Arterial Thrombosis Model Research Articles

Pharmacological profile of asundexian, a novel, orally bioavailable inhibitor of factor XIa

BackgroundActivated coagulation factor XI (FXIa) contributes to the development and propagation of thrombosis but plays only a minor role in hemostasis; therefore, it is an attractive antithrombotic target. ObjectivesTo evaluate the pharmacology of asundexian (BAY 2433334), a small molecule inhibitor targeting FXIa, in vitro and in various rabbit models. MethodsThe effects of asundexian on FXIa activity, selectivity versus other proteases, plasma thrombin generation, and clotting assays were evaluated. Antithrombotic effects were determined in FeCl2‐ and arterio‐venous (AV) shunt models. Asundexian was administered intravenously or orally, before or during thrombus formation, and with or without antiplatelet drugs (aspirin and ticagrelor). Potential effects of asundexian on bleeding were evaluated in ear‐, gum‐, and liver injury models. ResultsAsundexian inhibited human FXIa with high potency and selectivity. It reduced FXIa activity, thrombin generation triggered by contact activation or low concentrations of tissue factor, and prolonged activated partial thromboplastin time in human, rabbit, and various other species, but not in rodents. In the FeCl2‐injury models, asundexian reduced thrombus weight versus control, and in the arterial model when added to aspirin and ticagrelor. In the AV shunt model, asundexian reduced thrombus weight when administered before or during thrombus formation. Asundexian alone or in combination with antiplatelet drugs did not increase bleeding times or blood loss in any of the models studied. ConclusionsAsundexian is a potent oral FXIa inhibitor with antithrombotic efficacy in arterial and venous thrombosis models in prevention and intervention settings, without increasing bleeding.

Novel contact-kinin inhibitor sylvestin targets thromboinflammation and ameliorates ischemic stroke.

Ischemic stroke is a leading cause of death and disability worldwide. Increasing evidence indicates that ischemic stroke is a thromboinflammatory disease in which the contact-kinin pathway has a central role by activating pro-coagulant and pro-inflammatory processes. The blocking of distinct members of the contact-kinin pathway is a promising strategy to control ischemic stroke. Here, a plasma kallikrein and active FXII (FXIIa) inhibitor (sylvestin, contained 43 amino acids, with a molecular weight of 4790.4Da) was first identified from forest leeches (Haemadipsa sylvestris). Testing revealed that sylvestin prolonged activated partial thromboplastin time without affecting prothrombin time. Thromboelastography and clot retraction assays further showed that it extended clotting time in whole blood and inhibited clot retraction in platelet-rich plasma. In addition, sylvestin prevented thrombosis in vivo in FeCl3-induced arterial and carrageenan-induced tail thrombosis models. The potential role of sylvestin in ischemic stroke was evaluated by transient and permanent middle cerebral artery occlusion models. Sylvestin administration profoundly protected mice from ischemic stroke by counteracting intracerebral thrombosis and inflammation. Importantly, sylvestin showed no signs of bleeding tendency. The present study identifies sylvestin is a promising contact-kinin pathway inhibitor that can proffer profound protection from ischemic stroke without increased risk of bleeding.

Memory-like response in platelet attenuates platelet hyperactivation in arterial thrombosis

Recent studies showed that in responding of pathogens stimulation, immune cells and other cells display memory-like effects. Platelets are primary effectors of hemostasis and thrombosis which also participate in immune responses. However, there is no relevant research on whether memory-like effect exists in platelets. In our study after recovery from repetitive LPS stimulus, platelets aggregation, diffusion and clot retraction exhibit a significant reduction. It proves that memory-like response could be aroused in platelets. Furthermore, in the mouse arterial thrombosis model, LPS pretreated platelets showed lower integrin activation, shorter thrombus length and longer occlusion time, indicating that the memory-like response of platelet could alleviate arterial thrombosis. Moreover, memory-like response of platelets was also found to be related to PI3K/AKT signaling pathway. The decreased mitochondrial DNA methylation reveal that platelet memory-like responses may be produced from epigenetic reprogramming. Our research proves for the first time that memory-like response in platelets protects mice from arterial thrombosis, extends the understanding of trained memory.

Effects of MaiLiuPian on carotid thrombosis in rats and acute pulmonary embolism in mice and its antithrombotic mechanism.

