Goldsmith H L, McIntosh F A, Shahin J, Frojmovic M M
McGill University Medical Clinic, Montreal General Hospital Research Institute, Montreal, Quebec, Canada.
Biophys J. 2000 Mar;78(3):1195-206. doi: 10.1016/S0006-3495(00)76677-X.
We studied the shear-induced breakup of doublets of aldehyde/sulfate (A/S) latex spheres covalently linked with purified platelet GPIIb-IIIa receptor, and cross-linked by fibrinogen. Flow cytometry with fluorescein isothiocyanate-fibrinogen showed than an average of 22,500 molecules of active GPIIb-IIIa were captured per sphere, with a mean K(d) = 56 nM for fibrinogen binding. The spheres, suspended in buffered 19% Ficoll 400 containing 120 or 240 pM fibrinogen, were subjected to Couette flow in a counter-rotating cone-plate rheoscope. Doublets, formed by two-body collisions at low shear rate (G = 8 s(-1)) for < or =15 min, were subjected to shear stress from 0.6 to 2.9 Nm(-2), their rotations recorded until they broke up or were lost to view. Although breakup was time dependent, occurring mostly in the first 2 rotations after the onset of shear, the percentage of doublets broken up after 10 rotations were almost independent of normal hydrodynamic force, F(n): at 240 pN, 15.6, 16.0, and 17.0% broke up in the force range 70-150 pN, 150-230 pN, and 230-310 pN. Unexpectedly, at both [fibrinogen], the initial rate of breakup was highest in the lowest force range, and computer simulation using a stochastic model of breakup was unable to simulate the time course of breakup. When pre-sheared at low G for >15 min, no doublets broke up within 10 rotations at 70 < F(n) < 310 pN; it required >3 min shear (>1110 rotations) at F(n) = 210 pN for significant breakup to occur. Other published work has shown that binding of fibrinogen to GPIIb-IIIa immobilized on plane surfaces exhibits an initial fast reversible process with relative low affinity succeeded by transformation of GPIIb-IIIa to a stable high-affinity complex. We postulate that most doublet breakups observed within 10 rotations were from a population of young doublets having low numbers of bonds, by dissociation of the initial receptor complex relatively unresponsive to force. The remaining, older doublets with GPIIb-IIIa in the high-affinity complex were not broken up in the time or range of forces studied.
我们研究了与纯化的血小板糖蛋白IIb-IIIa受体共价连接并由纤维蛋白原交联的醛/硫酸盐（A/S）乳胶球双联体的剪切诱导破裂。用异硫氰酸荧光素-纤维蛋白原进行的流式细胞术显示，每个球平均捕获22,500个活性糖蛋白IIb-IIIa分子，纤维蛋白原结合的平均解离常数K(d)=56 nM。将球悬浮在含有120或240 pM纤维蛋白原的缓冲19%菲可400中，在反向旋转锥板流变仪中进行库埃特流。在低剪切速率（G = 8 s(-1)）下通过双体碰撞形成15分钟及以内的双联体，使其承受0.6至2.9 Nm(-2)的剪应力，记录其旋转直至破裂或不可见。尽管破裂与时间有关，大多发生在剪切开始后的前2次旋转中，但10次旋转后破裂的双联体百分比几乎与正常流体动力F(n)无关：在240 pN时，在70 - 150 pN、150 - 230 pN和230 - 310 pN的力范围内，分别有15.6%、16.0%和17.0%的双联体破裂。出乎意料的是，在两种[纤维蛋白原]浓度下，最低力范围内的初始破裂速率最高，并且使用破裂随机模型的计算机模拟无法模拟破裂的时间进程。当在低G下预剪切超过15分钟时，在70 < F(n) < 310 pN范围内，10次旋转内没有双联体破裂；在F(n)=210 pN时，需要超过3分钟的剪切（>1110次旋转）才会发生明显破裂。其他已发表的工作表明，纤维蛋白原与固定在平面表面的糖蛋白IIb-IIIa的结合表现出一个初始快速可逆过程，亲和力相对较低，随后糖蛋白IIb-IIIa转变为稳定的高亲和力复合物。我们推测，在10次旋转内观察到的大多数双联体破裂来自键数量较少的年轻双联体群体，是由对力相对不敏感的初始受体复合物解离所致。其余具有高亲和力复合物中糖蛋白IIb-IIIa的较老双联体在研究的时间或力范围内未破裂。
