Dissecting Clot Retraction and Platelet Aggregation

Abstract

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...

Fibrin Clot Retraction by Human Platelets Correlates with αIIbβ3Integrin-dependent Protein Tyrosine Dephosphorylation

“…This is consistent with the work by Cohen et al (7) who first showed that RGDS and 10E5 did not inhibit (but instead increased) isometric clot tension. Our results are also consistent with the work of Rooney et al (10), who showed that recombinant fibrinogen in which the ␥408 -411 region was deleted did support platelet retraction and not aggregation. The molecular mechanism underlying differential engagement of ␣ IIb ␤ 3 with fibrin in retraction versus fibrinogen in aggregation remains to be elucidated, although differences in avidity of polymeric(fibrin) versus monomeric (fibrinogen) ligands for ␣ IIb ␤ 3 may be at play.…”

“…We therefore considered our conditions nonlimiting and chose arbitrarily to perform experiments at concentrations of 1 mM Ca 2ϩ and 1 unit/ml thrombin. Previous studies have demonstrated ␣ IIb ␤ 3 integrin involvement in retraction of fibrin clot in plasma (7)(8)(9)(10). To verify the engagement of ␣ IIb ␤ 3 in our plasma-free conditions, we used various ␣ IIb ␤ 3 -specific inhibitors (Fig.…”

“…More direct evidence for a role of ␣ IIb ␤ 3 in fibrin clot retraction was gained from studies using ␣ IIb ␤ 3 -specific monoclonal antibodies and ␣ IIb ␤ 3 antagonist peptides (7)(8)(9). In addition, a differential engagement of ␣ IIb ␤ 3 in fibrin clot retraction versus aggregation has been suggested (10,11). Altogether, these studies have demonstrated that ␣ IIb ␤ 3 is an important component of fibrin clot retraction, but the presence of plasma in most studies has precluded more refined biochemical characterization as well as the study of retraction-associated platelet signaling.…”

A Structural and Dynamic Investigation of the Facilitating Effect of Glycoprotein IIb/IIIa Inhibitors in Dissolving Platelet-Rich Clots

“…Finally, it also emphasizes that plateletfibrin(ogen) interactions are extremely complex and may involve receptors other than GP IIb/IIIa, as recently reported with recombinant fibrinogen molecules lacking the specific GP IIb/IIIa binding sites. 28,29 FITC-rtPA and GP IIb/IIIa inhibitors underwent a diffusion-mediated transport, resulting in a slower delivery, especially within platelet-rich areas. This experimental condition represents an important limitation of this study.…”

Role of β1 and β3 Integrins in Human Smooth Muscle Cell Adhesion to and Contraction of Fibrin Clots In Vitro

“…The production, purification, and characterization of recombinant fibrinogen have already been described. 58,59 Human ␣-thrombin was purchased from Sigma Chemical Co.…”