Platelet Cytosolic Calcium Concentration Research Articles

Platelet Expression of a Genetically Encoded Calcium Indicator Reveals Platelet Activation Gradients in Real Time during Thrombus Formation in Vivo

Introduction: Recent studies have demonstrated that platelet activation during hemostatic thrombus formation in vivo is heterogeneous in time and space. We have previously shown that in response to a penetrating injury in the microcirculation platelet accumulation and activation are driven by gradients of soluble agonists emanating from the site of injury. These gradients result in a characteristic thrombus architecture in which a core of fully activated platelets that have released their alpha granules is overlaid by a shell of less activated platelets that frequently embolize. While dependent on ADP/P2Y12 signaling, the extent and dynamics of platelet activation in the shell region remain poorly understood. Here, we used intravital imaging of platelet cytosolic calcium concentration to examine platelet activation in real time following vascular injury in vivo.Methods: We generated transgenic mice expressing a genetically encoded calcium indicator (GCaMP3) specifically in megakaryocytes and platelets by crossing PF4-Cre mice with Ai38 mice carrying the GCaMP3 transgene. GCaMP3 fluorescence was visualized in platelets following laser-induced injury in mouse cremaster arterioles using spinning disk confocal intravital microscopy.Results: Rapid and dynamic changes in GCaMP3 fluorescence were observed during platelet accumulation following vascular injury. Platelets immediately adjacent to the site of injury exhibited a rapid and sustained increase in cytosolic calcium. Peak cytosolic calcium levels in the core region occurred within the first minute post-injury, prior to peak P-selectin expression. Platelets in the shell region were characterized by dynamic changes in cytosolic calcium at the level of individual platelets, with the appearance of transient “waves” of calcium signaling propagating among groups of platelets. At the population level, cytosolic calcium concentration was substantially lower in the shell region as compared to the core.Conclusions: Expression of a genetically encoded calcium indicator in platelets allows for the visualization of platelet activation events in real time in vivo. Platelets within the core region of a thrombus exhibit a rapid and sustained increase in cytosolic calcium concentration, indicating robust activation. In contrast, platelets in the shell region exhibit transient increases in cytosolic calcium indicating weak activation, consistent with their increased likelihood of embolization. The examination of platelet calcium signaling in vivo provides a sensitive readout of platelet activation that will shed new light on mechanisms regulating hemostasis and thrombosis, and may be useful in assessing the impact of anti-thrombotic agents on platelet function. Research support was provided by the American Heart Association and National Heart, Lung and Blood Institute. DisclosuresStalker:Medicines Company: Research Funding. Brass:Medicines Company: Research Funding.

Cytosolic Calcium Changes in a Process of Platelet Adhesion and Cohesion on a von Willebrand Factor-Coated Surface Under Flow Conditions

Recent flow studies indicated that platelets are transiently captured onto and then translocated along the surface through interaction of glycoprotein (GP) Ib with surface-immobilized von Willebrand factor (vWF). During translocation, platelets are assumed to be activated, thereafter becoming firmly adhered and cohered on the surface. In exploring the mechanisms by which platelets become activated during this process, we observed changes in platelet cytosolic calcium concentrations ([Ca(2+)]i) concomitantly with the real-time platelet adhesive and cohesive process on a vWF-coated surface under flow conditions. Reconstituted blood containing platelets loaded with the Ca(2+) indicators Fura Red and Calcium Green-1 was perfused over a vWF-coated glass surface in a flow chamber, and changes in [Ca(2+)]i were evaluated by fluorescence microscopy based on platelet color changes from red (low [Ca(2+)]i) to green (high [Ca(2+)]i) during the platelet adhesive and cohesive process. Under flow conditions with a shear rate of 1,500 s(-1), no change in [Ca(2+)]i was observed during translocation of platelets, but [Ca(2+)]i became elevated apparently after platelets firmly adhered to the surface. Platelets preincubated with anti-GP IIb-IIIa antibody c7E3 showed no firm adhesion and no [Ca(2+)]i elevation. The intracellular Ca(2+) chelator dimethyl BAPTA did not inhibit firm platelet adhesion but completely abolished platelet cohesion. Although both firm adhesion and cohesion of platelets have been thought to require activation of GP IIb-IIIa, our results indicate that [Ca(2+)]i elevation is a downstream phenomenon and not a prerequisite for firm platelet adhesion to a vWF-coated surface. After platelets firmly adhere to the surface, [Ca(2+)]i elevation might occur through the outside-in signaling from GP IIb-IIIa occupied by an adhesive ligand, thereby leading to platelet cohesion on the surface.

