Basis for the Specificity and Activation of the Serpin Protein Z-dependent Proteinase Inhibitor (ZPI) as an Inhibitor of Membrane-associated Factor Xa

Abstract

The serpin ZPI is a protein Z (PZ)-dependent specific inhibitor of membrane-associated factor Xa (fXa) despite having an unfavorable P1 Tyr. PZ accelerates the inhibition reaction ϳ2000-fold in the presence of phospholipid and Ca 2؉ . To elucidate the role of PZ, we determined the x-ray structure of Gladomainless PZ (PZ ⌬GD ) complexed with protein Z-dependent proteinase inhibitor (ZPI). The PZ pseudocatalytic domain bound ZPI at a novel site through ionic and polar interactions. Mutation of four ZPI contact residues eliminated PZ binding and membrane-dependent PZ acceleration of fXa inhibition. Modeling of the ternary Michaelis complex implicated ZPI residues Glu-313 and Glu-383 in fXa binding. Mutagenesis established that only Glu-313 is important, contributing ϳ5-10-fold to rate acceleration of fXa and fXIa inhibition. Limited conformational change in ZPI resulted from PZ binding, which contributed only ϳ2-fold to rate enhancement. Instead, template bridging from membrane association, together with previously demonstrated interaction of the fXa and ZPI Gla domains, resulted in an additional ϳ1000-fold rate enhancement. To understand why ZPI has P1 tyrosine, we examined a P1 Arg variant. This reacted at a diffusion-limited rate with fXa, even without PZ, and predominantly as substrate, reflecting both rapid acylation and deacylation. P1 tyrosine thus ensures that reaction with fXa or most other arginine-specific proteinases is insignificant unless PZ binds and localizes ZPI and fXa on the membrane, where the combined effects of Gla-Gla interaction, template bridging, and interaction of fXa with Glu-313 overcome the unfavorability of P1 Tyr and ensure a high rate of reaction as an inhibitor.Blood coagulation is a complex process requiring tight regulation through the use of feedback loops and the involvement of proteinase-specific inhibitors (1). fXa, 4 in complex with its cofactor Va, plays a crucial role in blood coagulation, being directly involved in the generation of the final proteinase thrombin from its zymogen prothrombin. This reaction takes place on the membrane surface of activated platelets, with all three protein components interacting with the membrane surface, either through Gla domains in the case of fXa and prothrombin or the two C-type domains of factor Va. In addition, fXa is itself formed at the membrane surface through the action of factor IXa, in complex with its cofactor VIIIa, on factor X.To help prevent clot formation away from the site of injury, any fXa that dissociates from the membrane and diffuses away in the bloodstream must be efficiently inhibited. This is accomplished primarily by the serpin antithrombin, which, when allosterically activated by being bound to heparan sulfate chains on the surface of endothelial cells, is a rapid inactivator of fXa, as well as of other circulating arginine-specific proteinases, such as thrombin and factor IXa (2). The P1 residue 5 of antithrombin is arginine, in keeping with the substrate specificity of each of these proteinases. Interestingly, ...

Thermodynamic and Kinetic Characterization of the Protein Z-dependent Protease Inhibitor (ZPI)-Protein Z Interaction Reveals an Unexpected Role for ZPI Lys-239

“…The protein cofactor circulates as a tight complex with ZPI (14) with PZ levels limiting the amount of complex formed (3,15). The tight binding of PZ to ZPI is thought to enable the ZPI-PZ complex to bind to a procoagulant membrane surface where it can encounter and rapidly inhibit membrane-bound factor Xa (1,4,16). ZPI-PZ complex structures have been solved by us and another group (16,17).…”

“…Recombinant wild-type and mutant ZPIs were engineered on a wild type-like ZPI background in which cysteines 169 and 264 were changed to alanine and serine, respectively (16). In the case of wild-type and K239C ZPIs, variants with the natural P1 Tyr as well as with the P1 Tyr changed to Arg were engineered to facilitate factor Xa cleavage and preparation of an RCLcleaved form as reported previously (18).…”

“…Lys-239 was changed to cysteine in a cysteine-free ZPI variant in which the two wild-type Cys residues were changed to Ser or Ala. The Ser/Ala substitutions do not affect ZPI functional properties (Table 1) (16). K239C ZPI was expressed and purified with a yield similar to that of the cysteine-free parent ZPI, and the mutant was labeled with iodoacetamido-NBD or -DANS fluorophores to give final NBD/ZPI or DANS/ZPI ratios close to 1 (1.1 Ϯ 0.1 for NBD-ZPI and 1.0 Ϯ 0.1 for DANS-ZPI).…”

“…The tight binding of PZ to ZPI is thought to enable the ZPI-PZ complex to bind to a procoagulant membrane surface where it can encounter and rapidly inhibit membrane-bound factor Xa (1,4,16). ZPI-PZ complex structures have been solved by us and another group (16,17). The structures show that PZ binds ZPI through extensive ionic and hydrophobic interactions centered on helix G, the C terminus of helix A, and sheet C of ZPI, a novel binding site for a serpin cofactor interaction.…”

“…Calcium and Lipid Effects-Because ZPI complexation with PZ enables ZPI to bind to a membrane through the Gla domain of PZ and this binding depends on calcium ions (16,26), the effect of calcium ions and phospholipid membrane vesicles on the ZPI-PZ interaction was studied. PZ bound NBD-ZPI ϳ8-fold more weakly in the presence of 5 mM Ca 2ϩ than in its absence.…”