Low-Density Lipoproteins Inhibit the Na+/H+ Antiport in Human Platelets
A Novel Mechanism Enhancing Platelet Activity in Hypercholesterolemia
Background LDL have been reported to augment platelet activation, and increased platelet reactivity has been observed in familial hypercholesterolemia. However, the underlying mechanism of this putatively atherogenic effect is unknown. Because intracellular pH (pHi) may play an important role in platelet function, we examined the influence of LDL on pHi and Na+/H+antiport activity in human platelets and compared it with the effect of [3-methylsulfonyl-4-piperidinobenzoyl] guanidine hydrochloride (HOE 694), a selective Na+/H+ antiport inhibitor.
Methods and Results Using a fluorescent dye technique, we demonstrated that incubation of platelets with physiological concentrations of LDL or with HOE 694 decreased pHi. In addition, both LDL and HOE 694 inhibited the Na+/H+ antiport in platelets treated with sodium propionate or thrombin. The inhibitory effect of LDL was observed both in normal and in glycoprotein (GP)IIb/IIIa- as well as in GPIIIb (CD36)-deficient platelets and was not influenced by the covalent modification of apolipoprotein B lysine residues, suggesting that specific LDL binding sites were not involved. Thrombin-induced phosphoinositide breakdown, diacylglycerol formation, and Ca2+ mobilization, as well as platelet aggregation and granule secretion, were potentiated by both LDL and HOE 694. pHi and Na+/H+ antiport activity were significantly reduced in platelets from patients with familial hypercholesterolemia. Both parameters were normalized after normalization of LDL levels by apheresis treatment.
Conclusions LDL inhibits the Na+/H+ antiport most likely via receptor-independent mechanisms, thereby augmenting platelet reactivity. This novel mechanism explains increased platelet reactivity in patients with familial hypercholesterolemia and may contribute to the atherogenic potential of LDL.
Both platelets and lipoproteins are intimately involved in the pathogenesis of atherothrombotic diseases.1 Moreover, platelet function is directly influenced by lipoproteins,2 and platelets from patients with hypercholesterolemia display enhanced platelet reactivity. The exact mechanism of this effect is unclear. Supraphysiological LDL concentrations have been demonstrated to enhance arachidonic acid mobilization and thromboxane B2 formation, suggesting the involvement of phospholipase A2.4 Furthermore, LDL increases DAG and polyphosphoinositide formation, indicating the possible involvement of PI-PLC.5 6
The activation of human platelets is associated with an initial decrease in pHi. This response is counteracted by Na+/H+ antiport, which is activated immediately after platelet stimulation.7 Here we demonstrate that LDL at physiologically relevant concentrations inhibit the Na+/H+ antiport in human platelets, thereby producing intracellular acidification.
All chemicals were from Sigma unless indicated otherwise. HOE 694 was kindly provided by Dr Lang, Hoechst, Frankfurt, Germany. HOE 694 is a new Na+/H+ antiport inhibitor possessing anti-ischemic properties on myocardial cells. HOE 694 inhibits competitively Na+/H+ antiport isoform 1 (NHE1) with a much greater affinity than NHE2 and NHE3.8
The characteristics of the 15 healthy subjects studied are given in Table 1⇓. None had taken any medication for 4 weeks before the study. Eight patients heterozygous for FH with initial plasma cholesterol >520 mg/L and LDL cholesterol >350 mg/L were included. The diagnosis was confirmed by establishing a reduced binding of LDL to cultured fibroblasts (<60% of normal). All patients received HMG-CoA reductase inhibitors. The characteristics of 1 patient with Glanzmann thrombasthenia type 1, whose GPIIb/IIIa content is <1%, and another patient with GPIIIb (CD 36) deficiency have been described previously.9 10
Platelet count, prothrombin time, partial thromboplastin time; cholesterol, triglyceride, apo B, and apo A-I concentrations; and arterial blood gas analysis were determined by routine laboratory methods. The concentration of HDL cholesterol was measured after precipitating apo B–containing lipoproteins with phosphotungstic acid/MgCl2. The LDL concentration was calculated using the Friedewald formula.
