, the CBP consists of a sequence with at least 70% sequence identity to SEQ ID NO: 1. In some embodiments of any of the aspects, the CBP consists of a sequence with at least 80% sequence identity to SEQ ID NO: 1. In some embodiments of any of the aspects, the CBP consists of a sequence with at least 85% sequence identity to SEQ ID NO: 1. In some embodiments of any of the aspects, the CBP consists of a sequence with at least 90% sequence identity to SEQ ID NO: 1. In some embodiments of any of the aspects, the CBP consists ofa sequence with at least 95% sequence identity to SEQ ID NO: 1 . In some embodiments of any of the aspects, the CBP consists of a sequence with at least 96% sequence identity to SEQ ID NO: 1. In some embodiments of any of the aspects, the CBP consists of a sequence with at least 97% sequence identity to SEQ ID NO: 1. In some embodiments of any of the aspects, the CBP consists of a sequence with at least 98% sequence identity to SEQ ID NO: 1 . In some embodiments of any of the aspects, the CBP consists of a sequence with at least 99% sequence identity to SEQ ID NO: 1. In some embodiments of any of the aspects, the CBP consists of the sequence of SEQ ID NO: 1.

[0043] In some embodiments of any of the aspects, the CBP comprises a sequence with at least 70% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the CBP comprises a sequence with at least 80% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the CBP comprises a sequence with at least 85% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the CBP comprises a sequence with at least 90% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the CBP comprises a sequence with at least 95% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the CBP comprises a sequence with at least 96% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the CBP comprises a sequence with at least 97% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the CBP comprises a sequence with at least 98% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the CBP comprises a sequence with at least 99% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the CBP comprises the sequence of SEQ ID NO: 5.

[0044] In some embodiments of any of the aspects, the CBP consists essentially of a sequence with at least 70% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the CBP consists essentially of a sequence with at least 80% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the CBP consists essentially of a sequence with at least 85% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the CBP consists essentially of a sequence with at least 90% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the CBP consists essentially of a sequence with at least 95% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the CBP consists essentially of a sequence with at least 96% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the CBP consists essentially of a sequence with at least 97% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the CBP consists essentially of a sequence with at least 98% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the CBP consists essentially of a sequence with at least 99% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the CBP consists essentially of the sequence of SEQ ID NO: 5.

[0045] In some embodiments of any of the aspects, the CBP consists of a sequence with at least 70% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the CBP consists of a sequence with at least 80% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the CBP consists of a sequence with at least 85% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the CBP consists of a sequence with at least 90% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the CBP consists of a sequence with at least 95% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the CBP consists of a sequence with at least 96% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the CBP consists of a sequence with at least 97% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the CBP consists of a sequence with at least 98% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the CBP consists of a sequence with at least 99% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the CBP consists of the sequence of SEQ ID NO: 5.

[0046] In some embodiments of any of the aspects, the CBP comprises a sequence with at least 70% sequence identity to SEQ ID NO: 6. In some embodiments of any of the aspects, the CBP comprises a sequence with at least 80% sequence identity to SEQ ID NO: 6. In some embodiments of any of the aspects, the CBP comprises a sequence with at least 85% sequence identity to SEQ ID NO: 6. In some embodiments of any of the aspects, the CBP comprises a sequence with at least 90% sequence identity to SEQ ID NO: 6. In some embodiments of any of the aspects, the CBP comprises a sequence with at least 95% sequence identity to SEQ ID NO: 6. In some embodiments of any of the aspects, the CBP comprises a sequence with at least 96% sequence identity to SEQ ID NO: 6. In some embodiments of any of the aspects, the CBP comprises a sequence with at least 97% sequence identity to SEQ ID NO: 6. In some embodiments of any of the aspects, the CBP comprises a sequence with at least 98% sequence identity to SEQ ID NO: 6. In some embodiments of any of the aspects, the CBP comprises a sequence with at least 99% sequence identity to SEQ ID NO: 6. In some embodiments of any of the aspects, the CBP comprises the sequence of SEQ ID NO: 6.

[0047] In some embodiments of any of the aspects, the CBP consists essentially of a sequence with at least 70% sequence identity to SEQ ID NO: 6. In some embodiments of any of the aspects, the CBP consists essentially of a sequence with at least 80% sequence identity to SEQ ID NO: 6. In some embodiments of any of the aspects, the CBP consists essentially of a sequence with at least 85% sequence identity to SEQ ID NO: 6. In some embodiments of any of the aspects, the CBP consists essentially of a sequence with at least 90% sequence identity to SEQ ID NO: 6. In some embodiments of any of the aspects, the CBP consists essentially of a sequence with at least 95% sequence identity to SEQ ID NO: 6. In some embodiments of any of the aspects, the CBP consists essentially of a sequence with at least 96% sequence identity to SEQ ID NO: 6. In some embodiments of any of the aspects, the CBP consists essentially of a sequence with at least 97%sequence identity to SEQ ID NO: 6. In some embodiments of any of the aspects, the CBP consists essentially of a sequence with at least 98% sequence identity to SEQ ID NO: 6. In some embodiments of any of the aspects, the CBP consists essentially of a sequence with at least 99% sequence identity to SEQ ID NO: 6. In some embodiments of any of the aspects, the CBP consists essentially of the sequence of SEQ ID NO: 6.

[0048] In some embodiments of any of the aspects, the CBP consists of a sequence with at least 70% sequence identity to SEQ ID NO: 6. In some embodiments of any of the aspects, the CBP consists of a sequence with at least 80% sequence identity to SEQ ID NO: 6. In some embodiments of any of the aspects, the CBP consists of a sequence with at least 85% sequence identity to SEQ ID NO: 6. In some embodiments of any of the aspects, the CBP consists of a sequence with at least 90% sequence identity to SEQ ID NO: 6. In some embodiments of any of the aspects, the CBP consists of a sequence with at least 95% sequence identity to SEQ ID NO: 6. In some embodiments of any of the aspects, the CBP consists of a sequence with at least 96% sequence identity to SEQ ID NO: 6. In some embodiments of any of the aspects, the CBP consists of a sequence with at least 97% sequence identity to SEQ ID NO: 6. In some embodiments of any of the aspects, the CBP consists of a sequence with at least 98% sequence identity to SEQ ID NO: 6. In some embodiments of any of the aspects, the CBP consists of a sequence with at least 99% sequence identity to SEQ ID NO: 6. In some embodiments of any of the aspects, the CBP consists of the sequence of SEQ ID NO: 6.

[0049] In some embodiments of any of the aspects, the CBP comprises a sequence with at least 70% sequence identity to at least one of SEQ ID NOS: 1, 5, or 6. In some embodiments of any of the aspects, the CBP comprises a sequence with at least 80% sequence identity to at least one of SEQ ID NOS: 1, 5, or 6. In some embodiments of any of the aspects, the CBP comprises a sequence with at least 85% sequence identity to at least one of SEQ ID NOS: 1, 5, or 6. In some embodiments of any of the aspects, the CBP comprises a sequence with at least 90% sequence identity to at least one of SEQ ID NOS: 1, 5, or 6. In some embodiments of any of the aspects, the CBP comprises a sequence with at least 95% sequence identity to at least one of SEQ ID NOS: 1, 5, or 6. In some embodiments of any of the aspects, the CBP comprises a sequence with at least 96% sequence identity to at least one of SEQ ID NOS: 1, 5, or 6. In some embodiments of any of the aspects, the CBP comprises a sequence with at least 97% sequence identity to at least one of SEQ ID NOS: 1, 5, or 6. In some embodiments of any of the aspects, the CBP comprises a sequence with at least 98% sequence identity to at least one of SEQ ID NOS: 1, 5, or 6. In some embodiments of any of the aspects, the CBP comprises a sequence with at least 99% sequence identity to at least one of SEQ ID NOS: 1, 5, or 6. In some embodiments of any of the aspects, the CBP comprises the sequence of at least one of SEQ ID NOS: 1, 5, or 6.

[0050] In some embodiments of any of the aspects, the CBP consists essentially of a sequence with at least 70% sequence identity to at least one of SEQ ID NOS: 1, 5, or 6. In some embodimentsof any of the aspects, the CBP consists essentially of a sequence with at least 80% sequence identity to at least one of SEQ ID NOS: 1, 5, or 6. In some embodiments of any of the aspects, the CBP consists essentially of a sequence with at least 85% sequence identity to at least one of SEQ ID NOS: 1, 5, or 6. In some embodiments of any of the aspects, the CBP consists essentially of a sequence with at least 90% sequence identity to at least one of SEQ ID NOS: 1, 5, or 6. In some embodiments of any of the aspects, the CBP consists essentially of a sequence with at least 95% sequence identity to at least one of SEQ ID NOS: 1, 5, or 6. In some embodiments of any of the aspects, the CBP consists essentially of a sequence with at least 96% sequence identity to at least one of SEQ ID NOS: 1, 5, or 6. In some embodiments of any of the aspects, the CBP consists essentially of a sequence with at least 97% sequence identity to at least one of SEQ ID NOS: 1, 5, or 6. In some embodiments of any of the aspects, the CBP consists essentially of a sequence with at least 98% sequence identity to at least one of SEQ ID NOS: 1, 5, or 6. In some embodiments of any of the aspects, the CBP consists essentially of a sequence with at least 99% sequence identity to at least one of SEQ ID NOS: 1, 5, or 6. In some embodiments of any of the aspects, the CBP consists essentially of the sequence of at least one of SEQ ID NOS: 1, 5, or 6.

[0051] In some embodiments of any of the aspects, the CBP consists of a sequence with at least 70% sequence identity to at least one of SEQ ID NOS: 1, 5, or 6. In some embodiments of any of the aspects, the CBP consists of a sequence with at least 80% sequence identity to at least one of SEQ ID NOS: 1, 5, or 6. In some embodiments of any of the aspects, the CBP consists of a sequence with at least 85% sequence identity to at least one of SEQ ID NOS: 1, 5, or 6. In some embodiments of any of the aspects, the CBP consists of a sequence with at least 90% sequence identity to at least one of SEQ ID NOS: 1, 5, or 6. In some embodiments of any of the aspects, the CBP consists of a sequence with at least 95% sequence identity to at least one of SEQ ID NOS: 1, 5, or 6. In some embodiments of any of the aspects, the CBP consists of a sequence with at least 96% sequence identity to at least one of SEQ ID NOS: 1, 5, or 6. In some embodiments of any of the aspects, the CBP consists of a sequence with at least 97% sequence identity to at least one of SEQ ID NOS: 1, 5, or 6. In some embodiments of any of the aspects, the CBP consists of a sequence with at least 98% sequence identity to at least one of SEQ ID NOS: 1, 5, or 6. In some embodiments of any of the aspects, the CBP consists of a sequence with at least 99% sequence identity to at least one of SEQ ID NOS: 1, 5, or 6. In some embodiments of any of the aspects, the CBP consists of the sequence of at least one of SEQ ID NOS: 1, 5, or 6.

[0052] As used herein, “von Willebrand factor binding peptide” or “VBP” refers to a peptide that binds to von Willebrand factor (vWF). In some embodiments, the VBP can comprise a sequence that is a peptide ligand for vWF. Such VBPs are described in the art, e.g,. in WO 2007052067; Moriki et al., Biochemistry Biophys Res Commun, 2010 391:783-788; Lisman et al., Blood 2006 108: 3753-56; and Huang, et al Bioorg Med Chem, 1996 May;4(5):699-708; each of which is incorporated byreference herein in its entirety.Exemplary VBPs include TRYLRIHPQSWVHQI (SEQ ID NO: 2), TRYLRIHPQSQVHQI (SEQ ID NO: 7), and RVRSFYK (SEQ ID NO: 8).

[0053] In some embodiments of any of the aspects, the VBP comprises a sequence with at least 70% sequence identity to SEQ ID NO: 2. In some embodiments of any of the aspects, the VBP comprises a sequence with at least 80% sequence identity to SEQ ID NO: 2. In some embodiments of any of the aspects, the VBP comprises a sequence with at least 85% sequence identity to SEQ ID NO: 2. In some embodiments of any of the aspects, the VBP comprises a sequence with at least 90% sequence identity to SEQ ID NO: 2. In some embodiments of any of the aspects, the VBP comprises a sequence with at least 95% sequence identity to SEQ ID NO: 2. In some embodiments of any of the aspects, the VBP comprises a sequence with at least 96% sequence identity to SEQ ID NO: 2. In some embodiments of any of the aspects, the VBP comprises a sequence with at least 97% sequence identity to SEQ ID NO: 2. In some embodiments of any of the aspects, the VBP comprises a sequence with at least 98% sequence identity to SEQ ID NO: 2. In some embodiments of any of the aspects, the VBP comprises a sequence with at least 99% sequence identity to SEQ ID NO: 2. In some embodiments of any of the aspects, the VBP comprises the sequence of SEQ ID NO: 2.

