Calcium Mobilization In Platelets Research Articles

Effects of Cyclic Nucleotides and Protein Kinases on Platelet Calcium Homeostasis and Mobilization

(1993). Effects of Cyclic Nucleotides and Protein Kinases on Platelet Calcium Homeostasis and Mobilization. Platelets: Vol. 4, No. 5, pp. 231-242.

Increased internal calcium mobilization in platelets of patients with chronic renal failure.

Platelet free calcium concentrations ([Ca2+]i) were measured with Fura-2 to elucidate the intracellular calcium kinetics in patients with renal disease. There were no significant differences of the resting [Ca2+]i among the control subjects (C) (n = 12), patients with chronic glomerulonephritis (CGN) (n = 8), and patients with chronic renal failure (CRF) (n = 12). In all groups, platelets [Ca2+]i were significantly increased by agonists (thrombin, adenosine diphosphate) compared with their respective basal level. Thrombin-induced [Ca2+]i rise was significantly higher in CRF (840 +/- 265 nM) than in C (600 +/- 163) and CGN (562 +/- 137). Also adenosine diphosphate elicited similar responses. In the presence of calcium chelator in the incubation buffer, the elevation of [Ca2+]i after thrombin stimulation was statistically higher in CRF (469 +/- 85 nM) than in C (275 +/- 60) and CGN (301 +/- 41). These findings suggest that platelets of CRF were capable of increasing [Ca2+]i in response to agonists, through further mobilization of calcium from the intracellular pool rather than the elevation of transmembrane calcium influx.

Thrombin-induced platelet calcium mobilization is enhanced in bipolar disorders

Thrombin-induced platelet calcium mobilization is enhanced in bipolar disorders

Calcium Entry in Nonexcitable Cells: Lessons from Human Platelets

A rise in cytosolic calcium concentration plays an important role in platelet activation. As well as helping define stimulus-response coupling in these intriguing and clinically important cells, the study of calcium mobilisation in platelets serves as an important model for elucidating calcium signaling mechanisms in general.

Calcium mobilization in platelets from schizophrenic and healthy subjects. Regulation by lithium and neuroleptics

Intracellular free calcium concentrations ([Ca(2+)](1)) were measured in platelets from healthy volunteers before and after adding thrombin, chlorpromazine, haloperidol and/or lithium, and in platelets from DSM-III-R diagnosed schizophrenic patients receiving neuroleptic medication. Thrombin increased [Ca(2+)]( 1) in a dose- dependent fashion. Chlorpromazine and haloperidol also mobilized Ca(2+) in a dose-dependent fashion, and augmented the response to low doses of thrombin without changing the maximal response to thrombin. The effects of all three drugs were not additive, suggesting that they affected the same intraplatelet calcium pool; most likely the dense tubular system. Lithium also increased [Ca(2+) ] but without affecting the response to thrombin, chlorpromazine or haloperidol. The effects of the latter three drugs were additive to that of lithium, suggesting that lithium was acting on a different calcium pool. The response to thrombin was significantly lower in platelets from schizophrenic patients than in platelets from healthy volunteers. Further studies are required to explore potential causes for this observation. Such causes include schizophrenia per se and chronic neuroleptic treatment.

Heterologous supersensitization between serotonin2 and alpha 2-adrenergic receptor-mediated intracellular calcium mobilization in human platelets.

Recent reports suggest that serotonin (5-HT)2 receptor-mediated second messenger systems are enhanced in platelets of affective disorders. To make the mechanism of the enhanced response clear, we investigated 5-HT2 and alpha (alpha) 2-adrenergic receptor-induced intracellular calcium (Ca2+) mobilization in platelets of healthy volunteers, using fura-2. 5-HT2 and alpha 2-adrenergic receptor-mediated Ca2+ mobilization was enhanced by prior exposure to the other type of agonist, so called "heterologous supersensitization." The supersensitization was due to the enhancement of maximal response without change in agonist affinity. Chelating extracellular Ca2+ did not diminish the supersensitization. This enhancement of Ca2+ mobilization was not inhibited by H-7, an inhibitor of protein kinase C. However, this supersensitization was inhibited by pretreatment with sodium fluoride which directly activates guanine nucleotide binding regulatory proteins (G proteins). These results suggest that the supersensitization was caused from intracellular Ca2+ storage sites through a G protein-coupled pathway.

