Antiaggregatory effects of thromboxane receptor antagonists in vivo

THROMBOSIS RESEARCH 40; 663-675, 1985

0049-3848/85 $3.00 t .OO Printed Copyright (c) 1985 Pergamon Press

ANTIAGGRECATORY

in the USA. Ltd. All rights

reserved.

EFFECTS OF THROMBOXANE RECEPTOR ANTAGONISTS IN VIVO

Harald Darius and Allan M. Lefer Department of Physiology Jefferson Medical College Thomas Jefferson University Philadelphia, Pennsylvania 19107 U.S.A.

(Received

13.8.1985; Accepted in revised by Editor P. Didisheim)

form 2.9.1985

ABSTRACT The antiaggregatory and antisecretory effects of two newly developed thromboxane receptor antagonists, BM-13,177 and SQ-29,548, were studied in an -in vivo model of platelet activation. Arterial platelet count and whole blood ATP concentrations were measured continuously on-line in the arterial blood of anesthetized rabbits. Injections of collagen decreased peripheral platelet count by 25% of initial value. ATP concentrations increased 50 to 100 nM during collagen challenge. SQ-29,548 and BM-13,177 dose-dependently reduced platelet loss to about 50% of that observed in vehicle treated Injection of arachidonic acid (AA) or 9,11-methanoepoxy-PCH2 animals. resulted in sudden death of the animals associated with a 67 to 69% decrease in platelet count and a marked release of ATP. Pretreatment with SQ29,548 or BM-13,177 increased survival rates from 0 to lOO%, and reduced or totally inhibited ATP secretion and decreases in platelet count. In contrast, the thromboxane synthetase inhibitor, dazoxiben, was effective in inhibiting AA induced sudden death, but was without any effect when 9,11methanoepoxy-PGH was used as the challenging agent. We conclude that SQ-29,548 and BM-13,177 are effective in antagonizing the effects of

KEY WORDS:

platelet aggregation in vivo, platelet secretion, thromboxane receptor antagonists, thromboxane synthetase inhibitors

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prostaglandin endoperoxides and thromboxane A2 in vivo but do not influence the prostaglandin independent component>f?&agen induced In eicosanoid induced sudden death models, both platelet activation. thromboxane antagonists are highly effective anti-platelet agents in Contrast to dazoxiben, which is without any effect on endoperoxide induced platelet activation or sudden death. INTRODUCTION Thromboxane A2 (TxA2) is recognized as a potent platelet proaggregatory agent and constrictor of vascular and pulmonary smooth muscle. Increased concentrations of TxA metabolites have been found in a variety of experimental and clinical situations invo T* vmg activation of platelets. In animal models of arterial thrombosis and acute myocardial ischemia and in patients with angina1 attacks and acute myocardial infarction, plasma TxA2 concentrations were shown to be elevated (1, 2). TxA2 also seems to play an important role in the pathophysiological sequelae of circulatory shock (3). One approach to limit platelet inactivation is by elevating the intracellular concentrations of CAMP by stimulators of the adenylate cyclase (e.g., prostacyclin or inhibitors of CAMP-phosphodiesterase). However, these drugs are not specific for platelet phosphodiesterases, also influencing the concentration of cyclic nucleotides in vascular smooth muscle resulting in a vasodilator effect (4). Abolition of thromboxane formation by cyclooxygenase inhibition may also reduce the formation of PC1 by the endothelium and thereby remove one of the physiologic modulators of TxA2 at & e same time. More promising approaches for therapeutic measures against the deleterious effects of TxA2 are inhibition of thromboxane synthetase and specific antagonism of the TxA2 receptor. Although thromboxane synthetase inhibitors reduce the formation of TxA , they do not prevent the synthesis of prostaglandin endoperoxides. This could lead of endoperoxides in the circulation, which are potent to 3 he accumulation vasoconstrictors and proaggregatory agents on their own. In addition, concentrations of endoperoxides which are not metabolized, may lead to the formation of other prostaglandins, particularly PGD2 and PGF2 cI which are either vasoconstrictor or cardiodepressant. These difficulties are circumvented by thromboxane receptor antagonists which inhibit the activation of endoperoxide-thromboxane receptors on platelets and vascular tissue. In contrast to the first generation of thromboxane receptor the formation of TxA2 at higher concentrations antagonists like PTA2, which inhibited (5), the newly introduced compounds BM-13,177 (6, 7) and SQ-29,548 (8, 9) lack thromboxane synthetase inhibitory activity. Both antagonists prevent the activation of vascular endoperoxide-thromboxane receptors in a competitive manner and inhibit the aggregation of platelets --in vitro induced by thromboxane dependent stimuli. The purpose of this study was to compare SQ-29,548 and BM-13,177 with the thromboxane synthetase inhibitor dazoxiben for their --in vivo antiaggregatory effects and for their ability to prevent endoperoxide induced sudden death. MATERIALS