[0054] In some embodiments of any of the aspects, the VBP consists essentially of a sequence with at least 70% sequence identity to SEQ ID NO: 2. In some embodiments of any of the aspects, the VBP consists essentially of a sequence with at least 80% sequence identity to SEQ ID NO: 2. In some embodiments of any of the aspects, the VBP consists essentially of a sequence with at least 85% sequence identity to SEQ ID NO: 2. In some embodiments of any of the aspects, the VBP consists essentially of a sequence with at least 90% sequence identity to SEQ ID NO: 2. In some embodiments of any of the aspects, the VBP consists essentially of a sequence with at least 95% sequence identity to SEQ ID NO: 2. In some embodiments of any of the aspects, the VBP consists essentially of a sequence with at least 96% sequence identity to SEQ ID NO: 2. In some embodiments of any of the aspects, the VBP consists essentially of a sequence with at least 97% sequence identity to SEQ ID NO: 2. In some embodiments of any of the aspects, the VBP consists essentially of a sequence with at least 98% sequence identity to SEQ ID NO: 2. In some embodiments of any of the aspects, the VBP consists essentially of a sequence with at least 99% sequence identity to SEQ ID NO: 2. In some embodiments of any of the aspects, the VBP consists essentially of the sequence of SEQ ID NO: 2.

[0055] In some embodiments of any of the aspects, the VBP consists of a sequence with at least 70% sequence identity to SEQ ID NO: 2. In some embodiments of any of the aspects, the VBP consists of a sequence with at least 80% sequence identity to SEQ ID NO: 2. In some embodiments of any of the aspects, the VBP consists of a sequence with at least 85% sequence identity to SEQ ID NO: 2. In some embodiments of any of the aspects, the VBP consists of a sequence with at least 90% sequence identity to SEQ ID NO: 2. In some embodiments of any of the aspects, the VBP consists ofa sequence with at least 95% sequence identity to SEQ ID NO: 2. In some embodiments of any of the aspects, the VBP consists of a sequence with at least 96% sequence identity to SEQ ID NO: 2. In some embodiments of any of the aspects, the VBP consists of a sequence with at least 97% sequence identity to SEQ ID NO: 2. In some embodiments of any of the aspects, the VBP consists of a sequence with at least 98% sequence identity to SEQ ID NO: 2. In some embodiments of any of the aspects, the VBP consists of a sequence with at least 99% sequence identity to SEQ ID NO: 2. In some embodiments of any of the aspects, the VBP consists of the sequence of SEQ ID NO: 2.

[0056] In some embodiments of any of the aspects, the VBP comprises a sequence with at least 70% sequence identity to SEQ ID NO: 7. In some embodiments of any of the aspects, the VBP comprises a sequence with at least 80% sequence identity to SEQ ID NO: 7. In some embodiments of any of the aspects, the VBP comprises a sequence with at least 85% sequence identity to SEQ ID NO: 7. In some embodiments of any of the aspects, the VBP comprises a sequence with at least 90% sequence identity to SEQ ID NO: 7. In some embodiments of any of the aspects, the VBP comprises a sequence with at least 95% sequence identity to SEQ ID NO: 7. In some embodiments of any of the aspects, the VBP comprises a sequence with at least 96% sequence identity to SEQ ID NO: 7. In some embodiments of any of the aspects, the VBP comprises a sequence with at least 97% sequence identity to SEQ ID NO: 7. In some embodiments of any of the aspects, the VBP comprises a sequence with at least 98% sequence identity to SEQ ID NO: 7. In some embodiments of any of the aspects, the VBP comprises a sequence with at least 99% sequence identity to SEQ ID NO: 7. In some embodiments of any of the aspects, the VBP comprises the sequence of SEQ ID NO: 7.

[0057] In some embodiments of any of the aspects, the VBP consists essentially of a sequence with at least 70% sequence identity to SEQ ID NO: 7. In some embodiments of any of the aspects, the VBP consists essentially of a sequence with at least 80% sequence identity to SEQ ID NO: 7. In some embodiments of any of the aspects, the VBP consists essentially of a sequence with at least 85% sequence identity to SEQ ID NO: 7. In some embodiments of any of the aspects, the VBP consists essentially of a sequence with at least 90% sequence identity to SEQ ID NO: 7. In some embodiments of any of the aspects, the VBP consists essentially of a sequence with at least 95% sequence identity to SEQ ID NO: 7. In some embodiments of any of the aspects, the VBP consists essentially of a sequence with at least 96% sequence identity to SEQ ID NO: 7. In some embodiments of any of the aspects, the VBP consists essentially of a sequence with at least 97% sequence identity to SEQ ID NO: 7. In some embodiments of any of the aspects, the VBP consists essentially of a sequence with at least 98% sequence identity to SEQ ID NO: 7. In some embodiments of any of the aspects, the VBP consists essentially of a sequence with at least 99% sequence identity to SEQ ID NO: 7. In some embodiments of any of the aspects, the VBP consists essentially of the sequence of SEQ ID NO: 7.

[0058] In some embodiments of any of the aspects, the VBP consists of a sequence with at least 70% sequence identity to SEQ ID NO: 7. In some embodiments of any of the aspects, the VBP consists of a sequence with at least 80% sequence identity to SEQ ID NO: 7. In some embodiments of any of the aspects, the VBP consists of a sequence with at least 85% sequence identity to SEQ ID NO: 7. In some embodiments of any of the aspects, the VBP consists of a sequence with at least 90% sequence identity to SEQ ID NO: 7. In some embodiments of any of the aspects, the VBP consists of a sequence with at least 95% sequence identity to SEQ ID NO: 7. In some embodiments of any of the aspects, the VBP consists of a sequence with at least 96% sequence identity to SEQ ID NO: 7. In some embodiments of any of the aspects, the VBP consists of a sequence with at least 97% sequence identity to SEQ ID NO: 7. In some embodiments of any of the aspects, the VBP consists of a sequence with at least 98% sequence identity to SEQ ID NO: 7. In some embodiments of any of the aspects, the VBP consists of a sequence with at least 99% sequence identity to SEQ ID NO: 7. In some embodiments of any of the aspects, the VBP consists of the sequence of SEQ ID NO: 7.

[0059] In some embodiments of any of the aspects, the VBP comprises a sequence with at least 70% sequence identity to SEQ ID NO: 8. In some embodiments of any of the aspects, the VBP comprises a sequence with at least 80% sequence identity to SEQ ID NO: 8. In some embodiments of any of the aspects, the VBP comprises a sequence with at least 85% sequence identity to SEQ ID NO: 8. In some embodiments of any of the aspects, the VBP comprises a sequence with at least 90% sequence identity to SEQ ID NO: 8. In some embodiments of any of the aspects, the VBP comprises a sequence with at least 95% sequence identity to SEQ ID NO: 8. In some embodiments of any of the aspects, the VBP comprises a sequence with at least 96% sequence identity to SEQ ID NO: 8. In some embodiments of any of the aspects, the VBP comprises a sequence with at least 97% sequence identity to SEQ ID NO: 8. In some embodiments of any of the aspects, the VBP comprises a sequence with at least 98% sequence identity to SEQ ID NO: 8. In some embodiments of any of the aspects, the VBP comprises a sequence with at least 99% sequence identity to SEQ ID NO: 8. In some embodiments of any of the aspects, the VBP comprises the sequence of SEQ ID NO: 8.

[0060] In some embodiments of any of the aspects, the VBP consists essentially of a sequence with at least 70% sequence identity to SEQ ID NO: 8. In some embodiments of any of the aspects, the VBP consists essentially of a sequence with at least 80% sequence identity to SEQ ID NO: 8. In some embodiments of any of the aspects, the VBP consists essentially of a sequence with at least 85% sequence identity to SEQ ID NO: 8. In some embodiments of any of the aspects, the VBP consists essentially of a sequence with at least 90% sequence identity to SEQ ID NO: 8. In some embodiments of any of the aspects, the VBP consists essentially of a sequence with at least 95% sequence identity to SEQ ID NO: 8. In some embodiments of any of the aspects, the VBP consists essentially of a sequence with at least 96% sequence identity to SEQ ID NO: 8. In some embodiments of any of the aspects, the VBP consists essentially of a sequence with at least 97%sequence identity to SEQ ID NO: 8. In some embodiments of any of the aspects, the VBP consists essentially of a sequence with at least 98% sequence identity to SEQ ID NO: 8. In some embodiments of any of the aspects, the VBP consists essentially of a sequence with at least 99% sequence identity to SEQ ID NO: 8. In some embodiments of any of the aspects, the VBP consists essentially of the sequence of SEQ ID NO: 8.

[0061] In some embodiments of any of the aspects, the VBP consists of a sequence with at least 70% sequence identity to SEQ ID NO: 8. In some embodiments of any of the aspects, the VBP consists of a sequence with at least 80% sequence identity to SEQ ID NO: 8. In some embodiments of any of the aspects, the VBP consists of a sequence with at least 85% sequence identity to SEQ ID NO: 8. In some embodiments of any of the aspects, the VBP consists of a sequence with at least 90% sequence identity to SEQ ID NO: 8. In some embodiments of any of the aspects, the VBP consists of a sequence with at least 95% sequence identity to SEQ ID NO: 8. In some embodiments of any of the aspects, the VBP consists of a sequence with at least 96% sequence identity to SEQ ID NO: 8. In some embodiments of any of the aspects, the VBP consists of a sequence with at least 97% sequence identity to SEQ ID NO: 8. In some embodiments of any of the aspects, the VBP consists of a sequence with at least 98% sequence identity to SEQ ID NO: 8. In some embodiments of any of the aspects, the VBP consists of a sequence with at least 99% sequence identity to SEQ ID NO: 8. In some embodiments of any of the aspects, the VBP consists of the sequence of SEQ ID NO: 8.

[0062] In some embodiments of any of the aspects, the VBP comprises a sequence with at least 70% sequence identity to at least one of SEQ ID NOS: 2, 7, or 8. In some embodiments of any of the aspects, the VBP comprises a sequence with at least 80% sequence identity to at least one of SEQ ID NOS: 2, 7, or 8. In some embodiments of any of the aspects, the VBP comprises a sequence with at least 85% sequence identity to at least one of SEQ ID NOS: 2, 7, or 8. In some embodiments of any of the aspects, the VBP comprises a sequence with at least 90% sequence identity to at least one of SEQ ID NOS: 2, 7, or 8. In some embodiments of any of the aspects, the VBP comprises a sequence with at least 95% sequence identity to at least one of SEQ ID NOS: 2, 7, or 8. In some embodiments of any of the aspects, the VBP comprises a sequence with at least 96% sequence identity to at least one of SEQ ID NOS: 2, 7, or 8. In some embodiments of any of the aspects, the VBP comprises a sequence with at least 97% sequence identity to at least one of SEQ ID NOS: 2, 7, or 8. In some embodiments of any of the aspects, the VBP comprises a sequence with at least 98% sequence identity to at least one of SEQ ID NOS: 2, 7, or 8. In some embodiments of any of the aspects, the VBP comprises a sequence with at least 99% sequence identity to at least one of SEQ ID NOS: 2, 7, or 8. In some embodiments of any of the aspects, the VBP comprises the sequence of at least one of SEQ ID NOS: 2, 7, or 8.

[0063] In some embodiments of any of the aspects, the VBP consists essentially of a sequence with at least 70% sequence identity to at least one of SEQ ID NOS: 2, 7, or 8. In some embodimentsof any of the aspects, the VBP consists essentially of a sequence with at least 80% sequence identity to at least one of SEQ ID NOS: 2, 7, or 8. In some embodiments of any of the aspects, the VBP consists essentially of a sequence with at least 85% sequence identity to at least one of SEQ ID NOS: 2, 7, or 8. In some embodiments of any of the aspects, the VBP consists essentially of a sequence with at least 90% sequence identity to at least one of SEQ ID NOS: 2, 7, or 8. In some embodiments of any of the aspects, the VBP consists essentially of a sequence with at least 95% sequence identity to at least one of SEQ ID NOS: 2, 7, or 8. In some embodiments of any of the aspects, the VBP consists essentially of a sequence with at least 96% sequence identity to at least one of SEQ ID NOS: 2, 7, or 8. In some embodiments of any of the aspects, the VBP consists essentially of a sequence with at least 97% sequence identity to at least one of SEQ ID NOS: 2, 7, or 8. In some embodiments of any of the aspects, the VBP consists essentially of a sequence with at least 98% sequence identity to at least one of SEQ ID NOS: 2, 7, or 8. In some embodiments of any of the aspects, the VBP consists essentially of a sequence with at least 99% sequence identity to at least one of SEQ ID NOS: 2, 7, or 8. In some embodiments of any of the aspects, the VBP consists essentially of the sequence of at least one of SEQ ID NOS: 2, 7, or 8.

