Vol. 10, pp* 135-145, Pergamon Press

THROMBOSIS RESEARCH

1977

THE EFFECTS OF pCO2 AND pH ON PLATELET SHAPE CHANGE AND AGGREGATION FOR HUMAN AND RABBIT PLATELET-RICH PLASMA

S.S.

Tang and M.M, Frojmovic Department of Physiology McGill University Mont rea I , Quebec Canada, H3G 1~6

(Received 8.6.1976; in revised form 19.11.1976. Accepted by Editor K.M. Brinkhous)

ABSTRACT The rapid changes in pH, pCO2 and pO2 of human and rabbit plateletr’ich plasma (PRP) were measured during processing and storage in air in closed tubes and cuvettes. The pH increased frcm 7.3 to 7.8 and pop from 20 to 130 mnHg, while pCO2 decreased from 55 to 12 mmHg, after post-bleeding times of 150 minutes. The effects of pCO2 and pH on platelet shape change and aggregation induced by ADP, serotonin and adrenaline were studied with controlled physiologically normal pCO2 (35 mnHg) and pH (7.4) versus low pCO2 (I5 rmdig) and high pH (7.8) conditions. At the control led physiologically normal pCO2 and pH conditions, rabbit PRP gave reduction in primary and secondary aggregation with serotonin, and reduction in primary aggregation with adrenaline, as compared to low pCO2 and high pH. However, ADP-induced primary aggregation was insensitive to these pCO2 and pH changes for human and rabbit PRP anticoagulated with heparin or with low concentration of sodium citrate, but became markedly inhibited with increasing citrate concentrations. At the normally high pCO2 and low pH, ADP- and serotonin-induced shape change were not significantly altered. These results suggest that the pCO2 and/or pH might be affecting the transmission process between surface receptors and final aggregation, with probable dependence on available calcium.

INTRODUCTION Platelet-rich plasma (PRP) preparations have extensively used for the including shape change, primary and study of platelet structure and function, with the turbidimetric techand the release reaction, secondary aggregation, It has been known for some time that nique (l-3) as the principal method. the pH of PRP can affect platelet aggregation in response to various aggregatInitial studies, conducted with variation of pH of ACD-PRP ing agents. through additions of Tris buffer or isotonic saline of different pH, indicated that ADP-induced human platelet aggregation was inhibited below pH 6.5 and

135

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EFFECTS:pC02 & pH ON PLATELETS

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above pH 10.0, with maximal aggregation at pH 8.0 (4,5). These studies also showed similar increased ADP- and adrenaline-induced release with increases in pH. It has recently been recognized that PRP becomes alkaline if permitted to lose CO2 to the surrounding atmosphere, and that this pH change can be prevented by replacing the atmosphere above PRP with 5% CO2 (6,7,8). However, no systemic study of the simultaneous effects of different aggregating agents on platelet shape change and aggregation as a function of pCO2 and pH had been conducted. Furthermore, the experiments where pH was controlled with CO2 gave (4,5,7,8).Finally, conflicting results no direct measurements of both pH and pCO2 have been reported to-date, while many labs have continued to simply store PRP in an air atmosphere. We have therefore set out to measure the changes in pH, pCO2 and pO2 during preparation and storage of blood samples, and to examine the effects of pCO2 and pH on platelet shape change and aggreSince platelet function can change with incubation time even when pH gation. is kept constant (4,8,g), we have compared samples at identical times in order to ascertain time-independent changes.