AND METHODS

Male New Zealand rabbits weighing 2.5 - 3.5 kg were anesthetized by intravenous injection of pentobarbital sodium (30 mg/kg, i.v.). A tracheal cannula was introduced, and the animals were allowed to breathe spontaneously. Polyethylene catheters were inserted into the right carotid artery for monitoring arterial blood pressure and into the left femoral vein for administration of drugs or their vehicle. Additionally, a special double-lumen cannula was introduced into the right femoral artery which allowed

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continuous withdrawal of blood at a rate of 70 ul/min. The construction of this cannula allowed immediate anticoagulation of the blood by sodium-citrate (7:1, v:v) without allowing the anticoagulant to reach the animal’s circulation. The anticoagulated blood was then mixed with luciferin-luciferase reagent (Du Pont, Wilmington, DE) at a ratio of 0.085:0.015 ml/min. The ATP concentration of the blood was measured continuously in a bioluminescence meter (Lumi-Flow, Chronolog Corp., Havertown, PA) (11) having a detection limit of 10 nM for ATP in whole blood. Blood leaving the luminescence device was mixed with a solution of 1% ammonium oxalate and 0.002% saponin to lyse the red cells and to continuously determine the platelet count on-line with a flow-through particle counter (Technicon Autocounter, Tarrytown, NY) (10). At the completion of surgery, a stabilization period of 20 min was allowed while the luminescence biometer was calibrated with exogenous ATP added to the blood at the level of the intra-arterial cannula. This ensured that the exogenous ATP had essentially the same contact time with blood and thereby the same rate of metabolism as ATP released _in vivo into the catheter. Following the stabilization period, platelet aggregation _in vivo was induced by two consecutive intravenous injections of collagen (80 ug/kg; Chronolog Corp., Havertown, PA) with a 40 min interval. Ten minutes before the second collagen, either SQ-29,548 (0.7 or 2 mg/kg; Squibb Institute, Princeton, NJ) or BM-13,177 (15 or 50 mg/kg; Boehringer-Mannheim, FRG) or their respective vehicles were injected intravenously. The vehicles were ethanol (200 ul) in sodium carbonate (10 mM; 600 ul) for SQ-29,548 and 0.9% NaCl adjusted to pH 10 by addition of sodium hydroxide for BM-13,177. The sudden death experiments were performed by rapid intravenous injection of sodium-arachidonate (AA) (1.1 mg/kg; Nu-Chek Corp., Elysian, MN) in Na CO (10 mM), or the prostglandin endoperoxide analog 9,11-methanoepoxy-PGH2 (U-44612 30 ug/kg; Upjohn Co., Kalamazoo, MI) in 0.9% NaCl containing 1% ethanol. These doses chosen represent a LDl o. Ten minutes before injection of the challenging agents, either SQ29,548 (2 mg/kgj), BM-13,177 (50 mg/kg) or dazoxiben (2 mg/kg dissolved in saline; Pfizer Inc., Groton, CT) or their respective vehicles were given intravenously. Most of the treated animals surviving AA and U-46,619 challenge showed a remarkable recovery within 15 min indicated by a blood pressure of >90% of initial value. In a few rabbits, we injected a second dose of inhibitor at 1 mg/kg for SQ-29,548 and dazoxiben or 25 mg/kg for BM-13,177, which was followed by a second injection of U-46,619. Statistical

