Fibrinogen Coating Density Affects the Conformation of Immobilized Fibrinogen: Implications for Platelet Adhesion and Spreading

Abstract

SummaryAdhesion of platelets to immobilized fibrinogen appears to play an important role in a variety of physiologic and pathologic phenomena. We previously observed that the fibrinogen concentration used to coat polystyrene wells affected the morphology and distribution of GP IIb/ IIIa receptors on the surface of platelets adherent to the fibrinogen. One possible explanation for these differences is that fibrinogen immobilized at high density adopts a different conformation than fibrinogen immobilized at low density. To address this possibility, we studied the binding of a panel of anti-fibrinogen monoclonal antibodies (mAbs) to fibrinogen immobilized at different coating densities. Three different patterns of binding were observed: 1) a linear increase in binding to wells coated with 1-10 μg/ml fibrinogen, followed by a lesser increase or plateau at higher fibrinogen concentrations (mAbs Fd4-4E1, Fd4-7B3, 1D4, 4-2); 2) minimal reactivity at all fibrinogen concentrations (mAbs GC4-1A12, 2C34); 3) a biphasic response, with a linear increase up to 10 μg/ml fibrinogen and then a significant decline in binding at higher fibrinogen concentrations (mAbs 311, 31A9, FPA 19/7, 9C3, 1C5-A5/2, 44-3). The patterns of mAb binding to fibrinogen immobilized from plasma were similar. Most mAbs that demonstrated a biphasic response bound poorly or not at all to soluble fibrinogen, while mAbs that demonstrated a linear/plateau response were able to bind soluble fibrinogen. At equal surface densities, mAbs that bound biphasically, particularly mAb 1C5-A5/2, were more reactive to urea-denatured than native fibrinogen. mAbs 1C5-A5/2 and 44-3 are specific for γ 1-78 and 95-265, respectively, suggesting that the fibrinogen γ-chain may be sensitive to changes in conformation induced by immobilization. In summary, these data suggest that fibrinogen immobilized at 1-10 μg/ml adopts a conformation unlike soluble fibrinogen, while fibrinogen immobilized at >30 μg/ml adopts a more solution-like conformation. These differences in fibrinogen conformation may partially account for the ability of platelets to bind to immobilized fibrinogen without the addition of agonist, as well as the differences in spreading and GPIIb/IIIa distribution on platelets adherent to high- versus low-density immobilized fibrinogen.

Exposure of acidic residues as a danger signal for recognition of fibrinogen and other macromolecules by integrin αXβ2

“…The sharp peak in cell binding at a coating concentration of Ϸ3 g Fg͞ml (Fig. 2 A and B) is in quantitative agreement with the finding that adsorption to surfaces at concentrations Ͻ10 g of Fg͞ml promotes loss in structure as shown by spectroscopy (13) and binding by conformation-specific mAbs (25). Guanidine treatment converted the native, nonbinding form adsorbed at higher Fg concentrations to a maximally adhesive form (Fig.…”

Effects of Limiting Extension at the αIIb Genu on Ligand Binding to Integrin αIIbβ3

“…A number of mechanisms have been proposed to account for the difference in requirements for ␣IIb␤3 binding of soluble fibrinogen versus mediating adhesion to immobilized fibrinogen, including conformational changes in fibrinogen induced by immobilization (47) and the potential for multivalent interactions between receptors and ligand molecules stemming from the higher density of fibrinogen achieved with immobilization (43). We noted a greater impact of clamping ␣IIb at the lower fibrinogen coating density, suggesting a correlation with ligand density, but these data need to be interpreted in light of the known impact of fibrinogen coating density on the conformational changes induced by immobilizing the ligand (43,47). The recent study by Zhu et al (12) provides an additional hypothesis, namely adhesion-mediated engagement of the cytoplasmic region of ␤3 with the cytoskeleton, leading to activation of the receptor via contractile traction forces initiating receptor extension and ␤3 swing-out.…”

Modulation of integrin activation and signaling by α1/α1′-helix unbending at the junction

“…It has been known that, unlike soluble ligand binding, aIIbb3 integrin can mediate cell adhesion to immobilized Fg without preactivation (Savage and Ruggeri, 1991). The conformational a1/a19-helix unbending in integrin activation 5743 change of Fg upon immobilization (Moskowitz et al, 1998), the high local ligand density (Jirousková et al, 2007), and a small subset of integrins on the cell surface that are readily accessible to bind ligand all possibly account for this phenomenon. Our data indicate that the initial or nascent adhesion to immobilized ligand does not require the high affinity of integrin.…”