Adenosine Diphosphate-induced Platelet Aggregation in the States of Hypercoagulability

Transcription

1 ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 7, No. 2 Copyright 1977, Institute for Clinical Science Adenosine Diphosphate-induced Platelet Aggregation in the States of Hypercoagulability TEH Y. WANG, Ph.D. and CLARA V. HUSSEY, M.D. Department of Pathology, The Medical College of Wisconsin, Milwaukee County Medical Complex, Milwaukee, WI ABSTRACT One hundred and seven patients with congestive heart failure, myocardial infarction and arteriosclerotic heart disease were studied by adenosine diphosphate-induced platelet aggregation, fibrinogen levels and ethanol gelation test. Both increases and decreases in platelet aggregation were observed. A significantly high percentage of patients showed a decreased platelet aggregation which was especially marked in the more acute as opposed to the less acute phase. In addition, most patients exhibited a marked shift from abnormal to normal platelet aggregation or vise-versa within a short time period. This pattern of platelet aggregation suggests an active role of platelets in the states of hypercoagulability. The hypercoagulability of these patients was further substantiated by a high percentage of positive ethanol gelation tests and high fibrinogen levels. Introduction The role of platelets in thromboembolic complications, particularly that relating to hypercoagulable states, has been frequently described. Platelet aggregates have been demonstrated in vivo in animals subjected to stress6 as well as hyperacute rejection,15 and in intramyocardial vessels of patients dying suddenly of coronary artery disease.5 In addition, platelet appearance in those patients with acute strokes show more spread forms when compared with those of healthy adults.4 Indeed, transient micro-emboli of platelet-fibrin undoubtedly exert a significant effect in cerebrovascular disease changes of the heart.10 and ischemic In in vivo studies, platelet aggregation induced by adenosine diphosphate (ADP) has been found to be highly elevated in myocardial infarction and ischemic heart disease.2,12 Paradoxically, both increased and decreased platelet stickiness in cerebrovascular disease and myocardial infarction has been reported with different methods.1,2,14,17,18 A set pattern of platelet aggregation induced by ADP in patients with hypercoagulability is reported here. The significance of these findings is discussed. Materials and Methods P a t i e n t s One hundred and seven patients with congestive heart failure (CHF), myocar- 152

2 ADP-INDUCED PLATELET AGGREGATION IN HYPERCOAGULABILITY 153 dial infarction (MI) or arteriosclerotic heart disease (ASHD) have been studied. They were further divided into three groups: (1) those in CHF, with hypertension; (2) those with CHF, without hypertension, and (3) those without CHF, but with ASHD or MI. Most patients in the third group also showed hypertension, currently or by past history. Diagnoses were made by physicians in charge and confirmed by X-ray, EKG or laboratory data. All acute Mis were confirmed by EKG findings and/or elevated SGOT and LDH. Stage I Ml is defined as five days within onset and State II MI as between 6 and 14 days after onset. It is apparent that these groups of patients have several factors associated with a hypercoagulable state. Therefore, the following tests were performed for each patient: ADP-induced platelet aggregation, plasma fibrinogen level and ethanol gelation test for three times during the period of approximately two weeks. P l a t e l e t R i c h P l a s m a Citrated whole blood was prepared by adding nine parts of whole blood to one part of 3.8 percent sodium citrate. Platelet rich plasma (PRP) was prepared by centrifuging freshly drawn citrated whole blood at 75 x g for 10 min. Final platelet counts were 3 to 5 x 108 per ml. Platelet poor plasma (PPP) was prepared by centrifuging PRP at 1,000 X g for 10 min. P l a t e l e t A g g r e g a t i o n T e s t A volume of 0.1 ml of aggregating agent was added to 2.4 ml of PRP in a siliconized curvet containing two siliconized stirring rods to initiate the aggregation test. The percentage aggregation was immediately recorded graphically and continuously after initiation as percent of light transmission,* with PRP set at 0 percent against PRP at 100 transmission.16! The temperature was set at 37, the magnetic stirrer motor was set at 1,100 rpm and the wavelength adjusted to 609 nm. All the aggregating agents and drugs were prepared in veronal acetate buffer-saline (0.05 M veronal acetate buffer in normal saline, ph 7.4), and the ph was adjusted to 7.4. The final concentration of ADP for each patient s study was 0.85 x 10-6 M. In studies involving the effect of drugs, 0.1 ml of the drug was added to the system before initiation. Control tubes contained 0.1 ml of veronal acetate buffer saline in place of the drug. The first wave of aggregation was defined as the primary aggregation whether it was followed by either deaggregation or another wave of aggregation which was defined as the secondary aggregation. F i b r i n o g e n D e t e r m i n a t i o n A biuret reaction to the fibrin clot as modified by Ware7 was followed with a normal range of 200 to 400 mg per dl. E t h a n o l G e l a t i o n T e s t Each patient s plasma was treated by the addition of ethanol reagent as described by Horne.9 A normal individual shows negative results for the ethanol gelation test. Results N o r m a l V a l u e o f A D P - i n d u c e d A g g r e g a t i o n From previous determinations in our laboratory, the normal value of ADPinduced aggregation was 20 to 45 percent. The percentage aggregation above 45 was defined as increased aggregation, whereas a value below 20 was defined as decreased aggregation. A g g r e g a t i o n P a t t e r n in MI The aggregation pattern in MI is summarized in table I. Forty-five patients * Bausch and Lomb Eecorder. t Chrono-Log Aggregometer.

