Platelet Calcium Research Articles

Properties and regulation of human platelet cation channels.

The stimulation of calcium influx by various human platelet agonists which differ in their activation pathways was investigated. ADP activates a receptor-operated cation channel (ROC) and stimulates a phospholipase C (PLC)/inositol-trisphosphate (IP3)-mediated calcium mobilization associated with a secondary calcium influx. Thrombin only stimulates the PLC/IP3-mediated calcium mobilization and associated calcium influx, perhaps followed by an additional phase of calcium influx. The platelet calcium response after incubation with the thromboxane A2 mimetic U 46619 is similar but more transient compared to that after thrombin stimulation. Tert-butylhydroquinone (an inhibitor of endoplasmatic reticulum Ca(2+)-ATPases and cyclooxygenase) elevates cytosolic calcium levels by emptying intracellular calcium stores and stimulates a biphasic calcium influx. Activation of platelet cAMP- and cGMP-dependent protein kinases inhibits the ADP- and thrombin-evoked, calcium store-associated cation influx, but not the fast receptor operated cation influx induced by ADP. Experiments with various ADP-analogs, ATP and ATP-gamma-S suggest that two different ADP-receptors may mediate the calcium responses in human platelets.

Effect of inhibition of Na, K-ATPase on cytosolic free sodium and calcium in platelets of spontaneously hypertensive rats.

Cytosolic free sodium concentrations ([Na+]i) in intact platelets of 18 spontaneously hypertensive rats (SHR) and of 18 age-matched normotensive Wistar-Kyoto rats (WKY) were measured using the sodium-sensitive fluorescent dye sodium-binding-benzofuran-isophthalate. In resting platelets [Na+]i tended to be higher in SHR compared to WKY (20.5 +/- 3.5 mmol/L v 15.1 +/- 1.9 mmol/L, mean +/- SEM), but the differences were not statistically significant. Stimulation of the Na-H-exchange by 1.0 U/mL thrombin increased [Na+]i in SHR by 22.9 +/- 4.3 mmol/L and in WKY by 35.0 +/- 5.6 mmol/L in a similar way. After inhibition of Na, K-ATPase by 1 mmol/L ouabain there was a significant rise of [Na+]i both in platelets of SHR to 38.0 +/- 5.1 mmol/L (P < .01 compared to resting platelets) and in platelets of WKY to 26.5 +/- 4.3 mmol/L (P < .01). However, no significant difference could be observed between these two groups. Using the calcium-sensitive dye fura-2, resting cytosolic free calcium concentrations ([Ca2+]i) were found to be significantly higher in platelets of SHR compared to WKY (171.9 +/- 21.5 nmol/L v 93.14 +/- 19.7 nmol/L, P < .05). After the addition of ouabain [Ca2+]i was significantly higher in SHR compared to WKY (245.5 +/- 32.6 nmol/L v 159.6 +/- 22.5 nmol/L, P < .05). The results do not support the hypothesis that altered sodium-calcium exchange causes elevated cytosolic free calcium in SHR.

Thrombin-induced platelet calcium mobilization is enhanced in bipolar disorders

Thrombin-induced platelet calcium mobilization is enhanced in bipolar disorders

Low sodium diet in essential hypertension. Effect on blood cell ions and hemodynamic parameters.

The influence of salt restriction for 3 months on blood pressure, peripheral vascular resistance (observed by occlusive plethysmography), erythrocyte sodium, platelet calcium, and pH, was studied in eight untreated essential hypertensive patients. A low salt diet decreases blood pressure, vascular resistances, erythrocyte sodium, and platelet calcium, but not platelet pH. A strong positive correlation was noted between baseline platelet calcium and vascular resistances (r = 0.95, P less than .01). But during the salt restriction period, a negative correlation has been observed between the changes in these parameters, which casts doubt on the use of the platelet as a model of the smooth muscle cell.

Pharmacodynamics of salmon calcitonin in humans: new markers of pharmacological activity.