Mailiupian (MLP) is a new patent functional food that consists of Crataegi Fructus, Notoginseng Radix, and Ginkgo Folium, which was reported to be active in improving the microcirculation based on formulation screening. However, whether it is effective in inhibiting thrombus and its mechanism has not been evaluated. Therefore, in the present study, the models of arterial thrombosis induced by FeCl3 and the models of APE by ADP were established to evaluate the antithrombosis effect of MLP. Results showed that MLP markedly reduced the weight and size of wet thrombosis in FeCl3 -induced rats and decreased the recovery time from symptoms of APE mice. MLP was proved to prolong APTT, PT, TT and improve the levels of t-PA and 6-keto-PGF1α significantly, meanwhile, PAI-1 and TXB2 were reduced apparently. By comparing tail bleeding time, MLP showed antithrombotic effects, but without the risk of bleeding, taking aspirin as a control. PRACTICAL APPLICATIONS: Our experiments proved that MLP, a new patent health food, acted on both coagulation and fibrinolytic systems and the platelet aggregation to play antithrombosis roles, providing a theoretical basis for applications of MLP in preventing or curing thrombosis diseases.

Establishment of a Modified and Standardized Ferric Chloride-Induced Rat Carotid Artery Thrombosis Model.

Background: Ferric chloride is widely utilized in inducing thrombosis in small vessels of experimental animals. However, the lack of its application in large blood vessels of experimental animals and inconsistent concentration has limited its application. Therefore, we systematically tested the most suitable concentration and reliable induction time in the experiment of using ferric chloride to induce rat carotid artery thrombosis. Methods: In this study, we selected the common carotid artery of 59 Sprague–Dawley rats as the target vessel. The exploration process was divided into three stages. First, to determine the optimum induction concentration, we compared the effects of 30–60% ferric chloride on thrombus formation within 24 h. Second, to confirm the handling time, we tested different induction times from 3 min to 10 min. Lastly, we used the thrombolytic drug rt-PA to detect whether the formed thrombus can be lysed. Doppler blood flow imaging and H–E staining were employed to estimate the blood flow and thrombus. The ATP levels in the brain were measured using a bioluminescence ATP assay kit. Results: We found that the application of 50% ferric chloride for 10 min was enough to successfully induce thrombosis in the rat carotid artery and without spontaneous thrombolysis after 24 h. It is better than other concentrations and will lead to the decline of the ATP content in the ischemic hemisphere. Conclusions: Our results indicate that the rat carotid artery thrombosis model induced by 50% ferric chloride for 10 min is stable and reliable.

The effect of ChAdOx1 nCov-19 vaccine on arterial thrombosis development and platelet aggregation in female rats

ChAdOx1 nCoV-19 adenoviral vector vaccine (ChAd) against coronavirus disease 2019 has been associated with vaccine-induced thrombosis and thrombocytopenia (VITT), especially in young women who have presented with unusual localized thrombosis after receiving the vaccine. The pathogenesis of VITT remains incompletely understood. We tried to provide new insights into mechanisms underlying this phenomenon in the model of arterial thrombosis electrically induced in the carotid artery of female rats. At 28 days post-vaccination, ChAd induced SARS-CoV-2-specific neutralizing antibody responses in all animals. The analysis of the blood vessel/thrombus area showed slight luminal narrowing of the carotid artery with extravasation of blood in vaccinated rats. These small changes were not accompanied by differences in thrombus weight and composition. The vaccinated animals presented a slight increase (by around 14–24%) in platelet aggregation. ChAd did not significantly affect blood coagulation, platelet counts, and their activation markers. Unaffected thrombus formation, the lack of thrombocytopenia and all the measured blood and hemostasis parameters that predominantly stayed unchanged, indicate that the ChAd does not increase the risk of arterial thrombosis development in female rats.

Antithrombotic Effect of Sorbus commixta in an Arterial Thrombosis Mouse Model

Sorbus commixta Hedlund ( S. commixta) is known to possess cardiovascular protective activity. However, its effect on thrombosis has not been reported, although thrombosis plays a primary role in diverse circulatory diseases. Thus, this study was performed to assess the antithrombotic activity of dried S. commixta fruit extract. Mice were fed with a diet containing 8.3% extract for 4 weeks, which corresponded to the consumption of approximately 1000 mg extract/kg/day. The antithrombotic effect was tested in a FeCl3-induced arterial thrombosis model. Additionally, the effects on platelet aggregation and plasma coagulation were examined in vitro using washed platelets (WP) and isolated blood plasma, respectively. Thrombotic vascular occlusion time was significantly prolonged by the 4-week administration of the extract. Treatment of WP with the extract resulted in a decrease in thrombin-induced aggregation, which was concentration-dependent over 10 to 100 μg/mL and was not attributed to the direct inhibition of thrombin. The extract did not affect blood plasma coagulation, which was assessed by measuring prothrombin time and activated partial thromboplastin time. S. commixta is capable of preventing arterial thrombosis and such an effect may be attributed to the inhibition of platelet aggregation.

