Calcium Handling Systems And Platelet Activation (quin 2, Chlorotetracycline, Hyperaggregation, Thrombosis, Channel)

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Doctor of Philosophy (Ph.D.)




Ca('2+) handling systems in platelets from normal donors and patients with vascular occlusive diseases were studied with fluorescent Ca('2+) probes chlorotetracycline (CTC) and Quin2. The CTC fluorescence signal is shown to be a linear measure of the level of free calcium in the dense tubules and in the mitochondria, with probe sensitivity in the millimolar range. The cytoplasmic free Ca('2+) concentration is measured with intracellularly trapped Quin 2. Experiments perturbing the system with Ca('2+) ionophore A23187 shows that the mitochondrial Ca('2+) uptake is slow, produces a large increase in CTC fluorescence and is inhibited by sodium azide plus oligomycin. Uptake by the dense tubules is more rapid, produces a smaller increase in CTC fluorescence and is inhibited by trifluoperazine.A quantitative comparison of the free cytoplasmic calcium concentration and the free Ca('2+) concentration in the lumen of the dense tubules of human platelets between normal donors and patients with vascular occlusive diseases have been made. Studies show that the resting cytoplasmic and dense tubular Ca('2+) concentrations were elevated in patients suffering from venous and arterial thrombosis and other diseases involving platelet hyperactivation. The Ca('2+) handling defect of platelets form thrombotic patients was identified as leakage through activated channels in the plasma membrane. The defect and the elevated resting Ca('2+) (,cyt) and Ca('2+) (,dt) are adequate to explain the observation of increased rates of collagen-activated platelet aggregation in the thrombotic patients.Upon thrombin stimulation, the calcium concentration in the cytoplasm rose rapidly from 0.1 (mu)M to approx. 1 (mu)M then slowly decayed to a lower level. The change of Ca('2+) (,cyt) induced by thrombin is due to the initially massive Ca('2+) influx and dense tubular Ca('2+) release followed by Ca('2+) sequestration, and Ca('2+) extrusion. In the presence of 2 mM of external Ca('2+), most of the elevated Ca('2+) in the cytoplasm is due to the thrombin induced Ca('2+) influx. Studies indicate that the thrombin induced Ca('2+) influx is mediated through the receptor operated Ca('2+) channels and can be enhanced by the thromboxane A(,2) which is formed by the trigger of elevated Ca('2+) (,cyt). The extent of platelet aggregation is shown to be closely dependent on the increase of Ca('2+) (,cyt).


Health Sciences, Pharmacology

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