In order to define the pharmacodynamic profile of salmon calcitonin (sCT) in humans, several markers of the biological activity of the drug have been studied, namely cAMP, adenosine and pO2 in venous blood, and the cytosolic free calcium level in circulating cells. Different dosages and routes of administration (1.5 IU.kg-1 and 0.75 IU kg-1 IM, and 1.5 IU.kg-1 via nasal spray) were compared. sCT caused an increase in cAMP, adenosine and pO2, and a decrease in cytosolic free calcium in neutrophils, lymphocytes and platelets. The peak times of all these parameters ranged between 109 and 182 min, and 101 and 168 min after IM and nasal spray administration respectively. There was greater variability in the values after IM than nasal spray of administration of sCT. It is concluded that adenosine and pO2 in venous blood, and cytosolic free calcium in circulating cells are valuable markers of the activity of sCT and that sCT decreases the cytosolic free calcium level in neutrophils, lymphocytes and platelets. Pharmacodynamic analysis of the biological effects of the drug is highly reliable and valuable in predicting its pharmacological profile. sCT administration via a nasal spray is able to produce significant biological effects, although they are less marked than after IM dosing.

Shear stress-induced von Willebrand factor binding to platelet glycoprotein Ib initiates calcium influx associated with aggregation

Platelets subjected to elevated levels of fluid shear stress in the absence of exogenous agonists will aggregate. Shear stress-induced aggregation requires von Willebrand factor (vWF) multimers, extracellular calcium (Ca2+), adenosine diphosphate (ADP), and platelet membrane glycoprotein (GP)Ib and GPIIb-IIIa. The sequence of interaction of vWF multimers with platelet surface receptors and the effect of these interactions on platelet activation have not been determined. To elucidate the mechanism of shear stress-induced platelet aggregation, suspensions of washed platelets were subjected to different levels of uniform shear stress (15 to 120 dyne/cm2) in an optically modified cone and plate viscometer. Cytoplasmic ionized calcium ([Ca2+]i) and aggregation of platelets were monitored simultaneously during the application of shear stress; [Ca2+]i was measured using indo-1 loaded platelets and aggregation was measured as changes in light transmission. Basal [Ca2+]i was approximately 60 to 100 nmol/L. An increase of [Ca2+]i (up to greater than 1,000 nmol/L) was accompanied by synchronous aggregation, and both responses were dependent on the shear force and the presence of vWF multimers. EGTA chelation of extracellular Ca2+ completely inhibited vWF-mediated [Ca2+]i and aggregation responses to shear stress. Aurin tricarboxylic acid, which blocks the GPIb recognition site on the vWF monomer, and 6D1, a monoclonal antibody to GPIb, also completely inhibited platelet responses to shear stress. The tetrapeptide RGDS and the monoclonal antibody 10E5, which inhibit vWF binding to GPIIb-IIIa, partially inhibited shear stress-induced [Ca2+]i and aggregation responses. The combination of creatine phosphate/creatine phosphokinase, which converts ADP to adenosine triphosphate and blocks the effect of ADP released from stimulated platelets, inhibited shear stress-induced platelet aggregation without affecting the increase of [Ca2+]i. Neither the [Ca2+]i nor aggregation response to shear stress was inhibited by blocking platelet cyclooxygenase metabolism with acetylsalicylic acid. These results indicate that GPIb and extracellular Ca2+ are absolutely required for vWF-mediated [Ca2+]i and aggregation responses to imposed shear stress, and that the interaction of vWF multimers with GPIIb-IIIa potentiates these responses. Shear stress-induced elevation of platelet [Ca2+]i, but not aggregation, is independent of the effects of release ADP, and both responses occur independently of platelet cyclooxygenase metabolism. These results suggest that shear stress induces the binding of vWF multimers to platelet GPIb and this vWF-GPIb interaction causes an increase of [Ca2+]i and platelet aggregation, both of which are potentiated by vWF binding to the platelet GPIIb-IIIa complex.

