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Influence of Low Density Lipoprotein from Insulin-Dependent Diabetic Patients on Platelet Functions

Department of Diabetology, INRCA Hospital; Institute of Biochemistry, University of Ancona School of Medicine (D.M., G.C., L.M.); and the Department of Clinical Chemistry, Torrette Hospital (F.R.), 60131 Ancona, Italy; and the Institute of Biochemistry, University of Rangueil (N.D.), Toulouse, France

Address all correspondence and requests for reprints to: Prof. L. Mazzanti, Institute of Biochemistry, Via Ranieri 65, 60131 Ancona, Italy.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
In the present work we studied in vitro the action of low density lipoproteins (LDL) isolated from normolipemic insulin-dependent diabetic (IDDM) patients on transmembrane cation transport, nitric oxide synthase (NOS) activity, and aggregating response to stimuli of platelets from healthy subjects to elucidate whether the modified interaction between circulating lipoproteins and cells might be one of the pathogenetic mechanisms of the increased platelet activation in IDDM. LDL were obtained by discontinuous gradient ultracentrifugation from 15 IDDM out-patients and 15 sex- and age-matched healthy subjects and used for incubation experiments with control platelets. Lipid composition and hydroperoxide concentrations were studied in LDL. Platelet aggregation responses to ADP, NOS activity, cytosolic Ca²⁺ concentrations, and platelet membrane Na⁺/K⁺-adenosine triphosphatase (Na⁺/K⁺-ATPase) and Ca²⁺-ATPase activities were measured after incubation. IDDM LDL showed an increased lysophosphatidylcholine content compared with that of control LDL. IDDM LDL significantly increased the platelet aggregating response to ADP, cytosolic Ca²⁺ concentrations, and plasma membrane Ca²⁺-ATPase activity and significantly reduced NOS activity and platelet membrane Na⁺/K⁺-ATPase activity compared with those of platelets incubated in buffer or cells incubated with control LDL. The effects exerted by IDDM LDL on platelet suspensions from healthy subjects mimic the alterations observed in platelets from diabetic subjects in basal conditions Both the decreased activity of NOS and the higher cytoplasmic concentrations of Ca²⁺ might cause increased platelet activation, as observed in IDDM. In conclusion, the present study suggests a new mechanism with a potential role in the early development of atherosclerosis in diabetic patients, i.e. an altered interaction between circulating lipoproteins and platelets.

	Introduction

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HIGH PLASMA levels of low density lipoprotein (LDL) have been identified as a primary risk factor in atherosclerosis. Oxidized LDL (ox-LDL) can be taken up by macrophages, giving rise to foam cells, the primary constituents of the fatty streak (1). Besides their effect on macrophages, it might be hypothesized that LDL interact with circulating cells, such as platelets, determining modifications potentially relevant in the development of the atherosclerotic lesion. In fact, oxidized LDL are able to increase platelet aggregation and thromboxane A2 release (2), whereas platelets, in turn, are able to produce a factor enhancing ox-LDL uptake by macrophages (3). Recent works suggested that the effect of ox-LDL on platelet activation might be dependent on an inhibition of the plasma membrane Ca²⁺-adenosine triphosphatase (Ca²⁺-ATPase) with subsequently higher cytoplasmic calcium levels and increased sensitivity to agonists (4) and/or on a reduction of platelet nitric oxide (NO) synthase (NOS) expression and activity (5).

NO prevents platelet activation and adhesion and decreases platelet thrombus formation (6). Human platelets possess both constitutive NOS and inducible NOS isoforms with mol wt and properties different from those of endothelial cell NOS (7) and dependent on the presence of Ca²⁺ (8). A reduced platelet NOS activity has been recently reported by our group in patients affected by insulin-dependent (IDDM) and noninsulin-dependent diabetes mellitus (9), which are pathological conditions characterized by the presence of platelet activation and hyperaggregability.

In the present work we studied in vitro the action of LDL isolated from normolipemic insulin-dependent diabetic patients on the transmembrane cation transport and NOS activity of platelets from healthy subjects to elucidate whether the modified interaction between circulating lipoproteins and cells might be one of the pathogenetic mechanisms of the increased platelet activation in IDDM.