Structure of shear-induced platelet aggregated clot formed in an in vitro arterial thrombosis model

AbstractThe structure of occlusive arterial thrombi is described herein. Macroscopic thrombi were made from whole blood in a collagen-coated, large-scale stenosis model with high shear flow similar to an atherosclerotic artery. The millimeter-sized thrombi were harvested for histology and scanning electron microscopy. Histological images showed 3 distinctive structures of the thrombus. (1) The upstream region showed string-like platelet aggregates growing out from the wall that protrude into the central lumen, with red blood cells trapped between the strings. The strings were >10 times as long as they were wide and reached out to join the strings from the opposite wall. (2) Near the apex, the platelet strings coalesced into a dense mass with microchannels that effectively occluded the lumen. (3) In the expansion region, the thrombus ended abruptly with an annulus of free blood in the flow-separation zone. Scanning electron microscopy showed dense clusters of spherical platelets upstream and downstream, with amorphous platelets in the occluded throat consistent with prior activation. The total clot is estimated to contain 1.23 billion platelets with pores 10 to 100 μm in diameter. The results revealed a complex structure of arterial thrombi that grow from their tips under high shear stress to bridge the 2.5-mm lumen quickly with von Willebrand factor platelet strings. The occlusion leaves many microchannels that allow for some flow through the bulk of the thrombus. This architecture can create occlusion or hemostasis rapidly with minimal material, yet can remain porous for potential delivery of lytic agents to the core of the thrombus.

Engineered platelets-based drug delivery platform for targeted thrombolysis

Thrombolytic agents have thus far yielded limited therapeutic benefits in the treatment of thrombotic disease due to their short half-life, low targeting ability, and association with serious adverse reactions, such as bleeding complications. Inspired by the natural roles of platelets during thrombus formation, we fabricated a platelet-based delivery system (NO@uPA/PLTs) comprising urokinase (uPA) and arginine (Arg) for targeted thrombolysis and inhibition of re-embolism. The anchoring of uPA to the platelet surface by lipid insertion increased the thrombotic targeting and in vivo circulation duration of uPA without disturbing platelet functions. Nitric oxide (NO) generated by the loaded Arg inhibited platelet aggregation and activation at the damaged blood vessel, thereby inhibiting re-embolism. NO@uPA/PLTs effectively accumulated at the thrombi in pulmonary embolism and carotid artery thrombosis model mice and exerted superior thrombolytic efficacy. In addition, the platelet delivery system showed excellent thrombus recurrence prevention ability in a mouse model of secondary carotid artery injury. The coagulation indicators in vivo showed that the platelet-based uPA and NO co-delivery system possessed a low hemorrhagic risk, providing a promising tool for rapid thrombolysis and efficient inhibition of posttreatment re-embolism.

Antithrombotic Activity of the Antiplatelet Agent Angipur on the Model of Arterial Thrombosis in Rats with Isoproterenol-Induced Myocardial Infarction.

We studied the effect of Angipur on the process of experimental thrombosis induced by damage to the carotid artery wall by surface application of 50% ferric chloride (III) solution in rats without comorbidities and with isoproterenol-induced myocardial infarction. In animals without comorbidities, Angipur administered intravenously was 1.2 times less effective, in terms of ED50, than the well-known inhibitor of GPIIb/IIIa platelet receptors tirofiban. However, under conditions of non-coronary myocardial infarction, Angipur significantly prolonged the time of thrombus formation and exhibited 1.4-fold higher activity than the reference drug tirofiban.

Characterization of the Role of Integrin α5β1 in Platelet Function, Hemostasis, and Experimental Thrombosis.

Objective Integrins are key regulators of various platelet functions. The pathophysiological importance of most platelet integrins has been investigated, with the exception of α5β1, a receptor for fibronectin. The aim of this study was to characterize the role of α5β1 in megakaryopoiesis, platelet function, and to determine its importance in hemostasis and arterial thrombosis. Approach and Results We generated a mouse strain deficient for integrin α5β1 on megakaryocytes and platelets (PF4Cre-α5 −/− ). PF4Cre-α5 −/− mice were viable, fertile, and presented no apparent signs of abnormality. Megakaryopoiesis appears unaltered as evidence by a normal megakaryocyte morphology and development, which is in agreement with a normal platelet count. Expression of the main platelet receptors and the response of PF4Cre-α5 −/− platelets to a series of agonists were all completely normal. Adhesion and aggregation of PF4Cre-α5 −/− platelets under shear flow on fibrinogen, laminin, or von Willebrand factor were unimpaired. In contrast, PF4Cre-α5 −/− platelets displayed a marked decrease in adhesion, activation, and aggregation on fibrillar cellular fibronectin and collagen. PF4Cre-α5 −/− mice presented no defect in a tail-bleeding time assay and no increase in inflammatory bleeding in a reverse passive Arthus model and a lipopolysaccharide pulmonary inflammation model. Finally, no defects were observed in three distinct experimental models of arterial thrombosis based on ferric chloride-induced injury of the carotid artery, mechanical injury of the abdominal aorta, or laser-induced injury of mesenteric vessels. Conclusion In summary, this study shows that platelet integrin α5β1 is a key receptor for fibrillar cellular fibronectin but is dispensable in hemostasis and arterial thrombosis.