Analyses

,411 results are given as means + SEM of n observations. Comparisons between groups were accomplished by Student’s t-test for paired data and pXO.05 was accepted as statistical significance. In multi-group comparisons, analysis of variance (ANOVA) was used to compare groups. RESULTS Collagen induced aggregation The initial platelet counts of all the rabbits involved in this study were 504 + 21 x lOOO/ul and no significant differences existed between the means of different groups. Two consecutive injections of collagen in rabbits led to decreases in platelet count of 25 + 2 and 24 + 2% of initial value. The collagen-induced decreases in platelet count were markedly attenuated when the animals were treated before the second challenge with the thromboxane receptor antagonists SQ-29,548 or BM-13,177, The effects were statistically

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THROMBOXANE RECEPTORANTAGONISTS

significant I).

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40, No. 5

for BM-13,177 at 15 and 50 mg/kg and for SQ-29,548 at 2 mg/kg (p
The increases in whole blood ATP concentrations observed simultaneously with the fall in platelet count were 100 + 28 and 46 2 12 nM in the vehicle treated animals. There is a significant difference between the ATP released by the first and the second collagen injections (pCO.05). However, pretreatment with the thromboxane receptor antagonists at high doses reduced the second ATP release in response to collagen injection even further. This reduced second ATP release was significantly lower than the ATP release during the first challenge but was not significantly different from vehicle treated controls (Table 1).

NS

30 7

1

T

20

10

0

-

Vehicle + Collagen

\

15 (mg/kg) T BM-13,177 CollZgen

50

L

0.7

hg/kg)2.0 T SQ-29,548 Colligen

Platelet aggregate formation measured as decrease in arterial platelet count after injection of collagen (80 &kg). The results of the two consecutive injections are presented as pairs of columns with intermediate injection of vehicle, B&13,177 (I5 or 50 mg/kg) or SQ-29,548 (0.7 or 2 mg/kg). The numbers inside the columns show the number of animals in each group. ** = pCO.01 second collagen versus first collagen. NS = not significantly different.

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Eicosanoid

THROMBOXANE RECEPTOR

667

ANTAGONISTS

induced sudden death

Intravenous injection of sodium arachidonate (1.1 mg/kg) into rabbits produced a rapid respiratory arrest, convulsions and an acute drop in mean arterial blood pressure which resulted in death of 6 out of 6 animals within 3-5 minutes. Pretreatment with BM13,177 (50 mg/kg), SQ-29,548 (2 mg/kg) or dazoxiben (2 mg/kg) protected the animals from AA-induced sudden death and resulted in survival of all the treated animals tested Lower doses of BM-13,177 tested at 3 and 15 mg/kg were only partially (Table 2). effective in protecting the animals against injected AA under our experimental The endoperoxide analog U-46,619, injected at a dose of 30 ug/kg, produced conditions. similar results to AA, and 5 out of 5 animals died within 2-3 minutes. In contrast to the thromboxane receptor antagonists BM-13,177 (50mg/kg) and SQ-29,548 (2 mg/kg) which endoperoxide induced sudden death, the effectively protected the animals from thromboxane synthetase inhibitor dazoxiben was totally ineffective against U-46,619 (Table 2).

Mean arterial blood pressures (MABP) of the rabbits were employed as an index of the overall hemodynamic situation. The initial MABP of the different groups ranged from 88 t 5 to 103 _L 3 mm Hg and were not significantly different from each other. Injection of AA resulted in the acute decrease in MABP to 5 mm Hg within 3-5 minutes Treatment with either of the thromboxane receptor resulting in death of the animals. antagonists or thromboxane synthetase inhibitors resulted in only transient hypotensive periods following AA injection with the lowest blood pressures between 30 and 40 mm Hg l-2 minutes after the AA challenge (Table 3). Thereafter the animals recovered quickly, and II out of 14 treated rabbits attained MABP of >90% of initial values within 15 minutes. The hypotensive period following eicosanoid challenge was totally abolished in the BM-13,177 treated animals challenged with U-46,619, while the MABP of the SQ29,548 treated rabbits decreased slightly. In contrast, all 7 rabbits pretreated with dazoxiben experienced acute decreases in MABP to 5 mm Hg and died within three minutes (Table 3).

