Circulating platelets play a critical role in normal homeostasis; they adhere and aggregate at sites of vascular injury to initiate thrombus formation. Platelets also play a central role in the pathogenesis of arterial thrombosis, which accounts for the clinical events associated with cardiovascular disease. They are involved in the haematogenous spread of cancerous cells during the metastatic cascade, and in inflammatory diseases. The study of the pathways leading to platelet activation and expression of adhesive ligands is very important to understand and regulate platelet interaction with other platelets, tumour cells or leukocytes. In order to study platelet contents, protein expression, and function, molecular biologists have numerous techniques. However, most of these techniques, such as aggregometry or protein gels, analyse bulk samples in which a large number of platelets are present, giving as results the mean response of all the cells and not the response of individual cells. In order to study different cell populations, techniques quantifying the response of individual cells are necessary. Single platelet analysis is commonly done by flow cytometry, which is an optical technique for particle quantification and sorting using fluorescent indicators. Typically 10,000 platelets are analysed per sample using diluted whole blood. However, this technique is only able to measure platelets remaining as single cells in solution, a condition where platelets would not be allowed to be activated in vivo and also where it is difficult to accurately visually monitor platelets.
In vivo platelet adhesion should be the primary event in platelet activation. Platelet adhesion (to e.g. von Willebrand factor (vWF) or collagen) triggers platelet activation and thus the coagulation cascade leading to thrombus formation. The study of platelet adhesion to these proteins is crucial to understand platelet function and regulation. Numerous studies have been carried out using platelet adhesion assays, but they involve the use of relatively large volumes of blood and variable platelet preparation steps which are time consuming and might induce platelet activation. These known platelet adhesion assays are practiced on surfaces that are homogeneously coated with platelet binding agents, or that include relatively large regions coated with platelet binding agents (such regions are typically circular with about a diameter of at least 300 μm—i.e. 70650 μm2 surface area, and not less than 119 μm in diameter—i.e. 11116 μm2 surface area). Consequently, the platelets to be analysed are bound to the surface in large agglomerates (a region coated with platelet binding agents that has a diameter of about 119 μm in diameter could bind about 400 platelets at one time).
The applicants have found that it is difficult to accurately analyse a large number of platelets using such known platelet adhesion assays; only the results obtained from manual counting in a few selected areas are usually achieved, and the way of choosing the areas for analysis is commonly not clearly stated or done in a reproducible and objective way. Nowadays there is no adhesion assay available that could analyse in an automatic way a large number of single platelets, like in flow cytometry.
Following extensive experimentation, the inventors have identified a new method for characterising platelets, which has utility in, for example, methods for diagnosis, methods for monitoring the progression of disease, methods for monitoring the efficacy of treatment, or for research purposes.