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(Hypertension. 1998;31:595-602.)
© 1998 American Heart Association, Inc.

Scientific Contributions

Ca²⁺ in the Dense Tubules

A Model of Platelet Ca²⁺ Load

Makoto Horiguchi; Masayuki Kimura; Jonathan Lytton; Joan Skurnick; Frederic Nash; Girgis Awad; Esteban Poch; ; Abraham Aviv

From the Hypertension Research Center and the Department of Preventive Medicine and Community Health, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ; and the Renal Division, Departments of Medicine, Brigham & Women's Hospital and Harvard Medical School, Boston, Mass (J.L., E.P.).

Correspondence to Abraham Aviv, MD, Hypertension Research Center, University of Medicine and Dentistry of New Jersey-NJ Medical School, 185 S Orange Ave, MSB F-464, Newark, NJ 07103-2714.

	Abstract

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Abstract—In this work, we explored the relationship between the freely exchangeable Ca²⁺ (FECa²⁺) in the dense tubules (DT) and the sarco(endo)plasmic reticulum (SER) Ca²⁺-ATPase (SERCA) in circulating human platelets and examined the relationship between blood pressure (BP) and these platelet parameters. Studying platelets from 32 healthy men, we showed that the maximal reaction velocity (V_max) of the SERCA significantly correlated with FECa²⁺ in the DT and with the protein expressions of SERCA 2 and 3. BP positively correlated with both the V_max of the SERCA (r=.462, P=.010) and the FECa²⁺ sequestered in the DT (r=.492, P=.005). The relationships between these platelet Ca²⁺ parameters and BP were in part confounded by increased levels of serum triglycerides and diminished HDL cholesterol with a higher BP. No correlation was observed between the resting cytosolic Ca²⁺ and BP. Collectively, these findings indicate that (1) an increase in the cellular Ca²⁺ load in platelets is expressed by a higher activity of the SERCA and an increase in the expressions of SERCA 2 and 3 proteins, coupled with an increase in the FECa²⁺ in the DT, and (2) a higher BP is associated with an increase in platelet Ca²⁺ load in human beings, expressed by a rise in the FECa²⁺ in the DT and the upregulation of SERCA activity.

Key Words: hypertension, essential • thapsigargin • lipids • calcium transport • sodium transport

	Introduction

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By measuring Ca²⁺ levels in circulating cells, primarily in platelets, previous studies have tested the premise that elevated BP in human beings is associated with an increase in the cellular Ca²⁺ load. A number of these studies have shown that the resting cytosolic free Ca²⁺ concentration ([Ca²⁺]_c) in platelets was increased in essential hypertension.^{1 2 3 4 5} However, others have failed to confirm this observation.^{6 7} Moreover, with few exceptions,¹ relatively weak^{8 9} or no ^{2 4 5 6 7 10} correlations have been observed between the BP and the resting [Ca²⁺]_c in platelets when the BP was treated as a quantitative trait with continuous distribution. One possible explanation for these findings is that the resting [Ca²⁺]_c is an insensitive indicator of the overall Ca²⁺ status in platelets, since a major component of the FECa²⁺ in these cells is sequestered within the DT, which are equivalent to the SER in nucleated cells.¹¹ The Ca²⁺ transport system responsible for Ca²⁺ sequestration in the DT is the SERCA. Another possibility is that platelet function is modified by plasma lipids.^{12 13 14 15 16} Because dyslipidemia is commonly associated with essential hypertension,^{17 18} variations in platelet Ca²⁺ regulation might reflect variations in serum lipid levels rather than the BP level.

In the present work, we proceeded to examine the relationship between the protein and functional expressions of the SERCA and the FECa²⁺ in the DT of circulating platelets. Based on the proposition that a higher BP in humans is associated with increased platelet Ca²⁺ load, we also examined whether the BP level is correlated with the activity of the SERCA and the FECa²⁺ in the DT in circulating platelets. Finally, we examined correlations between serum lipids and the above platelet parameters in the context of the relationship between platelet Ca²⁺ regulation and BP.

