Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.5 (thrombin)
 33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mechanisms are assumed to exist in the resting platelet which maintain the concentration of cytoplasmic free calcium below that level required to activate cellular responses. To assess such processes the porcine platelet plasma membrane was selectively lysed with digitonin and the uptake (or flux) of free calcium monitored by an extracellular calcium electrode. Lysis resulted in an immediate lowering of the extracellular free calcium, due to the action of intracellular organelle(s) acting on the extracellular space through the permeabilized plasma membrane. In resting platelets, the rate of calcium uptake was first order with respect to the extracellular prelytic calcium concentration, and hence the cytoplasmic free concentration was found to be 1 X 10(-7) M by extrapolation to a point of zero flux (i.e., the null point). This approach could not be used with thrombin-stimulated platelets, as external calcium was required for both secretion of ATP + ADP and aggregation. Nevertheless, evidence for an increase in cytoplasmic free calcium after thrombin stimulation was obtained. Metabolic inhibitors and agents known to inhibit calcium uptake by mitochondria had no effect on the calcium flux following lysis, indicating different mechanisms for calcium homeostasis in the platelet when compared with other cell types (e.g., liver). Levels of ionophore A23187, which caused platelet aggregation, gave a massive release of the nonmitochondrial pool of calcium into the cytoplasmic space. Thus, in porcine platelets an intracellular energy-requiring calcium pump, which sequesters calcium in a nonmitochondrial membranous compartment, is crucial for intracellular calcium homeostasis.
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PMID:Cytoplasmic free calcium concentration in porcine platelets. Regulation by an intracellular nonmitochondrial calcium pump and increase after thrombin stimulation. 620 90

Thrombin and related protease-activated receptors 1, 2, 3, and 4 (PAR1-4) play a multifunctional role in many types of cells including endothelial cells. Here, using RT-PCR and immunofluorescence staining, we showed for the first time that PAR1-4 are expressed on primary human brain microvascular endothelial cells (HBMEC). Digital fluorescence microscopy and fura 2 were used to monitor intracellular Ca2+ concentration ([Ca2+]i) changes in response to thrombin and PAR1-activating peptide (PAR1-AP) SFFLRN. Both thrombin and PAR1-AP induced a dose-dependent [Ca2+]i rise that was inhibited by pretreatment of HBMEC with the phospholipase C inhibitor U-73122 and the sarco(endo)plasmic reticulum Ca2+-ATPase inhibitor thapsigargin. Thrombin induced transient [Ca2+]i increase, whereas PAR1-AP exhibited sustained [Ca2+]i rise. The PAR1-AP-induced sustained [Ca2+]i rise was significantly reduced in the absence of extracellular calcium or in the presence of an inhibitor of store-operated calcium channels, SKF-96365. Restoration of extracellular Ca2+ to the cells that were initially activated by PAR1-AP in the absence of extracellular Ca2+ resulted in significant [Ca2+]i rise; however, this effect was not observed after thrombin stimulation. Pretreatment of the cells with a low thrombin concentration (0.1 nM) prevented [Ca2+]i rise in response to high thrombin concentration (10 nM), but pretreatment with PAR1-AP did not prevent subsequent [Ca2+]i rise to high PAR1-AP concentration. Additionally, treatment with thrombin decreased transendothelial electrical resistance in HBMEC, whereas PAR1-AP was without significant effect. These findings suggest that, in contrast to thrombin, stimulation of PAR1 by untethered peptide SFFLRN results in stimulation of store-operated Ca2+ influx without significantly affecting brain endothelial barrier functions.
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PMID:Differential Ca2+ signaling by thrombin and protease-activated receptor-1-activating peptide in human brain microvascular endothelial cells. 1294 24

Intracellular free Ca2+ concentration ([Ca2+]c) is finely regulated by several mechanisms that either increase or reduce [Ca2+]c. Two different Ca2+ pumps have been described so far as the main mechanisms for Ca2+ removal from the cytosol, either by its sequestration into the stores, mediated by the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) or by Ca2+ extrusion to the extracellular medium, by the plasma membrane Ca2+-ATPase (PMCA). We have used inhibitors of these pumps to analyze their Ca2+ clearance efficacy in human platelets stimulated by the physiological agonist thrombin. Results demonstrate that, after platelet stimulation with thrombin, activation of SERCA precedes that of PMCA, although the ability of PMCA to remove Ca2+ from the cytosol last longer than that of SERCA. The efficacy of SERCA and PMCA removing Ca2+ from the cytosol is reduced when the concentration of thrombin increases. This phenomenon correlates with the greater increase in [Ca2+]c induced by higher concentrations of thrombin, which further confirms that SERCA and PMCA activities are regulated by [Ca2+]c.
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PMID:Collaborative effect of SERCA and PMCA in cytosolic calcium homeostasis in human platelets. 1666 48