The effect of doxazosin on platelet aggregation in normotensive subjects and patients with hypertension: An in vitro study

An in vitro assay was used to investigate the effects of doxarosin on the platelet aggregation induced by epinephrine, collagen, and adenosine diphosphate. Platelet-rich plasma from normotensive subjects and patients with hypertension was compared. Doxatosin produced a concentration-dependent inhibition of platelet aggregation in both groups, but significantly lower concentrations were required to inhibit platelet aggregation in plasma taken from patients with hypertension. The concentrations of doxarosin that inhibited platelet aggregation in vitro were similar to those that are used clinically to control blood pressure in patients with hypertension. (AM HEART J lgg1;121:389-94.)

Rafael Hernandez Hernandez, MD, Atiff Rafael Carvajal, MSc, Jaime Guerrero Pajuelo, MD, MSc, Maria Jose Armas de Hernandez, MD, Maria Cristina Armas Padilla, MD, Oscar Barragan, MD, Jose Jesus Boada Boada, MD, and Emir Roa Barquisimeto, Venezuela

The pathophysiology of coronary heart disease (CHD) is closely related to platelet and endothelial function. In particular, mechanisms involving plate- let activity are related to atherosclerotic processes and thrombus formation, which are the ultimate cause of CHD and myocardial infarction.lm3 An in- crease in platelet aggregation has been reported in hypertensive patients compared with normotensive subjects.4a 5

In addition to reducing blood pressure, antihyper- tensive drugs should also modify other coronary risk factors. It has been reported that prazosin, a postsyn- aptic al-inhibitor, normalizes the platelet hyperag- gregability that is observed in hypertension.6 The purpose of this study was to investigate the effect of doxazosin, a selective postsynaptic cul-inhibitor,7s 8 on platelet aggregation in plasma taken from patients with hypertension and from healthy, normotensive volunteers.

METHODS

Study groups. Participants were divided into twogroups: healthy, normotensive subjects and patients with essential hypertension (sitting diastolic blood pressure [DBP] 96 to

From the School of Medicine.

Reprint requests: Rafael Hernandez Hernandez, MD, Clinical Pharmacol- ogy linit, School of Medicine, Centro Occidental “Lisandro Alvarado,” Rarquisimeto, Venezuela.

41Ql24892

30 ml blood Sodium citrate

sample \ / anticoagulant

mix

1 centrifuge

Platelet-rich plasma (PRP) Platelet-poor plasma (PPP)

+ methanol vehicle (1 -minute incubation)

+ collagen or

epinephrine or ADP

(5.minute incubation)

(1 -minute incubation)

+ collagen or epinephrine or ADP @minute incubation)

Aggregation Inhibition of

(control) aggregation

Fig. 1. Assessment of platelet aggregation.

116 mm Hg). All participants were nonsmokers. Exclusion criteria are given in Table I. Current antihypertensive treatment was suspended 4 weeks before the start of the study, and blood pressure was carefully monitored through- out this period. During the study, none of the participants took any drugs that could influence platelet aggregation (e.g., antithrombotic agents or cyclooxygenase inhibitors).

Assessment of platelet aggregation (Fig. 1). While the subjects were supine, a 30 ml sample of blood was taken from the antecubital vein. The blood was immediately transferred to a plastic tube and mixed with 3.8% sodium

389

390 Hernandez et al. January ,991

American Heart Journal

Control 100 , 160 rig/ml

doxazosin 80 i

6 500 rig/ml doxazostn

800 rig/ml doxazosin

I I I I I -1 0 1 2 3 4 5

t Doxazosin Time (minutes)

Fig. 2. Patient record of the effect of doxazosin on platelet aggregation induced by epinephrine (2.5 kmol/L) in a normotensive, 34-year-old man (incubation time with doxazosin 1 minute, platelet count

Table I. Exclusion criteria

Subjects with one or more of the following conditions were excluded from the stud) Sitting DBP <96 mm Hg and >116 mm Hg Grade III or IV hypertensive retinopathy (according to the

Keith-Wagener-Barker classification) Incidence of cerebrovascular disease < than 1 yr before the start

of the study Myocardial infarction <3 yr before the start of the study Grade II or III atrioventricular block Clinical or laboratory evidence of heart failure, renal failure,

liver failure, unstable angina pectoris, or pregnancy Insulin-dependent diabetes mellitus Dyslipidemia Mental disorders

citrate. A platelet-rich plasma was obtained by centrifug- ing the sample for 10 minutes at 160 X g at room temper- ature. The supernatant (platelet-rich plasma) was sepa- rated from the sediment and the platelet count was obtained.

