Haemostasis 8: 38-46 (1979)
Platelet Adhesiveness in Ischemic Heart Disease1 J. Aznar, P. Villa and E. Rueda Department of Clinical Pathology, Ciudad Sanitaria La F e\ Valencia
Key Words. Adhesiveness • Platelet • Collagen • Electrophoretic mobility • Acute myocardial infarction • Ischemic heart disease Abstract. The adhesiveness of platelets to collagen and glass was studied ex vivo in groups of patients with ischemic heart disease (IHD) and acute myocardial infarction (AMI). Adhesiveness was increased to glass and normal to collagen in both groups of patients. No relation was found between reinfarction rate and adhesiveness in the acute stage of AMI. Electrophoretic mobility of platelets was the same in the AMI patients and a normal control group. It is suggested that the increased ‘retention’ of platelets in IHD patients might be due more to platelet hyperaggregation or to hypersensitivity to platelet aggregating agents than to altered adhesiveness.
Introduction
The results of studies on platelets adhesiveness (PA) in acute myocardial infarction (AMI) and coronary heart disease are controversial, and it has been
found to be increased (1, 4, 5, 9 -1 1 , 13, 23, 27, 30-35, 37, 44), normal (15, 17, 24, 40, 43, 45, 46) or even decreased in some patients (45). This variability of results may be due either to the heterogeneity of the clinical material or to the diversity of techniques used in these studies (9, 16, 22, 24, 29, 37, 42, 47, 49).
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1 This investigation was supported by a grant from the ‘Instituto Nacional de Prevision’ (No. 12/929/74).
Platelet Adhesiveness
39
Bearing this in mind and also that PA to glass is very different from the adhesiveness that occurs in vivo, we thought it useful to study PA in a group of patients with IHD, using a test for PA to collagen (2), and at the same time comparing these results with those of another test for adhesiveness to glass (6). Few studies consider the prognostic value of increased PA in patients with IHD, although some of those that do agree that there is little relation between increased PA and the clinical prognosis of these patients (10, 36, 45, 46).
Materials and Methods A group of 72 patients with AMI, diagnosed clinically, enzymatically, and by electro cardiography, was used as clinical material. Blood was taken from the cubital vein either within 48 h of the acute event and before beginning any type of antithrombotic therapy, or at least 3 months after the onset of myocardial infarction, in patients who had not received any type of antithrombotic medication for at least 15 days. A group of subjects of similar age and sex, but without cardiac pathology, was used as controls. Heparin with a final concentration of 4 N1H U/ml was used for assessment of adhesive ness to glass and EDTA at 10% (1 vol of EDTA and 9 vol of blood) for adhesiveness to collagen. Bowie’s method (6), modified by Caen el al. (12), was used for evaluation of adhesiveness to glass and Aznar et al.'s (2) for adhesiveness to collagen. The electrophoretic mobility of platelets was measured in a Zeiss cytospherometer according to a previously described method (3). Differences between groups were tested for statistical significance by Student’s t test.
(1 ) PA was studied in the group of 72 patients during the first 48 h following the onset of symptoms. Compared to the control group, a significantly higher PA to glass was observed in the AMI patients (table I). No significant differences were found between the groups in adhesiveness to collagen (table I). (2) When adhesiveness was evaluated after the event (at least 3 months) in patients who had not received antithrombotic therapy for at least 15 days, results were similar to those of the acute stage (table II), i.e., increased adhesive ness to glass in IHD patients and normal adhesiveness to collagen. (3) Of the 72 patients with AMI, 16 (22%) died within the year and a half following the onset of myocardial infarction. Evaluation of the adhesiveness to both glass and collagen in these patients (table III) showed that the PA in the dead patients was slightly but not significantly higher than in those surviving.
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Results
Aznar/Villa/Rueda
40
Table I. PA to glass (whole blood) and to collagen (PRP) in AMI patients and a control group of similar age and sex but with no cardiac history: AP was assessed within the first 48 h after the AMI
Means SD n
Adhesiveness to collagen, %
Adhesiveness to glass, %
control group
patients
control group
patients
27.9 9.9 24
32.6 11.0 71
64.4 23.9 54
80.7 17.8 72
p 0.05
PA to glass (whole blood) and to collagen (PRP) in IHD patients: all patients had had the AMI at least 2 months before and adhesiveness was assessed when the patients had not received antithrombotic therapy for 15 days Table II.
