Abstract Number: OC 27.3
Meeting: ISTH 2021 Congress
Background: Heparin acts as an anticoagulant by enhancing the effects of antithrombin (AT). Through an AT-independent mechanism, heparin also accelerates APC-catalyzed proteolytic inactivation of FV, but not FVa. While FV, FVa, and APC all bind heparin, the molecular basis for the enhanced proteolysis of FV remains unknown. Conserved basic (BR, 963-1008) and acidic (AR2, 1493-1537) regions within FV B-domain preserve the FV procofactor state and obscure high affinity binding sites for proteases including APC.
Aims: To investigate the mechanism by which heparin potentiates APC proteolytic inactivation of FV.
Methods: Using a purified system, we examined the effect of pharmacologic heparin species on FV proteolysis. Immunoblotting with FV specific antibodies and peptidyl substrate hydrolysis were utilized to visualized cleavage products, and assess APC protease function, respectively.
Results: Unfractionated heparin (UFH) showed a dose-dependent increase in FV proteolysis by APC over its therapeutic range (0-1.0 U/mL). Pharmacologic heparin derivatives, enoxaprin (a low molecular weight heparin), and fondaparinux showed some enhanced effect, but 2-O, 3-O desulfated heparin (ODSH) had no effect on APC potentiation of FV proteolysis. Densitometric analyses showed that, proteolysis of FVa and FV-short were not significantly altered by 1.0 U/mL UFH. In contrast, heparin did markedly accelerate APC inactivation of FV-short when it was bound to either the FV-BR or TFPIα-BR fragment. These data suggest that heparin likely disrupts the BR-AR2 interaction allowing APC to more efficiently cleave FV-short. Our data show that increased proteolysis correlated with enhanced cleavage of the procofactor at Arg306. Taken together, these data support the hypothesis that intramolecular or intermolecular BR-AR2 complexes are amenable to heparin binding and also influence proteolysis by APC.
Conclusions: This study unveils the molecular basis by which heparin enhances proteolytic inactivation of FV and provides evidence that a charged small molecule ligand could modulate the procofactor regulatory region and alter the anticoagulant response during hemostasis.
To cite this abstract in AMA style:Ayombil F, Camire RM. Heparin Modulates FV Procofactor Regulatory Region and Susceptibility to APC [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). https://abstracts.isth.org/abstract/heparin-modulates-fv-procofactor-regulatory-region-and-susceptibility-to-apc/. Accessed September 24, 2023.
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