Abnormal intracellular calcium ion concentration in platelets and lymphocytes of bipolar patients

The authors measured intracellular Ca2+ concentrations in four manic and five bipolar depressed patients and seven comparison subjects. Platelet and lymphocyte intracellular Ca2+ concentrations were comparable. The patients' mean intracellular Ca2+ concentrations were higher than those of the comparison subjects and demonstrated more interindividual variation. These findings suggest a diffuse abnormality in mechanisms affecting intracellular calcium homeostasis in bipolar disorder.

External Na + is not required for Ca 2+ mobilization in platelets stimulated with rattlesnake lectin or α-thrombin

The effects of extracellular sodium on platelet aggregation and calcium mobilization in platelets stimulated with either rattlesnake ( Crotalus atrox ) lectin (RSL) or α-thrombin were compared. The absence of extracellular sodium had no effect on platelet aggregation or calcium mobilization in response to all levels of RSL tested. In contrast platelet aggregation was sodium-dependent in response to ≤.2 units/ml α-thrombin. Surprisingly, calcium mobilization occurred in platelets treated with a threshold level of α-thrombin in the absence of external sodium. Thus sodium-dependent platelet aggregation in response to a low dose of thrombin apparently is not the result of sodium-dependent calcium mobilization.

Calcium mobilization in human platelets by receptor agonists and calcium-ATPase inhibitors

Inhibitors of the endoplasmic reticulum Ca 2+-ATPase like thapsigargin (TG) and 2,5-di ( tert-butyl)-1,4-benzohydroquinone ( tBuBHQ) cause increases in cytosolic calcium in intact human platelets resulting from prevention of reuptake. A maximal concentration of TG (0.2 μM) mobilized 100% of sequestered Ca 2+ compared to the action of a receptor agonist like thrombin (0.1 U/ml). A maximal dose of tBuBHQ (50 μM) stimulated release of about 40% of intracellular calcium compared to thrombin and TG. The reduced ability of tBuBHQ to release calcium can be explained with an inhibitory effect on the cyclooxygenase pathway ( K i ≈ 7 μM). Thererore tBuBHQ is not able to cause platelet aggregation compared to TG. In the presence of a cyclooxygenase inhibitor or a thromboxane A 2 receptor antagonist the action of TG is identical to that observed with tBuBHQ. Generally, inhibition of calcium sequestration does not automatically result in platelet activation. In contrast to a receptor mediated activation Ca 2+-ATPase inhibitors require the self-amplification mechanism of endogenously formed thromboxane A 2 to cause a similar response. We conclude that the calcium store sensitive to Ca 2+-ATPase inhibitors is a subset of the receptor sensitive calcium pool.

Increased intracellular calcium mobilization in platelets from patients with Type 2 (non-insulin-dependent) diabetes mellitus

Enhanced platelet functions have been reported in patients with diabetes mellitus. Our recent study demonstrated that phosphoinositide turnover is increased in platelets from diabetic patients. In the present study, we evaluated the abnormality in platelet intracellular calcium mobilization in patients with Type 2 (non-insulin-dependent) diabetes mellitus using fura-2, a fluorescent calcium indicator. Washed platelets were prepared from six diabetic patients with increased platelet aggregation rates (DM-A group), seven diabetic patients with normal platelet aggregation rates (DM-B group), and eight age-matched healthy control subjects. The basal intracellular free calcium concentrations in platelets were similar among the three groups. Thrombin (0.025-0.1 U/ml) induced a dose-dependent increase in intracellular calcium in both the presence and the absence of extracellular calcium. This increase in the presence of extracellular calcium, which depends on calcium influx and release, was significantly higher in the DM-A group than in the DM-B and control groups. However, there was no significant difference between the control group and the DM-B group. In the absence of extracellular calcium, thrombin-induced calcium increase, which depends only on calcium release, was also significantly enhanced in the DM-A group. Furthermore, the calcium increase stimulated by platelet-activating factor (10 nmol/l) with and without extracellular calcium was significantly higher in the DM-A group than in the other groups. Additionally, calcium ionophore A23187 (100 nmol/l) caused a significantly higher calcium increase in the DM-A group with extracellular calcium, while the calcium increase without extracellular calcium showed no significant difference among the three groups. These observations suggest that enhanced intracellular calcium mobilization due to increased calcium influx and release may be closely related to platelet hyperfunctions in diabetes mellitus.

