Use of fstl1 protein in preparation of anti-platelet drug

Abstract

The application of Fstl1 protein in preparing anti-platelet drug. The recombinant Fstl1 protein is prepared by a prokaryotic expression system, and after being incubated with human platelets, the platelet aggregation induced by thrombin, dense granule ATP release, alpha granule P-selectin exposure, integrin alpha IIb beta 3 activation, fibrinogen binding, phosphatidylserine everted, spread adhesion and blood clot contraction function can be significantly inhibited. Animal experiments show that the Fstl1 protein can effectively inhibit the FeCl3-induced thrombosis of the mesenteric artery and carotid artery of mice, reduce the accumulation of platelets in vivo, and does not significantly prolong the tail bleeding time of mice. The application realizes the effective separation of the anti-thrombosis effect and the hemostatic function, and provides a candidate molecule for developing a new type of anti-platelet drug with low bleeding risk.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to antiplatelet or antithrombotic drugs, and particularly to the application of an Fstl1 protein in the preparation of antiplatelet drugs. Background Technology

[0002] Platelets are cell fragments formed from the cytoplasm of megakaryocytes in the bone marrow, and their core physiological functions lie in maintaining the integrity of blood vessel walls and participating in the coagulation and hemostasis process. When the vascular endothelium is damaged, circulating platelets rapidly adhere to the exposed collagen surface, recruiting more platelets by releasing active substances such as adenosine diphosphate (ATP) and thromboxane A2. Through the cross-linking of integrin αIIbβ3 and fibrinogen, platelet thrombi are formed to seal the damaged vessel wall and prevent blood loss. However, if this protective response of platelets is overactivated under pathological conditions, it becomes the initiating factor for arterial and microvascular thrombosis. Critical conditions such as acute coronary syndrome, ischemic stroke, peripheral artery occlusion, and even disseminated intravascular coagulation are all closely related to abnormal platelet aggregation and activation. Thromboembolic diseases remain the leading cause of death and disability worldwide.

[0003] Given the crucial role of platelets in thrombosis, currently used antithrombotic drugs are mainly divided into two categories: anticoagulants and antiplatelet drugs. Anticoagulants, such as heparin, warfarin, and novel oral anticoagulants, inhibit fibrin production by intervening in the coagulation cascade. Antiplatelet drugs, on the other hand, act directly on different nodes of the platelet activation pathway. Examples include cyclooxygenase inhibitors like aspirin, P2Y12 receptor antagonists like clopidogrel and ticagrelor, phosphodiesterase inhibitors like dipyridamole, and glycoprotein IIb / IIIa receptor antagonists like tirofiban. These drugs significantly reduce the recurrence rate and mortality of thrombotic events, but their common safety concern lies in interfering with the normal function of platelets and the coagulation system in physiological hemostasis. Because the signaling pathways blocked by existing antiplatelet drugs highly overlap in pathological thrombosis and physiological hemostasis, these drugs inevitably weaken the body's ability to stop microvascular damage while inhibiting thrombosis. This leads to gastrointestinal bleeding, intracranial hemorrhage, and even fatal massive hemorrhage, becoming a major bottleneck limiting their clinical application and dosage intensity. Therefore, finding novel targets or drug molecules that can uncouple antithrombotic effects from hemostatic functions has always been a major challenge in cardiovascular basic research and drug development.

[0004] Fstl1, also known as FRP, TSC36, or FSL1, is a secreted glycoprotein encoded by the Fstl1 gene. Its mature peptide contains 308 amino acid residues. As a multifunctional extracellular matrix-associated protein, Fstl1 is widely expressed in various tissues and plays an important regulatory role in embryonic development, immune inflammation regulation, tissue damage repair, and fibrosis. Its biological functions are mainly achieved by antagonizing the TGFβ superfamily signaling pathway or influencing downstream transcriptional events. However, in the field of platelet physiology and pathology, the expression status of Fstl1, its impact on platelet activation and aggregation, and its potential role in thrombosis have not yet been reported. In-depth investigation of the regulatory effects of Fstl1 on platelet function will not only broaden our understanding of the protein's biological function but may also provide new insights into solving the problem of bleeding risk in antithrombotic therapy. Summary of the Invention

