PB1108 - Ordered cleavage of prothrombin by Hopsarin D in the absence of membranes

Background: Thrombin is produced by the prothrombinase complex, composed of factor (f) Xa and fVa on a membrane surface. Snakes of the Elapidae family have venom versions of these factors that cause coagulopathy in prey. Type C snakes contain both fXa and fVa orthologues. Type D snake venom only contains a fXa orthologue and hijack fV(a) of the prey. Hopsarin D (HopD) is the venom fXa of the Stephen’s Banded snake.

Aims: We set out to address the following: Does HopD bind to human fVa with high affinity in the absence of membranes? Does it process prothrombin through the meizothrombin pathway, as does fully assembled human prothrombinase? Is the order of cleavage membrane dependent? How does it clot plasma when there is no fVa?

Methods: We produced and characterized full-length and truncated HopD (EGF2/catalytic domain).

Results: HopD is only able to clot plasma that contains fV, in contrast to human fXa. In a purified system it has limited prothrombin processing activity, unless fVa is present. HopD binds to both human fVa and fV with high affinity (Kd~10nM), in contrast to human fXa which binds to fVa with a Kd of ~1µM and has no detectable binding to fV. HopD processes prothrombin down meizothrombin route in the absence and presence of membranes. Although HopD can bind to fV, conversion to fVa is necessary for prothrombin processing. HopD is able to activate fV in plasma. HopD competes with human fXa for fVa binding.

Conclusion(s): HopD binds to fVa with high affinity and rapidly converts prothrombin to thrombin in the absence of membranes 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.