Influence of heparins on inositol 1,4,5-trisphosphate-induced calcium mobilization in permeabilized human platelets

Heparin has been shown to prevent inositol 1,4,5-trisphosphate (IP3) binding to its receptor and to inhibit IP3-induced calcium mobilization in a variety of cells. Heparin added to whole blood at a concentration of 1 U/ml prevented thrombin-induced secretion of granule contents and irreversible aggregation of platelets. Heparin (2-15 kDa) had no inhibitory effect on IP3-induced calcium mobilization in Fura 2-loaded, saponin (10-15 micrograms/ml)-permeabilized platelets. None of the commercially available heparin preparations can induce inhibition of agonist-induced calcium mobilization in intact platelets because they are not cell permeant. Mild saponin treatment makes the membrane permeable to IP3, but restricts the action of heparins. Recent observations suggesting heparin's affinity to IP3 binding sites will be of clinical interest if effective cell permeant analogs can be developed.

Quasi-simultaneous measurement of ionized calcium and alpha-granule release in individual platelets

The effect of alpha-thrombin and ADP on calcium mobilization and alpha-granule release in individual platelets was investigated by flow cytometry. alpha-Thrombin (4.5 nM) caused a uniform rise of free cytosolic calcium ([Ca2+]i) among indo-1-loaded human platelets. Despite the uniformity of this effect, approximately 20% of the cells failed to secrete alpha-granule content, as shown by binding of fluorescein isothiocyanate (FITC)-conjugated S12 monoclonal antibody. ADP (10 microM) caused a similar brisk and uniform rise of calcium but did not increase S12 binding to any platelets. On the other hand, with alpha-thrombin (0.5 nM), calcium mobilization was heterogeneous and paralleled granule release. [Ca2+]i increased rapidly in some platelets, while only slowly in others. When an electronic gate was set according to FITC-S12 fluorescence, cells with a greater secretory response proved to be those with a higher calcium level. With both alpha-thrombin and ADP, chelation of external calcium by EGTA (2 mM) reduced calcium response of individual cells. NiCl2 (1 mM) also inhibited calcium rise of individual platelets to the same extent as EGTA (2 mM) in spite of the presence of 1 mM CaCl2 in the extracellular media. The effects of EGTA and NiCl2 were not limited to a particular subpopulation of cells. These data suggest that the putative Ni2(+)-inhibitable divalent cation channel(s) may be responsible for the increased influx of calcium that occurs during platelet activation by alpha-thrombin and ADP. It appears that these calcium channels contribute to the elevation of [Ca2+]i among virtually all the platelets.

D-indobufen inhibits collagen-induced intracellular calcium mobilization and inositol phosphates formation in human platelets

We obtained evidence that the cyclooxygenase inhibitor d-indobufen, (+)2[p-(1-oxo-2-isoindolinyl)phenyl] buthylic acid has a potent and specific inhibitory effect on collagen-induced aggregation and 40K-protein phosphorylation (Mamiya, S., Hagiwara, M., Ishikawa, T., and Hidaka, H. Thromb. Res. 54, 447, 1989) (1). In Fura-2 loaded platelets, d-indobufen inhibited collagen-induced intracellular calcium mobilization in a dose dependent manner and this inhibitory effect on calcium mobilization paralleled that on aggregation, either in the presence or absence of extracellular free calcium ions. This compound inhibited inositol phosphates (IPs) formation in collagen-stimulated platelets. In arachidonic acid-stimulated platelets, d-indobufen caused a lag on calcium mobilization, as observed with arachidonic acid-induced aggregation. There was no significant effect on thrombin- or A23187-induced calcium mobilization or on aggregation. These observations suggest that the collagen receptor couples to a distinct intracellular calcium mobilization system possibly via inositol phospholipid metabolism and that d-indobufen blocks the collagen-induced aggregation by arresting mobilization of intracellular calcium.

