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Quantitation of in vivo platelet aggregation by the platelet aggregate ratio method
THROMBOSIS
RESEARCH
Volume
BRIEF
10,
Pages
629-633.Pergamon
Press,
1977.
Printed
in Gt. Britain,
COMMUNICATION
QUANTITATIONOF IN VIVO PLATELET AGGRBGATION BY TRB PLATELET AGGREGATE RATIO METHOD
Thomas S. Bums and Robert N. Saunders Department of BiologicalResearch, Searle Laboratories Chicago, Illinois 60680 USA
(Received 3.11.1976. Accepted by Editor L. Weiss. Received by Executive Editorial Office 25.2.1977)
INTRODUCTION
A new method for quantitativelydetecting platelet aggregates-in vivo has been reported by Wu and Roak (1). Utilizing this method, these investigators have demonstratedincreased circulatingplatelet aggregates in patients with acute myocardial infarction,transient ischwic attacks and acute peripheralarterial insufficiency(1,2). The return of reduced platelet aggregate ratios (increasedcirculatingaggregates) tomrds normal values in two patients utilizing antiplatelettherapy has been reported by Hoak (3). The use of the platelet aggregate ratio method appears to be advantageous in the selectionof patients for antiplatelettherapy and the waluation of the effectivenessof the therapeuticregimen.
The utilizationof a similar method for discovery of new antiplatelet agents may lead to an improved correlationbetween animal test results and clinical response to these agents. Such a method for evaluationof agents designed to reduce platelet aggregate formationutilizing prostaglsndinEl (PGEl),a known platelet aggregation inhibitor (3), as a positive control is described here.
629
IN VIVO PLATELET
630
AGGREGATION
Vol.lO,No.4
MBTRODS I The platelet aggregate ratio method of Wu and Hoak (1) for the estimationof circulatingplatelet aggregateswas slightlymodified. Trisodium citrate at a final concentrationof 0.38% was used instead of BDTA. Adenosine-5'-diphosphate (ADP) was used as the platelet aggregation-inducing agent.
Initially, the relationshipbetween ADP dose and platelet aggregate ratio was determined. hale Charles River CD rats weighing between 365-450 g were Infused intravanouslywith ADP in concentrationsfrom 1 to 15 me/ml in phosphate-bufferedsaline (PBS) at the rate of 0.1 mlfmin for one minute. Two 1 ml blood samples were withdrawn from the iaferior vena cava at the conclusion of the ADP infusion. One sample was drawn into a polypropylenesyringe containing 4 ml of buffered citrate/formaliasolution and the second sample into 4 ml of buffered citrate solution. Buffered citrate/formalinwas prepared by mixing 10 ml of 1 M PBS (pR 7.4), 0.475 g trisodium citrate, 2.7 ml of 37% formalin solution, and diluting vith distilled water to 100 ml.
Buffered
citrate was prepared the same way except the formalin was eliminated. Platelet-rich plasma (PRP) was prepared from each sample by centrifugingat 170 g for 14 min. at room temperature. Platelet counts were done microscopically using hemacytometers. The ratio of the platelet count of the citrate/formalin PRP to the platelet count of the citrate PRP was determined. Since circulating platelet aggregatesare fixed by the formalin and centrifugedout during PRP preparation,the ratio equals 1 when no circulatingaggregatesare present and decreases as the number of aggregates increase.
The effects of a known aggregation inhibitor,PGE1, on aggregate ratios were determinedby pretreatingthe rata with concentrationsof PGEl from 12.5 to 200 ug/ml in PBS Infused i.v. into the tail vein at the rate of 0.2 ml/min for 5 min. This was followed Wiatelp
by an infusion of 0.1 ml of ADP at
5 mglml for 1 min. The platelet aggregate ratios were determlned as deacribed above. Statisticalevaluationof the data ME tests.
done with two-tailedStudent t
Vol.lO,No.4
IN VIVO PLATELET
AGGREGATION
631
RESULTS
Circulatingplatelet aggregates as determined by reduced platelet aggregate ratios were produced by the infusion of ADP as indicated in Fig. 1. The mean of the control aggregate was observed In the aggregate than0.9mg/kg.
ratios
ratio
was 0.94 f S.&M. 0.02. A plateau
response with ADP concentrationsgreater
The increase In the ratio above the control value for the
lowest ADP concentration
(0.25 lag/kg)is not readily explainable. For 'drug
evaluationan ADP dose of 1.25 n&kg
(5 mg/ml) was selected. This amount of
ADP consistentlygave a low platelet aggregate ratio.
