Zwaginga J J, Sixma J J, de Groot P G
Department of Hematology, University Hospital, Utrecht, The Netherlands.
Arteriosclerosis. 1990 Jan-Feb;10(1):49-61. doi: 10.1161/01.atv.10.1.49.
Previous studies have indicated that activation of endothelial cells may lead to the production of tissue factor. We have studied the effect of endothelial cell activation and subsequent tissue factor synthesis on thrombus formation on the extracellular matrix in flowing blood. Endothelial cells were stimulated with tumor necrosis factor, endotoxin, or phorbol ester. Coverslips with activated cells or their extracellular matrix were introduced into a perfusion system and exposed to blood anticoagulated with 20 U/ml low molecular weight heparin. This concentration allowed manipulation of blood without activation of the coagulation cascade. Platelet deposition and fibrin formation were evaluated by morphometry, and fibrinopeptide A formation was assayed as a measure of thrombin generation. Activation of endothelial cells caused fibrinopeptide A generation in the perfusate and some deposition of fibrin on endothelial cells; however, platelets were not deposited. The matrix of the stimulated endothelium also caused enhanced fibrinopeptide A generation, and platelet aggregates and fibrin were deposited on the matrix. Maximal effects were observed with stimulation periods between 4 and 10 hours and were still clearly present after 18 hours. Increase in shear rate, perfusion time, and platelet number resulted in an increase in platelet adhesion, but platelet aggregate formation as a percentage of adhesion remained constant. Platelet aggregate formation and fibrinopeptide A generation were inhibited with antibodies against tissue factor or factor VIIa. Platelet aggregate formation alone was inhibited by antibodies against glycoprotein IIb/IIIa. Polymerization of fibrin on the matrix was best supported in perfusions at a low shear rate. The new in vitro thrombosis model presented here provides a powerful tool for study of the regulation of thrombogeneity by the vessel wall in response to various stimuli.
先前的研究表明，内皮细胞的激活可能导致组织因子的产生。我们研究了内皮细胞激活及随后的组织因子合成对流动血液中细胞外基质上血栓形成的影响。用肿瘤坏死因子、内毒素或佛波酯刺激内皮细胞。将带有活化细胞或其细胞外基质的盖玻片引入灌注系统，并使其暴露于用20 U/ml低分子量肝素抗凝的血液中。该浓度可在不激活凝血级联反应的情况下操控血液。通过形态计量学评估血小板沉积和纤维蛋白形成，并检测纤维蛋白肽A的形成以衡量凝血酶的产生。内皮细胞的激活导致灌注液中纤维蛋白肽A的产生以及一些纤维蛋白在内皮细胞上的沉积；然而，血小板并未沉积。受刺激内皮细胞的基质也导致纤维蛋白肽A的产生增加，并且血小板聚集体和纤维蛋白沉积在基质上。在4至10小时的刺激时间段内观察到最大效应，并且在18小时后仍明显存在。剪切速率、灌注时间和血小板数量的增加导致血小板黏附增加，但血小板聚集体形成占黏附的百分比保持恒定。针对组织因子或因子VIIa的抗体可抑制血小板聚集体形成和纤维蛋白肽A的产生。单独的血小板聚集体形成可被抗糖蛋白IIb/IIIa抗体抑制。在低剪切速率的灌注中，基质上纤维蛋白的聚合得到最佳支持。本文介绍的新的体外血栓形成模型为研究血管壁在各种刺激下对血栓形成性的调节提供了一个强大的工具。
