Why and how to eliminate spontaneous platelet aggregation in blood measured by multiple electrode aggregometry

Abstract

Numerous studies show that spontaneous platelet aggregation (SPA) measured in stirred platelet‐rich plasma (PRP) and blood is enhanced in patients with various disorders, including diabetes, acute coronary syndrome and myocardial infarction [1Suehiro A. Uedaa M. Suehiroh M. Ohe Y. Kakishitaa E. Evaluation of platelet hyperfunction in aged subjects using spontaneous platelet aggregation in whole blood.Arch Gerontol Geriatr. 1995; 21: 277-83Crossref PubMed Scopus (11) Google Scholar, 2Gray R.P. Hendra T.J. Patterson D.L. Yudkin J.S. ‘Spontaneous’ platelet aggregation in whole blood in diabetic and non diabetic survivors of acute myocardial infarction.Thromb Haemost. 1993; 70: 932-6Crossref PubMed Scopus (16) Google Scholar, 3Krause S. May J. Koslowski H. Heptinstall S. Losche W. Enhanced spontaneous platelet aggregation and red blood cell fragility in whole blood obtained from patients with diabetes.Platelets. 1991; 2: 203-6Crossref PubMed Scopus (4) Google Scholar, 4Casonato A. 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Further characterisation of the platelet defect.Thromb Haemost. 1979; 41: 512-22Crossref PubMed Scopus (50) Google Scholar, 8Komarnicki M. Kazmierczak M. Haertle D. Standard heparin and low molecular weight heparin effect on spontaneous platelet aggregation measured in whole blood and in platelet‐rich plasma.Acta Haematol Pol. 1993; 24: 263-5PubMed Google Scholar, 9Gabbianelli R. Falcioni G. Dow C.S. Vince F.P. Swoboda B. A new method to evaluate spontaneous platelet aggregation in type 2 diabetes by Cellfacts.Clin Chim Acta. 2003; 329: 95-102Crossref PubMed Scopus (11) Google Scholar, 10Lecrubier C. Conard J. Horellou M.H. Kher A. Samama M. Spontaneous platelet aggregation in heparin‐treated patients.Acta Haematol. 1984; 71: 63-5Crossref PubMed Scopus (18) Google Scholar, 11Fox S.C. Kilby M.D. Sanderson H.M. Heptinstall S. Investigations on spontaneous aggregation and platelet responses to streptokinase and to adrenaline during pregnancy.Platelets. 1994; 5: 139-44Crossref PubMed Scopus (6) Google Scholar, 12Paulsen E.P. McClung N.M. Sabio H. Some characteristics of spontaneous platelet aggregation in young insulin‐dependent diabetic subjects.Horm Metab Res Suppl. 1981; 11: 15-21PubMed Google Scholar, 13Iwase E. Tawata M. Aida K. Ozaki Y. Kume S. Satoh K. Qi R. Onaya T. A cross‐sectional evaluation of spontaneous platelet aggregation in relation to complications in patients with type II diabetes mellitus.Metabolism. 1998; 47: 699-705Abstract Full Text PDF PubMed Scopus (60) Google Scholar]. In two studies, SPA was found to be a predictive risk marker of arterial occlusions [14Trip M.D. Cats V.M. van Capelle F.J. Vreeken J. Platelet hyperreactivity and prognosis in survivors of myocardial infarction.N Engl J Med. 1990; 322: 1549-54Crossref PubMed Scopus (546) Google Scholar, 15Breddin H.K. Lippold R. Bittner M. Kirchmaier C.M. Krzywanek H.J. Michaelis J. Spontaneous platelet aggregation as a predictive risk factor for vascular occlusions in healthy volunteers? Results of the HAPARG Study. Haemostatic parameters as risk factors in healthy volunteers.Atherosclerosis. 1999; 144: 211-9Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar]. Thus, SPA may be a clinically relevant index of systemic prothrombotic activity. Studies of SPA in blood instead of PRP may be more relevant, because (i) in contrast to PRP, blood contains the whole platelet population, including the younger, larger, more reactive platelets; (ii) centrifugation of blood required for the preparation of PRP may alter platelet responsiveness; and (iii) red and white blood cells might modulate platelet reactivity. SPA observed in stirred or roller‐mixed blood has been shown to largely depend on ADP released from erythrocytes [16Gaarder A. Jonsen J. Laland S. Hellem A. Owren P.A. Adenosine diphosphate in red cells as a factor in the adhesiveness of human blood platelets.Nature. 