?? p*o.o5
0
1.0
21) ADP
Dose (mglkg)
3.0
1
Effect of ADP on the platelet aggregate ratio _in viva in rats. A total volume of 0.1 ml of ADP, in concentrationsranging from O-15 mg/ml, was infused for 1 min. Bach value is the mean f S.E.H. The number of obecrpations are Micated. Statisticalcomparisons made to ADP control ratio (0.94 f 0.02).
PGEl Increased the platelet aggregate
ratio towards norms1 in a dose-
related manner as illustratedin Fig. 2. ADP infusion at 1.25 mg/kg followingPBS (0.2 ml/mln. for 5 min.) withaut PGEl yielded a mean aggregate ratio of 0.47 f S.E.M. 0.02, which was used as the basis for statfstical comparison. PGEl at 12.8 Irg/kg/min. for 5 min. significantly(p (0.05) increased the mean aggregate ratio to 0.56 f S.E.M. 0.02. The infusion of PGEl at 73.7 ug/kg/min. for 5 min. returned the aggregate ratio to within the control 95% confidence limits of 0.88 to 0.98.
632
IN VIVO PLATELET
AGGREGATION
Vol.lO,No.4
1.a
20
40
60
80
100
PGE, Dose (ug/kg/min) PIG. 2
Effect of PGEl on ADP-inducedplatelet aggregate formation-in vivo In rats. PGEl concentrationsranging from G-200 pg/ml were infused for 5 min. at 0.2 ml/min. followed by 0.1 ml of ADP (5 mg/ml) for 1 min. Each value is the mean + S.E.M. of the number of observationsindicated. Statisticalcompariaons made to PGEl control ratio (0.47 f 0.02).
DISCUSSION
The platelet aggregate ratio method may become a valuable discriminator for patient selection for antiplatelettherapy. An animal model using the platelet aggregate ratio method for the preliminaryevaluationof antiplatelet agents may also be useful. Wu -et al. (5) have reported that the platelet aggregate ratio is decreased, Indicatingaggregate formation, in rhesus monkeys during the early phase of diet-inducedatherosclerosis. This model, although intriguing,would not lend itself to early evaluationof potential antiplateletagents. The method we report for waluatlon of antiplatelet agents allows an early determinationof potential activity with a minimal time and compound usage. The method has been proven reproduciblewith minimal animal variation using PGEl as the standard platelet antlaggregation compound. We suggest that the use of this test may potentiallyimprove the correlationbetween animal evaluationand clinical effectivenessof antiplatelet therapy.
Vol.lO,No.4
IN VIVO PLATELET
A&XEGATION
633
1. WC, K.K. and BOAK, J.C. A new method for the quantitativedetection of platelet aggregates in patients with arterial insufficieucy. Lancet 2, 924, 1974. 2. WU, K.K. and HOAK, J.C. Increased platelet aggregates in patients with transient ischemlc attacks. Stroke 6, 521, 1975. 3. HOAK, J.C. In discussion,Platelet aggregation secondary to coronary obstruction,by S. Moore. Circulation 53, Supp. I, I-69, 1976. 4.
BOUSSRR, M.
ProstaglandinEl and platelets. Biomedicine 18, 95, 1973.
5. WJ, K.K.. ARMSTRONG,XL., HOAK, J.C. and MEGAN, M.B. Platelet aggregates In hypercholesterolemicrhesus monkeys. Thromb. Res. 7, 917, 1975.