Immunoselective LDL Apheresis
The immunoselective apheresis was performed using the LDL-Therasorb system (Baxter), which consists of two columns with Sepharose CL-4B coupled to anti–apo B antibodies. After an initial bolus of heparin (1000 IU), blood was continuously mixed with acid citrate dextrose. LDL-containing plasma was separated by a rotating membrane separator and passed through the anti-LDL column at a flow rate of 30 to 45 mL/min. Plasma volume treated approached 4200 mL.
In two subjects, extracorporeal immunoadsorption was carried out. After an initial bolus of heparin (1000 IU) and blood anticoagulation with acid citrate dextrose, the procedure was performed using the rotating membrane separator for blood cell separation and the TPS A201 system (Baxter) endowed with IMPM350 adsorbing columns (Diamed) for the removal of autoantibodies. The running parameters were similar to those used in LDL apheresis.
Platelet Isolation and LDL Isolation and Modification
Human platelets were isolated as described earlier.9 11 LDL (d=1.019 to 1.063 g/mL) were isolated from human plasma by discontinuous KBr gradients.12 The lipoproteins were extensively dialyzed against 0.05 mmol/L Tris-HCl and 0.14 mol/L NaCl, pH 7.4. The protein content of lipoproteins was determined after TCA precipitation according to Lowry. Cyclohexanedione-modified LDL (LDL-CHD) and minimally oxidized LDL (LDL-MOX) were prepared as described earlier.13 14
Determination of Platelet Cytosolic pH (pHi), Cytosolic Na+, and Ca2+ Concentrations ([Na+]i, [Ca2+]i)
pHi was measured using BCECF-AM in a 1-mL platelet suspension (108 cells/mL) at 37°C using a fluorescence spectrophotometer (model F-2000, Hitachi Ltd).15 Calibration was done using the high K+/nigericin method.16 The rate of pHi recovery after stimulation with either 100 mmol/L sodium propionate or 0.05 U/mL thrombin was computed using GraphPAD-Inplot 4.02 software and expressed as dpHi/s (change in pHi/s). Rate constants of the pHi recovery were obtained through iterative curve fitting of the experimental data to an exponential curve.17 Cytosolic Na+ and Ca2+ were measured as previously described.18 The fluorescence excitation ratio F340/F385 was taken as a measure for [Na+]i.
Determination of Phospholipid Turnover, DAG Formation, Platelet Aggregation, and Dense Granule Secretion
Phospholipid turnover and DAG formation were determined as previously described.19 Platelet aggregation was monitored in the Elvi 611 aggregometer adjusted to its maximal sensitivity at 37°C and at a stirring speed of 900 rpm, as described by Born.20 The measurement of dense granule secretion was performed as described earlier21 using 2 μmol/L [14C]serotonin binoxalate (Amersham).
Expression of P-Selectin and Granulophysin on the Platelet Surface
Platelets were preincubated for 5 minutes at 37°C with 0.5 g/L LDL, 0.01 mmol/L HOE 694, or an equal volume of buffer. Platelets were then activated with α-thrombin without stirring, and after 3 minutes the reaction was stopped by addition of paraformaldehyde (final concentration, 0.5% wt/vol). Platelets were then incubated with the monoclonal antibodies against P-selectin (clone: CLB-thromb/b, Immunotech; Marseille, France), granulophysin (clone D545, Dr McNicol; Winnipeg, Canada), or isotype matched mouse IgG (Coulter Electronics; Hialeah, Fla). For fluorescence labeling, sheep anti-mouse F(ab)2-FITC fragments were added for a further 30 minutes. Fluorescence of 5×103 platelets was measured using a FACS-SCAN instrument (Becton-Dickinson).
