Acta Clinica Belgica International Journal of Clinical and Laboratory Medicine
ISSN: 1784-3286 (Print) 2295-3337 (Online) Journal homepage: http://www.tandfonline.com/loi/yacb20
Enhanced Platelet Aggregation As A Risk Factor For Vascular Disease And Its Thromboembolic Complications K. Breddin, H.J. Krzywanek & M. Ziemen To cite this article: K. Breddin, H.J. Krzywanek & M. Ziemen (1975) Enhanced Platelet Aggregation As A Risk Factor For Vascular Disease And Its Thromboembolic Complications, Acta Clinica Belgica, 30:3, 195-203, DOI: 10.1080/17843286.1975.11716997 To link to this article: https://doi.org/10.1080/17843286.1975.11716997
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195
ENHANCED PLATELET AGGREGATION AS A RISK FACTOR FOR VASCULAR DISEASE AND ITS THROMBO EMBOLIC COMPLI CATIO NS
K. BR EDDI N, H.J. KRZYWANE"- AN D M . ZIEMEN*
Large scale epidemiologic studies have established the role of several risk factors contribu ting to the development of atherosclerosis and its complications, e.g. high blood press ure, di abetes, cigarette smoking, high levels of cholesterol , triglycerides and oth er lipid fractions. Oth er factors like obesity, hyperuricemia, hypercoagulability, carbohydrate consumption and many more are still under discussion . A number of tests measuring platelet behaviour have been tried to establish enhanced platelet adhesiveness or aggregation as a risk factor for atherosclerosis or its complications with co nfli cting res ults. I. PLATELET RETENTION TESTS
In these tests platelets are counted before and after the passage of native blood, citrate blood or platelet rich plas ma through glass bead fil te rs or after defined contact with other surfaces. The difference of the counts is usuall y ex pressed as percent platelets retained. The different tests vary widely as fa·r as pretreatment of th e blood or preparation of the filters are concerned. The first investigators thought that th e results of the· tests were primaril y influenced by platelet adh esion . But since it is known th at platelet aggrega tion and the entrapment of aggregates in the filters have an even greater im portance the term '' retention tests" is being preferred. H.P. Wright (1942) was th e first to report enhanced "adhesiveness" in patients with venous * From the Center of Intern al Medici ne, Departmen t of Angiology. University of Frankfurt a. M.
th rombos is. Similar resu lts were published by Moolten (1949), Mc Donald and Edgi ll (1957), POeiderer (1964) and Easth am (1966) in pat ients with occlusive vasc ul ar disease, by Horlick (1 96 1) in ath erosclerotic subjects, by Slack et al. (1 964) and O'Brien (1966) in patie nts wi th recent myoca rd ial in fa rct ion. No difference between patients and controls was seen by Kirby ( 1966) in patients wi th vascu lar lesions, by Fitzgerald (1 970) and Steele (1 973) in patients with recent myoca rdi al in farctio n and by Heath (1971 ) in diabetics. Badaw i et al. (1 970), Sjogren (1970) and Hellem (1 97 1) descri bed a sign ificantl y enhanced platelet retention in diabetics. A major drawback of all retention tests is th e enormous variance of test results which makes th em unsuitable for clinical use. 2. ELECT ROPHORETIC MOB ILITY OF PLATELETS
Hampton et al. (1966) noticed a hig h sensitivity of th e platelets of patients with coronary heart disease and ath erosclerotic vascul ar occlusions to small doses of ADP and epinephrine. These substances induced an increased electrophoretic mobility of platelets at low co ncent rations. The authors ass umed th at a plasma fac tor is responsible for th e abnormal behaviour of the patients platelets. 3. PLATELET TU RNOVE R AND LI FE SPAN
Platelet turnover was found increased in patients with atherosclerosis by Mustard (1 96 1). Harker et Acta Clinica Be/gica, 30, 3 (19 75)
196 al. (1971) described a shortened platelet survival time in patients with acute venous thrombosis and an increased fibrinogen turnover. Steele et al. (1973) got similar results in patients with coronary heart disease and recurring venous thrombosis. 4. ADP-, COLLAGEN OR EPINEPHRINE-INDUCED AGGREGATION AND VASCULAR DISEASE
In these widely used tests the following parameters have been examined as possible signs of vascular disease or thrombotic tendency : I. Enhanced aggregation after addition of constant amounts of the inducing agent as compared with control PRP. 2. Aggregation induced by very low concentrations of the inducing agent which are ineffective in control PRP. 3. Reduced desaggregation (e.g. with ADP as inducing agent). 4. A shortened time interval between addition of collagen and onset of aggregation . 5. The appearance of a second wave of aggregation after addition of low amounts of ADP or epinephrine to PRP. Using ADP as inducing agent Zahavi et al. (1 969), Hassanein et al. and Passa et al. (1974) found a reduced desaggregation in PRP of diabeti~ patients and Kwaan et al. (1972) reported on the increased aggregation in a similar group of patients, McKee et al. (1969) in patients with coronary heart disease, and Carvalho in patients with a type II hyperlipoproteinemia using ADP, collagen and epinephrine. Steel (1973) was not able to detect any difference between patients with recent myocardial infarction and controls. 5. MEASUREMENT OF "SPONTANEOUS" AGGREGATION :
We developed a simple method to investigate "spontaneous" platelet aggregation which we called the platelet aggregation test (PAT I, Breddin, 1965). Platelet rich plasma is rotated at 37°C for IOmin. in asiliconized glass flask . Plastic slides are covered with the diluted plasma for 30 min. Adhering platelets and platelet aggregates are fixed , stained and studied under a microscope. Acta Clinlca Belgica, 30, 3 (1975)
PLATELET AGGREGATION AS A RISK FACTOR
In this test we distinguish between reversible (grade 3), irreversible (grades 4 and 5) and missing aggregation (grades I and 2). With this test many thousands of patients were investigated . In a group of clinically healthy subjects an increasing number of individuals with enhanced aggregation (PAT grades 3-5) was found with rising age. In the 50-59 years age group 113 of the volunteers showed a markedly enhanced platelet aggregation with PAT grades 45 (fig. 1). Two groups of diabetics were studied : 388 patients with diabetes of unknown duration (1963-1966) and 472 patients in whom diabetes was known for at least 5 years (1966-1969). Incidence of Enhanced Platelet Aggregation in
"HEALTHY SUBJECTS"
Fig. 1 :-- PAT I results in 545 clinically healthy volunteers. With rising age the percentage ofresults indicafing enhanced aggregation rises. About 30 % of persons in the 50-60 years age group had maximal aggregation (PAT-grades 415).
