Application of platelet Piezo1 protein inhibitor in preparation of medicine for reducing shear induced platelet activation and aggregation

Abstract

The application of the platelet Piezo1 protein inhibitor in preparation of drugs for reducing shear-induced platelet activation and aggregation proves that platelet Piezo1 significantly promotes high-shear-induced platelet activation and arterial thrombosis formation, and indicates that a Piezo1 channel is an effective target of an Rb1 antiplatelet effect; ginsenoside Rb1 shows antiplatelet and antithrombotic effects under the condition of pathological high shear rate through a targeted Piezo1 channel, Rb1 effectively inhibits high shear induced arterial thrombosis and platelet activation in vivo, is an effective drug for inhibiting shear induced platelet activation and arterial thrombosis, and is an inhibitor of Piezo1 protein.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of a platelet Piezo1 protein inhibitor in the preparation of a drug for reducing shear-induced platelet activation and aggregation. Background Art

[0002] Arterial thrombosis is one of the key mechanisms behind life-threatening diseases such as myocardial infarction and ischemic stroke. The latest data reported more than 190 million cases of ischemic heart disease and more than 70 million cases of ischemic stroke worldwide. Platelets play a dual role in hemostasis and thrombosis and are crucial in arterial thrombosis. Studies have shown that the hemodynamic environment during hemostasis and thrombosis is completely different. During hemostasis, due to the loss of pressure gradient inside and outside the blood vessels, platelets rapidly adhere to the site of vascular injury while the blood vessels are constricted. In contrast, during arterial thrombosis, platelets are activated by the high shear stress around atherosclerotic plaques. Different wall shear rates lead to different platelet thrombosis patterns. For example, 125-250 s -1 Shear rates of 500-1500 s -1 The shear rate will promote the growth of thin platelet thrombus. -1 The thrombus morphology becomes thicker when the shear stress is higher. The platelet thrombus induced by pathological high shear stress can easily form an embolus in the small artery along the direction of blood flow. -1 Compared with the shear rate of 3000 - 6000 s -1 The mean volume of platelet aggregates was significantly larger at shear rates between 2.5 and 1.5. The platelet aggregation rate in patients with acute myocardial infarction, cerebral infarction, atherosclerosis, and diabetes was significantly higher than that in healthy controls, which may be due to increased platelet reactivity caused by an abnormal shear stress environment. Targeting overactive platelets under pathological shear stress may provide a therapeutic strategy to prevent arterial thrombosis without affecting normal platelet hemostasis.

[0003] Piezo1 is a mechanically activated ion channel that plays a role in platelet activation through cellular mechanosignaling. High shear-induced Piezo1 activation triggers Ca2+ in platelets. 2+Influx of Piezo1 drives calpain-mediated talin cleavage and promotes integrin αIIbβ3 activation and P-selectin expression in platelets. Moreover, under pathological shear stress, Piezo1 mediates platelet activation responses within milliseconds, independent of platelet adhesion to the subendothelial matrix. Given the potential critical role of Piezo1 in platelet function, while its precise role in shear-induced platelet activation and arterial thrombosis remains unclear, new avenues for preventing and treating arterial thrombosis may be found by discovering potential pharmacological inhibitors of the Piezo1 channel. Summary of the invention

[0004] In order to solve the technical defects of the prior art, the present invention provides an application of a platelet Piezo1 protein inhibitor in the preparation of a drug for reducing shear-induced platelet activation and aggregation.

[0005] The technical solution adopted by the present invention is: application of platelet Piezo1 protein inhibitor in the preparation of drugs for reducing shear-induced platelet activation and aggregation.

[0006] The platelet Piezo1 protein inhibitor is a blocker of the Piezo1 calcium ion signaling channel.

[0007] The blocker of the Piezo1 calcium ion signaling channel is ginsenoside Rb1.

[0008] The concentration of ginsenoside Rb1 in the drug for reducing shear-induced platelet activation and aggregation is 5-80 μM.

