Cardiac Myosin Promotes Thrombin Generation and Coagulation In Vitro and In Vivo

Abstract

Objective: Cardiac myosin (CM) is structurally similar to skeletal muscle myosin, which has procoagulant activity. Here, we evaluated CM’s ex vivo, in vivo, and in vitro activities related to hemostasis and thrombosis. Approach and Results: Perfusion of fresh human blood over CM-coated surfaces caused thrombus formation and fibrin deposition. Addition of CM to blood passing over collagen-coated surfaces enhanced fibrin formation. In a murine ischemia/reperfusion injury model, exogenous CM, when administered intravenously, augmented myocardial infarction and troponin I release. In hemophilia A mice, intravenously administered CM reduced tail-cut-initiated bleeding. These data provide proof of concept for CM’s in vivo procoagulant properties. In vitro studies clarified some mechanisms for CM’s procoagulant properties. Thrombin generation assays showed that CM, like skeletal muscle myosin, enhanced thrombin generation in human platelet-rich and platelet-poor plasmas and also in mixtures of purified factors Xa, Va, and prothrombin. Binding studies showed that CM, like skeletal muscle myosin, directly binds factor Xa, supporting the concept that the CM surface is a site for prothrombinase assembly. In tPA (tissue-type plasminogen activator)-induced plasma clot lysis assays, CM was antifibrinolytic due to robust CM-dependent thrombin generation that enhanced activation of TAFI (thrombin activatable fibrinolysis inhibitor). Conclusions: CM in vitro is procoagulant and prothrombotic. CM in vivo can augment myocardial damage and can be prohemostatic in the presence of bleeding. CM’s procoagulant and antifibrinolytic activities likely involve, at least in part, its ability to bind factor Xa and enhance thrombin generation. Future work is needed to clarify CM’s pathophysiology and its mechanistic influences on hemostasis or thrombosis.

Novel blood coagulation molecules: Skeletal muscle myosin and cardiac myosin

“…Perfusion of fresh human blood over CM-coated surfaces caused extensive thrombus formation and fibrin deposition. 16 Addition of CM to blood enhanced fibrin formation 16,25 and enhanced thrombin generation in whole blood, in platelet-rich and platelet-poor plasmas, and in mixtures of purified factors Xa and Va plus prothrombin. 16 The procoagulant potency of CM, based on k cat values for prothrombin activation by FXa:FVa, is generally similar to that of SkM 16 (Table 1).…”

“…16 Addition of CM to blood enhanced fibrin formation 16,25 and enhanced thrombin generation in whole blood, in platelet-rich and platelet-poor plasmas, and in mixtures of purified factors Xa and Va plus prothrombin. 16 The procoagulant potency of CM, based on k cat values for prothrombin activation by FXa:FVa, is generally similar to that of SkM 16 (Table 1). Moreover, direct binding studies with purified proteins showed that CM binds FXa, supporting the model that CM, like SkM, binds FXa:FVa to F I G U R E 1 Conventional myosin structure and nomenclature.…”

“…After dialysis, particles causing turbidity were removed by high-speed centrifugation (21 130g for 1 minute), which removed visible aggregates activity for SkM's enhancement of prothrombin activation ( Figure 3D), and DG-FXa still binds to SkM. 14,16 Gilbert reported that DG-FXa had only 1% of FXa activity. 30 The assertion that contaminating PS-vesicles explain SkM's enhancement of prothrombin activation is contradicted by the facts ( Figure 3D and Table 2).…”

“…When wt-C57BL/6J mice (n = 6/group) subjected to myocardial ischemia reperfusion injury were given CM (5.4 mg/kg) or vehicle via intraarterial infusion at 15 minutes after initiation of reperfusion, serum cardiac troponin I levels measured at 3 hours after injury initiation were increased. 16 100% was defined as the median of control vehicle group values. patterns; and, currently, mechanisms for greater or lesser bleeding remain unclear.…”

“…was procoagulant similar to SkM by similar mechanisms and that CM could modify myocardial damage in murine myocardial ischemia reperfusion studies. 16 This review summarizes current knowledge regarding the procoagulant and anticoagulant properties of SkM and CM and also…”