[0005] Technical Problem Solved: To address the long-standing clinical bottleneck of existing antiplatelet drugs interfering with physiological hemostasis due to overlapping signal pathways during thrombus inhibition, leading to a significant increase in bleeding risk, this invention provides the application of Fstl1 protein in the preparation of antiplatelet drugs. This significantly inhibits multiple activation reactions, including platelet aggregation, particle release, integrin activation, spreading and adhesion, and clot contraction, without prolonging tail bleeding time or impairing normal hemostasis. It also effectively reduces arterial thrombosis and local platelet accumulation, thereby achieving effective separation of antithrombotic effects and hemostatic function. This provides a novel candidate molecule and target for developing new antiplatelet drugs with low bleeding risk.

[0006] Technical solution: The use of Fstl1 protein or its active fragment, with the amino acid sequence shown in SEQ ID NO: 1, in the preparation of a medicament for the prevention or treatment of thrombotic diseases by inhibiting platelet activation.

[0007] The above-mentioned inhibition of platelet activation includes at least one of the following: inhibition of platelet aggregation, inhibition of dense granule release, inhibition of α-granule release, inhibition of integrin αIIbβ3 activation, inhibition of fibrinogen binding, inhibition of phosphatidylserine eversion, inhibition of platelet spreading and adhesion, and inhibition of clot contraction.

[0008] The aforementioned thrombotic diseases include those related to arterial thrombosis.

[0009] The aforementioned drugs are antiplatelet or antithrombotic drugs that do not significantly prolong physiological hemostasis time.

[0010] Beneficial Effects: This invention is the first to discover that Fstl1 protein can directly act on platelets and inhibit multiple activation reactions. Experiments show that after co-incubating Fstl1 protein with human platelets, the thrombin-triggered aggregation amplitude significantly decreased, accompanied by reduced ATP release, decreased P-selectin exposure ratio, inhibited integrin αIIbβ3 activation, and weakened fibrinogen binding capacity. Further observation revealed that Fstl1-treated platelets showed reduced adhesion to fibrinogen-coated surfaces, smaller single-cell spreading area, fewer phosphatidylserine eversion events, and a corresponding slowdown in the speed and extent of clot retraction. These changes in functional indicators do not occur in isolation but cover the complete reaction chain of platelets from initial activation, release of granule contents, membrane receptor conformational switching, intracellular signal transduction, to terminal contraction. The cumulative effect of the simultaneous suppression of multiple links makes the overall antiplatelet effect more thorough, and the working concentration of the protein is maintained at a low level. The inhibitory trends under different detection methods are mutually corroborative and highly consistent. In animal experiments, mice injected with Fstl1 protein via the tail vein showed significantly reduced local platelet accumulation at the site of FeCl3-induced mesenteric artery injury and a significantly delayed interruption of carotid artery blood flow, suggesting that Fstl1 also has the ability to suppress arterial thrombosis in vivo. However, in a tail tip transection hemorrhage model, the same dose of Fstl1 did not change the bleeding duration, indicating that the protein has no significant effect on the physiological hemostatic response after acute injury. Most existing antiplatelet drugs directly block key receptors or enzymes in the thrombosis pathway, and these targets are precisely those essential for normal hemostasis; therefore, efficacy and bleeding risk often increase simultaneously. Fstl1 is unique in that it can appropriately separate its antithrombotic effect from its hemostatic function, preserving the body's basic hemostatic response while inhibiting pathological thrombosis. This characteristic makes it a novel candidate molecule that differs from traditional antiplatelet strategies and provides new ideas for developing safer antithrombotic drugs. Attached Figure Description

[0011] Figure 1 Fstl1 protein purity verification.

[0012] Figure 2 Fstl1 protein inhibits platelet aggregation. A: Platelet aggregation response in both groups after induction with low-concentration thrombin; B: Statistical results of platelet aggregation induced by low-concentration thrombin; C: Statistical results of ATP release induced by low-concentration thrombin. Data are expressed as mean ± SEM and analyzed using Student's t-test. ** P < 0.01, *** P < 0.001 were considered statistically significant.

[0013] Figure 3Fstl1 protein inhibits the release of platelet α-granules. A: Flow cytometry representations of platelet α-granule secretion in the control group and the Fstl1 protein-treated group under Thrombin (0.4 U / mL) stimulation; B: Statistical analysis graph. Data are expressed as mean ± SEM and analyzed using One-way ANOVA. **** P < 0.0001 was considered statistically significant.

