Kasirer-Friede A, Frojmovic M M
Dept. of Physiology, McGill University, Montreal, Que., Canada.
Thromb Haemost. 1998 Sep;80(3):428-36.
We recently reported that washed platelets (WP) activated with ADP and expressing surface-bound vWF aggregated in flow through small tubes or in a cylindrical couette device at physiological shear rates of G = 300 s(-1)-1000 s(-1) in the absence of exogenous ligands, with GPIb-vWF partially, and activated GPIIb-IIIa totally required for the aggregation. We have now extended these studies to aggregation of platelets "activated" with ristocetin or thrombin. Washed platelet suspensions with added soluble vWF and ristocetin (0.3-0.75 mg/ml), or activated with thrombin (0.01-0.5 U/ml) but no added ligand, were sheared in a coaxial cylinder device at uniform shear rate, G = 1000 s(-1). The collision capture efficiency (alphaG) with which small aggregates form (= experimental/calculated initial rates of aggregation) was correlated with vWF platelet binding assessed by flow cytometry. The vWF-GPIb interaction was exclusively able to support ristocetin-mediated shear aggregation of metabolically active platelets, with very few vWF monomer equivalents bound per platelet (representing < or = 10 molecules of 10 million Da) required to yield high capture efficiencies (alphaG = 0.38+/-.02; n = 11), suggesting rapid and stable bond formations between vWF and GPIb. However, platelet surface-expressed vWF, generated by addition of thrombin to washed platelets, was found to mediate platelet aggregation with alphaG = 0.08+/-.01 (n = 6), surprisingly comparable to that previously reported for WP and ADP activation. Blocking the GPIIb-Illa receptor decreased alphaG by 95+/-3% (n =3), while a monoclonal antibody to the vWF site on GPIb caused a 49+/-7% (n = 8) decrease in alphaG. The partial role for GPIb thus appears to reflect a facilitative function for increasing contact time between flowing platelets, and allowing engagement of the GPIIb-IIa receptor to yield stable attachment.
我们最近报道，经ADP激活并表达表面结合型vWF的洗涤血小板（WP），在不存在外源性配体的情况下，于生理剪切速率G = 300 s⁻¹ - 1000 s⁻¹下通过小管流动或在圆柱形库埃特装置中聚集，其中GPIb - vWF部分参与，而激活的GPIIb - IIIa对于聚集完全必需。我们现在已将这些研究扩展至用瑞斯托菌素或凝血酶“激活”的血小板聚集。添加可溶性vWF和瑞斯托菌素（0.3 - 0.75 mg/ml）的洗涤血小板悬浮液，或用凝血酶（0.01 - 0.5 U/ml）激活但未添加配体的悬浮液，在同轴圆柱装置中以均匀剪切速率G = 1000 s⁻¹进行剪切。小聚集体形成的碰撞捕获效率（alphaG）（=实验性/计算出的初始聚集速率）与通过流式细胞术评估的vWF与血小板的结合相关。vWF - GPIb相互作用能够专门支持代谢活跃血小板的瑞斯托菌素介导的剪切聚集，每个血小板结合极少的vWF单体当量（代表≤10个1000万Da的分子）就能产生高捕获效率（alphaG = 0.38±0.02；n = 11），这表明vWF与GPIb之间能快速形成稳定的键。然而，发现向洗涤血小板中添加凝血酶所产生的血小板表面表达的vWF能介导血小板聚集，alphaG = 0.08±0.01（n = 6），令人惊讶的是这与先前报道的WP和ADP激活的情况相当。阻断GPIIb - IIIa受体使alphaG降低95±3%（n = 3），而针对GPIb上vWF位点的单克隆抗体使alphaG降低49±7%（n = 8）。因此，GPIb的部分作用似乎反映了一种促进功能，即增加流动血小板之间的接触时间，并使GPIIb - IIIa受体能够结合以产生稳定附着。