Throughout the various age groups the incidence of PAT grades 4 and 5 was found to be I 0-15 % higher in diabetics of more than 5 years duration as compared with the unselected group. In all age groups the number of patients with enhanced aggregation was significantly higher than in the respective controls (fig. 2). Similar results were obtained from patients with ecent myocardial infarction or peripheral arterial occlusions. In 159 patients PAT has been done within 6 months prior to a thromboembolic episode. In most cases the test had been performed less
PLATELET AGGREGATION AS A RISK FACTOR Incidence of Enhanced Platelet Aggregation in DIABETES MELLITUS of unknown Duration 1A J and of > 5 Yea rs· Dural ion 1B J
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197 In this gro up 4! patients aged 50 to 69 yea rs were studied before th ey suffered myoca rdi al infarction. Only one of them had PAT grade 2. Grades 4 or 5 were found in 52 % and 74 % respecti vely (fi g. 4). Similar res ults were obtained in 24 patients prior to arterial or venous thrombosis (Jager et al. 1973). Incidence of Enhanced Platelet Aggregati on Q!lQr to
MYOCARDIAL INFARCTION and VENOUS or ARTERIAL THROMBOSIS
Fig. 2 - PAT I results in two groups of diabetic patients. In group A the onset of diabetes was not known. In group B only patients with a hist01y of diabetes of more than 5 years were in vestigated. In all age groups enhanced aggregation is more frequent than in the control group (see .fig. 1). In patients ofgroup B enhanced aggregation tendency 1's.found I 0-14 % more .frequently than in group A .
than 6 weeks before the thromboembolic event. The incid.ence of PAT grade 2 was strikingly low in all age groups. PAT grades 4 and 5 were found in 52-78 % of th ese patients (fig. 3).
lnctdence of Enhanced Platelet Aggregation in Persons prior to THROMBOEMBOLIC DISEASE
Fig. 3 - PAT I results of 148 patients, who were investigated up 6 months prior to a thromboem bolic attack. Vety rarely a missing aggregation (PAT grade 2) was r gistered. In 55-80 % max imal aggregation (PAT-grades 4 or 5) was found.
Fig. 4 - PAT I results of patients who were investigated prior to myocardial infa rction, venous or arterial Thrombosis. In one patient only PAT grade 2 was fou nd.
The PAT I shows th e extent of aggregation aft er 10 min . rotation of PRP in a glass flask. The velocity of the aggregation reaction and the time interval between onset of rotation and start of agg regation are not recorded. Therefore we developed a photomet ric test which we modified recentl y and which in its present form we named platelet aggregation test III (PAT III). In this test PRP rotates in a disk-shaped cuvette in the light beam of a photometer to which a recorder has been att ached. The ph ysical co nditions which enhance aggregation in this test and details of the procedure are reported elsewhere (Breddin et al. 1975). The aggregometer is commerci ally ava ilable from Braun-Melsungen as additional eq uipment to the Eppendorf-Photometer. Aggregation curves are evaluated using the followin g principles (fig. 5). Primary aggregation is recorded as angle a~> maximal aggregation as Acta C/inica Belgica, 30, 3 (1975)
198
PLATELET AGGREGATION AS A RISK FACTOR
errE"IO O IH
.) E
1,000 (
Original PAT 111-CurVPS
Int erpr etation of PAT - Ill - curves
Fig. 5 - Evaluation ofPAT Ill curves: 1. .angle a 1 between the tangent on the flat part of the curvefrom start o.f rotation to the steepest descent o.f the aggregation curve - and the horizontal line; 2. angle a1 (maximum aggregation speed) between the tangent on the steepest part ofthe curve and the horizontal line; 3. Tr - time in minutes from the start of rotation to the onset of maximum aggregation (intersection of a 1 and a~; 4. Am (maximum amplitude) Extinction difference between starting point and maximal deviation.
angle a 2, time between onset of rotation and start of maximal aggregation as Tr and the maximal amplitude (Am) between the extinction at the start of rotation and the minimal extinction reached. a2 and Tr are the most important parameters in clinical investigations. Figure 6 shows some examples of aggregation curves. As with PAT I studies of normal individuals showed an increase of enhanced aggregation with rising age and again in diabetics a high inActa Clinica Be/gica, 30, 3 (1975)
Fig. 6 - Examples o,[PAT Ill - curves: 1 no aggregation, 2 curve shape falsified by lipemia. 3 enhan ced aggregation with prolonged TR -time, 4 and 5 strongly enhanced aggregation.
cidence of maximal enhanced aggregation was found (fig. 7). · The photometric aggregation test allows a more subtle differentiation of the aggregating tendency in PRP than PAT I. Our preliminary experience makes it likely that the photometric aggregation test is a useful method to detect a thrombotic tendency. The aggregating tendency may have different causes like at herosclerotic vascular lesions, inflammatory reactions or autoaggressive mechanism . Activated plas ma proteins seem to be mainly responsible for the enhanced aggregating tendency.
PLATELET AGGREGATION ASA RISK FACTOR
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Fig. 7- PAT Ill results in clinically healthy volunteers and diabetic patients of the same age groups. The frequency of angle a2 and if this was missing of angle a 1 is recorded in percent. Low alpha values are mainly seen in healthy volunteers. In diabetics a peak of results with high a2 values is found. TIM E DEPENDENT CHANGES OF AGG REGATION TESTS
If the PAT III is perform ed a few minutes after blood sampling using PRP of a patient with vascular disease no aggregation occurs, while 45 or 60 minutes later an·agg regation curve ca n be registered. From the first hour after venepuncture until 4 hours later the max imal velocity of aggregation and the other parameters of the agg regation curves remain fairly constant. Later the agg regation tendency diminishes. The time dependent changes of angle a 2 in a series of multiple investigations are demonstrated in fig. 8. What changes in PRP may be responsible for the time dependent increase in aggregability? Native platelets fixed directly at venepuncture for instance by feeding blood into prewarmed, buffered (pH 7,3) 6% glutaraldehyde appear as flat discs under the microscope or the stereoscan microscope. During the first minutes after blood sampling platelets form protrusions and swell . If th e protrusions are counted in PRP after fi xation at different times aft er venepunc-
ture 25 % are found at ve nepuncture, 50% 10 min . later and 75 % at 30 minutes. Incubation ex periments with PRP showed th at lower temperatures accelerate th e formation of pseudopodes and swelling, whereas at 37°C these changes occurred more slowly (fig. 9). Aggregation is also enhanced in PAT III and in induced aggregation at low temperatures, whereas it is slowed down at 37°C. A continuous increase in the sensitivity of PRP to ADP, collagen or epinephrine is reco rded shortl y after blood sampling and during the following hours. It seems likely th at the rising number of" irritated " platelets is responsible for the changing response of PRP. If PRP of young healthy indi\liduals is tested simultaneo usly using PAT lfi and collageninduced aggregation (I ,ug/ml PRP) at different times after venepuncture and if the same procedure is performed with PRP of a patient with vascular disease the two methods show good correlation over a certain period of time. If aggregation occurs in PAT III, th e time between addition of collagen and onset of aggregation is shorter th an in " non-aggregating" plasma. Acla C/inica Be/Rica. 30. 3 (1975)
200
PLATELET AGGREGATION ASA RISK FACTOR
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Changes of Angle a2 in Relation to the time tag between Blood Drawing and f.otation at 20 rpm. !Changes in com pari son with the preceding value I
Fig. 8- Changes of angle a2 compared with the respective preceding value. In rhe.firsr 60 minures.following blood sampling a2 increases rapidly. Between 60 minutes and 90 min. a2 increases slightly: if remains.fairly constant .for 3-4 hours thereaft er.
