Stimulation of aggregation and thromboxane A2 formation of human platelets by hypoxia

Prostaglandins, Lcukotrienes and Medicine (1987) 29. 49-59 ‘(3 Longman Group UK Ltd 1987

STIMULATION FORMATION K.

Ponicke

OF AGGREGATION AND OF HUMAN PLATELET5 1

, R. Sternitzky

2

THROMBOXANE BY HYPOXIA

, and

H.-J.

A,

Mest

1

1

and Second Leninallee to KP)

Department of {harmacology and Toxicology Medical Clinic Martin Luther University, 40'20, GOR (reprint requests Halle,

4,

ABSTRACT The influence of hypoxia on the spontaneous gregation (SPA) and thromboxane formation analysis of aggregation curve was carried Breddin.

platelet agwas studied. The out according to

ihe hypoxia enhanced the aggregagility from Qznorm ?? 2.46 -0.40 (normoxia) to QZhyp ?? 4.39-0.39 (hypoxia), n = 52, 10 samples of those showed no SPA under equilip < 0.001. bration with air but the h+ypoxic stimulus provoked SPA ?? 1.19-60, n ? ? 10, p < 0.001). (Q2norm = 0, Qnhyp When the results were arranged according could the stimulation of SPA, two groups low and high response to hypoxia.

to be

the degree of separated with

The hypoxia caused also an augmentation of the TXl32 level in The stronger enhancement of the TXB? comparison to normoxia. formation during the incubation under hypoxic conditions was independent of the fact whether SPA took place or not. The present study suggests that hypoxic conditions alone may of the platelets be a reason for a stimulated TXAZ formation caused by hypoxia is and that the enhanced TXA2 formation possibly inducing or reinforcing the SPA. INTRODUCTION It has been demonstrated that hypoxic conditions in various tissues enhanced the level of prostanoids in perfusates and aggregability and plasma thromboxane B2 blood (1). Platelet

49

(TXB2)

disease

levels are increased in and peripheral vascular

But the mechanism hanced levels of The purpose fluence of thromboxane

by which prostanoids

patients disease

ischemia is not

with ischemic (2, 3, 4).

and hypoxia known.

heart

produce

of the present study was to investigate hypoxia on spontaneous aggregation (SPA) AS formation of human platelets.

the and

enin-

MATERIALS AND METHODS Subjects: 59 patients with clinically apparent periphersi vascular disease, ranging from 28 to 72 years of age (x-SO=50.6-8.0) were studied. None of them took cyclooxygenase inhibiting drugs for the last 14 days prior to the study. Platelet

rich

plasma

(PRP):

Venous blood was collected into 3.18 % citrate 9 : 1, v : v. PRP was prepared by centrifugation at 250 g and room tempera ture for 6 minutes. Platelet concentration was adjusted to 375 000 per pl by addition of platelet poor plasma. The PRP was stored at room temperature in a plastic syringe containing 2 ml air enriched with 5 % CO*. The platelet aggregation was studied 1 h after collection of blood. Spontaneous

platelet

aggregation

(SPA):

SPA was measured by the method of Breddin, PAT III (5) using a photometer “Spekol” (VEB Carl Zeiss Jena, GOR) equipped The aggregometer (designed in our with an aggregometer. department) included a rotating disc-shaped plastic cuvette, 8 mm thick. Oisc cuvette diameter was 17 mm; the rotation speed was 22 rpm (for SPA measurement) and 3 rpm at the beginning of the preincubation period, respectively. The rotation processing is demonstrated in fig. 1, together with the course of pH of the PRP. The sample volume aBounted to 500 1.11. After for 5 minutes at 37 C the PRP was incubated minutes for measuring of SPA.

a pregncubation at 37 C for 10

SPA was investigated by recording the changes in optical density of the PRP sample at 500 nm. The photometer was adjusted to a transmission of PRP (0 %> and PPP (100 %>. The analysis of the aggregation curve was carried out as described by Breddin (5) as demonstrated in fig. 2.

50

PH 9

f __-- 4

8

*-

I

,A

,,*----

It-

*_--

/*------*--

% a

I I

4 a2

Figure

1:

3

0

I

I

56

9

b

12

Emin

Rotation processing and influence of the incubation period on the plasma pH. (1 = 22 rpm, 2 = 3 rpm, 3 0 rpm, 4 = 22 rpm) ??

