EFFECT OF ISRADIPINE ON IN-VIVO PLATELET FUNCTION

ThrombosisResearch,Vol. 86, No. 5, pp. 363–371, 1997 CopyrightO 1997 EkvicrScicncc Ltd Rintcd in the USA. All rightsreserved 0049-3848/97 $17.(YJ+ .(S)

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EFFECT OF ISRADIPINE ON IN-VW(3 PLATELET FUNCTION John O’Grady’, Harald Kritz’, Peter Schmid’, Christian Pirichl and Helmut Sinzinger’3 1. Wilhelm Auerswald Atherosclerosis Research Group (ASF) Vienna, 2, Cardiovascular Rehabilitation Center Bad %hallerbach, Upper Austria. and 3 Department of Nuclear Medicine, University of Vienna, Austria (Received21 Januaty 1997by EditorD.L.Heene;revised/accepted4 April 1997)

Abstract

In animal studies calcium channel blockers (CCB’S) and especially isradipine. a second generation dihydropyridine, interrupt the sequence of events culminating in the formation of atherosclerotic lesions, The effect of 4 weeks isradipine treatment (5mg daily) on blood pressure and in-vivo platelet timction (measured with “]Indium-oxine labeled autologous platelets) were investigated in a randomized, double-blind and placebo controlled trial in 40 patients with mild to moderate hypertension and scintigraphically diagnosed active atherosclerotic lesions of the carotid arteries, The average supine systolic/diastolic blood pressure was significantly reduced at the end of the treatment period in the isradipine group (group I; p
Key words: Atherosclerosis, hemostasis, isradipine, platelet finction, in-vivo, hypertension. Corresponding author: Helmut Sinzinger, MD, Prof., Wilhelm-Auerswald Atherosclerosis Research Group (ASF), Vienna; Nadlergasse 1, A-109O Vienna, Austria; phone: +43-1-4082633; fax: +43-1-4081366, E-mail: Helmut. Sinzinger@j,akh-wien.ac.at;

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Acute thrombotic and thromboembolic occlusion of atherosclerotic vessels causes most myocardial infarctions and strokes which in turn are the first and third leading cause of mortality in the Western World, respectively (1). Calcium has been claimed to play a pathogenetic role in experimental atherosclerosis (2). Calcium-dependent processes are involved in the formation of atherosclerotic lesions including platelet aggregation, monocyte adhesion, release of growth factors, cell proliferation and migration, protein synthesis and secretion, and endothelial damage (3-6), The understanding of the basic molecular processes involved in thrombus formation in-vitro resulted in the development of new antithrombotic agents (6). Calcium channel blockers particularly second generation dihydropyridines such as felodipine, isradipine, Iacidipine, nicardipine, nimodipine and nitrendipine have properties that suggest their usefulness in patients with hypertension and concomitant left ventricular dysfunction as recently reviewed by Parmley (7). Dihydropyridines have been shown to exert potent antiatherosclerotic effects in animal studies (8, 9). Isradipine possesses a marked vascular selectivity with a strong vasodilatatory and minimal cardiac effects (10). Furthermore, this substance has been shown to be more potent in inhibiting platelet fhnction in-vitro and influencing the prostaglandin system than other calcium channel blockers (1 1). It enhances significantly vascular PGI,-formation in animals (12). While there is little experimental data on the ex-vivo platelet effects (13-17), no information regarding in-vivo effects is available yet. In-vivo platelet scintigraphy has been used for imaging of carotid artery disease since the studies of Goldman (18) and Kessler (19). The present study was intended to demonstrate the extent to which isradipine is able to influence local and systemic in-vivo platelet finction as determined by the platelet uptake ratio (PUR) and the platelet life span in patients with hypertension and with atherosclerotic lesions,