Abelacimab Reduces Thrombus Propagation in a Baboon Model of Vascular Graft Thrombosis

Background: Factor XI (FXI) inhibition demonstrated strong efficacy in preventing thrombus formation in preclinical and clinical models of arterial and venous thrombosis. However, the effect of FXI inhibition in halting the progression of a formed clot remains largely unknown.Aims: This study aims to test whether abelacimab, a dual-acting FXI and activated FXI (FXIa) monoclonal antibody, is effective in halting clot formation and downstream growth when administered before or during active clot formation in an established baboon femoral arterio-venous (AV) shunt model.Methods: Three baboons had a chronic femoral AV shunt put in place; platelet and fibrin deposition inside and distal to collagen- or collagen + tissue factor (TF)-coated vascular grafts were measured at baseline (control), in a therapeutic setting, where abelacimab (1 mg/kg, intravenously) was administered 30 minutes after thrombus initiation, and in a preventative setting within the first 48 h and 1 week (144 - 216 h) post-administration. Pharmacodynamic effect was measured by activated partial thromboplastin time (aPTT).Results: Consistent with its half-life of 20 to 30 days, single iv administration of abelacimab at a dose of 1 mg/kg resulted in long-lasting (> 4-week) aPTT prolongation (> 2-fold). Administration of abelacimab 30 minutes after initiation of thrombosis using grafts coated either with collagen or with collagen + TF quickly halted downstream propagation of platelet and fibrin deposition compared to control. Further, downstream propagation of platelet and fibrin deposition was markedly reduced when clotting was induced by collagen, or collagen + tissue factor after abelacimab administration.Conclusions: These data suggest that abelacimab, a dual-acting anti-FXI/FXIa monoclonal antibody with a single long-lasting iv injection has the potential to slow down the growth and reduce the size of thrombi when admistered before or after clot induction. Data indicate a potential for therapeutic benefit of targeting FXI both in therapeutic and preventive settings.Sponsored by: Anthos Therapeutics Inc., 55 Cambridge Parkway, Suite 103, Cambridge, MA 02142 [Display omitted] DisclosuresWallisch: Aronora Inc,: Current Employment. Khder: Anthos Therapeutics: Consultancy; Novartis: Current equity holder in publicly-traded company, Other: Retiree. Bloomfield: Anthos Therapeutics: Current Employment. Gruber: Aronora Inc.: Current Employment, Current equity holder in publicly-traded company; Oregon Health and Science University: Current Employment.

Investigating the Roles of Platelet PAR4 in Hemostasis, Thrombosis and Viral Infection Using a Newly Generated PAR4 Floxed Mouse

Introduction: Protease-activated receptor 4 (PAR4) is expressed by a wide variety of cells, including megakaryocytes/platelets, immune cells, cardiomyocytes and lung epithelial cells, and activated by multiple ligands including thrombin and cathepsin G. Importantly, PAR4 is the only functional thrombin receptor on murine platelets. A global deficiency of PAR4 is associated with impaired hemostasis and protection from thrombosis which are attributed to loss of platelet PAR4, but this has not been specifically demonstrated in mice. Additionally, global PAR4 deficiency increases mortality after influenza A virus infection, but the cell type/s responsible for the enhanced mortality have not been determined. Here, we describe the generation of PAR4 floxed (PAR4 fl/fl) mice that can be used to delete PAR4 in a cell type-specific manner, and examine the effect of megakaryocyte/platelet-specific deletion of PAR4 on hemostasis, thrombosis and viral infection using PAR4 fl/fl;PF4Cre + mice.Methods: PAR4global knockout (PAR4 -/-), MK/platelet-specific knockout (PAR4 fl/fl;PF4Cre +) and appropriate littermate control mice were used for experiments. Platelet function was determined by light transmission aggregometry and flow cytometry. Hemostasis was assessed in the saphenous vein laser injury model. Platelet plug formation was visualized by intravital microscopy following saphenous vein laser ablation (~50 μm diameter injury), followed by 2 subsequent ablations to reinjure the same site unless on-going bleeding was occurring. Mice were treated with ibrutinib (12.5 mg/kg) to inhibit GPVI signaling, or dabigatran etexilate (chow containing 10 mg/g) or recombinant hirudin (50 mg/kg) to inhibit thrombin activity. Thrombosis was assessed in the carotid artery FeCl 3 model. The carotid artery was exposed and 8% FeCl 3 applied for 3 mins. Blood flow was observed for 30 mins and occlusion was defined as no blood flow for 2 mins. To study susceptibility to viral infection, mice were challenged intranasally with a mouse-adapted H1N1 influenza A virus (H1N1 IAV PR8; 0.02 hemagglutination assay units), which induces mortality in 20% of WT mice. Mortality was defined as body weight loss greater than 25%, which required euthanasia.Results: As expected, PAR4 fl/fl;PF4Cre + platelets were unresponsive to thrombin or PAR4-specific stimulation, while the response to other agonists was retained. In the saphenous vein laser injury hemostasis model, PAR4 fl/fl;PF4Cre + mice were able to rapidly form a hemostatic platelet plug, but the majority of plugs (7/8) were unstable and re-opened after several minutes, leading to severely prolonged total bleeding times. We observed similar findings in global PAR4 -/- mice with 8/12 plugs re-opening. To investigate the mechanism mediating initial platelet plug formation, we inhibited GPVI signaling in PAR4 fl/fl;PF4Cre + mice using the Btk inhibitor ibrutinib. Ibrutinib administration shortened time to plug re-opening in PAR4 fl/fl;PF4Cre + mice but plugs were still able to form, which is likely mediated by GPIbα/VWF. We observed a similar phenotype to PAR4 fl/fl;PF4Cre + mice in mice treated with the direct thrombin inhibitor hirudin, suggesting thrombin is the primary activator of PAR4 during hemostatic plug formation. In the FeCl 3-induced carotid artery thrombosis model, both PAR4 fl/fl;PF4Cre + and PAR4 -/- mice were significantly protected compared to controls. Finally, when challenged with the mouse-adapted H1N1 IAV PR8, PAR4 fl/fl;PF4Cre + mice demonstrated similar body weight loss and survival as littermate controls.Conclusions: Our results in mice demonstrate that 1) platelet PAR4 is not required for initial hemostatic plug formation but is necessary for maintaining hemostatic plug stability, 2) loss of platelet PAR4 protects from arterial thrombosis, and 3) platelet PAR4 does not alter the course of H1N1 IAV infection, at least at the virus dose used in this study. In summary, we generated a novel mouse line carrying a floxed PAR4 allele which can be used to investigate cell-specific roles of PAR4 in disease. DisclosuresNo relevant conflicts of interest to declare.