[0009] Application of platelet Piezo1 protein inhibitors in the preparation of anti-arterial thrombotic drugs.

[0010] The platelet Piezo1 protein inhibitor is ginsenoside Rb1.

[0011] The concentration of ginsenoside Rb1 in the anti-arterial thrombotic drug is 5-80 μM.

[0012] The dosage of the anti-arterial thrombotic drug is 20-100 mg·kg-1.

[0013] Application of ginsenoside Rb1 in the preparation of blockers of Piezo1 calcium ion signaling channel.

[0014] Application of ginsenoside Rb1 in the preparation of platelet Piezo1 protein inhibitors.

[0015] The beneficial effects of the present invention are as follows: the present invention provides an application of a platelet Piezo1 protein inhibitor in the preparation of a drug for reducing shear-induced platelet activation and aggregation, proving that activation of platelet Piezo1 can significantly promote high shear-induced platelet activation and arterial thrombosis, indicating that the Piezo1 channel is an effective target for the antiplatelet effect of Rb1, and that ginsenoside Rb1 exhibits antiplatelet and antithrombotic effects under pathological high shear rate conditions by targeting the Piezo1 channel, and that Rb1 effectively inhibits high shear-induced arterial thrombosis and platelet activation in vivo, is an effective drug for inhibiting shear-induced platelet activation and arterial thrombosis, and is an inhibitor of Piezo1 protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Preparation of conditional platelet Piezo1 knockout mice.

[0018] Figure 2 Platelet Piezo1 plays an important role in regulating shear-induced platelet activation and aggregation.

[0019] Figure 3 Conditional knockout of platelet Piezo1 prevents high-shear-induced arterial thrombosis in the Folts mouse model.

[0020] Figure 4 PNS inhibits high shear-induced platelet calcium influx and platelet aggregation.

[0021] Figure 5 Ginsenoside Rb1 effectively inhibits high shear-induced platelet activation and aggregation.

[0022] Figure 6 Ginsenoside Rb1 inhibits shear-induced Ca2+ expression via Piezo1 2+ Dependent platelet activation and aggregation.

[0023] Figure 7 Ginsenoside Rb1 reduces high-shear-induced arterial thrombosis and platelet activation via Piezo1 channels in vivo.

[0024] Figure 8 In vivo effects of ginsenoside Rb1 on bleeding time in mice. DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Experimental methods Using platelet-specific Piezo1-deficient transgenic mice (Piezo1 ΔPlt ) to elucidate the role of platelet Piezo1 in SIPA and arterial thrombosis. A microfluidic channel system was used to apply shear stress to platelets to evaluate platelet aggregation, calcium influx, calpain activity, talin cleavage, integrin αIIbβ3 activation, and P-selectin expression. A cell thermal migration assay (CETSA) was used to detect the binding of Rb1 to Piezo1. A high shear-induced mouse arterial thrombosis model was used to evaluate the antithrombotic effect of Rb1.

[0027] Piezo1 is considered to be a mechanosensitive channel that can sense and conduct shear stress. In order to study the role of Piezo1 in shear-induced platelet aggregation (SIPA), the present invention constructed platelet-specific Piezo1 knockout mice ( Figure 1 a). PCR analysis showed that Piezo1 fl / fl and Piezo1 ΔPlt The genotype of mice ( Figure 1 b). Western blotting confirmed that Piezo1 ΔPlt Piezo1 protein deficiency in mouse platelets ( Figure 1 c). Then compare Piezo1 fl / fl and Piezo1 ΔPlt Expression of major platelet receptors on the mouse surface. Flow cytometry results showed that Piezo1 fl / fl and Piezo1 ΔPlt In mice, levels of platelet surface receptors such as αIIbβ3, GPVI, and GPIbα are comparable ( Figure 1 d). This suggests that the absence of Piezo1 in platelets does not affect the expression of these receptors responsible for platelet activation and aggregation.

