Fusing the C-terminal tridecapeptide of hirudin to α1-proteinase inhibitor M358R accelerates its rate of thrombin inhibition

Abstract

<p>The serpin α-1 proteinase inhibitor (API) normally only impacts the coagulation cascade through its ability to inactivate factor XIa. However, the point mutation (Met to Arg) at position 358 results in a potent thrombin inhibitor, API M358R. This mutation also enhances this serpin’s ability to inhibit the anticoagulant protein, activated protein C (APC) and hence this property limits its therapeutic potential. As a result, various modifications to this protein have been engineered in order to enhance its specificity towards thrombin. Previously, the Heparin Cofactor II (HCII) N-terminal tail, HCII 1-75, which binds exosite 1 of thrombin, was tethered to the N-terminus of API M358R, creating HAPI M358R. Although this change did not alter anti-APC activity, it did augment the anti-thrombin activity of API M358R. In addition, further changes in the reactive center loop, the region that interacts with the thrombin active site, resulted in a significant reduction in APC activity while maintaining antithrombotic activity similar to HAPI M358R; this variant was termed HAPI RCL5.</p> <p>Preliminary experiments were performed with the C-terminal tridecapeptide of Hirudin Variant 3 (HV3) to determine its exosite 1 binding capacity compared to HCII 1-75. Three different variants of this peptide were tested: one with a hexahistidine tag (H₆HV3_54-66), another that also had a hexa-glycine C-terminal addition (H₆HV3_54-66G₆) and a third without either addition. All were found to bind exosite 1 with a greater affinity than HCII 1-75. Thus, the H₆HV3_54-66G₆peptide was fused to API M358R and API RCL5 in hopes of creating an inhibitor with heightened specificity compared to HAPI M358R and HAPI RCL5, respectively.</p> <p>HV3API M358R and HV3API RCL5 were expressed in a bacterial system and purified by nickel-chelate and ion exchange chromatography. Second order rate constants for the inhibition of thrombin and APC by the API variants and fusion proteins were determined. The K₂ values for α-thrombin inhibition ranged from 186 M^-1min^-1 to 22 M^-1min^-1 with an order of inhibitory potency observed as follows: HAPI M358R > HAPI RCL5 > HV3API M358R > HV3API RCL5>API RCL5 > API M358R.</p> <p>The ability of recombinant chimeric serpins to bind thrombin exosite 1 in a manner independent of RCL-thrombin active site interactions was also investigated through competitive inhibition of the binding of active site-inhibited thrombin to immobilized HCII 1-75. It was found that the order of exosite 1 binding affinity was HV3API RCL5 > H₆HV3_54-66G₆> HCII 1-75 > HAPI RCL5. Our results indicate that fusing the C-terminal tridecapeptide of HV3 to API variants enhanced their ability to inhibit thrombin, but to a lesser extent than fusing the N-terminal 75 residues of HCII. This finding likely reflects a requirement for the exosite 1-binding motif of the fusion protein to bind exosite 1 in a way that allows for subsequent optimal active site attack on the RCL by the serpin moiety of the fusion protein. In general, this work provides a second novel example of how the activity of a thrombin-inhibitory serpin can be enhanced by fusion to an exosite-1 binding motif.</p>