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

Abstract

Prothrombin (FII) is activated to ␣-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 ( In the presence of a procoagulant membrane surface and divalent metal ions, factor Va (fVa) 3 binds factor Xa (fXa) to form prothrombinase. Prothrombinase is the two-subunit enzymatic complex where the non-enzymatic regulatory subunit (fVa) controls the rate and directs cleavage of prothrombin (FII) by the catalytic subunit (fXa) at two spatially distinct sites resulting in timely ␣-thrombin (IIa) formation at the place of vascular injury (1-3). Cleavage at Arg 271 and Arg 320 of FII is required to form the active serine protease IIa. The essential IIa molecule bears strong homology with other serine protease enzymes, such as activated protein C (APC), chymotrypsin, and fXa. Several different numberings of IIa residues appear in the literature based on either the chymotrypsin numbering (4) or IIa numbering (5, 6) or the entire FII sequence (7). The latter nomenclature is used herein with the appropriate chymotrypsin numbering in parentheses when required for comparison with the existing data in the literature.Historically, it has been shown that in the absence of fVa, initial cleavage at Arg 271 of FII results in the generation of the inactive intermediate prethrombin-2 and fragment 1⅐2. Further cleavage of prethrombin-2 at Arg 320 generates IIa (prethrombin-2 pathway) (8 -21). Concurrent with the appearance of excess fVa during clotting and in the presence of a procoagulant surface, the order of cleavages is reversed, and initial cleavage at Arg 320 generates a transient enzymatically active intermediate, meizothrombin, that has much higher catalytic efficiency than IIa toward chromogenic substrates usually employed to assess IIa activity (18,(22)(23)(24). Meizothrombin is next cleaved at Arg 271 resulting in the generation of IIa and fragment 1⅐2 (meizothrombin pathway). Although efficient cleavage at each site requires the presence of phospholipids, initial cleavage at Arg 320 is entirely fVa-dependent. In the absence of fVa, the two activation cleavage sites are not readily available, and FII is activated at a slow non-physiological rate by membrane-bound fXa alone. Interactions between fXa and FII are known to exist in the presence and absence of fVa; however, the enhanced activity of fXa within prothrombinase toward both activating cleavage sites is controlled solely by the membrane-bound non-enzymatic cofactor (3,16,17,25,26 3 The abbreviations used are: fVa, factor Va; fV, factor V; FII, prothrombin; fXa, factor Xa; IIa, ␣-thrombin; r, recombinant; PC, L-␣-phosphatidylcholine; PCPS, small unilamellar phospholipids vesicles composed of 75% PC and 25% L-␣-phosphatidylserine (w/w); rFII WT , recombinant...