Cytosolic Calcium Changes in a Process of Platelet Adhesion and Cohesion on a von Willebrand Factor-Coated Surface Under Flow Conditions: Presented in part at the American Society of Hematology Meeting in Miami Beach, FL, December 4-8, 1998 (abstr no. 1423).

AbstractRecent flow studies indicated that platelets are transiently captured onto and then translocated along the surface through interaction of glycoprotein (GP) Ib with surface-immobilized von Willebrand factor (vWF). During translocation, platelets are assumed to be activated, thereafter becoming firmly adhered and cohered on the surface. In exploring the mechanisms by which platelets become activated during this process, we observed changes in platelet cytosolic calcium concentrations ([Ca2+]i) concomitantly with the real-time platelet adhesive and cohesive process on a vWF-coated surface under flow conditions. Reconstituted blood containing platelets loaded with the Ca2+ indicators Fura Red and Calcium Green-1 was perfused over a vWF-coated glass surface in a flow chamber, and changes in [Ca2+]i were evaluated by fluorescence microscopy based on platelet color changes from red (low [Ca2+]i) to green (high [Ca2+]i) during the platelet adhesive and cohesive process. Under flow conditions with a shear rate of 1,500 s−1, no change in [Ca2+]i was observed during translocation of platelets, but [Ca2+]i became elevated apparently after platelets firmly adhered to the surface. Platelets preincubated with anti-GP IIb-IIIa antibody c7E3 showed no firm adhesion and no [Ca2+]i elevation. The intracellular Ca2+chelator dimethyl BAPTA did not inhibit firm platelet adhesion but completely abolished platelet cohesion. Although both firm adhesion and cohesion of platelets have been thought to require activation of GP IIb-IIIa, our results indicate that [Ca2+]i elevation is a downstream phenomenon and not a prerequisite for firm platelet adhesion to a vWF-coated surface. After platelets firmly adhere to the surface, [Ca2+]i elevation might occur through the outside-in signaling from GP IIb-IIIa occupied by an adhesive ligand, thereby leading to platelet cohesion on the surface.

Platelet cytosolic calcium concentration in patients with liver cirrhosis: Relationship with hepatic and systemic hemodynamics

Backgrouond/Aims: Due to structural and functional similarities between platelets and vascular smooth muscle cells, platelet cytosolic calcium concentration ([Ca 2+] 1) has been suggested to be a useful tool to study regulatory mechanisms of peripheral vascular tone. The aim of the present study was to investigate platelet [Ca 2+] i in patients with cirrhosis and whether this parameter is related with the systemic and splanchnic vasodilation found in these patients. Methods: Seventeen patients with cirrhosis and eight age- and sex-matched controls were studied. Mean arterial pressure, cardiac output, femoral blood flow and basal and thrombin-stimulated platelet [Ca 2+] i were measured. Cardiac output (thermal dilution), azygos blood flow, hepatic venous pressure gradient and hepatic blood flow were also measured in patients with cirrhosis. Results: Patients with cirrhosis had severe portal hypertension and a significantly higher cardiac output and femoral blood flow and a significantly lower systemic and femoral vascular resistance than controls. Patients with cirrhosis had a lower basal platelet [Ca 2+] i than normal subjects. However, there was no relationship between platelet [Ca 2+] i and any of the hemodynamic parameters that evaluate systemic or splanchnic vasodilatation. Conclusions: This study shows that cirrhotic patients with portal hypertension have a significant reduction in platelet basal [Ca 2+] i. The lack of correlation between platelet [Ca 2+] i and hepatic and systemic hemodynamics does not support the use of platelet [Ca 2+] i as a model to study mechanisms involved in the pathophysiology of the hyperdynamic circulation associated to portal hypertension.

Role of nitric oxide resistance in erythropoietin-induced hypertension in rats with chronic renal failure.

