American Journal of Hematology 19:21-25 (1985)

Platelet Activity in Mitral Valve Prolapse: A Study of Platelet Aggregation, Malondialdehyde Production, and Plasma P-Thromboglobulin Francisco Arocha, Maria Diez-Ewald, Ana I . Durango, and Tulio Sulbaran

Cardiology Service, Hospital Universitario (F.A., A. D., IS.) and lnstituto de lnvestigaciones Clinicas, Universidad del Zulia (M. D. -€.), Maracaibo, Venezuela

Platelet aggregation, malondialdehyde (MDA) production, and recovery after aspirin (ASA) administration and plasma levels of beta-thromboglobulin (BTG) were deter- mined in 40 asymptomatic patients with mitral valve prolapse (MVP) and 17 normal subjects. Platelet aggregation was similar in patients and controls, although two patients presented spontaneous aggregation. Production of MDA and plasma levels of BTG were higher in MVP than in controls; however, recovery after ASA was similar in both groups. The results further indicate that platelet hyperactivity is present in a significant number of asymptomatic patients with MVP.

Key words: mitral valve prolapse, malondialdehyde, /34hromboglobuh

INTRODUCTION

The association of thromboembolic manifestations with the existence of mitral valve prolapse (MVP) has been documented by several authors [l-31. The possible role of platelet activation in the pathogenesis of this complication has been investi- gated, and increased platelet coagulant activity [4] and elevated values of platelet factor 4 (pf4) in plasma [5] have been found in patients with the diagnosis of MVP with no evidence of ischemia. In this study we further evaluate platelet function in a group of MVP patients with no history of ischemic events in order to obtain more information that could eventually help to detect those patients at risk of developing thromboembolic episodes.

MATERIALS AND METHODS

Forty patients, 32 females and eight males, ages 4 to 38 years (mean 22 +_ lo), with echocardiographic confirmation of MVP were studied. The majority of the patients were high school or college students, with no previous symptomatology, who needed cardiac evaluation in order to enroll in sport activities, but whose physical examination indicated the presence of a cardiac systolic murmur. Other patients were seen in the out-patient cardiac clinic due to such manifestations as chest pains, palpitations, or dyspnea.

The control group consisted of 17 healthy persons, 12 females and five males between 19 and 30 years of age (mean 24.7 k 2.8 years), all of them with negative

Received for publication April 9, 1984; accepted September 27, 1984.

Address reprint requests to Dr. Maria Diez-Ewald, Instituto de Investigaciones Clinicas, Apartado 1151, Maracaibo 4001-A, Venezuela.

0 1985 Alan R. Liss, Inc.

22 Arochaet al

echocardiographic results. Neither patients nor controls had histories of diseases such as diabetes mellitus, coronary artery disease, cerebrovascular disease, or cancer.

Patients and controls were instructed to abstain from any medication or alcohol for 10 days prior to the study. An informed consent was obtained from each of the subjects included in the study.

The following determinations were made in each case: platelet aggregation, malondialdehyde (MDA) production, platelet survival time, and 0-thromboglobulin (PTG) levels in plasma. Blood collection was performed after overnight fasting, using a double plastic syringe technique, and the samples were handled according to the specific procedure for each determination.

Platelet counts in whole blood and platelet rich plasma (PRP) were performed in a Neubauer chamber with a phase-contrast microscope, following the technique of Brecher and Cronkite [6] .

Platelet aggregation was studied in citrated PRP using a platelet aggregometer (Chrono-Log Co., Havertown, PA) following a previously described methodology

Malondialdehyde production and platelet life-span were measured according to the method of Stuart et a1 [8], using 0.2 mM sodium arachidonate as an inducer. The authors relied upon the fact that aspirin inhibits the peroxidation of platelet lipids, normally induced by a variety of aggregating agents, with a gradual return to basal levels in a period of 10 days. Immediately after blood withdrawal, 3 mg/kg of body weight of ASA was administered to each subject, and production of MDA was subsequently studied at 2, 24, 72, 120, and 168 hr after ASA administration. Malondialdehyde values were plotted against time on linear graph paper and T 112 was calculated as the time necessary for recovery of 50% of the initial MDA values.

Plasma PTG was determined by means of an RIA Kit (Amersham International, HLC) .

Statistical analysis was performed by employing the Student’s t-test.

[71.

RESULTS

Platelet counts were between 2.5 X 105/pl and 3.5 X lo5/$ in all subjects. Platelet aggregation studies showed similar results in MVP patients and controls (Table l), although spontaneous platelet aggregation (SPA) was repeatedly found in

TABLE I. Platelet Aggregation in MVP Patients and Controls

Epinephrine 3 @Ma ADP 3 pM Collagen 6 ,ug/mla Platelets Maximal Initial Maximal Initial Maximal in PRP aggregation velocity aggregation velocity aggregation

X + _ S D X f S D X i S D X k S D X i S D (%/mi$, - (%I,

X i SD - (%I, ( - %/min), - (%I7 - (x - 1 0 ~ 4 4 ,

MVP

Control 40’ 348 f 88 85 f 12 46 + 17 80 12 95 + 10 83 i 11

17’ 327 f 94 87 f 9 45 + 16 77 & 8 95 * 9 85 f 16

aFinal concentration. ’Number of cases.

