Rapid and ordered cleavage of prothrombin by Hopsarin D in the absence of phospholipid membranes

Abstract

Background: Thrombin is produced by the prothrombinase complex, composed of factor (f) Xa and fVa on a phospholipid (PL) membrane surface. Snakes of the Elapidae family have venom versions of these factors that cause coagulopathy in prey. Group C venoms contain both fXa and fVa orthologues. Group D venoms only contain a fXa orthologue and hijack fV(a) of the prey. Hopsarin D (HopD) is the venom fXa of the Stephen’s Banded snake (Hoplocephalus stephensii). Objectives: We set out to address the following: Does HopD bind to human fVa with high affinity in the absence of PL? Does it process prothrombin through the meizothrombin pathway? Is the order of cleavage PL-dependent? Can HopD activate fV? Methods: We produced and characterized full-length and truncated HopD. Results: HopD is only able to clot plasma that contains fV, and competes with human fXa for fVa binding. HopD binds to both human fVa and fV with high affinity (Kd~10nM), in contrast to fXa. HopD processes prothrombin down the meizothrombin route in the absence and presence of PL. Although HopD can bind to fV, conversion to fVa is necessary for prothrombin processing. HopD initiates clotting in the blood of prey by activating fV. Conclusions: HopD binds to fVa with high affinity and rapidly activates prothrombin in the absence of PL, exclusively through the meizothrombin intermediate. HopD binds with high affinity to both fVa and fV, suggesting that the B-domain does not sterically block fXa binding, but inhibits productive interaction in another way, and additionally prevents prothrombin binding.