Abstract 1653: The Role of the Fibrinogen Alpha Chain in Controlling the Rate of Factor XIII Activation: Implications for Coronary Artery Thrombus Formation
Background: The development of an obstructive arterial thrombus is dependent on the generation of a platelet rich fibrin mesh secondary to plaque rupture. Fibrin itself is formed by the cleavage of fibrinogen by thrombin with subsequent fibrin cross-linking by activated Factor XIII. Critical to these processes, fibrinogen α-chain residues 242– 424 have a major regulatory role in the dissociation of coagulation Factor XIII A subunit (FXIIIA) from the Factor XIII B subunit (FXIIIB), however the mechanisms for this enhancing effect have not been determined.
Methods: α-Chain residues 242– 424 were expressed as a GST-fusion protein in E.coli, along with six further truncations; αC fragments α242– 402, α242–387, α242–374, α242–341, α242–289 and α242–265. FXIII-A and an inactive double thrombin cleavage mutant (R37A/K513A) were also expressed as GST-fusion proteins in E. coli.
Results: Using surface plasmon resonance (SPR) and ELISA we can confirm the presence of at least two binding sites on the αC for activated FXIII-A within residues α388 – 424 and α242–265 with an overall KD of 4.9 ± 2.29 μM. This interaction was specific for activated FXIII-A, as the inactive FXIII-A double thrombin cleavage mutant did not bind to the αC as indicated by SPR and ELISA. We demonstrated that the binding of the αC is dependent on a conformational change which occurs during activation of FXIII-A with calcium and thrombin. FXIII-A activated with thrombin alone did not bind αC as determined by SPR and ELISA. Competition studies showed that the αC competitively inhibits binding with a KI of 1.2 μM confirming the specificity of this interaction. Iodoacetamide blocking of FXIII-A active site cysteine did not prevent binding of the αC suggesting this interaction is independent of FXIII-A cross-linking. Furthermore we have identified a novel interaction between FXIIIB and α-chain by ELISA. Utilising the αC fragments we have localised two binding regions between α290–341 and α242–265 with a KD of 123nM ± 11 and 286nM ± 24.7 respectively.
Conclusions: The binding of both FXIII A- and B-subunits to this area of the fibrinogen α-chain suggests a crucial role in controlling the amount of activated FXIII generated for clot stabilisation and presents a potential target for therapeutic intervention.