[0028] Example 1 In the present invention, the Bioflux1000z microfluidic system was used and 500 s -1 and 4000 s -1 The shear rates of 500 s and 200 s respectively simulate the physiological normal and pathological high shear rates of human arteries. -1In comparison, in the 4000s -1 At a shear rate of fl / fl Platelets from mice rapidly adhered to the collagen-coated microfluidic channels and formed larger thrombi. Platelets from mice with conditional knockout of Piezo1 adhered to the microfluidic channels more rapidly than those from mice with conditional knockout of Piezo1. -1 At high shear rates, platelet aggregation was significantly reduced, with a reduction rate of about 80% ( Figure 2 a).

[0029] With 500 s -1 Compared with the normal shear rate of 4000 s -1 The high shear rate induces Piezo1 fl / fl More significant Ca in platelets 2+ internal flow ( Figure 2 b). Piezo1 deficiency in platelets (Piezo1 ΔPlt ) largely eliminated the Ca2+ induced by the high shear rate of 4000 s-1. 2+ internal flow ( Figure 2 b). Flow cytometry experiments showed that Piezo1 was activated at a high shear rate of 4000 s-1 compared with the normal shear rate of 500 s-1. fl / fl The activation of integrin αIIbβ3 and the expression of p-selectin in platelets were significantly increased ( Figure 2 c, d). At a high shear rate of 4000 s-1, Piezo1 ΔPlt The activation of integrin αIIbβ3 and the expression of p-selectin in platelets were significantly reduced ( Figure 2 c; d). These data indicate that platelet Piezo1 is essential in regulating shear-induced platelet activation and aggregation.

[0030] Example 2 The Folts mouse model uses silk thread ligation to cause vascular stenosis, increase local vascular shear stress, and mechanical compression to cause endothelial damage, simulating the platelet activation and thrombosis process in the human body. In order to study the role of platelet Piezo1 in high shear-related thrombosis in vivo, the present invention uses Piezo1 in the Folts mouse arterial thrombosis model. fl / fl and Piezo1 ΔPlt Transgenic mice. Left carotid artery (LCA) stenosis, blood flow reduction of 50%, forming a high shear stress area. Compared with the model group, Piezo1 fl / fl The Folts model in mice resulted in a decrease in circulating blood flow (CFR) of 11.5 ± 2.1 ( Figure 3 a, b). Piezo1 deficiency in platelets significantly reduced the incidence of CFR. ΔPltThe average CFR of mice after modeling was 0.7±0.5 ( Figure 3 a, b). in Piezo1 fl / fl In mice, H&E and IHC analysis revealed significant arterial thrombi composed mainly of platelets ( Figure 3 c). However, Piezo1 ΔPlt Mice showed almost no platelet-rich arterial thrombosis ( Figure 3 c).

[0031] In addition, compared with the sham operation group, Piezo1 fl / fl Folts-derived platelets from mice showed increased P-selectin expression and integrin αIIbβ3 activation ( Figure 3 d, e). In contrast, Piezo1 ΔPlt Mouse platelets showed reduced P-selectin expression and reduced integrin αIIbβ3 activation ( Figure 3 d, e). These findings suggest that platelet Piezo1 is essential for high shear stress-induced arterial thrombosis.

[0032] Example 3 The chemical quality control of PNS was detailed in the previous study of the present inventors, which reported that PNS contained 45.9% ginsenoside Rg1, 23.6% ginsenoside Rb1, 11.6% ginsenoside R1, 6.3% ginsenoside Re, and 1.7% ginsenoside Rd. PNS was found to affect Ca2+ in collagen-coated microfluidic channels. 2+ Signal transduction to inhibit high shear-induced platelet aggregation. In the present invention, the experimental scheme established by Ilkan et al. (2017) was used to study the effect of PNS on platelet calcium influx under single-factor shear stress conditions. Briefly, PECAM-1 antibody protein was coated on microfluidic channels to immobilize platelets, and the effect of PNS on shear-induced platelet aggregation was observed using Piezo1 protein blocker GsMTx-4 and agonist Yoda1.

