THROMBOSIS RESEARCH 64; 503-508,199l 0049-3848/91 $3.00 + .OO Printed in the USA. Copyright (c) 1991 Pergamon Press pk. All rights reserved.


Per L Katzman’,





Shaun Walker', Yvette Perry', Peter l30+'.

' Departments of Medicine and ' Pharmacology and Therapeutics, University of Manitoba, Winnipeg, Canada.




in revised form 9.9.1991

by Editor N.U. Bang)

ABSTRACT The effect of low density lipoprotein (LDL) on intracellular free calcium ion concentration ([Caz+li), taken as an index of the degree of platelet activation, was investigated in normal volunteers. At 37'C LDL, in a dose of 20 1.19of protein / ml, increased [Ca2+liin all subjects tested (basal 57211 to 113+19 nM). In contrast, when measurements were performed at 20°C, no effect on [Ca2+liwas seen following LDL. Thrombin (0.2 U / ml) increased [Ca2+lito 455f98 nM. When platelets had been exposed to LDL before thrombin stimulation, this increase was less pronounced (to 301+43 nM). Our finding of a temperature dependence of LDL induced increase in platelet [Ca'li supports the concept of a platelet-LDL receptor mediated mechanism. Furthermore, the lower thrombin response following LDL exposure suggests a LDL-thrombin interaction, possibly at the thrombin receptor level and/or calcium recruitment from the same stores.

INTRODUCTION Elevated plasma cholesterol concentration is an important risk factor for cardiovascular disease (l), and LDL concentration relates strongly to coronary heart disease events (2). Accordingly, LDL seems to play a key role in initiating and promoting the atherosclerotic / thrombotic process in the vessel wall (3)* Platelets have to be activated to release vasoactive substances

Key Words: LDL, thrombin, temperature, calcium, human platelets. 503


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such as platelet derived growth factor and thromboxane A2 (4, 5). This activation can be induced by a variety of agonists for which the platelets contain receptors. Although platelets become most importantly involved in advanced stages of atherosclerosis, the question arises whether they could participate in the atherogenic process through an LDL / platelet interaction at an earlier stage . . when the vessel wall is still morphologically intact. The iozsibility that platelets could contribute early to the atherogenesis stems from observations showing that increased plasma concentrations of LDL enhances platelet aggregation (6, 7, 8, 9), that LDL binds with high affinity to a platelet membrane receptor (10, 11, 12), and that this binding is temperature dependent (12). The aims of the present study were to establish whether LDL at physiological concentrations activated platelets (evaluated as [ca’+l i) I and whether this LDL induced activation of intact platelets was temperature dependent, compatible with a receptor mediated mechanism. METHODS Material: Dextrose/citrate VacutainerR tubes were obtained from Becton Dickinson, Missisauga, ON, Canada; Fura- pentaacetoxymethyl ester (AM) from Molecular Probes, Eugene, OR, USA: Sepharose ZB-CL from Pharmacia, Uppsala, Sweden; LDL, bovine thrombin, N-2hydroxyethylpiperazine-NI-2-ethansulfonicacid (HEPES), digitonin, dimethyl sulfoxide (DMSO), and other chemicals used from Sigma Chemical Co, St Louis, MO, USA. Healthy subjects: A total of 8 healthy volunteers, age 25-58 years, participated in the studies, which were approved by the faculty committee on the use of human subjects in research. Informed consent was obtained from all subjects and none had used any medication ~14 days prior to the studies. Platelet isolation: After overnight fasting, venous blood was drawn in dextrose/citrate VacutainerR tubes and immediately centrifuged for 20 min, 20°C, at 120 x g. Platelet-rich plasma (PRP) was aspirated with plastic pipettes and incubated with fura-2/AM, added for as a 300 PM stock solution in DMSO, at a concentration of 4 I.LM 30 min at 37OC. PRP was then filtrated on Sepharose 2B-CL equilibrated with elution buffer containing 145 mM NaCl, 5 mM KCl, 1 mM MgSO,, 0.5 mM NaH,PO,, 6 mM glucose and 10 mM HEPES, pH 7.4. After gel filtration CaCl was added (final concentration 1 mM) and the samples kept at 4%. Before measurements platelet count (Coulter CounterR) was adjusted to 3-5 x 10' / ml with elution buffer. [Ca2+li measurements: Measurements were performed in a Jasco CA-100 Ca2+ analyzer (Jasco Inc, Easton, MD, USA) equipped with thermostatically controlled cuvette (0,5 ml) holder and magnetic stirrer. Dual excitation wavelengths (340 and 380 nm, 40 Hz) were used and emission ratio (R) was recorded at 500 nm . [Ca2+liwas calculated using the equation (13): [Ca2+li= I$ x ( k - R,,,i,m/ Q, - R ) x B, where % is the dissociation constant of the fura-2/Ca2+ complex the ratio for calcium-free (224 nM at 37'C; (13)), qi igiSt onin 160 PM) fura- and B (EGTA 20 mM) and calcium-saturated the ratio of fluorescence between calcium-free and calcium-saturated fura- at 380 nm. R,,,iand &, were in each experiment determined without prior addition of LDL and/or thrombin.