MATERIALS

AND METHODS

Human and rabbit blood was collected by venipuncture and by ear arterial puncture respectively, mixed with anticoagulants and centrifuged, as previously described for the preparation of citrate-PRP (2), except that the citrate to maintain normal pH and was dissolved in Tyrode’s solution (pH 7.4) in order maintain normal concentrations of blood ions. Heparinized PRP was similarly prepared, using 9 vol. of blood mixed with 1 vol. of heparin-Tyrode’s solution, To keep constant pCO2 and with no evidence for any spontaneous aggregation. pH, we have modified a simple method (7) where the air over a given sample is displaced with appropriate concentrations of CO2, 02 and N2 under 1 atmosphere and the polypropylene tube (I cm in diameter and 7.3 cm in length) or siliconized cuvette (6.9 mm in diameter and 45 mm in length) is then covered with paraffin film. This procedure ailowed for constant pCO2 and pH for at least four hours. All the samples were kept at 37oC throughout the experiment. Platelet shape change and primary and secondary aggregation were assessed with an aggregometer (Payton Associates Ltd., Scarborough, Ontario, Canada), at 37oC, and with the Payton stir bar (6 mn in length and 1 nnn in diameter) at speed 1,000 r.p.m. (2). Samples of PRP (0.45 ml) were placed in the siliconized cuvettes, into which 5 ul of various concentrations of aggregating agents were added. To examine the relative effects of pCO2 and pH, these samples were compared with control samples kept under 1 atmosphere air and covered with paraffin film with post-bleeding times ranging fran 90 to 150 minutes. The pH, pCO2 and pO2 of a given sample was measured within 1 minute with the EMS-3MK2 Blood Along with every aggregometry Micro System (Radiometer, Copenhagen) at 39OC. an al iquot (0.1 ml) of one of twin samples was taken at the same measurement, time in a stoppered syringe and injected into the capillary tube of Blood Micro Since we found that the measurement was System for pH and gas measurement. not significantly different from that obtained by simply taking 0.1 ml of PRP frcnn the sample in the aggregometer after recording a response, sane data was collected in this simple way.

line were

Heparin (158 units/mg), (all from Sigma Chem. dissolved in Tyrode’s

adenosine diphosphate (ADP), serotonin, Co.), and trisodium citrate (J.T. Baker solution with a final pH of 7.4.

adrenaChem. Co.)

137

EFFECTS:pC02 & pH ON PLATELETS

Vol.10,No.l

RESULTS We found changes in pH, pCO2 and pO2 of blood samples in tubes and in covered with paraffin film at room temperature, especially following cuvettes, in which typically pH increased from centrifugation and removal of red cells, 7.3 to 7.4, pCO2 decreased from 55 to 40 mmhg, and pO2 jumped from 20 to I30 Greater changes occurred mmHg and then gradually continued to change (Fig. I). at 37W but the preparations were kept at this temperature in order to prevent spontaneous platelet shape change from discs to spiny spheres known to occur at room temperature (2,lO).

C-BLOOD

140

t 100

FIG.

2

E --t

The changes of pH, pCO2 and pop of the blood and PRP samoles. anticoaaulated with citrate-Tyrode’buffer, in tube or cuvette in air, covered with paraffin film during processing and storage at room temperature at 37oC. The dotted line indicates the measurement of titrated-PRP after centrifuqation.

0

a

1

50.

..~ 7.6 77I D

7.5 -

7.3 55-

p

40-

: 0” u,

..A

..

P

.-\.

25.

l2L

\ ?? \:z*

.‘\

0

4.

60 T,ME

90

120

150

,m,n,

Incubation of the whole blood and PRP samples with 5 to 7% CO2 in air or oxygen yielded constant pH, with no changes in pC02, for rabbit arterial and small decreases in pCO2 for human venous samples, independently samples, This method can allow for of large and predictable changes in pop (Table I). constant pH and pCO2 for at least four hours, similar to Han and Ardlie’s observation (8).

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EFFECTS:pCOZ & pH ON PLATELETS

138

TABLE

I

pH, pCO2 and pO2 Values of Human and Rabbit Blood and Platelet-rich Plasma Under Various Conditions. The platelet counts of human and rabbit c.PRP samples are 350-550 x 103 and 526-1006x 103 per ul respectively. The hematocrits are 39.5 f 3.X and 42.5 + 3.5%, correspondingly.

Source

Blood Donor

Human

Antecubi Vein

Samples

ta

I

(n)‘(

Post-bleeding Time(minute)

c. Blood # (5)

D

c. PRP in air (5) 120 CO2c. PRP in 5-p/o air mixture (3) 45-240

Rabbit

Ear

Artery

c. Blood (5) c. PRP in air (5) c. PRP in 5% CO2air mixture (3)

= number whole blood lent results.

of different donors. for 4 of 5 experiments, @ Mean + S.D.

