Razdan K, Hellums J D, Kroll M H
Veterans' Affairs Medical Center, Houston, TX.
Biochem J. 1994 Sep 15;302 ( Pt 3)(Pt 3):681-6. doi: 10.1042/bj3020681.
Pathological arterial blood flow generates fluid shear stresses that directly cause platelet aggregation. The mechanism of shear-induced platelet aggregation is incompletely understood, but involves von Willebrand factor (vWF) binding to platelet glycoprotein (GP) Ib and GP IIb-IIIa, leading to the transmembrane influx of Ca2+ and the activation of protein kinase C. To investigate this further, shear-stress-induced protein tyrosine phosphorylation (PTP) of washed platelets was studied in a cone-plate viscometer. A time- and shear-stress-dependent tyrosine phosphorylation of substrates with approx. M(r) 29,000-31,000, 36,000, 50,000, 58,000, 64,000, 76,000, 85,000 and 105,000 was observed. PTP in response to a threshold shear stress of 0.3 mN/cm2 (30 dyn/cm2) was enhanced in most cases by exogenous purified human vWF, and PTP in response to a pathological shear stress of 0.9 mN/cm2 (90 dyn/cm2) was inhibited in some cases by inhibiting vWF binding to GP Ib or GP IIb-IIIa, or by inhibiting Ca2+ responses with extracellular EGTA. Shear-induced PTP of a substrate of M(r) approximately 31,000 appeared to be independent of GP Ib, and PTP of a substrate(s) of M(r) approximately 29,000 was shear-stress-dependent but independent of extracellular Ca2+. Cytochalasin D, which inhibits GP Ib-cytoskeleton interactions, inhibits the PTP of a substrate of M(r) approximately 76,000. These results suggest that tyrosine phosphorylation may be involved in transmembrane signalling that mediates platelet adhesion and aggregation in response to pathological shear stresses generated at sites of arterial vaso-occlusion.
病理性动脉血流产生的流体剪切应力可直接导致血小板聚集。剪切诱导的血小板聚集机制尚未完全明确，但涉及血管性血友病因子（vWF）与血小板糖蛋白（GP）Ib及GP IIb-IIIa结合，进而导致Ca2+跨膜内流及蛋白激酶C激活。为进一步研究此机制，在锥板粘度计中对洗涤后的血小板进行了剪切应力诱导的蛋白酪氨酸磷酸化（PTP）研究。观察到约M(r) 29,000 - 31,000、36,000、50,000、58,000、64,000、76,000、85,000和105,000的底物出现时间和剪切应力依赖性酪氨酸磷酸化。在大多数情况下，外源性纯化的人vWF可增强对0.3 mN/cm2（30 dyn/cm2）阈值剪切应力的PTP反应，而在某些情况下，通过抑制vWF与GP Ib或GP IIb-IIIa结合，或用细胞外EGTA抑制Ca2+反应，可抑制对0.9 mN/cm2（90 dyn/cm2）病理性剪切应力的PTP反应。约M(r) 31,000底物的剪切诱导PTP似乎与GP Ib无关，约M(r) 29,000底物的PTP依赖于剪切应力但与细胞外Ca2+无关。抑制GP Ib - 细胞骨架相互作用的细胞松弛素D可抑制约M(r) 76,000底物的PTP。这些结果表明，酪氨酸磷酸化可能参与跨膜信号传导，介导血小板在动脉血管阻塞部位产生的病理性剪切应力作用下的黏附和聚集。