The sediment was centrifuged at 1500 X g for 15 minutes to separate out the platelet-poor plasma. This was used as a reference in subsequent aggregation assays to adjust the transmittance and platelet count of the platelet-rich plasma on dilution. Aggregation assays were performed at 37’ C in silicone glass trays with 0.4 ml of platelet-rich plasma. The rate and percentage of platelet aggregation induced by ad- enosine diphosphate (ADP), collagen, and epinephrine were assessed.g For each analysis, a standard curve was prepared with a concentration of 300,000 platelets per mi- croliter. This assay was carried out in the presence of methanol (the doxazosin vehicle), which, at a final concen- tration of no greater than 3 70, did not affect the initial standard curve.

Doxazosin was dissolved in 70% methanol to give final concentrations of 10 to 2000 rig/ml and incubated with the platelet-rich plasma for 1 minute. The inducers (ADP, 2.5 to 10 pmol/L; epinephrine, 0.5 to 20 pmol/L; collagen, 0.25 to 2.0 pg/ml) were then added at a constant concentration, and the medium was incubated for 5 minutes. The per- centage aggregation obtained was compared with that of the initial standard (70 t 10% 1. The assay was repeated with increasing doses of doxazosin until a maximal inhibi- tion was obtained, 100% if possible.

The concentration of doxazosin that produced 25%) 50 %,75 ‘, , and 100 % inhibition of platelet aggregation was estimated for each patient by extrapolation. The Student t test for paired data (two-tailed) was used to assess statis- tical significance; p I 0.05 was considered significant.

RESULTS Patient characteristics. The group of normotensive

subjects consisted of 12 (5 men and 7 women) healthy, nonsmoking volunteers, with a mean age of 31.1 years. The 10 patients (2 men and 8 women) with essential hypertension were nonsmokers and had a mean age of 47.5 years. All participants were over 18 and under 60 years of age.

Effect of doxarosin in normotensive subjects. The effect of doxazosin on the platelet aggregation in- duced by epinephrine and collagen in a normotensive subject is shown in Figs. 2 and 3, respectively. In each patient doxazosin produced a concentration-depen- dent inhibition of platelet aggregation. The concen- trations of doxazosin required to inhibit aggregation induced by collagen, epinephrine, and ADP are illus- trated in Fig. 4.

Effect of doxazosin in patients with hypertension. Figs. 5 and 6 show the effect of doxazosin on the

Volume 121 Number 1. Part 2 Platelet antiaggregatory effect of doxazasin in vitro 391

t Doxazosin Time (minutes)

Fig. 3. Patient record of the effect of doxazosin on platelet aggregation induced by collagen (2 rglml) in a normotensive, 34-year-old man (incubation time with doxazosin 1 minute, platelet count 300,000/~1).

platelet aggregation induced by epinephrine and col- lagen in patients with hypertension. With each in- ducer, doxazosin produced a concentration-depen- dent inhibition of aggregation at lower concentra- tions than in normotensive subjects. Fig. 7 illustrates the concentration of doxazosin producing 25 % ,50 % , 75 % , and 100 % inhibition of aggregation with colla- gen, epinephrine, and ADP.

Comparison between normotensive subjects and pa- tients with hypertension. To produce a similar degree of aggregation in normotensive and hypertensive pa- tients, higher concentrations were used in the latter (Table II). The same concentration was used for each subject throughout the study.

The pattern of platelet aggregation induced by collagen, epinephrine, and ADP was similar in nor- motensive subjects and patients with hypertension. However, the concentration of doxazosin needed to inhibit aggregation in hypertensive patients was sig- nificantly reduced (p < 0.001). The concentrations of doxazosin required to produce 50% inhibition of platelet aggregation in normotensive subjects and patients with hypertension are given in Table III. In normotensive subjects, the maximum inhibition (25 % ) of ADP-induced aggregation was achieved by 224.3 -t 43.9 rig/ml of doxazosin.