[0064] In some embodiments of any of the aspects, the VBP consists of a sequence with at least 70% sequence identity to at least one of SEQ ID NOS: 2, 7, or 8. In some embodiments of any of the aspects, the VBP consists of a sequence with at least 80% sequence identity to at least one of SEQ ID NOS: 2, 7, or 8. In some embodiments of any of the aspects, the VBP consists of a sequence with at least 85% sequence identity to at least one of SEQ ID NOS: 2, 7, or 8. In some embodiments of any of the aspects, the VBP consists of a sequence with at least 90% sequence identity to at least one of SEQ ID NOS: 2, 7, or 8. In some embodiments of any of the aspects, the VBP consists of a sequence with at least 95% sequence identity to at least one of SEQ ID NOS: 2, 7, or 8. In some embodiments of any of the aspects, the VBP consists of a sequence with at least 96% sequence identity to at least one of SEQ ID NOS: 2, 7, or 8. In some embodiments of any of the aspects, the VBP consists of a sequence with at least 97% sequence identity to at least one of SEQ ID NOS: 2, 7, or 8. In some embodiments of any of the aspects, the VBP consists of a sequence with at least 98% sequence identity to at least one of SEQ ID NOS: 2, 7, or 8. In some embodiments of any of the aspects, the VBP consists of a sequence with at least 99% sequence identity to at least one of SEQ ID NOS: 2, 7, or 8. In some embodiments of any of the aspects, the VBP consists of the sequence of at least one of SEQ ID NOS: 2, 7, or 8.

[0065] In some embodiments of any of the aspects, the CBP is N-terminal of the VBP. In some embodiments of any of the aspects, at least one CBP is N-terminal of at least one VBP.

[0066] In some embodiments of any of the aspects, the VBP is N-terminal of the CBP. In some embodiments of any of the aspects, at least one VBP is N-terminal of at least one CBP.

[0067] In some embodiments of any of the aspects, the polypeptide does not comprise intervening sequence between the VBP and the CBP. In some embodiments of any of the aspects, the polypeptide does not comprise intervening sequence between each VBP and each CBP.

[0068] In some embodiments of any of the aspects, the polypeptide comprises intervening sequence between the VBP and the CBP. In some embodiments of any of the aspects, the polypeptide comprises intervening sequence between each VBP and each CBP.

[0069] As used herein, “linker” refers to an oligo- or polypeptide region from about 2 to 100 amino acids in length, which links together any of the other domains or sequences of the polypeptides as described herein. In some embodiments of any of the aspects, linkers can include or be composed of flexible residues such as glycine and serine so that the adjacent protein domains are free to move relative to one another. Longer linkers may be used when it is desirable to ensure that two adjacent domains do not sterically interfere with one another. Linkers may be cleavable or non-cleavable.

[0070] In some embodiments of any of the aspects, the linker is at least 2 amino acids in length. In some embodiments of any of the aspects, the linker is 2-200 amino acids in length. In some embodiments of any of the aspects, the linker is 2-100 amino acids in length. In some embodiments of any of the aspects, the linker is 2-50 amino acids in length. In some embodiments of any of the aspects, the linker is 2-20 amino acids in length. In some embodiments of any of the aspects, the linker is 2-10 amino acids in length.

[0071] In some embodiments of any of the aspects, the linker comprises no more than 50% of the polypeptide by weight. In some embodiments of any of the aspects, the linker comprises no more than 40% of the polypeptide by weight. In some embodiments of any of the aspects, the linker comprises no more than 30% of the polypeptide by weight. In some embodiments of any of the aspects, the linker comprises no more than 20% of the polypeptide by weight. In some embodiments of any of the aspects, the linker comprises no more than 15% of the polypeptide by weight. In some embodiments of any of the aspects, the linker comprises no more than 10% of the polypeptide by weight. In some embodiments of any of the aspects, the linker comprises no more than 5% of the polypeptide by weight.

[0072] In some embodiments of any of the aspects, the linker comprises no more length than the CBP and the VBP combined. In some embodiments of any of the aspects, the linker comprises no more length than the CBP. In some embodiments of any of the aspects, the linker comprises no more length than the VBP. In some embodiments of any of the aspects, the linker comprises no more length than either of each of the CBP and the VBP.

[0073] Exemplary linker sequences are provided herein, e.g., SEQ ID NO: 3. In some embodiments of any of the aspects, the linker comprises the sequence of SEQ ID NO: 3. In some embodiments of any of the aspects, the linker domain consists essentially of the sequence of SEQ ID NO: 3. In some embodiments of any of the aspects, the linker domain consists of the sequence of SEQ ID NO: 3.

[0074] In some embodiments of any of the aspects, the polypeptide described herein comprises a sequence with at least 70% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the polypeptide described herein comprises a sequence with at least 80% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the polypeptide described herein comprises a sequence with at least 85% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the polypeptide described herein comprises a sequence with at least 90% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the polypeptide described herein comprises a sequence with at least 95% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the polypeptide described herein comprises a sequence with at least 96% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the polypeptide described herein comprises a sequence with at least 97% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the polypeptide described herein comprises a sequence with at least 98% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the polypeptide described herein comprises a sequence with at least 99% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the polypeptide described herein comprises the sequence of SEQ ID NO: 5.

[0075] In some embodiments of any of the aspects, the polypeptide described herein consists essentially of a sequence with at least 70% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the polypeptide described herein consists essentially of a sequence with at least 80% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the polypeptide described herein consists essentially of a sequence with at least 85% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the polypeptide described herein consists essentially of a sequence with at least 90% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the polypeptide described herein consists essentially of a sequence with at least 95% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the polypeptide described herein consists essentially of a sequence with at least 96% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the polypeptide described herein consists essentially of a sequence with at least 97% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the polypeptide described herein consists essentially of a sequence with at least 98% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the polypeptide described herein consists essentially of a sequence with at least 99% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the polypeptide described herein consists essentially of the sequence of SEQ ID NO: 5.

[0076] In some embodiments of any of the aspects, the polypeptide described herein consists of a sequence with at least 70% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the polypeptide described herein consists of a sequence with at least 80% sequence identity toSEQ ID NO: 5. In some embodiments of any of the aspects, the polypeptide described herein consists of a sequence with at least 85% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the polypeptide described herein consists of a sequence with at least 90% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the polypeptide described herein consists of a sequence with at least 95% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the polypeptide described herein consists of a sequence with at least 96% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the polypeptide described herein consists of a sequence with at least 97% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the polypeptide described herein consists of a sequence with at least 98% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the polypeptide described herein consists of a sequence with at least 99% sequence identity to SEQ ID NO: 5. In some embodiments of any of the aspects, the polypeptide described herein consists of the sequence of SEQ ID NO: 5.

[0077] In some embodiments of any of the aspects, the polypeptide is not conjugated or attached to a substrate. In some embodiments of any of the aspects, the polypeptide is not conjugated or attached to a polymer. In some embodiments of any of the aspects, the polypeptide is not conjugated or attached to a non-proteinaceous polymer.

[0078] In some embodiments of any of the aspects, the polypeptide is not conjugated to a substrate. In some embodiments of any of the aspects, the polypeptide is not conjugated to a polymer. In some embodiments of any of the aspects, the polypeptide is not conjugated to a non-proteinaceous polymer.

[0079] In some embodiments of any of the aspects, the polypeptide is not attached to a substrate. In some embodiments of any of the aspects, the polypeptide is not attached to a polymer. In some embodiments of any of the aspects, the polypeptide is not attached to a non-proteinaceous polymer.

[0080] In some embodiments of any of the aspects, the polypeptide is not conjugated or attached to a substrate or polymer. In some embodiments of any of the aspects, the polypeptide is not conjugated or attached to a substrate or non-proteinaceous polymer. In some embodiments of any of the aspects, the polypeptide is not conjugated to a substrate or polymer. In some embodiments of any of the aspects, the polypeptide is not conjugated to a substrate or non-proteinaceous polymer. In some embodiments of any of the aspects, the polypeptide is not attached to a substrate or polymer. In some embodiments of any of the aspects, the polypeptide is not attached to a substrate or non-proteinaceous polymer.

[0081] As used herein, “conjugated” refers to the attachment of at least two entities to form one entity. The joining of the two entities can be direct (e.g., via covalent or non-covalent bonds) or indirect (e.g., via linkers etc.). Thus, conjugation can be by means of linkers, chemical modification, peptide linkers, chemical linkers, covalent or non-covalent bonds, or protein fusion or by any meansknown to one skilled in the art. The joining or conjugation can be permanent or reversible. In some embodiments of any of the aspects, the conjugation is by a covalent bond.

[0082] As used herein, “attached” refers to the joining or connection of two entities.

[0083] Conjugated and attached do not encompass physical proximity or physical contact. For example, a polypeptide as described herein, when in a soluble form in a solution which is provided in a container, is not necessarily conjugated or attached to the container even if the polypeptide physically contacts a wall of the container.

[0084] As used herein, “substrate” refers to a structure, that provides a surface suitable for adherence of biological particles, e.g., polypeptides. The substrate can comprise patterns, grooves, channels, and / or supports. In some embodiments of any of the aspects, the substrate or polymer comprises a Fc backbone. In some embodiments of any of the aspects, the substrate or polymer comprises a lipid. In some embodiments of any of the aspects, the substrate or polymer comprises a lipid chain. In some embodiments of any of the aspects, the substrate or polymer comprises a Fc backbone, a lipid, and / or a lipid chain.

[0085] As used herein, “polymer” refers to refers to oligomers, co-oligomers, polymers and copolymers, e.g., random block, multiblock, star, grafted, gradient copolymers and combination thereof. The average molecular weight of the polymer, as determined by gel permeation chromatography, can range from 500 to about 500,000, e.g., from 20,000 to about 500,000. A non-proteinaceous polymer is a polymer in which the monomer units comprise moieties which are not amino acids.

[0086] Exemplary polymers include but are not limited to polysaccharides, polypeptides, polynucleotides, copolymers of fumaric / sebacic acid, poloxamers, polylactides, polyglycolides, polycaprolactones, copolymers of polylactic acid and polyglycolic acid, polyanhydrides, polyepsilon caprolactone, polyamides, polyurethanes, polyesteramides, polyorthoesters, polydioxanones, polyacetals, polyketals, polycarbonates, polyorthocarbonates, polydihydropyrans, polyphosphazenes, polyhydroxybutyrates, polyhydroxyvalerates, polyalkylene oxalates, polyalkylene succinates, poly(malic acid), poly(amino acids), polyvinylpyrrolidone, polyethylene glycol, polyhydroxycellulose, polymethyl methacrylate, chitin, chitosan, copolymers of polylactic acid and polyglycolic acid, poly(glycerol sebacate) (PGS), gelatin, collagen, silk, alginate, cellulose, poly-nucleic acids, cellulose acetates (including cellulose diacetate), polyethylene, polypropylene, polybutylene, polyethylene terphthalate (PET), polyvinyl chloride, polystyrene, polyamides, nylon, polycarbonates, polysulfides, polysulfones, hydrogels (e.g., acrylics), polyacrylonitrile, polyvinylacetate, cellulose acetate butyrate, nitrocellulose, copolymers of urethane / carbonate, copolymers of styrene / maleic acid, poly(ethylenimine), hyaluron, heparin, agarose, pullulan, and copolymers, terpolymers, and copolymers comprising any combinations thereof. Exemplary polymers can include, by way of non-limiting example polylactide (PLA): polyglycolide (PGA); poly-(s-caprolactonc) (PCL); polyvinyl alcohol (PVA), poly(lactic-co-caprolactone) (PLCL), hyaluronic acid (HA), gelatin, collagen; poly(glycerolsebacate) (PGS); polyphosphazenes; polyorthoesters; polyanhydrides; poly(a-hydroxy esters); poly(ether esters); copolymers comprising lactide of glycolide and s-caprolactonc or trimethylene carbonate; poly(polyol sebacate) elastomers; elastomers; poly(polyol citrate); polyesters; poly(glycolic acid); poly(lactic acid); poly(caprolactone); poly(lactic-co-glycolic acid); poly(butylene succinate); poly(trimethylene carbonate); poly(p-dioxanone); poly(butylene terephthalate); poly(ester amide)s; Hybrane™ S1200; DegraPol™; polyurethanes; polyanhydrides; poly[(caboxyphenoxy) propane -sebacic acid]; polyphsophoesters; poly[bis(hydroxyethyl) terephthalate-ethyl orthophosphorylate / terephthaloyl chloride]; poly(ortho esters); poly(alkyl cyanoacrylates); poly(butyl cyanoacrylate); polyethers; polyethylene glycol); poly(amino acids); tyrosine derived polycarbonate; microbial polyesters; poly(P-hydroxyalkanoate); poly (hydroxybutyrate); poly(hydroxybutyrate-co- hydroxy valerate); collagen; albumin; gluten; chitosan; hyaluronate; cellulose; alginate; and starch.

