Abstract Number: OC 27.4
Meeting: ISTH 2021 Congress
Background: The inactivation of coagulation factor (F)Va by activated protein C (APC) proceeds through cleavage at Arg306 and Arg506, resulting in A2-domain dissociation and loss of FVa cofactor function. In contrast, snake venom Pseudonaja textilis FVa (ptFVa) retains cofactor function despite APC-dependent proteolysis. However, ptFVa lacks an Arg306-like cleavage site and comprises a unique disulfide link connecting the A2-A3-domains.
Aims: Unravel the molecular basis of ptFVa’s functional APC-resistance by assessing the role of the unique disulfide bond and absence of the Arg306 cleavage site in ptFV in response to APC cleavage.
Methods: The human Arg306-region PKKTRNL was exchanged for the homologous ptFV sequence GNPDTLT, generating ptFV-h306 and hFV-pt306; recombinant removal of ptFV’s disulfide bond resulted in ptFV-h306-SS. Molecular Dynamics simulations were performed on reconstructed FVa models in which the APC-cleavage sites were disrupted to mimic APC-proteolysis.
Results: Introduction of the human Arg306 region in ptFVa resulted in APC-proteolysis at Arg306 and Lys507. Surprisingly, functional analysis demonstrated that in contrast to human FVa, APC-catalyzed proteolysis of ptFVa at Arg306 and Lys507 does not abrogate ptFVa function. Remarkably, analytical size-exclusion chromatography showed that the structural integrity of APC-treated ptFVa is maintained, indicating that stable non-covalent interactions prevent A2-domain dissociation. Using Molecular Dynamics simulations we uncovered a key loop structure in the ptFVa A2-domain that facilitates the formation of several stable hydrogen bonds with the A1-domain, likely preventing A2-domain dissociation.
Conclusions: These findings indicate that APC-proteolysis of ptFVa at Arg306 or deletion of the unique disulfide bond does not abrogate cofactor function, unlike human FVa. Remarkably, upon proteolysis the structural integrity of ptFVa is maintained, indicating a completely novel role for specific A1 and A2-domain structural regions that have been uniquely adapted in ptFVa in order to prevent A2-domain dissociation. As such, these results challenge our current understanding by which strict regulatory mechanisms control FVa activity.
To cite this abstract in AMA style:Schreuder M, Liu X, Cheung KL, Reitsma PH, Nicolaes GAF, Bos MHA. A Unique Loop Structure in the A2-domain of Pseudonaja textilis Factor Va Prevents Activated Protein C-catalyzed A2-domain Dissociation [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). https://abstracts.isth.org/abstract/a-unique-loop-structure-in-the-a2-domain-of-pseudonaja-textilis-factor-va-prevents-activated-protein-c-catalyzed-a2-domain-dissociation/. Accessed November 29, 2023.
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