60 min . after blood sampling the correlation between the two tests is less pronounced, at 180 min . it has disappeared . In principle very similar correlations can be obtained with PAT III and ADP · or epinephrine induced aggregation if low doses of the aggregating agents are used. These investigations lead to the following conclusions: I. The results of different aggregation tests may show good correlation if the effects of time dependent changes in the PRP on the tests are being taken into consideration. 2. Conflicting results obtained with the same method in similar patient groups can easily be explained if the tests were performed at different times after blood sampling. 3. The photometric PAT III seems especially suited for clinical investigations on platelet aggregation since constant results can be obtained over a period of more than three hours, while this time is limited to 3060 min. in ADP, collagen or epinephrine induced aggregation. 4. The enhanced aggregability found with the different aggregation tests is probably caused by the same changes in PRP. Time deActa Clinica Be/gica, 30, 3 (1975)
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Pr imary sh ape change ol pl at elets wi t h time alter blood s amp ling Citra t e blood kept at dlltere n t te mpe ra tures
Fig. 9 - Changes of prima1y "shape change " qf platelets with time after blood sampling. Citrate blood vas incubated af d([ferenf temperatures. A f 4°C all platelets are swollen and show tentacles after 10 minutes. At J7 oC about 60 % of the platelets show !his shape change 60 minules after venepuncture.
PLATEL ET AGGREGA TION ASA RISK FA CTOR
201
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Co tla gen · lnduced A~gregallon Change of TRwi l h li me after bloo d sampli ng
Fig. 10 - CollaRen-induced AJ;.f!,reRation: Tim e between addition of collagen and onset of aggregation- Tr is registered in PRP aR.gregatingspontaneously in PAT !11 (lowercurve)andq[PRP not aggregating(uppercurve). Tr is sign(/icantly shorter in PRP spontaneously aggregating. pendent changes after bl ood sampling are predispos ing fac tors for the varying resu lts with tim e. Aggregatin g plasma proteins seem to be respon sible in patients with vascular les ions for the enhanced aggrega ti on. K E Y WORDS Pl atelet aggregation - Vascular disease- Th ro mbosis- PAT - Aggregometer - A therosclerosis.
th e underl ying disease. A very high incidence of enhanced pl atelet aggregation and on ly rare cases with normal (= miss ing) aggregation were observed in patients prior to th romboembolic disease li ke myocardi al infarction and arteri al or venous thrombosis. Studi es with a recently developed aggregometer revealed the im port ance of time dependant changes of pl atelet morphology (pri mary shape changes) upon the shape of aggregati on curves in spontaneous aggrega tion (PAT III) and A DP-, collagen- or epin ephrine- induced aggregati on. If the vari ous aggregati on tests are bei ng performed at the right time after blood sampling, th ey wi ll yield very si milar results.
SUMMARY A number of pl atelet fun ction tests (retention tes ts, elec trophoreti c pl atelet mobilit y, platelet turnover, induced and spontaneous aggregat ion) have been used to confi rm th e role of enhanced thrombocy te adhesive ness or aggregation as risk factors for progressive ath erosclerosis and its co mpli cations, with co nnicting res ults . "Spontaneous" platelet aggrega ti on using PAT I has been studied in a great number of healthy persons and in patients with a wide vari ety of internal diseases, especially vasc ular and thrombo-embolic disease over th e past yea rs. Th e incidence of enh anced pl atelet aggregation in healthy subjects is increasing with age. In diabetes mellitus the incide nce of enh anced pl atelet aggregati on was greater th an in age matched controls, and there was ano ther 10% increase in diabetes of more th an fi ve years' duration. Thi s findin g is in good correlation with th e progression of diabetic angiopath y with the durati on of
SAM ENV A TTING Een aantal thrombocy tenfunctietesten (retentietes ten, electroforeti sche bloedplaatjesmobiliteit , pl aatjesoverl eving, ge"ind ucee rde en spontane aggregatie) werden - met tegenstrijd ige resultaten - aangewend om de rol va n verhoogde thrombocy tenadhesivitei t of aggregatie als risicofactor voor progrcss ieve atherosclerose en zij n verwikkelingen, te bevest igen. I n de loop van voorgaande j aren werd de ,spontane" bloedplaatsjesaggregatie, gebruik makend van de PAT I, bes tudeerd bij een groot aantal gezonde personen en bij patienten met een brede waaier va n interne aandoeningen, inzonderh eid vascu laire en thrombo-embolische pathologic. De incidentie van verhoogde bl oedplaatjesaggregatie bij gezonde subjecten neemt toe met de leeftijd . Bij diabetes mellitus was de incidentie va n verhoogde bloedplaatjesag-
Acta C/inica Belgica. 30. 3 (19 75)
202 gregatie grater dan bij controles van vergelijkbare leeftijd, en er was nag een 10% toename wanneer de diabetes reeds meer dan vijf jaar duurde. Deze bevinding is in goede correlatie met de diabetische angiopathie voortschrijdende met de duur van de onderliggende ziekte. Een zeer hoge incidentie van toegenomen thrombocytenaggregatie en· slechts zeldzame gevallen met normale (= ontbrekende) aggregatie werden waargenomen bij patienten v66r een thrombo-embolische aandoening zoals myocardinfarct en arteriele of veneuse thrombose. Studies met een recent ontwikkelde aggregometer toonden het belang van tijd afhankelijke veranderingen in bloedplaatjesmorfologie (hoofdzakelijk vormveranderingen) op de vorm van de aggregatiecurven bij spontane aggregatie (PAT III) en ADP-, collageen- of epinefrine-ge'induceerde aggregatie. Wanneer de verschillende aggregatietesten uitgevoerd worden op het goede ogenblik na de bloedname, zullen zij zeer vergelijkbare resultaten opleveren . RESUME Plusieurs tests de Ia fonction plaquettaire (tests de retention, mobilite des plaquettes a !'electrophorese, enumeration des plaquettes, agregation induite ou spontanee) ant ete utilises pour preciser le role de !'adherence ou de l'agregation thrombocytai re augmentee comme facteurs provocant l'arteriosclerose progressive et ses complications; les resultats en sont contradictoires. Nous avons etudie l'agregation «spontanee» des plaquettes par