The processes of Ca2+ influx and release from internal stores are central to the activation and aggression of blood platelets. The processes are triggered to varying extents by physiological activators. The activation sequence includes Ca2+ influx and release from the dense tubules, a shape change, exocytotic release of the contents of two types of secretion granule (alpha and dense granules), development of """"""""stickiness"""""""" as well as primary and secondary aggregation. The primary focus of the study will be the characterization of the fundamental properties of Ca2+ extrusion systems of the plasma membrane (PM) and the Ca2+ accumulation of the dense tubular (DT) pump system. This will be done in intact platelets and in preparations where the PM barrier has been removed (""""""""chemically skinned""""""""). Fluorescent indicators of the average free Ca2+ concentration in the cytoplasm (Quin 2) and of the free Ca2+ in the dense tubular lumen (chlortetracycline, CTC), will be used together with more conventional 45CA2+ techniques. Cytoplasmic pH will also be determined fluoimetrically. Clarification of controversies regarding indicator is an expected byproduct of they study. A method (Quin2 overload) for determination of mu mole Ca2+ moved per liter cell volume using these indicators is described. Quin 2 and CTC will be used for the in situ characterization of the kinetics of the Ca2+-ATPase pump (Vm, Km, Hill coefficient), determination of average (Ca2+)eyt and (Ca2+)dt levels obtained with the pumps operating against passive leaks (and opened channels), pump inhibition by transported Ca2+ and pump modulation by calmodulin binding and cAMP-stimulated or calmodulin-stimulated regulatory phosphorylation. The possible involvement of a Na+/Ca2+ exchange function of the PM, membrane potentials and pH shifts in activation process will be evaluated. The absolute potentials and pH shifts in activation process will be evaluated will be evaluated. The absolute rates of Ca2+ influx and dense tubular Ca2+ release will be determined for the following activators: thrombin, collagen, ADP, platelet activating factor, epinephrine and arachidonic acid/thromboxane A2. Positive and negative feedback interactions will be dissected out. The study will attempt to describe normal Ca2+ homeostasis as the interplay between pumps and channels using computer simulations based on the quantitative Vm, Km, n and channel permeability data obtained. Contributions to quantitative methodology involving in vivo use of Ca2+ and pH indicators is an expected byproduct of this research.