Means SD n
Adhesiveness to collagen, %
Adhesiveness to glass, %
control group
patients
control group
patients
27.9 9.9 24
31.2 10.1 31
64.4 23.8 54
90.5 6.7 32
p 0.05
Table III .
PA to glass (whole blood) and to collagen (PRP) in a group of IHD patients without thrombotic relapses after the first acute event and another group of patients who died within 18 months of the acute event: PA was evaluated on the patients 48 h after the acute event
Means SD n
Adhesiveness to glass, %
without relapse
deceased
without relapse
deceased
30.6 14.2 58
32.2 4.9 13
80.4 19.4 56
82 12 16
p >0.05
p >0.05 Downloaded by: King's College London 137.73.144.138 - 1/16/2019 10:22:42 AM
Adhesiveness to collagen, %
Platelet Adhesiveness
41
Platelet electrophoretic mobility in AMI patients and the control group: tests were made on the 1st, 2nd, 3rd, and 14th day after the onset of myocardial infarction
Table IV.
Electrophoretic mobility of platelets, um/sec/V/cm patients
Means SD n
control group
1 day
2 days
3 days
14 days
1 day
2 days
3 days
14 days
0.92 0.14 24
0.94 0.12 19
0.89 0.13 16
0.85 0.12 9
0.95 0.15 21
0.95 0.15 IS
0.93 0.10 11
0.82 0.08 9
p >0.05
p >0.05
(4) Finally, the electrophoretic mobility of the platelets (table IV) was studied 24 and 48 h, 3 and 14 days after the onset of myocardial infarction. No difference was observed in platelet electrophoretic mobility between AMI pa tients and the control group.
The differences in the PA in IHD patients, referred to earlier, may be due, on the one hand, to the different techniques used to test PA and, on the other, to the time after onset of the disease at which the tests were made. As regards the technique, PA appears increased in investigations using techniques proposed by Moolten and Vroman (29), Wright (49), McDonald and Edgill (28), Hutchin son etal. (24), Hellem (22), or a modification of Salzman’s method (35). These techniques use whole native blood (35), anticoagulated with citrate (22, 29,49), or with citrate/heparin (20). An increased PA also appears in the investigations by Nestel (3), Pfleiderer and Rucker (37), and Bygdeman and Eliasch (10) using platelet-rich plasma (PRP). In other investigations, also using PRP (30, 40, 43, 46) or anticoagulated whole blood with high doses of heparin (15, 45), the PA was normal. It seems to us that the discrepant results obtained using PRP may be due to different techniques of each method. Studies which found normal PA (30, 40, 43, 46) used a technique of positive pressure through a glass bead column (47), carried out at least 2 months after the onset of symptoms. Those which found an increased PA using PRP, were done shortly after the acute event (33, 37)
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Discussion
42
using a rotation technique in a glass centrifuge tube (24) or in a glass bottle (30). It is known that with rotation techniques in glass containers PA is normal even in patients with von Willebrand’s syndrome, in contrast to PA evaluated by nonrotation techniques (7). The technique of positive pressure through a glass bead column (47) used by Bygdeman and Eliasck (10), gave an increase in PA when the sample was obtained during the AMI whereas the results were normal 2 months later. With techniques that use whole blood to evaluate the PA, PA is increased in experiments using whole native blood (35), anticoagulated with citrate (4, 13, 23, 30—32), or with citrate/heparin (5, 27, 44), or with small quantities of heparin, as in our study. Only those (5, 27, 44) that use McDonald’s method (28), with wich increased PA was also found, use a larger dose of heparin, approximately 20 U/ml, and a rotation technique in a glass container. On the other hand, PA was normal in studies which use whole blood, but with a rotation method in a polystyrene tube (15), anticoagulating with a high concentration of heparin (100 U/ml) (16). PA was also normal in the study by Steele et al. (45) who used a glass bead column and a heparin concentration similar to that of McDonald and Edgill (28). The latter extracted 2 ml of the blood sample on syringes previously moistened with heparin, which according to our measurements gives an anticoagulant concentration between 20 and 30 U/ml. With regard to Steele et aids (45) results, it should be borne in mind that he does not specify the flow rate through the glass bead column and the rate of flow through the column is known to influence the percentage of platelets retained, the PA decreasing in proportion to the increase in flow rate ( 6 , 8 ). Basically, therefore, in chronic IHD patients PA appears increased when tested by any method using whole blood (except when a high heparin concentra tion is used and a rotation technique in a glass container is not used) or PRP (but with a rotation technique in a glass container). On the other hand, PA is normal when PRP and a technique of positive pressure are used. In our opinion, the increase in PA could be due to the fact that in IHD patients, there is an increase in platelet ‘retention’ in which, as we know, adhesiveness and aggregation coexist, the platelet hyperaggregation playing the predominant role in the intensity of ‘retention’. This agrees with the findings of increased platelet aggregation in IHD patients when stimulated with activators (14, 18, 19, 21, 39, 51, 52) or spontaneously (14, 50, 52), although this spontaneous hyperaggregation was not confirmed in Prazich et aids (38) recent study.