[0014] Figure 4 Fstl1 protein inhibits the activation of platelet integrin αIIbβ3. A: Flow cytometry representations of platelet PAC-1 in the control group and the Fstl1 protein-treated group under Thrombin (0.4 U / mL) stimulation; B: Statistical analysis graph. Data are expressed as mean ± SEM and analyzed using One-way Anova. **** P < 0.0001 was considered statistically significant.

[0015] Figure 5 Fstl1 protein inhibits platelet fibrinogen binding. A: Flow cytometry representations of platelet Fg binding in the control group and the Fstl1 protein-treated group under Thrombin (0.8 U / mL) stimulation; B: Statistical analysis graph. Data are expressed as mean ± SEM and analyzed using One-way Anova. * P < 0.05, **** P < 0.0001 were considered statistically significant.

[0016] Figure 6 Fstl1 protein inhibits platelet phosphatidylserine eversion. A: Thrombin (0.4 U / mL) and 2.5 mM Ca 2+ A: Flow cytometry representations of Annexin V in platelets in the control group and the Fstl1 protein-treated group under stimulation conditions; B: Statistical analysis plot. Data are expressed as mean ± SEM and analyzed using One-way ANOVA. **** P < 0.0001 was considered statistically significant.

[0017] Figure 7 Fstl1 inhibits platelet spreading. A: Representative images of platelet adhesion and spreading after fibrinogen coating; B: Statistical analysis of the number of platelets adhering per unit area in the two groups; C: Statistical analysis of the average spreading area of ​​platelets in the two groups. Data are expressed as mean ± SEM. Student t-test analysis was used. *** P < 0.001 was considered statistically significant.

[0018] Figure 8Fstl1 protein inhibits platelet clot contraction. A: Platelet clot retraction in the two groups at different time points; B: Statistical analysis results of platelet clot retraction in the two groups. Data are expressed as mean ± SEM. Student t-test was used for analysis. **** P < 0.0001 was considered statistically significant.

[0019] Figure 9 Fstl1 protein did not affect tail hemorrhage time in mice. Experimental mice were injected with 200 μg of Fstl1 protein via the tail vein, while control mice were injected with an equal volume of PBS via the tail vein. Tail hemorrhage time was measured. Data are expressed as mean ± SEM, and analysis using the Student t-test showed no statistically significant difference in ns.

[0020] Figure 10 Fstl1 protein negatively regulates platelet accumulation in vivo. A: Fluorescence images of platelet accumulation at vascular injury sites in the two groups of mice at different time points; B: Mean fluorescence density curves of platelet accumulation at vascular injury sites in the two groups of mice. The scale bar in the figure is 200 μm. Data are expressed as mean ± SEM. Student t-test analysis was used. **P<0.01, ****P<0.0001 were considered statistically significant.

[0021] Figure 11 Fstl1 protein negatively regulates thrombus formation in vivo. A: Real-time images of carotid artery occlusion in two groups of mice; B: Statistical graph of carotid artery occlusion time in two groups of mice. Data are expressed as mean ± SEM. Student t-test was used for analysis. * P < 0.05 was considered statistically significant.

[0022] Figure 12 Fstl1 protein inhibits U46619-induced platelet aggregation. Data are expressed as mean ± SEM. Student t-test analysis was performed. ** P < 0.01 was considered statistically significant.

[0023] Figure 13 Fstl1 protein inhibits U46619-induced platelet α-granule release. Data are expressed as mean ± SEM and analyzed using Student t-test. ** P < 0.01, * P < 0.05 were considered statistically significant.

[0024] Figure 14 Fstl1 protein inhibits U46619-induced activation of platelet integrin αIIbβ3. Data are expressed as mean ± SEM and analyzed using Student t-test. *** P < 0.001, **** P < 0.0001 were considered statistically significant.

[0025] Figure 15Fstl1 protein inhibits U46619-induced platelet fibrinogen binding. Data are expressed as mean ± SEM. Student t-test analysis was used. ** P < 0.01, **** P < 0.0001 were considered statistically significant.