[0033] The results showed that with 500 s -1 Compared with the normal shear rate of 4000 s -1 The high shear rate leads to Ca 2+ The influx increased significantly and platelet aggregation was obvious ( Figure 4 a). 0.15 g·L -1 or 0.6 g·L -1 PNS at high concentrations significantly reduced shear-induced platelet Ca influx 2+ ( Figure 4a). The Piezo1 agonist Yoda1 was also used to activate Piezo1 channels in platelets. Compared with the control group, 25 μM Yoda1 treatment increased the Piezo1 channel in platelets at 500 s. -1 The normal shear rate increases platelet Ca 2+ Influx of Ca in PNS was reduced in a dose-dependent manner. 2+ internal flow ( Figure 4 b).

[0034] In addition, with 500 s -1 Compared with the shear rate of 4000 s -1 Shear rate also leads to increased platelet calpainase activity and cleavage of the substrate protein talin ( Figure 4 c, d). PNS significantly reduced platelet calpain activity and talin cleavage ( Figure 4 c, d). Yoda1 significantly increased calpain activity and talin cleavage, while PNS reduced Yoda1-induced calpain activity and talin cleavage ( Figure 4 e, f).

[0035] 4000 s -1 Shear rate ratio 500 s -1 Shear rate significantly enhanced the activation of platelet integrin αIIbβ3. 0.15 g·L -1 and 0.6 g·L -1 The concentration of PNS can significantly reduce 4000 s -1 High shear rate-induced platelet integrin αIIbβ3 activation ( Figure 4 g). In addition, fibrinogen, a ligand for platelet integrin αIIbβ3, binds to integrin upon activation. -1 Compared with the shear rate of 4000 s -1 Platelet fibrinogen binding increased significantly at shear rates, but at 0.15 g·L -1 and 0.6 g·L -1 At high shear rates, PNS significantly inhibited the binding of platelet fibrinogen. The effect of Piezo1 channel blocker GsMTx-4 on platelet response under high shear rate stimulation was similar to that of PNS. Yoda1 can activate integrin αIIbβ3 on the platelet membrane surface, thereby binding to fibrinogen ( Figure 4 h). PNS also reduced Yoda1-induced integrin αIIbβ3 activation and platelet fibrinogen binding ( Figure 4 h). With 500 s -1 Compared with the shear rate of 4000 s -1Shear rate increased the expression of Piezo1 in platelets, while 0.15 g·L -1 and 0.6 g·L -1 PNS significantly reduced the high shear-induced Piezo1 expression ( Figure 4 i). Yoda1 also increased the expression of Piezo1 in platelets, but 0.15 g·L -1 and 0.6 g·L -1 PNS reduced Yoda1-induced Piezo1 expression ( Figure 4 j). These results suggest that PNS may reduce high shear-induced platelet calcium signaling and aggregation by affecting Piezo1.

[0036] PNS showed significant antithrombotic effects in the high shear-induced common carotid artery thrombosis model in mice. The LCA blood flow in the Folts mouse model was monitored for 20 minutes, and the average number of CFRs during this time frame was 14.2±4.7. Prophylactic administration of 75 mg kg -1 (PNS-L) and 225 mg kg -1 (PNS-H) can reduce CFR in a dose-dependent manner, with an average of 5.0±1.4 and 3.8±3.1, respectively. Both PNS-L and PNS-H can eliminate platelet-dominated arterial thrombosis in mouse LCA. PNS-L and PNS-H reduce the expression of p-selectin on platelets in Folts model mice, with inhibition rates of approximately 21% and 24%, respectively. PNS-L and PNS-H also reduce the activation of integrin αIIbβ3 in a dose-dependent manner, with inhibition rates of 37% and 46%, respectively.