Generation of a humanized FXII knock‐in mouse—A powerful model system to test novel anti‐thrombotic agents

BackgroundEffective inhibition of thrombosis without generating bleeding risks is a major challenge in medicine. Accumulating evidence suggests that this can be achieved by inhibition of coagulation factor XII (FXII), as either its knock‐out or inhibition in animal models efficiently reduced thrombosis without affecting normal hemostasis. Based on these findings, highly specific inhibitors for human FXII(a) are under development. However, currently, in vivo studies on their efficacy and safety are impeded by the lack of an optimized animal model expressing the specific target, that is, human FXII. ObjectiveThe primary objective of this study is to develop and functionally characterize a humanized FXII mouse model. MethodsA humanized FXII mouse model was generated by replacing the murine with the human F12 gene (genetic knock‐in) and tested it in in vitro coagulation assays and in in vivo thrombosis models. ResultsThese hF12KI mice were indistinguishable from wild‐type mice in all tested assays of coagulation and platelet function in vitro and in vivo, except for reduced expression levels of hFXII compared to human plasma. Targeting FXII by the anti‐human FXIIa antibody 3F7 increased activated partial thromboplastin time dose‐dependently and protected hF12KI mice in an arterial thrombosis model without affecting bleeding times. ConclusionThese data establish the newly generated hF12KI mouse as a powerful and unique model system for in vivo studies on anti‐FXII(a) biologics, supporting the development of efficient and safe human FXII(a) inhibitors.

Thrombolytic and anticoagulant effects of a recombinant staphylokinase-hirudin fusion protein

A pure recombinant staphylokinase-hirudin fusion protein (SFH) was obtained by recombinant genetic engineering and purification techniques. The thrombolytic and anticoagulant activities of SFH were investigated using in vitro coagulation models and chromogenic assays. The results showed that intact SFH had targeted thrombolytic activity, and gained anticoagulant activity when cleaved by FXa. In addition, we investigated the pharmacodynamics of SFH in vivo using a variety of animal models, including a rat inferior vena cava thrombosis model, a rat coronary thrombosis model, a rabbit carotid artery thrombosis model and a canine coronary thrombosis model. We found that SFH had an obvious thrombolytic effect and could prevent and reduce re-embolization after thrombolysis and reduce the serious bleeding side effects caused by the combination of thrombolytic and anticoagulant drugs. The results suggest that SFH can be used for thrombolytic therapy in thromboembolic diseases.

Evaluation of the prothrombotic potential of four-factor prothrombin complex concentrate (4F-PCC) in animal models.

