Endothelial Protein C Receptor Is Expressed on Procoagulant Platelets and Contributes to Factor VIIa Binding and Activity
Authors: Fager Ammon M. , Hoffman Maureane 
Published: October 2019

Abstract
Recombinant factor VIIa (rFVIIa) is routinely used as an effective bypassing agent to promote hemostasis in hemophilia patients with inhibitory antibodies that compromise factor replacement. In addition, rFVIIa is extensively used off-label as a hemostatic agent in cardiovascular surgery, trauma, and intracranial hemorrhage.
Attempts to improve the treatment for these patients have included the production of rFVIIa analogs such as V158D/E296V/M298Q-FVIIa (FVIIa-DVQ). Previous studies have shown that FVIIa-DVQ exhibits enhanced in vitro procoagulant and antifibrinolytic activity, greater factor Xa (FXa) and thrombin generation on activated platelets, and improved hemostatic efficacy relative to wild-type FVIIa in a mouse model of hemophilia. Surprisingly, while FVIIa-DVQ and rFVIIa bind similarly to phospholipid vesicles, FVIIa-DVQ exhibits greater binding to platelets than rFVIIa.
It has been established that only a fraction of activated platelets are able to bind high levels of coagulation factors. These highly procoagulant platelets, often called COATed platelets, also preferentially bind rFVIIa and FVIIa-DVQ in a Gla-domain dependent manner. However, the exact mechanism of this interaction remains largely unknown.
As previous studies have shown that the endothelial cell protein C receptor (EPCR) also functions as the endothelial cell receptor for FVIIa, the purpose of the current study was to determine whether an interaction with EPCR might be responsible for the increased platelet binding and hemostatic efficacy of FVIIa-DVQ.
Following their isolation from anticoagulated whole blood, human platelets were activated with a combination of thrombin plus the collagen receptor agonist convulxin, in order to generate highly procoagulant platelets. We then examined the binding of both rFVIIa and FVIIa-DVQ by flow cytometry in the presence and absence of excess protein C (PC) to determine its ability to compete for platelet binding.
As previously reported, maximal binding of FVIIa-DVQ was significantly higher than the maximal binding of rFVIIa in the absence of PC. Interestingly, the addition of PC inhibited the binding of FVIIa-DVQ to a greater extent than the binding of rFVIIa, thereby eliminating the difference in binding seen in the absence of PC. Using both FXa and thrombin generation assays we found that this competition for platelet binding resulted in a corresponding decrease in the procoagulant activity of FVIIa-DVQ.
Similar experiments were also performed to evaluate rFVIIa and FVIIa-DVQ binding to platelets in the presence and absence of a rabbit anti-EPCR antibody. Equivalent decreases in the platelet binding and activity of both molecules were seen in the presence of this antibody, thereby confirming that these results are indeed due to interactions with EPCR.
However, while mRNA encoding EPCR has been reported in genome-wide sequencing of the human platelet transcriptome, EPCR protein expression has not previously been shown in platelets. We therefore conducted flow cytometric analyses of unactivated, thrombin activated, and thrombin plus convulxin activated platelets to evaluate their expression of EPCR. We found that EPCR is not expressed on either unactivated or thrombin-activated platelets. Conversely, the highly procoagulant platelets do express EPCR. Dual-labeling studies confirmed that those platelets which express EPCR also bind the most rFVIIa.
To confirm platelet EPCR expression we first confirmed the presence of EPCR pre-mRNA in unactivated platelets by RT-PCR. We then determined the presence of EPCR protein by immunoprecipitation from unactivated platelet lysates followed by western blotting and mass spectrometric analyses. These data unambiguously demonstrate that EPCR is present in unactivated platelets, and is specifically expressed by the highly procoagulant platelet subpopulation.
This work represents the first demonstration that human platelets are capable of expressing EPCR, and suggests that EPCR plays a role in the efficacy of rFVIIa as a therapeutic agent by contributing to platelet-FVIIa interactions. A better understanding of the mechanism by which rFVIIa binds to the activated platelet will facilitate the development of new therapeutic agents to improve the treatment and quality of life for patients requiring emergency hemostasis.

Disclosures
Hoffman: CSL-Behring: Consultancy, Research Funding; Boehringer Ingelheim: Research Funding; Novo Nordisk: Honoraria, Research Funding.