TABLE1 Effects

Group

of Thromboxane Receptor Induced by Two Consecutive

_N

Increase

Antagonists on ATP Secretion In Vivo Injections of Collagen (80 ug/kg)

in Whole Blood ATP Concentration Collagen

I

r nM 1

Collagen

Vehicle

7

100 + 28

46 + 12*

B&13,177 15 mg/kg

6

87 _r 42

55 2 27*

50 mg/kg

8

96 + 26

19 _I IO”

E$g .

4

83 + 11

29 + II”

2.0 mg/kg

6

141 + 42

*pCO.O5 versus

Collagen

I. N = number

32 + 19” of experiments

II

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668

TABLE 2 Effects of Thromboxane Receptor Antagonists (SQ-29,548) and BM-13,177) and Thromboxane Synthetase Inhibitor (Dazoxiben) on Survival Rates in SodiumArachidonate and U-46,619-Induced Sudden Death in Rabbits Treatment

Survivors/Total -AA

% Survival

U-46,619

-AA

U-46,619

Vehicle

O/6

o/5

0

0

SQ-29,548 (2 mdkd

5/5

5/5

100

100

BM-13,177

515

5/5

100

100

414

o/7

100

0

(50 mg/kg) Dazoxiben (2 mgfkg) AA = arachidonic acid. Dose of AA = 1.1 mglkg; Dose of U-46,619 = 30 ug/kg.

TABLE 3 Mean Arterial Blood Pressures (MABP) in Response to Eicosanoid Challenge MABP (mm Hg) g

U-46,619 e control

n

0

6

102 ? 8

0

5

103 * 3

32 ? 7

5

96 t 7

71 Y!17

5

97 ? 5

38 + 3

5

88 ? 5

9625

5

101 + 10

38 + 6

4

93 ? 5

0

7

Treatment control Vehicle SQ-29,548

99 i 9

ti post

(2 mglkg) BM-13,177 (50 mg/kg) Dazoxiben (2 mglkg)

AA = arachidonic acid. Data given represent MABP before (control) and the lowest values measured within 10 minutes after challenge (post) with arachidonic acid (1.1 mg/kg) or U-46,619 (30 ng,/kg).

Vol.

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No.

THROMBOXANERECEPTOR ANTAGONISTS

5

80 80

E 3 0

40

H Q) z h

20 0

.-c

IL 6

AA

AA + 6th13,177

+ VEH

AA + SQ-29,548

AA + Daroxiben

20 0

-

5 U-46.6 + VEH

19

(5)

U-46,619 + 0M-13,177

7”

I

5

I

U-46,6 19 f SQ-29,548

U-46,6 19 + Dazoxiben

FIG. 2 Platelet aggregate formation measured as decrease in arterial platelet count after injection of arachidonic acid (1.1 mg/kg; upper panel) or The animals were pretreated with U-46,619 (30 @kg; lower panel). vehicle, BM-13,177 (50 mg/kg), SQ-29,548 (2 mg/kg) or dazoxiben (2 mg/kg) The numbers inside the columns represent the 10 min before challenge. number of animals in each group. ** = pCO.01 versus vehicle treated group. NS = not significantly different.

669

670

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THROMBOXANE RECEPTORANTAGONISTS

2000

1500 ll E c 1000 ._6

u

5 E

500

$

0

0g

6 AA + VW

AA

AA

+

+

-t

SO-29.548

Dazoxiben

W&13,177

AA

F

u

5OOr

u

INS------l

1

400 ~

300 -

5 5

200 100 -

46,619 VEH

M-13.177

SO-is.548

Dazoxiben

FIG. 3 Platelet ATP secretion measured as increase in whole blood ATP concentration after injection of arachidonic acid (1.1 mg/kg; upper panel) or The animals were pretreated with U-46,619 (30 ug/kg; lower panel). vehicle, BM-13,177 (50 mg/kg), SQ-29,548 (2 mg/kg), or dazoxiben (2 mg/kg) 10 min before challenge. The numbers inside the columns represent the number of animals in each group. ** = flO.01 versus vehicle treated group. NS = not significantly different.