action du PAT I sur un grand nombre de personnes bien portantes et sur des patients souffrant d'une grande variete de maladies internes et plus particulierement de maladies vasculaires et thromboemboliques. L'incidence de l'agregation plaquettaire augmentee croit avec I' age. Elle est plus elevee dans les diabetes «mellitus» que dans les controles du meme age, et de 10% plus elevee dans les diabetes durant depuis plus de 5 ans. Cette decouverte concorde bien avec Ia progression de l'angiopathie diabetique en fonction de Ia duree de Ia maladie sous-jacente. Une incidence tres elevee et quelques rares cas ·d'agregation normale (c.a.d. inexistante) ant ete observes sur des patients ayant souffert anterieurement de maladies thrombo-emboliques comme l'infarctus du myocarde et Ia thrombose arterielle ou veineuse. Les etudes effec'tuees avec Ia technique recente de l'agregometre revelent !'importance de !'influence des changements de Ia morphologie des piaqueues dus au temps (principaux changements de forme) sur Ia forme des courbes d'agregation dans l'agregation spontanee (PAT Ill) et dans l'agn!gation induite par l'ADP, le collagene ou !'epinephrine. Si les differents tests d'agregation avaient ete effectues au moment correct apres Ia prise de sang, ils auraient donnes des resultats tres semblables. REFERENCES BADAWI, H., EL SAWY, M., MIKHAIL, M., NOMEIR, A.M., TEWFIK, S.- Platelets, Coagulation and Fibrinolysis in Diabetic and Non-diabetic Patients with
Acta C/inica Belgica. 30. 3 (1975)
PLATELET AGGREGATION AS A RISK FACTOR
Quiescent Coronary Heart Disease. Angiology, 21. No.8, 511, 1970. BREDDIN, K. - Ober die gesteigerte Thrombozytenagglu tination bei Gefal3krankheiten. Schweiz. med. Wschr., 95, Nr. 20, 655, 1965. BREDDIN, K. et al. - Zur Messung der ,spontanen" Thrombozytenagg regation Pliittchenaggregationstest III. Methodik f!lin . Wschr .. 1975, 1m Druck. CARVALHO , A.C.A ., COLMAN, R.W., LEES, R.S.- Platelet Function in Hyperlipoproteinemia. New Engl., J. Med .. 290, 1974. EASTHAM, R.D.- Adhesive platelets and myocardial infarction. Geriatrics, 21, N. 7, 182, 1966. FITZGERALD, D.E., BUTIERFIELD, W.J.H ., SMINK, D., KRUISH EER, H.E.J . - Platelet Adhesiveness: Post-Myocardical infarction Patients compared with controls- Atherosclerosis, Elsevier Pub/. Amsterdam, 13, 217,1971. HAMPTON , J.R. , MITCHELL, J.R.A. - A Transferable Factor Causing Abnormal Platelet Behaviour in Vascu , lar Disease. Lancet, Oct. 8, 764, 1966. HAMPTON , J.R., MITCHELL, J.R.A.- Abnormalities in Platelet Behaviour in Acute Illness. Brit. med. J., / , 1078, 1966. HARKER, L.A. , SLICHTER, S.J. - Platelet-Fibrinogen Kinetic Studies in Arterial and Venous Thrombosis . Circulation , 44, No. 4, Suppl. 2, 11-68, 1971. HASSANEIN, A.A., EL-GARF, Th . A., EL-BAZ, Z. - Platelet Aggregation in Diabetes Mellitus and the Effect of Insulin in Vivo on Aggregation . Thrombos. Diathes. _. Haemorrh. (Stullgt.), 27, 114, 1972. HEATH , H., BRIDGEN, W.O., CANEVER, J.V., POLLOCK, J., HUNTER, P.R. , KELSEY , J. , BLOOM, A. - Platelet Adhesiveness in Relation to Diabetic Retinopath y. Diabetology, 7, 308, 1971 . HELLEM , J.- Adenosine diphosphate induced Platelet Adhesiveness in Diabetes Mellitus with Complications. A eta med. Scand.. /90, 291 , 1971. HORLICK, L. - Platelet Adhesiveness in Normal Persons and Subjects with Atherosclerosis. A mer. J. of Card., 3, 459, 1961. JAGER, W., PIETSCH, U., KRZYWANEK, H.J ., BREDDIN, K.- Die gesteigerte Plii.ttchenaggregation bei Diabetikern und ihre Bedeutung als Risikofaktor fUr Gefal3erkrankungen. 79. Kongrel3 flir lnnere Medizin, Verhandlungen, Bergmann Veri. Milnchen, 1360, 1973. KIRBY , J.C.Jr. , MARTIN, C.L. - Platelet Adhesiveness and Vascular Disease. Suppl. III to Circulation III-17, 1966. KWAAN, H.C., COLWELL, J.A., CRUZ, S., SUWANWELA , N., DOBBIE, J.G.- Increased platelet aggregation in diabetes mellitus. J. Lab. Clin . Med., 80, No. 2, 236, 1972. McDONALD, L., EDGILL, M.- Coagulability of the Blood in lschaemic Heart Disease, Lancet, I. 457, 1957. McKEE, P.A., KANNEL, W.B. - Fibrinolytic Activity and Platelet Aggregation in Coronary Artery Disease Pa-
PLATELET AGGREGATION AS A RISK FACTOR
tients: the Framingham Study. C/in. R es., 17, No. I, 61, 1969. MOOLTEN, S.E., VROMAN, L. - The ad hesiveness of blood platelets in th romboembolism and hemorrhagic disorders, I measurement of platelet adhesiveness by the glasswool filter. Amer. J. clin . Path. , 19, 701 , 1949. MUSTARD, I. F. - Platelets thrombosis and vascular disease. Canad. med. Ass .. I, 85 , 621, 1961. O' BRIEN , J.R., HEYWOOD, J.B., HEA DY , J.A. - The Quantitatio n of Platelet Aggregation Induced by Four Compounds: a Study in Relation to Myocardial In farction. Thromb . Dialh . haemorrh., 16, 752, 1966. PASSA, P., BENSOSSAN, D., LEVY-TOLEDANO , S., CAEN, J., CANIVET, J. - Etude de !'aggregation plaquettaire au course de Ia n!tinopathie diabetique. Atherosclerosis, 19, 277, 1974. PFLEIDERER, Th. , RUCKER, G. - Ober Kaliumgehalt und Adhasivitiit der Thromboyzten von gesunden Menschen und Patienten mit obliterierender Gefaf3Sklerose. Kl. WscJrr., 42. 1223, 1964. SCHARRER, 1., BREDDIN , K. - Vergleichende Untersuchungen zur Erfassung einer gesteigerten Thrombozytenaggregation . Bericht tiber zwei Workshops in Frankfurt in : Verh . Dtsch . Ges. Inn . Med., 80, 1432, 1974.
203 SJOGREN A., BOTTIGER, L. -E., BJORK , G., WAHLBERG, F. , CARLSON, L.A . - Adenosine Diphos-. phate-lnduced Platelet Adhesiveness in Patients wi th Ischaemic Heart Disease. Acta med. sca nd.. Vol. 187, 89, 1970. SLACK, J., SEYMOUR, J., McDONALD , L. , LOVE, F.Lipoprotein-Lipase Levels and Platelet Stickiness in Patients with Ischaemic Heart-Disease and in Controls, Distinguishing those with an affected First-Degree Relative. Lancet. I033, 1964. STEELE , P.P ., HUGH, S., WElL Y, H., DAVIES, H., GEBTON, E. - Platelet Function Studies in Coronary Artery Disease. Circulation, 1194, 1973. STEELE, P.P., WEILY, S., GENTON, E. - Platelet Survival and Adhesiveness in Recurrent Venous Thrombosis . New Engl. J. M ed., 288, 1973. WRIGHT, H.P., SCHOLAR, G.- Changes in the ad hesiveness of blood platelets following parturition and surgical operations. J. Path . Bact .. 54. 1942. ZAHA VI , J., DREYFUSS, F. - An Abnormal Pattern of Adenosine Diphosphate-Induced Platelet Aggregation in Acute Myocardial Infarction . Thromb . Diath . Haemorrh. , 21, 1969.
