The Dual Regulatory Role of Amino Acids Leu480 and Gln481 of Prothrombin

Abstract

Prothrombin (FII) is activated to α-thrombin (IIa) by prothrombinase. Prothrombinase is composed of a catalytic subunit, factor Xa (fXa), and a regulatory subunit, factor Va (fVa), assembled on a membrane surface in the presence of divalent metal ions. We constructed, expressed, and purified several mutated recombinant FII (rFII) molecules within the previously determined fVa-dependent binding site for fXa (amino acid region 473-487 of FII). rFII molecules bearing overlapping deletions within this significant region first established the minimal stretch of amino acids required for the fVa-dependent recognition exosite for fXa in prothrombinase within the amino acid sequence Ser(478)-Val(479)-Leu(480)-Gln(481)-Val(482). Single, double, and triple point mutations within this stretch of rFII allowed for the identification of Leu(480) and Gln(481) as the two essential amino acids responsible for the enhanced activation of FII by prothrombinase. Unanticipated results demonstrated that although recombinant wild type α-thrombin and rIIa(S478A) were able to induce clotting and activate factor V and factor VIII with rates similar to the plasma-derived molecule, rIIa(SLQ→AAA) with mutations S478A/L480A/Q481A was deficient in clotting activity and unable to efficiently activate the pro-cofactors. This molecule was also impaired in protein C activation. Similar results were obtained with rIIa(ΔSLQ) (where rIIa(ΔSLQ) is recombinant human α-thrombin with amino acids Ser(478)/Leu(480)/Gln(481) deleted). These data provide new evidence demonstrating that amino acid sequence Leu(480)-Gln(481): 1) is crucial for proper recognition of the fVa-dependent site(s) for fXa within prothrombinase on FII, required for efficient initial cleavage of FII at Arg(320); and 2) is compulsory for appropriate tethering of fV, fVIII, and protein C required for their timely activation by IIa.

Keywords: activation; blood; coagulation factor; factor Xa; factor v; mutation; prothrombin; prothrombinase; thrombin; thrombosis.