Moskowitz K A, Kudryk B, Coller B S
Department of Medicine, The Mount Sinai School of Medicine, New York, NY 10029-6574, USA.
Thromb Haemost. 1998 Apr;79(4):824-31.
Adhesion 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 microg/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 microg/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 gamma 1-78 and 95-265, respectively, suggesting that the fibrinogen gamma-chain may be sensitive to changes in conformation induced by immobilization. In summary, these data suggest that fibrinogen immobilized at 1-10 microg/ml adopts a conformation unlike soluble fibrinogen, while fibrinogen immobilized at > 30 microg/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.
血小板与固定化纤维蛋白原的黏附似乎在多种生理和病理现象中起重要作用。我们之前观察到，用于包被聚苯乙烯孔的纤维蛋白原浓度会影响黏附于纤维蛋白原的血小板表面GP IIb/IIIa受体的形态和分布。对这些差异的一种可能解释是，高密度固定化的纤维蛋白原与低密度固定化的纤维蛋白原具有不同的构象。为了探究这种可能性，我们研究了一组抗纤维蛋白原单克隆抗体（mAb）与不同包被密度固定化纤维蛋白原的结合情况。观察到三种不同的结合模式：1）与包被有1 - 10微克/毫升纤维蛋白原的孔的结合呈线性增加，在更高纤维蛋白原浓度时增加较少或达到平台期（mAb Fd4 - 4E1、Fd4 - 7B3、1D4、4 - 2）；2）在所有纤维蛋白原浓度下反应性极低（mAb GC4 - 1A12、2C34）；3）双相反应，在纤维蛋白原浓度达到10微克/毫升之前呈线性增加，然后在更高纤维蛋白原浓度时结合显著下降（mAb 311、31A9、FPA 19/7、9C3、1C5 - A5/2、44 - 3）。mAb与从血浆中固定化的纤维蛋白原的结合模式相似。大多数表现出双相反应的mAb与可溶性纤维蛋白原结合不良或根本不结合，而表现出线性/平台期反应的mAb能够结合可溶性纤维蛋白原。在相同表面密度下，表现出双相结合的mAb，特别是mAb 1C5 - A5/2，对尿素变性的纤维蛋白原比对天然纤维蛋白原更具反应性。mAb 1C5 - A5/2和44 - 3分别对γ1 - 78和95 - 265具有特异性，表明纤维蛋白原γ链可能对固定化诱导的构象变化敏感。总之，这些数据表明，1 - 10微克/毫升固定化的纤维蛋白原具有与可溶性纤维蛋白原不同的构象，而> 30微克/毫升固定化的纤维蛋白原具有更类似溶液的构象。纤维蛋白原构象的这些差异可能部分解释了血小板在不添加激动剂的情况下与固定化纤维蛋白原结合的能力，以及黏附于高密度与低密度固定化纤维蛋白原的血小板在铺展和GPIIb/IIIa分布上的差异。