1961; 192: 531-2Crossref PubMed Scopus (408) Google Scholar, 17Aursnes I. Gjesdal K. Abildgaard U. Platelet aggregation induced by ADP from unsheared erythrocytes at physiological Ca++‐concentration.Br J Haematol. 1981; 47: 149-52Crossref PubMed Scopus (30) Google Scholar, 18Saniabadi A.R. Lowe G.D. Barbenel J.C. Forbes C.D. A comparison of spontaneous platelet aggregation in whole blood with platelet rich plasma: additional evidence for the role of ADP.Thromb Haemost. 1984; 51: 115-8Crossref PubMed Scopus (74) Google Scholar, 19Saniabadi A.R. Lowe G.D. Barbenel J.C. Forbes C.D. Further studies on the role of red blood cells in spontaneous platelet aggregation.Thromb Res. 1985; 38: 225-32Abstract Full Text PDF PubMed Scopus (39) Google Scholar, 20Brown P. Harrison M.J. The role of red blood cells in platelet aggregation in whole blood.Atherosclerosis. 1988; 71: 261-2Abstract Full Text PDF PubMed Scopus (7) Google Scholar]. Erythrocyte fragility and deformability, which are increased in diabetic patients, are determinants of SPA [3Krause S. May J. Koslowski H. Heptinstall S. Losche W. Enhanced spontaneous platelet aggregation and red blood cell fragility in whole blood obtained from patients with diabetes.Platelets. 1991; 2: 203-6Crossref PubMed Scopus (4) Google Scholar, 21May J. Loesche W. Heptinstall S. Glucose increases spontaneous platelet aggregation in whole blood.Thromb Res. 1990; 59: 489-95Abstract Full Text PDF PubMed Scopus (21) Google Scholar]. In vivo the interaction of platelets with erythrocytes could be particularly relevant at sites of atherosclerotic stenotic arteries, where abnormal conditions of flow favour reversible deformation of red cells and ADP leakage [22Born G.V. Bergquist D. Arfors K.E. Evidence for inhibition of platelet activation in blood by a drug effect on erythrocytes.Nature. 1976; 259: 233-5Crossref PubMed Scopus (76) Google Scholar]. Of note, the clinical benefit of the antithrombotic drug dipyridamole might in part be attributable to its action on erythrocytes and subsequent inhibition of SPA [23Diener H.C. Cunha L. Forbes C. Sivenius J. Smets P. Lowenthal A. European Stroke Prevention Study. 2. Dipyridamole and acetylsalicylic acid in the secondary prevention of stroke.J Neurol Sci. 1996; 143: 1-13Abstract Full Text Full Text PDF PubMed Scopus (1747) Google Scholar, 24Halkes P.H. van Gijn J. Kappelle L.J. Koudstaal P.J. Algra A. Aspirin plus dipyridamole versus aspirin alone after cerebral ischaemia of arterial origin (ESPRIT): randomised controlled trial.Lancet. 2006; 367: 1665-73Abstract Full Text Full Text PDF PubMed Scopus (921) Google Scholar, 25Gresele P. Arnout J. Deckmyn H. Vermylen J. Mechanism of the antiplatelet action of dipyridamole in whole blood: modulation of adenosine concentration and activity.Thromb Haemost. 1986; 55: 12-8Crossref PubMed Scopus (108) Google Scholar, 26Saniabadi A.R. Lowe G.D. Barbenel J.C. Forbes C.D. Effect of dipyridamole on spontaneous platelet aggregation in whole blood decreases with the time after venepuncture: evidence for the role of ADP.Thromb Haemost. 1987; 58: 744-8Crossref PubMed Scopus (18) Google Scholar, 27Harrison M.J. Pollock S.S. Steiner M. Weisblatt E. Inhibitors of ‘spontaneous’ platelet aggregation in whole blood.Atherosclerosis. 1985; 58: 199-203Abstract Full Text PDF PubMed Scopus (16) Google Scholar]. Thus, a simple and reliable method to measure SPA, representing a functional parameter of the interaction of red blood cells with platelets in blood, could be clinically useful. We therefore carried out a methodological study of SPA determination in blood using multiple electrode aggregometry (MEA) [28Toth O. Calatzis A. Penz S. Losonczy H. Siess W. Multiple electrode aggregometry: a new device to measure platelet aggregation in whole blood.Thromb Haemost. 