3 154 WANG AND HUSSEY T A B L E I Adenosine Diphosphate-induced Platelet Aggregation Pattern in Patients with Myocardial Infarction # Patients Studied Total 45 Stage I 37 Stage II 38 Percent of patients + 25(90)* showing abnormal aggregation + 56(86)t t Percent of patients showing increased aggregation on at least one occasion during the period studied. 4- Percent of patients showing decreased aggregation on at least one occasion during the period. * Percent of patients of that particular group that showed normal and/or decreased aggregation on different occasions during the period of approximately two weeks, t Percent of patients of that particular group that showed normal and/or increased aggregation on different occasions during the period. with MI were studied. Twenty-five percent of the patients showed high aggregation, while 56 percent showed decreased aggregation on at least one occasion (table I). In a series of studies within a period of approximately two weeks, 90 and 86 percent of high and low platelet aggregation groups, respectively, also showed normal and/or an opposite pat- T A B L E II Comparison Among Groups of Total Patients Studied # Patients Group 1 Group 2 Group 3 Studied Percent of + 31(80)* 19(100)* 26(90)* patients showing 4-42 (100) t 33 (86) t 42(90) + normal aggregation Percent of patients showing positive ethanol gelation test + Percent of patients showing increased aggregation on at least one occasion during the period studied. + Percent of patients showing decreased aggregation on at least one occasion during the period. * Percent of patients of that particular group that showed normal and/or decreased aggregation on different occasions during the period of approximately two weeks. t Percent of patients of that particular group that showed normal and/or increased aggregation on different occasions during the period. tern on different occasions. The pattern of extensively shifting aggregation is also demonstrated in table III. This is quite opposite to what has been observed by the present authors with the normal population, which stays relatively stable on the same level. According to the time of onset, patients with MI were further divided in Stages 1 and 2. In Stage 1, 16 percent of the patients showed increased platelet aggregation by ADP on one or more occasions in comparison to 33 percent in Stage 2. The most distinct feature was that 51 percent of the patients in Stage 1 showed decreased aggregation on at least one occasion in contrast to 28 percent in Stage 2. Thus it was evident that in the more acute phase there was more low aggregations and fewer high aggregations. A g g r e g a t i o n P a t t e r n i n CHF It has been shown in table II that there was no significant difference in the aggregation patterns among Groups 1, 2, and 3 and between these groups and MI. Both increase and decrease in the extent of aggregation were observed among the 107 patients studied during a period of about two weeks after an exacerbation. Similarly, the shifting phenomenon of individuals was great. The occurence of secondary aggregation among the 107 patients at the final ADP concentration used (0.85 x 10-6 M) was 17 percent. Normal population showed only primary aggregation at this concentration. Nevertheless, it appears that there are less abnormal aggregations in Group 2 than in Groups 1 and 3. This may be related to the fact that patients in Groups 1 and 3 exhibited hypertension, thus exposed to stronger stress. Fibrinogen level of Group 1 was 400 mg per dl (S.D., 117) and Group 3 was 396 mg per dl (S.D., 103). These were significantly different from Group 2, 335 mg per dl (S.D., 90) (P<0.01). This dif