Acta Clinica Belgica, 30, 3 (1975)
ISSN: 1784-3286 (Print) 2295-3337 (Online) Journal homepage: http://www.tandfonline.com/loi/yacb20
Enhanced Platelet Aggregation As A Risk Factor For Vascular Disease And Its Thromboembolic Complications K. Breddin, H.J. Krzywanek & M. Ziemen To cite this article: K. Breddin, H.J. Krzywanek & M. Ziemen (1975) Enhanced Platelet Aggregation As A Risk Factor For Vascular Disease And Its Thromboembolic Complications, Acta Clinica Belgica, 30:3, 195-203, DOI: 10.1080/17843286.1975.11716997 To link to this article: https://doi.org/10.1080/17843286.1975.11716997
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195
ENHANCED PLATELET AGGREGATION AS A RISK FACTOR FOR VASCULAR DISEASE AND ITS THROMBO EMBOLIC COMPLI CATIO NS
K. BR EDDI N, H.J. KRZYWANE"- AN D M . ZIEMEN*
Large scale epidemiologic studies have established the role of several risk factors contribu ting to the development of atherosclerosis and its complications, e.g. high blood press ure, di abetes, cigarette smoking, high levels of cholesterol , triglycerides and oth er lipid fractions. Oth er factors like obesity, hyperuricemia, hypercoagulability, carbohydrate consumption and many more are still under discussion . A number of tests measuring platelet behaviour have been tried to establish enhanced platelet adhesiveness or aggregation as a risk factor for atherosclerosis or its complications with co nfli cting res ults. I. PLATELET RETENTION TESTS
In these tests platelets are counted before and after the passage of native blood, citrate blood or platelet rich plas ma through glass bead fil te rs or after defined contact with other surfaces. The difference of the counts is usuall y ex pressed as percent platelets retained. The different tests vary widely as fa·r as pretreatment of th e blood or preparation of the filters are concerned. The first investigators thought that th e results of the· tests were primaril y influenced by platelet adh esion . But since it is known th at platelet aggrega tion and the entrapment of aggregates in the filters have an even greater im portance the term '' retention tests" is being preferred. H.P. Wright (1942) was th e first to report enhanced "adhesiveness" in patients with venous * From the Center of Intern al Medici ne, Departmen t of Angiology. University of Frankfurt a. M.
th rombos is. Similar resu lts were published by Moolten (1949), Mc Donald and Edgi ll (1957), POeiderer (1964) and Easth am (1966) in pat ients with occlusive vasc ul ar disease, by Horlick (1 96 1) in ath erosclerotic subjects, by Slack et al. (1 964) and O'Brien (1966) in patie nts wi th recent myoca rd ial in fa rct ion. No difference between patients and controls was seen by Kirby ( 1966) in patients wi th vascu lar lesions, by Fitzgerald (1 970) and Steele (1 973) in patients with recent myoca rdi al in farctio n and by Heath (1971 ) in diabetics. Badaw i et al. (1 970), Sjogren (1970) and Hellem (1 97 1) descri bed a sign ificantl y enhanced platelet retention in diabetics. A major drawback of all retention tests is th e enormous variance of test results which makes th em unsuitable for clinical use. 2. ELECT ROPHORETIC MOB ILITY OF PLATELETS
Hampton et al. (1966) noticed a hig h sensitivity of th e platelets of patients with coronary heart disease and ath erosclerotic vascul ar occlusions to small doses of ADP and epinephrine. These substances induced an increased electrophoretic mobility of platelets at low co ncent rations. The authors ass umed th at a plasma fac tor is responsible for th e abnormal behaviour of the patients platelets. 3. PLATELET TU RNOVE R AND LI FE SPAN
Platelet turnover was found increased in patients with atherosclerosis by Mustard (1 96 1). Harker et Acta Clinica Be/gica, 30, 3 (19 75)
196 al. (1971) described a shortened platelet survival time in patients with acute venous thrombosis and an increased fibrinogen turnover. Steele et al. (1973) got similar results in patients with coronary heart disease and recurring venous thrombosis. 4. ADP-, COLLAGEN OR EPINEPHRINE-INDUCED AGGREGATION AND VASCULAR DISEASE
In these widely used tests the following parameters have been examined as possible signs of vascular disease or thrombotic tendency : I. Enhanced aggregation after addition of constant amounts of the inducing agent as compared with control PRP. 2. Aggregation induced by very low concentrations of the inducing agent which are ineffective in control PRP. 3. Reduced desaggregation (e.g. with ADP as inducing agent). 4. A shortened time interval between addition of collagen and onset of aggregation . 5. The appearance of a second wave of aggregation after addition of low amounts of ADP or epinephrine to PRP. Using ADP as inducing agent Zahavi et al. (1 969), Hassanein et al. and Passa et al. (1974) found a reduced desaggregation in PRP of diabeti~ patients and Kwaan et al. (1972) reported on the increased aggregation in a similar group of patients, McKee et al. (1969) in patients with coronary heart disease, and Carvalho in patients with a type II hyperlipoproteinemia using ADP, collagen and epinephrine. Steel (1973) was not able to detect any difference between patients with recent myocardial infarction and controls. 5. MEASUREMENT OF "SPONTANEOUS" AGGREGATION :
We developed a simple method to investigate "spontaneous" platelet aggregation which we called the platelet aggregation test (PAT I, Breddin, 1965). Platelet rich plasma is rotated at 37°C for IOmin. in asiliconized glass flask . Plastic slides are covered with the diluted plasma for 30 min. Adhering platelets and platelet aggregates are fixed , stained and studied under a microscope. Acta Clinlca Belgica, 30, 3 (1975)
PLATELET AGGREGATION AS A RISK FACTOR
In this test we distinguish between reversible (grade 3), irreversible (grades 4 and 5) and missing aggregation (grades I and 2). With this test many thousands of patients were investigated . In a group of clinically healthy subjects an increasing number of individuals with enhanced aggregation (PAT grades 3-5) was found with rising age. In the 50-59 years age group 113 of the volunteers showed a markedly enhanced platelet aggregation with PAT grades 45 (fig. 1). Two groups of diabetics were studied : 388 patients with diabetes of unknown duration (1963-1966) and 472 patients in whom diabetes was known for at least 5 years (1966-1969). Incidence of Enhanced Platelet Aggregation in
"HEALTHY SUBJECTS"
Fig. 1 :-- PAT I results in 545 clinically healthy volunteers. With rising age the percentage ofresults indicafing enhanced aggregation rises. About 30 % of persons in the 50-60 years age group had maximal aggregation (PAT-grades 415).