	Methods

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Subjects, BP Measurements, and Blood Collection
Thirty-two men (16 blacks and 16 whites) donated blood. All subjects were without any apparent disease (including sickle-cell trait in blacks) and received no medication (including antiplatelet agents) for at least 2 weeks before the study. Manual BP measurements and blood collections were performed between 8 and 9 AM after an overnight fast. Each subject signed an informed consent form approved by the institutional review board for human investigations. After subjects rested for 10 minutes in the sitting position, BP measurements were obtained from the nondominant arm using a mercury sphygmomanometer. Three BP measurements were taken at 2-minute intervals between measurements (DBP=fifth Korotkoff sound). The mean of the three measurements was used for the study. Thereafter, height and weight (subject wearing light clothing and no shoes) were measured. Fasting venous blood (70 mL) was obtained. The initial 60 mL was taken (into a buffer [20:1] comprising [in mmol/L] 14 Na citrate, 11.8 citric acid, 18 dextrose, pH 6.5) for measurements of parameters of Ca²⁺ regulation in circulating platelets; the subsequent 10 mL was taken for determination of blood chemistry levels that included serum lipids, fasting glucose, and creatinine. Systemic parameters of these subjects are presented in Table 1.

View this table: [in this window] [in a new window]   Table 1. Subject Characteristics

Platelet Preparation
Platelets were isolated by differential centrifugation as described.¹⁹ Briefly, platelet-rich plasma was obtained by centrifugation at 200g for 10 minutes at room temperature. After treatment with 0.1 mmol/L aspirin for 20 minutes, platelet-rich plasma was centrifuged at 1000g, and the pellet was washed three times in a buffer consisting of (in mmol/L) 140 NaCl, 5 KCl, 10 glucose, 0.3 EGTA, and 10 HEPES. Bovine serum albumin (0.1%) was added to the third washing, and EGTA was deleted. Platelets were kept in Ca²⁺-free buffer until loading with fura 2, and Ca²⁺ was repleted by adding 1 mmol/L CaCl₂ during the loading with fura 2 (see below).

Membrane Preparation
Platelets were washed twice with buffer consisting of (in mmol/L) 140 NaCl, 5 KCl, 10 glucose, 3 EDTA, and 10 HEPES, plus 0.005 U/mL aprotinin and 20 µmol/L PMSF (pH 7.5). Washed cells were sonicated in a buffer consisting of (in mmol/L) 100 KCl, 15 NaCl, 12 sodium citrate, 2 MgSO₄, 10 glucose, and 25 HEPES, plus 0.2 mmol/L PMSF, 0.5 µg/mL leupeptin, 0.7 µg/mL pepstatin A, 0.05 U/mL aprotinin, and 1 mmol/L DTT (pH 7.5). Thereafter, cells were centrifuged at 19 000g for 25 minutes. The supernatant was further centrifuged at 100 000g for 60 minutes, and the pellet was suspended in the appropriate buffers for the kinetics of the SERCA and protein expressions of SERCA 2b and SERCA 3. The protein content was measured by the Bio-Rad method using immunoglobulin as the protein standard.

Monitoring of the Cytosolic Ca²⁺ Concentration ([Ca²⁺]_c)
Platelets were incubated for 30 minutes at 37^oC with 5 µmol/L fura 2-AM in HEPES buffer consisting of (in mmol/L) 140 NaCl, 5 KCl, 1 MgCl₂, 1 CaCl₂, 10 HEPES, 10 glucose plus 0.1% bovine serum albumin. The extracellular dye was removed by centrifugation. [Ca²⁺]_c measurements were performed at 37^oC under constant stirring in SPEX Fluoromax. Excitation wavelengths were set at 340 and 380 nm, and emission wavelength was set at 505 nm. R_max and R_min were determined by the addition of 1 mmol/L CaCl₂ and 20 µmol/L digitonin followed by 10 mmol/L EGTA (final pH 8.5). Autofluorescence was determined at the end of each experiment by the addition of 1 mmol/L MnCl₂ and 20 µmol/L digitonin.

Measurements of the Kinetics of Activation of the SERCA
Platelet membranes (10 to 20 µg protein) were incubated for 15 minutes at 37 °C in 72 µL HEPES buffer consisting of (in mmol/L) 95 NaCl, 31 KCl, 20 HEPES, 5 Na₂-ATP, 3.75 MgCl₂, 2 phosphoenolpyruvate, 1 ascorbic acid, 0.1 ouabain, 1 NADH, plus 5 µmol/L ionomycin, 14 U/mL pyruvate kinase, 20 U/mL lactate dehydrogenase, with or without 500 nmol/L Tg (to inhibit the SERCA) (pH 7.0 at 37°C). The reaction was linear within the 15-minute incubation period. To determine the kinetics of Ca²⁺ activation of the SERCA, external Ca²⁺ concentrations were adjusted by EGTA and CaCl₂, yielding concentrations of up to 7.8 µmol/L ionized Ca²⁺. The reaction was stopped by the addition of 36 µL 1 N HCl. An aliquot of 45 µL acidified incubation medium was added to 1.5 mL of 6 N NaOH and incubated at 60°C for 20 minutes in the dark. The fluorescence of NAD was read at excitation 340 nm and emission 460 nm. Calibration was performed by the addition of ADP instead of membranes to the reaction medium.