[0087] In some embodiments of any of the aspects, the polymer is hyaluronic acid, polyvinyl alcohol, and / or polylactic-co-glycolic acid.

[0088] In one aspect of any of the embodiments, described herein is a pharmaceutical composition comprising a polypeptide as described herein. In some embodiments of any of the aspects, the composition described herein does not comprise a substrate. In some embodiments of any of the aspects, the composition described herein does not comprise a polymer. In some embodiments of any of the aspects, the composition described herein does not comprise a non-proteinaceous polymer. In some embodiments of any of the aspects, the composition does not comprise a protein- polymer bilayer. In some embodiments of any of the aspects, the composition does not comprise a substrate, polymer, or protein-polymer bilayer.

[0089] As used herein, “protein-polymer bilayer” refers to a bilayer (e.g., a bilayer structure or bilayer substrate) comprising protein and polymeric material. In some embodiments of any of the aspects, the bilayer is a phospholipid bilayer. In some embodiments of any of the aspects, the bilayer is series of layers, e.g., as constructed by layer-by-layer deposition.

[0090] In one aspect of any of the embodiments, described herein is a pharmaceutical composition comprising a nucleic acid encoding a polypeptide as described herein. In some embodiments of any of the aspects, the composition described herein does not comprise a substrate. In some embodiments of any of the aspects, the composition described herein does not comprise a polymer. In some embodiments of any of the aspects, the composition described herein does not comprise a non-proteinaceous polymer. In some embodiments of any of the aspects, the composition does not comprise a protein-polymer bilayer. In some embodiments of any of the aspects, the composition does not comprise a substrate, polymer, or protein-polymer bilayer.

[0091] In some embodiments, the technology described herein relates to a nucleic acid encoding a polypeptide as described herein. In some embodiments, the nucleic acid is a cDNA.

[0092] In some embodiments, a nucleic acid encoding a peptide as described herein is comprised by a vector. In some of the aspects described herein, a nucleic acid sequence encoding a polypeptide is operably linked to a vector.

[0093] A nucleic acid molecule, such as DNA, is said to be “capable of expressing” a polypeptide if it contains nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are “operably linked” to nucleotide sequences which encode the polypeptide. An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene expression as polypeptides in recoverable amounts. The precise nature of the regulatory regions needed for gene expression may vary from organism to organism, as is well known in the analogous art.

[0094] In one aspect of any of the embodiments, described herein is a cell comprising a polypeptide as described herein, or a nucleic acid encoding such a polypeptide. The cell can be either a prokaryotic or eukaryotic cell. In some embodiments, the nucleotide sequence is incorporated into a plasmid or viral vector capable of autonomous replication in the recipient cell. Any of a wide variety of vectors can be employed for this purpose and are known and available to those or ordinary skill in the art. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to “shuttle” the vector between host cells of different species.

[0095] In some embodiments, the technology described herein relates to a syringe or catheter, comprising a therapeutically effective amount of a composition described herein.

[0096] In one aspect, described herein is a kit comprising a composition as described herein, e.g., a composition comprising a polypeptide as described herein. A kit is any manufacture (e.g., a package or container) comprising at least one reagent, e.g., a polypeptide , the manufacture being promoted, distributed, or sold as a unit for performing the methods described herein. The exact nature of the components configured in the kit depends on its intended purpose. In some embodiments of any of the aspects, a kit includes instructions for use. “Instructions for use” typically include a tangible expression describing the technique to be employed in using the components of the kit, e.g., to treat a subject in need of hemostatic treatment. Still in accordance with the present invention, “instructions for use” may include a tangible expression describing the preparation of at least one reagent described herein, such as dilution, mixing, or dosing instructions, and the like, typically for an intended purpose. Optionally, the kit also contains other useful components, such as, measuring tools, diluents, buffers, syringes, pharmaceutically acceptable carriers, or other useful paraphernalia as will be readily recognized by those of skill in the art.

[0097] The materials or components assembled in the kit can be provided to the practitioner stored in any convenient and suitable ways that preserve their operability and utility. For example, the components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room, refrigerated or frozen temperatures. The components are typically contained in suitable packaging material(s). As employed herein, the phrase “packaging material” refers to one or more physical structures used to house the contents of the kit, such as inventive compositions and the like. The packaging material is constructed by well-known methods, preferably to provide a sterile, contaminant-free environment. The packaging may also preferably provide an environment that protects from light, humidity, and oxygen. As used herein, the term “package” refers to a suitable solid matrix or material such as glass, plastic, paper, foil, polyester (such as polyethylene terephthalate, or Mylar) and the like, capable of holding the individual kit components. Thus, for example, a package can be a glass vial used to contain suitable quantities of a composition containing a volume of at least one reagent described herein. The packaging material generally has an external label which indicates the contents and / or purpose of the kit and / or its components.

[0098] In some embodiments of any of the aspects, a polypeptide as described herein is engineered. In some embodiemnts of any of the aspects, a polypeptide as described herein is not naturally occurring. In some embodiemnts of any of the aspects, a polypeptide as described herein comprises a CBP and VBP that do not naturally occur in the same polypeptide. In some embodiemnts of any of the aspects, a CBP as described herein is not naturally occurring. In some embodiemnts of any of the aspects, a VBP as described herein is not naturally occurring.

[0099] In some embodiments of any of the aspects, the polypeptide present in a composition, or combination, of the disclosure exhibit an increased utility that is not exhibited when said a VBP or CBP sequence occurs alone or are present at a naturally occurring concentration. In some embodiments of any of the aspects, compositions of the disclosure, comprising a polypeptide comprising at least one VBP and at least one CBP as taught herein, exhibit a synergistic effect on imparting at least one improved trait when the VBP and CBP are present in the same polypeptide. In some embodiments of any of the aspects, the compositions of the disclosure— comprising a polypeptide comprising at least one VBP and at least one CBP as taught herein— exhibit markedly different characteristics / properties compared to their closest naturally occurring counterpart. That is, the compositions of the disclosure exhibit markedly different functional and / or structural characteristics / properties, as compared to their closest naturally occurring counterpart. For instance, the polypeptides comprising at least one VBP and at least one CBP of the disclosure are structurally different from a VBP or CBP as it naturally exists in a cell or polypeptide, for at least the following reasons: said polypeptide can be isolated and purified, such that it is not found in the milieu of the cell or naturally occurring polypeptides, said polypeptide comprising at least one VBP and at least one CBP can be present at concentrations that do not occur in the cell or natural polypeptide environment,said polypeptide comprising at least one VBP and at least one CBP can be associated with acceptable carriers that do not occur in the cell or natural polypeptide environment, said polypeptide comprising at least one VBP and at least one CBP can be formulated to be shelf-stable and exist outside the cell or natural polypeptide environment, and said polypeptide comprising at least one VBP and at least one CBP can be combined with other therapeutic agents at concentrations that do not exist in the cell or natural polypeptide environment. Further, the polypeptide comprising at least one VBP and at least one CBP of the disclosure are functionally different from a VBP or CBP as it naturally exists in a cell or natural polypeptide environment, for at least the following reasons: said polypeptide comprising at least one VBP and at least one CBP when applied in an isolated and purified form can lead to effective clotting and / or reductions in bleeding time and / or blood loss, said polypeptide comprising at least one VBP and at least one CBP can be formulated to be shelf-stable and able to exist outside the cell or natural polypeptide environment, such that the polypeptide comprising at least one VBP and at least one CBP now has a new utility as a therapeutic agent capable of administration to a subject, wherein the CBP and / or VBP could not have such a utility in it's natural state in the cell or natural polypeptide environment, as the VBP and / or CBP would be unable to survive outside the cell or natural polypeptide environment without the intervention of the hand of man to formulate the polypeptide comprising at least one VBP and at least one CBP into a shelf-stable state and impart this new utility that has the aforementioned functional characteristics not possessed by the VBP and / or CBP in it's natural state of existence in the cell or natural polypeptide environment.

[0100] In some embodiments, the methods described herein relate to treating a subject having or diagnosed as having a hemostatic disorder or injury with a polypeptide comprising at least one VBP and at least one CBP. The compositions and combinations described herein have the effect of slowing or stopping bleeding (a hemostatic effect), e.g., by promoting clot formation. Accordingly, in one aspect of any of the embodiments, provided herein is a method comprising administering the composition or combination of described herein to a subject in need of hemostatic treatment. A subject in need of hemostatic treatment is a subject who is bleeding or at risk of bleeding a volume of blood that would be deleterious. That is, a subject suffering from uncontrolled or suboptimally controlled bleeding. Such subjects may have bleeding or coagulation disorders, or be suffering a trauma or wound (e.g, an injury or surgical intervention). For example, the disorder can be a coagulation defect (e.g., disseminated intravascular coagulation (difibrination syndrome)), hereditary factor VIII deficiency (hemophilia A), hereditary factor IX deficiency (hemophilia B, Christmas disease), and other coagulation defects such as Von Willebrand's disease, hereditary factor Xi deficiency (hemophilia C), and purpura (e.g., qualitative platelet defects and Glanzmann's disease). In some embodiments of any of the aspects, the subject is a subject having or diagnosed as having trauma or other serious wounds, hemorrhage, internal wounds, surgery-associated hermorrhage, postpartum hemorrhage, oncologic clotting disorders, chemo / radiotherapy-induced myelosuppression,or hemophilia. In some embodiments of any of the aspects, the subject is a subject having or diagnosed as having hemophilia. In some embodiments of any of the aspects, the subject is a subject having or diagnosed as having hemophilia or Glanzmann thrombasthenia. In some embodiments of any of the aspects, the subject is a subject having a trauma or wound causing bleeding at a deleterious volume.

[0101] In one aspect of any of the embodiments, the compositions described herein are for use in a method of treating a hemostatic disorder or injury. In one aspect of any of the embodiments, the compositions described herein are for use in a method of treating a hemostatic disorder. In one aspect of any of the embodiments, the compositions described herein are for use in a method of treating a hemostatic injury. In some embodiments of any of the aspects, the hemostatic disorder can be a coagulation defect (e.g., disseminated intravascular coagulation (difibrination syndrome)), hereditary factor VIII deficiency (hemophilia A), hereditary factor IX deficiency (hemophilia B, Christmas disease), and other coagulation defects such as Von Willebrand's disease, hereditary factor Xi deficiency (hemophilia C), and purpura (e.g., qualitative platelet defects and Glanzmann's disease). In some embodiments of any of the aspects, the hemostatis disorder or injury is trauma or other serious wounds, hemorrhage, internal wounds, surgery-associated hemorrhage, postpartum hemorrhage, oncologic clotting disorders, chemo / radiotherapy-induced myelosuppression, or hemophilia. In some embodiments of any of the aspects, the hemostatis disorder is hemophilia. In some embodiments of any of the aspects, the hemostatis disorder is hemophilia or Glanzmann thrombasthenia. In some embodiments of any of the aspects, the hemostatis injury is a trauma or wound causing bleeding at a deleterious volume.

[0102] The compositions and methods described herein can be administered to a subject having or diagnosed as having hemostatic disorder or injury. In some embodiments, the methods described herein comprise administering an effective amount of compositions described herein, e.g. a polypeptide comprising at least one VBP and at least one CBP to a subject in order to alleviate a symptom of a hemostatic disorder or injury. As used herein, "alleviating a symptom" is ameliorating any condition or symptom associated with the condition, disorder, or injury. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique. A variety of means for administering the compositions described herein to subjects are known to those of skill in the art. Such methods can include, but are not limited to oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, topical, injection, or intratumoral administration. Administration can be local or systemic. In some embodiments of any of the aspects, the administration is topical. In some embodiments of any of the aspects, the administration is parenteral. In some embodiments of any of the aspects, the administration is intravenous.