[0037] Example 4 To evaluate the effectiveness of compounds in PNS in inhibiting SIPA, the present invention evaluated the inhibitory effects of five major compounds in PNS (ginsenoside Rg1, ginsenoside Rb1, ginsenoside Rd, ginsenoside Re and notoginsenoside R1) on shear-induced platelet Ca 2+ Among them, ginsenoside Rb1 has a -1 Platelet Ca 2+ The strongest inhibitory effect on influx ( Figure 5 a). That is, 40 μM Rb1 reduced platelet Ca at this shear rate. 2+ The internal flow is about 64%, and 0.15 g·L -1 The inhibitory effect of PNS at the concentration was comparable ( Figure 5 a). Therefore, ginsenoside Rb1 may be the most effective compound in inhibiting shear-induced platelet activation and aggregation via Piezo1.

[0038] With 500s -1 Compared with the normal shear rate of 4000s -1 The high shear rate induced strong platelet aggregation. Rb1 reduced this high shear-induced platelet aggregation in a concentration-dependent manner. 50 10.8 μM ( Figure 5 b, c). In addition, Rb1 significantly inhibited platelet Ca 2+ influx, integrin αIIbβ3 activation and p-selectin expression, IC 50 The values ​​were 35.5 μM, 24.7 μM and 19.4 μM ( Figure 5 di). Rb1 also significantly reduced high shear-induced platelet calpainase activity and talin cleavage ( Figure 5 j, k). Overall, these data suggest that Rb1 -1 Under high shear rates, platelet Ca 2+ Influx, P-selectin expression, calpain activation, talin cleavage, and activation of integrin αIIbβ3 inhibit shear-induced platelet activation.

[0039] Furthermore, Rb1 significantly reduced Yoda1-induced platelet aggregation in a concentration-dependent manner. 50 The value was 21.0 μM. Rb1 also inhibited Yoda1-induced Ca 2+ influx, p-selectin expression and integrin αIIbβ3 activation, IC 50 The values ​​were 40.8 μM, 551.7 μM, and 114.4 μM, respectively. Yoda1-stimulated Piezo1 expression was significantly reduced after Rb1 treatment. These data suggest that Rb1 inhibits Yoda1-induced platelet activation and aggregation, but the inhibitory effect is weak.

[0040] Example 5 The cell thermal migration method was used to evaluate the targeting effect of drugs in cells. The thermal stability of Piezo1 in platelets was evaluated by CETSA-WB experiment ( Figure 6 a). The results showed that Rb1 treatment of platelets resulted in a significant change in the melting curve of Piezo1 compared with the control group, especially in the temperature range of 52 to 68°C. Therefore, these data indicate that Rb1 treatment increases the thermal stability of Piezo1 in platelets, indicating that Rb1 binds to the Piezo1 channel ( Figure 6 a, b).

[0041] As mentioned above, the mechanosensitive Piezo1 channel is crucial in SIPA. To determine the mechanism by which Rb1 inhibits SIPA, the present invention uses the Piezo1 fl / fl Mice and Piezo1 ΔPlt Mouse platelets, the results showed that 10 μM concentration of Rb1 significantly reduced 4000 s -1 High shear rate-induced Piezo1 fl / fl Platelet aggregation inhibition rate is about 55% ( Figure 6 c, d). High shear rates hardly induce Piezo1 ΔPlt Platelet aggregation, Rb1 also had no additional inhibitory effect on shear-induced platelet aggregation ( Figure 6 c, d).

[0042] Rb1 significantly decreased by 4000 s -1 Piezo1 under shear rate fl / fl Ca in platelets 2+ Inflow, while Piezo1 ΔPlt Ca in platelets 2+ There is no further decrease in inflow ( Figure 6 e). With 4000s -1 Piezo1 under shear rate fl / fl Compared with platelets, Rb1 treatment significantly reduced Piezo1 fl / fl Calpain activity in platelets ( Figure 6 f). Under the same shear rate, Piezo1 ΔPlt Calpain activity in platelets was almost eliminated, and Rb1 did not further reduce calpain activity in these platelets ( Figure 6 f). In addition, at 4000 s -1 At the shear rate, 10 μM Rb1 significantly reduced the expression of Piezo1 fl / fl Cleavage of talin in platelets ( Figure 6 g). Piezo1 ΔPlt Platelets showed reduced talin cleavage, and Rb1 did not have any additional effect on talin cleavage in these platelets ( Figure 6 g).