Data are mean±SEM. The groups were compared with the Wilcoxon-Mann-Whitney test with the use of the computer software Instat 2.02 (GraphPAD) unless indicated otherwise. Two-tailed values of P<.05 were considered significant. Original tracings shown in the figures were computed from measured data with the use of locally weighted scatterplot smoothing (GraphPAD Prism 1.0) and are representative for a minimum of 5 similar experiments.
LDL Inhibits Sodium Propionate–Activated and Thrombin-Activated Na+/H+ Antiport
Resting platelets had a pHi of 7.35±0.02 (n=17). LDL 0.5 g/L decreased pHi by 0.17±0.06 pH unit within 5 minutes (Fig 1⇓). Likewise, 0.01 mmol/L HOE 694 decreased resting pHi by 0.21±0.02 pH unit within 5 minutes (Fig 1⇓). Next, the effect of LDL and HOE 694 on sodium propionate–activated or thrombin-activated Na+/H+ antiport was tested. After administration of sodium propionate, pHi decreased rapidly to a minimum value (pHi,min) and then slowly recovered, reaching a new steady state after ≈100 seconds (Fig 2A⇓). The pHi increase (DpHi) and the initial Na+/H+ antiport rate (dpHi/s) after acidification were used as a measure of Na+/H+ antiport activity. Both LDL and HOE 694 significantly reduced the Na+/H+ antiport activity and the pHi recovery after acidification (Fig 2A⇓ and Table 2⇓). Fig 2B⇓ demonstrates the dose-dependent effect of LDL on the initial rate of pHi recovery.
Thrombin (0.05 U/mL) slightly and transiently decreased pHi by 0.03 unit followed by a pHi rise above the resting level within 100 seconds (Fig 2C⇑). The effects of LDL and HOE 694 on the thrombin-stimulated changes in pHi and Na+/H+ antiport activity are demonstrated in Fig 2C⇑ and Table 2⇑. As with the sodium propionate–induced changes, LDL inhibited the thrombin-induced Na+/H+ antiport stimulation in a concentration-dependent fashion (Fig 2D⇑). Resting [Na+]i increased after addition of both LDL and HOE 694 (data not shown). Expectedly, the pretreatment of platelets with LDL or HOE 694 for 5 minutes markedly reduced the sodium propionate–induced or thrombin-induced increases in [Na+]i (Fig 3⇓).
Involvement of LDL-Specific Binding Sites
To investigate whether or not the inhibitory effect of LDL on the Na+/H+ antiport is receptor specific, we measured the sodium propionate–induced Na+/H+ antiport activity in the presence of LDL-CHD. This modification has been shown to abolish specific binding of LDL to platelets.13 Preincubation of 0.5 g/L LDL-CHD for 5 minutes with platelets decreased pHi by 0.29±0.08 pH unit. The inhibitory effect of 0.5 g/L LDL-CHD on the sodium propionate–induced Na+/H+ antiport activity was comparable to that of native LDL (Table 3⇓).
Since GPIIb/IIIa is a putative binding protein for LDL in human platelets, we also examined the effect of LDL on sodium propionate–activated Na+/H+ antiport in GPIIb/IIIa-deficient platelets. Resting pHi in GPIIb/IIIa-deficient platelets (7.41±0.01) was similar to that of control platelets. Furthermore, the inhibitory effects of LDL and HOE 694 were also seen in platelets lacking GPIIb/IIIa (Table 3). However, the fall in pHi induced by incubation of GPIIb/IIIa-deficient platelets with 0.5 g/L LDL or 0.01 mmol/L HOE 694 amounted to 0.57±0.03 and 0.45±0.07, respectively, and was much more pronounced than in control platelets. Moreover, the initial Na+/H+ antiport rates were significantly lower in GPIIb/IIIa-deficient platelets than in normal platelets.
