Activated Platelets Protect Factor VIIIa from Degradation by Activated Protein C and from Subunit Dissociation.

Abstract

Activated platelets support factor VIII (FVIII) activity at 100-fold lower FVIII concentration than phospholipid vesicles (PLV) but the mechanism(s) remain unknown. Therefore, we compared activity of FVIIIa vs. emicizumab using platelets and PLV. We also utilized engineered B-domain deleted human factor VIII (FVIIIBDD) with degradation-resistant primary APC cleavage sites R336Q/R562Q (FVIIIQQ) or a stabilized A2 domain, D519V/E665V (FVIIIVV), or both (FVIIIQQVV). FVIII-dependent clotting was faster with platelets than PLV. The relative platelet:PLV FVIII procoagulant activity varied from 40:1 to 2:1; the highest ratios achieved with lower FVIII concentrations. In contrast, activity of Emi was similar with platelets and PLV. Steady state kinetic experiments indicated that platelets do not convey superior Xase activity. This indicated that FVIII binding sites protect or stabilize FVIII(a). FVIIIVV and FVIIIQQVV supported equivalent clot times on platelets and PLV suggesting that platelets slow dissociation of the A2 subunit. Studies with purified proteins confirmed that FVIIIa dissociates more slowly on platelets than on PLV. Addition of APC prevented PLV-supported clotting but modestly slowed clotting with FVIIIQQ and PLV or with FVIIIBDD and platelets. This suggested that platelets protect FVIII from APC. Defined studies confirmed that platelets slowed APC degradation of FVIII(a) to about the same extent as inactivation of FVIIIQQ with PLV. Platelet microparticles slowed FVIII degradation to a degree that was intermediate between PLV and platelets. Together, these data indicate that platelets support greater activity of FVIII than PLV through stabilization against dissociation of the A2 domain and protection from degradation by APC.