ObjectivesAcquired coagulopathy may be associated with bleeding risk. Approaches to restore haemostasis include administration of coagulation factor concentrates, but there are concerns regarding potential prothrombotic risk. The present study assessed the prothrombotic potential of four-factor prothrombin complex concentrate (4F-PCC) versus activated PCC (aPCC) and recombinant factor VIIa (rFVIIa), using three preclinical animal models.MethodsThe first model was a modified Wessler model of venous stasis-induced thrombosis in rabbit, focusing on dilutional coagulopathy; the second model employed the same system but focused on direct oral anticoagulant reversal (i.e. edoxaban). The third model assessed the prothrombotic impact of 4F-PCC, aPCC and rFVIIa in a rat model of ferric chloride-induced arterial thrombosis.ResultsIn the first model, thrombi were observed at aPCC doses ≥10 IU/kg (therapeutic dose 100 IU/kg) and rFVIIa doses ≥50 μg/kg (therapeutic dose 90 μg/kg), but not 4F-PCC 50 IU/kg (therapeutic dose 50 IU/kg). The impact of 4F-PCC (up to 300 IU/kg) on thrombus formation was evident from 10 minutes post-administration, but not at 24 hours post-administration; this did not change with addition of tranexamic acid and/or fibrinogen concentrate. 4F-PCC-induced thrombus formation was lower after haemodilution versus non-haemodilution. In the second model, no prothrombotic effect was confirmed at 4F-PCC 50 IU/kg. The third model showed lower incidence of thrombus formation for 4F-PCC 50 IU/kg versus aPCC (50 U/kg) and rFVIIa (90 μg/kg).ConclusionsThese results suggest that 4F-PCC has a low thrombotic potential versus aPCC or rFVIIa, supporting the clinical use of 4F-PCC for the treatment of coagulopathy-mediated bleeding.

Thrombolytic Effect of Nattokinase and Its Mechanism

Objective: Exploring the anti-thrombotic effect and mechanism of nattokinase in vitro and vivo. Methods: The thrombolytic effect of NK on old thrombus was studied by blood clot lysis method in vitro, the rat carotid artery thrombosis model induced by ferric chloride and the rat venous thrombosis model induced by inferior vena cava ligation method were used to study the thrombolytic effect of NK in vivo. TXB2, 6-k, PGE2, t-PA, \begin{document}$\cdot {\rm{O}}_2^- $\end{document} , ·OH, XOD and other indicators were detected to explore the antithrombotic mechanism of NK. Results: Nattokinase could significantly dissolve old thrombus (P \begin{document}$\cdot {\rm{O}}_2^- $\end{document} , ·OH and XOD. The values of TXB2, PEG2, \begin{document}$\cdot {\rm{O}}_2^- $\end{document} , ·OH and XOD were increased significantly, while the values of 6-K-PGF1 α and t-PA were decreased significantly. Conclusion : NK had strong thrombolytic activity in vivo and vitro, and had effect on fresh thrombus and old thrombus. NK could inhibit thrombosis by inhibiting platelet aggregation, reduce oxidative damage and reducing the level of coagulation factors. NK could also play a direct thrombolytic role by stimulating the production of t-PA, dissolving fibrin, and might play an indirect thrombolytic role by inhibiting the production of PAI-1.

SYNTHESIS, ANTIAGGREGATION AND ANTITROMBOTIC ACTIVITIES OF NEW DERIVATIVES OF HYDROXYBENZOIC ACIDS WITH TAURIC FRAGMENT

A high prevalence of thrombotic disorders, insufficient effectiveness or safety of antithrombotic therapy is an urgent problem of modern healthcare. The main means of preventing thrombosis is acetylsalicylic acid. Despite its long history, aspirin attracts researchers in the fields of medicinal chemistry, biology, and medicine. The development of new antiplatelet agents, including chemical modification of the acetylsalicylic acid molecule, remains relevant. Modification of the acetylsalicylic acid molecule using amino acids and obtaining their salt forms makes it possible to maintain antiplatelet or antithrombotic properties, as well as to impart additional pharmacodynamic effects. In modern science, a lot of attention is paid to the sulfur-containing amino acid taurine. An analysis of modern scientific literature revealed the protective effect of taurine in diabetes mellitus and cardiovascular diseases, liver dysfunction, gastrointestinal tract, and kidney diseases.The aim of the article is to study synthesis of new compounds, determination of their physical characteristics and assessment of their antiplatelet and antithrombotic activities in vitro and in vivo.Materials and methods. To confirm the structure of the synthesized new derivatives of hydroxybenzoic acids with a taurine fragment by the acelation method, thin layer chromatography and NMR spectra were used. In vitro studies were carried out on the model of ADP-induced platelet aggregation according to the Born G. methods modified by V.A. Gabbasov. In vivo, the studies were carried out on the model of arterial thrombosis induced by the application of iron chloride in the following groups of animals: intact, with experimental diabetes mellitus and three-year-olds; the rate of bleeding from the tail vein was also evaluated.Results. New compounds – derivatives of ortho-, meta- and para-hydroxybenzoic acids with a taurine residue – were synthesized. A procedure for the preparation of N-hydroxybenzoyl taurine compounds and their salt forms have been described; their spectral characteristics and melting points have been determined. The synthesized compounds are superior to acetylsalicylic acid in solubility and are not inferior to it in antiplatelet and antithrombotic activities. The results of the in vitro antiplatelet activity assessment in a wide concentration range from 10-4M to 10-8M, are presented. It has been revealed that the dipotassium salt of N-(2-hydroxybenzoyl)taurine exhibits a less antiplatelet activity than the dipotassium salt of N-(3-hydroxybenzoyl)taurine. The most pronounced antiplatelet activity is exhibited by the compound N-(4-hydroxybenzoyl)taurine. In in vivo experiments on the model of arterial thrombosis in 3-year-olds or animals with experimental diabetes mellitus, carotid artery thrombosis occurred faster than in young or intact animals. A single preliminary oral administration of the test compounds prolonged the time of the thrombus formation, which makes it possible to conclude that they have an antithrombotic effect. In this study, the dipotassium salt of N-(3-hydroxybenzoyl)taurine exhibits a more pronounced activity than that of acetylsalicylic acid.Conclusion. Against the background of the modeled pathologies, the studied drugs showed the expected antithrombotic activity, in terms of the severity not inferior to that found in acetylsalicylic acid.