Influence of LDL Oxidation
Oxidation renders lipoproteins more reactive to platelets.22 Therefore, we investigated the effect of LDL-MOX on sodium propionate–induced Na+/H+ antiport activity. LDL-MOX (0.5 g/L) decreased pHi by 0.20±0.02 pH units. The sodium propionate–induced Na+/H+ antiport activity was decreased similarly as with native LDL (Table 3⇑)
To further examine the possible role of LDL oxidation for the inhibitory effect of LDL on the Na+/H+ antiport activity, we investigated the effect of LDL on the sodium propionate–induced Na+/H+ antiport activity in platelets lacking GPIIIb (CD 36), a putative receptor for oxidized LDL.23 Resting pHi in GPIIIb-deficient platelets (7.42±0.05) was similar to that of control platelets. Furthermore, the inhibitory effect of LDL on Na+/H+ antiport was also seen in GPIIIb-deficient platelets (Table 3⇑). We concluded that the effect exerted by LDL on Na+/H+ antiport is not dependent on the presence of oxidized LDL.
Involvement of PI-PLC and Prostaglandins
Since activation of prostaglandins and PI-PLC was postulated to be involved in LDL-platelet interactions,4 5 6 we evaluated the effect of the respective inhibitors on LDL-induced inhibition of Na+/H+ antiport. Pretreatment of the cells for 5 minutes with the cyclooxygenase inhibitor indomethacin (0.1 mmol/L) or with the PI-PLC inhibitor U73122 (0.05 mmol/L) did not affect the resting pHi. In indomethacin-pretreated platelets the activity of sodium propionate–stimulated Na+/H+ antiport in the absence or presence of 0.5 g/L LDL averaged 10.84±1.86×10−3 pHi/s (n=6) and 5.96±2.80×10−3 pHi/s (n=9, P<.02), respectively. Likewise, inhibition of platelet cyclooxygenase with 0.1 mmol/L indomethacin had no effect on LDL-induced inhibition of the thrombin-stimulated Na+/H+ antiport activity (9.85±0.73×10−3 pHi/s, n=6, versus 6.75±0.61×10−3 pHi/s, n=8; P<.05). Finally, pretreatment of platelets with U73122 failed to affect LDL-induced inhibition of the sodium propionate–stimulated Na+/H+ antiport activity (9.42±0.23×103 versus 5.71±0.23×103, n=8; P<.05). These data suggest that neither prostaglandins nor PI-PLC are involved in the effects of LDL on the Na+/H+ antiport.
Effect of Na+/H+ Antiport Inhibition on Thrombin-Induced Cellular Signaling
Inhibition of Na+/H+ antiport is known to affect the phosphoinositide cycle in activated platelets. Therefore, we tested the effect of LDL and HOE 694 on the thrombin-induced PIP2 turnover in platelets prelabeled with [32P]orthophosphate. As shown in Fig 4A⇓, thrombin induced a rapid breakdown of [32P]PIP2. The initial fall in PIP2-associated radioactivity occurred within 15 seconds after stimulation and was followed by a rapid resynthesis, which peaked 60 seconds after stimulation. After the pretreatment of platelets with 0.5 g/L LDL for 5 minutes, the thrombin-induced breakdown of [32P]PIP2 was considerably augmented. The initial fall of the PIP2-associated radioactivity was more pronounced and the apparent PIP2 resynthesis was decelerated. Similarly, administration of 0.01 mmol/L HOE 694 5 minutes before stimulation of platelets with thrombin resulted in an enhanced [32P]PIP2 breakdown.
Since DAG is the direct product of PIP2 breakdown, we investigated the effect of LDL and HOE 694 on the thrombin-induced [14C]DAG production in platelets prelabeled with [14C]arachidonic acid. Thrombin induced the formation of [14C]DAG, which peaked 30 seconds after stimulation (Fig 4B⇑). Pretreatment of platelets with 0.5 g/L LDL for 5 minutes did not alter the [14C]DAG resting level but considerably enhanced the thrombin-stimulated [14C]DAG production. Its maximum was noted 15 seconds after stimulation. Likewise, pretreatment of platelets with 0.01 mmol/L HOE 694 resulted in an increased [14C]DAG production (Fig 4B⇑). The enhancing effects of LDL and HOE 694 on DAG production remained unchanged in the presence of 0.1 mmol/L indomethacin (data not shown).