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Aznar/Villa/Rueda
Platelet Adhesiveness
43
That increase platelet ‘retention’ in I HD patients may be due more to increased aggregation than to increased adhesiveness agrees with the normal adhesiveness to collagen found in our study, since the technique we used evaluated only platelet adhesiveness with no interference from aggregation (2). The electrical surface charge undoubtedly plays an interesting role in PA to the healthy or damaged vascular endothelium and among the platelets them selves, negativity favouring their maintenace in suspension in the bloodstream (3,21). In our study and in agreement with other authors (20, 43), but contradic tory to Larcan et al.'s (26) results, we found no changes in platelet electro phoretic mobility in AMI patients. This supports our hypothesis that in these patients PA is normal and increased platelet retention must be related more directly to platelet hyperreactivity to aggregating stimuli than to increased adhesiveness. With regard to the possible prognostic value of increased ‘retention’, our results agree with previous studies (10, 36,45, 46) suggesting a weak correlation between increased PA and the danger of relapse. Regarding the pathogenesis of the hyperaggregation found in I HD patients, it seems relevant to consider the changes in the lipid components of platelets which may make them more sensitive to the thrombin stimulus (39). Thus, we have found that IHD patients have an increase in saturated fatty acids of their platelets (48). It is also known that glycoproteins of the platelet membrane play an important role in platelet adhesiveness to collagen (25) but in a group of IHD patients we found no deviation from normal in the composition of the glyco proteins (Cubillo and Aznar, unpubl. results). Acknowledgements We wish to thank Miss Maria José Muñoz for her invaluable technical assistance. References
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1 Ardlie, N.G.; Kinlough, R.L., and Schwartz, C.J.: In vitro thrombosis and platelet aggregation in myocardial infarction. Br. med. J. i: 888 (1966). 2 Aznar, J.; Jimenez, C., and Villa, P.: A technique for the evaluation of platelet-collagen adhesiveness in vitro. Haemostasis 5: 318 (1976). 3 Aznar, J.; Martinez Sausor, V.; Valles, J., and Villa, P.: Estudio de la movilidad electroforética de las plaquetas. Breve revisión bibliográfica y aportación de la experi encia personal. Sangre 23: 13 (1978). 4 Baumgartner, H.R.; Cronquist, M.; Wobman, P.; Streuli, F. und Duckert, F.: Die
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Messung der Thrombozytenadhâsivitàt. Erste Erfahrungen mit neueren Methoden. Schweiz, med. Wschr. 97: 1647 (1967). 5 Besterman, E.; Myat, G., and Travadi, V.: Diurnal variations of platelet stickiness compared with effects produced by adrenaline. Br. med. J. i: 597 (1967). 6 Bowie, E.J.W.; Owen, C.A., jr.; Thompson, J.,jr., and Didisheim, P.: Platelet adhesive ness in von Willebrand’s disease. Am. J. clin. Path. 52: 69 (1969). 7 Bowie, E.J.W.; Didisheim, P.; Thrompson, J.H., jr., and Owen, C.A., jr.: The spectrum of von Willebrand’s disease. Thromb. Diath. haemorrh. 18: 40 (1967). 8 Breda, R.; Musumeci, V., and Zuppi, G: A new approach to the use of glass bead platelet-retention techniques in the diagnosis of thrombosis. An attempt to investigate the platelet population pattern with reference to glass adhesiveness; in Caprino and Rossi, Platelet aggregation and drugs, p. 251 (Academic Press, London 1974). 9 Breddin, K.: Über die gesteigerte Thrombocytenagglutination bei Gcfasskrankheiten. Schweiz, med. Wschr. 95: 655 (1965). 