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The amount of extraneous fura-2, estimated using a method previously described in detail by Pollock and Rink (14) and Oshima et al (15), contributed to less than 3 %, and autofluorescence from unloaded platelets and test agents at concentrations used was less than 5 % of the total fluorescence signal throughout the studies. Study protocol: Fluorescence was continuously recorded. Basal [Ca2+liwas registered after 5 min prewarming of the samples. Based on a preliminary dose finding study, where LDL at a dose of 20 1.19 of protein / ml consistently increased [Ca2*li(37OC),this LDL-dose was used in the present study, where measurements were performed at 20' and 37OC. The platelets were stimulated with thrombin (final concentration 0.2 U / ml) with and without prior stimulation (1 min) with LDL (figure 1). Experiments were at each temperature made in duplicate, and mean values were used in the further evaluation. At 37OC the intra individual coefficient of variance for basal measurement of [Ca2+liwas 11 %. Statistical evaluation: The level of statistical difference was accessed using the Wilcoxon's matched-pairs signed-ranks test, and a P-value less than 0.05 was considered statistically significant. All values are given as mean + SEM. RESULTS As shown in figure 1, measurements performed at 20°C showed virtually no change in fluorescence ratio following LDL (20 pg of protein / ml, final concentration), whereas at 37Oc, this LDL concentration increased the fluorescence ratio in all subjects, corresponding to an mean increase in [Ca2+liof 57+12 nM.

37 O c







1 min

Figure 1. Typical tracings of 340/380 nm fluorescence ratios recorded at 37OC (upper) and 2o"c (lower) following LDL (L; 20 pg of protein / ml) and thrombin (T; 0.2 U / ml) added as indicated by arrows.



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Basal [Ca*+].. was57?rll nM. Exposure of platelets to thrombin (0.2 increased [Ca*+]. by 396+100 nM. This increase was U / ml) significantly smaller (by ;44+40 nM; P c 0.05) when the same dose of thrombin was added following exposure of platelets to LDL (figure 2). a,b nM







Figure 2. Intracellular free calcium ion concentration (nM). Bas = basal, LDL = after LDL (20 c(gof protein / ml), LDL + Thr = after LDL (20 1.19of protein / ml) and 60 seconds later followed by thrombin (0.2 U / ml), Thr = after thrombin alone (0.2 U / ml)b Mean f SEM. a denotes P c 0.01 as compared to basal values, denotes P < 0.02 as compared to LDL + Thr.

DISCUSSION A methodological limitation measuring [Ca2+liin vitro using furais, that platelets are exposed to LDL in an environment without plasma proteins. Therefore findings cannot be fully extrapolated to the clinical setting. However, in the present study a low (considered physiological) concentration of LDL of 20 of protein / ml induced an increase in platelet [Ca*'].in all subjects studied when measurements were performed at 37*C. This finding is in agreement with those of Knorr et al (16), who reported increased [Ca2+liin human platelets exposed to LDL using another fluorescent dye (Quin-2). In this study a slower but more pronounced response to LDL as well as thrombin was reported. This could possibly be explained by their method including an incubation with calcium at 37'C for 1 hour prior to measurements, by a possible leak of Quin2, a higher basal [Ca2+liand/or altered platelet viability all of which may have affected estimations of calcium responses (16). In our study the amount of extraneous fura- following gel filtration was < 3 %, and no further fura- leak could be registered within the time-frame of the experiments performed. At these low of protein / ml of LDL concentrations of fura-2, addition of 20 1.19