RESEARCH
Volume
BRIEF
10,
Pages
629-633.Pergamon
Press,
1977.
Printed
in Gt. Britain,
COMMUNICATION
QUANTITATIONOF IN VIVO PLATELET AGGRBGATION BY TRB PLATELET AGGREGATE RATIO METHOD
Thomas S. Bums and Robert N. Saunders Department of BiologicalResearch, Searle Laboratories Chicago, Illinois 60680 USA
(Received 3.11.1976. Accepted by Editor L. Weiss. Received by Executive Editorial Office 25.2.1977)
INTRODUCTION
A new method for quantitativelydetecting platelet aggregates-in vivo has been reported by Wu and Roak (1). Utilizing this method, these investigators have demonstratedincreased circulatingplatelet aggregates in patients with acute myocardial infarction,transient ischwic attacks and acute peripheralarterial insufficiency(1,2). The return of reduced platelet aggregate ratios (increasedcirculatingaggregates) tomrds normal values in two patients utilizing antiplatelettherapy has been reported by Hoak (3). The use of the platelet aggregate ratio method appears to be advantageous in the selectionof patients for antiplatelettherapy and the waluation of the effectivenessof the therapeuticregimen.
The utilizationof a similar method for discovery of new antiplatelet agents may lead to an improved correlationbetween animal test results and clinical response to these agents. Such a method for evaluationof agents designed to reduce platelet aggregate formationutilizing prostaglsndinEl (PGEl),a known platelet aggregation inhibitor (3), as a positive control is described here.
629
IN VIVO PLATELET
630
AGGREGATION
Vol.lO,No.4
MBTRODS I The platelet aggregate ratio method of Wu and Hoak (1) for the estimationof circulatingplatelet aggregateswas slightlymodified. Trisodium citrate at a final concentrationof 0.38% was used instead of BDTA. Adenosine-5'-diphosphate (ADP) was used as the platelet aggregation-inducing agent.
Initially, the relationshipbetween ADP dose and platelet aggregate ratio was determined. hale Charles River CD rats weighing between 365-450 g were Infused intravanouslywith ADP in concentrationsfrom 1 to 15 me/ml in phosphate-bufferedsaline (PBS) at the rate of 0.1 mlfmin for one minute. Two 1 ml blood samples were withdrawn from the iaferior vena cava at the conclusion of the ADP infusion. One sample was drawn into a polypropylenesyringe containing 4 ml of buffered citrate/formaliasolution and the second sample into 4 ml of buffered citrate solution. Buffered citrate/formalinwas prepared by mixing 10 ml of 1 M PBS (pR 7.4), 0.475 g trisodium citrate, 2.7 ml of 37% formalin solution, and diluting vith distilled water to 100 ml.
Buffered
citrate was prepared the same way except the formalin was eliminated. Platelet-rich plasma (PRP) was prepared from each sample by centrifugingat 170 g for 14 min. at room temperature. Platelet counts were done microscopically using hemacytometers. The ratio of the platelet count of the citrate/formalin PRP to the platelet count of the citrate PRP was determined. Since circulating platelet aggregatesare fixed by the formalin and centrifugedout during PRP preparation,the ratio equals 1 when no circulatingaggregatesare present and decreases as the number of aggregates increase.
The effects of a known aggregation inhibitor,PGE1, on aggregate ratios were determinedby pretreatingthe rata with concentrationsof PGEl from 12.5 to 200 ug/ml in PBS Infused i.v. into the tail vein at the rate of 0.2 ml/min for 5 min. This was followed Wiatelp
by an infusion of 0.1 ml of ADP at
5 mglml for 1 min. The platelet aggregate ratios were determlned as deacribed above. Statisticalevaluationof the data ME tests.
done with two-tailedStudent t
Vol.lO,No.4
IN VIVO PLATELET
AGGREGATION
631
RESULTS
Circulatingplatelet aggregates as determined by reduced platelet aggregate ratios were produced by the infusion of ADP as indicated in Fig. 1. The mean of the control aggregate was observed In the aggregate than0.9mg/kg.
ratios
ratio
was 0.94 f S.&M. 0.02. A plateau
response with ADP concentrationsgreater
The increase In the ratio above the control value for the
lowest ADP concentration
(0.25 lag/kg)is not readily explainable. For 'drug
evaluationan ADP dose of 1.25 n&kg
(5 mg/ml) was selected. This amount of
ADP consistentlygave a low platelet aggregate ratio.