[0043] Rb1 significantly decreased by 4000 s -1 Shear rate-induced Piezo1 fl / fl Activation of integrin αIIbβ3 in platelets. ΔPlt Compared with platelets, Rb1 has no additional inhibitory effect on integrin αIIbβ3 activation ( Figure 6h). In addition, Rb1 effectively reduced the Piezo1 fl / fl Expression of p-selectin in platelets ( Figure 6 i). In contrast, Rb1 did not further degrade Piezo1 ΔPlt Expression of p-selectin in platelets ( Figure 6 i). in Piezo1 fl / fl In platelets, Rb1 also showed a concentration-dependent response to shear-induced platelet Ca 2+ Inhibition of influx, integrin αIIbβ3 activation, and P-selectin expression. -1 After shear rate stimulation, Rb1 also inhibits the expression of Piezo1 in platelets ( Figure 6 j, k). In summary, ginsenoside Rb1 inhibits shear-induced platelet activation and aggregation by affecting the Piezo1 calcium signaling pathway.

[0044] Example 6 Using Piezo1 fl / fl and Piezo1 ΔPlt Transgenic mice were used to observe the effects of ginsenoside Rb1 on arterial thrombosis and platelet activation in the Folts model. fl / fl In mice, the average CFR was 11.5±2.1. Rb1-L (20 mg kg -1 ) and Rb1-H (50 mg kg -1 ) preventive treatment significantly reduced CFR in a dose-dependent manner, with an average of 4.8±1.6 and 3.2±1.0 ( Figure 7 a, b). H&E and immunohistochemical staining showed that both Rb1-L and Rb1-H could effectively eliminate Piezo1 fl / fl Platelet-rich thrombus formation in mice ( Figure 7 c). In addition, Rb1-L and Rb1-H significantly reduced the expression of p-selectin in a dose-dependent manner, with inhibition rates of 20% and 26%, respectively ( Figure 7 d). Rb1-L and Rb1-H also significantly reduced the activation of integrin αIIbβ3, with inhibition rates of 43% and 54%, respectively ( Figure 7 e). These findings suggest that Rb1 effectively attenuates arterial thrombosis and platelet activation in the Folts mouse model.

[0045] Importantly, give Piezo1 ΔPlt Injection of mice with Rb1-H had no additional effect in reducing the number of CFRs ( Figure 7 f, g). Histological analysis showed that in Piezo1 ΔPltIn the Folts model of mice and the Rb1-H treatment group, platelet thrombi were eliminated ( Figure 7 h). In addition, with Piezo1 ΔPlt Compared with the mouse Folts model, Rb1-H did not further reduce Piezo1 ΔPlt p-selectin expression or integrin αIIbβ3 activation in mice ( Figure 7 i, j). In addition, in the tail bleeding assay, fl / fl Compared with mice, Piezo1 ΔPlt Mice showed similar tail bleeding times. Notably, 50 mg kg -1 dose of Rb1 did not prolong tail bleeding time ( Figure 8 ). Taken together, these data suggest that ginsenoside Rb1 inhibits high shear-induced platelet activation and arterial thrombosis via the Piezo1 channel but does not affect hemostasis.