MATERIAL and METHODS After a two weeks single blinded wash-out period the influence of isradipine on platelet function in 40 patients was assessed in a randomized double-blind placebo-controlled clinical study over a 4-weeks period. After the wash-out period all patients received double-blind randomized once daily 5 mg isradipine (Lomir SROW, Sandoz”, Vienna) or placebo for 4 weeks. The patients included were 25 males (62.5°/0) and 15 females (37.50/0). The mean age was 45+8 years, the mean height 173 cm, the mean weight 72 kg, the mean heart rate 77 counts/minute, the mean systolic blood pressure 135 mm Hg and the mean diastolic blood pressure 100 mm Hg (Table 1) There was no significant difference between the placebo (P) and isradipine group (I) relative to age, height, body weight, heart rate and blood pressure. 9 patients in the I group and 4 patients in the P group additionally had a metabolic disease (Table 1). Treatment regimes (Table 1) did not change throughout the study, All patients had active atherosclerotic lesions of the carotid vessels characterized by scintigraphically visible lesions and a PUR above 1.15. Treatment with other antihypertensive drugs, steroids, digitalis, nitrates and other substances with known effects on platelets like acetylsalicylic acid, prostaglandins, anticoagulants, dipyridamole and any non-steroidal antiinflammatory drug were criteria for exclusion. Also patients with known allergy to dihydropyridines, pregnancy, secondary hypertension, recent cerebrovascular or cardiovascular events were excluded, All participants fhlfilled the inclusion criteria and gave their written informed consent according to the declaration of Helsinki. Blood for platelet labeling and determination of safety parameters (creatinine, blood urea nitrogen, glucose, bilirubine) was drawn at the beginning, at the end of the washout period and at the end of the treatment period, In addition, blood pressure, heart rate, body weight and side effects were documented,

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Table I. Patients Characteristics of the Isradipine- and the Placebo-Treated-Group

Female Male

Placebo (groupP; n=20)

Isradipine (group 1;n=20)

9

6

11

14

Age (years)

44.4 (32-56)

45.8 (28-68)

Height (cm)

173(159-187)

173 (158-184)

Weight (kg)

71 (58-84)

73 (56-87)

HR

76*5

77+3

BP systolic

136*9

134*1O

BP diastolic

100*4

100+4

Diabetes

1

2

Hyperuricemia

1

3

Hyperlipidemia

2

4

Sulfonylurea

0

1

Insulin

1

I

Uric acid lowering drugs

1

3

Lipid lowering drugs

2

4

Differences are not significant; values are given as mean and range (in parenthesis); HR=heart rate in counts/minute; BP=blood pressure in mm Hg; Platelet labeling The radiolabeling of platelets with “’ln(indium)-oxine was performed as introduced by Thakur 1976 (20) and fimther modified by Sinzinger (21) to a relatively simple (45 minutes for labeling) routine procedure using a commercially available kit (KarmedQ, Klosterneuburg, Austria). This technique requires 16 mL of blood anticoagulated with 4 mL acid citrate dextrose (ACD). After a short sedimentation (5 minutes, 22°C ) platelet rich plasma (PRP) was prepared by centrifbgation (150xg for 5 minutes at 22[’C), PRP was removed and the platelet pellet was resuspended in 100 VL freshly prepared tyrode buffer (pH 6.2). Platelet poor plasma (PPP) was obtained by a fi,u-ther centrifbgation (500xg). 100 ~1 (100 pCi) ‘‘‘In-oxine (Department of Chemistry, Research Center Seibersdorf, Austria) were added and incubated at 37°C for 5 minutes in a stirred water bath. The labeled platelets were then resuspended in autologous PPP and reinfected immediately. Labeling ef$ciencyhcovery AIiquots were taken after labeling to calculate labeling efficiency (LE), that is cell bound activity compared to the total radioactivity. 60 minutes atler reinfection of labeled platelets, 2 mL blood were taken from each patient for the calculation of recovery (REC). Calculation ofplatelet survival For determination of platelet survival 2 mL blood samples were taken three times daily for the initial 4 days after radiolabeling of platelets. Cell bound radioactivity was measured in a

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gamma-counter and survival assessed by the multiple hit model (22) (using a computer program kindly provided by Anthon du P, Heyns, MD, Prof, Johannesburg, South Africa) according to the standards given by the International Society of Hematology (ISH) (23). The normal value is above 180 hours. The the interassay variation in healthy volunteers was 0.75+0. 14°/0(in patients 0.71+0, 140A), the intraassay variation in healthy volunteers was O.12+0.02°A (in patients 0.39+0,12’?40). Gamma camera imaging and calculation qf the platelet uptake ratio (P[JR) Gamma camera imaging and quantitative uptake measurement was done before and afler therapy. The patients were placed under a large field-of-view-gamma camera (Siemensc’, Erlangen, Germany) connected to a data processor, 24 hours ailer reinfection of the autologous radiolabeled platelets daily images (matrix 64x64, exposure time 10 minutes) of the carotid arteries during 4 consecutive days were taken and the mean value of the platelet uptake per pixel afler background subtraction was used to calculate the PUR (24),

STATISTICAL ANALYSIS Calculations for significance of differences within subjects of each group or between subject groups (repeated measures with a single variable) were performed using the analysis of variance (ANOVA). A level of significance less than 0.05 was taken as a minimum. Data are given as arithmetic mean+standard deviation (SD). RESULTS The PUR measured on 4 consecutive days before (week 2) and at the end of the treatment period (week 6) was diminished significantly in group I from 1.20 to 1.15 (within group: p
Week 2

Week 6

Differences

Level of significance*

P

I

P

I

P

I

P

I

1

1,21

1.20

1.21

1.15

0

0.05

n.s.

