This study investigates three aspects of the adhesive interaction operating between platelet glycoprotein Ib/IX and integrin ␣ IIb  3 . These include the following: 1) examining the sufficiency of GPIb/IX and integrin ␣ IIb  3 to mediate irreversible cell adhesion on immobilized von Willebrand factor (vWf) under flow; 2) the ability of the vWf-GPIb interaction to induce integrin ␣ IIb  3 activation independent of endogenous platelet stimuli; and 3) the identification of key second messengers linking the vWf-GPIb/IX interaction to integrin ␣ IIb  3 activation. By using Chinese hamster ovary cells transfected with GPIb/IX and integrin ␣ IIb  3 , we demonstrate that these receptors are both necessary and sufficient to mediate irreversible cell adhesion under flow, wherein GPIb/IX mediates cell tethering and rolling on immobilized vWf, and integrin ␣ IIb  3 mediates cell arrest. Moreover, we demonstrate direct signaling between GPIb/IX and integrin ␣ IIb  3 . Studies on human platelets demonstrated that vWf binding to GPIb/IX is able to induce integrin ␣ IIb  3 activation independent of endogenous platelet stimuli under both static and physiological flow conditions (150 -1800 s ؊1 ). Analysis of the key second messengers linking the vWf-GPIb interaction to integrin ␣ IIb  3 activation demonstrated that the first step in the activation process involves calcium release from internal stores, whereas transmembrane calcium influx is a secondary event potentiating integrin ␣ IIb  3 activation.
Fibrinogen mediates the processes of platelet aggregation and clot retraction. Previous studies have demonstrated that fibrinogen binding to the platelet receptor ␣ IIb  3 requires the C-terminal residues of the fibrinogen ␥ chain. We made a recombinant human fibrinogen that lacks the ␥ chain C-terminal four residues (AGDV). As expected this fibrinogen did not support platelet aggregation. Unexpectedly, this variant did support clot retraction that was indistinguishable from retraction 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 recombinant 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 recognition 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 aggregation (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 retraction to the same extent as normal recombinant and plasma fibrinogen. These findings suggest that other domains in fibrinogen participate in clot retraction. MATERIALS AND METHODS Construction of ExpressionVectors-pMLP-A␣, pMLP-B, and pMLP-␥ encoding the A␣, B, and ␥ chains of human fibrinogen have been described previously (9, 10). The vector pMLP-␥407 was constructed from pMLP-␥ and p647, described in Hettasch et al. (4). The C-terminal nucleotides from the Escherichia coli e...
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.
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