100

*

PPP

1

\ \ opgrqobility

P2 = ff-

C I

Figure

2:

.

15

PRP

.-I

5

0 min

3

Evaluation of aggregation tion of aggregability Q,.

curve

and

determina-

The following parameters were measured: Gradient al gradient aggregation” and corresponds to the “primary a2 to the maximum speed of aggregation. Both parameters were expressed as percentage changes of transmission per minute.

51

The reaction time Tr is defined as the point of the gradients al and a2. The spontaneous of the PRP was defined which is as Q,, tion. Oxygen

of intersection aggregability the a2/Tr rela-

content:

The polarographic measurements were made with MF 65 (VEB Metra Radebeul, GDR) equipped with Clark-type electrode (11).

a pOz-Meter a micro

The 0,/l

oxygen content of the PRP was adjusted to 209 nmol (normoxia) and to 113 nmol 02/1 (hypoxia), respectiof air or nitrogen (1 ml per min) during vely , by a stream preincubation and incubation (aggregation) time. The oxygen content during the hypoxia is shown in fig. 3.

pm0l.l-1

f

.4---___Q

* 0.2

Figure

3:

Determination

I

I

3

5

I 1

6

9

The time course of during the hypoxia of

12

the

plasma

15 min

oxygen

content

TXB*:

Thromboxane AZ was determined as the stable hydrolysis incubation, the metabolism of arachiproduct TXBZ. After donic acid was terminated by transfering 400 pl of the sample from the disc cuvette into a tube containing 400 pl ethanol/saline mixture (3 : 7) and 100 pg.2,.3-dinorTXBz as internal GLC standard. Extraction? esterlflcatlon, separation by TLC,silylation, and quantiflcatron of TX82 by using GLC were as described earlier (6). were kindly provided by Or. J.E. TXB2 and 2,3-dinor-TXB2 Upjohn Company (Kalamazoo, Michigan, U.S.‘A.). Pike, 52

STATISTICS Significant Wilcoxon’s

differences were calculated by means of signed rank test for paired samples (PI. The slopes of the regression lines were compared by means of the method of Sachs (7) (Z>. All values in the paper are e?pressed as the mean and the standard error of the mean (x-SEM). RESULTS Spontaneous

platelet

aggregation:

In 42 of 59 patients spontaneous platelet aggregation was observed under control condition (normoxia, 209 nmol 0,/l) In contrast to this, the reduction of the oxygen content of the PRP to 113 nmol 02/1 (fig. 3) caused SPA in 52 of PRP preparations. 10 samples of these showed consequently negative SPA under equilibration with air, but hypoxia SPA. The hypoxi? stimulus increased the aggregabi provoked lity from Qznorm = 2.455-0.400 (normoxia) to Qphyp = 4.387-0.724 (hypoxia) n = 52, p < 0.0001 (fig. 4).

EGG n=52

ICI normoxia

F igure

4:

Influence

of

El hypoxia

hypoxia

on the

aggregab

lility

Q2

When the results were arranged according to the degree could be separated. the stimulation of SPA, two groups with relative changes of SPA Group A, n = 37, patients response to hypoxia less than 1, (Qzhyp - Qznorm)/Qznorm Cl, and additionally the patients with Q,hyp < 1 and Qznorm = o, fig. 5.

53

of in

yx1.2103r.0.1S57 r.O.S751

Figure

5:

Correlation between the aggregability under normoxic (Qznorm) and hypoxic (Qzhyp) conditions. Group A, patients with low response to hypoxia.

Group 8, n = 15, patients with relative changes of SPA in response to hypoxia larger than 1, (Qzhyp - Q2norm)/Q2norm > 1, and additionally, the patients with Q,hyp > 1 and Q2norm = 0, fig. 6. In both groups, a statistically significant correlation (p < 0.001) between the aggregability Q2norm (under control conditions) and the aggregability Qnhyp (under hypoxia) was Pound.