SERCA activity was measured as the Tg-sensitive component of ATP hydrolysis (Fig 1A). The Tg-resistant component (reflecting plasma membrane [PM] Ca²⁺-ATPase [PMCA] activity) was a small fraction (5%) of total Ca²⁺-ATPase activity. This level was insufficient for accurate assessment of the kinetics of PMCA activation in membrane preparation for most individuals.

View larger version (12K): [in this window] [in a new window]   Figure 1. Methodological tools for measuring SERCA activity, SERCA protein expressions, and FECa2+ in the dense tubules. A, An illustration of the kinetics of Ca2+ activation of Ca2+-ATPase from platelet membranes of one donor. Open circles indicate Tg-sensitive activity; closed circles, Tg resistant activity. The Tg-sensitive component was considered an indicator of SERCA activity. Lines show the fit of the model (described in "Analysis of the Data") to the results. B, Standards showing the optical density (O.D.) of immunoblots for SERCA 2 (open circles), SERCA 3 recognized by the PL/IM430 antibody (closed circles), and SERCA 3 recognized by the N89 antibody (open triangles) as a function of platelet membrane proteins. C, Illustrations of the ionomycin-evoked increases in [Ca2+]c. Platelets were treated with 5 µmol/L ionomycin (plus 500 nmol/L Tg). For non–ouabain-treated platelets, R1 and P1 denote the resting [Ca2+]c before the addition of ionomycin and the peak of the ionomycin-evoked increase in [Ca2+]c, respectively. For ouabain-treated platelets, R2 and P2 denote the resting [Ca2+]c and the ionomycin-evoked [Ca2+]c response of ouabain-treated platelets, respectively. P1-R1 and P2-R2 are values that represent the ionomycin-induced Ca2+ release in non–ouabain-treated and ouabain-treated platelets, respectively.

Western Immunoblots of SERCA Proteins
Platelet membrane proteins were electrophoresed on SDS-polyacrylamide gels (7.5%) according to Laemmli²⁰ and electrophoretically transferred to nitrocellulose membranes. After the nitrocellulose membrane was blocked with 5% milk in Tris-buffered saline (TBS) (150 mmol/L NaCl, 10 mmol/L Tris) for 30 minutes, nitrocellulose membranes were incubated with a specific monoclonal antibody (Ab), IID8 (catalog No. MA3–910, Affinity BioReagents) against SERCA 2, and two antibodies against SERCA 3, ie, a monoclonal antibody, PL/IM430 (catalog No. RDI-CBL226, Research Diagnostics, Inc), and a polyclonal antibody, N89 (kindly provided by F. Wuytack, Katholieke University, Leuven, Belgium). The specificity of these antibodies is addressed in "Discussion." Nitrocellulose membranes were then washed three times in TBS/0.1% Tween for 5 minutes, rinsed with TBS, and incubated with horseradish peroxidase–conjugated secondary antibody in 1% milk/TBS for 1 hour. Nitrocellulose membranes were then washed three times in TBS/0.1% Tween and rinsed with TBS. The blots were developed with ECL (catalog No. RPN2106, Amersham) and quantified by densitometry (Molecular Dynamics, Computing Densitometer model 300B, Image Quant version 3.3). To standardize the gels for SERCA expressions in platelets, 2 to 6 µg platelet proteins from the same reference subject were loaded on each gel (Fig 1B).