[0103] The term “effective amount" as used herein refers to the amount of a polypeptide comprising at least one VBP and at least one CBP needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect. The term "therapeutically effective amount" therefore refers to an amount of a polypeptide comprising at least one VBP and at least one CBP that is sufficient to provide a particular anti -bleeding effect when administered to a typical subject. An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not generally practicable to specify an exact “effective amount". However, for any given case, an appropriate “effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.

[0104] In some embodiments of any of the aspects, an amount is therapeutically effective or a treatment is effective when the rate of bleeding is decreased as compared to prior to the treatment being administered. In some embodiments of any of the aspects, an amount is therapeutically effective or a treatment is effective when the rate of bleeding is decreased as compared to a subject with the same condition not receiving the treatment. In some embodiments of any of the aspects, an amount is therapeutically effective or a treatment is effective when the total amount of bleeding is decreased as compared to prior to the treatment being administered. In some embodiments of any of the aspects, an amount is therapeutically effective or a treatment is effective when the total amount of bleeding is decreased as compared to a subject with the same condition not receiving the treatment. In some embodiments of any of the aspects, an amount is therapeutically effective or a treatment is effective if it stops the bleeding.

[0105] Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50 / ED50. Compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (z.e., the concentration of a polypeptide comprising at least one VBP and at least one CBP, which achieves a half-maximal inhibition of symptoms) as determined in cell culture, or in an appropriate animal model. Levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by asuitable bioassay, e.g., assay for bleeding time, blood loss, or clotting, among others. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.

[0106] In some embodiments, the technology described herein relates to a pharmaceutical composition comprising a polypeptide as described herein, and optionally a pharmaceutically acceptable carrier. In some embodiments, the active ingredients of the pharmaceutical composition comprise a polypeptide as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist essentially of a polypeptide as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist of a polypeptide as described herein.

[0107] Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, ionic liquid-based formulations, solvents and / or dispersion media. The use of such carriers and diluents is well known in the art. Some non-limiting examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) semm component, such as semm albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as "excipient", "carrier", "pharmaceutically acceptable carrier" or the like are used interchangeably herein. In some embodiments, the carrier inhibits the degradation of the active agent, e.g. a polypeptide as described herein.

[0108] In some embodiments, the pharmaceutical composition comprising a polypeptide as described herein can be a parenteral dose form. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensionsready for injection, and emulsions. In addition, controlled-release parenteral dosage forms can be prepared for administration of a patient, including, but not limited to, DUROS®-type dosage forms and dose-dumping.

[0109] Suitable vehicles that can be used to provide parenteral dosage forms of a polypeptide as disclosed within are well known to those skilled in the art. Examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, com oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. Compounds that alter or modify the solubility of a pharmaceutically acceptable salt of a polypeptide as disclosed herein can also be incorporated into the parenteral dosage forms of the disclosure, including conventional and controlled-release parenteral dosage forms.

[0110] Pharmaceutical compositions comprising a polypeptide can also be formulated to be suitable for oral administration, for example as discrete dosage forms, such as, but not limited to, tablets (including without limitation scored or coated tablets), pills, caplets, capsules, chewable tablets, powder packets, cachets, troches, wafers, aerosol sprays, or liquids, such as but not limited to, syrups, elixirs, solutions or suspensions in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil emulsion. Such compositions contain a predetermined amount of the pharmaceutically acceptable salt of the disclosed compounds, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams, and Wilkins, Philadelphia PA. (2005).

[0111] Conventional dosage forms generally provide rapid or immediate drug release from the formulation. Depending on the pharmacology and pharmacokinetics of the drug, use of conventional dosage forms can lead to wide fluctuations in the concentrations of the drug in a patient's blood and other tissues. These fluctuations can impact a number of parameters, such as dose frequency, onset of action, duration of efficacy, maintenance of therapeutic blood levels, toxicity, side effects, and the like. Advantageously, controlled-release formulations can be used to control a drug's onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels. In particular, controlled- or extended-release dosage forms or formulations can be used to ensure that the maximum effectiveness of a drug is achieved while minimizing potential adverse effects and safety concerns, which can occur both from under-dosing a drug (i.e., going below the minimum therapeutic levels) as well as exceeding the toxicity level for the drug. In some embodiments, the polypeptide can be administered in a sustained release formulation.

[0112] Controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled release counterparts. Ideally, the use of an optimallydesigned controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled-release formulations include: 1) extended activity of the drug; 2) reduced dosage frequency; 3) increased patient compliance; 4) usage of less total drug; 5) reduction in local or systemic side effects; 6) minimization of drug accumulation; 7) reduction in blood level fluctuations; 8) improvement in efficacy of treatment; 9) reduction of potentiation or loss of drug activity; and 10) improvement in speed of control of diseases or conditions. Kim, Chemg-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).

[0113] Most controlled-release formulations are designed to initially release an amount of drug (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled-release of an active ingredient can be stimulated by various conditions including, but not limited to, pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological conditions or compounds.

[0114] A variety of known controlled- or extended-release dosage forms, formulations, and devices can be adapted for use with the salts and compositions of the disclosure. Examples include, but are not limited to, those described in U.S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5674,533; 5,059,595; 5,591 ,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 Bl ; each of which is incorporated herein by reference. These dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS® (Alza Corporation, Mountain View, Calif. USA)), or a combination thereof to provide the desired release profde in varying proportions.

[0115] In one aspect, provided herein the polypeptide or composition described herien is formulated for topical administration, e.g., for applying to the skin of a subject. In some embodiments, the topical formulation is in the form of a lotion, cream, solution, gel, emugel, oil, serum, powder, ointment, suspension, slurry, paste, spray, dispersion, or foam. For example, the topical formulation is in form of a dermatological ointment. In some embodiments, the topical formulation is in the form of a transdermal patch or a controlled-release patch. A topical formulation for use in the present invention may be in any form suitable for application to the body surface, such as a cream, lotion, sprays, solution, gel, ointment, paste, plaster, paint, bioadhesive, suspensions or the like, and / or may be prepared so as to contain liposomes, micelles, and / or microspheres. Such a formulation may be used in combination with an occlusive overlayer so that moisture evaporatingfrom the body surface is maintained within the formulation upon application to the body surface and thereafter.

[0116] Methods of producing topical pharmaceutical compositions such as creams, ointments, lotions, sprays and sterile aqueous solutions or suspensions are well known in the art. Suitable methods of preparing topical pharmaceutical compositions are described, for example in WO 95 / 10999, U.S. Pat. No. 6,974,585, WO 2006 / 048747 (incorporated herein by reference), as well as in documents cited in any of these references.

[0117] Formulations of the invention may optionally contain a pharmaceutically acceptable viscosity enhancer and / or film former. A viscosity enhancer increases the viscosity of the formulation so as to inhibit its spread beyond the site of application. Balsam Fir (Oregon) is an example of a pharmaceutically acceptable viscosity enhancer.

[0118] A film former, when it dries, forms a protective film over the site of application. The film inhibits removal of the active ingredient and keeps it in contact with the site being treated. An example of a film former that is suitable for use in this invention is Flexible Collodion, USP. As described in Remington: The Science and Practice of Pharmacy, 19th Ed. (Easton, Pa.: Mack Publishing Co., 1995), at page 1530, collodions are ethyl ether / ethanol solutions containing pyroxylin (a nitrocellulose) that evaporate to leave a film of pyroxylin. A film former may act additionally as a carrier. Solutions that dry to form a film are sometimes referred to as paints.

[0119] Creams, as is well known in the arts of pharmaceutical formulation, are viscous liquids or semisolid emulsions, either oil-in-water or water-in-oil. Cream bases are water-washable, and contain an oil phase, an emulsifier, and an aqueous phase. The oil phase, also called the “internal” phase, is generally comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol. The aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant. The emulsifier in a cream formulation is generally a nonionic, anionic, cationic or amphoteric surfactant.

[0120] Lotions are preparations to be applied to the skin surface without friction, and are typically liquid or semiliquid preparations in which particles, including the active agent, are present in a water or alcohol base. Lotions are usually suspensions of solids, and preferably, comprise a liquid oily emulsion of the oil-in-water type. It is generally necessary that the insoluble matter in a lotion be finely divided. Lotions will typically contain suspending agents to produce better dispersions as well as compounds useful for localizing and holding the active agent in contact with the skin, e.g., methylcellulose, sodium carboxymethyl-cellulose, or the like.

[0121] Solutions are homogeneous mixtures prepared by dissolving one or more chemical substances (solutes) in a liquid such that the molecules of the dissolved substance are dispersed among those of the solvent. The solution may contain other pharmaceutically or cosmeticallyacceptable chemicals to buffer, stabilize or preserve the solute. Common examples of solvents used in preparing solutions are ethanol, water, propylene glycol or any other acceptable vehicles.

[0122] As is well known, gels are semisolid, suspension-type systems. Single-phase gels contain organic macromolecules distributed substantially uniformly throughout the carrier liquid, which is typically aqueous, but also, preferably, contain an alcohol, and, optionally, an oil. Preferred “organic macromolecules,” i.e., gelling agents, are crosslinked acrylic acid polymers such as the “carbomer” family of polymers, e.g., carboxypolyalkylenes that may be obtained commercially under the Carbopol® trademark. Also preferred are hydrophilic polymers such as polyethylene oxides, polyoxyethylene-polyoxypropylene copolymers and polyvinylalcohol; cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and methyl cellulose; gums such as tragacanth and xanthan gum; sodium alginate; and gelatin. In order to prepare a uniform gel, dispersing agents such as alcohol or glycerin can be added, or the gelling agent can be dispersed by trituration, mechanical mixing or stirring, or combinations thereof.

[0123] Ointments, as also well known in the ert, are semisolid preparations that are typically based on petrolatum or other petroleum derivatives. The specific ointment base to be used, as will be appreciated by those skilled in the art, is one that will provide for a number of desirable characteristics, e.g., emolliency or the like. As with other carriers or vehicles, an ointment base should be inert, stable, nonirritating, and nonsensitizing. As explained in Remington: The Science and Practice of Pharmacy, 19th Ed. (Easton, Pa.: Mack Publishing Co., 1995), at pages 1399-1404, ointment bases may be grouped in four classes: oleaginous bases; emulsifiable bases; emulsion bases; and water-soluble bases. Oleaginous ointment bases include, for example, vegetable oils, fats obtained from animals, and semisolid hydrocarbons obtained from petroleum. Emulsifiable ointment bases, also known as absorbent ointment bases, contain little or no water and include, for example, hydroxystearin sulfate, anhydrous lanolin, and hydrophilic petrolatum. Emulsion ointment bases are either water-in-oil (W / O) emulsions or oil-in- water (O / W) emulsions, and include, for example, acetyl alcohol, glyceryl monostearate, lanolin, and stearic acid. Preferred water-soluble ointment bases are prepared from polyethylene glycols of varying molecular weight; again, see Remington: The Science and Practice of Pharmacy for further information.

[0124] Pastes are semisolid dosage forms in which the active agent is suspended in a suitable base. Depending on the nature of the base, pastes are divided between fatty pastes or those made from single-phase aqueous gels. The base in a fatty paste is generally petrolatum or hydrophilic petrolatum or the like. The pastes made from single-phase aqueous gels generally incorporate carboxymethylcellulose or the like as a base.

[0125] Formulations may also be prepared with liposomes, micelles, and microspheres.

[0126] Liposomes are microscopic vesicles having a lipid wall comprising a lipid bilayer, and, in the present context, encapsulate one or more components of the formulations.Liposomal preparations herein include cationic (positively charged), anionic (negatively charged), and neutral preparations. Cationic liposomes are readily available. For example, N[l-2,3- dioleyloxy)propyl]-N,N,N-triethyl -ammonium (DOTMA) liposomes are available under the tradename Lipofectin® (GIBCO BRL, Grand Island, N.Y.). Similarly, anionic and neutral liposomes are readily available as well, e.g., from Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using readily available materials. Such materials include phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), and dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with DOTMA in appropriate ratios. Methods for making liposomes using these materials are well known in the art.

[0127] Micelles are known in the art as comprised of surfactant molecules arranged so that their polar headgroups form an outer spherical shell, while the hydrophobic, hydrocarbon chains are oriented towards the center of the sphere, forming a core. Micelles form in an aqueous solution containing surfactant at a high enough concentration so that micelles naturally result. Surfactants useful for forming micelles include, but are not limited to, potassium laurate, sodium octane sulfonate, sodium decane sulfonate, sodium dodecane sulfonate, sodium lauryl sulfate, docusate sodium, decyltrimethylammonium bromide, dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, tetradecyltrimethyl-ammonium chloride, dodecylammonium chloride, polyoxyl-8 dodecyl ether, polyoxyl-12 dodecyl ether, nonoxynol 10, and nonoxynol 30.