2006; 96: 781-8Crossref PubMed Scopus (481) Google Scholar], a method that is now widely used to determine stimulated platelet aggregation in blood in patients with various diseases [29Elalamy I. Galea V. Hatmi M. Gerotziafas G.T. Heparin‐induced multiple electrode aggregometry: a potential tool for improvement of heparin‐induced thrombocytopenia diagnosis.J Thromb Haemost. 2009; 7: 1932-4Crossref PubMed Scopus (29) Google Scholar, 30Siller‐Matula J.M. Christ G. Lang I.M. Delle‐Karth G. Huber K. Jilma B. Multiple electrode aggregometry predicts stent thrombosis better than the vasodilator‐stimulated phosphoprotein phosphorylation assay.J Thromb Haemost. 2010; 8: 351-9Crossref PubMed Scopus (122) Google Scholar]. MEA has been used up to now in over 200 scientific and clinical studies [31Multiplate in the literature. Version 21.0. http://www.multiplate.net/; 25 August 2011.Google Scholar]. For MEA, aliquots (0.6 mL) of whole blood diluted 1:1 with physiological saline are stirred with a Teflon‐coated magnetic bar rotating at 900 rpm in a test cuvette, and the increase of electrical impedance due to adhesion and aggregation of platelets on silver‐coated electrodes is measured (Multiplate®, Dynabyte GmbH, Munich, Germany). Platelet adhesion/aggregation is expressed as the mean of the areas under the impedance curves, determined over time (AU*min) from two electrode pairs in each cuvette [28Toth O. Calatzis A. Penz S. Losonczy H. Siess W. Multiple electrode aggregometry: a new device to measure platelet aggregation in whole blood.Thromb Haemost. 2006; 96: 781-8Crossref PubMed Scopus (481) Google Scholar]. SPA was studied in hirudin‐anticoagulated blood of a large cohort of healthy volunteers during a period of 5 years. SPA ranged from 0 to 330 AU*min (n= 81 donors), with a mean (± SD) value of 63 (±61) AU*min (Fig. 1A). SPA measured within the same experiment several times (2–19‐fold) showed a large variability: the mean coefficient of variation (CV) in 140 experiments was 66% (SD:44%) (see also Fig. 1A). Platelet concentration and the time of blood testing after venipuncture (10–150 min) did not influence SPA (data not shown). The intra‐individual %CV of donors who were tested repeatedly (n= 2–9 times) over a 6–60‐month period was 55 ± 35% (mean ± SD; n= 35 donors); thus the biological variability was similar to the analytical variability. We observed in some experiments that SPA values were not only higher than the stimulated platelet aggregation values (> 100 AU*min), but also widely scattered. Two examples to illustrate this point are SPA values (in AU*min; range, mean ± SD, n determinations in parenthesis) of experiment A (44–273, 135 ± 83 (19)) and of experiment B (11–488, 294 ± 169 (16)). No explanations (such as type of Multiplate® model, batch of MEA cuvettes, conditions of venipuncture or source of hirudin) could be found for this variability. By addressing the mechanism of SPA, we found that acetylsalicylic acid was without effect on SPA (data not shown). However, addition of apyrase (10U mL−1) to blood before start of stirring almost completely eliminated SPA (see insert in Fig. 1A). Apyrase degrades extracellular ATP (activating platelets through binding to P2X1 receptors) as well as ADP (activating platelet P2Y1 and P2Y12 receptors), leading to an increase of adenosine, a platelet inhibitor [32Hu H. Hoylaerts M.F. The P2X1 ion channel in platelet function.Platelets. 2010; 21: 153-66Crossref PubMed Scopus (16) Google Scholar, 33Gachet C. Regulation of platelet functions by P2 receptors.Annu Rev Pharmacol Toxicol. 2006; 46: 277-300Crossref PubMed Scopus (239) Google Scholar]. In order to distinguish between these possibilities of SPA inhibition by apyrase, we studied the effect of specific ADP‐receptor antagonists, and found that the combined blood incubation with the P2Y1 receptor antagonist MRS2179 (100 μm) and the P2Y12 receptor antagonist AR‐C69931MX (1 μm) reduced aggregation from 40 ± 33 AU*min to 5 ± 8 AU*min (mean ± SD; n= 6). The results indicate that SPA is mainly mediated by extracellular ADP present in stirred blood. To address this point further, we determined the concentration and kinetic of ATP in stirred blood, which is released (probably along with ADP) from erythrocytes [34Sprague R.S. Ellsworth M.L. Stephenson A.H. Lonigro A.J. ATP: the red blood cell link to NO and local control of the pulmonary circulation.Am J Physiol. 