Shear Stress-Induced von Willebrand Factor Binding to Platelet Glycoprotein lb Initiates Calcium Influx Associated With Aggregation

Abstract Platelets subjected to elevated levels of fluid shear stress in the absence of exogenous agonists will aggregate. Shear stress-induced aggregation requires von Willebrand factor (vWF) multimers, extracellular calcium (Ca2+), adenosine diphosphate (ADP), and platelet membrane glycoprotein (GP)Ib and GPIIb-IIIa. The sequence of interaction of vWF multimers with platelet surface receptors and the effect of these interactions on platelet activation have not been determined. To elucidate the mechanism of shear stress-induced platelet aggregation, suspensions of washed platelets were subjected to different levels of uniform shear stress (15 to 120 dyne/cm2) in an optically modified cone and plate viscometer. Cytoplasmic ionized calcium ([Ca2+]i) and aggregation of platelets were monitored simultaneously during the application of shear stress; [Ca2+]i was measured using indo-1 loaded platelets and aggregation was measured as changes in light transmission. Basal [Ca2+]i was approximately 60 to 100 nmol/L. An increase of [Ca2+]i (up to greater than 1,000 nmol/L) was accompanied by synchronous aggregation, and both responses were dependent on the shear force and the presence of vWF multimers. EGTA chelation of extracellular Ca2+ completely inhibited vWF-mediated [Ca2+]i and aggregation responses to shear stress. Aurin tricarboxylic acid, which blocks the GPIb recognition site on the vWF monomer, and 6D1, a monoclonal antibody to GPIb, also completely inhibited platelet responses to shear stress. The tetrapeptide RGDS and the monoclonal antibody 10E5, which inhibit vWF binding to GPIIb-IIIa, partially inhibited shear stress-induced [Ca2+]i and aggregation responses. The combination of creatine phosphate/creatine phosphokinase, which converts ADP to adenosine triphosphate and blocks the effect of ADP released from stimulated platelets, inhibited shear stress-induced platelet aggregation without affecting the increase of [Ca2+]i. Neither the [Ca2+]i nor aggregation response to shear stress was inhibited by blocking platelet cyclooxygenase metabolism with acetylsalicylic acid. These results indicate that GPIb and extracellular Ca2+ are absolutely required for vWF-mediated [Ca2+]i and aggregation responses to imposed shear stress, and that the interaction of vWF multimers with GPIIb-IIIa potentiates these responses. Shear stress-induced elevation of platelet [Ca2+]i, but not aggregation, is independent of the effects of release ADP, and both responses occur independently of platelet cyclooxygenase metabolism. These results suggest that shear stress induces the binding of vWF multimers to platelet GPIb and this vWF-GPIb interaction causes an increase of [Ca2+]i and platelet aggregation, both of which are potentiated by vWF binding to the platelet GPIIb-IIIa complex.

Cyclic nucleotides and intracellular-calcium homeostasis in human platelets.

The relationship between agonist-sensitive calcium compartments and those discharged by the Ca(2+)-ATPase inhibitor thapsigargin were studied in human platelets. In this context, calcium mobilization from intracellular pools and manganese influx was investigated in relation to the effect of altered cyclic-nucleotide levels. For maximal calcium release from intracellular stores, thapsigargin, compared to a receptor agonist like thrombin, requires the platelet's self-amplification mechanism, known to generate thromboxane A2. With this lipid mediator formed, thapsigargin released calcium and stimulated manganese influx in a manner similar to thrombin. Blocking the thromboxane receptor by addition of sulotroban (BM13.177) or, alternatively, increasing platelet cAMP or cGMP using prostacyclin or sodium nitroprusside, dramatically reduced the ability of thapsigargin to release calcium from intracellular compartments. The same experimental conditions significantly reduced the rate of manganese influx initiated by thapsigargin compared to thrombin. The experiments indicate that thapsigargin-sensitive compartments play only a minor role in inducing manganese influx compared to the receptor-sensitive compartment. Cyclic nucleotides accelerate the redistribution of an agonist-elevated platelet calcium into the thapsigargin-sensitive compartment, from which calcium can be released by inhibition of the Ca(2+)-ATPase. In human platelets, thapsigargin-induced calcium increase and influx were responsible for only part the calcium release resulting from inhibition of the corresponding ATPase; another part results from the indirect effect of thapsigargin acting via thromboxane-A2-receptor activation. Cyclic nucleotides are therefore an interesting regulatory device which can modify the thapsigargin response by not allowing the self-amplification mechanism of platelets to operate.