#

7.30-1 .07@ 54 + 7 7.752 .05 7.352

23 + 3

I4 f 4

105 _I I5

41 + 1

100 + IO

32

300

120 f

.05

0 120

7.36+ .I0 7.752 .05

30 f 3 12 _r2

45-240

7.35+ .I5

29 f 4

100 + IO IO

127 k 2

c. PRP in 5% CO295% 02 mixture (2)

“n

PH

c = citrate and 0.10

250 + 50 of M in

0.13 M final concentration one experiment with equiva-

in

Figures 2 and 3 show typical results for heparinized PRP. Figure 2 illustrates that the controlled normal pCO2 (35 mmHg) and pH (7.45) conditions gave decreases selectively in primary aggregation induced by 7 uM adrenaline and in secondary aggregation by 20 uM serotonin for human heparinized PRP, as compared to low pCO2 and high pH conditions. In the case of rabbit heparinized PRP where much higher doses were needed to produce aggregation, a similar inhibition of primary aggregation with adrenaline was observed, while marked inhibition of both primary and secondary aggregation was observed for serotonin. However, ADP-induced primary aggregation was insensitive to the pC02 and pH changes in the rabbit for similar studies with heparinized PRP or titrated (0.2%) PRP preparations (Fig. 4-(l), (2)), with parallel results observed for correspond i ng human p repa rat i ons (not shown) . Interestingly, increasing the concentration of sodium citrate in the blood preparations leads to marked inhibition of ADP-induced primary aggregation for rabbit PRP (Fig. 4-(3),(4)). Much smaller differences were observed for human titrated (0.3%) PRP unless the comparison was made with higher concentrations of CO2 (not shown). The extent and rate of initial decrease in light transmission following additions of ADP and serotonin, believed to be associated with shape changes in platelets were not significantly altered in these samples where inhibition (2,3,11,12), of primary and/or secondary aggregation occured (Figs. 2-4).

Vol.10,No.l

EFFECTS:pC02 & pH ON PLATELETS

HUMAN

hcp.

PRP

(5

139

U ml-’ )

A

t

7 pM

ADRENALINE

PC02

20 PM SEROTONIN

1

18.3

7.72

Ii

3’LO

1.45

I

TIME

FIG.

PH

A

I min

2

Aggregometry tracings showing platelet shape change and aggregation responses of human heparinized (final concentration: 5 units/ml) PRP for (A) low pCO2 and high pH and (B) physiologically normal pCO2 and pH. (pCO2 is shown in Arrows indicate the addition of agents, with final concentrations mmHg). indicated.

RABBIT

L

hep.

PRP

(5

)

u ml-’

00 A

i 50 c

b

[+

B

PC02

PH

16.8

7.69

37.5

7.36

FIG.3 t 200 @,I ADRENALINE

PC02 25.3

7.57

B

44.0

7.29

i mM SEROTONIN TIME

PH

A

I min

Aggregomet ry t raci ngs for rabbit heparinized PRP (as Fig. 2).

Vol.10,No.l

EFFECTS:pC02 & pH ON PLATELETS

140

: -ADP

RABBIT

PRP

I)hep.

(Nml-‘1

( 2 PM)

2) c. (0.29%)

t 4)

c.

( 0.43 % )

‘TF A

B

:

:

I

TIME

I

min

FIG.

A

PC02 18.0 24.0

PH 7.70 2.04

B

36.0 2 8.0

7.39 2.06

4

Platelet shape change and aggregation response induced by 2 uM ADP (addition at arrows) for (A) low pCO2 and high pH and (B) physiologically normal pCO2 heparinized (5 units/ml) PRP, and (2) - (4): PRP at increasand pH for (1): ing citrate concentrations (final concentrations in whole blood shown in pH was measured immediately after a response, brackets).

When rabbit titrated PRP at low pCO2 and high pH was successively exposed the primary aggregation resulting from potentiato adrenaline and serotonin, tion by adrenaline was observed (Fig. s-sample A). At the controlled normal pCO2 and pH conditions, however, only the similar decrease in light transmission associated with the platelet shape change was observed (Fig. S-sample Addition of 5 ul of I mM sodium hydroxide solution to the PRP of twin B). seconds before introducing serotonin, led to the same shape sample 8, thirty This was change and primary aggregation as observed in sample A (Fig. SC). associated however with a rise in pH and a decrease in pCO2, with no signifiessentially identical cant change in ~02, making pCO2, pH and ~02 conditions for samples A and C.

RABBIT

h 9 e

50,

141

EFFECTS:pC02 & pH ON PLATELETS

Vol.10,No.l

c.