54

I :, I

group .B

26

I

y= 2.403x + 1.630

I

I I I I I I

22

r-0.9662 P-opm n=lS

18

14 I I

10

6

i

Figure

6:

.

ii

ua

lb

2 norm -

Correlation between the aggregability under normoxic (Qznorm) and hypoxic (Qzhyp) condiGroup 8, patients with high response tions. to hypoxia.

The slopes of both regression lines were also statistically significantly different, z = 8.423, p < 0.0001, and therefore the distribution of the patients into a group of high sensitivity (5) and into a group of low sensitivity (A) to the hypoxic stimulus appears justified. Changes

of

pH :

The observed rises in pH values 1) were in agreement tests (fig. Breddin in spite of the streaming the surface of the sample.

55

during the aggregation with the findings by of air or nitrogen over

TXB?

formation:

TXBl level aggregation 10 minutes

was determined in test was started under normoxic or

25 PRP preparations and after a rotation hypoxic conditions.

before the time of

nglml

100 f

pGYGsGo1 p*O.OOl n = 25 before after

oggregotion

r-l

oggregotion normoxio

Figure

7:

Influence

As shown in statistically

from the under

respectively.

of

hypoxio

hypoxia

on

the

TXB,

level

of

the

PRP.

fig. 7, in the significantly

basal

control

El

value

of

condition

plasma sample the TXB;! level was gnhanced during th$ aggregation 39.4-4.5 ngiml to 58.3-8.4 ng/ml and to 81.9-9.7 ng/ml under hypoxia,

Fig. 8 demonstrates the subdivision of the samples with respect to their spontaneous platelet aggregability into three subgroups. In seven PRP withoyt SPA, the TX82 level was significantly increased to 46.7-7.7 ng/ml within 10 min of hypoxic processing (from 36.6-9.0 ng TXB2/ml before the rotation was started). In contrast to thig, normoxia caused no stimulation of TXB2 formation (36.9-10.2 ng/ml). 4 samples without SPA under normoxic conditions but with positive SPA under hypoxia showed particularly enhanced TXBz levels under hypoxia. In 14 samples with positive SPA under normoxia as well as under hypoxia, statistically significant differences were found also in the TXB2 formation during aggregation. SPA

56

undef normoxia caused $ rise 38.1-5.7 ng/ml to 69.9-12.9 hypoxia enhanced the plasma

without

SPA

in the plasma TXBz level from ng/ml, whereas SPA stimulated by TXB2 level to 89.0+12.2 ng/ml.

SPA

only at hypoxia

SPA

at normoxia and

at

hypoxia

TXB;Z- level ng /ml 100

/

P*O.OOl

PeO.02 n=4

n=7

II Figure

8:

before oggregotion

n=l& after aggregation

after aggregation

normoxia

hypoxia

Influence of hypoxia on the TXB2 level of PRP after the rotation of the disc cuvette. The samples are subdivided into three groups with and without SPA under normoxia and hypoxia, respectively.

0IscussIori The results show that even mild hypoxia - a reduced oxygen content by about 50 96 - caused a significant stimulation of is remarkable that the PRP samples SPA by 79 % (n = 52). It could be divided on the basis of their hypoxic response into But the arrangement in groups of high and low two groups. sensitivity is only possible with respect to the aggregabiliwas not significantly different ty. The production of TXA2 in these groups during aggregation under hypoxia. We also observed a significantly stronger enhancement of the TXAZ formation in the PRP under hypoxic conditions in comparison to normoxia.

57

Our findings are in agreement with the previous studies, stressing that ischemia or hypoxia cause an enhanced level of AA-metabolites (2, 3, 4, 8, 9). Nakano has suggested (10) that the decreased metabolism of prostanoids due to inhibition of 15-prostaglandin-dehydrogenase may cause a rise of the prostanoid level. Another possible way for enhancing the level is the stimulation of the biosynthesis of arachidonic acid metabolites. But the causative mechanism of this rise is not jet known. The present study suggests that hypoxic conditions alone may be a reason for the stimulated TXA, formation of the platelets. It is particularly relevant that also in samples without SPA the TXB2 levels were weakly, but significantly enhanced (n = 7) and that hypoxia may be inducing SPA in samples without SPA under normoxic conditions. The major finding of this study is that the enhanced TXA2 formation caused by hypoxia induces SPA and is responSPA. sible.for the stimulation of the basal REFERENCES 1.

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2.

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