Eriksson Andreas C, Whiss Per A
Division of Pharmacology, Department of Medicine and Care, Faculty of Health Sciences, Linköping University, SE-581 85 Linköping, Sweden.
J Pharmacol Toxicol Methods. 2005 Nov-Dec;52(3):356-65. doi: 10.1016/j.vascn.2005.06.002. Epub 2005 Jul 7.
Platelet adhesion is an initial, crucial and complex event for inhibiting blood loss upon vascular injury. Activation and adhesion of platelets also play a fundamental role in the development of thrombosis. A combination of exposed extracellular matrix proteins in the vascular wall and release of activating compounds from the participating cells activate the platelets. New potent anti-platelet agents are in progress but there is a shortage of methods that measure the concerted action of adhesive surfaces and soluble compounds upon platelet adhesion in vitro. The aim of this work was to develop a method to measure adhesion of platelets in plasma with standard laboratory equipment.
Platelet-rich plasma from healthy humans was used in studies to optimise the conditions of the present assay. Different proteins were coated in microplate wells and various soluble platelet activators and inhibitors were added to establish the ability of the current method to detect increased as well as decreased platelet adhesion. The amount of platelet adhesion was measured by the reaction between p-nitrophenyl phosphate and the intracellular enzyme acid phosphatase.
Adhesion of platelets in plasma to microplate wells coated with albumin, collagen, fibrinogen and activated plasma showed significant surface dependency. The known soluble platelet activators adenosine diphosphate, adrenaline and ristocetin enhanced the levels of adhesion. Available anti-platelet agents such as prostacyclin, forskolin, acetylsalicylic acid and RGD containing peptides caused dose-dependent inhibition of platelet adhesion.
This report describes a further development of a previously described method and offers the advantage to use platelets in plasma to measure platelet adhesion to protein surfaces. The assay is simple and flexible and is suitable in basic research for screening and characterisation of platelet adhesion responsiveness.
血小板黏附是血管损伤时抑制失血的首个关键且复杂的事件。血小板的激活和黏附在血栓形成过程中也起着重要作用。血管壁中暴露的细胞外基质蛋白与参与细胞释放的激活化合物共同作用激活血小板。新型强效抗血小板药物正在研发中，但缺乏在体外测量黏附表面和可溶性化合物协同作用对血小板黏附影响的方法。本研究的目的是开发一种利用标准实验室设备测量血浆中血小板黏附的方法。
使用健康人富含血小板的血浆进行研究，以优化本检测方法的条件。在微孔板孔中包被不同蛋白质，并添加各种可溶性血小板激活剂和抑制剂，以确定当前方法检测血小板黏附增加和减少的能力。通过对硝基苯磷酸与细胞内酶酸性磷酸酶之间的反应来测量血小板黏附量。
血浆中的血小板黏附于包被有白蛋白、胶原蛋白、纤维蛋白原和活化血浆的微孔板孔时，表现出显著的表面依赖性。已知的可溶性血小板激活剂二磷酸腺苷、肾上腺素和瑞斯托霉素可提高黏附水平。现有的抗血小板药物如前列环素、福斯可林、乙酰水杨酸和含RGD的肽可引起血小板黏附的剂量依赖性抑制。
本报告描述了对先前所述方法的进一步改进，具有利用血浆中的血小板测量血小板对蛋白质表面黏附的优势。该检测方法简单灵活，适用于基础研究中血小板黏附反应性的筛选和表征。