1996; 271: H2717-22Crossref PubMed Google Scholar, 35Rozier M.D. Zata V.J. Ellsworth M.L. Lactate interferes with ATP release from red blood cells.Am J Physiol Heart Circ Physiol. 2007; 292: H3038-42Crossref PubMed Scopus (27) Google Scholar]. These cells contain about 10 × more intracellular ATP (approximately 1–2 mm) than ADP [34Sprague R.S. Ellsworth M.L. Stephenson A.H. Lonigro A.J. ATP: the red blood cell link to NO and local control of the pulmonary circulation.Am J Physiol. 1996; 271: H2717-22Crossref PubMed Google Scholar, 36Dean B.M. Perrett D. Studies on adenine and adenosine metabolism by intact human erythrocytes using high performance liquid chromatography.Biochim Biophys Acta. 1976; 437: 1-5Crossref PubMed Scopus (46) Google Scholar]. ATP was measured after addition of luciferase‐luciferin reagent in the Lumi‐aggregometer® (Chrono‐log Corp., Havertown, PA, USA) [37Dwivedi S. Pandey D. Khandoga A.L. Brandl R. Siess W. Rac1‐mediated signaling plays a central role in secretion‐dependent platelet aggregation in human blood stimulated by atherosclerotic plaque.J Transl Med. 2010; 8: 128Crossref PubMed Scopus (33) Google Scholar]. We found that stirring of blood diluted 1:1 with saline (i.e. under the conditions of MEA) induces an ATP increase, which was dependent on the speed of stirring (Fig. 1B). To confirm that the stirring‐induced increase of ATP originates from red cells, and not from platelets, blood with normal platelet count and reduced platelet count was prepared by reconstituting centrifuged blood (160 g for 20 min at room temperature) with PRP and PPP, respectively. There was no difference in stirring‐induced increase of ATP between blood with normal platelet counts and platelet‐poor blood; moreover, stirring of PRP did not induce an increase of ATP (Fig. 1C). Also RGDS (2 mm), which eliminated SPA, had no effect on the stirring‐induced increase of ATP (Fig. 1C). These findings indicate that the stirring‐induced increase of ATP does not derive from platelets, but from erythrocytes. We further monitored extracellular ATP in more detail under the conditions of MEA measurements. According to the instructions of the manufacturer of the Multiplate®, the samples are stirred in the MEA cuvettes for 3 min at 37 °C before addition of the platelet stimulus and start of the measurements. Thus, the samples are simultaneously stirred and warmed to 37 °C for 3 min, which might cause a release of ATP/ADP from red blood cells. By monitoring ATP continuously over time we found that in the absence of stirring during the warm‐up of the samples, ATP was degraded continuously over the 3‐min incubation period, whereas in the presence of stirring the concentration of ATP initially increased, and then decreased much less, leading to a plateau of constant high ATP levels (see ATP tracings in Fig. 1D). Consistently, the ATP concentration after 3 min was significantly lower in the non‐stirred blood samples as compared with the stirred blood samples (see bar diagram in Fig. 1D). We reasoned then that omitting the stir bar during the 3‐min pre‐incubation period should decrease SPA. Indeed, when the stir bar was added to the cuvettes only after the 3‐min incubation period, just before the MEA measurement was started, SPA was not only decreased, but completely eliminated (Fig. 1D). We therefore conclude that platelets during the 3‐min period of stirring get exposed to ADP released from red cells and are therefore pre‐activated, when the MEA measurements start. This leads to SPA in the pre‐stirred blood samples. Thus the results in the pre‐stirred samples confirm previous findings of the mechanism of SPA in blood measured by other methods [17Aursnes I. Gjesdal K. Abildgaard U. Platelet aggregation induced by ADP from unsheared erythrocytes at physiological Ca++‐concentration.Br J Haematol. 