Lahav Judith, Jurk Kerstin, Hess Oded, Barnes Michael J, Farndale Richard W, Luboshitz Jacob, Kehrel Beate E
Coagulation Laboratory, Institute of Hematology, Rabin Medical Center-Beilinson Campus, Petah-Tiqva, Israel.
Blood. 2002 Oct 1;100(7):2472-8. doi: 10.1182/blood-2001-12-0339.
Studies have suggested a pivotal role for free sulfhydryls in platelet integrin function, and enzyme-mediated reduction of disulfide bonds on platelets has been implicated. The platelet fibrinogen receptor alpha(IIb)beta(3) is the best-studied platelet integrin and serves as a model system for studying the structure-function relation in this family of adhesion receptors. The demonstration of free sulfhydryls on the exofacial domain of purified alpha(IIb)beta(3), specifically in its activated conformation, prompted us to explore the potential for activation-dependent, enzymatically catalyzed thiol expression on intact platelets and the possible role of surface-associated protein disulfide isomerase (PDI) in alpha(IIb)beta(3) ligation. Using the membrane-impermeant sulfhydryl blocker para-chloromercuriphenyl sulfonate, the inhibitor of disulfide exchange bacitracin, and the monoclonal anti-PDI antibody RL90, we examined fibrinogen binding to alpha(IIb)beta(3) as well as ligation-induced allosteric changes in the conformation of alpha(IIb)beta(3). We sought to distinguish the possible involvement of disulfide exchange in agonist-induced platelet stimulation from its role in integrin ligation. Analysis of the role of free thiols in platelet aggregation suggested a thiol-independent initial ligation followed by a thiol-dependent stabilization of binding. Flow cytometric analysis showed that sustained binding of fibrinogen, as well as expression of ligand-induced binding site epitopes and ligand-bound conformation, depended on free thiols and disulfide exchange. Expression of P-selectin was minimally affected, even with complete inhibition of alpha(IIb)beta(3) function. These data indicate that although agonist-induced platelet stimulation is independent of ecto-sulfhydryls, engagement of integrin alpha(IIb)beta(3) on the intact platelet depends totally on their enzymatically catalyzed surface expression.
研究表明游离巯基在血小板整合素功能中起关键作用，并且酶介导的血小板二硫键还原也与之相关。血小板纤维蛋白原受体α(IIb)β(3)是研究最为深入的血小板整合素，可作为研究该粘附受体家族结构-功能关系的模型系统。纯化的α(IIb)β(3)胞外结构域上存在游离巯基，特别是在其活化构象中，这促使我们探索完整血小板上依赖活化的、酶催化的硫醇表达潜力，以及表面相关蛋白二硫键异构酶（PDI）在α(IIb)β(3)连接中的可能作用。使用膜不透性巯基阻断剂对氯汞苯磺酸盐、二硫键交换抑制剂杆菌肽和单克隆抗PDI抗体RL90，我们检测了纤维蛋白原与α(IIb)β(3)的结合以及连接诱导的α(IIb)β(3)构象变构变化。我们试图区分二硫键交换在激动剂诱导的血小板刺激中的可能参与与其在整合素连接中的作用。对游离硫醇在血小板聚集中作用的分析表明，初始连接不依赖硫醇，随后结合的稳定依赖硫醇。流式细胞术分析表明，纤维蛋白原的持续结合以及配体诱导结合位点表位的表达和配体结合构象取决于游离硫醇和二硫键交换。即使α(IIb)β(3)功能完全被抑制，P-选择素的表达也受到最小影响。这些数据表明，尽管激动剂诱导的血小板刺激不依赖胞外巯基，但完整血小板上整合素α(IIb)β(3)的结合完全依赖于其酶催化的表面表达。
