Abstract
Protein Z-dependent protease inhibitor (ZPI) is a recently identified member of the serpin superfamily that functions as a cofactor-dependent regulator of blood coagulation factors Xa and XIa. Here we provide evidence that, in addition to the established cofactors, protein Z, lipid, and calcium, heparin is an important cofactor of ZPI anticoagulant function. Heparin produced 20-100-fold accelerations of ZPI reactions with factor Xa and factor XIa to yield second order rate constants approaching the physiologically significant diffusion limit (k(a) = 10(6) to 10(7) M(-1) s(-1)). The dependence of heparin accelerating effects on heparin concentration was bell-shaped for ZPI reactions with both factors Xa and XIa, consistent with a template-bridging mechanism of heparin rate enhancement. Maximal accelerations of ZPI-factor Xa reactions required calcium, which augmented the heparin acceleration by relieving Gla domain inhibition as previously shown for heparin bridging of the antithrombin-factor Xa reaction. Heparin acceleration of both ZPI-protease reactions was optimal at heparin concentrations and heparin chain lengths comparable with those that produce physiologically significant rate enhancements of other serpin-protease reactions. Protein Z binding to ZPI minimally affected heparin rate enhancements, indicating that heparin binds to a distinct site on ZPI and activates ZPI in its physiologically relevant complex with protein Z. Taken together, these results suggest that whereas protein Z, lipid, and calcium cofactors promote ZPI inhibition of membrane-associated factor Xa, heparin activates ZPI to inhibit free factor Xa as well as factor XIa and therefore may play a physiologically and pharmacologically important role in ZPI anticoagulant function.
Highlights
Protein Z-dependent protease inhibitor (ZPI)2 is one of the more recently characterized serpin superfamily proteins encoded by the human genome [1,2,3]
Heparin Accelerating Effects on the ZPI-Factor Xa Reaction— Previous studies have shown that heparin accelerates the inhibition of factor Xa by the serpin, ZPI, to a modest extent (ϳ3fold) as compared with the ϳ2000-fold acceleration produced by the cofactors, protein Z, phospholipid, and calcium ions [5, 7]
To determine the potential physiologic relevance of the heparin accelerating effect, we studied its dependence on heparin concentration and heparin chain length as well as whether it was affected by calcium, protein Z, and lipid cofactors using recombinant ZPI
Summary
Proteins—Recombinant human ZPI was expressed in baculovirus-infected insects cells and purified by successive SPSepharose, Mono S, and Sephacryl S200 chromatography steps, as in previous studies [5, 6]. Full reaction progress curves were measured for ZPI-protease reactions in the absence and presence of optimal heparin concentrations at fixed 0.1– 0.2 nM protease concentrations, as a function of the ZPI concentration in the range 5–200 nM, with or without protein Z equimolar with ZPI, with or without 5 mM CaCl2, and with or without 25 M lipid. In this case, identical reaction mixtures were quenched at varying reaction times with substrate, and the time course of protease inactivation was measured from the decrease in initial rates of substrate hydrolysis. Where Vo, Ve, and Vf are the volume at the start of the gradient, the volume corresponding to the elution peak of the protein, and the volume at the end of the gradient, respectively
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