Throughout the various age groups the incidence of PAT grades 4 and 5 was found to be I 0-15 % higher in diabetics of more than 5 years duration as compared with the unselected group. In all age groups the number of patients with enhanced aggregation was significantly higher than in the respective controls (fig. 2). Similar results were obtained from patients with ecent myocardial infarction or peripheral arterial occlusions. In 159 patients PAT has been done within 6 months prior to a thromboembolic episode. In most cases the test had been performed less
PLATELET AGGREGATION AS A RISK FACTOR Incidence of Enhanced Platelet Aggregation in DIABETES MELLITUS of unknown Duration 1A J and of > 5 Yea rs· Dural ion 1B J
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197 In this gro up 4! patients aged 50 to 69 yea rs were studied before th ey suffered myoca rdi al infarction. Only one of them had PAT grade 2. Grades 4 or 5 were found in 52 % and 74 % respecti vely (fi g. 4). Similar res ults were obtained in 24 patients prior to arterial or venous thrombosis (Jager et al. 1973). Incidence of Enhanced Platelet Aggregati on Q!lQr to
MYOCARDIAL INFARCTION and VENOUS or ARTERIAL THROMBOSIS
Fig. 2 - PAT I results in two groups of diabetic patients. In group A the onset of diabetes was not known. In group B only patients with a hist01y of diabetes of more than 5 years were in vestigated. In all age groups enhanced aggregation is more frequent than in the control group (see .fig. 1). In patients ofgroup B enhanced aggregation tendency 1's.found I 0-14 % more .frequently than in group A .
than 6 weeks before the thromboembolic event. The incid.ence of PAT grade 2 was strikingly low in all age groups. PAT grades 4 and 5 were found in 52-78 % of th ese patients (fig. 3).
lnctdence of Enhanced Platelet Aggregation in Persons prior to THROMBOEMBOLIC DISEASE
Fig. 3 - PAT I results of 148 patients, who were investigated up 6 months prior to a thromboem bolic attack. Vety rarely a missing aggregation (PAT grade 2) was r gistered. In 55-80 % max imal aggregation (PAT-grades 4 or 5) was found.
Fig. 4 - PAT I results of patients who were investigated prior to myocardial infa rction, venous or arterial Thrombosis. In one patient only PAT grade 2 was fou nd.
The PAT I shows th e extent of aggregation aft er 10 min . rotation of PRP in a glass flask. The velocity of the aggregation reaction and the time interval between onset of rotation and start of agg regation are not recorded. Therefore we developed a photomet ric test which we modified recentl y and which in its present form we named platelet aggregation test III (PAT III). In this test PRP rotates in a disk-shaped cuvette in the light beam of a photometer to which a recorder has been att ached. The ph ysical co nditions which enhance aggregation in this test and details of the procedure are reported elsewhere (Breddin et al. 1975). The aggregometer is commerci ally ava ilable from Braun-Melsungen as additional eq uipment to the Eppendorf-Photometer. Aggregation curves are evaluated using the followin g principles (fig. 5). Primary aggregation is recorded as angle a~> maximal aggregation as Acta C/inica Belgica, 30, 3 (1975)
198
PLATELET AGGREGATION AS A RISK FACTOR
errE"IO O IH
.) E
1,000 (
Original PAT 111-CurVPS
Int erpr etation of PAT - Ill - curves
Fig. 5 - Evaluation ofPAT Ill curves: 1. .angle a 1 between the tangent on the flat part of the curvefrom start o.f rotation to the steepest descent o.f the aggregation curve - and the horizontal line; 2. angle a1 (maximum aggregation speed) between the tangent on the steepest part ofthe curve and the horizontal line; 3. Tr - time in minutes from the start of rotation to the onset of maximum aggregation (intersection of a 1 and a~; 4. Am (maximum amplitude) Extinction difference between starting point and maximal deviation.
angle a 2, time between onset of rotation and start of maximal aggregation as Tr and the maximal amplitude (Am) between the extinction at the start of rotation and the minimal extinction reached. a2 and Tr are the most important parameters in clinical investigations. Figure 6 shows some examples of aggregation curves. As with PAT I studies of normal individuals showed an increase of enhanced aggregation with rising age and again in diabetics a high inActa Clinica Be/gica, 30, 3 (1975)
Fig. 6 - Examples o,[PAT Ill - curves: 1 no aggregation, 2 curve shape falsified by lipemia. 3 enhan ced aggregation with prolonged TR -time, 4 and 5 strongly enhanced aggregation.
cidence of maximal enhanced aggregation was found (fig. 7). · The photometric aggregation test allows a more subtle differentiation of the aggregating tendency in PRP than PAT I. Our preliminary experience makes it likely that the photometric aggregation test is a useful method to detect a thrombotic tendency. The aggregating tendency may have different causes like at herosclerotic vascular lesions, inflammatory reactions or autoaggressive mechanism . Activated plas ma proteins seem to be mainly responsible for the enhanced aggregating tendency.
PLATELET AGGREGATION ASA RISK FACTOR
199
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Fig. 7- PAT Ill results in clinically healthy volunteers and diabetic patients of the same age groups. The frequency of angle a2 and if this was missing of angle a 1 is recorded in percent. Low alpha values are mainly seen in healthy volunteers. In diabetics a peak of results with high a2 values is found. TIM E DEPENDENT CHANGES OF AGG REGATION TESTS
If the PAT III is perform ed a few minutes after blood sampling using PRP of a patient with vascular disease no aggregation occurs, while 45 or 60 minutes later an·agg regation curve ca n be registered. From the first hour after venepuncture until 4 hours later the max imal velocity of aggregation and the other parameters of the agg regation curves remain fairly constant. Later the agg regation tendency diminishes. The time dependent changes of angle a 2 in a series of multiple investigations are demonstrated in fig. 8. What changes in PRP may be responsible for the time dependent increase in aggregability? Native platelets fixed directly at venepuncture for instance by feeding blood into prewarmed, buffered (pH 7,3) 6% glutaraldehyde appear as flat discs under the microscope or the stereoscan microscope. During the first minutes after blood sampling platelets form protrusions and swell . If th e protrusions are counted in PRP after fi xation at different times aft er venepunc-
ture 25 % are found at ve nepuncture, 50% 10 min . later and 75 % at 30 minutes. Incubation ex periments with PRP showed th at lower temperatures accelerate th e formation of pseudopodes and swelling, whereas at 37°C these changes occurred more slowly (fig. 9). Aggregation is also enhanced in PAT III and in induced aggregation at low temperatures, whereas it is slowed down at 37°C. A continuous increase in the sensitivity of PRP to ADP, collagen or epinephrine is reco rded shortl y after blood sampling and during the following hours. It seems likely th at the rising number of" irritated " platelets is responsible for the changing response of PRP. If PRP of young healthy indi\liduals is tested simultaneo usly using PAT lfi and collageninduced aggregation (I ,ug/ml PRP) at different times after venepuncture and if the same procedure is performed with PRP of a patient with vascular disease the two methods show good correlation over a certain period of time. If aggregation occurs in PAT III, th e time between addition of collagen and onset of aggregation is shorter th an in " non-aggregating" plasma. Acla C/inica Be/Rica. 30. 3 (1975)
200
PLATELET AGGREGATION ASA RISK FACTOR
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Changes of Angle a2 in Relation to the time tag between Blood Drawing and f.otation at 20 rpm. !Changes in com pari son with the preceding value I
Fig. 8- Changes of angle a2 compared with the respective preceding value. In rhe.firsr 60 minures.following blood sampling a2 increases rapidly. Between 60 minutes and 90 min. a2 increases slightly: if remains.fairly constant .for 3-4 hours thereaft er.
