THE CONTRIBUTION OF THE 3 HYPOTHESIZED INTEGRIN-BINDING SITES IN FIBRINOGEN TO PLATELET-MEDIATED CLOT RETRACTION

Fibrinogen is a plasma protein that interacts with integrin alpha(IIb) beta(3) 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-V al (AGDV) sequence at the C-terminus of the gamma chain and two Arg-Gl y-Asp (RGD) sequences in the A alpha chain. Recent data showed that AG DV is critical for platelet adhesion and aggregation, but not retracti on, suggesting that either one or both of the RGD sequences are involv ed in clot retraction. Here we provide evidence, using engineered reco mbinant fibrinogen, that no one of these sites is critical for clot re traction; fibrinogen lacking all three sites still sustains a relative ly normal, albeit delayed, retraction response. Three fibrinogen varia nts with the following mutations were examined: a substitution of RGE for RGD at position A alpha 95-97, a substitution of RGE for RGD at po sition A alpha 572-574, and a triple substitution of RGE for RGD at bo th A alpha positions and deletion of AGDV from the gamma chain. Retrac tion rates and final clot sizes after a 20-minute incubation were indi stinguishable when comparing the A alpha D97E fibrinogen or A alpha D5 74E fibrinogen with normal recombinant fibrinogen. However, with the t riple mutant fibrinogen, clot retraction was delayed compared with nor mal recombinant fibrinogen. Nevertheless, the final clot size measured after 20 minutes was the same size as a clot formed with normal recom binant fibrinogen. Similar results were observed using platelets isola ted from an afibrinogenemic patient, eliminating the possibility that the retraction was dependent on secretion of plasma fibrinogen from pl atelet cu-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, b ut not for a subsequent step. With the triple mutant fibrinogen, the s econd step of clot retraction, possibly the development of clot tensio n, proceeds with a rate similar to that observed with normal recombina nt fibrinogen. These results are consistent with a mechanism where a n ovel site on fibrin is involved in the second step of clot retraction. (C) 1998 by The American Society of Hematology.