Identification of Receptors and Signaling Pathways Mediating the Platelet Contribution to Clotting Parameters in Thromboelastography (TEG)

Viscoelastic testing is used for real-time determination of hemostatic status in patients with acute risk of bleeding. Clot formation and contraction in thromboelastography (TEG) are assumed to be driven by thrombin through its two main actions, the conversion of fibrinogen to fibrin and activation of platelets through protease activated receptors (PARs). Platelet stimulation leads to inside-out activation of αIIbβ3 integrin, a process regulated by the small GTPase Rap1, and fibrin binding to activated αIIbβ3 triggers outside-in signaling and platelet contraction. However, studies have shown no impact of PAR inhibitors on viscoelastic clotting parameters, and the impact of defects in Rap1 signaling on TEG have not been determined. Furthermore, the role of novel ligand-receptor interactions such as fibrin-GPVI on TEG parameters have not been studied. Here we investigated the specific receptors and signaling pathways required for platelet function in standard (kaolin-triggered) TEG using genetic or pharmacological inhibition of platelet proteins in murine and human blood samples. We confirmed the requirement of platelets and αIIbβ3 integrin for mouse whole blood clot formation/contraction in TEG. Loss of the integrin adaptor Talin1 in mouse platelets impaired clotting parameters, but to a lesser extent than αIIbβ3 blockade. We observed no changes in clotting parameters using blood from mice lacking Rap1b, or with combined deficiency of CalDAG-GEFI and P2Y12, the two main pathways mediating Rap1 activation. Deficiency of PAR4, which fully mediates the platelet response to thrombin in mice, only partially reduced the speed of clot formation (α-angle) with no impact on maximal clot strength (maximal amplitude, MA). However, TEG parameters similar to samples lacking platelets were observed for blood from PAR4-deficient mice treated with inhibitors of the GPVI receptor or a key downstream signaling protein Syk. These results were reproduced in human blood samples, where inhibition of both PAR1 and PAR4 combined with Syk inhibition robustly reduced α-angle and MA. In summary, our results suggest that global hemostasis assays such as TEG, which are driven by high concentrations of thrombin, are not sensitive to platelet signaling pathways critical for integrin inside-out activation. Furthermore, we provide evidence that platelet-mediated clot contraction in TEG is dependent on both the thrombin-PAR interaction and GPVI, likely interacting with fibrin. Our findings suggest that current interpretation of platelet function in TEG may not reflect platelet function in vivo, and that novel interactions need to be integrated into clinical assays.