Abstract 36: Uncovering Lipid-Independent Prothrombinase Function Using Human-Snake Chimeras of Blood Coagulation Factor V
The prothrombinase complex, which converts prothrombin to the key regulatory enzyme thrombin, consists of the serine protease Xa (FXa) and cofactor Va (FVa; A1-A2-A3-C1-C2 domains). The latter dramatically enhances the rate of prothrombin catalysis and exclusively associates with FXa on negatively charged phospholipid surfaces through its lipid-binding C-domains. Interestingly, we have previously shown that the FVa and FXa homologues found in the venom of the Australian snake Pseudonaja textilis bypass the requirement for a membrane surface to achieve complexation. Here we investigated whether the C-domains of venom-derived P. textilis factor V (ptFV) drive its lipid-independent cofactor function and, if so, can sustain this unique property in the setting of human FV. Therefore, we swapped the C-domains of constitutively active B-domainless human FV (hFV) and ptFV and expressed and purified the chimeric variants hFV-ptC and ptFV-hC. Using a purified prothrombinase assay with the lipid-independent substrate prethrombin-1, FV cofactor activity was examined with the corresponding human or venom-derived P. textilis FXa species in the presence or absence of lipids. We hypothesized that the C-domains constrain ptFV in a conformation optimal for lipid-independent cofactor function. Surprisingly, ptFV-hC displayed full cofactor activity, irrespective of the availability of anionic membranes. Similarly, hFV-ptC functioned equivalent to hFV as no gain-of-function in the absence of lipids was observed. Subsequent lipid-titration experiments indicated that whereas both C-domain species promoted maximum rates of substrate conversion at identical phospholipid concentrations, the lipid-affinity of the ptFV C-domains was at least 10-fold reduced as compared to that of hFV. These results demonstrate that the P. textilis C-domains do not play a role in the unique capacity of ptFV to function in the absence of lipids, suggesting that they may be of little relevance to ptFV cofactor function. As such, our findings imply an alternative mode of macromolecular complex assembly for P. textilis venom FVa and FXa and their natural substrate prothrombin, thereby potentially challenging the current paradigm of human prothrombinase assembly and function.
Author Disclosures: D. Verhoef: None. R.M. Camire: None. P.H. Reitsma: None. M.H.A. Bos: Research Grant; Significant; Bayer Early Career Award, Dutch Thrombosis Foundation, American National Blood Foundation, European Commission, Marie Curie Fellowship.
- © 2014 by American Heart Association, Inc.