Platelets enhance factor VIII activity through protection from activated Protein C Free

Introduction

Phospholipid membranes provide a catalytic surface for factor VIII (FVIII) activity in the blood clotting cascade. Thrombin-stimulated platelets also provide specific FVIII binding sites, predominantly through soluble fibrin ligated to the αIIbβ3 integrin (Phillips et al 2004 J Thromb Haemost; Gilbert et al 2015 Blood) rather than phosphatidylserine (PS)-rich membrane. In this study, we seek to determine the effect of platelet FVIII binding sites on the inactivation of FVIII by the anticoagulant activated protein C (APC) with or without the cofactor protein S (ProtS).

Methods

We utilized wild type FVIII or FVIII engineered to resist APC cleavage. Site-directed mutagenesis of recombinant B-domain-deleted human FVIII (FVIII-WT) replaced the APC cleavage sites R336 and R562 with Q (FVIII-QQ).

Plasma clotting assays were performed using delipidated FVIII-deficient plasma treated with corn trypsin inhibitor against contact pathway activation. FVIII was added to the plasma with either PAR1/PAR4 activated platelets (apheresis platelets in 5% DMSO stored at -80C and then purified via density gradient) vs. 10 μM 20% PS lipid vesicles (PLV). Previous experiments have shown that cryo-platelets have equivalent activity in the plasma clotting assay as fresh platelets. For some experiments, microparticles (MPs) were purified from platelet-poor plasma via ultracentrifugation and used as a lipid source. Clotting was initiated with 5 mM Ca⁺⁺ and either 1 pM factor XIa (intrinsic pathway) or 0.1 pM factor Xa (extrinsic pathway) and the time to clot was measured with turbidity.

A two-stage Xase activity assay was used to directly test inactivation of FVIII by APC. Thrombin-activated FVIII was preincubated with APC ± ProtS in the presence of either PLV, activated platelets, or MPs followed by mixing with fIXa, fX, and Ca⁺⁺. FXa was measured with a chromogenic substrate.

Results

FVIII-QQ had 10% and 20% shorter PLV-supported clot times compared to FVIII-WT when clotting was initiated through the intrinsic and extrinsic pathway, respectively. This suggests that a small quantity of APC or APC-like activity is generated in the clotting assay. However, platelet-supported clotting times of FVIII-QQ and FVIII-WT were equivalent, suggesting that FVIII-WT was protected from the anticoagulant activity when supported by platelets.The addition of 20 nM exogenous APC to the clotting assay eliminated measurable FVIII-WT activity on PLV whereas the clot times of FVIII-QQ increased by 70%. In stark contrast, when clotting was supported by platelets, APC did not lead to prolonged FVIII-WT or QQ clotting times. When clotting was triggered through the extrinsic pathway, APC increased platelet-supported clotting times modestly for FVIII-WT and FVIII-QQ (23-35%). The results suggest that binding of factor VIII to platelets attenuates FVIII cleavage by APC, particularly when clotting is driven by the intrinsic pathway.