[0026] Figure 16 Fstl1 protein inhibits U46619-induced phosphatidylserine outwards movement of platelets. Data are expressed as mean ± SEM and analyzed using Student t-test. ** P < 0.01, **** P < 0.0001 were considered statistically significant. Detailed Implementation

[0027] Those skilled in the art should understand that various equivalent substitutions or modifications can be made to the above specific embodiments without departing from the spirit and essence of the present invention, and all such substitutions or modifications fall within the protection scope of the present invention.

[0028] Example 1:

[0029] Fstl1 protein preparation

[0030] Using the wild-type Fstl1 cDNA sequence as a template, the product was digested with restriction endonucleases. The digested PCR product was ligated into a cloning vector and transformed. Single clones were selected for sequencing identification, and the successfully sequenced plasmids were transformed into E. coli BL21. The transformed competent cells were added to 200 μL of antibiotic-free LB broth and cultured at 37°C and 150 rpm for 60 minutes. After culturing, single bacterial clones were picked and added to LB broth containing 100 μg / mL ampicillin, and cultured overnight at 37°C and 250 rpm. Isopropyl-β-D-thiogalactoside (IPTG) was added to the cultured solution to a working concentration of 0.5 mM, and the mixture was shaken at 25°C and 200 rpm for 16 hours. Protein expression was induced, and the bacterial cells were collected by centrifugation. The bacterial lysate was treated with sonication. The treated bacterial lysate was purified using a NiFocurose 6FF (IDA) affinity column, and the Fstl1 protein was obtained by dialysis and refolding. Protein concentration was determined using a Bradford protein assay kit, and protein purity was assessed using Coomassie blue staining. Results showed a clear single band before and after protein concentration. Figure 1 ).

[0031] Platelet sample processing

[0032] Platelet samples were collected from individuals, resuspended in Tyrode's buffer, and incubated with Fstl1 protein at 37°C for 10 min. The control group was incubated with an equal amount of PBS.

[0033] Under thrombin stimulation, Fstl1 protein inhibits platelet aggregation and dense granule secretion.

[0034] Human platelet suspensions were incubated with 5 μM Fstl1 and placed in a platelet aggregator. Aggregation was induced under calcium ion-assisted and thrombin-stimulated conditions. Aggregation curves were continuously recorded over 5 minutes, and the maximum aggregation rate was calculated. ATP release levels were monitored simultaneously. Results showed that, compared with the control group, platelet aggregation function was significantly decreased in the Fstl1 protein treatment group. Figure 2 The results (AB) indicate that Fstl1 can inhibit platelet aggregation. Furthermore, ATP released from dense granules during platelet activation is a key indicator for assessing the level of activation. Our analysis of ATP levels in the supernatant after the aggregation reaction revealed that, compared to the control group, the Fstl1 protein-treated group showed a significant decrease in platelet ATP release (AB). Figure 2 (C), the results suggest that Fstl1 negatively regulates platelet ATP release.

[0035] Under thrombin stimulation, Fstl1 protein inhibits the release of platelet α-granules.

[0036] Platelets are rich in secretory granules, with α-granules being the most abundant. To investigate whether Fstl1 protein regulates the release of platelet α-granules, we incubated human platelets treated with 5 μM Fstl1 and control platelets with PE-labeled Anti-Human P-selectin antibody for 10 min, followed by stimulation with Thrombin (0.4 U / mL) to activate the platelets. The results showed that the fluorescence intensity of P-selectin antibody bound to platelets treated with Fstl1 protein was significantly lower than that of control platelets. Figure 3 ).

[0037] Under thrombin stimulation, Fstl1 protein inhibits the activation of platelet integrin αIIbβ3.

[0038] PAC-1 is a specific marker of platelet activation resulting from conformational changes in platelet membrane glycoproteins IIb / IIIa, directly reflecting platelet activation status. We incubated 5 μM Fstl1-treated human platelets and control platelets with FITC-labeled Anti-Human PAC-1 (platelet membrane glycoprotein complex) antibody for 10 min, followed by stimulation with Thrombin (0.4 U / mL) to activate the platelets. The results showed that the fluorescence intensity of PAC-1 antibody binding on platelets treated with Fstl1 was significantly lower than that on control platelets. Figure 4 This indicates that Fstl1 inhibits platelet activation.

[0039] Under thrombin stimulation, Fstl1 protein inhibits platelet fibrinogen binding.