4 ADP-INDUCED PLATELET AGGREGATION IN HYPERCOAGULABILITY 155 ference also may be due to the hypertension factor. No significant shifting of plasma fibrinogen levels as observed in ADP-induced aggregation was noted. In addition, no direct correlation between the extent of platelet aggregation and fibrinogen level was observed. The evidence showing that these groups of platelets were in hypercoagulable states was further substantiated by the high percentage of positive ethanol gelation tests (table II). However, no direct correlation between the extents of platelet aggregation and the ethanol gelation test was observed. E f f e c t o f D r u g s o n A g g r e g a t i o n In order to find out whether or not the low aggregation was affected by drugs taken by patients during the period of study, effects of drugs on aggregation were studied at the concentration compatible with the calculated blood levels. Drugs were incubated at 37 C for five min with unwashed platelets prepared from normal individuals who had not taken aspirin for at least seven days. Aggregation was initiated by the addition of ADP and epinephrine, respectively, at the concentrations that induced the secondary phase of aggregation (ADP 2 x 10 em and epinephrine 1 x 10~5 M). All the drugs studied did not affect the primary phase of aggregation. However, the secondary phase of aggregation was inhibited by few drugs (table IV). Discussion In the present study, a pattern of variability in the ADP-induced aggregation has been studied in patients with hypercoagulability. The present authors were not successful in consistently demonstrating an increased or decreased platelet aggregation in our patients. On the contrary, an extensive shifting of the platelet aggregation pattern was demonstrated. T A B L E III Some Representative Patterns of Adenosine Diphosphate-Induced P latelet Aggregation in Patients with Congestive Heart Failure, A rteriosclerotic Heart Disease, Myocardial In farctio n, Etc. Days After Onset or Exacerbation Patient E.S L.S M. J /76,* F.R A. V C.T J.S M.U D.D /56 W.D H.M E.F C.U /62 56 H.G J.B V.C E.M V.B D.O *Second wave of aggregation. Drugs T A B L E IV Effect of Drugs on Adenosine Diphosphate-Induced Aggregation Final Concentration Primary Phase Aggregation Secondary Phase Atropine 0.03 mg/ml * * Compazine 0.62 mg/ml * * Coumadin 1.20 yg/ml * * Dilaudid 0.50 yg/ml * * Inderal 0.18 mg/ml * * Lasix 0.25 mg/ml * * Librium 2.00 yg/ml * * Seconal 0.62 mg/ml * * Valium 1.20 yg/ml * * Aspirin 0.02 mg/ml * + Esidrix 0.84 mg/ml * Pronestyl 0.24 mg/ml * + Aldomet 0.21 mg/dl * S1+ Apresoline 1.68 mg/ml * S1 + Benadryl 0.12 mg/ml * s u Chlortrimeton 0.90 yg/ml * S14 Reserpine 1.40 yg/ml * S U Vistaril 0.12 mg/ml * SI* The final concentration of adenosine diphosphate was 2 ym/1. *No effect. ^Complete inhibition of the secondary phase of aggregation. S1+ Prolongation of the secondary wave of aggregation only.