Preferential Inhibition of Fibrin Mediated Thrombosis in Rodent Models By JNJ-9375, a Novel exosite1 Thrombin Inhibitor

We have previously reported an improved therapeutic index (TI) for JNJ-9375 vs apixaban in rat and cynomolgus monkey models of venous thrombosis and bleeding. We have also reported that JNJ-9375 preferentially inhibited the fibrin component of the thrombus without significant impact on the platelet component under low and high shear conditions in a human translational model of thrombosis (Badimon chamber model). We hypothesized that the novel mode of action of JNJ-9375 which by selectively inhibiting exosite 1 substrates from binding to thrombin (such as fibrinogen and PAR-1) may allow for other thrombin interactions at the active site and exosite 2 including platelet activation by PAR-4 and GP1b.In the present studies, we have compared the antithrombotic effects of JNJ-9375, the active site thrombin inhibitor dabigatran and the factor Xa inhibitor apixaban in well characterized and widely used rat AV-shunt and carotid ferric chloride models of venous and arterial thrombosis, respectively. JNJ-9375, dabigatran and apixaban all maximally inhibited thrombus formation in a dose dependent manner in the rat AV-shunt model of venous thrombosis. Significant inhibition of thrombus weights of 50%, 44% and 57% were observed at 0.3 mg/kg, i.v., 0.1 mg/kg, i.v. and 1 mg/kg, i.v. with JNJ-9375, dabigatran and apixaban respectively. Dose dependent prolongation in coagulation parameters were also observed ex-vivo with JNJ-9375 (TT, ECT, aPTT and PT), dabigatran (TT, ECT, aPTT) and apixaban (aPTT and PT). In addition, there were dose dependent increases in drug exposure.In the rat ferric chloride model of arterial thrombosis, JNJ-9375, dabigatran and apixaban administration also resulted in dose dependent inhibition of thrombus formation as assessed by the time to occlusion (TTO) of the carotid artery and area under the curve (AUC) for carotid blood flow. However, the dose required to inhibit arterial thrombosis Vs venous thrombosis varied substantially for the different agents. With JNJ-9375 we only observed a significant inhibition of arterial thrombosis (TTO and AUC) at a dose of 10 mg/kg, i.v. (Vs 0.3 mg/kg in the AV-shunt model) which represents a 33-fold shift in the dose required to inhibit arterial thrombosis (Vs venous thrombosis). In contrast, we observed a significant inhibition of arterial thrombosis with dabigatran (AUC) and apixaban (TTO and AUC) at doses of 0.3 mg/kg, i.v. and 3 mg/kg, i.v. (Vs 0.1 mg/kg and 1 mg/kg in the AV-shunt model) respectively. These doses of dabigatran and apixaban represent a 3-fold shift in the dose required to inhibit arterial thrombosis (Vs venous thrombosis). We have also documented dose dependent prolongations in coagulation parameters and drug exposure with all three agents in the these studies. In addition, we performed ex-vivo platelet aggregation in platelet rich plasma with various platelet agonists (ADP, collagen, AA, rat and human thrombin) in those animals dosed with JNJ-9375. As anticipated and consistent with its mechanism of action, JNJ-9375 only inhibited platelet aggregation to thrombin stimulation but not with other platelet agonists confirming the specificity of JNJ-9375. We observed about 50%, >95% and >95% inhibition of thrombin-induced platelet aggregation at 1, 3 and 10 mg/kg, i.v. with JNJ-9375 respectively. However, we only observed inhibition of arterial thrombosis at 10 mg/kg.In summary, we have demonstrated dose dependent inhibition of thrombosis with JNJ-9375, dabigatran and apixaban in venous and arterial model of thrombosis in rats. However, we have observed a larger difference in the dose required for inhibition of arterial Vs venous thrombosis with JNJ-9375 (33-fold) compared to a more modest 3-fold shift observed for dabigatran and apixaban. These findings with JNJ-9375 demonstrate an anticoagulant profile that preferentially inhibits fibrin mediated clot formation with a more modest impact on platelet driven thrombosis in the present models. This differential profile may account in part for the improved TI over apixaban we had reported earlier in animal models. This profile suggests JNJ-9375 which is currently in early clinical development may also be more amenable to dosing in combination with antiplatelet therapy for indications such as acute coronary syndromes and needs to be tested experimentally. DisclosuresChintala:Janssen Research & Development: Employment. Li:Janssen R&D: Employment. Huang Devine:Janssen Research & Development: Employment. Bunce:Janssen R&D: Employment.