The breakdown of PIP2 results in the formation of inositol(1,4,5)trisphosphate (InsP3), which in turn mobilizes Ca2+ from internal stores. Therefore, we investigated the effect of LDL and HOE 694 on the thrombin-induced Ca2+ mobilization. Resting [Ca2+]i (78±4 nmol/L, n=10) rose after stimulation with thrombin by 56±8 nmol/L. Neither 0.5 g/L LDL nor 0.01 mmol/L HOE 694 significantly affected resting [Ca2+]i, but both markedly enhanced the thrombin-induced [Ca2+]i increase (Fig 4C⇑; LDL, 104±4 nmol/L; n=7, P<.05; HOE 694; 99±2 nmol/L; n=5; P<.05). On the other hand, neither LDL nor HOE 694 significantly enhanced the [Ca2+]i response to sodium propionate. The potentiating effects of LDL and HOE 694 were not affected by 0.1 mmol/L indomethacin (data not shown).
To confirm the potentiating effect of the pHi lowering on the DAG formation and the mobilization of intracellular Ca2+, we examined the [14C]DAG and [Ca2+]i response to thrombin after adjusting pHi to 6.3. This was accomplished by adjusting the extracellular pH to 6.3 in the presence of 0.01 mmol/L nigericin, a K+/H+ ionophore, and 0.01 mmol/L HOE 694 to eliminate the counteracting activity of Na+/H+ antiport. The thrombin-induced DAG and [Ca2+]i response was markedly enhanced after lowering pHi (increase in DAG, 117±11%, n=3; [Ca2+]i increase, 332±33 nmol/L, n=5; P<.001).
Effect of LDL and HOE 694 on Platelet Aggregation and Granule Secretion
Next we investigated whether the potentiating effect of LDL and HOE 694 on DAG formation and intracellular Ca2+ mobilization was accompanied by the enhancement of thrombin-induced aggregation and secretory responses. Both LDL at concentrations up to 1 g/L and 0.01 mmol/L HOE 694 failed to induce platelet aggregation and granule secretion. By contrast, preincubation of platelets with LDL or HOE 694 for 5 minutes significantly augmented platelet aggregation and [14C]serotonin secretion induced by thrombin (Fig 5⇓, A and B, and Table 4⇓). Both LDL and HOE 694 failed to affect aggregation and [14C]serotonin secretion induced by 1 μmol/L A23187 (Table 4⇓).
Furthermore, the influence of LDL and HOE 694 on granule secretion was monitored by flow cytometry. Expression of granulophysin and P-selectin on the platelet surface was used as a measure for dense granule and α-granule secretion, respectively. Neither 0.01 mmol/L HOE 694 nor LDL alone in concentrations up to 1 g/L caused platelet granule secretion. Preincubation of platelets with 0.5 g/L LDL or 0.01 mmol/L HOE 694 for 5 minutes potentiated secretion of both dense granule and α-granule (Fig 5⇑, C and D).
Determination of pHi and Na+/H+ Antiport Activity in Platelets From FH Patients
To further examine the effect of LDL on Na+/H+ antiport activity, we determined pHi as well as DpHi and dpHi/s in platelets obtained from patients with FH. pHi in FH platelets was 7.16±0.05 (n=8) and was significantly lower than in platelets from control subjects (7.35±0.02, n=17; P<.001). After acidification with 0.1 mol/L sodium propionate, pHi dropped to 6.73±0.06 (n=8) versus control, 6.91±0.02 (n=17) (P<.01) and then rose due to activation of Na+/H+ antiport by 0.19±0.04 pH units (n=8) versus control, 0.26±0.02 (n=17) (P<.01). The dpHi/s in platelets from FH patients was 3.77±0.73×10−3 dpHi/s (n=8) and was significantly lower than in platelets from control subjects (9.13±0.74×10−3 dpHi/s; P<.001).
