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

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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 r HCII 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 × 106 M-1 min-1) than native rHCII (k2 = 6.3 × 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 × 108 M-1 min-1) than for native rHCII (k2 = 2.2 × 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 Pi 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.

Original languageEnglish
Pages (from-to)5623-5628
Number of pages6
JournalJournal of Biological Chemistry
Issue number10
StatePublished - 1990


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