A 40 rig/ml concentration of doxazosin completely inhibited the secondary phase and almost totally in- hibited the primary phase of epinephrine-induced aggregation in one of the hypertensive patients (Fig. 5). A concentration of 800 rig/ml was required in one of the normotensive subjects to achieve a similar ef- fect (Fig. 2). Doxazosin concentration/effect ratios for normotensive subjects and patients with hyper-

c%lia&l Eplk*fin& ADP

Fig. 4. The concentration of doxazosin required to inhibit by 25 ?0,50?), 75 %, and 100 !% the platelet aggregation in- duced by collagen (0.25 to 2.0 pg/ml), epinephrine (0.5 to 20.0 pmol/L), and ADP (2.5 to 10.0 pmol/L) in normoten- sive subjects (n = 12).

tension are presented in Figs. 8 and 9 for aggregation induced by epinephrine and collagen, respectively. In normotensive subjects the inhibitory mean concen- trations of doxazosin were greater than in patients with hypertension; this was statistically significant.

DISCUSSION

Doxazosin inhibited the in vitro platelet aggrega- tion induced by epinephrine, collagen, and ADP, with the platelets suspended in plasma, pretreated, and incubated for 1 minute with the drug. This was observed in both healthy normotensive subjects and patients with hypertension.

It has been shown that doxazosin has a dose- dependent antihypertensive effect in patients with mild or moderate hypertension, with the once-daily

392 Hernandez et al. January 1991

American Heart Journal

60

Control

20 rig/ml doxazosm

40 rig/ml doxazosin

I ~~- -1 0 1 2 3 4 5

t Doxazosin Time (minutes)

Fig. 5. Patient record of the effect of doxazosin on platelet aggregation induced by epinephrine (0.5 pmol/L) in a 49-year-old man with hypertension (incubation time with doxazosin 1 minute, platelet count 300,000/~1).

r; 8 c a

90 ! I

80

70

60

50

40

30

20

10

0

Control 20 ngiml doxazosin 40 rig/ml doxazosin 80 nglml doxazosin

120 ngiml doxazosin

f Doxazosin Time (minutes)

Fig. 6. Patient record of the effect of doxazosin on platelet aggregation induced by collagen (0.5 pg/ml) in a 49-year-old man with hypertension (incubation time with doxazosin 1 minute, platelet count 300,000/ /.a.

Table II. Concentration of inducers and basal platelet aggregation for normotensive subjects and hypertensive patients

Normotensives (n = 12) Hypertensives (n = 10)

Collagen Epinephrine ADP Collagen Epinephrine ADP (helm 1) I/.lmollLi fp?wllLi fielml) (pmollL1 IwmollLi

Mean inducer 0.82 2 0.14 3.40 t 1.72 4.75 i 0.84 0.52 + 0.06 1.15 2 0.27 ‘2 I. 75 f 0.25 concentration ? SEM

Range 0.25 - 2.00 0.5 - 20.0 2.5 - 10.0 0.25 - 1.00 0.5 - 2.5 2.5 - 5.0 Mean cc, platelet aggregation -t SEM 71.4 k 4.3 75.6 i 4.9 72.8 zk 3.7 70.8 i- 3.9 67.3 i 3.5 75.5 XL 4.4

Volume 121 Number 1. Part 2 Platelet antiaggregatory effect of doxazosin in oitro 393

.r : Y 3o01 cl 25% inhibition w 8 50% inhibition

B 75% inhibition 5 200 100% inhibition

.s -h mE 2’ sE z 100 8 ,x 8 P c 0 s

Collagen Epinephrine ADP

Fig. 7. The concentration of doxazosin required to inhibit by 25’; ,50’;, 75’;) and 100”;’ the platelet aggregation in- duced by collagen (0.25 to 1.0 pg/ml), epinephrine (0.5 to 2.5 pmol/L), and ADP (2.5 to 5.0 pmol/L) in patients with hypertension (n = 10).