[0046] in conclusion Mechanical forces, such as blood shear stress, are the main triggers for Piezo1 activation. Platelet mechanotransduction is now considered a new therapeutic target for hemorrhagic or thrombotic diseases. Piezo1 pharmacology highlights the clinical prospects of developing Piezo1 agonists or inhibitors. Given that shear-induced platelet activation is crucial in the development of arterial thrombosis, the present invention hypothesizes that the Piezo1 channel may play an important role in shear-induced platelet activation and aggregation, thereby promoting thrombosis. The present invention demonstrates for the first time that platelet Piezo1 significantly promotes high shear-induced platelet activation and arterial thrombosis. In addition, ginsenoside Rb1 exhibits antiplatelet and antithrombotic effects under pathological high shear rate conditions by targeting the Piezo1 channel.

[0047] Platelet-specific knockout mice provide consistent protein-modified platelets useful for both in vitro and in vivo studies. This technique has been widely used to study the role of specific proteins in platelet function, and in our study, we bred transgenic mice with Piezo1 deficiency in the megakaryocyte lineage to obtain uniform Piezo1-deficient platelets to study their role in high shear-induced platelet aggregation and the potential targeting of Rb1. Microfluidic shearing was used to study the effects of shear on platelet activation and aggregation in vitro. 4000 s -1 High shear rates induce platelet aggregation; however, Piezo1 ΔPlt SIPA was significantly reduced in platelets, indicating that Piezo1 channels are essential for SIPA. ΔPlt In platelets, the present invention observed that the -1 Compared with the shear rate of 4000 s-1 Ca under shear rate 2+ This suggests that other mechanosensitive channels may also contribute to shear-induced Ca 2+ Inflow, such as TRPC6, TRPV4 or Orai1. The role of Piezo1 in high shear-induced arterial thrombosis was further investigated in the Folts mouse model. The Folts model combines high shear stress of blood flow and endothelial injury to induce platelet thrombosis, which has good clinical relevance. Computational fluid simulations of mouse carotid artery stenosis showed that in the Folts mouse model, a 50% reduction in blood flow could result in a shear rate of more than 4600 s -1 The shear rate of the blood vessel wall is as high as 1×10 5 / s. It is worth noting that, with Piezo1 fl / fl Compared with mice, Piezo1 ΔPlt The incidence of CFR was significantly reduced in mice, indicating that Piezo1 deficiency in platelets could significantly inhibit SIPA and thus reduce thrombosis.

[0048] The present invention utilizes a microfluidic channel coated with PECAM-1 antibody to immobilize platelets on the surface of the microfluidic channel without chemically induced platelet activation. The present invention uses the Piezo1 channel blocker GsMTx-4 and the agonist Yoda1 to demonstrate that PNS inhibits shear-induced platelet aggregation. At a normal shear rate of 500 s -1 , and 4000 s -1 Compared with the high shear rate of 2+ The internal flow increased significantly. 0.15 g·L -1 and 0.6 g·L -1 Effect of different concentrations of PNS on high shear-induced platelet Ca 2+ The influx was significantly inhibited, with an inhibition rate of about 72%.

[0049] The present invention shows for the first time that ginsenoside Rb1 has an effect on platelet Ca 2+ At a concentration of 40 μM, Rb1 had the most potent inhibitory effect on high shear-induced platelet Ca influx. 2+ The inhibitory effect on influx was more than 60%, showing the highest efficacy among the five tested ingredients. -1 Rb1 also significantly reduced shear-induced platelet aggregation, with an IC50 of 10.8 μM as measured by microfluidic shear assay. -1 Inhibits platelet Ca under high shear rates 2+Rb1 significantly reduced platelet calpain activity and talin cleavage induced by high shear. Rb1 showed an inhibitory effect on Yoda1-induced platelet activation and aggregation. Rb1 significantly reduced Yoda1-induced platelet aggregation with an IC50 of 21.0 μM. Rb1 inhibited Yoda1-induced Ca 2+ The IC50 values ​​for influx, p-selectin expression, and integrin αIIbβ3 activation were 40.8 μM, 551.7 μM, and 114.1 μM, respectively. Thus, Rb1 has a stronger inhibitory effect on shear-induced platelet activation than Yoda1-induced platelet activation. In general, Rb1 exhibits a selective role in shear-induced platelet activation and aggregation.

