Platelet Intracellular Free Calcium Concentration Research Articles

Platelet Hyperactivation in Maintained Growth Hormone-Deficient Childhood Patients after Therapy Withdrawal as a Putative Earlier Marker of Increased Cardiovascular Risk

GH deficiency (GHD) is associated with a higher risk of vascular disease, whose pathophysiological mechanisms remains not yet fully elucidated. This study aimed to assess the main cardiovascular risk indexes, plasma catecholamines content, and the platelet function in childhood-onset GHD patients. Some of the main clinical examinations related with cardiovascular risk, plasma catecholamines content, as well as platelet intracellular free calcium concentration ([Ca(2+)](i)), whole-blood aggregation, and morphology were evaluated in childhood-onset GHD patients treated with GH for a variable period and off GH therapy for at least 2 yr before entry into study and in sex-, age-, and body mass index-matched control groups. Among the patients, group 1 (GHD-1) has recovered GH levels after withdrawal, whereas group 2 (GHD-2) has remained GH deficient. Minor differences on the cardiovascular risk indexes were observed between the groups. Plasma catecholamine concentrations in the GHD groups did not statistically differ from the control group, but higher adrenaline content was observed in the GHD-2 group when compared with the GHD-1 one. Basal and thrombin-evoked [Ca(2+)](i) and platelet aggregation were identical between the GHD-1 group and the matched control. However, the GHD-2 group has increased thrombin-evoked [Ca(2+)](i) (297.0 +/- 15.7 Deltanmol/liter; P < 0.01), collagen, and ADP-induced platelet aggregation (33.3 +/- 4.3 and 12.5 +/- 2.1 Omega, respectively; P < 0.05) vs. the control-2 group (Delta[Ca(2+)](i): 102.1 +/- 13.6 Deltanmol/liter; aggregation: 19.6 +/- 2.9 and 6.2 +/- 0.8 Omega). The platelet hyperreactivity state in the GHD-2 was reinforced by morphologic studies of electron microscopy. In conclusion, there were minor differences between the GHD-1 group and the controls, which might be due to the recovery of GH levels after therapy withdrawal. However, the maintained GHD group, despite minor cardiovascular risk index differences, has increased [Ca(2+)](i) and aggregation, which could indicate a hyperactivation state that might be viewed as an earlier marker of cardiovascular disturbances.

Role of Protein Kinase C in the Anti-Aggregatory Effects of Endothelin-1 on Human Platelets

1. Endothelin-1 has anti-aggregatory properties, but the mechanism underlying this inhibitory action is unknown. This in vitro study investigates effects of endothelin-1 on thrombin-stimulated aggregation and intracellular free calcium concentration in human platelets and assesses the role of protein kinase C in the interactions between endothelin-1 and thrombin. Aggregation was measured turbidometrically and the intracellular free calcium concentration was determined with the fluorescent indicator fura 2-acetoxymethyl ester. 2. Endothelin-1 at concentrations from 10(-11) to 10(-6) mol/l had no effect on platelet aggregation or intracellular free calcium concentration but inhibited in a dose-dependent manner aggregation induced by 0.05 unit/ml thrombin (pD2 for inhibition by endothelin = 8.1 +/- 0.12). 3. Endothelin-1 at 10(-9) mol/l significantly decreased (P < 0.01) thrombin-stimulated aggregation from 81.4 +/- 1.5% (in the absence of endothelin-1) to 53.5 +/- 1.1% (in the presence of endothelin) and thrombin-stimulated intracellular free calcium concentration from 179 +/- 1.7 nmol/l to 140 +/- 1.8 nmol/l. 4. Preincubation of platelets with 10(-7) mol/l staurosporine (protein kinase C inhibitor), calphostin C (highly selective protein kinase C inhibitor) or 5-(N,N-hexamethylene) amiloride (highly selective Na(+)-H+ exchange blocker) significantly inhibited (P < 0.01) thrombin-stimulated platelet responses and suppressed the inhibitory effect of endothelin-1 on thrombin-induced aggregation and intracellular free calcium concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Platelet calcium homeostasis is abnormal in patients with severe arteriosclerosis.