We studied the mechanism of erythropoietin (EPO)-induced hypertension (HTN) in rats with chronic renal failure (CRF). After partial nephrectomy, rats were randomized into four groups. Group A received EPO, 150 U/kg, two times weekly for 6 wk to prevent anemia; group B received placebo injections and became anemic; group C received EPO but was kept anemic by dietary iron deficiency; and group D received placebo and regular transfusions to match hematocrit (Hct) in group A. Blood pressure (BP), Hct, platelet cytosolic calcium ([Ca2+]i) and magnesium concentration, and pressor and vasodilatory responses were determined. By design, Hct in groups A and D were comparable and significantly greater (P < 0.01) than in groups B and C. Despite divergent Hct values, the EPO-treated groups A and C showed a significant rise in BP compared with the placebo-treated groups B and D. HTN occurred whether EPO therapy was begun immediately or 4 wk after nephrectomy. EPO therapy augmented the elevation of basal [Ca2+]i and restored the defective thrombin-mediated rise of platelet [Ca2+]i in CRF animals. EPO therapy did not alter caudal artery contraction in response to either 68 mM K(+)-induced depolarization, angiotensin II or alpha 1-agonist, methoxamine in vitro, or the pressor response to angiotensin II in vivo. However, EPO therapy impaired the hypotensive response to nitric oxide (NO) donors, sodium nitroprusside and S-nitroso-N-acetyl-D,L-penicillamine, and reversed the CRF-induced upregulation of guanosine 3',5'-cyclic monophosphate production by thoracic aorta in vitro. Thus EPO-induced HTN in CRF rats is Hct independent and is associated with and perhaps causally related to increased basal and stimulated [Ca2+]i and impaired vasodilatory response to NO.

Serotonin-induced increases in platelet cytosolic calcium concentration in depressed, schizophrenic, and substance abuse patients

Exaggerated intracellular calcium responses to challenges with serotonin (5-HT) have been reported in depression. In our studies, consistent with previous reports, patients with depression exhibited an exaggerated increase in 5-HT-stimulated intracellular calcium concentration ([Ca++]i). Basal cytosolic calcium was elevated in both calcium-free and 1 mM calcium media in depressed patients. the increased responsiveness to 5-HT was seen in both conditions. Patients with schizophrenia and substance abuse did not differ from normal controls. The 5-HT response was correlated with diastolic blood pressure (r = 0.33, p = 0.02): however, this association did not fully account for the exaggerated [Ca++]i responses in the depressed group. These findings suggest that exaggerated increases in [Ca++]i in response to serotonin are a characteristic of depressed patients not shared with schizophrenic and substance abuse patients. The relationship of depression to hypertension, two conditions that share abnormalities of calcium homeostasis, warrants further study.

The Antiplatelet Effects of a New Nitroderivative of Acetylsalicylic Acid - An In Vitro Study of Inhibition on the Early Phase of Platelet Activation and on TXA2 Production

We studied in vitro the antiplatelet activity of a new nitroderivative chemically related to acetylsalicylic acid: 2 acetoxybenzoate 2-[1-nitroxy-methyl]-phenyl ester (NCX 4016), in order to identify any effects due to the release of nitric oxide and the blockade of cyclo-oxygenase. The effects of scalar doses of NCX 4016 on the early phase of platelet activation, platelet aggregation and thromboxane A2 production were investigated. We observed inhibitory effects of NCX 4016 on platelet adhesion (IC50 = 7.3 x 10(-5) M), platelet cytosolic calcium concentration, assayed by fluorescent probe Fura 2, and the expression of glycoprotein IIb/IIIa (CD41/alpha IIb beta 3) (IC50 = 3.4 x 10(-5) M) and P-selectin (CD62/GMP-140) (IC50 = 4.9 x 10(-5) M) measured by flow cytometry. NCX 4016 also prevented thrombin-induced platelet aggregation (IC50 = 3.9 x 10(-5) M). None of these parameters were affected by acetylsalicylic acid. These inhibitory activities of NCX 4016 were abolished by oxyhaemoglobin and methylene blue. Intracellular cyclic GMP observed during thrombin-induced aggregation was increased by incubation with NCX 4016. These results appear to be attributable to the release of nitric oxide, which activates soluble platelet guanylylcyclase and promotes intracellular cyclic GMP increase. NCX 4016 almost completely inhibited platelet thromboxane A2 production and arachidonic acid-induced platelet aggregation. This also occurred in the presence of oxyhaemoglobin and methylene blue, indicating that its antiplatelet activity can be attributed not only to nitric oxide release but also to cyclo-oxygenase inhibition.