?? p*o.o5
0
1.0
21) ADP
Dose (mglkg)
3.0
1
Effect of ADP on the platelet aggregate ratio _in viva in rats. A total volume of 0.1 ml of ADP, in concentrationsranging from O-15 mg/ml, was infused for 1 min. Bach value is the mean f S.E.H. The number of obecrpations are Micated. Statisticalcomparisons made to ADP control ratio (0.94 f 0.02).
PGEl Increased the platelet aggregate
ratio towards norms1 in a dose-
related manner as illustratedin Fig. 2. ADP infusion at 1.25 mg/kg followingPBS (0.2 ml/mln. for 5 min.) withaut PGEl yielded a mean aggregate ratio of 0.47 f S.E.M. 0.02, which was used as the basis for statfstical comparison. PGEl at 12.8 Irg/kg/min. for 5 min. significantly(p (0.05) increased the mean aggregate ratio to 0.56 f S.E.M. 0.02. The infusion of PGEl at 73.7 ug/kg/min. for 5 min. returned the aggregate ratio to within the control 95% confidence limits of 0.88 to 0.98.
632
IN VIVO PLATELET
AGGREGATION
Vol.lO,No.4
1.a
20
40
60
80
100
PGE, Dose (ug/kg/min) PIG. 2
Effect of PGEl on ADP-inducedplatelet aggregate formation-in vivo In rats. PGEl concentrationsranging from G-200 pg/ml were infused for 5 min. at 0.2 ml/min. followed by 0.1 ml of ADP (5 mg/ml) for 1 min. Each value is the mean + S.E.M. of the number of observationsindicated. Statisticalcompariaons made to PGEl control ratio (0.47 f 0.02).
DISCUSSION
The platelet aggregate ratio method may become a valuable discriminator for patient selection for antiplatelettherapy. An animal model using the platelet aggregate ratio method for the preliminaryevaluationof antiplatelet agents may also be useful. Wu -et al. (5) have reported that the platelet aggregate ratio is decreased, Indicatingaggregate formation, in rhesus monkeys during the early phase of diet-inducedatherosclerosis. This model, although intriguing,would not lend itself to early evaluationof potential antiplateletagents. The method we report for waluatlon of antiplatelet agents allows an early determinationof potential activity with a minimal time and compound usage. The method has been proven reproduciblewith minimal animal variation using PGEl as the standard platelet antlaggregation compound. We suggest that the use of this test may potentiallyimprove the correlationbetween animal evaluationand clinical effectivenessof antiplatelet therapy.
Vol.lO,No.4
IN VIVO PLATELET
A&XEGATION
633
1. WC, K.K. and BOAK, J.C. A new method for the quantitativedetection of platelet aggregates in patients with arterial insufficieucy. Lancet 2, 924, 1974. 2. WU, K.K. and HOAK, J.C. Increased platelet aggregates in patients with transient ischemlc attacks. Stroke 6, 521, 1975. 3. HOAK, J.C. In discussion,Platelet aggregation secondary to coronary obstruction,by S. Moore. Circulation 53, Supp. I, I-69, 1976. 4.
BOUSSRR, M.
ProstaglandinEl and platelets. Biomedicine 18, 95, 1973.
5. WJ, K.K.. ARMSTRONG,XL., HOAK, J.C. and MEGAN, M.B. Platelet aggregates In hypercholesterolemicrhesus monkeys. Thromb. Res. 7, 917, 1975.