Platelet interaction with collagens, via von Willebrand factor, is a potent trigger of shear-dependent thrombus formation mediated by subsequent engagement of the signaling collagen receptor glycoprotein (GP)VI, enforced by integrin α₂β₁. Protein tyrosine kinase Syk is central in the GPVI-induced signaling pathway, leading to elevated cytosolic Ca²⁺. We aimed to determine the Syk-mediated thrombogenic activity of several collagen peptides and (fibrillar) type I and III collagens. High-shear perfusion of blood over microspots of these substances resulted in thrombus formation, which was assessed by eight parameters and was indicative of platelet adhesion, activation, aggregation, and contraction, which were affected by the Syk inhibitor PRT-060318. In platelet suspensions, only collagen peptides containing the consensus GPVI-activating sequence (GPO)_n and Horm-type collagen evoked Syk-dependent Ca²⁺ rises. In whole blood under flow, Syk inhibition suppressed platelet activation and aggregation parameters for the collagen peptides with or without a (GPO)_n sequence and for all of the collagens. Prediction models based on a regression analysis indicated a mixed role of GPVI in thrombus formation on fibrillar collagens, which was abolished by Syk inhibition. Together, these findings indicate that GPVI-dependent signaling through Syk supports platelet activation in thrombus formation on collagen-like structures regardless of the presence of a (GPO)_n sequence.

Platelet and coagulation activation are highly reciprocal processes driven by multi-molecular interactions. Activated platelets secrete several coagulation factors and expose phosphatidylserine, which supports the activation of coagulation factor proteins. On the other hand, the coagulation cascade generates known ligands for platelet receptors, such as thrombin and fibrin. Coagulation factor (F)Xa, (F)XIIIa and activated protein C (APC) can also bind to platelets, but the functional consequences are unclear. Here, we investigated the effects of the activated (anti)coagulation factors on platelets, other than thrombin. Multicolor flow cytometry and aggregation experiments revealed that the ‘supernatant of (hirudin-treated) coagulated plasma’ (SCP) enhanced CRP-XL-induced platelet responses, i.e., integrin α_IIbβ₃ activation, P-selectin exposure and aggregate formation. We demonstrated that FXIIIa in combination with APC enhanced platelet activation in solution, and separately immobilized FXIIIa and APC resulted in platelet spreading. Platelet activation by FXIIIa was inhibited by molecular blockade of glycoprotein VI (GPVI) or Syk kinase. In contrast, platelet spreading on immobilized APC was inhibited by PAR1 blockade. Immobilized, but not soluble, FXIIIa and APC also enhanced in vitro adhesion and aggregation under flow. In conclusion, in coagulation, factors other than thrombin or fibrin can induce platelet activation via GPVI and PAR receptors.

• Multiple mouse genes regulating bleeding and platelet traits contribute to human hemostasis. • Combined assessment of orthologous human and mouse platelet transcriptomes and proteomes reveals their roles in hemostasis.

Pharmacologic modifiers of fibrinolysis are used to modulate bleeding and thrombosis. Characterization of a sensitive plasmin generation (PG) assay to identify abnormal fibrinolytic states and monitor antifibrinolytic medication may improve clinical care. To establish reference values of PG parameters, investigate sex-related differences, and study different regulators of fibrinolysis, using a PG assay. PG was assessed in plasma of 130 healthy individuals, normal pooled plasma, or factor-deficient plasma, using tissue factor or Russell viper venom-factor (F)X activator, with tissue plasminogen activator or the novel antifibrin urokinase plasminogen activator. Reference values for PG parameters were established in plasma from healthy individuals. No PG was observed in plasminogen- or fibrinogen-deficient plasma. Thrombomodulin (TM), α₂-antiplasmin, and tranexamic acid reduced PG. Inhibitory effects of TM in PG were counteracted using carboxypeptidase inhibitor, an inhibitor specific to active thrombin-activatable fibrinolysis inhibitor, confirming a role for thrombin-activatable fibrinolysis inhibitor and TM in modulating PG. Although the PG assay triggered with tissue plasminogen activator was insensitive to plasminogen activator inhibitor-1, it became sensitive to it when induced with antifibrin urokinase plasminogen activator. PG measurements in plasma of healthy individuals showed elevated PG levels in 11 of 13 female healthy donors taking oral contraceptives and an increased resistance to TM in females compared with that in males. The PG assay is a specific and efficient tool to assess modulators of fibrinolysis and to study sex-related differences and has potential as a clinical tool to identify abnormal fibrinolytic profiles in patients and monitor antifibrinolytic drugs, such as tranexamic acid.