Platelet cytosolic calcium concentration, plasma lipids and hypertension

To study the relationship between high blood pressure and hyperlipidaemia and the cytosolic calcium concentration in unstimulated platelets, focusing on the effects of an alteration in membrane dynamics. Basal cytosolic calcium concentrations were determined in the presence and the absence of a significant calcium influx in platelets of 47 untreated hypertensive patients and 26 normotensive subjects. Membrane microviscosity was investigated by fluorescence depolarization of diphenylhexatriene and trimethylaminodiphenylhexatriene. To study the influence of plasma factors, unstimulated platelets were loaded in the presence of plasma with Quin-2, which forms a relatively strong intracellular calcium buffer. The cytosolic calcium concentration was then determined at two extracellular calcium concentrations (1 mmol/l and in the absence of a Ca2+ influx). Irrespective of the external calcium concentration, the cytosolic calcium concentration increased significantly with diastolic blood pressure (P = 0.026 in the presence and P = 0.003 in the absence of Ca2+ influx) and with plasma triacylglycerols (P = 0.03 and 0.001, respectively). Multiple regression analysis indicated that the cytosolic Ca+ concentration was independently related to these two factors [Ca2+ = 35 + (18.6 +/- 4.6). In triacylglycerols (mmol/l) + (0.45 +/- 0.15) mmHg diastolic blood pressure; P < 0.001]. The relationship between the cytosolic calcium concentration and diphenylhexatriene or trimethylaminodiphenylhexatriene anisotropies was not independent of blood pressure and plasma triacylglycerol levels. The present results confirm the link between blood pressure and the platelet cytosolic calcium concentration and indicate that plasma triacylglycerols directly or indirectly modulate the ex vivo efficacy of platelet calcium storage and/or extrusion mechanisms. They could facilitate cell stimulation.

Serotonin-induced increases in platelet cytosolic calcium concentration in depressed, schizophrenic, PTSD, and substance abuse patients

Serotonin-induced increases in platelet cytosolic calcium concentration in depressed, schizophrenic, PTSD, and substance abuse patients

Parathyroid hormone and platelet cytosolic calcium concentration in essential hypertension

To investigate the possible relationships between blood pressure, parathyroid hormone (PTH) and platelet cytosolic Ca2+ concentration ([Ca2+]i) in patients with essential hypertension, we studied 17 patients with this disease aged 48 +/- 2 years and 17 normotensive controls aged 44 +/- 3 years. Platelet [Ca2+]i was measured by spectrofluorimetry using the dye Fura-2 acetoxymethylester. Patients with essential hypertension displayed lower levels of serum ionized Ca2+ (0.99 +/- 0.03 versus 1.1 +/- 0.01 mmol/l, P < 0.05) and higher serum intact PTH levels (37 +/- 3 versus 26 +/- pg/ml, (P < 0.01) than the normotensive controls. Although serum levels of intact PTH were significantly correlated with mean arterial pressure (MAP) in the combined group of subjects (r = 0.42, P < 0.05), there was no correlation when each group was considered separately. Resting platelet [Ca2+]i was also higher in patients than in controls (57 +/- 3 versus 48 +/- 2 nmol/l, P < 0.005). When platelets were stimulated in vitro with thrombin, the increment in [Ca2+]i was also greater in patients than in controls in the presence of extracellular Ca2+ (264 +/- 24 versus 194 +/- 19 nmol/l, P < 0.05) but not in its absence (123 +/- 12 versus 112 +/- 10 nmol/l). The thrombin-induced increment in [Ca2+]i was correlated with MAP in the hypertensive patients (r = 0.64, P < 0.01) and in the combined group of subjects (r = 0.42, P < 0.05). There was no relationship between resting or thrombin-induced [Ca2+]i and serum PTH in either group of patients or in the combined group of subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Dietary calcium reduces blood pressure, parathyroid hormone, and platelet cytosolic calcium responses in spontaneously hypertensive rats.