60 min . after blood sampling the correlation between the two tests is less pronounced, at 180 min . it has disappeared . In principle very similar correlations can be obtained with PAT III and ADP · or epinephrine induced aggregation if low doses of the aggregating agents are used. These investigations lead to the following conclusions: I. The results of different aggregation tests may show good correlation if the effects of time dependent changes in the PRP on the tests are being taken into consideration. 2. Conflicting results obtained with the same method in similar patient groups can easily be explained if the tests were performed at different times after blood sampling. 3. The photometric PAT III seems especially suited for clinical investigations on platelet aggregation since constant results can be obtained over a period of more than three hours, while this time is limited to 3060 min. in ADP, collagen or epinephrine induced aggregation. 4. The enhanced aggregability found with the different aggregation tests is probably caused by the same changes in PRP. Time deActa Clinica Be/gica, 30, 3 (1975)
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Pr imary sh ape change ol pl at elets wi t h time alter blood s amp ling Citra t e blood kept at dlltere n t te mpe ra tures
Fig. 9 - Changes of prima1y "shape change " qf platelets with time after blood sampling. Citrate blood vas incubated af d([ferenf temperatures. A f 4°C all platelets are swollen and show tentacles after 10 minutes. At J7 oC about 60 % of the platelets show !his shape change 60 minules after venepuncture.
PLATEL ET AGGREGA TION ASA RISK FA CTOR
201
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Co tla gen · lnduced A~gregallon Change of TRwi l h li me after bloo d sampli ng
Fig. 10 - CollaRen-induced AJ;.f!,reRation: Tim e between addition of collagen and onset of aggregation- Tr is registered in PRP aR.gregatingspontaneously in PAT !11 (lowercurve)andq[PRP not aggregating(uppercurve). Tr is sign(/icantly shorter in PRP spontaneously aggregating. pendent changes after bl ood sampling are predispos ing fac tors for the varying resu lts with tim e. Aggregatin g plasma proteins seem to be respon sible in patients with vascular les ions for the enhanced aggrega ti on. K E Y WORDS Pl atelet aggregation - Vascular disease- Th ro mbosis- PAT - Aggregometer - A therosclerosis.
th e underl ying disease. A very high incidence of enhanced pl atelet aggregation and on ly rare cases with normal (= miss ing) aggregation were observed in patients prior to th romboembolic disease li ke myocardi al infarction and arteri al or venous thrombosis. Studi es with a recently developed aggregometer revealed the im port ance of time dependant changes of pl atelet morphology (pri mary shape changes) upon the shape of aggregati on curves in spontaneous aggrega tion (PAT III) and A DP-, collagen- or epin ephrine- induced aggregati on. If the vari ous aggregati on tests are bei ng performed at the right time after blood sampling, th ey wi ll yield very si milar results.
SUMMARY A number of pl atelet fun ction tests (retention tes ts, elec trophoreti c pl atelet mobilit y, platelet turnover, induced and spontaneous aggregat ion) have been used to confi rm th e role of enhanced thrombocy te adhesive ness or aggregation as risk factors for progressive ath erosclerosis and its co mpli cations, with co nnicting res ults . "Spontaneous" platelet aggrega ti on using PAT I has been studied in a great number of healthy persons and in patients with a wide vari ety of internal diseases, especially vasc ular and thrombo-embolic disease over th e past yea rs. Th e incidence of enh anced pl atelet aggregation in healthy subjects is increasing with age. In diabetes mellitus the incide nce of enh anced pl atelet aggregati on was greater th an in age matched controls, and there was ano ther 10% increase in diabetes of more th an fi ve years' duration. Thi s findin g is in good correlation with th e progression of diabetic angiopath y with the durati on of
SAM ENV A TTING Een aantal thrombocy tenfunctietesten (retentietes ten, electroforeti sche bloedplaatjesmobiliteit , pl aatjesoverl eving, ge"ind ucee rde en spontane aggregatie) werden - met tegenstrijd ige resultaten - aangewend om de rol va n verhoogde thrombocy tenadhesivitei t of aggregatie als risicofactor voor progrcss ieve atherosclerose en zij n verwikkelingen, te bevest igen. I n de loop van voorgaande j aren werd de ,spontane" bloedplaatsjesaggregatie, gebruik makend van de PAT I, bes tudeerd bij een groot aantal gezonde personen en bij patienten met een brede waaier va n interne aandoeningen, inzonderh eid vascu laire en thrombo-embolische pathologic. De incidentie van verhoogde bl oedplaatjesaggregatie bij gezonde subjecten neemt toe met de leeftijd . Bij diabetes mellitus was de incidentie va n verhoogde bloedplaatjesag-
Acta C/inica Belgica. 30. 3 (19 75)
202 gregatie grater dan bij controles van vergelijkbare leeftijd, en er was nag een 10% toename wanneer de diabetes reeds meer dan vijf jaar duurde. Deze bevinding is in goede correlatie met de diabetische angiopathie voortschrijdende met de duur van de onderliggende ziekte. Een zeer hoge incidentie van toegenomen thrombocytenaggregatie en· slechts zeldzame gevallen met normale (= ontbrekende) aggregatie werden waargenomen bij patienten v66r een thrombo-embolische aandoening zoals myocardinfarct en arteriele of veneuse thrombose. Studies met een recent ontwikkelde aggregometer toonden het belang van tijd afhankelijke veranderingen in bloedplaatjesmorfologie (hoofdzakelijk vormveranderingen) op de vorm van de aggregatiecurven bij spontane aggregatie (PAT III) en ADP-, collageen- of epinefrine-ge'induceerde aggregatie. Wanneer de verschillende aggregatietesten uitgevoerd worden op het goede ogenblik na de bloedname, zullen zij zeer vergelijkbare resultaten opleveren . RESUME Plusieurs tests de Ia fonction plaquettaire (tests de retention, mobilite des plaquettes a !'electrophorese, enumeration des plaquettes, agregation induite ou spontanee) ant ete utilises pour preciser le role de !'adherence ou de l'agregation thrombocytai re augmentee comme facteurs provocant l'arteriosclerose progressive et ses complications; les resultats en sont contradictoires. Nous avons etudie l'agregation «spontanee» des plaquettes par action du PAT I sur