To evaluate platelet calcium homeostasis in a typical thrombosis-prone clinical condition, 14 patients with severe arteriosclerosis and 11 healthy control subjects were studied. Platelet intracellular free calcium concentration ([Ca2+]i) was evaluated by means of the fluorescent probe fura 2 under resting conditions and after challenge with 0.05, 0.1, and 0.5 U/mL thrombin (final concentrations). Three different concentrations of extracellular ionized calcium ([Ca2+]e) were used: 1 mmol/L, 1 mumol/L, and < 1 nmol/L. Resting platelet [Ca2+]i was significantly higher (P < .001) in patients than in control subjects. After addition of 0.05 U/mL thrombin, the relative increase of [Ca2+]i was lower in patients than in control subjects in each of the three [Ca2+]e conditions (P = .05 at 1 mmol/L, P = .02 at 1 mumol/L, and P = .04 at < 1 nmol/L). After addition of 0.1 U/mL thrombin, the relative increase of [Ca2+]i was lower in patients than in control subjects under two [Ca2+]e conditions, 1 mumol/L and < 1 nmol/L (P = .04 and P = .03 respectively). With 0.5 U/mL thrombin, a trend toward lower values in patients than in control subjects was observed, reaching statistical significance (P = .03) only at < 1 nmol/L [Ca2+]e. These results suggest that calcium homeostasis is abnormal in platelets from patients with severe arteriosclerosis and probably reflects a chronic activation.

Effect of Prostacyclin on Platelet Intracellular Free Calcium Concentration of Hypertensive and Normotensive Humans

Platelet intracellular free calcium concentration ([Ca2+]i) has been reported to be increased in essential hypertensive patients (EHT) as compared with normotensive controls (NT). Prostacyclin (PGI2), which influences cellular Ca2+, has been reported to be reduced in EHT. This study tested the hypothesis that the resting level of platelet [Ca2+]i in humans is influenced by PGI2. We also investigated the role of PGI2 in regulating platelet [Ca2+]i of 28 EHT subjects compared to 28 NT controls. Platelet [Ca2+]i was measured using the fluorescent Ca2+ probe fura-2 under control conditions and a 10-min preincubation with PGI2. Simultaneous measurement of platelet cyclic-adenosine 3':5'-monophosphate (cAMP) was performed by radioimmunoassay. The resting level of platelet [Ca2+]i was significantly higher in EHT than in NT (32.7 +/- 1.4 v 28.3 +/- 0.9 nmol/L; P < .01). PGI2 from 30 nM to 1 mumol/L lowered the resting level of platelet [Ca2+]i in a dose-dependent manner (EHT -22.2 +/- 2.4, NT -22.9 +/- 2.3%, 1 mumol/L PGI2); however, no significant difference in platelet [Ca2+]i was observed between NT and EHT. While prostacyclin induced a transient rise in platelet cAMP, the magnitude of PGI2-induced cAMP level was similar between the two groups. These results do not support the hypothesis that endogenous PGI2 activity contributes to the increased level of platelet [Ca2+]i in EHT, although PGI2 incubation lowered the resting level of platelet [Ca2+]i.

