Heparin cofactor II. Purification and properties of a heparin-dependent inhibitor of thrombin in human plasma

We have isolated a previously unrecognized heparin-dependent inhibitor of thrombin from human plasma. The inhibitor, designated heparin cofactor II (HCII), was purified to homogeneity with sulfated-dextran, DEAE-Sepharose, heparin-Sepharose and Sephadex G-150. HCII is a glycoprotein consisting of a single polypeptide chain with a M(r)=65,600 as determined by sedimentation equilibrium analysis. Other physical properties include: s₂₀(w)=4.31 S; Stokes radius=34 Å; E₂₈₀(1%)=11.7; and pI=4.95 to 5.15. The purified inhibitor is not precipitated by antibodies directed against seven other plasma protease inhibitors, including antithrombin III (ATIII). HCII blocks the proteolytic and amidolytic activities of thrombin by forming a covalent, 1:1 molar complex with the protease. The second-order rate constant for inhibition of thrombin by purified HCII increases from 5.0 x 10⁵ M^-1 min^-1 in the absence of heparin to 4.5 x 10⁸ M^-1 min^-1 at optimal heparin concentrations of 0.8 x 1.0 unit/ml. In comparison with ATIII, HCII is a relatively ineffective inhibitor of coagulation factor X(a).