Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation.

Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or in buffer (washed platelets), by the use of one or a combination of agonists. The use of washed platelets separated from their plasma environment and in the absence of anticoagulants allows for studying intrinsic platelet properties. Among the large panel of agonists, arachidonic acid (AA), adenosine di-phosphate (ADP), thrombin and collagen are the most frequently used. The aggregation response is quantified by measuring the relative optical density (OD) over time of platelet suspension under continuous stirring. Platelets in homogeneous suspension limit the passage of light after the addition of an agonist, platelet shape change occurs producing a small transitory increase in OD. Following this initial activation step, platelet clots form gradually, allowing the passage of light through the suspension as a result of decreased OD. The aggregation process is ultimately expressed as a percentage, compared to the OD of platelet-poor plasma or buffer. Rigorous calibration is thus essential at the beginning of each experiment. As a general rule: calibration to 0% is set by measuring the OD of a non-stimulated platelet suspension while measuring the OD of the suspension medium containing no platelets represents a value of 100%. Platelet aggregation is generally visualized as a real-time aggregation curve. Turbidimetry is one of the most commonly used laboratory techniques for the investigation of platelet function and is considered as the historical gold standard and used for the development of new pharmaceutical agents aimed at inhibiting platelet aggregation. Here, we describe detailed protocols for 1) preparation of human washed platelets and 2) turbidimetric analysis of collagen-induced aggregation of human washed platelets pretreated with the food dye Brilliant Blue FCF that was recently identified as an inhibitor of Pannexin1 (Panx1) channels.