Pm 2

1,21

1.20

1,21

1.14

0

0.06

n.s,

<0.0001 < (),()()01

Pm 3

1.21

1.21

1.21

1.15

0

0.06

n.s.

<0,0001

PUR 4

1,21

1.20

1.22

1.15

0.1

0.06

n.s.

<0.0001

Pm

Values are given as arithmetic mean; *=Anova-repeated measures within groups; n,s.=not significant; P=placebo treated group; I=isradipine treated group;

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Table 111. Platelet Uptake Ratio (PUR) on 4 consecutive days (PUR 1,2,3 and 4) before and after 4 weeks of isradipine (I) or placebo (P) treatment, The measurements were done hetwwn the groups. Platelet uptake ratio

Week 2

Week 6

Level of significance*

P/I

P/I

1

1.21+0.05

1.18+0.05

P/l <0.001

Pm 2

1.21+0.04

1,18+0,06

<0.001

PUR 3

1.21*0.05

1.18+0.05

<0.001

Pm 4

1.21+0.04

1.18+0.05

<0.001

Pm

Values are given as arithmetic mean+standard deviation; *=Anova-repeated measures between groups: P=placebo treated group; I=isradipine treated group; The protocol was completed by all 40 patients included without any serious side-effects. In 3 patients treated with isradipine ankle edema appeared 2-3 days after initiation of treatment, No other side effects occured. The heart rate was not significantly altered in either group. As shown in Figure 1, a significant reduction in systolic (-10 mm Hg) and diastolic blood pressure (-12 mm Hg) was achieved during the treatment period in the I group, while the values in the P group remained unchanged

FIG. 1. Blood pressure at the beginning of the wash-out period (week O), after 2 weeks (week 2) and 4 weeks later (week 6) for patients with placebo or isradipine treatment. Values are given as mean+SD, n.s.=not significant, ***=p
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EKEm

180 175 ~ s 170 2 w

165 1

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[

I

FIG. 2 Platelet survival (hours) before (week 2) and after 4 weeks (week 6) for the isradipine and the placebo treated group. Values are given as mean+SD; n.s.= not significant; ***=P
Platelet survival (Figure 2) after “’In-oxine labeling of platelets increased from 162.5 hours before to 168.2 hours (p
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detects the activity of the pathogenetic process rather than its extent (3 1). Follow-up controls over several months indicate that atherosclerotic lesion sites with pathologically augmented platelet deposition are quite stable both as to localization and intensity of uptake. Thus, radioisotopic follow-up, is a valuable model for assessing and monitoring the eflicacy of any kind of therapeutic intervention. Fetkovska et al. (32) observed in 17 patients with essential hypertension that isradipine significantly inhibited the ex-vivo platelet aggregation induced by IOW density lipoprotein(LDL)-cholesterol and serotonin. Ding et al. (33) found in thirty hypertensive patients treated with isradipine for sixteen weeks a significantly reduced epinephrine- and ADP-induced platelet aggregation. In a clinical study in hypertensive patients we found that therapeutic doses of isradipine favorably affected ex-vivo platelet aggregation, lowering exercise-induced TX% -formation and plasma 13-thromboglobulinlevels ( 16). Recent experimental investigations using a cross perlision model in rabbits revealed that the vessel wall rather than the platelets is responsible for vascular thromboresistance (34). PGIZ and EDRF act synergistically in the inhibition of platelet activity via cAMP- and cGMP-elevation (4, 35), isradipine markedly stimulating the release of both compounds (36). The local and the systemic hemostatic balance is impaired in patients even with early atherosclerotic lesions. AS shown in animal studies the sequence of events culminating in the formation of atherosclerotic lesions can be interrupted, mainly through enhancement of vascular PGI,-production and decreasing platelet acitivity (37). Our data suggest that in-vivo treatment with isradipine causes a significant decrease in the PUR and a significant increase in platelet survival, therefore improving in-vivo platelet fimction. In conclusion the results demonstrate, that isradipine treatment prevents platelet mediated growth of atherosclerotic lesions and may thus be beneficial in delaying atherosclerotic lesion development and/or progression.

Acknowledgements The authors gratefully acknowledge the valuable help of Judith Bednar, T.A,, Ingrid Blazek, T.A., Bettina Mosing, T.A,, and Susanne Granegger, T.A, for technical assistance and G. Nimberger, M.D., for statistical analysis.

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