Increase in the cytosolic concentration of calcium in platelets of diabetics type II

THROMBOSIS RESEARCH 62; 421-428,199l 0049-3848/91 $3.00 + .OO Printed in the USA. Copyright (c) 1991 Pergamon Press pk. All rights reserved.

INCREASE IN THE CYTOSOLIC CONCENTRATION OF CALCIUM OF DIABETICS TYPE II.

IN PLATELETS

D.Tschope, P.Rosen*, F.A.Gries Diabetes Research Institute, Auf' m Hennekamp D-4000 Dusseldorf, FRG

(Received

27.12.1990;

accepted

in revised form 15.2.1991

65,

by Editor H. Graeff)

ABSTRACT The intracellular concentration of calcium (Ca.) was control subjec 'ts and measured in platelets of healthy in platelets of diabetics type II. Cai was elevated diabetics type II under basal conditions, after stimulation by collagen, in the presence and absence of calcium in Cai after in the incubation medium. The increase stimulation by collagen was inhibited by nitrendipine in both kinds of platelets, however Cai remained elevated in platelets of diabetics. Our data and findings of others may be understood as indications of disturbances in the handling of Cai in diabetes.

INTRODUCTION The vascular risk of diabetic patients is largely increased as indicated by the augmented mortality and morbidity of diabetics from cardiovascular complications (1). This high vascular risk is partly due to the accelerated development of atherosclerotic lesions of coronary and peripheral vessels in diabetes (1). Additionally, the increased incidence of thromboembolic complications in diabetes might be due to hematological abnormalities which can aggravate the vascular risk of diabetics furtheron (2-4). Inplatelet diabetes Keywords: aggregation, galcium antagonists to whom correspondence should be addressed

421

mellitus,

calcium,

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creased activity of platelets in diabetic patients has been described by enhanced spontaneous aggregation, the increased sensitivity of platelets to stimuli such as ADP, collagen and adrenaline, and the accelerated release of thromboxane A2, PF4 and B-thromboglobuline (for review 2-5). The underlying mechanisms are only partly understood. Whereas there is only minor evidence for alterations in the terminal effector systems of platelets (proteinkinase C, myosin light chain kinase, phospholipase A2, phospholipase C), recent studies indicate subtle changes in the number of receptors and glycoproteins on the surface of platelets (5-8). Since the regulation of intracellular calcium (Cai) is a critical step in platelet activation (9-11) and seems to be disturbed in a variety of tissues in diabetes (12), it was intriguing to prove whether the handling of calcium by platelets is affected in diabetes. Bergh et al. (13) provided very recently evidence that the exchange of calcium across the plasma membrane is increased in diabetics type I. Furthermore elevated basal concentrations of intracellular cytosolic calcium have been described in platelets of hypertensive A direct link between thrombosis and calcium patients (14). handling by platelets has been demonstrated by Shanbaky et al. (15). Therefore we proved in this study whether the handling of calcium is altered in platelets of type II diabetics. METHODS In healthy and diabetic subjects anticoagulated (9:l with 3.13% sodium citrat) whole blood was taken from an undwelled peripheral vein. Platelet rich plasma (PRP) was obtained by centrifugation and platelet poor plasma (PPP) as described previously (8,9). Calcium was measured in the basal state and after stimulation of platelets by collagen. For measurement of intracellular calcium (Cai) platelet counting was adjusted to l-l.5 x lOa platelets/ml. Cai was determined by the fluorescence dye indo 1 (17,18) using an Perkin-Elmer fluorimeter (650-40, Perkin-Elmer, tiberlingen, FRG) at an excitation wavelength of 331 nm and emission wavelength of 401 nm. PRP was incubated with indo 1 for 30 min at 37OC in a final concentration of 5 /.4moles/land 5 x lo8 platelets/ ml. Thereafter, platelets were washed by gel filtration on Sepharose 2B to separate dye loaded platelets from dye not taken up by platelets (19-21). Platelets were eluted from the column with a buffer containing 145 mM NaCl, 5 mM KCl, 1 mM MgS04, 10 mM Hepes and 10 mM glucose, and a pH of 7.4. Basal and stimulated fluorescence after addition of collagen were recorded under continuous stirring of the platelet suspension in the presence and absence of extracellular calcium (Fca). TO determine the maximal fluorescence (Fmax) platelets were permeabilized by 60 /.JMdigitonin and if necessary saturated with 1 mM Minimal fluorescence (Fmin was measured after quenching CaCl the %'luorescence by 0.1 M MnC12. s he free calicum concentration was calculated according to the following equation:

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Ca2+=Kd

X

(Fca-Fmin)/(Fmax-Fca)

with Kd= 250 nM (22). Indo l/AM was obtained from Calbiochem (Frankfurt, FRG), Sepharose 2B from Pharmacia (Upsalla, Sweden), collagen from Hormon Chemie (Munich, FRG), nitrendipine from Bayer (Leverkusen, FRG). All other reagents were of p.a. grade and purchased from Merck, Darmstadt, FRG. All data are given as mean +SEM of (n) patients. Comparison between groups was performed using Student's t-test after log transformation of data when appropriate. Significance threshold was set at p q0.05 STUDIED SUBJECTS: Patients of the clinical department of the Diabetes Research Institute with manifest type II diabetes (NIDDM) were included in this study. These patients were under continuous control of their metabolic state receiving insulin and partially additionally sulphonylureas as oral antidiabetics. Subjects exposed to medication affecting platelets 10 days prior to blood sampling were excluded. All patients suffered from one diabetic late complication such as retinopathy, peripheral vascular diseases, and neuropathy as followed from clinical inspection of the patients. RESULTS Basic clinical data are volunteers were selected.

given

in

Tab.1.

As

Clinical data of diabetic patients Healthy subjects Sex

Age (years) Duration of diabetes (years) Blood glucose (mg/dl) Triglycerides (mg/dl) Cholesterol (mg/dl) HbAl (%) Hematokrit (%)

4f, 5m 37 + 6 (19-81)

reference

normal

(type II) Patients

2f, 6m 58.5 + (47-72) ' 17.8 + 1.7 143 + 15

1 1

90.7 + 9.1

149

+ 22

1

171.5 + 5.7

258

+ 19

1

Cl10

<5% 43.7 + 1.6

10.6 + 0.6 41.9 + 1.0

1

Tab.1: Clinical data of control subjects and diabetic patients. Means + SEM of (n) subjects are given. p < 0.05 diabetics vs controls

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z

700 +

*

-

1 cg

collagen *

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2 )co

-5 E 600 5

500

?J 400 i

300

i

200

8 5

160 0 control club]ecte : - calcium ISLY + calcium m dlabetlc patlenb: - calcium m + calcium [x)

Fig-l: Intracellular calcium in platelets of control subjects and diabetics type II. Plateletes were isolated and loaded with Indo-l as described in METHODS. Intracellular calcium was determined fluorimetrically after stimulation of platelets with 1 and 2 mg collagen in the presence and absence of 1 mmol/ calcium in the incubation medium. Intracellular calcium is significantly increased (p -C 0.05) in the presence of extracellular calcium as compared to controls without calcium: column 1 vs 2, 3 vs 4, 5 vs 6, 7 vs a. The difference between diabetic and control samples is significant: column 2 vs 4, 5 vs 7, 6 vs 8.

INFLUENCEOF NlTftENDlPlNEON COLLAGENINDUCED CHANGESIN INTRACELLUURCALCIUM

5

700

E e 5

600

3

400

$

300

j

200

1 =

100

*

Control

*

500

0 nitrendlplns (pmol/l):

0 LS9, 5 m,

10 BBI,

20 IZ

basal, wlthout collagen 0

Fig.2: Inhibition of the collagen induced increase in intracellulular calcium by nitrendipine. Platelets were stimulated by 2 pg collagen in the presence of extracellular calcium, and the intracellular calcium concentration determined as described in METHODS.