1981; 47: 149-52Crossref PubMed Scopus (30) Google Scholar, 18Saniabadi A.R. Lowe G.D. Barbenel J.C. Forbes C.D. A comparison of spontaneous platelet aggregation in whole blood with platelet rich plasma: additional evidence for the role of ADP.Thromb Haemost. 1984; 51: 115-8Crossref PubMed Scopus (74) Google Scholar, 19Saniabadi A.R. Lowe G.D. Barbenel J.C. Forbes C.D. Further studies on the role of red blood cells in spontaneous platelet aggregation.Thromb Res. 1985; 38: 225-32Abstract Full Text PDF PubMed Scopus (39) Google Scholar, 20Brown P. Harrison M.J. The role of red blood cells in platelet aggregation in whole blood.Atherosclerosis. 1988; 71: 261-2Abstract Full Text PDF PubMed Scopus (7) Google Scholar]. The observation that the absence of stirring in the pre‐incubation phase quasi completely eliminated SPA is surprising. One would have expected SPA to occur during the subsequent 5‐min period of MEA measurement involving stirring. Our results showing that extracellular ATP decreases rapidly during the warming‐up phase in non‐stirred blood indicate that ecto‐nucleoside triphosphate‐diphosphohydrolases present on the surface of blood cells and degrading ATP and ADP are more active, and/or erythrocytes leak less ATP and ADP, once the blood is at 37 °C. Further experiments were then carried out to investigate the possibility that SPA might be a confounding factor for the MEA measurement of stimulated platelet aggregation. We found that for all stimuli tested, platelet aggregation was lower, when SPA was eliminated by omitting the stir bar during the 3‐min pre‐incubation period (Fig. 1E). These results also indicate that desensitization of platelet ADP receptors by ADP released from erythrocytes during the 3‐min stirring phase does not occur, otherwise the values of stimulated platelet aggregation, particularly after addition of ADP, would have been lower in pre‐stirred blood samples. Together we conclude that (i) SPA can be measured in stirred blood by MEA, and it is mediated by ADP released from red blood cells. However, SPA shows a large analytical variability, which precludes the identification of differences between individuals. Measurement of SPA in PRP, which has been shown to be a predictive risk marker for arterial occlusions [14Trip M.D. Cats V.M. van Capelle F.J. Vreeken J. Platelet hyperreactivity and prognosis in survivors of myocardial infarction.N Engl J Med. 1990; 322: 1549-54Crossref PubMed Scopus (546) Google Scholar, 15Breddin H.K. Lippold R. Bittner M. Kirchmaier C.M. Krzywanek H.J. Michaelis J. Spontaneous platelet aggregation as a predictive risk factor for vascular occlusions in healthy volunteers? Results of the HAPARG Study. Haemostatic parameters as risk factors in healthy volunteers.Atherosclerosis. 1999; 144: 211-9Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar], may be a more direct and hence better index of platelet reactivity than measurement of SPA in blood. (ii) SPA is a confounding factor of stimulated platelet aggregation measured in blood by MEA for two reasons: SPA values can unpredictably be higher than stimulated platelet aggregation values, and SPA leads to an increase of stimulated platelet aggregation values. In order to obtain more valuable data on agonist‐induced platelet aggregation, SPA should be prevented by a suitable method such as the protocol described in our study. This is important, because MEA is now increasingly used to determine platelet reactivity in patients. The authors state that they have no conflict of interest. We thank K. von Oheimb for her technical assistance. The study was supported by grants from the Deutsche Forschungsgemeinschaft (DFG Si 274/11), and the August‐Lenz‐Stiftung. The results are part of the doctoral thesis of S.A.B. at the University of Munich.