Table III. Concentrations of doxazosin required to produce 50”;) inhibition of platelet aggregation in normotensive subjects and patients with hypertension

Concentration of doxazosin (nglml) producing 505 inhibition of

platelet aggregation (ID&

Inducer Normotensive Patients with

subjects hypertension

0 ; 0 100 200 300 400 500 600 700

Mean (k SEM) mhlbltory concentration of doxarosln (ng’ml)

l Patients Wh hypertension Normotenstve subjects

Fig. 8. Doxazosin concentration/effect ratios for aggre- gation induced by epinephrine in normotensive subjects and patients with hypertension.

* p < 0 05. StatIstIcally slgnlflcant between- group difference

Collagen Epinephrine ADPt

339.2 2 74.3 80.0 t 10.9' 432.8 zt 120.3 25.7 k 6.3*

126.6 i 12.4

*p 5 0.001, statistically significant between-group difference. +The maximum inhibition obtained by doxazosin (224.3 + 43.9 rig/ml) for ADP-induced aggregation in normotensive subjects was 25”,

administration of 1, 2, 4, and 8 mg.l” With these an- tihypertensive doses, plasma levels of 13 to 80 rig/ml have been reported. 11, l2 In the group of hypertensive patients and at the concentrations used in this study, we observed an in vitro inhibition of platelet aggre- gation. Thus we can assume that the doxazosin- induced inhibition of platelet aggregation occurs at therapeutic antihypertensive doses of the drug.

In the study significantly greater concentrations of doxazosin were required to observe a similar effect in the group of healthy normotensive subjects. This could be explained by the fact that platelets in hypertensive subjects are much more reactive than in normotensive subjects. 4s 5 Alternatively, according to Ikeda et a1.,6 al-inhibitors could normalize the plate- let hyperfunction in patients with essential hyper- tension and reduce the very high P-thromboglobulin values of these patients.

In animal models and healthy volunteers, dox- azosin shifts the dose-response curve of norepineph-

700

+ Patients with hypertension -1 Normotensive subjects

Fig. 9. Doxazosin concentration/effect ratios for aggre- gation induced by collagen in normotensive subjects and patients with hypertension.

rine and phenylephrine to the right in a competitive fashion. Minimal changes in heart rate are seen.7as This suggests that doxazosin selectively inhibits al- adrenergic receptors. However, using a radioligand technique, Newman et a1.13 and Hoffmann et a1.14, l5 classified the adrenergic receptor of human platelets as the cr2 type.

In this study doxazosin inhibited the platelet aggregation of normotensive subjects and hyperten- sive patients, but important differences with regard to the median infective dose (ID& were observed, particularly in the aggregation induced by epineph- rine (IDso: 25.7 rig/ml for hypertensive subjects and 432.8 rig/ml for normotensive subjects). This could indicate that although the adrenergic receptors of platelets are normally of the CYZ type, the receptors may undergo a degree of modification at the platelet

394 Hernandez et al. January 1991

American Heart Journal

level during hypertension. Because most studies that have characterized the platelet receptor have used normotensive subjects with no specification of arte- rial pressure, it is not known whether platelet recep- tors differ in normotensive and hypertensive persons.

Doxazosin also inhibited the platelet aggregation induced by ADP and collagen. This could suggest that doxazosin inhibits different intraplatelet meta- bolic pathways and not those that are specifically adrenergically related. Because the effects observed were concentration dependent, this suggests the in- volvement of a specific rather than a nonspecific pathway.

In conclusion, in a group of hypertensive patients, doxazosin inhibited platelet aggregation in vitro at concentrations that produce an antihypertensive ef- fect in vivo. The inhibitory effect of doxazosin on platelet aggregation was concentration dependent. In healthy volunteers a lo- to 20-fold increase in the concentration of doxazosin was required to produce a similar effect. A drug such as doxazosin that reduces not only blood pressure but also inhibits platelet ag- gregation could represent a therapeutic advantage. In addition to lowering blood pressure, doxazosin could reduce the thrombotic and atherogenic pro- cesses that often lead to cerebrovascular disease and CHD. REFERENCES

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2. Weiss H. Platelets and ischemic heart disease. N Engl J Med 1980;302:4:225.

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