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THROMBOXANE RECEPTOR ANTAGONISTS

FIG. 4

Original tracings of alterations in arterial platelet count (upper panels) and whole blood ATP concentrations (lower panels) in rabbits pretreated with SQ-29,548 (2 mg/kg; upper panels) or dazoxiben (2 mg/kg; lower panels) following injection of arachidonic acid (1.1 mg/kg; 0) or U-46,619 (30 ug/kg; t).

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Sudden death induced by AA and U-46,619 resulted in marked platelet activation J-J vivo resulting in formation of platelet aggregates as indicated by decreases in circulating platelet count and in release of adenine nucleotides as evidenced by the elevation of whole blood ATP concentrations. In the animals injected with AA, platelet count was decreased by 67 t 3% whereas in rabbits pretreated with BM-13,177, SQ-29,548 or dazoxiben the platelet count decreased by only 12 to 16% (Figure 2). In animals given only U-46,619, platelet count decreased by 69 _I 4%. Pretreatment with BM-13,177 totally inhibited aggregate formation and the platelet count was basically unaltered, while SQ29,548 also significantly reduced the decrease in platelet count to only 8 2 4%. However, dazoxiben was totally ineffective in preventing platelet aggregation induced by U-46,619 (Figure 2). The increase in whole blood ATP concentrations in vehicle treated animals was 1.43 + 0.56 uM and 0.32 + 0.10 ~_IMfollowing injection of AA or U-46,619, respectively. The thromboxane receptor antagonists effectively reduced ATP secretion induced by either of these stimuli. In contrast, dazoxiben inhibited the ATP release induced by AA but not to U-46,619 (Figure 3). Figure 4 illustrates typical platelet count and ATP recordings from rabbits pretreated with SQ-29,548 (2 mg/kg) and dazoxiben (2 mg/kg), receiving injections of AA followed by U-46,619. Both animals survived the AA challenge and showed only minor alterations in platelet count with only a moderate release of ATP. Thereafter, a recovery period was allowed until the MABP and platelet count recovered to at least 90% of the initial values. The animals were then challenged with U-46,619. The rabbit pretreated with the thromboxane receptor antagonist SQ-29,548 survived the challenge without major alterations in platelet count and ATP secretion. Although not shown, The same response was seen in animals MABP also did not change significantly. pretreated with BM-13,177. When AA was given as a second challenge instead of U46,619, all the animals survived, indicating that the dazoxiben concentration in the circulation was sufficiently high to prevent thromboxane synthesis and thromboxane A2 In contrast, animals receiving the thromboxane synthetase induced sudden death. inhibitor, dazoxiben, died immediately upon U-46,619 challenge, and showed marked decreases in platelet count and release of ATP, (Figure 4). This is due to the activation of the “endoperoxide/thromboxane” receptor by the U-46,619, an endoperoxide analog. DISCUSSION The model of _in vivo platelet aggregation employed in this study allowed the continuous measurement of whole blood ATP concentrations as well as the number of The basal whole blood ATP concentrations in circulating platelets in peripheral blood. anesthetized rabbits and guinea pigs are in the range of the detection limit of our method This shows that no artificial activation of platelets or mechanical about 10 n-M. destruction of platelets takes place during the normal withdrawal process by the double Platelet aggregation in vivo induced by pharmacologic stimuli is lumen cannula. measured as a decrease in the number ofcmating platelets because the circulating platelet aggregates are trapped in the microcirculation (e.g., of the lungs). Injection of platelet activating stimuli like collagen, AA or U-46,619 results in a decreased platelet count and increased whole blood ATP concentrations as a measure of platelet adenine nucleotide secretion. Our thromboxane ineffective

findings show that BM-13,177 and SQ-29,548 are able to inhibit the dependent component of the collagen induced aggregation, while being against the previously described thromboxaneindependent component of

