A Potentiometric Method for Studying the Platelet Release Reaction

A Potentiometrie method which permits the continuous recording of small changes in extracellular calcium (Ca) concentration (1-10 nmoles/ml) is used to study the thrombin-induced release of Ca from human platelets. The instrumentation consists of a Radiometer Ca ion electrode and pH meter, and a chart recorder. Platelets are suspended in Tris-buffered saline, pH 7.4, containing 50 μM Ca. The electrode is placed in a 2 ml aliquot of the platelet suspension in a beaker, thrombin is added, and the change in extracellular Ca (in mV) accompanying the release of Ca is recorded. Recorder tracings of the release reaction show three phases: 1) an initial lag period inversely related to thrombin concentration; 2) an exponential phase during which most of the Ca release occurs; and 3) a final linear phase during which a slow release of Ca is observed. The amount of Ca released is a direct function of both platelet and thrombin concentration. Because an open system is used and release is recorded continuously, considerable flexibility in experimental design is possible. Platelet and thrombin concentrations can be varied over a wide range; thrombin can be added in single or multiple increments and at different time intervals; fresh platelets can be introduced to a completed release reaction; and inhibitors can be added at various times relative to thrombin. Results obtained by using some of these variations suggest that platelets undergo a partial release reaction and become refractory to repeated low level stimuli. The method is both sensitive and inexpensive, and provides a rapid, non-traumatic means of studying temporal and quantitative aspects of the release reaction, and the effects of pathology, drugs or storage on this aspect of platelet function.