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The blood platelets have many of the characteristics of living cells including an active metabolism. As in other cells, the concentration of potassium within platelets is high in comparison to that of extracellular fluid and-is similar to that of red blood cells (1). ATP also is present in high concentration within platelets (2) and is considered to provide the energy necessary for clot retraction, a process that requires metabolically active platelets (3). Unlike other cells, platelets are uniquely susceptible to the effects of thrombin, a proteolytic enzyme formed during clotting of blood. During coagulation, the platelets undergo a complex series of morphologic and biochemical changes (4), and there is a decrease in the concentration of some of the constituents of platelets, including potassium (1) and ATP (5). The present studies were designed to investigate some of the factors that influence changes in potassium and ATP within platelets. MethodsAll glassware with which platelets had contact was coated with silicone.' Blood was drawn from normal human donors through 15-gauge, uncoated, disposable metal needles attached to plastic tubing and was collected in tubes containing an aqueous solution of the disodium * Submitted for publication February 26, 1964; accepted July 7, 1964. This investigation was carried out under contract salt of EDTA as an anticoagulant (1 part EDTA: 9 parts blood). The final concentration of EDTA in blood was 0.005 M. Platelet-rich plasma was obtained by differential centrifugation (6). Unless specified otherwise, platelets from 3-ml portions of platelet-rich plasma were sedimented (6) and washed twice with and finally resuspended in 1.8 ml of Tris-buffered saline, pH 7.5 (6), containing potassium chloride (0.004 M), glucose (0.0055 M), and EDTA (0.0013 M). The washing procedures were performed at 40 C and required approximately 30 minutes. Platelet counts were performed on these suspensions by the method of Brecher and Cronkite (7). The final concentration of platelets was approximately 250,000 per mm'. The washed platelets then were incubated without agitation at 370 C under various experimental conditions. After incubation, the platelets to be analyzed for potassium and ATP were washed once with Tris-buffered saline containing EDTA but no potassium or glucose. Those to be analyzed for sodium were washed once with Tris-buffered potassium chloride (0.155 M) containing EDTA. The final washing procedure was performed at 40 C and required approximately 10 minutes. The platelets then were sedimented by centrifugation at 22,000 g for 5 minutes at 40 C and lysed in 3 ml of a 3% solution of trichloroacetic acid. Potassium and sodium in the lysates were measured by use of a flame photometer employing an internal lithium standard. ATP, in most experiments, was measured after fractionation of the lysate on a Dowex-1 anion exchange column by use of a Beckman model DU spectrophotometer reading at 260 mg (8). In other experiments ATP was measured either by the firefly luminescence method or e...

The morphologic, functional, and biochemical changes produced by hematoporphyrin and light in human platelets have been characterized. By phase microscopy the cells appeared swollen and resembled signet rings; by electron microscopy they showed considerable loss of cytoplasm and their contour was smoother than normal. Irradiated platelets were not aggregable by thrombin and calcium chloride, although they contained clottable protein, and were incapable of supporting clot retraction. A linear relationship was demonstrated between the per cent depletion of serotonin from irradiated platelets and the log dose of hematoporphyrin. The depletion of serotonin from these platelets was related linearly to the log of time of exposure to light during the initial six minutes of exposure; but thereafter continued at a constant rate. The temperature of incubation influenced directly the rate of depletion of serotonin from irradiated platelets but did not influence the movement of serotonin into these platelets,. ATP was diminished considerably in irradiated platelets. These changes are attributable to damage to the membrane of the platelet by hematoporphyrin and light.These studies provide additional information about the blood platelet in terms of its response to photodynamic action.

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