10 Bygdeman, S. and Eliasch, H.: Platelet adhesiveness in myocardial infarction in relation to clinical course. Acta. med. scand. 199: 475 (1976). 11 Bygdeman, S. and Wells, R.: Studies on platelet adhesiveness, blood viscosity and the microcirculation in patients with thrombotic disease. J. Atheroscler. Res. 10: 33 (1969). 12 Caen, J.; Larrieu, M.J. et Samama, M.: Retention des plaquettes sur billes de verre sur sang heparinisé: in L’hcmostase, p. 66 (Expansion scientifique française, Paris 1975). 13 Chaudhuri, S.: Platelet adhesiveness in the assessment of ischaemic heart diseases. Thromb. Res. 6: 209 (1975). 14 Dreyfuss, F. and Zahavi, J.: Adenosine diphosphate induced platelet aggregation in myocardial infarction and ischaemic heart disease Atherosclerosis 1 7: 107 (1973). 15 Eastham, R.D.: Attempted reduction of adhesive platelets after cardiac infarction. Lancet ii: 541 (1964). 16 Eastham, R.D.: Rapid adhesive platelet count in whole blood. J. clin. Path. 17: 45 (1964). 17 Fitzgerald, D.E.; Butterfield, W.J.H.; Smink, D., and Kruisheer, H.E.J.: Platelet adhe siveness in post-myocardial infarction patients compared with controls. Atherosclerosis 13: 217 (1971). 18 Fleischeman, A.I.; Bierenbaum, M.L.; Justice, D.; Stier, A., and Sullivan, A.: In vivo platelet function in acute myocardial infarction acute cerebrovascular accidents and following surgery. Thromb. Res. 6: 205 (1975). 19 Goldenfarb, P.B.; Cathey, M.H.; Zucker, S.; Wilburg, P., and Corrigan, J.J.: Changes in the hemostatic mechanism after myocardial infarction. Circulation 43: 538 (1971). 20 Grottum, K.A.: Influence of aggregating agents on electrophoretic mobility of blood platelets from healthy individuals and from patients with cardiovascular diseases. Lancet/'.' 1406 (1968). 21 Hampton, J.R. and Mitchell, J.R.: Platelet electrophoresis: the present position. Thromb. Diath. haemorrh. 31: 204 (1974). 22 Hellem, A.J.: The adhesiveness of human blood platelets in vitro. Scand. J. clin. Lab. Invest., suppl. 51 (1960). 23 Horlick, L.: Platelet adhesiveness in normal persons and subjects with atherosclerosis:
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Platelet Adhesiveness
25 26
27 28 29
30
31 32 33 34
35
36
37
38
39
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effect of high fat meal and anticoagulants on the adhesive index. Am. J. Cardiol. 8: 459 (1961). Hutchinson, H.E.; Stark, J.M., and Chapman, J.A : Platelet serotonin and normal haemostasis. J. clin. Path. 12: 265 (1959). Jamieson, G.A.; Urban, C.L., and Barber, A.J.: Enzymatic basis for platelet collagen adhesion as a primary step in haemostasis. Nature new Biol. 234: 5 (1971). Larcan, A.; Stoltz, J.F. et Volary, A.M.: Les altérations de la charge électrique plaquettaire au cours de diverses aggressions (thromboses - shocks endotoxiniques hémolyses aiguës). Presse méd. 78: 2379 (1970). McDonald, L. and Edgill, M.: Changes in coagulability of blood during various phases of ischaemic heart disease. Lancet i: 1115 (1959). McDonald, L. and Edgill, M.: Coagulability of the blood in ischaemic heart disease. Lancet ii: 457 (1957). Moolten, S.E. and Vroman, L.: Adhesiveness of blood platelets in thromboembolism and hemorrhagic disorders; measurement of platelet adhesiveness by the glass-wool filter. Am. J. din. Path. 19: 701 (1949). Moolten, S.E.; Vroman, L., and Vroman, G.M.S.: Adhesiveness of blood platelet in thromboembolism and hemorrhagic disorders. 11. Diagnostic and prognostic signifi cance of platelet adhesiveness. Am. J. clin. Path. 19: 814 (1949). Moolten, S.E.; Jennings, P.B., and Solden, A.: Dietary fat and platelet adhesiveness in arteriosclerosis and diabetes. Am. J. Cardiol. 