NaOH (10s3 M) ( B-K

PRP

( 0.43% )

)

1

PCO*

1

zI_

3o T;

ADRENALINE

z

PH

PO2

C 23.0 A 22.0

7.56 7.58

92.3 89.9

6

7.34

88.4

40.0

I

I

TIME

I

I min

SEROTONIN

FIG.5 Reversibility of effects of low pCO2 and high pH on serotonin-induced platelet aggregation potentiated with adrenaline, Addition of 5 ul of 1 mM NaOH to the identical sample B, 30 seconds before addition of serotonin (shown by arrow), resulted in conversion to the new pCO2 and pH values shown for C. Gas and pH measurements were made immediately after the observed responses.

DISCUSSION The changes in pH, pCO2 and pO2 of human and rabbit plasma were greatest for PRP exposed to air following removal of the red cells, which can otherwise act as effective pH and pCO2 buffers. It is important to note that sealing PRP in a tube or a cuvette containing air did not effectively prevent the pCO2, pH and pO2 changes with time, which were almost identical to changes observed for tubes and cuvettes kept open to the air (not shown in Fig. 1 or Table I). The simplest way to control pH and pCO2 is to displace the air over the blood and PRP with a 5 to 7% C02-air mixture which can allow for constant pCO2 and pH for at least four hours. Variations in pO2 obtained by using air or pure oxygen with 5 to 7% CO2 did not affect the pCO2 and pH (Table 1). The simultaneous increase in pH and decrease in pCO2 observed upon addition of sodium hydroxide solution to titrated PRP at physiologic pCO2 and pH, with identical platelet responses to the aggregating agents as observed for PRP maintained in air for two hours (Fig. 5), suggests that the simultaneous effects of pH and pCO2 changes from physiologic conditions might indeed be readily studied by direct additions of acid or base to anticoagulated PRP, in the manner first suggested by Zucker (5). However, further studies to distinguish the effects of pCO2 from those of pH will be conducted with suitable buffer systems. When compared at low pCO2, high pH conditions, the controlled normal pCO2 and pH inhibited the primary aggregation by adrenaline and the secondary aggregation by serotonin in the human heparinized PRP (Fig. 2), and reduced the primary aggregation by adrenaline and the primary and secondary aggregation

EFFECTS:pC02

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& pH ON PLATELETS

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by serotonin in the rabbit heparinized PRP (Fig. 3). Although the secondary aggregation induced by serotonin is unusual (13), we added high concentrations of serotonin to the heparinized PRP and measured the stable phase of aggregation, which was similar to some other reports (14-16). Interestingly, ADPinduced primary aggregation in rabbit (Fig. 4-(l), (2)) and human heparinized or titrated (0.29%) PRP was insensitive to the pCO2, pH changes. Rabbit titrated (0.38%) PRP, however, was sensitive to these changes (Fig. 4-(3)) while the corresponding human preparation was insensitive unless a higher pCO2 was introduced. Most studies on platelet physiology have been conducted with 0.38% titrated PRP. Thus, Skoza et al (4) observed that the aggregation of human titrated PRP induced by 5 uM ADP was increased as pH rose fran 7 to 8. Oppositely, Han and Ardlie (8) reported that a decrease in pH from 8.3 to 7.2 was associated with an increased responsiveness to ADP. Nevertheless, Rogers (7) found that adrenaline-induced aggregation was decreased but ADP-induced primary aggregation was almost unchanged with pH of human titrated PRP varying from 7.6 to 7.2, which was similar to our results. The inhibitory effect of controlled normal pCO2 and pH on the ADP-induced primary aggregation of rabbit platelets was not observed for normal extracellular calcium and magnesium associated with the heparinized PRP (17) nor for the lower citrate concentrations in PRP (Fig. 4-(l), (2)), but was indeed observed with increasing citrate concentrations (Fig. 4-(3), (4)). Sodium citrate chelates ionic extracellular calcium and magnesium (17) and was recently reported to facilitate ADP-induced release reaction (18), although its actions in detail It has been shown that the binding of membrane bound calcium are sti I1 unknown. and magnesium increases with pH (19,20), because of decreased competitive bindthe binding of calcium and magnesium to ing by hydrogen ions (21). However, citrate would not be expected to change significantly between pH 7 to 8, since Based on the observathe dissociation constants of citric acid are below 6.4. tions that sufficient EDTA can prevent platelet aggregation but cannot significantly affect platelet shape change (4,ll ,22), the marked inhibition of ADPinduced aggregation at higher citrate concentration (Fig. 4-(3), (4)) might be related to the critical dependence of the pH, pC0 effects on a sufficient decrease in ionized calcium as found in the more hig i?ly titrated plasma. Magnesium can facilitate the disaggregation of rabbit platelets (23,24) but its role in the present experiments remains to be defined. The calculated osmolarity for our PRP preparations are: iso-osmolar for heparin (Tyrode’s)-PRP, where the average molecular weight of heparin is taken as 10,000 Dalton Units and average number of negative charges is 60 per mole (25); 1.30, 1.25 and 1.23 times iso-osmolar respectively for the 0.43%, 0.38% in contrast to 1.13 times iso-osmolar for and 0.29% ci trated (Tyrode’s)-PRP, However, platelet aggregation with ADP, 0.38% titrated (no Tyrode’s)-PRP. thrcnnbin and collagen have been found unchanged for osmolarity variations at least 1.30 times iso-osmolar (Mason, R.G., private canmunication; 26). We therefore feel we need not consider the effects of these osmolarity changes in the different PRP preparations. It does not appear that the effects of pCO2 and pH upon aggregation induced by different agonists studied can be adequately explained by one mechanism. The direct inhibitory effect of CO2 has been reported for airway smooth muscle CO2 relaxed the serotonin-induced muscle preparations by Duckles et al (27). These contraction but could not affect the acetylcholine-induced contraction. authors suggested that the site of action of CO2 was not on the intrinsic excitation coupling per se, but probably on the binding of drug to receptors or on the specific transmission process from the receptors to the contractile elements.

