The protease thrombin is required for normal hemostasis and pathologic thrombogenesis. Since the mechanism of coagulation factor XI (FXI)-dependent thrombus growth remains unclear, we investigated the contribution of FXI to thrombus formation in a primate thrombosis model. Pretreatment of baboons with a novel anti-human FXI monoclonal antibody (aXIMab; 2 mg/kg) inhibited plasma FXI by at least 99% for 10 days, and suppressed thrombin-antithrombin (TAT) complex and ␤-thromboglobulin (␤TG) formation measured immediately downstream from thrombi forming within collagen-coated vascular grafts. FXI inhibition with aXIMab limited platelet and fibrin deposition in 4-mm diameter grafts without an apparent increase in D-dimer release from thrombi, and prevented the occlusion of 2-mm diameter grafts without affecting template bleeding times. In comparison, pretreatment with aspirin (32 mg/kg) prolonged bleeding times but failed to prevent graft occlusion, supporting the concept that FXI blockade may offer therapeutic advantages over other antithrombotic agents in terms of bleeding complications. In whole blood, aXIMab prevented fibrin formation in a collagencoated flow chamber, independent of factor XII and factor VII. These data suggest that endogenous FXI contributes to arterial thrombus propagation through a striking amplification of thrombin generation at the thrombus luminal surface. (Blood. 2009;113:936-944) IntroductionBlood coagulation during hemostasis is initiated by the tissue factor (TF)/factor VIIa complex (the extrinsic pathway) that activates factors IX and X, and ultimately produces thrombin at sites of vascular injury. 1 In thrombosis, intravascular blood coagulation may also be initiated by the extrinsic pathway. 2,3 However, impairment of the TF/factor VIIa pathway does not provide full protection from thrombosis, since symptomatic factor VII deficient subjects can develop concurrent thrombosis and severe bleeding. 4 The functions of the contact proteins (factor XI, factor XII, prekallikrein, and high-molecular-weight kininogen) in hemostasis are less clear. The physiologic role of factor XI (FXI) has been difficult to determine because of the variable bleeding disorder associated with FXI deficiency, 5 and because monospecific FXI inhibitors have not been widely available for experimental investigation. FXI activation is thought to proceed through thrombin-and/or factor XIIdependent mechanisms, and activated FXI (FXIa) contributes to sustained thrombin generation after initiation of blood clotting by activating factor IX. These activities ultimately promote coagulation, platelet activation, and preservation of fibrin clot integrity. 6,7 Thrombin also increases the density of fibrin networks 8 and indirectly inhibits fibrinolysis through activation of carboxypeptidase B (thrombin-activatable fibrinolysis inhibitor, TAFI). 9 Thus, FXI may support thrombus propagation and clot stability by increasing thrombin generation. 10,11 Compelling circumstantial evidence suggests a contributory role for FXI in the p...

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.

Objective-Thrombin containing the mutations Trp215Ala and Glu217Ala (WE) selectively activates protein C and has potent antithrombotic effects in primates. The aim of this study was to delineate the molecular mechanism of direct WE-platelet interactions under static and shear conditions. Methods and Results-Purified platelets under static conditions bound and spread on immobilized wild-type but not WE thrombin. In PPACK-anticoagulated blood under shear flow conditions, platelets tethered and rolled on both wild-type and WE thrombin, and these interactions were abrogated by the presence of a glycoprotein Ib (GPIb)-blocking antibody. Platelet deposition on collagen was blocked in the presence of WE, but not wild-type thrombin or prothrombin. WE also abrogated platelet tethering and rolling on immobilized von Willebrand factor in whole blood under shear flow. Conclusions-These observations demonstrate that the thrombin mutant WE, while not activating platelets, retains the ability to interact with platelets through GPIb, and inhibits

The minor gammaA/gamma' fibrinogen isoform contains a high affinity binding site for thrombin exosite II that is lacking in the major gammaA/gammaA fibrinogen isoform. We therefore investigated the biological consequences of the gamma' chain binding to thrombin. Thrombin-induced platelet aggregation was inhibited by gammaA/gamma' fibrinogen. Carboxyl terminal peptide fragment gamma'410-427 from the gamma' chain was also inhibitory, with an IC(50) of approximately 200 microM in whole plasma. Deletion of the peptide from either the amino or carboxyl end significantly decreased inhibition. In contrast to thrombin-induced platelet aggregation, aggregation induced by epinephrine, ADP, arachidonic acid, or SFLLRN peptide showed little inhibition by the gamma' peptide. The inhibition of thrombin-induced platelet aggregation was not due to direct inhibition of the thrombin active site, since cleavage of a small peptidyl substrate was 91% of normal even in the presence of 1 mM gamma'410-427. The gamma'410-427 peptide blocked platelet adhesion to immobilized thrombin under both static and flow conditions, blocked soluble thrombin binding to platelet GPIbalpha, and inhibited PAR1 cleavage by thrombin. These results suggest that the gamma' chain of fibrinogen inhibits thrombin-induced platelet aggregation by binding to thrombin exosite II. Thrombin that is bound to the gamma' chain is thereby prevented from activating platelets, while retaining its amidolytic activity.

Subendothelial collagen plays an important role, via both direct and indirect mechanisms, in the initiation of thrombus formation at sites of vascular injury. Collagen binds plasma von Willebrand factor, which mediates platelet recruitment to collagen under high shear. Subsequently, the direct binding of the platelet receptors glycoprotein VI and α2β1 to collagen is critical for platelet activation and stable adhesion. Leeches, have evolved a number of inhibitors directed towards platelet–collagen interactions so as to prevent hemostasis in the host during hematophagy. In this article, we describe the molecular mechanisms underlying the ability of the leech product saratin to inhibit platelet binding to collagen. In the presence of inhibitors of ADP and thromboxane A2, both saratin and 6F1, a blocking α2β1 mAb, abrogated platelet adhesion to fibrillar and soluble collagen. Additionally, saratin eliminated α2β1‐dependent platelet adhesion to soluble collagen in the presence of an Src kinase inhibitor. Moreover, saratin prevented platelet‐rich plasma adhesion to fibrillar collagen, a process dependent upon both α2β1 and von Willebrand factor binding to collagen. Furthermore, saratin specifically inhibited the binding of the α2 integrin subunit I domain to collagen, and prevented platelet adhesion to collagen under flow to the same extent as observed in the presence of a combination of mAbs to glycoprotein Ib and α2β1. These results demonstrate that saratin interferes with integrin α2β1 binding to collagen in addition to inhibiting von Willebrand factor–collagen binding, presumably by binding to an overlapping epitope on collagen. This has significant implications for the use of saratin as a tool to inhibit platelet–collagen interactions.