Substitution of arginine for Leu⁴⁴⁴ in the reactive site of heparin cofactor II enhances the rate of thrombin inhibition

Heparin cofactor II (HCII), a member of the 'serpin' family of serine protease inhibitors, is a 65,600-Da plasma glycoprotein that inhibits thrombin and chymotrypsin. The rate of thrombin inhibition is stimulated ~1000-fold by heparin or dermatan sulfate. Thrombin and chymotrypsin cleave the Leu⁴⁴⁴-Ser⁴⁴⁵ bond (designated P₁-P₁') in the reactive site of HCII, forming a stable equimolar complex in which the protease is inactive. In this study, we have determined the effects of substituting an arginine for Leu⁴⁴⁴ in recombinant HCII (rHCII). The rHCII was expressed in Escherichia coli and partially purified by heparin-Sepharose chromatography. Apparent second-order rate constants (k₂) for inhibition of thrombin, coagulation factor Xa, kallikrein, plasmin, and chymotrypsin by rHCII were determined using appropriate chromogenic substrates. In the absence of a glycosaminoglycan, rHCII(Leu⁴⁴⁴→Arg) inhibited thrombin at a 98-fold higher rate (k₂ = 6.2 x 10⁶ M^-1 min^-1) than native rHCII (k₂ = 6.3 x 10⁴ M^-1 min^-1). Dermatan sulfate accelerated thrombin inhibition by both forms of rHCII, but the maximum rate constant in the presence of dermatan sulfate was only 2-fold higher for rHCII(Leu⁴⁴⁴→Arg) (k₂ = 5.3 x 10⁸ M^-1 min^-1) than for native rHCII (k₂ = 2.2 x 10⁸ M^-1 min^-1). Heparin was less effective than dermatan sulfate in stimulating both forms of rHCII. Factor Xa, kallikrein, and plasmin were inhibited more rapidly and chymotrypsin more slowly by rHCII(Leu⁴⁴⁴→Arg) than by native rHCII. These effects are qualitatively similar to those observed with the natural mutant α₁-antitrypsin Pittsburgh (Met³⁵⁸→Arg at the P₁ position) and strengthen the hypothesis that the P₁ residue is a major determinant of protease specificity in the serpins. Furthermore, the rapid rate of inhibition of thrombin by rHCII(Leu⁴⁴⁴→Arg) in the absence of heparin or dermatan sulfate suggests that this variant may be useful as a therapeutic agent.