Experimental Manipulations to Alter the FECa²⁺ in the DT and Test SERCA Capacity to Sequester Ca²⁺
The rapid increase in [Ca²⁺]_c of fura 2–loaded platelets in response to ionomycin and Tg (to inhibit Ca²⁺ reuptake by the DT) in Ca²⁺-free HEPES buffer was used as an indicator of FECa²⁺ in the DT. Two experimental protocols were performed to evaluate the FECa²⁺ in the DT and its relation to SERCA function and protein expressions. In the first protocol, FECa²⁺ in the DT was assessed in unchallenged platelets. In the second protocol, platelets were acutely challenged with an increase in Ca²⁺ load by treatment with ouabain (100 nmol/L) for 60 minutes, of which the last 30 minutes were during fura 2 loading (in 1 mmol/L Ca²⁺ HEPES buffer). Treatment with ouabain inhibits the Na⁺-pump, reduces the Na⁺ gradient across the plasma membrane, and diminishes Ca²⁺ extrusion via the Na⁺/Ca²⁺ exchanger.

Ionomycin-Evoked Rise in [Ca²⁺]_c in Platelets
Experiments were performed in Ca²⁺-free medium. Because Tg inhibits the SERCA, differences in the decay of [Ca²⁺]_c after the peak response to ionomycin plus Tg are related to different activities of the PMCA, the Na⁺/Ca²⁺ exchanger, and possibly redistribution within cellular compartments other than the DT. Treatment of platelets with ionomycin (plus Tg) without a prior exposure to ouabain resulted in a sharp increase in [Ca²⁺]_c (P₁-R₁; first phase), followed by a gradual rise in the [Ca²⁺]_c (second phase) that was observed throughout the remaining period of monitoring (Fig 1C). The rapid increase in [Ca²⁺]_c during the first phase was taken as an indicator of FECa²⁺ in the DT. Treatment of platelets with ionomycin after 60 minutes of preincubation with ouabain resulted in an abrupt increase in the [Ca²⁺]_c(P₂-R₂; first phase), followed by a decline in the [Ca²⁺]_c (second phase) during the remainder of the monitoring (Fig 1C). The peak of the ionomycin-evoked [Ca²⁺]_c response was taken as the FECa²⁺ in the DT. The difference in the peak of the ionomycin-evoked [Ca²⁺]_c response in ouabain-preincubated platelets minus that of the ionomycin-evoked [Ca²⁺]_c response in non–ouabain-treated platelets (P₂-P₁; Fig 1C) was taken as a criterion for the magnitude of the ouabain-induced acute Ca²⁺ sequestration in the DT.

Ca²⁺ activation of the SERCA in platelet membrane was fitted to the following model (Fig 1A): V=V_maxx[Ca²⁺]^N_ext/Km^N+ [Ca²⁺]^N_ext, where V_max is maximal reaction velocity, V is reaction velocity at a given external Ca²⁺ concentration ([Ca²⁺]_ext), Km is the equilibrium dissociation constant, and N is the Hill coefficient.

Other Measurements
Levels of total serum cholesterol, HDL cholesterol, triglycerides, serum glucose, and creatinine were measured with a Kodak Ectachem DT 60 analyzer.

Analysis of the Data
Statistical analyses utilized the Student t test and Pearson correlation analysis. In addition, multiple linear regressions were used to analyze the contributions of platelet parameters to predictions of SBP and DBP. SBP and DBP were regressed in separate models on the platelet parameters P_2-P₁, V_max of the SERCA, and SERCA 2 protein. For each of the platelet parameters, SBP and DBP were regressed on the best combination of that platelet parameter plus one of the following: BMI, LDL cholesterol, HDL cholesterol, triglycerides, glucose, ratio of total lipids to HDL cholesterol, or ratio of triglycerides to HDL cholesterol. The contribution of the platelet parameter to the variability of BP was assessed, after accounting for variability attributed to BMI, glucose, or the lipid variable. Evaluation of the contributions of the platelet parameters after adjustment for BMI or lipids gives a conservative assessment of the importance of platelet parameters in jointly predicting SBP or DBP.

Because of technical problems, not all platelet variables were obtained for all subjects. For this reason, whenever necessary, we indicate the number of observations (n) in the text, legends to the figures, and in the tables. Data in tables and text are presented as mean±SEM.

	Results

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Parameters of Platelet Ca²⁺ and SERCA Kinetics
Table 2 presents parameters of Ca²⁺ and SERCA kinetics of platelets. Platelets from blacks showed lower resting [Ca²⁺]_c than platelets from whites; this was shown in Ca²⁺ containing medium and in Ca²⁺-free medium. However, these differences were not statistically significant. Pretreatment of platelets for 60 minutes with ouabain resulted in increases of roughly 12 nmol/L in the resting [Ca²⁺]_c and about 68 nmol/L in the ionomycin(+Tg)-evoked [Ca²⁺]_c response. There were no significant racial differences in these parameters or in the kinetic parameters of SERCA activation. Therefore, data from both racial groups were pooled for further analyses.