11: 290 (1963). Murphy, C.A. and Mustard, J.F.: Coagulation tests and platelet economy in athero sclerotic and control subjects. Circulation 24: 114 (1962). Nestel, P.J.: A note on platelet adhesiveness in ischaemic heart disease. J. clin. Path. 14: 150 (1961). O’Brien, J.R.; Heywood, J.B., and Heady, J.A.: The quantitation of platelet aggrega tion induced by four compounds: a study in relation to myocardial infarction. Thromb. Diath. haemorrh. 16: 752 (1966). O’Brien, J.R.; Etherington, M.; Jamieson, S.; Klaber, M.R., and Ainsworth, J.F.: Stressed template bleeding-time and other platelet-function tests in myocardial infarc tion. Lancet «V694 (1973). Pfleiderer, T.; Weber, E., and Walter, E.: Platelet function and myocardial infarction; in Gerlach, Moser, Deutsch and Wilmans, Erythrocytes, thrombocytes, leucocytes, p. 270 (Thieme, Stuttgart 1973). Pfleiderer, T. und Rucker, G.: Über Kaliumgehalt und Adhäsivität der Thrombocyten von gesunden Menschen und Patienten mit obliterierender Gefassklerose. Klin. Wschr. 42: 82 (1964). Prazich, J.A.; Rapaport, S.I.; Samples, J.R., and Engler, R.: Platelet aggregate ratios. Standardization of technique and test results in patients with myocardial ischaemia and patients with cerebrovascular disease. Thromb. Haemost. 38: 597 (1977). Renaud, S.; Kuba, K.; Goulet, G; Lemire, Y., and Allard, C : Relationship between fatty-acid composition of platelets and platelet aggregation in rat and man. Circulation Res. 26: 553 (1970). Rozenberg, M.C. and Stormorken, H.: Comparison of glass adhesiveness and rate of aggregation of blood platelets. Scand. J. clin. Lab. Invest. 19: 82 (1967). Downloaded by: King's College London 137.73.144.138 - 1/16/2019 10:22:42 AM
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44
45 46 47
48 49 50 51 52
Ruhenstoth-Brauer, G.; Straub, E.; Schtleben, P., and Fuhrmann, G.F.: Electrophoretic mobility of blood cells in normal and sick persons, Münch, med. Wschr. 103: 794 (1961). Salzman, E.: Measurement of platelet adhesiveness. J. Lab. clin. Med. 62: 724 (1963). Sjogren, A.; Bottiger, L.E.; Biörck, G.; Wahlberg, F., and Carlson, L.A.: Adenosine diphosphate induced platelet adhesiveness in patients with ischaemic heart diesase. Acta med. scand. 187: 89 (1970). Slack, J.; Seymour, J.; McDonald, L., and Love, F.: Lipoprotein-lipase levels and platelet stickiness in patients with ischaemic heart disease and in controls, distin guishing those with an affected first degree relative. Lancet ii: 1033 (1964). Steele, P.P.; Weily, H.S.; Davies, H., and Genton, E.: Platelet function studies in coronary artery disease. Circulation 48: 1194 (1973). Stormorken, H.: Platelet adhesiveness in coronary heart disease. Evaluation of the platelet rich plasma-ADP method. Acta. med. scand. 188: 339 (1970). Stormorken, H.; Lund-Ruse, A., and Rorvik, T.O.: Platelet adhesiveness to glass beads: methodological investigation using automatic particle counting. Scand. J. clin. Lab. Invest. 84: suppl., p. 183 (1965). Valles, J.; Aznar, J., and Santos, M.T.: Platelet fatty acids in acute myocardial infarction. Thromb. Res. (in press). Wright, H.P.: The adhesiveness of blood platelets in normal subjects with varying concentrations of anticoagulants. J. Path. Bact. 53: 255 (1941). Wu, K.K. and Hoak, J.C.: Platelet aggregation in arterial insufficiency. Mechanisms and implications. Thromb. Haemost. 35: 702 (1976). Yamazaki, H.; Takahashi, T., and Sano, T.: Hyperaggregability of platelets in thrombo embolic disorders. Thromb. Diath. haemorrh. 34: 94 (1975). Zahavi, J. and Dreyfuss, F.: An abnormal pattern of adenosine diphosphate induced platelet aggregation in acute myocardial infarction. Thromb. Diath. haemorrh. 21: 76 (1969).