un grand nombre de personnes bien portantes et sur des patients souffrant d'une grande variete de maladies internes et plus particulierement de maladies vasculaires et thromboemboliques. L'incidence de l'agregation plaquettaire augmentee croit avec I' age. Elle est plus elevee dans les diabetes «mellitus» que dans les controles du meme age, et de 10% plus elevee dans les diabetes durant depuis plus de 5 ans. Cette decouverte concorde bien avec Ia progression de l'angiopathie diabetique en fonction de Ia duree de Ia maladie sous-jacente. Une incidence tres elevee et quelques rares cas ·d'agregation normale (c.a.d. inexistante) ant ete observes sur des patients ayant souffert anterieurement de maladies thrombo-emboliques comme l'infarctus du myocarde et Ia thrombose arterielle ou veineuse. Les etudes effec'tuees avec Ia technique recente de l'agregometre revelent !'importance de !'influence des changements de Ia morphologie des piaqueues dus au temps (principaux changements de forme) sur Ia forme des courbes d'agregation dans l'agregation spontanee (PAT Ill) et dans l'agn!gation induite par l'ADP, le collagene ou !'epinephrine. Si les differents tests d'agregation avaient ete effectues au moment correct apres Ia prise de sang, ils auraient donnes des resultats tres semblables. REFERENCES BADAWI, H., EL SAWY, M., MIKHAIL, M., NOMEIR, A.M., TEWFIK, S.- Platelets, Coagulation and Fibrinolysis in Diabetic and Non-diabetic Patients with
Acta C/inica Belgica. 30. 3 (1975)
PLATELET AGGREGATION AS A RISK FACTOR
Quiescent Coronary Heart Disease. Angiology, 21. No.8, 511, 1970. BREDDIN, K. - Ober die gesteigerte Thrombozytenagglu tination bei Gefal3krankheiten. Schweiz. med. Wschr., 95, Nr. 20, 655, 1965. BREDDIN, K. et al. - Zur Messung der ,spontanen" Thrombozytenagg regation Pliittchenaggregationstest III. Methodik f!lin . Wschr .. 1975, 1m Druck. CARVALHO , A.C.A ., COLMAN, R.W., LEES, R.S.- Platelet Function in Hyperlipoproteinemia. New Engl., J. Med .. 290, 1974. EASTHAM, R.D.- Adhesive platelets and myocardial infarction. Geriatrics, 21, N. 7, 182, 1966. FITZGERALD, D.E., BUTIERFIELD, W.J.H ., SMINK, D., KRUISH EER, H.E.J . - Platelet Adhesiveness: Post-Myocardical infarction Patients compared with controls- Atherosclerosis, Elsevier Pub/. Amsterdam, 13, 217,1971. HAMPTON , J.R. , MITCHELL, J.R.A. - A Transferable Factor Causing Abnormal Platelet Behaviour in Vascu , lar Disease. Lancet, Oct. 8, 764, 1966. HAMPTON , J.R., MITCHELL, J.R.A.- Abnormalities in Platelet Behaviour in Acute Illness. Brit. med. J., / , 1078, 1966. HARKER, L.A. , SLICHTER, S.J. - Platelet-Fibrinogen Kinetic Studies in Arterial and Venous Thrombosis . Circulation , 44, No. 4, Suppl. 2, 11-68, 1971. HASSANEIN, A.A., EL-GARF, Th . A., EL-BAZ, Z. - Platelet Aggregation in Diabetes Mellitus and the Effect of Insulin in Vivo on Aggregation . Thrombos. Diathes. _. Haemorrh. (Stullgt.), 27, 114, 1972. HEATH , H., BRIDGEN, W.O., CANEVER, J.V., POLLOCK, J., HUNTER, P.R. , KELSEY , J. , BLOOM, A. - Platelet Adhesiveness in Relation to Diabetic Retinopath y. Diabetology, 7, 308, 1971 . HELLEM , J.- Adenosine diphosphate induced Platelet Adhesiveness in Diabetes Mellitus with Complications. A eta med. Scand.. /90, 291 , 1971. HORLICK, L. - Platelet Adhesiveness in Normal Persons and Subjects with Atherosclerosis. A mer. J. of Card., 3, 459, 1961. JAGER, W., PIETSCH, U., KRZYWANEK, H.J ., BREDDIN, K.- Die gesteigerte Plii.ttchenaggregation bei Diabetikern und ihre Bedeutung als Risikofaktor fUr Gefal3erkrankungen. 79. Kongrel3 flir lnnere Medizin, Verhandlungen, Bergmann Veri. Milnchen, 1360, 1973. KIRBY , J.C.Jr. , MARTIN, C.L. - Platelet Adhesiveness and Vascular Disease. Suppl. III to Circulation III-17, 1966. KWAAN, H.C., COLWELL, J.A., CRUZ, S., SUWANWELA , N., DOBBIE, J.G.- Increased platelet aggregation in diabetes mellitus. J. Lab. Clin . Med., 80, No. 2, 236, 1972. McDONALD, L., EDGILL, M.- Coagulability of the Blood in lschaemic Heart Disease, Lancet, I. 457, 1957. McKEE, P.A., KANNEL, W.B. - Fibrinolytic Activity and Platelet Aggregation in Coronary Artery Disease Pa-
PLATELET AGGREGATION AS A RISK FACTOR
tients: the Framingham Study. C/in. R es., 17, No. I, 61, 1969. MOOLTEN, S.E., VROMAN, L. - The ad hesiveness of blood platelets in th romboembolism and hemorrhagic disorders, I measurement of platelet adhesiveness by the glasswool filter. Amer. J. clin . Path. , 19, 701 , 1949. MUSTARD, I. F. - Platelets thrombosis and vascular disease. Canad. med. Ass .. I, 85 , 621, 1961. O' BRIEN , J.R., HEYWOOD, J.B., HEA DY , J.A. - The Quantitatio n of Platelet Aggregation Induced by Four Compounds: a Study in Relation to Myocardial In farction. Thromb . Dialh . haemorrh., 16, 752, 1966. PASSA, P., BENSOSSAN, D., LEVY-TOLEDANO , S., CAEN, J., CANIVET, J. - Etude de !'aggregation plaquettaire au course de Ia n!tinopathie diabetique. Atherosclerosis, 19, 277, 1974. PFLEIDERER, Th. , RUCKER, G. - Ober Kaliumgehalt und Adhasivitiit der Thromboyzten von gesunden Menschen und Patienten mit obliterierender Gefaf3Sklerose. Kl. WscJrr., 42. 1223, 1964. SCHARRER, 1., BREDDIN , K. - Vergleichende Untersuchungen zur Erfassung einer gesteigerten Thrombozytenaggregation . Bericht tiber zwei Workshops in Frankfurt in : Verh . Dtsch . Ges. Inn . Med., 80, 1432, 1974.
203 SJOGREN A., BOTTIGER, L. -E., BJORK , G., WAHLBERG, F. , CARLSON, L.A . - Adenosine Diphos-. phate-lnduced Platelet Adhesiveness in Patients wi th Ischaemic Heart Disease. Acta med. sca nd.. Vol. 187, 89, 1970. SLACK, J., SEYMOUR, J., McDONALD , L. , LOVE, F.Lipoprotein-Lipase Levels and Platelet Stickiness in Patients with Ischaemic Heart-Disease and in Controls, Distinguishing those with an affected First-Degree Relative. Lancet. I033, 1964. STEELE , P.P ., HUGH, S., WElL Y, H., DAVIES, H., GEBTON, E. - Platelet Function Studies in Coronary Artery Disease. Circulation, 1194, 1973. STEELE, P.P., WEILY, S., GENTON, E. - Platelet Survival and Adhesiveness in Recurrent Venous Thrombosis . New Engl. J. M ed., 288, 1973. WRIGHT, H.P., SCHOLAR, G.- Changes in the ad hesiveness of blood platelets following parturition and surgical operations. J. Path . Bact .. 54. 1942. ZAHA VI , J., DREYFUSS, F. - An Abnormal Pattern of Adenosine Diphosphate-Induced Platelet Aggregation in Acute Myocardial Infarction . Thromb . Diath . Haemorrh. , 21, 1969.
Acta Clinica Belgica, 30, 3 (1975)