View this table: [in this window] [in a new window]   Table 2. Platelet Ca2+ Parameters

Correlations Among Parameters of Platelet Ca²⁺ Regulation
The V_max of the SERCA demonstrated significant, positive correlations with (1) the ionomycin(+Tg)-evoked Ca²⁺ response in non–ouabain-treated platelets, ie, P₁-R₁ (r=.417, P=.024), (2) the ionomycin(+Tg)-evoked Ca²⁺ response in ouabain-treated platelets, ie, P₂-R₂ (r=.583, P=.0009), and (3) the magnitude of the ouabain-induced Ca²⁺ sequestration in the DT, ie, P₂-P₁ (r=.598, P=.0006) (Fig 2A, 2B, and 2C).

View larger version (12K): [in this window] [in a new window]   Figure 2. Relationships between the Vmax of the SERCA and (A) the ionomycin-evoked [Ca2+]c in non–ouabain-treated platelets (P1-R1) (n=29), (B) the ionomycin-evoked [Ca2+]c in ouabain-treated platelets (P2-R2) (n=29), and (C) the ouabain-dependent component of the ionomycin-evoked [Ca2+]c response (P2-P1) (n=29). Open symbols indicate whites; closed symbols, blacks.

Positive correlation was observed for the V_max of the SERCA with SERCA 2 protein (r=.547, P=.002) (Fig 3A) and SERCA 3 protein (Fig 3B and 3C). It is noteworthy, however, that depending on the antibody used, substantial differences were observed in the correlation between the V_max of the SERCA and SERCA 3 protein expression. Robust correlation was observed between the V_max of the SERCA and SERCA 3 protein expression (r=.717, P=.001) when the N89 antibody was used (Fig 3B). However, only a trend was observed between the V_max of the SERCA and SERCA 3 protein expression (r=.353, P=.055) when the PL/IM430 antibody was used (Fig 3C).

View larger version (14K): [in this window] [in a new window]   Figure 3. Relationships for the Vmax of the SERCA of platelet membranes with (A) SERCA 2 protein (n=30), (B) SERCA 3 protein using the N89 antibody, and (C) SERCA 3 protein using the PL/IM430 antibody (n=30). Open symbols indicate whites; closed symbols, blacks.

Both SERCA 2 and SERCA 3 proteins exhibited significantly positive correlations with the FECa²⁺ in the DT (Figs 4 and 5). For SERCA 2 protein, for instance, significant correlations were seen with (1) the ionomycin(+Tg)-evoked [Ca²⁺]_c increase in non–ouabain-treated platelets (r=.366, P=.043), (2) the ionomycin(+Tg)-evoked [Ca²⁺]_c increase in ouabain-treated platelets (r=.479, P=.006), and (3) the magnitude of the ouabain-induced Ca²⁺ sequestration in the DT (r=.472, P=.007) (Fig 4 A, 4B, and 4C). Similar correlations were observed for SERCA 3 (recognized by the N89 antibody) with the FECa²⁺ in the DT (Fig 5A, 5B, and 5C). There were no correlations between the resting [Ca²⁺]_c and any of the other platelet parameters.

View larger version (12K): [in this window] [in a new window]   Figure 4. Relationship of SERCA 2 protein with (A) the ionomycin-evoked [Ca2+]c increase in non–ouabain-treated platelets (P1-R1) (n=31), (B) the ionomycin-evoked increase in ouabain-treated platelets (P2-R2) (n=31), and (C) the ouabain-dependent component of the ionomycin-evoked [Ca2+]c response (P2-P1) (n=31). Open symbols indicate whites; closed symbols, blacks.

View larger version (12K): [in this window] [in a new window]   Figure 5. Relationships of SERCA 3 protein recognized by the N89 antibody with (A) the ionomycin-evoked [Ca2+]c increase in non–ouabain-treated platelets (P1-R1) (n=31), (B) the ionomycin-evoked increase in ouabain-treated platelets (P2-R2) (n=31), and (C) the ouabain-dependent component of the ionomycin-evoked [Ca2+]c response (P2-P1) (n=31). Open symbols indicate whites; closed symbols, blacks.