Received: June 15, 1978; accepted in revised form by Editor G. de Gaetano: October 9, 1978 Dr. J. Aznar, Departamento de Biopatologia Clínica, Ciudad Sanitaria 'La Fe’, Valencia (Spain) Downloaded by: King's College London 137.73.144.138 - 1/16/2019 10:22:42 AM
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46
Platelet Adhesiveness in Ischemic Heart Disease1 J. Aznar, P. Villa and E. Rueda Department of Clinical Pathology, Ciudad Sanitaria La F e\ Valencia
Key Words. Adhesiveness • Platelet • Collagen • Electrophoretic mobility • Acute myocardial infarction • Ischemic heart disease Abstract. The adhesiveness of platelets to collagen and glass was studied ex vivo in groups of patients with ischemic heart disease (IHD) and acute myocardial infarction (AMI). Adhesiveness was increased to glass and normal to collagen in both groups of patients. No relation was found between reinfarction rate and adhesiveness in the acute stage of AMI. Electrophoretic mobility of platelets was the same in the AMI patients and a normal control group. It is suggested that the increased ‘retention’ of platelets in IHD patients might be due more to platelet hyperaggregation or to hypersensitivity to platelet aggregating agents than to altered adhesiveness.
Introduction
The results of studies on platelets adhesiveness (PA) in acute myocardial infarction (AMI) and coronary heart disease are controversial, and it has been
found to be increased (1, 4, 5, 9 -1 1 , 13, 23, 27, 30-35, 37, 44), normal (15, 17, 24, 40, 43, 45, 46) or even decreased in some patients (45). This variability of results may be due either to the heterogeneity of the clinical material or to the diversity of techniques used in these studies (9, 16, 22, 24, 29, 37, 42, 47, 49).
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1 This investigation was supported by a grant from the ‘Instituto Nacional de Prevision’ (No. 12/929/74).
Platelet Adhesiveness
39
Bearing this in mind and also that PA to glass is very different from the adhesiveness that occurs in vivo, we thought it useful to study PA in a group of patients with IHD, using a test for PA to collagen (2), and at the same time comparing these results with those of another test for adhesiveness to glass (6). Few studies consider the prognostic value of increased PA in patients with IHD, although some of those that do agree that there is little relation between increased PA and the clinical prognosis of these patients (10, 36, 45, 46).
Materials and Methods A group of 72 patients with AMI, diagnosed clinically, enzymatically, and by electro cardiography, was used as clinical material. Blood was taken from the cubital vein either within 48 h of the acute event and before beginning any type of antithrombotic therapy, or at least 3 months after the onset of myocardial infarction, in patients who had not received any type of antithrombotic medication for at least 15 days. A group of subjects of similar age and sex, but without cardiac pathology, was used as controls. Heparin with a final concentration of 4 N1H U/ml was used for assessment of adhesive ness to glass and EDTA at 10% (1 vol of EDTA and 9 vol of blood) for adhesiveness to collagen. Bowie’s method (6), modified by Caen el al. (12), was used for evaluation of adhesiveness to glass and Aznar et al.'s (2) for adhesiveness to collagen. The electrophoretic mobility of platelets was measured in a Zeiss cytospherometer according to a previously described method (3). Differences between groups were tested for statistical significance by Student’s t test.