[0050] The present invention uses a cell thermal migration assay (CETSA) to measure the binding of Rb1 to the Piezo1 channel in platelets. The results show that treatment of platelets with Rb1 enhances the thermal stability of Piezo1, indicating the binding of Rb1 to platelet Piezo1 protein. It is worth noting that 10 μM Rb1 significantly inhibits the Piezo1 fl / fl SIPA in platelets and reduced high shear-induced Ca 2+ influx, calpain activation, talin cleavage, P-selectin expression, and integrin αIIbβ3 activation. Importantly, Piezo1 ΔPlt Compared with platelets, Rb1 treatment did not affect Piezo1 ΔPlt Platelets produced an additional inhibitory effect, suggesting that the Piezo1 channel is a valid target for the antiplatelet effects of Rb1.

[0051] According to the present study, mice with specific Piezo1 deficiency showed reduced high shear stress-induced platelet activation, thereby reducing thrombus formation. Administration of Rb1-L (20 mg kg -1 ) and Rb1-H (50 mg kg -1 ) significantly reduced Piezo1 fl / fl The number of CFRs and thrombus formation in mice was significantly reduced in a dose-dependent manner. Notably, the inhibition rate of Rb1-L was similar to that of PNS (58% vs. 65%). Both Rb1-L and Rb1-H significantly reduced the fl / flIn the Folts mouse model of thrombosis, the average inhibition rates of P-selectin expression and integrin αIIbβ3 were 20% and 43% for Rb1-L and 26% and 54% for Rb1-H, respectively. These results suggest that Rb1 effectively inhibits high shear-induced arterial thrombosis and platelet activation in vivo. ΔPlt Administration of Rb1 to mice did not produce additional inhibitory effects on CFR and platelet activation. Our data suggest that Rb1 is an effective inhibitor of shear-induced platelet activation and arterial thrombosis and is an inhibitor of Piezo1.

[0052] The results showed that the lack of Piezo1 in platelets did not affect the hemostasis of mice. The effective antithrombotic dose of Rb1 was selected to evaluate the bleeding time of mice, and the results showed that Rb1 did not prolong the bleeding time. As an antiplatelet drug, Rb1 has good antithrombotic effect and safety.

[0053] In summary, we demonstrated that Piezo1 plays an important role in shear-induced platelet activation and arterial thrombosis, and that ginsenoside Rb1 exerts antiplatelet and antithrombotic effects under pathological high shear conditions through Piezo1. This study provides new insights into antiplatelet therapeutic strategies under pathological mechanical conditions.

Claims

1. Application of platelet Piezo1 protein inhibitors in the preparation of drugs that reduce shear-induced platelet activation and aggregation.

2. The use according to claim 1, characterized in that: The platelet Piezo1 protein inhibitor is a blocker of the Piezo1 calcium ion signaling channel.

3. The use according to claim 2, characterized in that: The blocker of the Piezo1 calcium ion signaling channel is ginsenoside Rb1.

4. The use according to claim 3, characterized in that: The concentration of ginsenoside Rb1 in the drug for reducing shear-induced platelet activation and aggregation is 5-80 μM.

5. Application of platelet Piezo1 protein inhibitors in the preparation of anti-arterial thrombotic drugs.

6. The use according to claim 5, characterized in that: The platelet Piezo1 protein inhibitor is ginsenoside Rb1.

7. The use according to claim 6, characterized in that: The concentration of ginsenoside Rb1 in the anti-arterial thrombotic drug is 5-80 μM.

8. The use according to claim 6, characterized in that The dosage of the anti-arterial thrombotic drug is 20-100 mg·kg-1.

9. Application of ginsenoside Rb1 in the preparation of blockers of Piezo1 calcium ion signaling channel.

10. Application of ginsenoside Rb1 in the preparation of platelet Piezo1 protein inhibitors.

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