Next, the effect of LDL reduction by immunoselective LDL apheresis in FH patients on Na+/H+ antiport activity was investigated. This treatment led to a >60% reduction of LDL-C, total cholesterol, and apo B (Fig 6⇓ and Table 5⇓). The marked prolongation of partial thromboplastin time and prothrombin time was related to the anticoagulation upon the treatment. Fig 6⇓ illustrates that the reduction of total cholesterol and LDL-C was accompanied by an increase in platelet pHi and sodium propionate–induced Na+/H+ antiport activity. As the result of LDL apheresis, pHi increased to 7.28±0.06 (n=8, NS compared with pretreatment values) and Na+/H+ antiport activity increased to 6.28±0.89×10−3 pHi/s (n=8, P<.01 versus pretreatment values).
In two subjects, the effect of extracorporeal immunoadsorption on pHi and Na+/H+ antiport activity was determined. This intervention does not affect plasma LDL and apo B concentrations (Table 5⇑) but, like LDL apheresis, involves anticoagulation therapy and artificial surfaces. The pHi and Na+/H+ antiport activity before immunoadsorption were 7.43±0.06 (n=2) and 7.99±1.37×10−3 pHi/s (n=2), respectively. After the intervention, pHi and Na+/H+ antiport activity decreased to 7.35±0.08 and 7.33±1.07×10−3, respectively.
This study demonstrates that LDL causes intracellular acidification and decreases maximal Na+/H+ antiport activity in human platelets. In this respect, LDL resembles HOE 694, the selective inhibitor of Na+/H+ antiport. The LDL-associated effects were concentration dependent, occurred at physiologically relevant concentrations, and were confined solely to this lipoprotein class, as VLDL failed to affect Na+/H+ antiport and even opposite effects were observed with HDL3.11
Our results argue against a role of specific LDL binding sites in the inhibitory effect of LDL on Na+/H+ antiport. Modification of LDL with cyclohexanedione, otherwise known to abolish LDL binding to platelets, did not affect the Na+/H+ antiport inhibition. Furthermore, the results obtained with GPIIb/IIIa- or with GPIIIb-deficient platelets clearly demonstrate that the inhibitory effect of LDL on Na+/H+ antiport is not mediated via binding to GPIIb/IIIa or GPIIIb. Rather, the present data suggest receptor-independent mechanisms by which LDL inhibits Na+/H+ antiport. This notion is supported by findings indicating that the enrichment of platelet membranes with cholesterol inhibits the Na+/H+ antiport in human platelets.24 Recently, the modulation of transmembrane signaling by cholesterol-rich domains has been postulated.25
Whereas the inhibition of the Na+/H+ antiport by LDL was not changed in GPIIb/IIIa-deficient platelets, the degree of intracellular acidification in the presence of LDL or HOE 694 was much more pronounced in GPIIb/IIIa- and GPIIIb-deficient platelets than in normal platelets. Moreover, in both defects, a decreased maximal Na+/H+ antiport activity was observed. Either these glycoproteins indeed modulate Na+/H+ exchange, as suggested earlier for GPIIb/IIIa,26 or Na+/H+ exchange activation is secondary to another defect yet to be discerned.