(1 ) PA was studied in the group of 72 patients during the first 48 h following the onset of symptoms. Compared to the control group, a significantly higher PA to glass was observed in the AMI patients (table I). No significant differences were found between the groups in adhesiveness to collagen (table I). (2) When adhesiveness was evaluated after the event (at least 3 months) in patients who had not received antithrombotic therapy for at least 15 days, results were similar to those of the acute stage (table II), i.e., increased adhesive ness to glass in IHD patients and normal adhesiveness to collagen. (3) Of the 72 patients with AMI, 16 (22%) died within the year and a half following the onset of myocardial infarction. Evaluation of the adhesiveness to both glass and collagen in these patients (table III) showed that the PA in the dead patients was slightly but not significantly higher than in those surviving.
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Results
Aznar/Villa/Rueda
40
Table I. PA to glass (whole blood) and to collagen (PRP) in AMI patients and a control group of similar age and sex but with no cardiac history: AP was assessed within the first 48 h after the AMI
Means SD n
Adhesiveness to collagen, %
Adhesiveness to glass, %
control group
patients
control group
patients
27.9 9.9 24
32.6 11.0 71
64.4 23.9 54
80.7 17.8 72
p 0.05
PA to glass (whole blood) and to collagen (PRP) in IHD patients: all patients had had the AMI at least 2 months before and adhesiveness was assessed when the patients had not received antithrombotic therapy for 15 days Table II.
Means SD n
Adhesiveness to collagen, %
Adhesiveness to glass, %
control group
patients
control group
patients
27.9 9.9 24
31.2 10.1 31
64.4 23.8 54
90.5 6.7 32
p 0.05
Table III .
PA to glass (whole blood) and to collagen (PRP) in a group of IHD patients without thrombotic relapses after the first acute event and another group of patients who died within 18 months of the acute event: PA was evaluated on the patients 48 h after the acute event
Means SD n
Adhesiveness to glass, %
without relapse
deceased
without relapse
deceased
30.6 14.2 58
32.2 4.9 13
80.4 19.4 56
82 12 16
p >0.05
p >0.05 Downloaded by: King's College London 137.73.144.138 - 1/16/2019 10:22:42 AM
Adhesiveness to collagen, %
Platelet Adhesiveness
41
Platelet electrophoretic mobility in AMI patients and the control group: tests were made on the 1st, 2nd, 3rd, and 14th day after the onset of myocardial infarction
Table IV.
Electrophoretic mobility of platelets, um/sec/V/cm patients
Means SD n
control group
1 day
2 days
3 days
14 days
1 day
2 days
3 days
14 days
0.92 0.14 24
0.94 0.12 19
0.89 0.13 16
0.85 0.12 9
0.95 0.15 21
0.95 0.15 IS
0.93 0.10 11
0.82 0.08 9
p >0.05
p >0.05
(4) Finally, the electrophoretic mobility of the platelets (table IV) was studied 24 and 48 h, 3 and 14 days after the onset of myocardial infarction. No difference was observed in platelet electrophoretic mobility between AMI pa tients and the control group.
