Factor Va Cofactor Activity Is Dependent on the Interaction of Prothrombin with the Membrane Surface.

Activation of human prothrombin (Pro) occurs because of two sequential cleavages in prothrombin by membrane-bound factor Xa (fXa) (Arg²⁷¹ followed by Arg³²⁰, prethrombin 2 pathway). However, the catalytic efficiency of membrane-bound fXa is poor in the absence of factor Va (fVa), and the overall reaction is incompatible with efficient thrombin formation required for the timely arrest of bleeding. Binding of fVa to fXa on a membrane surface in the presence of divalent metal ions results in the formation of prothrombinase. This complex catalyzes the activation of Pro following the opposite pathway (cleavage at Arg³²⁰ followed by Arg²⁷¹, meizothrombin pathway). This pathway results in a dramatic increase in the catalytic efficiency of fXa. It is well established that initial cleavage of prothrombin at Arg³²⁰ is strictly dependent on the incorporation of fVa into prothrombinase and that the increase in the overall efficiency of the enzymatic reaction is solely credited to the interaction of the cofactor molecule with discrete amino acid residues from both membrane-bound enzyme and membrane-bound substrate. Thus, the activity of fXa within prothrombinase is controlled by the presence of the soluble, non-enzymatic cofactor, fVa. We have shown that a pentapeptide with the sequence DYDYQ specifically inhibits this pathway. It has been also established that Hir^54–65(SO₃⁻) is a specific inhibitor of prothrombinase. To understand the role of fVa within prothrombinase at the molecular level, we have studied thrombin formation by prothrombinase in the presence of various Pro-derived fragments alone or in combination. Activation of prethrombin 1 is slow with cleavages at Arg³²⁰ and Arg²⁷¹ occurring with similar rates. Addition of purified fragment 1 to prethrombin 1 accelerates both the rate of cleavage at Arg³²⁰ and thrombin formation. Both reactions were inhibited by Hir^54–65(SO₃⁻) while DYDYQ had no significant inhibitory effect on prethrombin 1 cleavage in the absence or presence of fragment

1. Similarly, activation of prethrombin 2 by prothrombinase, which is notably slow and inhibited by Hir^54–65(SO₃⁻), is not affected by DYDYQ. Addition of purified fragment 1·2 to prethrombin 2 accelerates the rate of cleavage at Arg³²⁰ by prothrombinase resulting in a rate of thrombin formation comparable to the rate of Pro activation by prothrombinase. This addition results in a significant inhibition of thrombin formation by DYDYQ and is concurrent with the elimination of the inhibitory effect of Hir^54–65(SO₃⁻) on the same reaction. Finally, a membrane-bound ternary complex composed of prethrombin 2/fragment 1·2/Hir^54–65(SO₃⁻) is inhibited by DYDYQ. These data demonstrate that DYDYQ and Hir^54–65(SO₃⁻) inhibit prothrombinase activity through different mechanisms because the peptides most likely interact with distinct portions of prethrombin

2. Altogether, the data demonstrate that membrane-bound fragment 1 is required to promote optimum fVa cofactor activity which in turn is translated by efficient initial cleavage of Pro by prothrombinase at Arg³²⁰.

Therefore, our findings put in the context of the literature suggest that

1. fVa incorporation into prothrombinase equalizes the rate of both Pro activating cleavages, and

2. it is the interaction of membrane-bound fragment 1 with fVa and/or fXa within prothrombinase that promotes a further accelerating effect of the rate of cleavage at Arg³²⁰, resulting in meizothrombin generation.