Humoral factor, intraplatelet calcium, and hypertension.

Plasma humoral factors which modulate the transmembrane distribution of sodium and calcium have been identified in hypertensive patients and have been hypothesized to be involved in the etiology of essential hypertension. In cross-incubation experiments, we have found that plasma of hypertensive subjects elevated basal and stimulated intraplatelet calcium levels, while plasma of normal subjects has an opposite effect on platelets from hypertensives. Basal intraplatelet calcium in normal platelets was 108 +/- 5 nmol/L and rose to 142 +/- 3 nmol/L (P less than .001) after incubation in plasma from hypertensive patients. Platelets from hypertensive patients had basal calcium levels of 182 +/- 11 nmol/L which fell to 127 +/- 11 nmol/L (P less than .01) after incubation in normal plasma. Hypertensive plasma potentiated the rise in intraplatelet calcium in response to ADP and PAF. Hypertensive patients treated experimentally with plasmapheresis exhibited a disappearance of the plasma factor responsible for elevating intraplatelet calcium. These results indicate the presence of a plasma humoral factor in hypertensives which elevates intraplatelet calcium and sensitizes platelets to agonist stimuli.

Influence of calcium antagonists on thrombin-induced calcium mobilization and platelet-vessel wall interactions.

Elevation of cytosolic ionized calcium plays a critical role in human platelet activation. We have evaluated three well-characterized calcium antagonists for their ability to prevent thrombin-induced calcium mobilization in Fura 2 AM-loaded platelets and also their ability to inhibit platelet-vessel wall interactions. Thrombin (0.2 U/ml) caused significant elevation of cytosolic calcium (basal 84 +/- 18, activated 546 +/- 76 nM; n = 3). Verapamil, diltiazem, and nifedipine (100 microM) did not exert any inhibitory effect on thrombin-mediated calcium elevation. Untreated platelets perfused through a Baumgartner chamber containing a rabbit aorta preparation reacted with exposed and denuded subendothelium. The percentage of the total area covered by control platelet thrombi was 39.6 +/- 3.4. Diltiazem and Nifedipine significantly reduced the percentage of area covered by platelet thrombi, but the drugs were not as effective as aspirin (8.2 +/- 1.4). Calcium antagonists studied did not inhibit thrombin-stimulated elevation of cytosolic calcium in blood platelets. Although these drugs have been shown to prevent in vitro platelet aggregation and offer some protection against risks for atherosclerosis and thrombosis, they failed to significantly inhibit platelet-vessel wall interactions leading to formation of spread platelets and aggregates.

Platelet—neutrophil interactions: Their significance

In the vicinity of an acute inflammatory response both cellular and non-cellular elements may interact to modify the overall response. Evidence suggests that leukocytes may play an active role in the modulation of platelet function and vice-versa. This interaction may be abnormal in certain pathological states. Neutrophils have been found to alter platelet behaviour by several mechanisms. These include transcellular metabolism of eicosanoids. Neutrophils utilize platelet-derived arachidonate to increase leukotriene synthesis. Other arachidonate metabolites result from platelet-neutrophil interaction and these differ quantitatively and qualitatively from those arising from either cell-type alone. Another mechanism is the release of a nitric oxide-like factor by neutrophils. Nitric oxide inhibits platelet adhesion and aggregation via guanylate cyclase stimulation. Neutrophils, under different conditions, are potent inducers of platelet calcium flux, aggregation and secretion. This activity is mediated by a neutrophil-derived protease, most likely to be cathepsin G. The interaction of platelets with neutrophils may help to explain some of the pathophysiological events associated with different clinical states.