	Subjects and Methods

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The study was performed on 15 IDDM out-patients (7 men and 8 women) and 15 healthy volunteers (7 men and 8 women). Their clinical characteristics are shown in Table 1. Each subject gave informed consent before the investigation. The diabetic subjects were normoalbuminuric and normotensive. All subjects showed plasma lipid levels and body mass indexes within the normal ranges. Three IDDM patients were affected by background retinopathy. The patients were not receiving any treatment except for insulin. The study was performed in accordance with the principles of the Declaration of Helsinki as revised in 1996, and informed consent was obtained from the patients.

The incubation was performed with buffer, native LDL from healthy subjects, and native LDL from IDDM subjects (100 µg LDL protein/mL) in Tyrode’s buffer (137 mmol/L NaCl, 2.7 mmol/L KCl, 1 mmol/L MgCl₂, 1 mmol/L CaCl₂, 0.35 mmol/L NaH₂PO₄, 11.9 mmol/L NaHCO₃, and 5.5 mmol/L glucose, pH 7.5) for 3 h at 37 C. Additional experiments were performed with different concentrations of LDL (100–200-300 µg LDL protein/mL) obtained again from six healthy subjects and six IDDM subjects of the two groups previously described and immediately used for further incubation experiments with platelets from control subjects under the conditions described above. All of the incubation experiments were mixed, i.e. platelets were always incubated with LDL from a different donor.

After the incubation, NOS activity and cytoplasmic Ca²⁺ levels were determined in platelets, whereas platelet membranes were prepared for the assay of Na⁺/K⁺-ATPase and Ca²⁺-ATPase activities. Platelet plasma membranes were obtained according to the method of Enouf et al. (11). Moreover, platelet aggregation responses to ADP (2 µmol/L) were determined before and after the incubation with buffer, LDL from healthy subjects, and LDL from IDDM patients. The platelet aggregation studies were carried out according to Born’s method (12) using the photometric system Packs-4 (Helena Laboratories, Beaumont, TX). The aggregating response to ADP was evaluated as the reduction of light absorption and quantified as maximal aggregation (percentage) using the formula of Weiss and Rogers (13).

Data are expressed as the mean ± SD. Statistical analysis was carried out using Student’s t test for paired data. The differences observed in the paired data were normally distributed.

Preparation and characterization of LDL

Native lipoproteins (LDL; density between 1.025–1.063 g/mL) were isolated from plasma by discontinuous density gradient ultracentrifugation as described by Chen et al. (5). The concentrations of triglycerides, phospholipids, and cholesterol were determined in LDL as previously described (14). LDL lipid hydroperoxide concentrations were measured by the ferrous oxidation-xylenol orange assay according to the method of Jiang et al. (15). Lipids were extracted from LDL as described by Folch et al. (16), and the phospholipid composition was analyzed by thin layer chromatography according to the method of Watala and Jozwiak (17).

Na⁺/K⁺-ATPase activity

Na⁺/K⁺-ATPase activity was determined in platelet membranes by a modification of the Kitao method (18), as previously described (19). Briefly, ATPase activity was assayed by incubating membranes at 37 C in 5 mmol/L MgCl₂, 140 mmol/L NaCl, 14 mmol/L KCl, and 40 mmol/L Tris-HCl, pH 7.7. The ATPase reaction was started by the addition of 3 mmol/L Na₂ATP. Inorganic phosphate (Pi) hydrolyzed from this reaction was measured by the method of Fiske and Subbarow (20). The ATPase activity assayed in the presence of 10 mmol/L ouabain was subtracted from the total Mg²⁺-dependent ATPase activity to calculate the activity of Na⁺/K⁺-ATPase. Protein concentration was determined as described by Lowry et al. (21) using serum albumin as a standard.

Ca²⁺-ATPase activity

Ca²⁺-ATPase activity was determined in platelet membranes according to the method of Davis et al. (22) by measuring the Pi hydrolyzed from 1 mmol/L Na₂-ATP at 37 C in the presence and absence of 0.15 mmol/L Ca²⁺. The ATPase activity determined in the absence of Ca²⁺ was subtracted from total ATPase activity to calculate Ca²⁺-ATPase activity.