5 156 WANG AND HUSSEY Twenty-five and 56 percent of the patients with acute MI showed increased and decreased platelet aggregation, respectively. A similar pattern was also observed on the three groups of patients studied. These figures would vary more if the aggregation test had been studied only once for each patient (table III). Regrettably, it was not possible to study the aggregation test for the patients on a day to day basis. Nevertheless, a general aggregation pattern was demonstrated of patient s platelets in response to ADP in a period of about two weeks. From studying the pattern, it appears that the test may not reveal useful information if performed only once but can serve as a useful diagnostic tool if performed consecutively within a period of time. In contrast to the concept that platelet aggregation is usually high in hypercoagulable states, it has been demonstrated that a greater percentage of patients showed low aggregation on at least one occasion. The low aggregation is not due to the effect of drugs, since the primary phase of aggregation is not inhibited, nor is it due to variations in fibrinogen levels since no correlation with aggregation was found. More likely such platelets have been subjected to stimulation in vivo and have gone extensively into an aggregation-deaggregation process. This may be compatible with the in vitro phenomenon of platelets refractoriness that platelets become less responsive upon reexposure to ADP.3 11,13 This is also compatible with the idea that there are two populations of platelets, one predisposed to ADP stimulation and one which is not.8 Thus, after the one population has been knocked out of circulation, the remainder will respond poorly to ADP. Low aggregation thus indicates that platelets have been subjected to in vivo activation. Our findings may also explain why a larger proportion of patients exhibited low aggregation in Stage 1 MI. On the other hand, an increase in aggregation also was observed. Increased aggregation might be due to plasma factors, e.g., plasma catecholamines, or to platelet membrane change. Therefore, increases and decreases of platelet aggregation as well as the shifting phenomenon observed in short time periods demonstrate the active role of platelets in hypercoagulability states. References 1. D a n t a, G.: Platelet adhesiveness in cerebrovascular disease. Atherosclerosis 11: , D r e y f u s s, F., Z a h a v i, J.: Adenosine diphosphate induced platelet aggregation in myocardial infarction and ischemic heart disease. Atherosclerosis J 7: , E lk A, C.: Platelet refractory state induced by heparin. Scand. J. Haematol 9: , G i l r o y, T., B a r n h a r t, M. L., M e y e r, J. S.: Dextran 40 used in treatment of acute stroke. Mod. Med. 38:156, H a e r e m, J. W.: Platelet aggregates in intramyocardial vessels of patients dying suddenly and unexpectedly of coronary artery disease. Atherosclerosis 15: , H a f t, J. T. and F a n i, K.: Intravascular platelet aggregation in the heart induced by stress. Circulation 47: , H e n r y, R. J.: Clinical Chemistry, Principles and Technics. Hoeber, New York, pp , H i r s h, J.: The relevance of tests of platelet function for monitoring the antithrombotic effects of drugs. Canad. Med. Assoc. J. 108: , H o r n e, M. M.: Investigation of the significance and clinical usefulness of the ethanol gelation test. Thesis under Dr. C. V. Hussey, The Medical College of Wisconsin, Milwaukee, W I, MUSTARD, J. F.: The fete of thrombi. Thrombosis. National Academy of Science, Washington, pp , M u s t a r d, J. F. and P a c k h a m, M. A.: Factors influencing platelet function: Adhesion, release, and aggregation. Pharmacol. Rev. 22:97-187, NILSSON, I. M. and ISACSON, S.: New aspects of the pathogenesis of thrombo-embolism. Prog. Surg. 11:46-48, P a c k h a m, M. A., G u c c i o n e, M. A., C h a n g, P. L., a n d M u s t a r d, J. F.: P latelet aggregation and

6 ADP-INDUCED PLATELET AGGREGATION IN HYPERCOAGULABILITY 157 release: effects of low concentrations of thrombin or collagen. Amer. J. Physiol. 225:38-47, Sa n o, T., B o x e r, G. J., B o x e r, L. A., and Y o k o y a m a, M.: Platelet sensitivity to aggregation in normal and disease groups. A method for assessment of platelet aggregability. Thromb. Diath. Haemorrh. 25: , S h a rm a, H. M., M o o r e, S., M e r r ic k, H. W., and S m ith, M. B.: Platelets in early hyperacute allograft rejection in kidneys and their m odificatio n b y s u lfin p y r a z o n e (A n tu ra n ) th e ra p y. A m er. J. Path. 66: , W a n g, T. Y., H u s s e y, C. V., and G a r a n c is, J. C.: Effect of DBcAMP and PGE on platelet aggregation and shape changes. Amer. J. Clin. Path, (in press). 17. Y a m a z a h, H., O d a k u r a, T., Ta k e u c h i, K., and SANO, T.: Adhesive platelet count and blood coagulability in myocardial infarction and cerebral thrombosis. Thromb. Diath. Haemorrh. 24: , Z b in d e n, G., G r im m, L., and M u h e i m, M.: Aggregation of platelets remaining in circulation after acute thrombosis. Thromb. D iath. Haemorrh. 25: , 1971.