Correlations Between Platelet Ca²⁺ Parameters and BP
A weak correlation was observed between the DBP and the ionomycin(+Tg)-evoked [Ca²⁺]_c increase in non–ouabain-treated platelets (r=.379, P=.035; n=31). A much stronger correlation was shown between the DBP and the ionomycin(+Tg)-evoked [Ca²⁺]_c increase in ouabain-treated platelets (r=.481, P=.006; n=31). The SBP also positively correlated with the ionomycin(+Tg)-evoked [Ca²⁺]_c rise in ouabain-treated platelets (r=.422, P=.018; n=31).

Highly significant and robust correlations were observed between the magnitude of the ouabain-induced Ca²⁺ sequestration in the DT and the BP parameters (r=.498, P=.004; r=.492, P=.005; for SBP and DBP, respectively) (Fig 6A and 6B). In addition, the DBP positively correlated with the V_max of the SERCA (r=.462, P=.010; n=30) (Fig 7), whereas only borderline significance was shown between the SBP and the V_max of the SERCA (r=.348, P=.060; n=30). There were no correlations between the BP parameters and the resting [Ca²⁺]_c.

View larger version (11K): [in this window] [in a new window]   Figure 6. Relationships between the ouabain-dependent component of the ionomycin-evoked [Ca2+]c response (P2-P1) with (A) SBP (n=31) and (B) DBP (n=31). Open symbols indicate whites; closed symbols, blacks.

View larger version (18K): [in this window] [in a new window]   Figure 7. Relationship between the DBP and the Vmax of the SERCA (n=30). Open symbols indicate whites; closed symbols, blacks.

Correlations Among Systemic Variables
The SBP, DBP, and BMI positively correlated with serum LDL cholesterol and triglyceride levels but negatively correlated with HDL cholesterol. In addition, the BMI positively correlated with the SBP and DBP, and the fasting blood glucose positively correlated with the BMI (Table 3).

View this table: [in this window] [in a new window]   Table 3. Statistically Significant Correlations Among Relevant Systemic Parameters

Correlations of Platelet Ca²⁺ Parameters With Other Systemic Variables
Table 4 summarizes additional correlations between platelet Ca²⁺ parameters and systemic variables. The BMI positively correlated with the ionomycin(+Tg)-evoked [Ca²⁺]_c response in (1) non–ouabain-treated platelets (r=.371, P=.040), (2) ouabain-treated platelets (r=.491, P=.005), (3) the magnitude of the ouabain-induced Ca²⁺ sequestration in the DT (r=.534, P=.002), and (4) the V_max of the SERCA (r=.363, P=.049). Additionally, the V_max of the SERCA showed positive correlation with serum triglycerides (r=.425, P=.024) and negative correlation with HDL cholesterol (r=-.466, P=.011).

View this table: [in this window] [in a new window]   Table 4. Statistically Significant Correlations of Platelet Parameters With BMI and Serum Lipids

Joint Regressions of BP on Platelet Parameters and Systemic Parameters
Tables 3 and 4 show strong correlations of BMI and lipids with platelet parameters as well as SBP and DBP. To further evaluate the relationships of the platelet parameters to SBP and DBP, considering their mutual correlations with systemic variables, regressions of SBP and DBP on P₂-P₁ and V_max of the SERCA were expanded to include the systemic variable that gave the best two-predictor model for BP. Table 5 presents these multiple regression models. Because neither SERCA 2 protein nor SERCA 3 protein became a significant predictor of BP by inclusion of lipids in the regression models (data not shown), these platelet parameters are not included in Table 5.

View this table: [in this window] [in a new window]   Table 5. Linear Regression of SBP and DBP on P2-P1 or Vmax and Systemic Variables

The strongest systemic correlates of DBP were triglycerides, ratio of triglycerides to HDL, and the BMI (Table 3). The best regression model of DBP on P₂-P₁ and one systemic variable was the model that included triglycerides. Both were significant predictors, taken separately. With triglycerides in the model, the incremental contribution of P₂-P₁ remained significant (P=.045). Triglycerides remained a significant predictor in the joint model (P=.010).

The best regression model of DBP on V_max of the SERCA and one systemic variable included the BMI. Both were significant predictors, taken separately. Once BMI was in the model, the contribution of V_max of the SERCA was borderline (P=.058). BMI remained significant in the presence of V_max of the SERCA (P=.016).