NOS activity

NOS activity was measured spectrophotometrically by following the oxidation of oxyhemoglobin ( maximum, 426 nm) to methemoglobin ( maximum, 405 nm) by NO at 37 C, as previously described (23). Oxy-hemoglobin was prepared by reduction. Briefly, human hemoglobin (60 mg; Sigma Chemical Co., St. Louis, MO) and sodium dithionite (120 mg) were dissolved in distilled water (2 mL) and gently agitated in a flat dish for 15 min. The hemoglobin mixture was then chromatographed on a column of Sephadex G-25 (1.5 x 30 cm). The reduced fraction, with its characteristic bright red color, was isolated and stored frozen (-20 C). The NOS assay mixture consisted of 200 µmol/L CaCl₂, 1 mmol/L MgCl₂, 100 µmol/L L-arginine, 100 µmol/L NADPH, 1.6 µmol/L oxyhemoglobin, 12 µmol/L (6R)-5,6,7,8-tetrahydro-L-biopterin dihydrochloride. The reaction was initiated with 50 µL platelet homogenate, which was mixed with the reaction medium with the aid of a glass plumper. The amount of NO produced was calculated from the decrease in absorbance at 426 nm (with an estimated extinction coefficient of 98,000 L/mol·cm). NOS activity was expressed as picomoles of NO produced per min/mg protein.

Platelet Ca²⁺ concentrations

Ca²⁺ concentrations were measured in intact platelets using the fluorescent probe fura-2/AM, as previously described (19). Determinations were performed in a Perkin Elmer Corp. MPF-66 spectrofluorometer (Branchburg, NJ) at 37 C according to the method of Rao (10). Fluorescence intensity was read at a constant emission wavelength (490 nm), with changes in the excitation wavelength (340 and 380 nm).

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
No significant difference was observed in triglycerides, phospholipid, and cholesterol concentrations and in lipid hydroperoxide levels in LDL obtained from IDDM patients compared with LDL isolated from healthy subjects (Table 2). A significant change in the phospholipid composition was observed in LDL from IDDM patients, which showed an increased content of lysophosphatidylcholine (LPC) compared with that in healthy subjects (Table 3; P < 0.01).

The aggregating response to ADP (2 µM) quantified as maximal aggregation (percentage) was significantly increased in platelets incubated with LDL from diabetic patients compared with that in cells incubated in buffer and with platelets incubated with control LDL (Table 4; P < 0.01).

The results obtained with experiments using different concentrations of LDL are shown in Table 5. The highest concentration of control LDL used (300 µg LDL protein/mL) caused a significant reduction in platelet NOS activity with increased maximal aggregation compared with platelets incubated alone and platelets incubated with the lowest control LDL concentration. Cytoplasmic Ca²⁺ concentrations were also significantly increased compared with those in platelets incubated in buffer. On the contrary, the use of higher concentrations of IDDM LDL did not cause further significant changes in the parameters studied compared with the modifications obtained with IDDM LDL at 100 µg LDL protein/mL. The comparison of the effect of control and IDDM LDL showed that even at the highest concentrations of control LDL, all of the parameters studied were significantly different from the modifications observed after incubation with the lowest concentrations of IDDM LDL, except for cytosolic Ca²⁺.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

The effects exerted by IDDM LDL on platelet suspensions from healthy subjects mimic the alterations observed in platelets from diabetic subjects in basal conditions. In fact, we reported in previous works that compared with normal subjects, insulin-dependent diabetic patients show both reduced platelet Na⁺/K⁺-ATPase and NOS activities and increased platelet cytosolic Ca²⁺ concentrations and plasma membrane Ca²⁺-ATPase activity (9, 19). It might be hypothesized that these alterations are dependent on the constant exposure of diabetic platelets to the action of modified LDL or on the basal presence of the same membrane lipid alterations caused in healthy platelets by the interaction with IDDM LDL. Further experiments on platelets isolated from diabetic patients and incubated alone and with diabetic or nondiabetic LDL might support the hypothesis of a central role of the interaction of lipoprotein with circulating cells.