Changes in platelet intracellular free calcium in normal pregnancy

To determine if platelet intracellular free calcium concentration (p.(Ca2+)i) changes with gestation in normotensive, uncomplicated pregnancy. A prospective, longitudinal study of primigravid pregnant women compared to a large control group consisting of nulliparous nonpregnant volunteers. The antenatal clinic at University Hospital, Queen's Medical Centre, Nottingham. Two groups of women were studied. Thirty-one nulliparous, nonpregnant women not using oral contraception were investigated in the early follicular phase of their menstrual cycles. Also, 24 primigravid women with normotensive, uncomplicated pregnancies were investigated on eight separate occasions during their pregnancies. A significant increase in mean p.(Ca2+)i was demonstrated by 28 weeks gestation in the pregnant women as compared to the nonpregnant control group and the same individuals when investigated at 12 weeks postpartum (P < 0.05; ANOVA). This change was maximal at 36 weeks gestation (P < 0.001; ANOVA); concentrations had returned to those not significantly different from nonpregnant women by six weeks postpartum. Basal platelet (Ca2+)i increases significantly by the third trimester of normal, primigravid pregnancy as compared to pregnancy values and postnatal values. These data mirror the previously described observations of platelet behaviour noting increased activity at this gestation of pregnancy. It may be that the increased basal p.(Ca2+)i indicates that smaller transient signals have to be generated to induce platelet activity, such as shape change, aggregation and exocytosis.

The effect of angiotensin II on platelet intracellular free calcium concentration in human pregnancy.

The primary objective of this study was to determine the effect of i.v. angiotensin II infusion on platelet intracellular free calcium concentration in potentially hypertensive primigravid women. Patients at 28-32 weeks gestation were selected on the basis of a diastolic pressure less than or equal to 80 mmHg taken at the antenatal clinic visit. An angiotensin II infusion test (4-16 ng/kg per min) was performed in 13 women. Platelet intracellular free calcium concentration, platelet angiotensin II binding site density and plasma angiotensin II concentration were measured before and at the end of the infusion. Pregnancy outcome (normotension or hypertension) was recorded. Platelet intracellular free calcium concentration rose in 12 of 13 women during angiotensin II infusion. This rise was not directly correlated with initial or final platelet angiotensin II binding site density, plasma angiotensin II concentration or the evoked change in systolic or diastolic blood pressure. Five women subsequently developed pregnancy-induced hypertension. Although basal platelet intracellular free calcium concentration did not differ between the two groups, platelet intracellular free calcium concentration rose twice as much in response to angiotensin II in the five hypertensive pregnant women. There was a 12-fold increase in platelet angiotensin II binding in the future hypertensives, although basal angiotensin II was the same in the two groups. The enhanced rise in platelet intracellular free calcium concentration in response to angiotensin II administration in women who subsequently became hypertensive, together with their increased angiotensin II binding site density suggest a possible enhanced stimulus-effect coupling. Pregnancy-induced hypertension is a state of marked vasoconstriction, with an enhanced pressor response to angiotensin II. These data supply grounds for a hypothesis concerning the mechanism.

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)

The effects of age on platelet intracellular free calcium concentrations in normotensives and hypertensives.

We have investigated relationships between age, blood pressure and intracellular calcium concentration in platelets from normotensives and hypertensives. In normotensives, there were positive correlations between age and platelet intracellular calcium concentration (r = 0.76, P less than 0.001), age and mean arterial pressure (MAP; r = 0.55, P less than 0.01) and MAP and platelet intracellular calcium concentration (r = 0.45, P less than 0.01). Multiple regression analysis revealed that age was the primary determinant of platelet intracellular calcium concentration in normotensives. The effect of MAP on platelet intracellular calcium concentration when adjusted for age was not significant (P = 0.73). In hypertensives, there was no significant relationship between age and platelet intracellular calcium concentration (r = 0.15, P = 0.43), age and MAP (r = 0.17, P = 0.37) or MAP and platelet intracellular calcium concentration (r = -0.27, P = 0.15). Overall, platelet intracellular calcium concentration was significantly higher in hypertensives than in age-matched normotensives (P less than 0.05). Within the age groups examined, platelet intracellular calcium concentration was significantly higher only in younger hypertensives when compared with controls of a similar mean age (P less than 0.001). Thus, age, in addition to hypertension, is an important determinant of platelet intracellular calcium concentration.

