Frojmovic M M, Kasirer-Friede A, Goldsmith H L, Brown E A
Departments of Physiology and Medicine, McGill University, Montreal, Canada.
Thromb Haemost. 1997 Mar;77(3):568-76.
We previously showed that ADP activation of washed human platelets in plasma-free suspensions supports aggregation at moderate shear stress (0.4-1.6 Nm-2) in Poiseuille flow. Although most activated platelets expressed maximal fibrinogen-occupied GPIIb-IIIa receptors, aggregation appeared to be independent of bound fibrinogen, but blocked by the hexapeptide GRGDSP. Here, we tested the hypothesis that von Willebrand factor (vWF) secreted and expressed on activated platelets mediates aggregation at moderate shear rates from 300 to 1000 s-1 corresponding to shear stresses from 0.3 to 1.1 Nm-2. Relatively unactivated platelets (< 15% expressing prebound fibrinogen) were prepared from acidified citrated platelet rich plasma (cPRP) by single centrifugation with 50 nM stable prostacyclin derivative ZK 36374 and resuspended in Tyrodes-albumin at 5 x 10(4) cells microliter-1. Flow cytometric measurements with monoclonal antibody (mAb) 2.2.9 reporting on surface-bound vWF, and with mAb S12 reporting on alpha-granule secreted P-selectin, showed that 65% and 80%, respectively, of all platelets were maximally activated with respect to maximal secretion and surface expression of these proteins. "Resting" washed platelets exhibited both surface-bound vWF and significant P-selectin secretion. We showed that mAbs 6D1 and NMC4, respectively blocking the adhesive domains on the GPIb receptor recognizing vWF, and on the vWF molecule recognizing the GPIb receptor, partially inhibited ADP-induced aggregation under shear in Couette flow, the degree of inhibition increasing with increasing shear stress. In contrast, mAb 10E5, blocking the vWF binding domain on GPIIb-IIIa, essentially blocked all aggregation at the shear rates tested. We conclude that vWF, expressed on ADP-activated platelets, is at least the predominant cross-bridging molecule mediating aggregation at moderate shear stress. There is an absolute requirement for free activated GPIIb-IIIa receptors, postulated to interact with platelet-secreted, surface bound vWF. The GPIb-vWF cross-bridging reaction plays a facilitative role becoming increasingly important with increasing shear stress. Since aurin tricarboxylic acid, which blocks the GPIb binding domain on vWF, was also found to completely block aggregation in Poiseuille flow, we conclude that it too affects the GPIIb-IIIa interaction.
我们之前发现，在无血浆悬浮液中，ADP激活洗涤后的人血小板，可在泊肃叶流中中等剪切应力（0.4 - 1.6 Nm-2）下支持聚集。尽管大多数活化血小板表达了被纤维蛋白原完全占据的GPIIb-IIIa受体，但聚集似乎与结合的纤维蛋白原无关，却被六肽GRGDSP阻断。在此，我们检验了这样一个假设：活化血小板分泌并表达的血管性血友病因子（vWF）在300至1000 s-1的中等剪切速率下介导聚集，该剪切速率对应0.3至1.1 Nm-2的剪切应力。通过用50 nM稳定的前列环素衍生物ZK 36374单次离心，从酸化的枸橼酸化富血小板血浆（cPRP）中制备相对未活化的血小板（<15%表达预结合的纤维蛋白原），并以5×10(4)个细胞微升-1的浓度重悬于Tyrodes-白蛋白中。用报告表面结合vWF的单克隆抗体（mAb）2.2.9以及报告α-颗粒分泌的P-选择素的mAb S12进行流式细胞术测量，结果显示，就这些蛋白的最大分泌和表面表达而言，分别有65%和80%的所有血小板被最大程度激活。“静息”洗涤后的血小板既表现出表面结合的vWF，也有显著的P-选择素分泌。我们发现，分别阻断GPIb受体上识别vWF的黏附结构域以及vWF分子上识别GPIb受体的mAb 6D1和NMC4，在Couette流剪切条件下部分抑制了ADP诱导的聚集，抑制程度随剪切应力增加而增大。相比之下，阻断GPIIb-IIIa上vWF结合结构域的mAb 10E5在测试的剪切速率下基本阻断了所有聚集。我们得出结论，ADP活化血小板上表达的vWF至少是在中等剪切应力下介导聚集的主要交联分子。绝对需要游离的活化GPIIb-IIIa受体，推测其与血小板分泌的、表面结合的vWF相互作用。GPIb-vWF交联反应起促进作用，随着剪切应力增加其重要性日益凸显。由于发现阻断vWF上GPIb结合结构域的金精三羧酸也能完全阻断泊肃叶流中的聚集，我们得出结论，它也影响GPIIb-IIIa的相互作用。
