Abstract Number: OC 05.2
Meeting: ISTH 2021 Congress
Background: The conversion of prothrombin (proT) to thrombin by the prothrombinase complex (PTC) is a key step of blood coagulation, responsible for controlling the rate at which fibrin clots form at the site of injury. Mechanistic insights into this reaction have been challenging to obtain for two reasons. First, the complex is short-lived. Second, assembly of the complex occurs on negatively charged phospholipids, requiring highly coordinated interactions between the phospholipid-bound components.
Aims: Develop a new methodology based on Fluorescence Correlation Spectroscopy (FCS) to gain mechanistic insights into the assembly of blood coagulation complexes.
Methods: Recombinant factor Xa (fXa) and proT were expressed in mammalian cells. Site-specific fluorescent labeling of proT was achieved by maleimide chemistry after replacing the native serine residue 478 with a cysteine. Catalytically inactive fXa was produced by mutating the catalytic serine 419 to alanine (fXaS419A). Factor Va (fVa) was from human plasma. Liposomes were prepared by extrusion. Data was collected on a customized MicroTime 200 confocal microscope.
Results: Addition of increasing concentrations of phosphatidylserine-containing liposomes to a solution of proT labeled with Atto-655 produced a progressive ~5-fold increase of the diffusion time, enabling accurate determination of the interaction’s strength. Binding of proT to sub-saturating concentrations of liposome was possible only in the presence of fXaS419A and fVa but not in the presence of the individual factors. This indicates that assembly of a fully functional PTC is necessary to keep proT bound to the lipid phase. Experiments designed to displace proT bound to PTC using unlabeled proT fragments and proT variants stabilized in the open and closed conformations revealed competition by the N-terminal fragment and a strong preference of PTC for the closed form.
Conclusions: We successfully applied FCS to study the mechanism of proT recognition by PTC. Our results suggest a probable mechanism of thrombin generation.
To cite this abstract in AMA style:Chinnaraj M, Pozzi N, Di Cera E. Fluorescence Correlation Spectroscopy Provides New Insights into the Mechanism of Prothrombin Recognition by the Prothrombinase Complex [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). https://abstracts.isth.org/abstract/fluorescence-correlation-spectroscopy-provides-new-insights-into-the-mechanism-of-prothrombin-recognition-by-the-prothrombinase-complex/. Accessed November 29, 2023.
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