IDENTIFICATION AND CHARACTERIZATION OF VARIANTS OF TICK ANTICOAGULANTPEPTIDE WITH INCREASED INHIBITORY POTENCY TOWARD HUMAN FACTOR XA

Tick anticoagulant peptide (TAP) is a specific and potent inhibitor of factor Xa (fXa), a central enzyme in the blood clotting cascade. As s uch, TAP is a potential antithrombotic agent. Site-directed mutagenesi s studies were undertaken to determine the feasibility of increasing t he inhibitory potency of TAP toward fXa. The amino acid substitutions Tyr-1 to Trp (Y1W) and Asp-10 to Arg (D10R) increased inhibitory poten cy toward human fXa by 2.5- and 4-fold, respectively. The increased in hibitory potency reflected a decrease in the rate constant for dissoci ation of the final fXa-TAP inhibitory complex. The double mutant, Y1W/ D10R, exhibited an inhibition constant of 10 pM, a 37-fold enhancement of inhibitory potency toward human fXa. The improvement in inhibitory potency was less pronounced (12-fold) with dog fXa wherein K(i)s of 2 20 and 18 pM were observed for wild-type TAP and the double mutant, re spectively. Mutation of Tyr-1 to Glu (Y1E) generated a weaker inhibito r (K-i = 2 nM) that bound human fXa more slowly. However, no change in inhibitory potency toward human fXa was observed when Tyr-1 was repla ced by Phe. Taken together, these observations are consistent with the view that a hydrophobic amino acid at the N-terminus of TAP may be a determinant of inhibitory potency. Decreases by 3-4 orders of magnitud e in inhibitory potency were noted upon mutation of Asn-2 and Leu-4 of TAP, further implicating the N-terminal domain as an important determ inant of inhibitory potency. A peptide, TAP(1/9), corresponding to the first nine residues of TAP (with a Cys-5 do Ala replacement) was a co mpetitive inhibitor of fXa-catalyzed hydrolysis of a small chromogenic substrate (K-i = 100 mu M). This result suggests that the N-terminal domain of TAP interacts with the active site of fXa.