Utility of thromboelastography with platelet mapping for monitoring platelet transfusion in qualitative platelet disorders

Abstract

Background

Patients with pathogenic variants in RASGRP2 (inherited platelet disorder (IPD)-18) exhibit normal platelet counts but impaired platelet aggregation and αIIbβ3 activation. Moderate-to-severe bleeding episodes require patients to be transfused with platelets and/or pro-hemostatic agents. We recently demonstrated that 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. Thus, higher amounts of transfused platelets were required to outcompete these dysfunctional cells and to reverse bleeding.

Objective

We here studied the usefulness of thromboelastography with platelet mapping (TEG-PM) for ex vivo monitoring of the hemostatic potential in Rasgrp2−/− mice transfused with various amounts of wild-type (WT) platelets.

Methods

Whole blood (WB) samples from WT and Rasgrp2−/− mice were tested in TEG-PM and parameters for clot formation and contraction (K time, α-angle, maximum amplitude [MA]) were measured.

Results

Rasgrp2−/− WB samples did not contract in TEG-PM, consistent with a critical role of this protein in αIIbβ3 activation. Addition of WT platelets improved TEG parameters 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. K and α values were identified as better predictors of transfusion efficacy than MA, the most platelet-dependent TEG parameter.

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.

Introduction

Platelet transfusion therapy is instrumental in preventing major hemorrhage in patients with hypoproliferative thrombocytopenia [1]. One transfusion unit can increase the circulating platelet count by more than 30 000/μL, easily maintaining patients above a critical threshold of 10 000/μL [2]. In these patients, platelet recovery in circulation after transfusion is often monitored by methods that approximate the increase in platelet count, such as corrective count increment and percent platelet recovery [3]. However, these methods have limited accuracy in patients with higher pretransfusion platelet counts, such as patients with platelet function disorders, and they do not determine the origin of the platelets measured (ie, endogenous vs transfused).
Platelet function disorders include acquired disorders such as from antiplatelet therapy or inherited disorders such as Glanzmann thrombasthenia (GT) or inherited platelet disorder (IPD)-18. IPD-18 is caused by pathogenic variants of RASGRP2, the gene encoding for calcium- and diacylglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI), a protein that is highly expressed in platelets and plays a critical role in inside-out activation of integrin αIIbβ3 [4]. IPD-18 patients can suffer from severe spontaneous and surgical bleeding requiring prohemostatic therapies such as recombinant activated factor (F)VII, antifibrinolytics, and platelet transfusion [5,6]. Interestingly, several groups have reported an inability of platelet transfusions to control bleeding [[6], [7], [8]], potentially due to the low transfused to endogenous platelet ratio, as has been suggested in GT patients [9,10]. We recently performed mechanistic studies in Rasgrp2−/− mice, which demonstrated that endogenous, dysfunctional platelets in these mice impair the hemostatic activity of transfused, healthy platelets by 2 mechanisms: 1) competition for primary adhesion sites and 2) impaired clot contraction. As a result, a 2:1 ratio of dysfunctional to healthy platelets was required for hemostatic success [11]. In that study, platelet ratios were monitored by flow cytometry, a technique that can readily identify platelets lacking surface glycoproteins (GPs) but requires more extensive protocols for disorders of intracellular proteins such as CalDAG-GEFI [12]. We here investigated whether thromboelastography (TEG) with platelet mapping (TEG-PM), a method that evaluates platelet function in the context of clot contraction, can be used to monitor platelet ratios and hemostatic potential in Rasgrp2−/− mice following platelet transfusion.