Abstract Number: LPB0100
Meeting: ISTH 2021 Congress
Background: Prothrombinase (Ptase) is the enzyme complex that converts prothrombin to thrombin. It is composed of factor (f) Va, and fXa, assembled on the surface of negatively charged membranes in the presence of calcium ions. Pseutarin C is an orthologue of human (h) Ptase and processes prothrombin in the same way as hPtase, predominantly through initial cleavage at R320 (meizothrombin intermediate), even in the absence of membranes. The molecular basis of Ptase assembly and function is of central importance to understanding haemostasis.
Aims: To investigate the interaction between fVa and prothrombin by mutating residues within and outside the proposed binding site on Pseutarin C and assessing the effects on prothrombin processing.
Methods: Residues on the A1 and A2 domains and a1-loop were mutated to Cys and modified with a polyethylene glycol (PEG) moiety. The complete a1-loop was also swapped with that of hfV and hfVIII to assess its role in prothrombin recognition.
Results: As a control, Ser578, located in the fXa interface in Pseutarin C, was mutated to Cys and PEGylated. The Cys mutation had no effect on prothrombin processing, but PEGylation completely abolished activity. As predicted by our model, PEGylation of residues 134, 168 and 540 had no effect on thrombin generation, whereas 276, 302, 441 and 455 slowed thrombin generation by 7-, 12.6-, 408- and 12.4-fold, respectively. PEGylation of individual a1-loop residues reduced the rate of thrombin generation to a similar extent. Swapping the a1-loop for that of hfVIII reduced the rate by 6.6-fold. Swapping the a1-loop for that of hfV increased the rate by 3.6-fold, and channeled processing exclusively through the meizothrombin intermediate, with no evidence of prethrombin-2.
Conclusions: These results support our proposed prothrombin binding site on Pseutarin C. The composition of the a1-loop plays a key role in prothrombin binding and in presentation of the R320 site for cleavage.
To cite this abstract in AMA style:Ustok FI, Huntington JA. Mapping Prothrombin Binding on Pseutarin C by Site-directed PEGylation [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). https://abstracts.isth.org/abstract/mapping-prothrombin-binding-on-pseutarin-c-by-site-directed-pegylation/. Accessed November 29, 2023.
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