The Contribution of the Three Hypothesized Integrin-Binding Sites in Fibrinogen to Platelet-Mediated Clot Retraction

Abstract

Fibrinogen is a plasma protein that interacts with integrin αIIbβ3 to mediate a variety of platelet responses including adhesion, aggregation, and clot retraction. Three sites on fibrinogen have been hypothesized to be critical for these interactions: the Ala-Gly-Asp-Val (AGDV) sequence at the C-terminus of the γ chain and two Arg-Gly-Asp (RGD) sequences in the Aα chain. Recent data showed that AGDV is critical for platelet adhesion and aggregation, but not retraction, suggesting that either one or both of the RGD sequences are involved in clot retraction. Here we provide evidence, using engineered recombinant fibrinogen, that no one of these sites is critical for clot retraction; fibrinogen lacking all three sites still sustains a relatively normal, albeit delayed, retraction response. Three fibrinogen variants with the following mutations were examined: a substitution of RGE for RGD at position Aα 95-97, a substitution of RGE for RGD at position Aα 572-574, and a triple substitution of RGE for RGD at both Aα positions and deletion of AGDV from the γ chain. Retraction rates and final clot sizes after a 20-minute incubation were indistinguishable when comparing the Aα D97E fibrinogen or Aα D574E fibrinogen with normal recombinant fibrinogen. However, with the triple mutant fibrinogen, clot retraction was delayed compared with normal recombinant fibrinogen. Nevertheless, the final clot size measured after 20 minutes was the same size as a clot formed with normal recombinant fibrinogen. Similar results were observed using platelets isolated from an afibrinogenemic patient, eliminating the possibility that the retraction was dependent on secretion of plasma fibrinogen from platelet α-granules. These findings indicate that clot retraction is a two-step process, such that one or more of the three putative platelet binding sites are important for an initial step in clot retraction, but not for a subsequent step. With the triple mutant fibrinogen, the second step of clot retraction, possibly the development of clot tension, proceeds with a rate similar to that observed with normal recombinant fibrinogen. These results are consistent with a mechanism where a novel site on fibrin is involved in the second step of clot retraction.