[0040] Fibrinogen binding primarily reflects the conformational changes and functional state of the αIIbβ3 integrin receptor after platelet activation; therefore, it is an important indicator for assessing platelet aggregation function. Results showed that, compared to the control group, under Thrombin (0.4 U / mL) stimulation, the platelet fibrinogen binding level in the 5 μM Fstl1 protein treatment group was significantly decreased. Figure 5 The combined results of P-selectin and PAC-1 suggest that Fstl1 protein negatively regulates the release of platelet α-granules and inhibits platelet activation.

[0041] Under thrombin stimulation, Fstl1 protein inhibits platelet phosphatidylserine eversion.

[0042] Annexin V specifically binds to phosphatidylserine (PS) that flips to the outer layer of the cell membrane, making it an important biomarker for assessing the transition of platelets from a resting to a procoagulant state. Platelets treated with 5 μM Fstl1 showed significant changes upon stimulation with Thrombin (0.4 U / mL) and 2.5 mM Ca2+. 2+ Under the auxiliary effect, the surface phosphatidylserine exposure level of platelets was significantly lower than that of the control group. Figure 6 This indicates that Fstl1 inhibits the procoagulant state transition of platelets.

[0043] Fstl1 protein inhibits platelet spreading and adhesion.

[0044] During platelet aggregation, the binding of integrin αIIbβ3 to fibrinogen not only mediates intercellular connections but also induces cytoskeleton remodeling through "outside-inside" signal transduction, thereby regulating subsequent functions such as platelet spreading. The spreading response is a sensitive indicator of the activity of early αIIbβ3 "inside-outside" signal transduction. To investigate the effect of Fstl1 on this process, this study first coated coverslips with 20 μg / mL fibrinogen. Then, platelet suspensions treated with Fstl1 and those from the control group were added to the coverslips, and the adhesion and spreading of the two groups were compared. The results showed that compared with the control group, the number of adhered platelets in the Fstl1-treated group was significantly reduced (…). Figure 7 :AB), but its spreading level was significantly lower than that of the control group ( Figure 7 (C). The results suggest that Fstl1 negatively regulates αIIbβ3-mediated early adhesion signaling and spreading function.

[0045] Fstl1 protein inhibits platelet clot contraction.

[0046] Integrin αIIbβ3-mediated "outside-inside" signaling induces platelet-mediated clot retraction, a key manifestation of late-stage signaling, and participates in thrombus stability and wound repair. To investigate the effect of Fstl1 protein on this function, human platelet suspension was incubated with 2.5 μM Fstl1, followed by the addition of fibrinogen and a thrombin stimulator. Clot retraction was recorded at different time points. ImageJ was used to measure the two-dimensional area of ​​the clot, and the percentage of area (or volume) relative to the initial area (or volume) at each time point was calculated to evaluate the differences in clot retraction ability among the groups. Results showed that compared with the control group, the Fstl1 protein treatment group had prolonged platelet retraction time and reduced retraction intensity. Figure 8 This suggests that Fstl1 negatively regulates the late-stage αIIbβ3 “outside-inside” signal transduction.

[0047] Fstl1 protein does not affect physiological hemostasis in mice.

[0048] To assess the effect of Fstl1 on physiological hemostasis, this study measured tail bleeding time in mice. Mice were anesthetized after intravenous injection of 200 μg Fstl1 into the tail vein, and the tail was immediately severed 3 mm from the tip. The tail was then immersed in 37°C saline solution. Timing was recorded from the onset of persistent bleeding at the tail tip until complete cessation of bleeding (a clear white line was visible at the tail tip). The duration was recorded as bleeding time. There was no significant difference in tail bleeding time between the treatment group and the control group. Figure 9 This suggests that Fstl1 does not affect the physiological hemostasis process in mice.

[0049] Fstl1 inhibits thrombus formation in vivo.

[0050] To investigate the role of Fstl1 in thrombosis, a mouse mesenteric artery injury model induced by FeCl3 was established by tail vein injection of 200 μg Fstl1 protein. Platelet accumulation at the vascular injury site was observed in real time using in vivo fluorescence microscopy. Results showed that, compared with the control group, platelet accumulation at the injury site was significantly reduced in the Fstl1 protein treatment group. Figure 10 This indicates that Fstl1 negatively regulates platelet accumulation and thrombus formation in the body.

