Effects of Thrombin on the Potassium and ATP Content of Platelets *

Abstract

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 intracellular pH of the human platelet.

“…It has been stated that cell membranes are rela- (13) and to release potassium from a compartment within the cell (14).…”

The stimulation by thrombin of glucose oxidation in human platelets.

“…This change in the platelet membrane renders it more permeable to serotonin, ADP, protein, and ions such as potassium and calcium (33,36), but the increased permeability may be relatively specific, since quite different proportions of various intracellular substances escape (33). If the oxidative pathway for glucose were unsaturated due to limited transport of substrate across the cell membrane, reduction by thrombin of the relative barrier to the entry of glucose would allow its increased oxidation.…”

“…After the exposure of platelets to thrombin there are marked changes in the movement of cations into and out of the cell. Zieve, Gamble, and Jackson (36) showed that thrombin causes a greater permeability of the platelet membrane to potassium with resultant potassium loss from the cell. Grette (33) proposed that calcium ions enter the thrombin-treated platelet and trigger the release of intracellular contents.…”

The effect of 5‐hydroxytryptamine on the potassium exchange of human platelets

“…Platelets would be expected, therefore, to contain potassium at about the same concentration as the cytoplasm of other cells; this has been found to be so (Hartmann, Auditore & Jackson, 1958). Little is known, however, about the movements of this potassium (Zieve, Gamble & Jackson, 1964;Carroll & Gorstein, 1964) and whether, under normal conditions, it exchanges across the outer membrane of platelets in a manner similar to the exchange of potassium across the membranes of whole cells. Platelets have, moreover, the unusual property of being able to accumulate 5-HT against very steep concentration gradients; this amine affects the movement of potassium across other cell membranes (Pickles, 1956(Pickles, , 1957.…”