Correction: Platelet Calcium Metabolism in the Prediction of Preeclampsia

Correction: Platelet Calcium Metabolism in the Prediction of Preeclampsia

Protein phosphorylation and intracellular free calcium in platelets of patients with essential hypertension.

Platelet intracellular free calcium concentration [Ca2+]i from patients with essential hypertension has been found to be elevated, but the intracellular effects of this increase are still unclear. As protein phosphorylation is an important regulatory step in cell activation and increased protein phosphorylation has been demonstrated in platelets from hypertensive animals, we investigated protein phosphorylation and [Ca2+]i in platelets from patients with essential hypertension and age-matched normotensives. We measured the 32P incorporation into a 20 kDa protein and a 47 kDa protein in 17 hypertensive patients and 20 normotensive, age-matched subjects. The [Ca2+]i was measured with the fluorescent dye fura-2. Protein phosphorylation and [Ca2+]i were assessed in unstimulated platelets and after exposure of the cells to 0.1 and 0.25 U/mL thrombin at 20, 60, and 300 sec. In addition we assessed the activity of protein kinase C by incubating the platelets with phorbol-ester TPA at 20, 60, and 300 sec. Basal phosphorylation of the two proteins was not different between the two groups. After exposure of the platelets to thrombin 32P, incorporation into the 20 kDa protein and the 47 kDa protein was significantly increased in platelets from hypertensive patients at all times. Furthermore, the specific stimulation of protein kinase C with TPA resulted in a significantly higher phosphorylation of the 47 kDa protein, whereas the 20 kDa protein was not phosphorylated after incubation with TPA for 1 min. Basal [Ca2+]i was higher in platelets from hypertensive patients (124 +/- 7 nmol/L v 104 +/- 5 nmol/L, P less than .05), although there was a wide overlap between the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of prostaglandins with adenosine diphosphate induced increase in cytosolic free calcium in human platelets.

The interaction of prostaglandins with changes in cytosolic Ca2+ concentration ([Ca2+]) and aggregation of human platelets induced by adenosine diphosphate (ADP) were investigated. Cytosolic [Ca2+] was measured with the fluorescent dye Quin2. Addition of ADP (0.25-2.5 mumol l-1) to platelet suspensions produced a dose dependent increase in cytosolic [Ca2+] from a basal level of 51 +/- 1 nmol l-1 to maximum levels exceeding 1 mumol l-1 and induced platelet aggregation. Chelation of extracellular calcium with 100 mumol l-1 EGTA markedly reduced the increase in cytosolic [Ca2+] induced by 0.25 mumol l-1 ADP, while pretreatment with the calcium entry blocker verapamil was without effect. Stimulation of cyclic AMP with prostaglandins (PGD2, PGE1, PGE2, PGI2, but not PGF2 alpha) and forskolin, or incubation with dibutyryl-cAMP, inhibited the rise in cytosolic [Ca2+] and platelet aggregation following ADP. We conclude that prostaglandins inhibit the increase in cytosolic [Ca2+] and aggregation of human platelets induced by ADP, probably by stimulation of cyclic AMP generation, thereby opposing the mechanism by which ADP increases cytosolic [Ca2+] and subsequently induces platelet aggregation.

Pharmacological interactions between serotonin and calcium in blood platelets

Pharmacological interactions between serotonin and calcium in blood platelets

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.

Evaluation of platelet calcium ion mobilization by the use of various divalent ions

Divalent ion mobilization in human platelets was evaluated with Fura-2 fluorescence changes induced by Ca 2+, Sr 2+, Ba 2+ and Mn 2+. Extracellular Ca 2+, Sr 2+ and Ba 2+ all entered thrombin-stimulated platelets. These divalent ions were also able to refill the intracellular Ca 2+ storage sites which had been depleted of Ca 2+ by ionomycin treatment, and were released from the storage sites upon thrombin stimulation. However, only the refilling of the storage sites with Ca 2+ and Sr 2+, but not with Ba 2+, were capable of suppressing the opening state of Ca 2+ channels assessed with Mn 2+ influx. Efflux of intracellularly accumulated divalent ions was observed with Ca 2+ and Sr 2+ but not with Ba 2+. These findings indicate that there are subtle differences in the Ca 2+-binding domains of the various systems involved in Ca 2+ mobilization in platelets, some of which discriminate Ba 2+ while accepting Sr 2+.