The strongest systemic correlates of SBP were triglycerides, HDL cholesterol, and ratios of triglycerides or total cholesterol to HDL cholesterol (Tables 3 and 4). The best regression model of SBP on P₂-P₁ and one systemic variable was the model that included HDL cholesterol. Both P₂-P₁ and HDL were significant predictors, taken separately. Once triglyceride level was in the model, the contribution of P₂ -P₁ remained significant (P=.014). HDL cholesterol remained significant in the presence of P₂-P₁ (P=.004).

The best regression model of SBP on V_max of the SERCA and one systemic variable included the ratio of total cholesterol to HDL cholesterol. V_max of the SERCA was borderline (P=.060) as a separate predictor. Once the ratio of cholesterol to HDL cholesterol was in the model, the contribution of V_max of the SERCA was not significant (P=.280). The ratio of cholesterol to HDL cholesterol remained a strong predictor (P=.004) in the presence of V_max of the SERCA.

	Discussion

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[Ca²⁺]_c is the penultimate signal in platelet activation. Platelets should therefore manifest increased activity in essential hypertension, assuming that elevated BP is associated with an increase in platelet FECa²⁺. This concept is supported by findings of platelet hyperactivity^{21 22} and predisposition to thromboembolic events in essential hypertension.^{23 24 25 26} Moreover, increased platelet Ca²⁺ is associated with a heightened risk for arterial thrombosis.²⁷ Exploring the relationship between platelet Ca²⁺ homeostasis and BP is therefore important in and of itself, regardless of the validity of platelets as a paradigm expressing the relationship between BP and cellular Ca²⁺ status.

Relevant observations of the present work are that in a heterogeneous population of humans, the BP positively correlated with the FECa²⁺ in the DT, and in particular, Ca²⁺ sequestered in this compartment by an acute treatment of the platelets with ouabain (P₂-P₁). As important are the findings that in this population the V_max of the SERCA positively correlated with FECa²⁺ in the DT; this was apparent in non–ouabain-treated platelets and particularly in ouabain-treated platelets. Treatment with ouabain exerted a relatively small effect on the resting [Ca²⁺]_c but a profound influence on the FECa²⁺ in the DT, as was demonstrated in our previous work.²⁸ The inhibition of the Na⁺/Ca²⁺ exchanger that resulted from ouabain treatment served to magnify the effects of variations in SERCA activity so that platelets with high SERCA activity sequestered more Ca²⁺ into the DT after this treatment. Additionally, ouabain treatment demonstrated that changes in the overall FECa²⁺ are to a large extent reflected in the FECa²⁺ in the DT and in the capacity of the SERCA to sequester Ca²⁺ into this cellular compartment. In itself, the observation that ouabain treatment induced a greater Ca²⁺ sequestration in the DT in platelets of individuals with a higher BP is an insufficient indicator that the SERCA is upregulated with increased BP. However, such a concept is further supported by observations that the V_max of the SERCA positively correlated not only with BP but also with SERCA protein expressions. What is clear is that the resting [Ca²⁺]_c in platelets is neither an indicator of the BP level nor of the platelet Ca²⁺ status.

The V_max of the SERCA expresses the contributions of SERCA 2 and SERCA 3.²⁹ Because the SERCA isoforms were quantified using only relative values for the respective proteins, we could not assess the magnitude of contributions of each SERCA isoform to the V_max of the SERCA in platelet membranes. This may be the reason why the protein expression of the SERCA isoforms did not correlate, whereas the V_max of the SERCA—an indicator of the overall SERCA function—did correlate with the BP. It is noteworthy that the SERCA 2 antibody used in this work (IID8) cannot distinguish between SERCA 2a and SERCA 2b isoforms. However, there is very little or no SERCA 2a in human platelets.^{29 30} Thus, increased SERCA 2 expression in these cells reflects an increase in SERCA 2b expression. In contrast, there is a lack of consensus whether the PL/IM430 antibody recognizes SERCA 3 or another novel SERCA isoform.^{31 32 33} This may be the reason why we could only observe a weak relationship between the V_max of the SERCA and SERCA 3 protein when this antibody was used. In contrast, a robust relationship was noted between the V_max and the SERCA 3 when we used the N89 antibody. The reasons for these differences in the context of this work are not clear.

