Dissecting Clot Retraction and Platelet Aggregation

Abstract

Fibrinogen mediates the processes of platelet aggre-gation and clot retraction. Previous studies have dem-onstrated that fibrinogen binding to the platelet recep-tor ␣ IIb ␤ 3 requires the C-terminal residues of the fibrinogen ␥ chain. We made a recombinant human fi-brinogen that lacks the ␥ chain C-terminal four residues (AGDV). As expected this fibrinogen did not support platelet aggregation. Unexpectedly, this variant did sup-port clot retraction that was indistinguishable from re-traction with normal recombinant or plasma fibrinogen. These results suggest that the site on fibrinogen that is required for platelet aggregation differs from the site on fibrin that is required for clot retraction. The processes of platelet aggregation and clot retraction are mediated by fibrinogen in vitro and play a role in maintaining hemostasis in vivo. Fibrinogen is a large (340 kDa) plasma protein consisting of two sets of A␣, B␤, and ␥ chains. The protein is arranged in a symmetrical fashion with two lateral D domains and a central E domain (1). Previous work employing electron microscopy (2), synthetic peptides (3), and recombi-nant proteins (4, 5) has implicated the D domain, in particular residues ␥408 – 411 (AGDV), as the site on fibrinogen that interacts with platelets. This interaction involves the platelet integrin ␣ IIb ␤ 3 , a heterodimeric transmembrane receptor. Antibodies and peptides that prevent fibrinogen binding to ␣ IIb ␤ 3 also prevent platelet aggregation and clot retraction (6 – 8). Together, these findings predict that the ␣ IIb ␤ 3 recogni-tion site on fibrinogen (i.e. residues ␥408 – 411) must be present and accessible for aggregation or clot retraction to occur. Two studies support this prediction with respect to platelet aggre-gation (4, 5), but both have limitations. In one (4), recombinant ␥ chains lacking residues 408 – 411 were unable to support platelet aggregation; however, this variant ␥ chain was studied outside the context of the entire molecule. In the other (5), recombinant fibrinogen with a 20-amino acid insert in place of these four residues was unable to support platelet aggregation. However, the lack of aggregation cannot be directly attributed to the loss of the AGDV residues, as the additional 20 amino acids may sterically impair aggregation. Moreover, neither study explored the effects that these alterations may have had on clot retraction. We report that an intact recombinant fibrinogen lacking only residues ␥408 – 411 does not support aggregation, consistent with previous work (4, 5), but unexpectedly supports clot re-traction to the same extent as normal recombinant and plasma fibrinogen. These findings suggest that other domains in fibrin-ogen participate in clot retraction.