Whereas LDL inhibited Na+/H+ antiport, the thrombin-induced PIP2 breakdown and, consequently, the thrombin-induced DAG formation and [Ca2+]i mobilization were markedly enhanced in the presence of LDL. HOE 694 increased the thrombin-induced DAG formation and [Ca2+]i response similarly to LDL. It is unlikely that HOE 694, specifically inhibiting Na+/H+ antiport, affects the DAG formation and [Ca2+]i response to thrombin by other mechanisms. With respect to the effects analogous to those of HOE 694, LDL may also affect platelet DAG and Ca2+ handling by inhibition of Na+/H+ antiport, although for LDL, other additional mechanisms cannot be dismissed. Na+/H+ antiport inhibition may affect DAG production and cellular Ca2+ handling either by changes in [Na+]i or in pHi. Na+/H+ antiport inhibition decreases the thrombin-induced [Na+]i increase. On the other hand, the effect of [Na+]i on DAG formation has not been examined to date, and enhanced [Ca2+]i responses to various agonists have been described as a consequence of increased, not decreased, [Na+]i.27 28 Rather, a decreased pHi caused by Na+/H+ antiport inhibition could enhance the DAG and [Ca2+]i response to agonists. Intracellular acidification was found to promote phosphoinositide breakdown, DAG production, and inositol phosphate formation in various cells.29 30 Also, the functional coupling between [Ca2+]i responses and pHi is well documented. In human platelets, intracellular acidification was reported to favor [Ca2+]i mobilization both in the resting state as well as after stimulation with agonists.31 Furthermore, the present study revealed that lowering of pHi markedly augmented the DAG and [Ca2+]i response to thrombin. Therefore, it is conceivable that the LDL-induced inhibition of the Na+/H+ antiport and lowering of pHi mediates the LDL effects on the thrombin-induced phosphoinositide turnover, DAG production, and cellular Ca2+ handling.
Platelet reactivity to thrombin, eg, platelet aggregation, serotonin secretion, and expression of granulophysin and P-selectin, was enhanced in the presence of LDL. The slight difference in the sensitivity of P-selectin and granulophysin secretion compared with that of aggregation and serotonin secretion may be explained by the different preparation of platelets in these experiments. Whereas for measurements of aggregation and serotonin secretion the platelets were centrifuged, in the other experiments gel filtration had been used. By contrast, no sensitizing effect of LDL was noted when a Ca2+ ionophore, A23187, was used as an agonist. Hence, it appears that LDL-induced potentiation of platelet reactivity depends on upstream Ca2+-related effects and probably is associated with signal transduction events such as phosphoinositide turnover and DAG production. Furthermore, the potentiating effect of LDL was mimicked by HOE 694, suggesting that similar mechanisms operate in both cases. It was demonstrated that the inhibition of the Na+/H+ antiport as well as intracellular acidification greatly increases platelet secretion in response to various agonists.32 Thus, inhibition of Na+/H+ antiport and intracellular acidification may represent a novel mechanism by which LDL potentiates platelet responses to agonists.
The present study demonstrates that platelet pHi homeostasis is impaired in FH subjects as manifested by significantly lowered pHi and severely impaired activation of Na+/H+ antiport. Moreover, reduction of plasma LDL by means of immunoselective LDL apheresis increased pHi and normalized Na+/H+ antiport. By contrast, no significant changes in pHi and Na+/H+ antiport were observed after extracorporeal immunoadsorption, which, similar to LDL apheresis, involves artificial surfaces and anticoagulation. Hence, it is unlikely that LDL apheresis affects platelet pHi and Na+/H+ antiport independent of the LDL reduction. Taken together, the present results support the contention that abnormal functional responses encountered in platelets from FH subjects are related to the dysfunctional regulation of intracellular pH homeostasis.
The LDL-induced Na+/H+ inhibition represents a novel mechanism by which LDL enhances platelet reactivity in vitro and in vivo and thereby possibly contributes to the progression of atherothrombotic vascular disease.
Selected Abbreviations and Acronyms
|HOE||=||(3-methylsulfonyl-4-piperidinobenzoyl) guanidine hydrochloride|
|PI-PLC||=||phosphoinositide-specific phospholipase C|
- Received July 11, 1996.
- Revision received November 21, 1996.
- Accepted December 15, 1996.
- Copyright © 1997 by American Heart Association
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