Dietary calcium effects on blood pressure, parathyroid hormone (PTH), and platelet cytosolic calcium concentrations were investigated. The dietary calcium (low, 0.2%; medium, 0.5%; and high, 2.0% wt/wt) was supplemented in spontaneously hypertensive rats from 6 through 22 weeks of age. Mean systolic blood pressure was decreased by age 12 weeks with calcium supplementation (low, 227 +/- 6 mm Hg; medium, 211 +/- 6 mm Hg; and high, 182 +/- 7 mm Hg; P < .001). By the 10th week of age, the low calcium group had significantly (P < .05) more elevated (44 +/- 2.3 pg/ml) plasma PTH compared with the high calcium-supplemented group (15 +/- 4.5 pg/mL). Regression analysis showed a significant (P < .001) positive correlation (r = 0.3) between systolic blood pressure and PTH. The platelet cytosolic calcium concentration was determined using the fura-2 method. The basal calcium was 134 +/- 5.5 nmol/L for the low calcium group and thrombin increased to 228 +/- 8 nmol/L (P < .0001; +70% change). The normal calcium group had 202 +/- 8 nmol/L; thrombin increased to 239 +/- 10 nmol/L (P < .0026; +19% change). The high calcium group had basal levels 145 +/- 7 nmol/L, with thrombin stimulating to 212 +/- 8 nmol/L (P < .0001; +46% change). Although thrombin increased platelet cytosolic calcium concentration in all groups, normal and high dietary calcium groups had smaller percentage increases (51% and 24% lesser, respectively) compared with the low dietary calcium group.(ABSTRACT TRUNCATED AT 250 WORDS)

Neonatal and maternal platelet cytosolic calcium in normotensive and hypertensive pregnancies.

A prospective study investigated platelet cytosolic calcium in non-pregnant volunteers (n = 30) and samples from the umbilical veins of babies from both normotensive (n = 18) and hypertensive (n = 15) primigravidae, and their mothers. There was no significant difference between the neonatal umbilical venous platelet cytosolic calcium concentration (p[Ca2+]i) in babies born to normotensive primigravidae or to those whose pregnancies were complicated by gestational hypertension (88 x 9 (SE) 2 x 5) in normotensive primagravidae, 80 x 6 (2 x 8) in pregnancy induced hypertension without proteinuria, and 89 x 3 (3 x 2) nmol/l in pre-eclampsia. There was also no significant difference in the p[Ca2+]i from the umbilical veins of the pregnancies studied and those of non-pregnant female volunteers in the follicular phase of their menstrual cycle. This was despite a gradual and significant rise in p[Ca2+]i with increasing severity of disease in the mothers of the babies studied (119 x 9 (4 x 1) in normotensive primagravidae, 130 x 8 (7 x 3) in pregnancy induced hypertension without proteinuria, and 148 x 2 (4 x 5 ) nmol/l in pre-eclampsia). The mean maternal p[Ca2+]i in the three samples returned to concentrations comparable with those in non-pregnant subjects by 12 weeks after birth. These data demonstrate no significant difference between the mean p[Ca2+]i in non-pregnant women and those obtained from the umbilical venous blood of normotensive or hypertensive primigravidae. They suggest that the functional hypoactivity of neonatal platelets is probably not secondary to a decrease in basal p[Ca2+]i. They also suggest that the progressively raised p[Ca2+]i in normal and hypertensive pregnancies might be due to a pregnancy specific factor that does not cross the placenta,

Effects of suramin on increases in cytosolic calcium and on inhibition of adenylate cyclase induced by adenosine 5′-diphosphate in human platelets