Influence of nitric oxide on agonist-mediated calcium mobilization in platelets

Recent studies have characterized endothelium-derived relaxing factor as nitric oxide. It appears to exert its effect by elevating intracellular levels of cyclic GMP. In this study we confirm that nitric oxide is a potent inhibitor of agonist-induced irreversible aggregation. At the concentrations tested nitric oxide effectively blocked thrombin-stimulated mobilization of cytosolic-free calcium in Fura 2-loaded platelets. In addition, nitric oxide prevented the inositol 1,4,5-trisphosphate-stimulated calcium rise in cytosolic calcium in saponin-permeabilized Fura 2-loaded platelets. Similar to the action of adenylate cyclase stimulators, nitric oxide facilitated lowering of calcium levels raised by the action of agonists. The specific mechanism by which it exerts its effect on intracellular levels of calcium is not clear.

Recombinant Human Erythropoietin Increases Blood Pressure, Platelet Aggregability and Platelet Free Calcium Mobilisation in Uraemic Children: A Possible Link?

Recombinant human erythropoietin was administered to 10 uraemic children on chronic haemodialysis, all of whom responded by correcting their haemoglobin. In addition, they showed an increase in blood pressure; platelet aggregations, subnormal before therapy, improved during treatment. The intracellular free calcium concentration in platelets after thrombin stimulation also increased significantly during erythropoietin administration. We hypothesize that the effect of erythropoietin on platelet aggregability and on blood pressure may be due to an increase in the intracellular free calcium mobilisation in platelets and possibly in smooth muscle cells respectively.

Calcium Mobilization in Human Platelets Using Indo-1 and Flow Cytometry

Abstract Regulation of cytoplasmic free calcium concentration is believed to be important in the response of platelets to external stimuli. A relatively new fluorescent calcium indicator, indo-1, has properties by which alterations of cytoplasmic calcium can be evaluated in single platelets by flow cytometry. Activation of platelets at a temperature lower than 37 degrees C allows examination of the heterogeneity of intracellular free calcium levels and can distinguish variations among platelets in the initiation, duration, and magnitude of calcium fluxes. The clear advantage of flow cytometric analysis of platelet cytosolic calcium is that stimulus-response coupling can now be studied on a single cell basis. Platelets were activated by addition of human alpha- thrombin or ADP at 37 degrees C or at room temperature (22 degrees C). Activation at 37 degrees C approaches more closely an in vivo response and, as expected, increases in cytosolic calcium occurred within seconds of agonist addition. Transient increases in cytoplasmic calcium levels occurred when platelets were challenged with a low concentration of agonist. Heterogeneity in cytoplasmic calcium levels was also observed at 10(-5) mol/L ADP and 0.1 U/mL alpha-thrombin. Some of this heterogeneity was no longer observed at higher concentrations of agonist (10(-4) mol/L ADP and 0.5 U/mL thrombin), suggesting that a sufficient magnitude of signal is required to induce changes in platelet cytosolic calcium. Light-scatter properties of the activated platelets were also monitored simultaneously and showed changes in response to both agonists. The ability to measure changes in cytoplasmic free calcium by ratio flow cytofluorimetry provides a new approach to study of the role of alterations in intracellular calcium in response to agonists acting through different membrane receptors as well as providing a sensitive technique to detect functional subpopulations of platelets.

Calcium mobilization in human platelets using indo-1 and flow cytometry

Regulation of cytoplasmic free calcium concentration is believed to be important in the response of platelets to external stimuli. A relatively new fluorescent calcium indicator, indo-1, has properties by which alterations of cytoplasmic calcium can be evaluated in single platelets by flow cytometry. Activation of platelets at a temperature lower than 37 degrees C allows examination of the heterogeneity of intracellular free calcium levels and can distinguish variations among platelets in the initiation, duration, and magnitude of calcium fluxes. The clear advantage of flow cytometric analysis of platelet cytosolic calcium is that stimulus-response coupling can now be studied on a single cell basis. Platelets were activated by addition of human alpha-thrombin or ADP at 37 degrees C or at room temperature (22 degrees C). Activation at 37 degrees C approaches more closely an in vivo response and, as expected, increases in cytosolic calcium occurred within seconds of agonist addition. Transient increases in cytoplasmic calcium levels occurred when platelets were challenged with a low concentration of agonist. Heterogeneity in cytoplasmic calcium levels was also observed at 10(-5) mol/L ADP and 0.1 U/mL alpha-thrombin. Some of this heterogeneity was no longer observed at higher concentrations of agonist (10(-4) mol/L ADP and 0.5 U/mL thrombin), suggesting that a sufficient magnitude of signal is required to induce changes in platelet cytosolic calcium. Light-scatter properties of the activated platelets were also monitored simultaneously and showed changes in response to both agonists. The ability to measure changes in cytoplasmic free calcium by ratio flow cytofluorimetry provides a new approach to study of the role of alterations in intracellular calcium in response to agonists acting through different membrane receptors as well as providing a sensitive technique to detect functional subpopulations of platelets.