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in the expected range (61 + 2 nM) In control platelets Ca. was (Tab.2). Incubation wi&I 1 mM calcium in the incubation medium led to an increase of Ca. to about 84 nM (+ 37%) in controls. A close inspection of the tndividual data showed no dependency of the intracellular calcium concentrations on age of the control subjects or the diabetic patients so that the data of the groups can be compared directly. In platelets of diabetics type II the basal calcium accounted to 88 + 3 in the absence of extracellular calcium and reached values of 127 nM with 1mM calcium in the medium (+44%, Tab.2). Thus, under both conditions the basal cytosolic calcium is elevated in platelets of diabetics as compared to platelets of healthy subjects. Concentration of cytosolic and diabetic subjects Conditions - calcium + calcium

calcium

(nM) in platelets

Healthy subjects

(1 mM)

Patients 88+3 ' 127+7 'I2

61+2 84+4 '

of healthy

Change (% of control) 144 151

Tab.2: The intracellular concentration in platelets of control and diabetic subjects was measured by fluorimetry using indo-l as intracellular indicator. I P < 0.05 control vs. diabetes, 2 p < 0.05 platelets incubated without vs with calcium (1 mM) in the incubation medium. Stimulation of platelets by collagen caused an elevation of Ca. in both kinds of platelets dependent on the dose of collagen use a and on the concentration of calcium in the incubation medium. In platelets of diabetics the absolute values of Ca. were, however, generally higher than in control platelets. ti urthermore, the percentual increase in Ca. after stimulation of platelets by collagen was larger in plitelets of diabetics which was most obvious if platelets were activated by 2 I.rgcollagen (Fig.1). Nitrendipine effectively inhibited the increase in Cai in platelets of controls and diabetics after stimulation by collagen at concentrations of nitrendipine higher than 5 PM. However, the absolute concentrations of Ca. in platelets of diabetics after stimulation by collagen as we 11 as the percentual increase of Cai over the basal state remained higher in platelets of diabetics than in platelets of healthy subjects (Fig.2). DISCUSSION The intracellular calcium in gel filtered platelets of control subjects was in the range as determined by others, and exposure of unstimulated platelets to calcium in the incubation medium led

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to an enlarged increase of Cai as described previously (23-25). As compared to controls intracellular concentrations of calcium in platelets of diabetics type II were increased under basal conditions and after stimulation by collagen. This difference was pronounced in presence of extracellular calcium. Thus, our data suggest that the regulation of the cytosolic concentration of calcium is altered in platelets of diabetics type II. The observed alterations in the cytosolic calcium of platelets of diabetics type II might reflect either changes in the calcium uptake across the plasma membrane or in the discharge of calcium from intracellular stores such as the dense tubular system (DTS). Such alterations have not yet been described for type II diabetics, however, there is some evidence for an impaired exchange of calcium across the plasma membrane in type I diabetes. Bergh et al. (13) reported an increased influx rate and a diminished efflux rate. Additionally, it has been concluded from studies using chlortetracycline that the ratio between the fast and the slow phases of CTC fluorescence is strongly increased in platelets of diabetics type I indicating indirectly an elevation of free cytoplasmic calcium (26). These and our data suggest therefore that in both types of diabetes the exchange of calcium across the plasma membrane is altered resulting in an elevation of the free calcium concentration. This conclusion is further supported by the inhibition of the collagen induced increase in intracellular calcium by nitrendipine (Fig.2) and the diminuition of the sensititivity of platelets for activation by various stimuli by calcium antagonists (16,27). The biochemical defects causing these changes in calcium fluxes across the plasma membrane in platelets of diabetics are not known. It has been shown previously that the glycoproteins IIb/IIIa are involved or associated with the transport of calcium across the plasma membrane (6, 9) and the number of these glycoproteins is elevated in platelets of diabetics type II (8). Thus it is tempting to speculate that both alterations in platelets of diabetics type II are linked and that the increased concentration of cytosolic calcium in platelets reflects an enlarged exchange of calcium across the plasma membrane facilitated by increase in the number of glycoprotein IIb/IIIa. Interestingly, similar changes in free cytosolic calcium as discussed here have also been described in other disorders with enhanced thromboembolic risk such as hypertension, hypercholesterolemia, vasculitis (14,26). Since changes in the regulation of cytosolic calcium might be involved in the activation of platelets in diabetes, it is intriguing to suggest that reduction of the uptake and the mobilization of intracellular calcium by calcium antagonists might be helpful to diminish the thromboembolit and vascular risk in diabetes. Acknowledgements: This work was partly supported by the Bundesministerium fur Jugend, Familie, Frauen und Gesundheit and the Forschungsministerium of NRW.

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