The effects of the P 2-purinoceptor antagonist, suramin, on ADP-induced increases in human platelet cytosolic calcium concentration ([Ca 2+] i) and inhibition of prostaglandin E 1 (PGE 1)-stimulated adenylate cyclase activity were investigated. Suramin (50–200 μM) acted as an antagonist of ADP-induced increases in [Ca 2+] i, causing parallel, rightward shifts of the log concentration-response curve to ADP with no apparent depression of the maximal response. However, the slope of the Schild plot was 2.3 ± 0.3, similar to that obtained in previous studies on aggregation, indicating that the antagonism was not simply competitive. The apparent pA 2 for suraminn, taken from the Schild plot, was 4.63, similar to that for suramin's inhibition of aggregation, which suggests that these two effects are closely related. Suramin was not specific for the ADP receptor, however, as it was also able to inhibit, non-competitively, increases in [Ca 2+] i induced by 5-hydroxytryptamine. Suramin (50–400 μM) also inhibited the effect of ADP on PGE 1-stimulated accumulation of cyclic AMP, causing parallel shifts of the log concentration-response curve to ADP, with a Schild plot slope of 1.00 ± 0.10, suggesting competitive antagonism, and a pA 2 value of 5.09. Suramin (400 μM) did not reduce the inhibition of cyclic AMP accumulation by adrenaline, although it was able to inhibit the accumulation of cyclic AMP caused by PGE 1, again showing that suramin has some non-specific effects. These data suggest that suramin is an antagonist at the platelet ADP receptor mediating increases in [Ca 2+] i and inhibition of adenylate cyclase, but that it also shows non-specific effects and can depress platelets responses to other agonists. In addition, the similar pA 2 value of suramin for the two effects of ADP does not support suggestion that they are mediated by two different receptors on human platelets.

Platelet cytosolic calcium, peripheral hemodynamics, and vasodilatory peptides in liver cirrhosis

Background: Platelet cytosolic calcium concentration ([Ca2+]I) has been proposed to reflect changes in vascular smooth muscle cells. This study investigated if the platelet [Ca2+]I is altered in cirrhotic patients and determined its relationship with peripheral hemodynamics and peptide levels. Methods: Fourteen patients with cirrhosis and 11 healthy, age- and sex-matched controls had blood samples taken for determining platelet [Ca2+]I and glucagon, substance P (SP), and vasoactive intestinal peptide (VIP) values. Mean arterial pressure (MAP) and forearm blood flow (FBF) were measured on the same day of blood sampling. Forearm vascular resistance (FVR) was calculated. Results: Patients with cirrhosis had lower platelet [Ca2+]I (50.1 ± 2 vs. 73.0 ± 5 nmol/L; P < 0.001) than normal controls. Glucagon levels were significantly higher in patients with cirrhosis, but there was no difference in SP or VIP levels in both groups. MAP (80.5 ± 3 vs. 94.7 ± 3; P < 0.005) and FVR (20.1 ± 1 vs. 36.3 ± 2; P < 0.001) were significantly lower in patients with cirrhosis. A significant correlation was observed between platelet [Ca2+]I and MAP in patients with cirrhosis (r = 0.81; P < 0.001), between platelet [Ca2+]I and FVR (r = 0.87; P < 0.001), and between platelet [Ca2+]I and diastolic blood pressure (r = 0.78; P < 0.001). No correlation was found between platelet [Ca2+]I and peptide levels. Conclusions: Patients with cirrhosis have a significant reduction in the platelet [Ca2+]I. This finding correlates well with peripheral hemodynamics. Platelets may be a useful tool to study the etiologic mechanisms leading to the vasodilation of chronic liver disease.

Platelet cytosolic free calcium concentration and parathyroid hormone: changing relationships with haemodialysis in end-stage renal disease.

1. Twelve patients receiving haemodialysis for end-stage renal failure were studied at a single dialysis session. Platelet cytosolic calcium concentration, plasma ionized calcium concentration and serum parathyroid hormone concentration were measured before dialysis, mid-dialysis and 30 min after dialysis. 2. Plasma ionized calcium concentration increased towards dialysate calcium concentrations, falling insignificantly after cessation of dialysis. Serum parathyroid hormone concentration fell by 39% during dialysis, with incomplete recovery afterwards. There was no overall change in platelet cytosolic calcium concentration. 3. Patients were divided into two subgroups: low parathyroid hormone (serum parathyroid hormone concentration less than 10 pmol/l) and high parathyroid hormone (serum parathyroid hormone concentration greater than 10 pmol/l). Before dialysis, values of platelet cytosolic calcium concentration or plasma ionized calcium concentration were not statistically different between the subgroups, but the platelet cytosolic calcium concentration was higher in the high-parathyroid hormone subgroup during and after dialysis. 4. Before haemodialysis there was a linear correlation between plasma ionized calcium concentration and platelet cytosolic calcium concentration, which disappeared during dialysis. In contrast, there was no relationship between serum parathyroid hormone concentration and platelet cytosolic calcium concentration before dialysis, but after dialysis a hyperbolic relationship was evident. 5. These results suggest that uraemic toxins may interfere with cytosolic calcium homoeostasis, allowing passive diffusion of extracellular calcium to influence the resting concentration, and that this effect is reversible by haemodialysis.