Effect of a high concentration perfluorocarbon emulsion on platelet function.

Perfluorocarbon (PFC) and lipid emulsions (eg. Fluosol, Intralipid,) containing phospholipid have been reported to modify platelet function after intravenous infusion. Platelet activation might be responsible for the generation of mediators responsible for PFC-induced side effects. In view of this, we investigated the effect of a highly concentrated perfluorocarbon emulsion containing 90% (w/v) perfluorooctylbromide (perflubron; PFOB), on porcine and human platelet activation and function. We measured both: a) stimulated ex-vivo porcine platelet aggregation pre and post infusion of either perflubron or control (vehicle only) emulsions, and b) stimulated in vitro human platelet calcium flux in the presence of perflubron emulsion or control emulsions. Platelet aggregation stimulated by collagen, ADP or arachidonic acid (AA) was inhibited in ex-vivo porcine platelets following infusion of perflubron emulsion at a dose of 3 ml/kg (0.2 ml/kg/min). Inhibition was dose dependent and was decreased when the dose of perflubron emulsion was reduced to 1.0 or 0.3 ml/kg. Infusion of saline or "vehicle" emulsions had little or no effect on stimulated ex-vivo pig platelet aggregation. Fluosol, infusion was associated with inconsistent inhibition of platelet aggregation. A23187- or AA-stimulated calcium flux of human platelets in vitro was inhibited in the presence of 1% (v/v) perflubron emulsion. Similar effects were seen with Fluosol, or Intralipid. This inhibition was agonist dose-dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

Recent developments in pregnancy-induced hypertension

Pregnancy-induced hypertension (PIH) still remains an area in obstetrics of active research and investigation. Despite widespread academic attention, the cause of this disorder still remains unknown. The purpose of this paper is to review the important contributions to the literature during the period of July, 1990 through June, 1991. Elucidation of the pathophysiology of PIH has been enhanced by investigations of altered platelet calcium metabolism, the renin-aldosterone-angiotensin system, and other potent vasopressors. Recent reports of clinical management for eclampsia, liver rupture, HELLP syndrome, severe PIH in the second trimester, severe hypertension, and magnesium toxicity are presented.

Significance of intracellular free calcium and magnesium and calcium-regulating hormones with sodium chloride loading in patients with essential hypertension.

This study was designed: (1) to test the hypothesis that the pressor response to sodium chloride loading in patients with essential hypertension is associated with disorder of divalent cations (calcium and magnesium); and (2) to clarify the relationship between intracellular free calcium concentration and serum levels of calcium-regulating factors and intracellular magnesium concentration. With sodium chloride loading, mean blood pressure, urinary calcium and magnesium excretions and platelet calcium concentration were increased, and serum total and ionized calcium, magnesium concentrations and erythrocyte magnesium concentration were decreased. Change in mean blood pressure was correlated with changes in parathyroid hormone (r = 0.49, P less than 0.05), serum total and ionized calcium (r = -0.50, P less than 0.05) and erythrocyte magnesium (r = -0.56, P less than 0.05) and platelet calcium concentrations (r = 0.46, P less than 0.05). In addition, change in platelet calcium concentration was related to changes in parathyroid hormone (r = 0.44, P = 0.05), serum total and ionized calcium (r = -0.66, P less than 0.01) and erythrocyte magnesium concentration (r = -0.49, P less than 0.05). It is concluded that the pressor effect of excessive sodium chloride intake on blood pressure in patients with essential hypertension is associated with a disorder of divalent cations and that alteration of the intracellular free calcium concentration with sodium chloride loading may occur through handling of serum total and ionized calcium, parathyroid hormone and/or intracellular magnesium concentration.