Factor Xa (FXa) hydrolyzes two peptide bonds in prothrombin having (Glu/Asp)-Gly-Arg-(Thr/Ile) for P 3 -P 2 -P 1 -P 1 residues, but the exact preferences of its catalytic groove remain largely unknown. To investigate the specificity of FXa, we synthesized full sets of fluorescence-quenched substrates carrying all natural amino acids (except Cys) in P 3 , P 2 , P 1 , P 2 , and P 3 and determined the k cat /K m values of cleavage. Contrary to expectation, glycine was not the "best" P 2 residue; peptide with phenylalanine was cleaved slightly faster. In fact, FXa had surprisingly limited preferences, barely more pronounced than trypsin; in P 2 , the ratio of the k cat /K m values for the most favorable side chain over the least was 289 (12 with trypsin), but in P 1 , this ratio was only 30 (versus 80 with trypsin). This unexpected selectivity undoubtedly distinguished FXa from thrombin, which exhibited ratios higher than 19,000 in P 2 and P 1 . Thus, with respect to the catalytic groove, FXa resembles a low efficiency trypsin rather than the highly selective thrombin. The rates of cleavage of the peptidyl substrates were virtually identical whether or not FXa was in complex with factor Va, suggesting that the cofactor did not exert a direct allosteric control on the catalytic groove. We conclude that the remarkable efficacy of FXa within prothrombinase originates from exosite interaction(s) with factor Va and/or prothrombin rather than from the selectivity of its catalytic groove.At the confluence of the formerly named intrinsic and extrinsic pathways, factor Xa (FXa) 1 is the midway protease of the blood clotting waterfall (1). FXa belongs to clan SA of the S1 family of serine peptidases along with thrombin and trypsin (2-5). Without cofactors, activation of prothrombin by FXa is slow; it becomes efficient only when FXa complexes factor Va to form prothrombinase (6). Rapid inhibition of FXa by antithrombin also requires heparin as cofactor (7). However, tissue factor pathway inhibitor (TFPI) does not require any cofactor to rapidly neutralize FXa (8). FXa catalyzes a number of other reactions: activation of factor VII in a positive feedback within the tissue factor pathway (9), activation of factors V (10) and VIII (11), cleavage of protease-activated receptor 2 (12), and neutralization of protein S, albeit only in the presence of phospholipid and calcium (13). Thrombin (14) requires a cofactor for activation of protein C and factor XI, as well as for its inhibition by antithrombin and heparin cofactor II. In contrast to FXa, however, thrombin alone rapidly catalyzes a number of critical reactions in the cascade: cleavage of fibrinogen, activation of factors V and VIII, and activation of protease-activated receptor 1 (6, 10, 15). Trypsin, the archetypal endopeptidase of the digestive tract, does not require cofactors to rapidly hydrolyze (in appropriate conditions) most peptide bonds that follow an arginine or a lysine. The notable specificity of the blood coagulation peptidases result from at least four molecul...

The control of coagulation enzymes by antithrombin is vital for maintenance of normal hemostasis. Antithrombin requires the co-factor, heparin, to efficiently inhibit target proteinases. A specific pentasaccharide sequence (H5) in high affinity heparin induces a conformational change in antithrombin that is particularly important for factor Xa (fXa) inhibition. Thus, synthetic H5 accelerates the interaction between antithrombin and fXa 100-fold as compared with only 2-fold versus thrombin. We built molecular models and identified residues unique to the active site of fXa that we predicted were important for interacting with the reactive center loop of H5-activated antithrombin. To test our predictions, we generated the mutants E37A, E37Q, E39A, E39Q, Q61A, S173A, and F174A in human fXa and examined the rate of association of these mutants with antithrombin in the presence and absence of H5. fXa Q61A interacts with antithrombin alone with a nearly normal k ass ; however, we observe only a 4-fold increase in k ass in the presence of H5. The x-ray crystal structure of fXa reveals that Gln 61 forms part of the S1 and S3 pocket, suggesting that the P region of the reactive center loop of antithrombin is crucial for mediating the acceleration in the rate of inhibition of fXa by H5-activated antithrombin.The serine protease, factor Xa (fXa), 1 is a central enzyme in the coagulation cascade. The extrinsic and intrinsic pathways converge at the point of the prothrombinase complex, of which fXa is a key component along with factor Va and phospholipids (1). The serpin antithrombin (ATIII) controls a number of important coagulation enzymes including fXa and thrombin with the aid of the co-factor, heparin (physiologically represented by heparan sulfate chains) (2). In the absence of heparin, ATIII is a relatively ineffective inhibitor of fXa and thrombin (k ass [fXa] ϭ 2.6 ϫ 10. Heparin accelerates the interaction between ATIII and target proteinases by two distinct mechanisms. First, long chain heparin is able to act as a "template," to which both ATIII and the proteinase bind, bringing inhibitor and proteinase into close proximity (2-4). Second, a specific pentasaccharide sequence present in high affinity heparin (5) is able to induce a unique conformational change throughout ATIII, culminating in exposure of the reactive center loop (RCL), the region of the serpin responsible for primary interaction with the target proteinase (6 -9). The template mechanism has been shown to be important for accelerating the interaction between ATIII and both thrombin and fXa (2, 5). In contrast, the conformational change induced by heparin pentasaccharide (H5) results in a 100-fold increase in the rate of interaction between ATIII and fXa, compared with only a 2-fold increase in the rate of interaction versus thrombin (2). Thus, synthetic H5 is able to "target" ATIII to fXa, and therefore this molecule is an important potential therapeutic that has just successfully completed phase II clinical trials for treatment of deep vein thrombosis ...

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