[0128] Microspheres, similarly, may be incorporated into the present formulations. Like liposomes and micelles, microspheres essentially encapsulate one or more components of the present formulations. They are generally although not necessarily formed from lipids, preferably charged lipids such as phospholipids. Preparation of lipidic microspheres is well known in the art and described in the pertinent texts and literature.

[0129] Various additives, known to those skilled in the art, may be included in the topical formulations. For example, solvents, including relatively small amounts of alcohol, may be used to solubilize certain formulation components.

[0130] Various other additives may be included in the compositions of the present invention in addition to those identified above. These include, but are not limited to, antioxidants, preservatives, and emollients, as well as other classes of materials whose presence may be pharmaceutically or otherwise desirable. Typical examples of optional additives for inclusion in the formulations of the invention are as follows: preservatives such as sorbate; solvents such as isopropanol and propylene glycol; emollients such as polyalkylene methyl glucosides; and emulsifiers such as glycerol stearate, PEG- 100 stearate, polyglyceryl-3 hydroxylauryl ether, and polysorbate 60; sorbitol and otherpolyhydroxyalcohols such as polyethylene glycol. In certain embodiments, other agents may also be added, such as antimicrobial agents, to prevent spoilage upon storage, i.e., to inhibit growth of microbes such as yeasts and molds. Suitable antimicrobial agents are typically selected from the group consisting of the methyl and propyl esters of p-hydroxybenzoic acid (i.e., methyl and propyl paraben), sodium benzoate, sorbic acid, imidurea, and combinations thereof.

[0131] Im some embodiments of any of the aspects, the polypeptide described herein is administered as a monotherapy, e.g., another treatment for the hemostatic injury or disorder is not administered to the subject.

[0132] In some embodiments of any of the aspects, the methods described herein can further comprise administering a second agent and / or treatment to the subject, e.g. as part of a combinatorial therapy. Non-limiting examples of a second agent and / or treatment can include a hemostatic agent.

[0133] Hemostatic agents are known in the art. As used herein, “hemostatic agent” refers to an agent that promotes clotting or coagulation and / or stops bleeding. Exemplary hemostatic agents can include microfibrillar collagen, gelatin, factor concentrators (e.g., QuikClot™ (Z-Medica LLC., Newington, CT, USA), QuikClot ACS™ (advanced clotting sponge) (Z-Medica LLC., Newington, CT, USA), TraumaDex™ (Medafor Inc, Minneapolis, MN, USA), and self-expanding hemostatic polymer (Payload Systems Inc., Cambridge, MA, USA)), muscoadhesive agents (e.g., HemCon™ (HemCon Medical Technologies Inc. Portland, OR, USA) and Celox™ (Medtrade Products Ltd. Crewe, UK)), procoagulatn suppiementors (e.g., dry fribrin sealant dressing), fibrin, thrombin, collagent, adrenaline, VBP, CBP, factor VIII, Factor IX, and agents as described in US Patent Publication 2018 / 0311378, which is incorporated by reference herein in its entirety. Exemplary hemostatic agents can include but are not limited to Factor VII, Factor Vila, Factor IX, fibrinogen, prothrombin, tranexamic acid, 8-aminocaproic acid, and anti -inhibitor coagulant complex (FEIBA™).

[0134] In some embodiments of any of the aspects, a composition comprising a polypeptide described herein is administered with a further hemostatic agent, wherein the further hemostatic agent is Factor VII. In some embodiments of any of the aspects, a composition comprising a polypeptide described herein is administered with a further hemostatic agent, wherein the further hemostatic agent is tranexamic acid. In some embodiments of any of the aspects, a composition comprising a polypeptide described herein is administered with a further hemostatic agent, wherein the further hemostatic agent is Factor VII. In some embodiments of any of the aspects, a composition comprising a polypeptide described herein is administered with at least one further hemostatic agent, wherein the at least one further hemostatic agent is selected from: Factor VII and tranexamic acid.

[0135] In certain embodiments, an effective dose of a composition comprising a polypeptide as described herein can be administered to a patient once. In certain embodiments, an effective dose of a composition comprising a polypeptide as described herein can be administered to a patient repeatedly. For systemic administration, subjects can be administered a therapeutic amount of a compositioncomprising a polypeptide as described herein such as, e.g. 0. 1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, or more.

[0136] In some embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after treatment biweekly for three months, treatment can be repeated once per month, for six months or a year or longer. Treatment according to the methods described herein can reduce levels of a marker or symptom of a condition, e.g. clotting by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 % or at least 90% or more.

[0137] The dosage of a composition as described herein can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume treatment, or make other alterations to the treatment regimen. The dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to a polypeptide as described herein. The desired dose or amount of activation can be administered at one time or divided into subdoses, e.g., 2-4 subdoses and administered over a period of time, e.g., at appropriate intervals through the day or other appropriate schedule. In some embodiments, administration can be chronic, e.g., one or more doses and / or treatments daily over a period of weeks or months. Examples of dosing and / or treatment schedules are administration daily, twice daily, three times daily or four or more times daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, or more. A composition comprising a polypeptide as described herein can be administered over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period.

[0138] The dosage ranges for the administration of a polypeptide as described herein, according to the methods described herein depend upon, for example, the form of the polypeptide, its potency, and the extent to which symptoms, markers, or indicators of a condition described herein are desired to be reduced, for example the percentage reduction desired for bleeding time and / or volume or the extent to which, for example, clotting are desired to be induced. The dosage should not be so large as to cause adverse side effects, such as thrombosis. Generally, the dosage will vary with the age, condition, and sex of the patient and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of any complication.

[0139] The efficacy of a polypeptide as described herein in, e.g. the treatment of a condition described herein, or to induce a response as described herein (e.g. clotting) can be determined by the skilled clinician. However, a treatment is considered “effective treatment," as the term is used herein,if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 10% following treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and / or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate, e.g. bleeding time and / or blood loss. Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions (i.e., progression of the disease is halted). Methods of measuring these indicators are known to those of skill in the art and / or are described herein. Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the disease, e.g., preventing a worsening of symptoms (e.g. pain or inflammation); or (2) relieving the severity of the disease, e.g., causing regression of symptoms. An effective amount for the treatment of a disease means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that disease. Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response, (e.g. clotting). It is well within the ability of one skilled in the art to monitor efficacy of administration and / or treatment by measuring any one of such parameters, or any combination of parameters. Efficacy can be assessed in animal models of a condition described herein, for example treatment of tail vein lacterations. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a marker is observed, e.g. bleeding time and / or blood loss.

[0140] In vitro and animal model assays are provided herein which allow the assessment of a given dose of a polypeptide as described herein. By way of non-limiting example, the effects of a dose of a polypeptide as described herein can be assessed in a murine tail vein lacteration model.

[0141] In one respect, the present invention relates to the herein described compositions, methods, and respective component(s) thereof, as essential to the technology, yet open to the inclusion of unspecified elements, essential or not ("comprising). In some embodiments of any of the aspects, other elements to be included in the description of the composition, method or respective component thereof are limited to those that do not materially affect the basic and novel characteristic(s) of the technology (e.g., the composition, method, or respective component thereof “consists essentially of’ the elements described herein). This applies equally to steps within a described method as well as compositions and components therein. In other embodiments of any of the aspects, the compositions, methods, and respective components thereof, described herein are intended to be exclusive of any element not deemed an essential element to the component, composition or method (e.g., the composition, method, or respective component thereof “consists of’ the elements described herein). This applies equally to steps within a described method as well as compositions and components therein.

[0142] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

[0143] For convenience, certain terms employed herein, in the specification, examples and appended claims are collected here.

[0144] The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction" or “decrease" or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.

[0145] The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statistically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, a “increase” is a statistically significant increase in such level.

[0146] As used herein, a "subject" means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “individual,” “patient” and “subject” are used interchangeably herein.

[0147] Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of a hemostatic disorder or injury. A subject can be male or female.

[0148] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g. a hemostatic disorder or injury) or one or more complications related to such a condition, and optionally, have already undergone treatment for the condition or the one or more complications related to the condition. Alternatively, a subject can also be one who has not been previously diagnosed as having a condition or one or more complications related to the condition. For example, a subject can be one who exhibits one or more risk factors for the condition or one or more complications related to the condition or a subject who does not exhibit risk factors.

[0149] A “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.

[0150] As used herein, the terms “protein" and “polypeptide" are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms "protein", and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. "Protein" and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing. The terms also refer to fragments or variants of the polypeptide that maintain at least 50% of the activity or effect, e.g. collagen binding of a CBP or vWF binding of a VBP. Conservative substitution variants that maintain the activity of wildtype will include a conservative substitution as defined herein. The identification of amino acids most likely to be tolerant of conservative substitution while maintaining at least 50% of the activity of the wildtype is guided by, for example, sequence alignment with CBP or VBP homologs or paralogs from other species. Amino acids that are identical between CBP or VBP homologs are less likely to toleratechange, while those showing conservative differences are obviously much more likely to tolerate conservative change in the context of an artificial variant. Similarly, positions with non-conservative differences are less likely to be critical to function and more likely to tolerate conservative substitution in an artificial variant. Variants, fragments, and / or fusion proteins can be tested for activity, for example, by administering the variant to an appropriate animal model of a hemostatic disorder or injury as described herein. Further discussion of the structure of CBPs and VBPs can be found in the art, as discussed above herein.

[0151] In some embodiments, a polypeptide can be a variant of a sequence described herein, e.g. a variant of a polypeptide comprising the amino acid sequence of SEQ ID NO: 5. In some embodiments, the variant is a conservative substitution variant. Variants can be obtained by mutations of native nucleotide sequences, for example. A “variant,” as referred to herein, is a polypeptide substantially homologous to a native or reference polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions or substitutions. Polypeptide-encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a variant protein or fragment thereof that retains the relevant biological activity relative to the reference protein, e.g., can bind a target at least 50% as well as wildtype. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage, (i.e. 5% or fewer, e.g. 4% or fewer, or 3% or fewer, or 1% or fewer) of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. It is contemplated that some changes can potentially improve the relevant activity, such that a variant, whether conservative or not, has more than 100% of the activity of a wildtype e.g. 110%, 125%, 150%, 175%, 200%, 500%, 1000% or more.

[0152] One method of identifying amino acid residues which can be substituted is to align, for example, human CBP or VBP to a homolog from one or more non-human species. Alignment can provide guidance regarding not only residues likely to be necessary for function but also, conversely, those residues likely to tolerate change. Where, for example, an alignment shows two identical or similar amino acids at corresponding positions, it is more likely that that site is important functionally. Where, conversely, alignment shows residues in corresponding positions to differ significantly in size, charge, hydrophobicity, etc., it is more likely that that site can tolerate variation in a functional polypeptide. The variant amino acid or DNA sequence can be at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to a native or reference sequence or a nucleic acid encoding one of those amino acid sequences. The degree of homology (percent identity) between a native and a mutant sequence can be determined, for example, by comparing the twosequences using freely available computer programs commonly employed for this purpose on the world wide web. The variant amino acid or DNA sequence can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, similar to the sequence from which it is derived (referred to herein as an “original” sequence). The degree of similarity (percent similarity) between an original and a mutant sequence can be determined, for example, by using a similarity matrix. Similarity matrices are well known in the art and a number of tools for comparing two sequences using similarity matrices are freely available online, e.g. BLASTp or BLASTn (available on the world wide web at blast.ncbi.nlm.nih.gov), with default parameters set.

[0153] In the various embodiments described herein, it is further contemplated that variants (naturally occurring or otherwise), alleles, homologs, conservatively modified variants, and / or conservative substitution variants of any of the particular polypeptides described are encompassed. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid and retains the desired activity of the polypeptide. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure.

[0154] A given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as He, Vai, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known. Polypeptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired activity, e.g. binding activity and specificity of a native or reference polypeptide is retained.

[0155] A given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as He, Vai, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known. Polypeptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired activity of a native or reference polypeptide is retained. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure.

[0156] Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Particular conservative substitutions include, for example; Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into His; Asp into Glu; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; He into Leu or into Vai; Leu into He or into Vai; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into He; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and / or Phe into Vai, into He or into Leu. Typically conservative substitutions for one another also include: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

[0157] In some embodiments, the polypeptide described herein (or a nucleic acid encoding such a polypeptide) can be a functional fragment of one of the amino acid sequences described herein. As used herein, a “functional fragment” is a fragment or segment of a peptide which retains at least 50% of the wildtype reference polypeptide’s activity according to the assays described below herein. A functional fragment can comprise conservative substitutions of the sequences disclosed herein.