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The thromboxane synthetase inhibitor, collagen induced aggregation in vivo (13). dazoxiben, was reported to exert a comparable degree of inhibition of collagen induced The thromboxane independent component may be due to aggregate formation (14). release of other proaggregatory and vasoactive mediators by platelets including ADP, ATP, and serotonin. In this regard, the ATP release during the second challenge was somewhat diminished, but not significantly lowered by BM-13,177 or SQ-29,548 (Table 1). This is different from our experience with PG12 infusion which significantly reduced ATP release during the second collagen challenge as compared to the response observed in vehicle treated animals (12). PG12 treatment leads to CAMP elevation and a more general inactivation of platelets which is also effective against non-eicosanoid stimuli (e.g., ADP). The second part of this study compares the effectiveness of SQ-29,548 and BM13,177 with the thromboxane synthetase inhibitor dazoxiben in eicosanoid induced sudden death. Initially, AA induced sudden death was described in rabbits (15) and only later was as the primary mediator responsible for pulmonary thrombosis, TxA2 identified bronchoconstriction and vasoconstriction (16). Thus, in contrast to the collagen induced platelet activation a model almost entirely based on --in vivo, AA and U-46,619 represent prostaglandin dependent platelet activation. Eicosanoid induced sudden death is characterized by an immediate respiratory arrest attributable to marked bronchoconstriction and a decrease in MABP to 5 mm H within 2 - 3 min. This is accompanied by a 67 and 69% decrease in platelet count an % increases in ATP concentrations to 1.2 and 0.3 u! with AA and U-46,619 as the platelet activating stimuli. The thromboxane receptor antagonists BM-13,177 and SQ-29,548 were highly effective in protecting the rabbits from sudden death. The decrease in platelet and platelet ATP secretion was largely abolished. count was significantly reduced, Simultaneously, the decreases in MABP were either markedly diminished or totally inhibited, indicating that vasoactive agents released from platelets may be responsible for the observed hypotension. The highly effective protection by thromboxane receptor antagonists is in sharp contrast to the effects seen with the thromboxane synthetase inhibitor dazoxiben. Although dazoxiben protected the animals from AA induced sudden death as already described previously (171, it was totally ineffective against the endoperoxide analog, U-46,619. This lack of efficacy cannot be explained by a possible short plasma half-life of dazoxiben, since there was enough circulating dazoxiben to protect against a second injection of arachidonic acid. The dose of BM-13,177 (50 mg/kg) required to protect against AA induced death is rather high in comparison to another similar study in which 5 However, this difference in potency can be (7). mg/kg of BM-13,177 was effective explained by the more potent stimulus used in our model with AA, 1.1 mg/kg of >99% purity injected in a volume of 300 ul rapidly over 5 seconds into anesthetized rabbits, resulting in death of 100% of the animals. Stegmeier et al. (7) employed 1.0 mg/kg of AA of 90% purity which resulted in an LD The AA was given over 1 min in a volume of 2.1 to 3.9 ml in conscious animals after ora8P’pretreatment with BM-13,177. The results presented in this study clearly show the broader based efficacy of thromboxane receptor antagonists over thromboxane synthetase inhibitors in the eicosanoid induced sudden death model. The lack of protective effects of thromboxane synthetase inhibitors in vivo could be due to accumulation of platelet derived endoperoxides and posszly metabolism into other prostaglandins (e.g., PGD2, PGF2 o). In our experiments, it is apparent that the endoperoxide actions on pulmonary and vascular smooth muscle and on platelets are sufficient to induce lethality without their

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subsequent conversion to thromboxane A . BM-13,177 and SQ-29,548 are generally considered as specific antagonists of endo Beroxide/thromboxane receptors on platelets Preliminary clinical studies with BM-13,177 showed a and smooth muscles in vitro. -7 marked inhibition of -ex VIVO platelet aggregation in human volunteers and patients (18, encourage further study of thromboxane receptor antagonists as 19). These reports effective anti-thrombotic drugs in the experimental and clinical setting.

