BackgroundThe generation of thrombin is a critical process in the formation of venous thrombi. In isolated plasma under static conditions, phosphatidylserine (PS)-exposing platelets support coagulation factor activation and thrombin generation; however, their role in supporting coagulation factor binding under shear conditions remains unclear. We sought to determine where activated factor X (FXa), (pro)thrombin, and fibrin(ogen) are localized in thrombi formed under venous shear.Methodology/Principal FindingsFluorescence microscopy was used to study the accumulation of platelets, FXa, (pro)thrombin, and fibrin(ogen) in thrombi formed in vitro and in vivo. Co-perfusion of human blood with tissue factor resulted in formation of visible fibrin at low, but not at high shear rate. At low shear, platelets demonstrated increased Ca2+ signaling and PS exposure, and supported binding of FXa and prothrombin. However, once cleaved, (pro)thrombin was observed on fibrin fibers, covering the whole thrombus. In vivo, wild-type mice were injected with fluorescently labeled coagulation factors and venous thrombus formation was monitored in mesenteric veins treated with FeCl3. Thrombi formed in vivo consisted of platelet aggregates, focal spots of platelets binding FXa, and large areas binding (pro)thrombin and fibrin(ogen).Conclusions/SignificanceFXa bound in a punctate manner to thrombi under shear, while thrombin and fibrin(ogen) distributed ubiquitously over platelet-fibrin thrombi. During thrombus formation under venous shear, thrombin may relocate from focal sites of formation (on FXa-binding platelets) to dispersed sites of action (on fibrin fibers).

The anticoagulant and anti-inflammatory enzyme, activated protein C (APC), naturally controls thrombosis without affecting hemostasis. We therefore evaluated whether the integrity of primary hemostasis was preserved during limited pharmacological antithrombotic protein C activator (PCA) treatment in baboons. The double-mutant thrombin (Trp215Ala/ Glu217Ala) with less than 1% procoagulant activity was used as a relatively selective PCA and compared with systemic anticoagulation by APC and low-molecular-weight heparin (LMWH) at doses that inhibited fibrin deposition on thrombogenic segments of arteriovenous shunts. As expected, both systemic anticoagulants, APC (0.028 or 0.222 mg/kg for 70 minutes) and LMWH (0.325 to 2.6 mg/kg for 70 minutes), were antithrombotic and prolonged the template bleeding time. In contrast, PCA at doses (0.0021 to 0.0083 mg/kg for 70 minutes) that had antithrombotic effects comparable with LMWH did not demonstrably impair primary hemostasis. PCA bound to platelets and leukocytes, and accumulated in thrombi. APC infusion at higher circulating APC levels was less antithrombotic than PCA infusion at lower circulating APC levels. The observed dissociation of antithrombotic and antihemostatic effects during PCA infusion thus appeared to emulate the physiological regulation of intravascular blood coagulation (thrombosis) by the endogenous protein C system. Our data suggest that limited pharmacological protein C activation might exhibit considerable thrombosis specificity. IntroductionSystemic anticoagulants can completely interrupt thrombus formation, but their usefulness is limited because their antithrombotic and antihemostatic activities are mechanistically tied. Highly effective plasma concentrations of systemic anticoagulants, such as those used for temporary thrombo-prophylaxis in interventional cardiology, prevent thrombin generation in both the blood vessel and the wound and can paralyze hemostasis with potentially fatal consequences. Due to safety considerations, the vast majority of patients who receive antithrombotic treatment are not anticoagulated to full efficacy. Thrombotic blood vessel occlusions causing myocardial infarction and ischemic stroke thus continue to contribute to mortality statistics. 1 Until more thrombosis-specific intravascular anticoagulants become available, balancing the potential benefits and risks of systemic anticoagulation remains a critical hurdle for the clinician.Occlusive thrombus formation is naturally down-regulated without bleeding complications most of the time following thrombogenic stimuli, such as blood vessel injuries or infections. It thus seems possible that selective pharmacological enhancement of the natural antithrombotic systems could achieve thrombosis specificity. Enhancement of the endogenous protein C pathway in the vicinity of thrombus formation might offer this wider therapeutic window. Thrombin is the natural protein C activator enzyme that activates protein C on such anatomic surfaces as the endothelial lining of blood vessels. ...

Summary.  Background: Activated protein C (APC) regulates thrombin generation and inhibits apoptosis. Endothelial protein C receptor (EPCR)‐bound protein C is activated by thrombomodulin‐bound thrombin. APC inactivates coagulation factors (F)Va/VIIIa and generates cytoprotective signaling downstream of protease‐activated receptor‐1 (PAR‐1). Binding of APC to EPCR both modifies and induces PAR‐1 signaling, but it is unknown if protein C interacts with cells in an alternative manner. Aim: To determine whether platelets possess receptors for protein C that can generate intracellular signals. Results: Immobilized protein C or APC supported platelet adhesion, lamellipodia formation and elevation of intracellular Ca2+. Adhesion of platelets to protein C or APC was inhibited by soluble recombinant apolipoprotein E receptor 2’ (ApoER2′) and by receptor‐associated protein (RAP), an inhibitor of the low‐density lipoprotein receptor family. Under shear, surface‐bound protein C supported platelet adhesion and aggregation in a glycoprotein (GP)Ibα‐dependent manner, and adhesion of platelets to immobilized protein C was abrogated by the addition of soluble forms of ApoER2′ or RAP. APC bound to purified recombinant ApoER2′ or GPIbα. Conclusions: Our data demonstrate that activation of platelets with rapid intracellular signaling caused by binding to immobilized protein C or APC occurs via mechanisms that require ApoER2 and GPIbα and that APC directly binds to purified ectodomains of the receptors ApoER2 and GPIbα. These findings imply that protein C and APC may directly promote cell signaling in other cells by binding to ApoER2 and/or GPIbα.

Vartanian KB, Berny MA, McCarty OJ, Hanson SR, Hinds MT. Cytoskeletal structure regulates endothelial cell immunogenicity independent of fluid shear stress.