Because platelets are nonnucleated cells and lack an appreciable Golgi apparatus, they are incapable of significant protein synthesis. Adaptation to an increase in the cellular Ca²⁺ load is likely to occur at the megakaryocytic level. An increase in the FECa²⁺ in the SER of these cells is therefore expected to take place in concert with the upregulation of the protein expressions of the SERCA isoforms. Conversely, conditions that favor a decrease in the cellular Ca²⁺ load may result in the downregulation of SERCA function, protein expressions, and FECa²⁺ in the SER, as has been shown in cell lines that overexpress the PMCA.^{34 35 36} Megakaryocytes might therefore shape the behavior of circulating platelets by the up-and-down regulation of SERCA expression and activity in response to circumstances that chronically alter the cellular Ca²⁺ load. Accordingly, if a rise in BP is associated with processes that tend to increase platelet Ca²⁺ load, then increased SERCA activity represents a common pathway of these processes. The apparent conclusion derived from the present study is that platelet Ca²⁺ status and its relation to BP might be further understood by exploring the dynamics of Ca²⁺ regulation in the DT in platelets and the SER in megakaryocytes.

A recent study has demonstrated that the V_max of the SERCA was not different between platelets of humans with essential hypertension and normotensive control subjects.³⁷ In that study, the Tg-sensitive component was approximately 55% of total Ca²⁺-ATPase activity. This observation contrasts with our findings and observations by others³¹ that the Tg-sensitive component of Ca²⁺-ATPase activity is more than 90% of total Ca²⁺-ATPase activity in platelets. Consequently, we cannot formulate a conclusion with respect to the findings of that study. Another work did not find a correlation between Ca²⁺ uptake by the DT and of BP in saponin-permeabilized platelets from patients with essential hypertension.³⁸ The indirect methodology for the evaluation of the Ca²⁺ uptake by the DT, platelet permeabilization, and the use of unphysiological Ca²⁺ concentration for cytosolic Ca²⁺ (1 mmol/L) in the assay buffer cast doubt about the physiological implications of this finding.

A fundamental question regarding the relationship between BP and platelet Ca²⁺ parameters is whether this relationship reflects dyslipidemia, which is commonly associated with a higher BP. For instance, it is well established that increased LDL level enhances platelet aggregation via the Ca²⁺ signaling system,^{13 14} whereas increased HDL level exerts the opposite effect.^{15 16} We therefore evaluated whether the relationship between platelet parameters and BP can be explained by the serum lipids and the BMI, which often also correlates with blood lipids. BMI and lipid levels explained slightly more variability in BP than platelet parameters. However, despite the correlations between platelet parameters and BMI and lipid levels, platelet parameters provided additional information (although not always at a significant level of P<.05). This is not to imply that lipid levels, and particularly the levels of triglycerides, determine platelet Ca²⁺ parameters, which in turn correlate with BP. It is possible that variations in platelet parameters and lipid levels are shaped by different factors than those responsible for the variations in BP. Alternatively, variations in serum lipid levels, BP, and perhaps platelet Ca²⁺ might be pleiotropic expressions of the same genetic variants in humans. Such a concept is supported by the familial aggregation of dyslipidemia and hypertension^{17 18} and the recent finding of genetic linkage of hypertension to the gene locus of lipoprotein lipase—the enzyme that hydrolyzes serum triglycerides, thereby regulating their levels.³⁹ The more closely correlated the platelet parameters and serum lipid levels are with each other, the more difficult it is to quantify the relations of each with BP.

	Selected Abbreviations and Acronyms

 

BMI = body mass index BP = blood pressure DBP = diastolic blood pressure DT = dense tubules FECa2+ = freely exchangeable Ca2+ SER = sarco(endo)plasmic reticulum SERCA = SER Ca2+-ATPase SBP = systolic blood pressure Tg = thapsigargin

	Acknowledgments

 
This work was supported by National Institutes of Health grants HL47906 (Dr Aviv) and DK42879 (Dr Lytton). Dr Lytton is an Established Investigator of the American Heart Association and a Scholar of the Alberta Heritage Foundation for Medical Research. Dr Poch was supported by grant CIRIT BE94/1-552. Dr Horiguchi's postdoctoral fellowship was supported by the American Heart Association, NJ Affiliate. We thank Patricia A. Peluso for her excellent secretarial help.

Received June 5, 1997; first decision June 23, 1997; accepted September 4, 1997.

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