LDL from IDDM patients significantly reduced NOS activity within platelets. Such a reduction might be dependent on a decreased NOS expression, as previously reported by Chen et al. during the incubation of platelets with ox-LDL (5).

The increased cytosolic Ca²⁺ levels after IDDM LDL incubation are not dependent on the inhibition of Ca²⁺-ATPase activity, as this enzyme was stimulated under the same conditions. Therefore, this phenomenon can be related to an increased ion influx across the membrane or to a higher release from internal stores. A link with the decreased Na⁺/K⁺-ATPase activity might be suggested, as the sodium pump inhibition might secondarily determine a higher Na⁺/Ca²⁺ exchange, with increased intracellular calcium (24).

It must be underlined that both the decreased activity of NOS and the higher cytoplasmic concentrations of Ca²⁺ are able to cause an increased platelet activation, as observed in IDDM patients. In fact, the biochemical alterations found in the present work after the interaction between platelets and IDDM LDL are accompanied by a functional effect, i.e. an increased platelet aggregation in response to ADP.

The actions observed were exerted by LDL, which did not show increased hydroperoxide levels or modifications in triglycerides, cholesterol, and phospholipid content compared with control LDL. The only modification detected in lipoproteins from IDDM patients was an increased content of LPC, which might be ascribed to a mild LDL oxidative process associated with the formation of LPC and the release of the oxidized fatty acid at the 2 position by the action of a specific phospholipase A₂ (25).

It might be hypothesized that after LDL binding to the specific receptor at the membrane surface (26) the microenvironment surrounding the receptor is modified either because of the direct transfer of lipid molecules into the membrane bilayer or because of conformational alterations caused by the binding itself. Both conditions might alter the activity of integral membrane proteins, such as Na⁺/K⁺-ATPase and Ca²⁺-ATPase, and/or the functions of transmembrane channels, such as calcium channels. Modifications in the LDL LPC content occurring in diabetic patients, not grossly detectable by the determination of the main lipid classes, might strongly affect the platelet membrane level during the incubation through these mechanisms of action.

The hypothesis suggesting a central role of LDL LPC content in determining the observed alterations in platelet functions is intriguing because it is consistent with previous works demonstrating that LPC is able to inhibit Na⁺/K⁺-ATPase activity in erythrocytes (27) and to cause intracellular Ca²⁺ accumulation (28), as found in the present work after the incubation of platelets with LDL from IDDM patients. Chen hypothesized a role of LPC in the inhibitory action of ox-LDL on platelet NOS (5). Increased LPC concentrations have been described in the plasma of diabetic patients (27) and in LDL obtained from IDDM patients in the present work and might be at the basis of the action of IDDM LDL on both platelet NOS and membrane enzymatic activities. However, a difference in glycosylation between IDDM and control LDL might also be taken into consideration, as glycated LDL have been reported to promote platelet dysfunction (29). Further works are currently in progress to clarify the role of LDL glycosylation both directly in platelet dysfunction and in the mechanisms regulating phospholipase A2 activity and therefore LPC concentrations.

The data on the concentration dependence of the observed effects indicate that the modifications detected after incubation with IDDM LDL can not be simply obtained using higher concentrations of control LDL. In fact, a different behavior was observed in the parameters studied: Na⁺/K⁺-ATPase and Ca²⁺-ATPase activities were not changed even after incubation with the highest concentrations of LDL from healthy subjects, whereas NOS activity and maximal aggregation were significantly modified, but did not reach the values obtained after incubation with LDL from diabetic patients. These data suggest a qualitative difference in the effects exerted on platelets by LDL from healthy and diabetic subjects.

In conclusion, the present study suggests a new mechanism with a potential role in the early development of atherosclerosis in diabetic patients, i.e. an altered interaction between circulating lipoproteins and platelets.

Received March 2, 1999.

Revised June 17, 1999.

Accepted June 30, 1999.

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