RESUMEN
Patients with pathogenic variants in RASGRP2 (inherited platelet disorder (IPD)-18) have normal platelet counts but show impaired platelet aggregation due to diminished activation of αIIbß3 integrin. This defect results in moderate to severe bleeding episodes, especially following surgical procedures, which require patients to be transfused with platelets and/or pro-hemostatic agents. We recently demonstrated that the hemostatic efficacy of transfused platelets is limited by dysfunctional endogenous platelets in a mouse model of IPD-18 ( Rasgrp2 -/- mice), as dysfunctional platelets were recruited to the forming hemostatic plug but did not participate in clot contraction. Consequently, higher amounts of transfused platelets were required to outcompete these dysfunctional cells and to reverse bleeding. We here studied the usefulness of thromboelastography with platelet mapping (TEG-PM), a method to evaluate platelet-dependent clot contraction, for ex vivo monitoring of the hemostatic potential in Rasgrp2 -/- mice transfused with various amounts of wild-type (WT) platelets. Rasgrp2 -/- whole blood samples did not contract in TEG-PM, consistent with a critical role of this protein in αIIbß3 activation. Addition of WT platelets improved TEG parameters (K time, α-angle, MA) in a ratio dependent manner, consistent with our recent in vivo studies showing impaired hemostasis at a 5:1, but not at a 2:1 ratio of mutant to WT platelets. Interestingly, K and α values were identified as better predictors of transfusion efficacy than MA, the most platelet-dependent TEG parameter. In conclusion, this proof-of-concept study supports the use of TEG-PM to monitor platelet transfusion ratios and hemostatic potential in IPD-18 and potentially other platelet disorders.
RESUMEN
BACKGROUND: Thromboelastography (TEG) is used for real-time determination of hemostatic status in patients with acute risk of bleeding. Thrombin is thought to drive clotting in TEG through generation of polymerized fibrin and activation of platelets through protease-activated receptors (PARs). However, the specific role of platelet agonist receptors and signaling in TEG has not been reported. OBJECTIVES: Here, we investigated the specific receptors and signaling pathways required for platelet function in TEG using genetic and pharmacologic inhibition of platelet proteins in mouse and human blood samples. METHODS: Clotting parameters (R time, α-angle [α], and maximum amplitude [MA]), were determined in recalcified, kaolin-triggered citrated blood samples using a TEG 5000 analyzer. RESULTS: We confirmed the requirement of platelets, platelet contraction, and αIIbß3 integrin function for normal α and MA. Loss of the integrin adaptor Talin1 in megakaryocytes/platelets (Talin1mKO) also reduced α and MA, but only minimal defects were observed in samples from mice lacking Rap1 GTPase signaling. PAR4mKO samples showed impaired α but normal MA. However, impaired TEG traces similar to those in platelet-depleted samples were observed with samples from PAR4mKO mice depleted of glycoprotein VI on platelets or with addition of a Syk inhibitor. We reproduced these results in human blood with combined inhibition of PAR1, PAR4, and Syk. CONCLUSION: Our results demonstrate that standard TEG is not sensitive to platelet signaling pathways critical for integrin inside-out activation and platelet hemostatic function. Furthermore, we provide the first evidence that PARs and glycoprotein VI play redundant roles in platelet-mediated clot contraction in TEG.
