Department of Bioengineering, Rhodes Engineering Center, Clemson University, SC 29634, USA.
Biomaterials. 2010 Feb;31(5):832-9. doi: 10.1016/j.biomaterials.2009.10.008. Epub 2009 Oct 21.
While platelet adhesion to biomaterial surfaces is widely recognized to be related to adsorbed fibrinogen (Fg), it has remained controversial whether platelet adhesion is in response to the adsorbed amount or the adsorbed conformation of this protein. To address this issue, we designed a series of platelet adhesion studies to clearly separate these two factors, thus enabling us to definitively determine whether it is the amount or the conformation of adsorbed Fg that mediates platelet response. Fg was adsorbed to a broad range of surface chemistries from a wide range of solution concentrations, with the amount and conformation of adsorbed Fg determined by absorbance and circular dichroism (CD) spectropolarimetry, respectively. Platelet adhesion response was determined by lactate dehydrogenase (LDH) assay and scanning electron microscopy (SEM). Our results show that platelet adhesion is strongly correlated with the degree of adsorption-induced unfolding of Fg (r(2)=0.96) with essentially no correlation with the amount of Fg adsorbed (r(2)=0.04). Platelet receptor inhibitor studies using an RGDS peptide reduced platelet adhesion by only about 50%, and SEM results show that adherent platelets after RGDS blocking were much more rounded with minimal extended filopodia compared with the unblocked platelets. These results provide definitive proof that the conformation of adsorbed Fg is the critical determinant of platelet adhesion, not the amount of Fg adsorbed, with adsorption-induced unfolding potentially exposing two distinctly different types of platelet binding sites in Fg; one that induces platelet adhesion alone and one that induces both platelet adhesion and activation.
虽然血小板黏附到生物材料表面与吸附的纤维蛋白原（Fg）广泛相关，但血小板黏附是响应于蛋白质的吸附量还是吸附构象仍存在争议。为了解决这个问题，我们设计了一系列血小板黏附研究来明确分离这两个因素，从而能够确定是吸附 Fg 的量还是构象介导血小板反应。Fg 从广泛的溶液浓度被吸附到各种表面化学物质上，吸附 Fg 的量和构象分别通过吸光度和圆二色性（CD）光谱偏振法确定。血小板黏附反应通过乳酸脱氢酶（LDH）测定和扫描电子显微镜（SEM）确定。我们的结果表明，血小板黏附与 Fg 的吸附诱导去折叠程度强烈相关（r(2)=0.96），与吸附 Fg 的量几乎没有相关性（r(2)=0.04）。使用 RGDS 肽的血小板受体抑制剂研究将血小板黏附减少了约 50%，SEM 结果表明，与未阻断的血小板相比，经 RGDS 阻断后的黏附血小板更圆，延伸的丝状伪足最少。这些结果提供了确凿的证据，证明吸附 Fg 的构象是血小板黏附的关键决定因素，而不是吸附的 Fg 量，吸附诱导的去折叠可能会在 Fg 中暴露两种截然不同的血小板结合位点；一种单独诱导血小板黏附，另一种则诱导血小板黏附和激活。