In our

studies

on platelets,

the

high

pCO2 and

low pH conditions

EFFECTS:pCOZ

Vol.10,No.l

& pH ON PLATELETS

143

inhibited adrenalineand serotonin-induced primary and/or secondary aggregation as well as adrenaline-potentiated, serotonin-induced aggregation, but could not significantly affect ADP-induced primary aggregation. Based on the model proposed by Duckles et al, we suggest that the pCO2 and pH, within the range studied, does not alter any of the intrinsic primary and secondary aggregation mechanistic steps, common to ADP, adrenaline and serotonin. Platelet membrane surface receptors which lead to platelet shape change and in turn lead to aggregation have been proposed, based on studies of agonist binding, antagonism and refractoriness (28,29). The platelet shape change from discs to spheres is associated with the decrease of light transmission and disappearance of oscillations on aggregometer tracings (11,12). It is noted that the apparent shape change observed for adrenaline at both normal and high pH (Fig. 2 & 3) is in accord with a literature review (30) that adrenaline may have induced platelet aggregation with at least a partial shape change. However, since the mode of shape change by adrenaline may be different from that by ADP and serotonin (30). we do not consider it here. The rate and extent of ADPand serotonin-induced shape change reflected by the changes in the aggregometer tracings were not significantly changed at the controlled normal pCO2 and pH Therefore we further suggest that the inhibitory effect of normal (Fig. 2-5). pCO2 and pH does not change the binding of aggregating agents to their receptors, nor reduces the sensitivity of the receptors to aggregating agents leading to but possibly affects the transmission process shape change and aggregation, between the receptors and the final aggregation step. This may involve a decrease in membrane bound calci urn. Excepting serotonin-induced shape change, rabbit titrated (0.38%) PRP was unresponsive to serotonin or adrenaline (31 ,32) unless the anticoagulant was heparin (Fig.,3) or low concentrations of sodium citrate, or unless exogenous calcium was added (32). This transmission process may be calcium dependent with different thresholds depending on the aggregating agents and platelet species studied, Suzuki et al (33) observed different degrees of platelet aggregation for rabbit portal and arterial blood where there were no differences in the concenThey were seeking to isolate an trations of proteins, serotonin, and ions. inhibitory substance from the portal blood or an aggregation-promoting substance from the arterial blood. The usually higher pCO2 in venous blood might contribute directly to the observed inhibition of platelet aggregation.

ACKNOWLEDGEMENT We for the and Mr. Council

gratefully acknowledge assistance from Mr. M. Day and Dr. R. Williams use of their BMS-3MK2 Blood Micro System Radiometer; Dr. R. Panjwani C. Becker for technical assistance; and the Canadian Medical Research for a studentship (S.S. Tang) and grant MA-3612.

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The effects of pCO2 and pH on platelet shape change and aggregation for human and rabbit platelet-rich plasma.

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