[0158] In some embodiments, the polypeptide described herein can be a variant of a sequence described herein. In some embodiments, the variant is a conservatively modified variant. Conservative substitution variants can be obtained by mutations of native nucleotide sequences, for example. A “variant," as referred to herein, is a polypeptide substantially homologous to a native or reference polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions or substitutions. Variant polypeptide- encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a variant protein or fragment thereof that retains activity. A wide variety of PCR-based site-specific mutagenesis approaches are known in the art and can be applied by the ordinarily skilled artisan.

[0159] In some embodiments, a polypeptide can comprise one or more amino acid substitutions or modifications. In some embodiments, the substitutions and / or modifications can prevent or reduce proteolytic degradation and / or prolong half-life of the polypeptide in a subject. In someembodiments, a polypeptide can be modified by conjugating or fusing it to other polypeptide or polypeptide domains such as, by way of non-limiting example, transferrin (WO06096515A2), albumin (Y eh et al., 1992), growth hormone (US2003104578AA); cellulose (Levy and Shoseyov, 2002); and / or Fc fragments (Ashkenazi and Chamow, 1997). The references in the foregoing paragraph are incorporated by reference herein in their entireties.

[0160] In some embodiments, a polypeptide as described herein can comprise at least one peptide bond replacement. A polypeptide as described herein can comprise one type of peptide bond replacement or multiple types of peptide bond replacements, e.g. 2 types, 3 types, 4 types, 5 types, or more types of peptide bond replacements. Non-limiting examples of peptide bond replacements include urea, thiourea, carbamate, sulfonyl urea, trifluoroethylamine, ortho-(aminoalkyl)-phenylacetic acid, para-(aminoalkyl)-phenylacetic acid, meta-(aminoalkyl)-phenylacetic acid, thioamide, tetrazole, boronic ester, olefinic group, and derivatives thereof.

[0161] In some embodiments, a polypeptide as described herein can comprise naturally occurring amino acids commonly found in polypeptides and / or proteins produced by living organisms, e.g. Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M), Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q), Asp (D), Glu (E), Lys (K), Arg (R), and His (H). In some embodiments, a polypeptide as described herein can comprise alternative amino acids. Non-limiting examples of alternative amino acids include, D-amino acids; beta-amino acids; homocysteine, phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gamma-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylic acid, statine, l,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, penicillamine (3-mercapto-D-valine), ornithine, citruline, alpha-methyl-alanine, parabenzoylphenylalanine, para-amino phenylalanine, p-fluorophenylalanine, phenylglycine, propargylglycine, sarcosine, and tert-butylglycine), diaminobutyric acid, 7-hydroxy- tetrahydroisoquinoline carboxylic acid, naphthylalanine, biphenylalanine, cyclohexylalanine, aminoisobutyric acid, norvaline, norleucine, tert-leucine, tetrahydroisoquinoline carboxylic acid, pipecolic acid, phenylglycine, homophenylalanine, cyclohexylglycine, dehydroleucine, 2,2-diethylglycine, 1- amino-1 -cyclopentanecarboxylic acid, 1-amino-l -cyclohexanecarboxylic acid, amino-benzoic acid, amino-naphthoic acid, gamma-aminobutyric acid, difluorophenylalanine, nipecotic acid, alpha-amino butyric acid, thienyl-alanine, t-butylglycine, trifluoro valine; hexafluoroleucine; fluorinated analogs; azide-modified amino acids; alkyne -modified amino acids; cyano-modified amino acids; and derivatives thereof.

[0162] In some embodiments, a polypeptide can be modified, e.g. by addition of a moiety to one or more of the amino acids that together comprise the peptide. In some embodiments, a polypeptide as described herein can comprise one or more moiety molecules, e.g. 1 or more moiety molecules per polypeptide, 2 or more moiety molecules per polypeptide, 5 or more moiety molecules per polypeptide, 10 or more moiety molecules per polypeptide or more moiety molecules per polypeptide.In some embodiments, a polypeptide as described herein can comprise one more types of modifications and / or moieties, e.g. 1 type of modification, 2 types of modifications, 3 types of modifications or more types of modifications. Non-limiting examples of modifications and / or moieties include PEGylation; glycosylation; HESylation; ELPylation; lipidation; acetylation; amidation; end-capping modifications; cyano groups; phosphorylation; albumin, and cyclization. In some embodiments, an end-capping modification can comprise acetylation at the N-terminus, N- terminal acylation, and N-terminal formylation. In some embodiments, an end-capping modification can comprise amidation at the C-terminus, introduction of C-terminal alcohol, aldehyde, ester, and thioester moieties. The half-life of a polypeptide can be increased by the addition of moieties, e.g. PEG, albumin, or other fusion partners (e.g. Fc fragment of an immunoglobin).

[0163] Any cysteine residue not involved in maintaining the proper conformation of the polypeptide also can be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) can be added to the polypeptide to improve its stability or facilitate oligomerization.

[0164] Alterations of the native amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. Techniques for making such alterations are very well established. Alterations of the original amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites permitting ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. Techniques for making such alterations include those disclosed by Khudyakov et al. “Artificial DNA: Methods and Applications” CRC Press, 2002; Braman “In Vitro Mutagenesis Protocols” Springer, 2004; and Rapley “The Nucleic Acid Protocols Handbook” Springer 2000; which are herein incorporated by reference in their entireties. In some embodiments, a polypeptide as described herein can be chemically synthesized and mutations can be incorporated as part of the chemical synthesis process.

[0165] As used herein, the term “nucleic acid” or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single -stranded or double-stranded. A single-stranded nucleic acid can be one nucleic acid strand of a denatured double- stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double -stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable DNA can include, e.g., genomic DNA or cDNA. Suitable RNA can include, e.g., mRNA.

[0166] The term "expression" refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. Expression can refer to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid fragment or fragments of the invention and / or to the translation of mRNA into a polypeptide.

[0167] "Expression products" include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" means the nucleic acid sequence which is transcribed (DNA) to RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. The gene may or may not include regions preceding and following the coding region, e.g. 5’ untranslated (5’UTR) or "leader" sequences and 3’ UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0168] “Operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, control elements operably linked to a coding sequence are capable of effecting the expression of the coding sequence. The control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0169] In some embodiments of any of the aspects, a polypeptide, nucleic acid, or cell as described herein can be engineered. As used herein, “engineered" refers to the aspect of having been manipulated by the hand of man. For example, a polypeptide is considered to be “engineered" when at least one aspect of the polypeptide, e.g., its sequence, has been manipulated by the hand of man to differ from the aspect as it exists in nature. As is common practice and is understood by those in the art, progeny of an engineered cell are typically still referred to as “engineered" even though the actual manipulation was performed on a prior entity.

[0170] In some embodiments of any of the aspects, the polypeptide described herein is exogenous. In some embodiments of any of the aspects, the polypeptide described herein is ectopic. In some embodiments of any of the aspects, the polypeptide described herein is not endogenous.

[0171] The term "exogenous" refers to a substance present in a cell other than its native source. The term "exogenous" when used herein can refer to a nucleic acid (e.g. a nucleic acid encoding a polypeptide) or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is not normally found and one wishes to introduce the nucleic acid or polypeptide into such a cell or organism. Alternatively, “exogenous” can refer to a nucleic acid or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is found in relatively low amounts and one wishes to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to create ectopic expression or levels. In contrast, the term "endogenous" refers to a substance that is native to the biological system or cell. As used herein, “ectopic” refers to a substance that is found in an unusual location and / or amount. An ectopic substance can be one that is normally found in a given cell, but at a much lower amount and / or at a different time. Ectopic also includes substance, such as a polypeptide or nucleic acid that is not naturally found or expressed in a given cell in its natural environment.

[0172] In some embodiments, a nucleic acid encoding a polypeptide as described herein is comprised by a vector. In some of the aspects described herein, a nucleic acid sequence encoding a given polypeptide as described herein, or any module thereof, is operably linked to a vector. The term "vector", as used herein, refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be viral or non-viral. The term “vector” encompasses any genetic element that is capable of replication when associated with the proper control elements and that can transfer gene sequences to cells. A vector can include, but is not limited to, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc.

[0173] In some embodiments of any of the aspects, the vector is recombinant, e.g., it comprises sequences originating from at least two different sources. In some embodiments of any of the aspects, the vector comprises sequences originating from at least two different species. In some embodiments of any of the aspects, the vector comprises sequences originating from at least two different genes, e.g., it comprises a fusion protein or a nucleic acid encoding an expression product which is operably linked to at least one non-native (e.g., heterologous) genetic control element (e.g., a promoter, suppressor, activator, enhancer, response element, or the like).

[0174] In some embodiments of any of the aspects, the vector or nucleic acid described herein is codon-optomized, e.g., the native or wild-type sequence of the nucleic acid sequence has been altered or engineered to include alternative codons such that altered or engineered nucleic acid encodes the same polypeptide expression product as the native / wild-type sequence, but will be transcribed and / or translated at an improved efficiency in a desired expression system. In some embodiments of any of the aspects, the expression system is an organism other than the source of the native / wild-typesequence (or a cell obtained from such organism). In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in a mammal or mammalian cell, e.g., a mouse, a murine cell, or a human cell. In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in a human cell. In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in a yeast or yeast cell. In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in a bacterial cell. In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in an E. coli cell.

[0175] As used herein, the term "expression vector" refers to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector. The sequences expressed will often, but not necessarily, be heterologous to the cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification.

[0176] As used herein, the term “viral vector" refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle. The viral vector can contain the nucleic acid encoding a polypeptide as described herein in place of non-essential viral genes. The vector and / or particle may be utilized for the purpose of transferring any nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.

[0177] It should be understood that the vectors described herein can, in some embodiments, be combined with other suitable compositions and therapies. In some embodiments, the vector is episomal. The use of a suitable episomal vector provides a means of maintaining the nucleotide of interest in the subject in high copy number extra chromosomal DNA thereby eliminating potential effects of chromosomal integration.

[0178] As used herein, the terms "treat,” "treatment," "treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder, e.g. a hemostatic disorder or injury. The term “treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with a hemostatic disorder or injury. Treatment is generally “effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective" if the progression of a disease is reduced or halted. That is, “treatment" includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviationof one or more symptom(s), diminishment of extent of disease, stabilized (z.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and / or decreased mortality, whether detectable or undetectable. The term "treatment" of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).

[0179] In some embodiments of any of the aspects, described herein is a prophylactic method of treatment. As used herein “prophylactic” refers to the timing and intent of a treatment relative to a disease or symptom, that is, the treatment is administered prior to clinical detection or diagnosis of that particular disease or symptom in order to protect the patient from the disease or symptom. Prophylactic treatment can encompass a reduction in the severity or speed of onset of the disease or symptom, or contribute to faster recovery from the disease or symptom. Accordingly, the methods described herein can be prophylactic relative to bleeding or severe blood loss. In some embodiments of any of the aspects, prophylactic treatment is not prevention of all symptoms or signs of a disease.

[0180] As used herein, the term “pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a carrier other than water. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and / or ointment. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be an artificial or engineered carrier, e.g., a carrier that the active ingredient would not be found to occur in in nature.

[0181] As used herein, the term “nanoparticle” refers to particles that are on the order of about 1 to 1,000 nanometers in diameter or width. The term “nanoparticle” includes nanospheres; nanorods; nanoshells; and nanoprisms; these nanoparticles may be part of a nanonetwork. The term “nanoparticles” also encompasses liposomes and lipid particles having the size of a nanoparticle. Exemplary nanoparticles include lipid nanoparticles or ferritin nanoparticles. Lipid nanoparticles can comprise multiple componenents, including, e.g., ionizable lipids (such as MC3, DLin-MC3-DMA, ALC-0315, or SM-102), pegylated lipids (such as PEG2000-C-DMG, PEG2000-DMG, ALC-0159), phospholipids (such as DSPC), and cholesterol.

[0182] Exemplary liposomes can comprise, e.g., DSPC, DPPC, DSPG, Cholesterol, hydrogenated soy phosphatidylcholine, soy phosphatidyl choline, methoxypolyethylene glycol(mPEG-DSPE) phosphatidyl choline (PC), phosphatidyl glycerol (PG), distearoylphosphatidylcholine, and combinations thereof.

[0183] As used herein, the term "administering," refers to the placement of a compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject. In some embodiments, administration comprises physical human activity, e.g., an injection, act of ingestion, an act of application, and / or manipulation of a delivery device or machine. Such activity can be performed, e.g., by a medical professional and / or the subject being treated.