Platelet Activity in Mitral Valve Prolapse 23

: 12- Y A

W c Q

J L

0 0- \

1

U 0

c

two female patients-a 13-year-old girl, whose platelets aggregated spontaneously within 4 min of incubating the PRP in the aggregometer, and in an 18-year-old female, with SPA within 15 min of incubation of her PRP. Three aliquots of normal PRP (4.3 x lo5 platelets/pl) were adjusted to 2.5 x lo5 platelets/pl, one with homologous PRP and the other two with platelet poor plasma from one of the two patients with SPA. No significant differences in platelet aggregation were observed among the three suspensions with any of the inducers.

The values of platelet MDA and plasma PTG are shown in Figure 1 and Table 11. Patients showed higher levels of MDA than controls (8.8 4 2.9 nM/lOy platelets and 6.4 k 1.7 nM/109 platelets, respectively) with 30% of patients having values above 2 SD of the control mean.

Plasma PTG was also increased in the MVP group with relation to controls (42.3 k 8.5 ng/ml and 30.0 k 10 ng/ml, respectively). In 15% of the patients, PTG levels were higher than the control mean plus 2 SD.

No significant difference was found in the platelet lifespan of the two study groups. The T 112 was 4.2 days in MVP patients, and 4.9 days in controls, although

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PATIENT8 CONTROL1 PAT I EN T 8 CO NT ff OL 1

Fig. 1. and normal controls.

Values and means of platelet MDA production and plasma 0-thrombogIobulin in MVP patients

TABLE II. Platelet MDA Production and Recovery After ASA, and Plasma BTG in MVP Patients and Controls

MDA Production MDA Recovery after ASA BTG in plasma

X SD) X k SD) X k SD) (nM/109 - platelets, (T - 112 days, - ( n g / d ,

MVP

Control 40a 8.8 2.9 4.2 k 0.8 42.3 8.5

17a 6.4 1.7 4.9 f 1.0 30.0 10.0 < 0.001 NS < 0.01 P

aNumber of cases.

24 Arochaetal

in two patients (9- and 13-year-old girls), platelet T 1/2 was 2.9 and 2.3 days, respectively. No correlation was found between MDA and BTG levels or MDA and platelet life-span either in patients or in controls. There was a significant inverse correlation (r = -0.437, p < 0.01) between platelet life-span and BTG levels in patients but not in controls (r = -0.244).

Although both patients with SPA had a grade I11 MVP, there was no correlation between the severity of MVP and the parameters studied.

The individual results of patients with SPA and those with decreased platelet life-span are presented in Table 111.

DISCUSSION

Platelet activation seems to play an important role in the development of thrombotic episodes in patients with MVP [4,5]. A high percentage of cases without ischemic manifestations was found to have increased platelet coagulant activity [4] and elevated pf4 in plasma [5]. However, it is still difficult to separate the patients who are at risk of having an ischemic episode from those who are not.

In the present study, although there was no difference in platelet aggregation between patients and controls, there were two patients ( 5 % ) who showed SPA on repeated occasions. In both cases, PTG and platelet life-span were within normal limits, and in one of them MDA levels were slightly elevated. Spontaneous aggrega- tion has been reported in patients suffering from cerebral ischemic attacks, angina, diabetes mellitus, and in recurrent deep-vein thrombosis [9-13]. However, the inci- dence of SPA found in this study is similar to that found by Hoogendijk et a1 [ 131 in a normal population.

The finding of an augmented production of MDA and normal platelet life-span in MVP seems to be paradoxical, since increased MDA has been found in situations where platelet turnover is accelerated [ 141. However, during this investigation, it was observed that the recuperation of platelet aggregation after the administration of ASA occurred before MDA reached the original basal levels, which, as Reimers et a1 [I51 have pointed out, could mean that activated platelets may continue to circulate, thus maintaining their normal life-span. Increased MDA with normal platelet T 1/2 was also found by Sullivan et a1 [14] in patients with prosthetic heart valves and other cardiac diseases.

PTG is a specific platelet protein which is stored in the (Y granules and released in plasma along with pf4, platelet-derived growth factor, and other proteins that are also present in plasma during platelet aggregation. Its increased amount in plasma either means platelet activation or in vitro release [16], and both stituations may be

TABLE 111. Platelet Studies in Two Patients with Spontaneous Aggregation (SPA) and Two Patients With Shortened Platelet Life-Span

Age MDA Production MDA recovery after ASA, PTG in plasma Patient (years) SPA (nM/109 platelets) T 1/2 (days) ( n g W

BG 13 + 8.3 4.0 30 ML 18 + 10.0 5.0 34 AA 9 - 9.0 2.9 61 LR 13 - 6.7 2.3 55

Platelet Activity in Mitral Valve Prolapse 25

present in our patients. The significant statistical difference with the control group suggests that platelet hyper-reactivity is the main cause of this result. Elevated PTG has been found previously in MVP patients with cerebral ischemia, a common finding in most CVA cases but not in asymptomatic patients [ 171. The present results support those of Fisher et a1 [ S ] , who found high plasma levels of pf4 in 36 % of “asympto- matic patients. ”

The above results further indicate that platelet hyper-reactivity is present in a significant number of MVP patients with no specific symptomatology, and that they can be at risk of developing a cerebrovascular accident.

ACKNOWLEDGMENTS

We would like to thank Miss Xiomara Raleigh, Mrs. Trina Castellanos, and

This work has been supported in part by a grant from Consejo de Desarrollo Mrs. Carmen Pirela for their skillful technical assistance.

Cientifico y Humanistic0 (CONDES).

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