A cross-sectional study of basal platelet intracellular free calcium concentration in normotensive and hypertensive primigravid pregnancies

1. The intracellular free calcium concentration ([Ca2+]i) in washed human platelets was measured using the fluorescent indicator, fura-2, in a cross-sectional study of 36 normotensive, primigravid volunteers, 12 in each trimester of pregnancy and a further 12 at 6 weeks post partum. The results were compared with those obtained from 30 normal female volunteers not using oral contraception. 2. The mean basal [Ca2+]i in the platelets of the pregnant women in the first two trimesters (115.6 +/- 6.7 and 120.1 +/- 5.7 nmol/l, respectively) was not shown to differ significantly from that of normal non-pregnant volunteers (112.3 +/- 2.9 nmol/l). However, during the third trimester a significant increase in [Ca2+]i was noted (134.0 +/- 4.9 nmol/l; P less than 0.05), with a return to normal values in the post-partum period (108.2 +/- 6.1 nmol/l). 3. [Ca2+]i was also measured in the platelets of a group of 12 primigravid pregnant women in the third trimester whose pregnancies were complicated by gestational hypertension (pregnancy-induced hypertension and pre-eclampsia). A significant rise in basal [Ca2+]i was noted in the platelets of primigravidae whose pregnancies were complicated by pre-eclampsia (163.6 +/- 8.8 nmol/l) as compared with normotensive, third-trimester primigravidae (P less than 0.02). However, no correlation could be demonstrated between [Ca2+]i and systemic blood pressure.

Platelet intracellular free calcium concentration in normotensive and hypertensive pregnancies in the human.

1. Basal and stimulated platelet intracellular free calcium concentrations were measured in non-pregnant women and in third trimester patients who were either normotensive or who had pregnancy-induced hypertension or pre-eclampsia. There were 15 subjects in each group. 2. A trend for a reduction of the maximal response of platelet calcium levels to stimulation by 5-hydroxytryptamine was seen in pregnant groups compared with nonpregnant subjects, but this was significant only in pre-eclampsia. 3. No significant differences in basal or adenosine 5'-pyrophosphate-stimulated levels of platelet intracellular free calcium concentration were observed between the four groups. 4. These results illustrate that basal platelet calcium levels are unchanged in hypertension of pregnancy. Alterations in basal platelet calcium levels may not be involved in the platelet activation that is a feature of pre-eclampsia.

Exercise and platelet intracellular free calcium concentration.

1. A positive correlation between blood pressure and platelet intracellular free calcium concentration ([Ca2+]i) has been reported. We examined the effect of acute changes in blood pressure associated with exercise on platelet [Ca2+]i. 2. Twenty-one subjects had blood pressure and heart rate readings taken after 60 min rest, and subjects were then exercised on a bicycle ergometer at 120 W for 30 min. Blood pressure and heart rate readings were repeated immediately after exercise, 30 min after exercise, and then after a further hour. Blood samples were taken after each set of blood pressure and heart rate readings for catecholamine, lactate and platelet [Ca2+]i estimations. 3. There were significant increases in systolic and diastolic blood pressure, heart rate, and plasma lactate and catecholamine levels during the course of the study. There were no significant changes in platelet [Ca2+]i. 4. These results suggest that the acute blood pressure changes related to exercise are not associated with a change in platelet [Ca2+]i.

Exercise and Platelet Intracellular Free Calcium Concentration

1. A positive correlation between blood pressure and platelet intracellular free calcium concentration ([Ca2+]i) has been reported. We examined the effect of acute changes in blood pressure associated with exercise on platelet [Ca2+]i. 2. Twenty-one subjects had blood pressure and heart rate readings taken after 60 min rest, and subjects were then exercised on a bicycle ergometer at 120 W for 30 min. Blood pressure and heart rate readings were repeated immediately after exercise, 30 min after exercise, and then after a further hour. Blood samples were taken after each set of blood pressure and heart rate readings for catecholamine, lactate and platelet [Ca2+]i estimations. 3. There were significant increases in systolic and diastolic blood pressure, heart rate, and plasma lactate and catecholamine levels during the course of the study. There were no significant changes in platelet [Ca2+]i. 4. These results suggest that the acute blood pressure changes related to exercise are not associated with a change in platelet [Ca2+]i.