[0184] As used herein, “contacting" refers to any suitable means for delivering, or exposing, an agent to at least one cell. Exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, perfusion, injection, or other delivery method well known to one skilled in the art. In some embodiments, contacting comprises physical human activity, e.g., an injection; an act of dispensing, mixing, and / or decanting; and / or manipulation of a delivery device or machine.

[0185] The term “statistically significant" or “significantly" refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.

[0186] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%.

[0187] As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation.

[0188] The term "consisting of refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0189] As used herein the term "consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0190] As used herein, the term “corresponding to” refers to an amino acid or nucleotide at the enumerated position in a first polypeptide or nucleic acid, or an amino acid or nucleotide that is equivalent to an enumerated amino acid or nucleotide in a second polypeptide or nucleic acid. Equivalent enumerated amino acids or nucleotides can be determined by alignment of candidate sequences using degree of homology programs known in the art, e.g., BLAST.

[0191] As used herein, the term “specific binding” refers to an interaction between two molecules, compounds, cells and / or particles wherein the first entity binds to the second entity withgreater specificity and affinity than it binds to a third entity. A first entity specifically bound to a second entity is not displaced by a non-similar competitor. In certain embodiments, a first entity is said to specifically bind a second entity when it preferentially recognizes the second entity in a complex mixture of proteins and / or macromolecules. In some embodiments, specific binding can refer to an affinity of the first entity for the second entity which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or greater than the affinity for the third entity. In some embodiments, specific binding refers to the ability of a first entity to bind to a second entity with a KD 10 M (10000 nM) or less, e.g., 10 " M. 107M. 107M. 10 ' M. 10|UM. 10~nM, 10l 2M. or less. The person of ordinary skill in the art can determine appropriate conditions under which a first entity selectively binds a second entity using any suitable methods, such as titration of an entityy in a suitable binding assay. In some embodiments, specific binding does not refer to covalent bonding.

[0192] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

[0193] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0194] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Bruce Alberts et al., Molecular Biology of the Cell, published by W.W. Norton & Company, 2022 (ISBN 0393884821, 978-0393884821); John M. Lackie eat al.(eds.), The Dictionary of Cell and Molecular Biology, 5thEdition, published by Academic Press, 2013 (ISBN 0123849314, 978-0123849311); Nalini Chandar et al., Lippincott Illustrated Reviews: Cell andMolecular Biology, 3rdEdition, published by LWW, 2023 (ISBN 1975180895, 978-1975180898); Teresa Atwood et al., Oxford Dictionary of Biochemistry and Molecular Biology, 2ndEdition, published by Oxford University Press, 2006; Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); Johnathan Law et al., (eds.), A Dictionary of Chemistry, 8thEdition, published by Oxford University Press, 2020 (ISBN 9780198841227, 9780191876783); Robert C. King et al. (eds.), A Dictionary of Genetics, 8thEdition, published by Oxford University Press, 2013 (ISBN 9780199766444, 9780199376865); Richard Cammack et al. (eds.), Oxford Dictionary of Biochemistry and Molecular Biology, 2ndEdition, published by Oxford University Press, 2006 (ISBN 9780198529170, 9780191727641); John Lackie et al. (eds.), A Dictionary of Biomedicine, 2ndEdition, published by Oxford University Press, 2019 (ISBN 9780191829116); Lodish et al., Molecular Cell Biology, 8thEdition, published by W.H. Freeman, 2016 (ISBN 1464183392, 978- 1464183393); Abul K. Abbas et al., Cellular and Molecular Immunology, 10thEdition, published by Elsevier, 2021 (ISBN 0323757480, 978-0323757485); Kenneth M. Murphy et al., Janeway's Immunobiology, 10thEdition, published by W. W. Norton & Company, 2022 (ISBN 0393884899, 978-0393884890); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN- 1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 0444569464); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Frederick M. Ausubel (ed.), Current Protocols in Molecular Biology (CPMB), John Wiley and Sons, 1987-2010 (ISBN 047150338X, 9780471503385); Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties.

[0195] Other terms are defined herein within the description of the various aspects of the invention.

[0196] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is basedon the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0197] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0198] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[0199] In some embodiments, the present technology may be defined in any of the following numbered paragraphs:1. A polypeptide comprising: a) a collagen-binding peptide (CBP); and b) a von Willebrand factor binding peptide (VBP).2. The polypeptide of paragraph 1, further comprising a linker located between the CBP and the VBP.3. The polypeptide of any one of the preceding paragraphs, comprising, from N-terminus to C- terminus: a) a CBP; b) a linker; and c) a VBP.4. The polypeptide of any one of the preceding paragraphs, comprising, from N-terminus to C- terminus: a) a VBP;b) a linker; and c) a CBP. The polypeptide of any one of the preceding paragraphs, wherein the CBP comprises, consists of, or consists essentially of the sequence of SEQ ID NO: 1(GP {Hyp } GP {Hyp } GP {Hyp } GP {Hyp } GP {Hyp } GP {Hyp } GP {Hyp } ) . The polypeptide of any one of the preceding paragraphs, wherein the VBP comprises, consists of, or consists essentially of the sequence of SEQ ID NO: 2 (TRYLRIHPQSWVHQI) or SEQ ID NO: 7 (TRYLRIHPQSQVHQI). The polypeptide of any one of the preceding paragraphs, wherein the linker comprises, consists of, or consists essentially of the sequence of SEQ ID NO: 3 (GGGSGGGS). The polypeptide of any one of the preceding paragraphs, comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO: 4 (GP{Hyp}GP{Hyp}GP{Hyp}GP{Hyp}GP{Hyp}GP{Hyp}GP{Hyp}GGGSGGGSTRYLRI HPQSWVHQI) or GP{Hyp}GP{Hyp}GP{Hyp}GP{Hyp}GP{Hyp}GP{Hyp}GP{Hyp}GGGSGGGSTRYLRIH PQSQVHQI (SEQ ID NO: 9). The polypeptide of any one of the preceding paragraphs, not conjugated or attached to a substrate. The polypeptide of any one of the preceding paragraphs, not conjugated or attached to a polymer. A pharmaceutical composition comprising the polypeptide of any one of the preceding paragraphs. The pharmaceutical composition of paragraph 11, not comprising a substrate, polymer, or protein-polymer bilayer. The polypeptide of any one of the preceding paragraphs, wherein the polymer comprises hyaluronic acid, polyvinyl alcohol, or polylactic-co-glycolic acid. The polypeptide or composition of any of the preceding paragraphs, wherein the polymer is hyaluronic acid. A method comprising administering the polypeptide of composition of any one of the preceding paragraphs to a subject in need of hemostatic treatment. The method of paragraph 15, wherein the subject is a subject having or diagnosed as having hemophilia, thrombocytopenia, or Glanzmann thrombasthenia. The polypeptide or composition of any one of the preceding paragraphs, for use in treating a subject in need of hemostatic treatment. The polypeptide or composition of paragraph 17, wherein the subject is a subject having or diagnosed as having hemophilia, thrombocytopenia, or Glanzmann thrombasthenia.

[0200] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.EXAMPLESExample 1

[0201] Described herein is the synthesis of a single peptide with CBP on one end and VBP on the other end. The linker between CBP and VBP is another peptide GGGSGGGS (SEQ ID NO: 3). The peptide has the sequence of:GP{Hyp}GP{Hyp}GP{Hyp}GP{Hyp}GP{Hyp}GP{Hyp}GP{Hyp}GGGSGGGSTRYLRIHPQSWV HQI (SEQ ID NO: 4).This peptide is 44 amino acids in length, where Hyp is hydroxyproline.

[0202] The peptide is made recombinantly and is a new peptide drug which works well in reducing blood loss. There are several advantages to this approach: (i) it's a single peptide, so it can be entirely made recombinantly (as was done herein); (ii) since it is a single recombinant peptide, it is extremely well-defined and addresses polydispersity questions; and (iii) being a recombinant peptide, it can leverage translational advantages.

[0203] Structural analysis of the peptide by electron miscropscopy revealed the peptide spontaneously forms fibrils (Figs. 2-3).

[0204] The ability of the peptide to reduce blood loss was test in vivo in mouse tail vein laceration models according to the protocol depicted in Fig. 4A. A significant reduction in blood loss was observed for healthy mice (Fig. 4B), thrombocytopenic mice (Figs. 5A-5B), and hemophilic mice (Figs. 7A-7B). The bleeding kinetics of thrombocytopenic mice (Fig. 6) and hemophilic mice (Fig. 7C) also showed significant improvement.Example 2

[0205] An exemplary embodiment of the peptides described herein, comprising a single peptide with CBP on one end and VBP on the other end, connected by a linker was prepared and is referred to herein as covodutide, e.g., SEQ ID NO: 4.

[0206] Covodutide colocalizes with platelets and binds preferentially to activated platelets, enhancing platelet aggregation and platelet plugs (Figs. 9-1 IB). Covodutide does not activate resting platelets (Fig. 12).

[0207] In vivo, covodutide exhibits favorable pharmacokinetics and biodistribution (Figs. BABB). It does not exhibit toxicity (Figs. 14A-14B).

[0208] The performance of covodutide in bleeding models was tested. It reduced total blood loss in healthy mice (Fig. 16), thrombocytopenic mice (Figs. 17A-17B), and hemophilic mice (Figs. 18A-18B) using tail vein laceration models. It also reduces total blood loss in femoral artery incision models (Fig. 19-20). Covodutide functions as a prophylactic hemostat (Fig. 22).

Claims

1. What is claimed herein is:

1. A polypeptide comprising: a) a collagen-binding peptide (CBP); and b) a von Willebrand factor binding peptide (VBP).

2. The polypeptide of claim 1, further comprising a linker located between the CBP and the VBP.

3. The polypeptide of any one of the preceding claims, comprising, from N-terminus to C- terminus: a) a CBP; b) a linker; and c) a VBP.

4. The polypeptide of any one of the preceding claims, comprising, from N-terminus to C- terminus: a) a VBP; b) a linker; and c) a CBP.

5. The polypeptide of any one of the preceding claims, wherein the CBP comprises, consists of, or consists essentially of the sequence of SEQ ID NO: 1(GP {Hyp } GP {Hyp } GP {Hyp } GP {Hyp } GP {Hyp } GP {Hyp } GP {Hyp }) .

6. The polypeptide of any one of the preceding claims, wherein the VBP comprises, consists of, or consists essentially of the sequence of SEQ ID NO: 2 (TRYLRIHPQSWVHQI) or SEQ ID NO: 7 (TRYLRIHPQSQVHQI).

7. The polypeptide of any one of the preceding claims, wherein the linker comprises, consists of, or consists essentially of the sequence of SEQ ID NO: 3 (GGGSGGGS).

8. The polypeptide of any one of the preceding claims, comprising, consisting of, or consisting essentially of the sequence of SEQ ID NO: 4 (GP{Hyp}GP{Hyp}GP{Hyp}GP{Hyp}GP{Hyp}GP{Hyp}GP{Hyp}GGGSGGGSTRYLRI HPQSWVHQI) orGP{Hyp}GP{Hyp}GP{Hyp}GP{Hyp}GP{Hyp}GP{Hyp}GP{Hyp}GGGSGGGSTRYLRIH PQSQVHQI (SEQ ID NO: 9).

9. The polypeptide of any one of the preceding claims, not conjugated or attached to a substrate.

10. The polypeptide of any one of the preceding claims, not conjugated or attached to a polymer.

11. A pharmaceutical composition comprising the polypeptide of any one of the preceding claims.

12. The pharmaceutical composition of claim 11, not comprising a substrate, polymer, or protein- polymer bilayer.

13. The polypeptide of any one of the preceding claims, wherein the polymer comprises hyaluronic acid, polyvinyl alcohol, or polylactic-co-glycolic acid.

14. The polypeptide or composition of any of the preceding claims, wherein the polymer is hyaluronic acid.

15. A method comprising administering the polypeptide of composition of any one of the preceding claims to a subject in need of hemostatic treatment.

16. The method of claim 15, wherein the subject is a subject having or diagnosed as having hemophilia, thrombocytopenia, or Glanzmann thrombasthenia.

17. The polypeptide or composition of any one of the preceding claims, for use in treating a subject in need of hemostatic treatment.

18. The polypeptide or composition of any of the preceding claims, for use in treating excessive bleeding in a subject.

19. The polypeptide or composition of claim 17 or 18, wherein the subject is a subject having or diagnosed as having hemophilia, thrombocytopenia, or Glanzmann thrombasthenia.

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