Heparin Is a Major Activator of the Anticoagulant Serpin, Protein Z-dependent Protease Inhibitor

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 10The 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 ZPIprotease 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.

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

“…The differential effects of calcium on NBD-ZPI interactions with intact and Gla/EGF1-domainless PZ suggested that specific calcium interactions with the PZ Gla domain were responsible for the loss of complex affinity. Similar calcium-Gla domain interactions affect the reactions of factor Xa with its serpin inhibitors, ZPI and antithrombin, in the presence of the cofactor heparin (27,28). In this case, calcium is thought to displace the Gla domain from an intramolecular interaction with the heparin binding site in the protease catalytic domain that blocks heparin bridging of the serpin-protease interactions.…”

Basic Residues of β-Sheet A Contribute to Heparin Binding and Activation of Vaspin (Serpin A12)

“…Three independent selective labeling experiments resulted in the repeated identification of matching peptides and confirmed the selectivity and specificity of the labeling approach. Lys 22 and Lys 31 are located in the flexible N terminus (not shown in the structure), Lys 46 and Lys 62 in helix A, Lys 296 in helix H, and Lys 321 in helix I (Fig. 5C).…”

“…Because all experiments were performed using buffers with pH Ͼ7.8 and thus above the pK a values for histidine resi- dues (pK a 6.0 -7.0), a significant influence of the poly-His tag can be excluded. To determine whether basic N-terminal residues Lys 22 , Lys 31 , and Arg 28 participate in heparin binding, we generated the triple mutant K22A/R28A/K31A. Heparin affinity of this mutant was as wild type (Fig.…”

“…11D) (25), and also cleavage of vaspin N-terminal residues resulted in a marked decrease in heparin affinity. But both mutation of all basic N-terminal residues of vaspin in question (Lys 22 , Arg 28 , and Lys 31 ) and cleavage of the His tag sequence did not alter heparin binding. Vaspin N-terminal residues are highly flexible (4), and the presence of these additional nine residues may alter heparin affinity by supporting or facilitating Colors relate to the electrostatic surface potential (blue is positive, and red is negative, Ϫ8 to ϩ8 k B T) calculated by APBS (37).…”

Quantification of clusterin in paired cerebrospinal fluid and plasma samples

“…The direct interaction of clusterin with heparin has previously been shown to affect ELISA signals. 34 Additionally, heparin activates multiple proteases and inhibitors which may interact with clusterin in plasma, [35][36][37] influencing its detectability in commercially available (Biovendor -Laboratorni medicina, a.s.) immunoassays. These findings of the effects of freezing and thawing and anticoagulant type are important considerations when undertaking any studies of clusterin as a biomarker for disease.…”