Correlation of Blood Pressure in End-Stage Renal Disease with Platelet Cytosolic Free Calcium Concentration during Treatment of Renal Anemia with Recombinant Human Erythropoietin

The hemodynamic hallmark of hypertension complicating the treatment of renal anemia with recombinant human erythropoietin (rHu-EPO) is increased total peripheral vascular resistance, but the mechanisms underlying the arteriolar vasoconstriction are still an enigma. We studied body fluid volumes, plasma renin activity, plasma norepinephrine, and calcium metabolism in platelets in 40 previously normotensive hemodialysis patients before and after 12 weeks of rHu-EPO treatment. Partial correction of anemia caused a rise in arterial pressure (94 +/- 6 mmHg vs 124 +/- 7 mmHg, p less than 0.05) and in platelet cytosolic calcium concentration (113 +/- 5 nM vs 171 +/- 18 nM, p less than 0.05) in eight patients. Hypertensive patients had significantly higher plasma noradrenaline concentrations, but they did not differ significantly in body fluid volumes and plasma renin activities. There was a close correlation between free calcium concentration in platelets and mean arterial pressure in patients developing rHu-EPO-induced-hypertension (r = 0.95). Short-term antihypertensive treatment resulted in a reduction of free calcium concentrations in platelets and a concomitant fall in blood pressure. The main results of the present studies suggest that rHu-EPO-induced hypertension might be associated with altered cellular calcium homeostasis and hyperactivity of the sympathetic nervous system. If rHu-EPO therapy induces alterations of pressor factors or the hormone itself raises the cytosolic calcium not only in platelets but also in vascular smooth muscle cells, altered cellular calcium influx may contribute to the arteriolar vasoconstriction.

Role of platelet cytosolic calcium in regulation of blood pressure in patients on chronic hemodialysis.

We measured blood pressure, platelet cytosolic calcium concentration ([Ca2+]i) and vasoactive hormones in 20 anuric normotensive patients on chronic hemodialysis (HD) to clarify the role of cytosolic calcium in regulation of blood pressure. All measurements were performed at the end of HD (post-HD) and at the beginning of the next HD (pre-HD). During the HD-HD interval, mean blood pressure increased from 85 +/- 4 to 94 +/- 4 mm Hg (mean +/- SEM, p less than 0.05), but the median [Ca2+]i in platelets did not show any significant change (133 +/- 5 vs. 136 +/- 5 nmol/l). Although all patients were within their ideal body weight at post-HD, platelet [Ca2+]i did not correlate with mean blood pressure. By contrast, platelet [Ca2+]i correlated with mean blood pressure (r = 0.489, p less than 0.05) at pre-HD. Moreover, platelet [Ca2+]i at post-HD also correlated with changes in mean blood pressure during the HD-HD interval (r = 0.532, p less than 0.05). In addition, there were negative correlations between platelet [Ca2+]i and plasma renin activity both post- and pre-HD (r = -0.652, p less than 0.005; r = -0.673, p less than 0.005; respectively). These results suggest that cytosolic calcium plays important roles in regulation of renin secretion and in the elevation of blood pressure induced by volume expansion in patients on chronic HD.

