Nematode Anticoagulant Protein c2 Reveals a Site on Factor Xa That Is Important for Macromolecular Substrate Binding to Human Prothrombinase

Abstract

The binding of recombinant nematode anticoagulant protein c2 (NAPc2) to either factor X or Xa is a requisite step in the pathway for the potent inhibition of VIIa tissue factor. We have used NAPc2 as a tight binding probe of human Xa to investigate protein substrate recognition by the human prothrombinase complex. NAPc2 binds with high affinity (K d ϳ1 nM) to both X and Xa in a way that does not require or occlude the active site of the enzyme. In contrast, NAPc2 is a tight binding, competitive inhibitor of protein substrate cleavage by human Xa incorporated into prothrombinase with saturating concentrations of membranes and Va. By fluorescence binding studies we show that NAPc2 does not interfere with the assembly of human prothrombinase. These are properties expected of an inhibitor that blocks protein substrate recognition by targeting extended macromolecular recognition sites (exosites) on the enzyme complex. A weaker interaction (K d ‫؍‬ 260-500 nM) observed between NAPc2 and bovine X was restored to a high affinity one in a recombinant chimeric bovine X derivative containing 25 residues from the COOH terminus of the proteinase domain of human X. This region implicated in binding NAPc2 is spatially adjacent to a site previously identified as a potential exosite. Despite the weaker interaction with bovine Xa, NAPc2 was a tight binding competitive inhibitor of protein substrate cleavage by bovine prothrombinase as well. Extended enzymic surfaces elucidated with exosite-directed probes, such as NAPc2, may define a unique region of factor Xa that is modulated following its assembly into prothrombinase and in turn determines the binding specificity of the enzyme complex for its protein substrate.The proteolytic activation of prothrombin is catalyzed by the prothrombinase complex of coagulation (2-5). Prothrombinase assembles through membrane-dependent interactions between the serine proteinase, factor Xa, and the protein cofactor, factor Va (2, 3). Although solution-phase Xa is a competent enzyme, its incorporation into prothrombinase yields a profound increase in the rate of thrombin formation (2, 3).Prothrombin is the only known protein substrate cleaved efficiently by prothrombinase (2, 6). Such stringent selectivity is not evident in the action of factor Xa on oligopeptidyl substrates, nor is the rate of peptidyl substrate hydrolysis significantly enhanced upon assembly of factor Xa into prothrombinase (7,8). Thus, the narrow and defined specificity of prothrombinase toward its protein substrate is unlikely to be solely explained by the specific recognition of residues surrounding the scissile bond by the active site of factor Xa within the enzyme complex.Mechanistic studies of bovine prothrombinase function have borne out this suggestion (9 -11). A series of studies indicate that recognition of the biological substrate is achieved through stepwise interactions of the protein substrate at an extended macromolecular recognition site (exosite) in prothrombinase removed from the catalytic site of Xa, followed b...

The Conformational Switch from the Factor X Zymogen to Protease State Mediates Exosite Expression and Prothrombinase Assembly

“…rFXa S195A ) at 25°C in 1 ϫ 1-cm 2 stirred quartz cuvettes, and steady-state fluorescence intensity was measured using ex ϭ 480 and em ϭ 520 nm with a long pass filter (KV500, Schott, Duryea, PA) in the emission beam essentially as described (30).…”

“…Human FVa was prepared by proteolytic activation of FV by thrombin and purified as described before (36). Oregon Green 488 -FXa (OG 488 -FXa) was prepared as described (30). Molecular weights and extinction coefficients (E 0.1% 280 nm) of the various proteins used were taken as follows: RVV X-CP , 93,000 and 1.18 (37); prothrombin, 72,000 and 1.47 (33); prethrombin-1, 49,900 and 1.78 (33); thrombin, 37,500 and 1.94 (38); FVa, 173,000 and 1.78 (39); FX, 59,000 and 1.16 (40); and FXa, 46,000 and 1.16 (40).…”

Fate of Membrane-bound Reactants and Products during the Activation of Human Prothrombin by Prothrombinase

“…Factors Xa and Va were prepared and characterized as described before (20,24). The derivatives F12, F2, P2, mIIa, and IIa were prepared by preparative cleavage of prothrombin purified from plasma (II PL ) and subsequent repurification using established procedures (25).…”

A Bipartite Autoinhibitory Region within the B-domain Suppresses Function in Factor V

“…Reaction mixtures (2.5 ml) in a 1 ϫ 1-cm 2 quartz cuvette contained 20 or 30 nM OG 488 -FXa and 50 M PCPS in assay buffer to which increasing concentrations of FV/V(a) derivative (0.5-100 nM) were added. Fluorescence anisotropy or intensity measurements, including controls, were performed as described (29,34).…”