CD36 Promotes Platelet Phosphatidylserine Exposure through MAP Kinase ERK5

Dyslipidemic conditions can promote atherosclerotic plaque instability and rupture, leading to thrombosis that can cause life threatening complications. Thrombosis in this setting is augmented by circulating oxidized lipids on oxidized low density lipoprotein particles (oxLDL) that are recognized by key scavenger receptors. CD36 is a major platelet scavenger receptor that recognizes oxLDL and consequently promotes a prothrombotic platelet phenotype through multiple signaling pathways. We previously reported that recognition of oxLDL by CD36 generates specific reactive oxygen species through Src family kinases and NADPH oxidase. These species modulate redox sensitive signaling, such as the activation of the oxidant sensor MAP kinase ERK5, leading to platelet integrin activation, aggregation, and thrombus formation. Although ERK5 was a key effector for CD36 to promote thrombosis, the downstream signaling mechanisms are unclear. We hypothesize that CD36 promotes a procoagulant phenotype that requires ERK5. Platelets isolated and washed from healthy human donors were stimulated with buffer, native low-density lipoprotein (LDL), or oxLDL, and were stained with fluorophore-conjugated Annexin V or Lactadherin to detect exposure of procoagulant phosphatidylserine (PS). OxLDL-stimulated platelets showed dose-dependent 13.4 ± 2.8% and 18.5 ± 3.5% increases in Lactadherin binding at 50 and 100μg/mL oxLDL compared to control LDL (p=0.026; p<0.01), and a rapid binding within 5 min (11.0±0.35%, p<0.001). Furthermore, pre-treatment with oxLDL followed by addition of convulxin, a snake venom that activates platelets through the collagen signaling receptor GPVI, showed a 58.1 ± 4.62% increase in Lactadherin binding compared to oxLDL and convulxin alone (p<0.0001). Addition of ADP or thrombin, agonists that activates specific G-protein coupled receptors, following pre-treatment with oxLDL did not enhance Lactadherin binding. Platelets treated with FA6, a monoclonal CD36 antibody which prevents oxLDL binding, or BIX02188, an ERK5 inhibitor, showed an 80-90% decrease in Annexin V binding (p=0.039; p=0.02) after oxLDL exposure compared to a control non-immunizing IgG or DMSO. To link known pathways for PS exposure with oxLDL-induced PS exposure, platelets were stimulated with oxLDL followed by treatment with Z-VAD-FMK, a “pan” caspase inhibitor, or cyclosporin A, a cyclophilin D/mitochondrial permeability transition pore inhibitor. Z-VAD-FMK treated platelets showed 68% decrease in Lactadherin binding compared to control DMSO (p=0.0196), whereas cyclosporin A did not inhibit Lactadherin binding. Relevance of CD36/ERK5-mediated PS exposure was demonstrated using an in vivo arterial thrombosis model that relies on autologous transplantation of the epigastric artery, which has an adventitial surface that serves as a collagen-rich thrombogenic substrate for thrombosis, into the carotid artery. This model of thrombosis was chosen due to the potential impact of free radical generation in the standard ferric chloride or Rose Bengal-mediated arterial thrombosis models. Bone marrows from platelet ERK5-deficient mice (ERK5 flox/Pf4-cre+) or control mice (ERK5 flox/flox) were transplanted into irradiated atherogenic apoE null mice fed a high fat or control diet. Fibrin deposition, detected by a fluorophore-conjugated antibody to fibrin, was enhanced in hyperlipidemic conditions for mice with platelet ERK5. High fat diet-fed chimeric mice without ERK5 or animals on control diet do not show enhanced fibrin deposition. In conclusion, oxLDL promoted PS exposure through a CD36-Src family kinases-ERK5-caspase-dependent mechanism. PS exposure by this pathway potentially enhances fibrin deposition in an atherogenic arterial in vivo thrombosis model, which suggests a role for CD36 and ERK5 in thrombosis and coagulation in dyslipidemic conditions. DisclosuresNo relevant conflicts of interest to declare.