Chow T W, Hellums J D, Moake J L, Kroll M H
Biomedical Engineering Laboratory, Rice University, Houston, TX.
Blood. 1992 Jul 1;80(1):113-20.
Platelets subjected to elevated levels of fluid shear stress in the absence of exogenous agonists will aggregate. Shear stress-induced aggregation requires von Willebrand factor (vWF) multimers, extracellular calcium (Ca2+), adenosine diphosphate (ADP), and platelet membrane glycoprotein (GP)Ib and GPIIb-IIIa. The sequence of interaction of vWF multimers with platelet surface receptors and the effect of these interactions on platelet activation have not been determined. To elucidate the mechanism of shear stress-induced platelet aggregation, suspensions of washed platelets were subjected to different levels of uniform shear stress (15 to 120 dyne/cm2) in an optically modified cone and plate viscometer. Cytoplasmic ionized calcium ([Ca2+]i) and aggregation of platelets were monitored simultaneously during the application of shear stress; [Ca2+]i was measured using indo-1 loaded platelets and aggregation was measured as changes in light transmission. Basal [Ca2+]i was approximately 60 to 100 nmol/L. An increase of [Ca2+]i (up to greater than 1,000 nmol/L) was accompanied by synchronous aggregation, and both responses were dependent on the shear force and the presence of vWF multimers. EGTA chelation of extracellular Ca2+ completely inhibited vWF-mediated [Ca2+]i and aggregation responses to shear stress. Aurin tricarboxylic acid, which blocks the GPIb recognition site on the vWF monomer, and 6D1, a monoclonal antibody to GPIb, also completely inhibited platelet responses to shear stress. The tetrapeptide RGDS and the monoclonal antibody 10E5, which inhibit vWF binding to GPIIb-IIIa, partially inhibited shear stress-induced [Ca2+]i and aggregation responses. The combination of creatine phosphate/creatine phosphokinase, which converts ADP to adenosine triphosphate and blocks the effect of ADP released from stimulated platelets, inhibited shear stress-induced platelet aggregation without affecting the increase of [Ca2+]i. Neither the [Ca2+]i nor aggregation response to shear stress was inhibited by blocking platelet cyclooxygenase metabolism with acetylsalicylic acid. These results indicate that GPIb and extracellular Ca2+ are absolutely required for vWF-mediated [Ca2+]i and aggregation responses to imposed shear stress, and that the interaction of vWF multimers with GPIIb-IIIa potentiates these responses. Shear stress-induced elevation of platelet [Ca2+]i, but not aggregation, is independent of the effects of release ADP, and both responses occur independently of platelet cyclooxygenase metabolism. These results suggest that shear stress induces the binding of vWF multimers to platelet GPIb and this vWF-GPIb interaction causes an increase of [Ca2+]i and platelet aggregation, both of which are potentiated by vWF binding to the platelet GPIIb-IIIa complex.
在没有外源性激动剂的情况下，受到高水平流体剪切应力作用的血小板会发生聚集。剪切应力诱导的聚集需要血管性血友病因子（vWF）多聚体、细胞外钙（Ca2+）、二磷酸腺苷（ADP）以及血小板膜糖蛋白（GP）Ib和GPIIb-IIIa。vWF多聚体与血小板表面受体相互作用的顺序以及这些相互作用对血小板活化的影响尚未确定。为了阐明剪切应力诱导血小板聚集的机制，在经过光学改良的锥板粘度计中，将洗涤过的血小板悬液置于不同水平的均匀剪切应力（15至120达因/平方厘米）下。在施加剪切应力的过程中，同时监测血小板的细胞质游离钙（[Ca2+]i）和聚集情况；使用indo-1负载的血小板测量[Ca2+]i，通过光透射变化测量聚集情况。基础[Ca2+]i约为60至100纳摩尔/升。[Ca2+]i的增加（高达大于1000纳摩尔/升）伴随着同步聚集，并且这两种反应都依赖于剪切力和vWF多聚体的存在。细胞外Ca2+的EGTA螯合完全抑制了vWF介导的[Ca2+]i和对剪切应力的聚集反应。金精三羧酸可阻断vWF单体上的GPIb识别位点，6D1是一种针对GPIb的单克隆抗体，它们也完全抑制了血小板对剪切应力的反应。抑制vWF与GPIIb-IIIa结合的四肽RGDS和单克隆抗体10E5部分抑制了剪切应力诱导的[Ca2+]i和聚集反应。磷酸肌酸/肌酸磷酸激酶的组合可将ADP转化为三磷酸腺苷，并阻断受刺激血小板释放的ADP的作用，抑制剪切应力诱导的血小板聚集，而不影响[Ca2+]i的增加。用乙酰水杨酸阻断血小板环氧化酶代谢既不抑制[Ca2+]i也不抑制对剪切应力的聚集反应。这些结果表明，GPIb和细胞外Ca2+对于vWF介导的[Ca2+]i和对施加的剪切应力的聚集反应是绝对必需的，并且vWF多聚体与GPIIb-IIIa的相互作用增强了这些反应。剪切应力诱导的血小板[Ca2+]i升高而非聚集与释放ADP的作用无关，并且这两种反应均独立于血小板环氧化酶代谢而发生。这些结果表明，剪切应力诱导vWF多聚体与血小板GPIb结合，这种vWF-GPIb相互作用导致[Ca2+]i增加和血小板聚集，两者均因vWF与血小板GPIIb-IIIa复合物的结合而增强。