Influence of sodium conductances on platelet activation

The effects of extracellular Na + and tetrodotoxin on resting membrane potential, cytosolic free Ca 2+ levels and aggregation of human platelets have been studied. Neither the decrease in extracellular Na +-concentration (from 140 mmol/l to 0 mmol/l) nor the addition of tetrodotoxin (10 −7 to 10 −5 mol/l) modified the platelet membrane potential. Zero extracellular Na + concentration or the presence of tetrodotoxin in the medium inhibited platelet aggregation; however, K +-depolarized platelets showed an unchanged aggregation induced by ADP or thrombin in media with zero or low extracellular Na + concentrations or in the presence of tetrodotoxin. Moreover, zero extracellular Na + concentration or tetrodotoxin inhibited calcium mobilization in platelets during activation induced by thrombin. Hence, voltage-dependent activation linked to Na + influx appears to be necessary for ADP- and thrombin-induced platelet aggregation under control conditions. Mechanisms for the role of Na + conductances in platelet function are discussed.

Nitroprusside differentially inhibits ADP-stimulated calcium influx and mobilization in human platelets

1. The effect of nitroprusside on cGMP concn., cAMP concn., shape change, aggregation, intracellular free Ca2+ concn. (by quin-2 fluorescence) and Mn2+ entry (by quenching of quin-2) was investigated in human platelets incubated with 1 mM-Ca2+ or 1 mM-EGTA. 2. Nitroprusside (10 nM-10 microM) caused similar concentration-dependent increases in platelet cGMP concn. and was without effect on cAMP concn. in the presence of extracellular Ca2+ or EGTA. 3. In ADP (3-6 microM)-stimulated platelets, nitroprusside caused 50% inhibition of shape change at 0.4 microM (+Ca2+) or 1.3 microM (+EGTA), aggregation at 0.09 microM (+Ca2+) and of increased intracellular Ca2+ at 0.02 microM (+Ca2+) or 2.1 microM (+EGTA). Entry of 1 mM-Mn2+ (-Ca2+) was inhibited by 80% by 5 microM-nitroprusside. 4. In ionomycin (20-500 nM)-stimulated platelets, nitroprusside (10 nM-100 microM) did not inhibit shape change or intracellular-Ca2+-increase responses, and only partially inhibited aggregation. 5. In phorbol myristate acetate (10 nM)-stimulated platelets, neither shape change nor aggregation was inhibited by 5 microM-nitroprusside. 6. The data demonstrate that nitroprusside inhibits ADP-mediated Ca2+ influx more potently than Ca2+ mobilization. Nitroprusside appears not to influence Ca2+ efflux or sequestration and not to affect the sensitivity of the activation mechanism to intracellular Ca2+ concn. or activation of protein kinase C.

Altered hemodynamic response to dobutamine in relation to the degree of preoperative β-adrenoceptor blockade: Tarnow J, Komar K. Anesthesiology 68:912, 1988

The effect of picotamide (G137 or N,N′-bis-3-picolyl-4-methoxy-isophthalamide), a dual thromboxane A2 (TxA2) synthetase inhibitor/TxA2 endoperoxide receptor antagonist, on the phospholipase C (PLC) activation and calcium mobilization in human platelets stimulated by arachidonic acid (AA) and TxA2 receptor synthetic agonist U46619, has been studied. Preincubation with picotamide (10−4 M) for 1 and 3 min significantly reduced (p<0.03 and p<0.005 respectively) the calcium concentration changes induced in gel-filtered platelets (GFPs) by U46619 125 nM and 250 nM. Picotamide also reduced the calcium concentration changes induced in GFPs by AA 75 and 150 μM and by ADP 5 and 10 μM. In thrombin degranulated platelets picotamide inhibited the effect of U46619 up to 500 nM. The PLC activation, as indicated by inositol-1,3,4 P3 (Ins 1,3,4 P3) formation in response to U46619 250 nM and AA 150 μM was also inhibited by picotamide. These results may suggest a dual effect of picotamide on the receptor/effector systems through which TxA2 mediates platelet activation.