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will contribute to a negligible fluorescence signal (data not shown). Thus, in the present study the increase in fluorescence ratio following LDL presumably solely reflects an increase in platelet [Ca2+li. When measurements were performed at 20°C, no response in [Ca2+lito LDL could be registered. The marked temperature dependence of LDLplatelet interaction most likely reflects a receptor mediated increase in [Ca*'].. Thus, our observation demonstrates the functional implicat:on of the finding of Curtiss and Plow (12) of a strictly temperature dependent binding of LDL to its platelet receptor. In their experiment LDL binding did not occur at 4' and 22O, but high affinity binding was present at 37'C. However, the LDL preparation used might be considered partly auto-oxidised (as determined by a slightly increased mobility on lipoproteinelectrophoresis and a malondialdehyde content of 0.6 nmoles / mg LDL protein), and our findings do not discriminate between a possible LDL binding to the classical or the scavenger type I/II LDL receptors (17,18). The thrombin induced increase in [Ca*'].was significantly less pronounced following prior exposure of platelets to LDL; this was observed at 20' as well as 37'C. The reason for this is not quite obvious. It could involve recruitment of calcium from the same stores, but since this was observed also at 20°C, this possibility seems less likely. Alternatively, our finding could be explained by some LDL-thrombin interaction, perhaps at the thrombin receptor level. In conclusion, our finding of a temperature dependence of LDL induced increase in platelet [Ca2*lisupports the concept of a platelet-LDL receptor mediated mechanism. The lower thrombin response in [Ca*+].following prior LDL exposure suggests a LDLthrombin interaction, possibly at the thrombin receptor level and/or calcium recruitment from the same stores. ACKNOWLEDGEMENTS This work was supported by grants from the Swedish Medical Research Council, the Swedish Society of Medicine, Hassle AB (Per L Katzman) and from the P Thorlakson Foundation, Manitoba Health Research Council, and the Kidney Foundation of Canada. REFERENCES 1. KANNEL, W.B., CASTELLI, W.P., GORDON, T., MCNAMARA, P.M. Serum cholesterol, lipoproteins, and the risk of coronary heart disease. Ann Int Med 74, 1-12, 1971. 2. BILHEIMER, D.W., HO, Y.K., BROWN, M.S., ANDERSON, R.G.W., GOLDSTEIN, J.L. Genetics of the low density lipoprotein receptor. J Clin Invest, 61, 678-696, 1978. 3. ROSS, R. The pathogenesis of atherosclerosis - an update. N Engl J Med, 314, 488-500, 1986. 4. ROSS, R., VOGEL, A. The platelet-derived growth factor. Cell, 14, 203-210, 1978 5. AVIRAM, M., SIRTORI, C.R., COLLI, S., MADERNA, P., MORAZZONI,



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TREMOLI, E. Plasma lipoproteins affect platelet malondialdehyde G and thromboxane B, production. Biochem Med, 34, 29-36, 1985. 6. CARVALHO, A.C. COLMAN, R.W., LEES, R.S. Platelet function in hyperlipoproteinemia. N Engl J Med, 290, 434-438, 1974. 7. JOIST, J.H., BAKER, R.K., SCHONFELD, G. Increased in vivo and in function in vitro platelet type IIand type IVhyperlipoproteinemia. Thromb Res, 15, 95-108, 1979. 8. BRADLOW, B.A., CHETTY, N., BIRNBAUM, M., BAKER, S.G., SEFTEL, H.C. Platelet function in familial hypercholesterolaemia in South Africa and the effects of probucol. Thromb Res, 26, 91-99, 1982. 9. AVIRAM, M., BROOK, G.J. The effect of human plasma on platelet function in familial hypercholesterolemia. Thromb Res, 26, 101-109, 1982. 10. AVIRAM, M., BROOK, J.G. Platelet interaction with high and low density lipoproteins. Atherosclerosis, 46, 259-268, 1983. 11. KOLLER, E., KOLLER, F., DOLESCHEL, W. Specific binding sites on human blood platelets for plasma lipoproteins. Hoppe-Seyler 2 Physiol Chem, 363, 395-405, 1982. 12. CURTISS, L.K., PLOW, E.F,. Interaction of plasma lipoproteins with human platelets. Blood, 64, 365-374, 1984. 13. GRYNKIEWICZ, G., POENIE, M., TSIEN, R.Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem, 260, 3440-3450, 1985. 14. POLLOCK, W.K., RINK, T.J. Thrombin and ionomycin can rise platelet cytosolic Ca2+ to micromolar levels by discharge of internal Ca2+ stores: studies using Fura-2. Biochem Biophys Res Comm, 139, 308-314, 1986. 15. OSHIMA, T., YOUNG, E.W., BUKOSI, R.D., MCCARRON, D.A. Abnormal Calcium handling by platelets of spontaneously hypertensive rats. Hypertension, 5, 606-611, 1990. 16. KNORR, M., LOCHER, R., VOGT, E., VETTER, W., BLOCK, L.H., FERRACIN, F., LEFKOVITS, H., PLETSCHER, A. Rapid activation of human platelets by low concentrations of low-density lipoprotein via phosphatidylinositol cycle. Eur J Biochem 172, 753-759, 1988. 17. KODAMA, T., FREEMAN, M., ROHRER, L., ZABRECKY, J., MATSUDAIRA, P KRIEGER, M. Type I'macrophage scavenger receptor cantains (Yhelical and collagen-like coiled coils. Nature 343, 531-535, 1990. 18. ROHRER, L., FREEMAN, M., KODAMA, T., PENMAN, M., KRIEGER, M. Coiled-coil fribrous domains mediate ligand binding by macrophage scavenger receptor type II. Nature 343, 570-572, 1990. Correspondence: Peter Bolli, Health Science Centre, Department of Medicine, Section of Nephrology, Room GE 421, 820 Sherbrook Street, Winnipeg, Manitoba, R3A lR9 Canada.

Temperature-dependence of LDL binding and activation of human platelets.

The effect of low density lipoprotein (LDL) on intracellular free calcium ion concentration ([Ca2+]i), taken as an index of the degree of platelet act...
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