The differences in the PA in IHD patients, referred to earlier, may be due, on the one hand, to the different techniques used to test PA and, on the other, to the time after onset of the disease at which the tests were made. As regards the technique, PA appears increased in investigations using techniques proposed by Moolten and Vroman (29), Wright (49), McDonald and Edgill (28), Hutchin son etal. (24), Hellem (22), or a modification of Salzman’s method (35). These techniques use whole native blood (35), anticoagulated with citrate (22, 29,49), or with citrate/heparin (20). An increased PA also appears in the investigations by Nestel (3), Pfleiderer and Rucker (37), and Bygdeman and Eliasch (10) using platelet-rich plasma (PRP). In other investigations, also using PRP (30, 40, 43, 46) or anticoagulated whole blood with high doses of heparin (15, 45), the PA was normal. It seems to us that the discrepant results obtained using PRP may be due to different techniques of each method. Studies which found normal PA (30, 40, 43, 46) used a technique of positive pressure through a glass bead column (47), carried out at least 2 months after the onset of symptoms. Those which found an increased PA using PRP, were done shortly after the acute event (33, 37)
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Discussion
42
using a rotation technique in a glass centrifuge tube (24) or in a glass bottle (30). It is known that with rotation techniques in glass containers PA is normal even in patients with von Willebrand’s syndrome, in contrast to PA evaluated by nonrotation techniques (7). The technique of positive pressure through a glass bead column (47) used by Bygdeman and Eliasck (10), gave an increase in PA when the sample was obtained during the AMI whereas the results were normal 2 months later. With techniques that use whole blood to evaluate the PA, PA is increased in experiments using whole native blood (35), anticoagulated with citrate (4, 13, 23, 30—32), or with citrate/heparin (5, 27, 44), or with small quantities of heparin, as in our study. Only those (5, 27, 44) that use McDonald’s method (28), with wich increased PA was also found, use a larger dose of heparin, approximately 20 U/ml, and a rotation technique in a glass container. On the other hand, PA was normal in studies which use whole blood, but with a rotation method in a polystyrene tube (15), anticoagulating with a high concentration of heparin (100 U/ml) (16). PA was also normal in the study by Steele et al. (45) who used a glass bead column and a heparin concentration similar to that of McDonald and Edgill (28). The latter extracted 2 ml of the blood sample on syringes previously moistened with heparin, which according to our measurements gives an anticoagulant concentration between 20 and 30 U/ml. With regard to Steele et aids (45) results, it should be borne in mind that he does not specify the flow rate through the glass bead column and the rate of flow through the column is known to influence the percentage of platelets retained, the PA decreasing in proportion to the increase in flow rate ( 6 , 8 ). Basically, therefore, in chronic IHD patients PA appears increased when tested by any method using whole blood (except when a high heparin concentra tion is used and a rotation technique in a glass container is not used) or PRP (but with a rotation technique in a glass container). On the other hand, PA is normal when PRP and a technique of positive pressure are used. In our opinion, the increase in PA could be due to the fact that in IHD patients, there is an increase in platelet ‘retention’ in which, as we know, adhesiveness and aggregation coexist, the platelet hyperaggregation playing the predominant role in the intensity of ‘retention’. This agrees with the findings of increased platelet aggregation in IHD patients when stimulated with activators (14, 18, 19, 21, 39, 51, 52) or spontaneously (14, 50, 52), although this spontaneous hyperaggregation was not confirmed in Prazich et aids (38) recent study.
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That increase platelet ‘retention’ in I HD patients may be due more to increased aggregation than to increased adhesiveness agrees with the normal adhesiveness to collagen found in our study, since the technique we used evaluated only platelet adhesiveness with no interference from aggregation (2). The electrical surface charge undoubtedly plays an interesting role in PA to the healthy or damaged vascular endothelium and among the platelets them selves, negativity favouring their maintenace in suspension in the bloodstream (3,21). In our study and in agreement with other authors (20, 43), but contradic tory to Larcan et al.'s (26) results, we found no changes in platelet electro phoretic mobility in AMI patients. This supports our hypothesis that in these patients PA is normal and increased platelet retention must be related more directly to platelet hyperreactivity to aggregating stimuli than to increased adhesiveness. With regard to the possible prognostic value of increased ‘retention’, our results agree with previous studies (10, 36,45, 46) suggesting a weak correlation between increased PA and the danger of relapse. Regarding the pathogenesis of the hyperaggregation found in I HD patients, it seems relevant to consider the changes in the lipid components of platelets which may make them more sensitive to the thrombin stimulus (39). Thus, we have found that IHD patients have an increase in saturated fatty acids of their platelets (48). It is also known that glycoproteins of the platelet membrane play an important role in platelet adhesiveness to collagen (25) but in a group of IHD patients we found no deviation from normal in the composition of the glyco proteins (Cubillo and Aznar, unpubl. results). Acknowledgements We wish to thank Miss Maria José Muñoz for her invaluable technical assistance. References
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Received: June 15, 1978; accepted in revised form by Editor G. de Gaetano: October 9, 1978 Dr. J. Aznar, Departamento de Biopatologia Clínica, Ciudad Sanitaria 'La Fe’, Valencia (Spain) Downloaded by: King's College London 137.73.144.138 - 1/16/2019 10:22:42 AM
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