Blood pressure, left ventricular mass and intracellular calcium in primary hyperparathyroidism

1. Blood pressure, left ventricular mass and platelet cytosolic free calcium concentrations were measured in 23 patients with untreated primary hyperparathyroidism, 30 normotensive control subjects and 23 control subjects matched for age, sex and blood pressure. In 12 patients measurements were repeated after parathyroidectomy. 2. Patients with primary hyperparathyroidism had significantly elevated blood pressures (139 +/- 6/86 +/- 3 mmHg, mean +/- SEM) compared with control subjects (125 +/- 2/78 +/- 1 mmHg), but high values persisted after hypercalcaemia was corrected. 3. Despite chronic extracellular hypercalcaemia, intracellular free calcium levels were lower in patients with hyperparathyroidism than in controls matched for age, sex and blood pressure (median concentrations 81.5 nmol/l vs 93 nmol/l, 95% confidence interval 0.1 to 20.1; P less than 0.05) and values tended to increase after parathyroidectomy. 4. Left ventricular mass index was increased in the primary hyperparathyroid group as compared with control subjects matched for age, sex and blood pressure (123 g/m2 vs 100 g/m2, 95% confidence interval -36.1 to -3.1; P = 0.03). Parathyroidectomy resulted in a small reduction of the left ventricular mass index (123.5 g/m2 vs 104 g/m2, 95% confidence interval 46.5 to 2.5; P = 0.1) but no change in blood pressure. 5. Hypertension and left ventricular hypertrophy in primary hyperparathyroidism are associated with relatively low levels of free calcium in platelets.

Platelet cytosolic proton and free calcium concentrations in essential hypertension

Alterations in the metabolism of intracellular messengers, such as calcium and cyclic adenosine 5'-phosphate (cAMP), have been reported in essential hypertension. Since intracellular pH (pHi) participates in the control of fundamental cell functions, we looked for changes in platelet cytosolic H+ concentration [( H+]i) in hypertension and investigated whether or not its impaired metabolism is linked to the calcium handling abnormalities. The fluorescent pH indicator BCECF has been used to evaluate intracellular H+ concentration in platelets, unstimulated ex vivo, from normotensive (n = 20) and hypertensive patients (n = 20). Cytosolic [H+] was 20% lower in hypertensive than in normotensive subjects (49.5 +/- 3.4 and 61.8 +/- 2.2 nmol/l cells, respectively, P less than 0.005; mean pHi values were 7.21 and 7.33, respectively). Platelet cytosolic H+ and free Ca2+ concentrations ([Ca2+]i) were determined in parallel in 15 normotensive and 15 hypertensive patients. [Ca2+]i was found to be 19% higher (P less than 0.01), and [H+]i 22% lower (P less than 0.02), in the hypertensive patients compared with the normotensive subjects. Platelet pHi and [Ca2+]i were increased simultaneously in some hypertensive patients. These results are compatible with the hypothesis of an in vivo activation of platelets in hypertension. If a similar alkalinization exists in smooth muscle cells, it may participate in cell proliferation and in an enhanced sensitivity to agonists, two parameters thought to be involved in blood pressure elevation.

The influence of epinephrine on washed human platelets: shape change and protein phosphorylation.

Protein phosphorylation is an important regulator of the properties or functions of many proteins and is associated with the platelet activation response to a number of chemically and functionally different agents such as thrombin, platelet-activating factor, serotonin and collagen. The physiological responses of platelets to these agents are similar, and the common intracellular messenger for activation is an elevated concentration of calcium. Platelets possess alpha-2-receptors, and treatment with epinephrine produces an elevation in platelet cytosolic free calcium concentrations. Methods are described for studying hormone sensitive shape change and protein phosphorylation in washed human platelets. Epinephrine induces platelet shape change, and this process is independent of extracellular calcium. Treatment of [32P]-orthophosphate-labelled platelets with epinephrine produces an increase in 32P-incorporation into two platelet proteins with molecular weights of 47000 and 20000. This phosphorylation response is both dose and time dependent. Extracellular calcium is not absolutely essential for epinephrine-induced phosphorylation, but does enhance the maximum levels of 32P-incorporation. Epinephrine sensitive phosphorylation is completely inhibited following pretreatment with verapamil or nitrendipine. Shape change in response to epinephrine occurs in the absence of enhanced protein phosphorylation. The data suggest that epinephrine mobilizes intracellular calcium, and induces platelet shape change and phosphorylation responses characteristic of platelet activation.