Characterisation of the Fibrinogen RGD Sequence in Erythrocyte Binding and Clot Structure

Background: Fibrinogen is a large protein dimer that is composed of three polypeptide chains Aα, Bβ and γ and plays a vital role in haemostasis. Fibrinogen is suggested to be a risk factor for cardiovascular disease. The Aα-chain contains two RGD sequences, which provide the interaction sites of fibrinogen with integrins present on cells such as platelets and endothelial cells. New studies suggested that RBCs are not innocent bystanders in thrombus formation suggesting presence of polyhedral shaped RBCs inside the clot. An integrin-type receptor interaction between erythrocytes and fibrinogen was proposed but some controversy is still present regarding the possible receptor on the surface of the RBC involved in this interaction between fibrinogen and RBCs. Hypothesis: Interaction between fibrinogen and RBCs is mediated by the RGD sequence in the coiled-coil of the Aα-chain of fibrinogen through αIIbβ3-like integrin receptor. Mutating this RGD site may inhibit the interaction between fibrinogen and RBCs and subsequently thrombosis. Methods: Six recombinant fibrinogens variants (R95E, R95Q, G96V, D97K, D97N, and F98I) were generated by Site-Directed Mutagenesis. The integrity of the proteins were tested by SDS-PAGE and circular dichroism. Clots formed by variant fibrinogens were evaluated by clotability, turbidity and lysis, and laser scanning confocal microscopy. Interaction between fibrinogen and RBCs were studied by Plate-binding assay, solution-binding assay and flow cytometry. Involvement of the Aα-chain RGD sequence in binding between fibrinogen and platelets was tested by platelet spreading assay. Results: Clot structure studies showed thinner, shorter fibres but no effect on fibrinolysis. Fibrinogen binding assays with both RBCs and platelets showed no effects of the mutations in the Aα-chain RGD sequence on cell binding. Conclusion: Aα-95-98 RGD has no obvious effect on the binding between red blood cells and fibrinogen, or platelets and fibrinogen. Nonetheless, mutations at this site change clot structure and polymerisation profile