[0051] Fstl1 inhibits platelet aggregation in the body.

[0052] To further determine the effect of Fstl1 on in vivo thrombosis, mice were anesthetized and their common carotid artery was exposed. Filter paper soaked in 7.5% FeCl3 was applied to the adventitia of the blood vessel for 3 minutes and then removed. Blood flow was monitored using Doppler flowmeter, and the time for blood flow to decrease to zero (vascular occlusion time) and the area under the flow curve were recorded. The results showed that treatment with 200 μg of Fstl1 protein injected into the tail vein significantly prolonged the time required for complete occlusion of the carotid artery. Figure 11 This suggests that Fstl1 negatively regulates thrombus formation in the body.

[0053] Example 2

[0054] Under U46619 (9,11-dideoxy-9A,11A-methylene epoxide prostaglandin F2A solution, Glpbio) stimulation, Fstl1 protein inhibits platelet aggregation and dense granule secretion.

[0055] After washing, human platelet suspension was incubated with 10 μM Fstl1, and then stimulated with U46619 (10 μM) in the presence of calcium ions. Aggregation curves were continuously recorded for 5 minutes using a platelet aggregator, and the maximum aggregation rate was calculated. The results showed that, consistent with the control group, platelet aggregation function in the Fstl1-treated group was still significantly decreased. Figure 12 This indicates that Fstl1 can inhibit platelet aggregation.

[0056] Under U46619 stimulation, Fstl1 protein inhibits the release of platelet α-granules.

[0057] Platelets from the 10 μM Fstl1 treatment group and the control group were incubated with PE-labeled Anti-Human P-selectin antibody for 10 min, and then stimulated with U46619 (20 μM) for activation. The results showed that the fluorescence intensity of P-selectin antibody binding in the Fstl1 treatment group was still significantly lower than that in the control group. Figure 13 ).

[0058] Under U46619 stimulation, Fstl1 protein inhibits platelet integrin αIIbβ3 activation.

[0059] Platelets from both the Fstl1-treated group and the control group were incubated with FITC-labeled Anti-Human PAC-1 antibody for 10 min, followed by activation stimulation with U46619 (20 μM). Results showed that the fluorescence intensity of PAC-1 antibody binding in the Fstl1-treated group was still significantly lower than that in the control group. Figure 14 This indicates that Fstl1 inhibits platelet activation.

[0060] Under U46619 stimulation, Fstl1 protein inhibits platelet fibrinogen binding.

[0061] Platelet fibrinogen binding levels in the 10 μM Fstl1 treatment group and the control group were measured under U46619 (10 μM) stimulation. Results showed that the fibrinogen binding level in the Fstl1 treatment group was still significantly lower than that in the control group. Figure 15 The combined results of P-selectin and PAC-1 suggest that Fstl1 protein negatively regulates the release of platelet α-granules and inhibits platelet activation.

[0062] Under U46619 stimulation, Fstl1 protein inhibits platelet phosphatidylserine eversion.

[0063] Platelets from the 10 μM Fstl1 treatment group and the control group were stimulated with U46619 (20 μM) and 10 mM Ca 2+ Annexin V binding levels were detected in the presence of [a specific substance / condition]. Results showed that the exposure level of phosphatidylserine (PS) on the platelet surface in the Fstl1-treated group was still significantly lower than that in the control group. Figure 16 This indicates that Fstl1 inhibits the conversion of platelets to a procoagulant state.

Claims

1. Application of Fstl1 protein with amino acid sequence as shown in SEQ ID NO: 1 in the preparation of antithrombotic drugs.

2. The application according to claim 1, characterized in that, The drug is an antithrombotic agent that inhibits platelet activation. The inhibition of platelet activation includes at least one of the following: inhibiting platelet aggregation, inhibiting dense granule release, inhibiting α-granule release, inhibiting integrin αIIbβ3 activation, inhibiting fibrinogen binding, inhibiting phosphatidylserine eversion, inhibiting platelet spreading and adhesion, and inhibiting clot contraction.

3. The application according to claim 1, characterized in that, The thrombus includes arterial thrombus.

4. The application according to any one of claims 1 to 3, characterized in that, The drug in question is an antithrombotic drug that does not significantly prolong physiological hemostasis time.

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