ACKNOWLEDGEMENTS This work was supported by Research Grant No. HL-25575 from the National Heart Lung and Blood Institute of the NIH. Dr. Darius is a recipient of a Research Fellowship of the Deutsche Forschungsgemeinschaft, Bonn, Federal Republic of Germany.

REFERENCES 1.

Clinical aspects of MITCHELL, J.R.A. pathway. Brit. Med. Bull. -39:289-295, 1983.

the

arachidonic

acid-thromboxane

2.

GRANSTROM, E., DISZFALUSY, U. and HAMBERG, M. The thromboxanes, IN: Pace-Asciak/Granstrom teds.1 Prostaglandins and Related Substances, Elsevier Science Publishers BV, Amsterdam, 1983, pp. 45-94.

3.

LEFER, A.M. Role of prostaglandins Lefer and Schumer (eds.) Handbook pp. 355-376.

4.

DARIUS, H., LEFER, A.M., LEPRAN, prostacyclin with an inhibitor of cyclic J. Pharmacol -84:735-741, 1985.

5.

NICOLAOU, K.C., MACOLDA, R.L., SMITH, J.B., AHARONY, D., SMITH, E.F. III, and LEFER, A.M. Synthesis and biological properties of pinane thromboxane A2, a selective inhibitor of coronary artery constriction, platelet aggregation and thromboxane formation. Proc. Natl. Acad. Sci. USA -76:2566-2570, 1979.

6.

PATSCHEKE, thromboxane 1984.

7.

STEGMEIER, K., PILL, J., MULLER-BECKMANN, B., SCHMIDT, F.H,, WITTE, E.C., WOLFF, H.P. and PATSCHEKE, H. The pharmacological profile of the thromboxane A antagonist BM-13,177. A new anti-platelet and anti-thrombotic drug. Thromb. kes. -’ 350379-395, 1984.

8.

OGLETREE, NAKANE, thromboxane

9.

DARIUS, H., SMITH, J.B. and LEFER, A.M. Beneficial effects of a new potent and specific thromboxane receptor antagonist (SQ-29,548) J--in vitro and --in vivo. Pharmacol. Exp. Ther. (In Press, 1985).

and thromboxanes in shock states. IN: Altura, of Shock and Trauma, Raven Press, NY, 1983,

I. and SMITH, J.B. --In vivo interaction of nucleotide phosphodiesterase, HL 725. Brit.

H. and STEGMEIER, K. Investigations on a selective non-prostanoic antagonist, BM-13,177, in human platelets. Thromb. Res. -33:277-288,

M.L., HARRIS, D.N., GREENBERG, R., HASLANGER, M. Pharmacological actions of SQ-29,548, a novel antagonist. J. Pharmacol. Exp. Ther. (In Press, 1985).

M.F. and selective

Vol.

40,

No.

5

THROMBOXANERECEPTOR ANTAGONISTS

675

10.

SMITH, C.M. and FREULER, continuous platelet counting.

II.

SMITH, J.B., measurements

12.

DARIUS, H., LEPRAN, I., SMITH, secretion and platelet aggregation by in vivo methodology. IN: Kharasch, Basicand Clinical Studies. (In Press,

13.

VARGAFTIG, B.B., LEFORT, J., JOSEPH, D. and FOUQUE, F. Mechanisms of bronchoconstriction and thrombocytopenia induced by collagen in the guinea pig. Eur. J. Pharmacol. 1979. -58:273-284,

14.

MALLARKEY, G. and SMITH, GM. A comparative study of the involvement of the prostaglandin H2/thromboxane A2 pathway in intravascular platelet aggregation in guinea-pigs and rats. Br. J. Pharmacol. -84:425-430, 1985.

15.

SILVER, M.J., HOCH, Arachidonic acid causes

16.

LEFER, A.M., BURKE, S.E. and SMITH, J.B. prostaglandin endoperoxides in the pathogenesis death. Thromb. Res. -32:311-320, 1983.

17.

LEFER, A.M., OKAMATSU, S., SMITH, E.F. III and SMITH, J.B. Beneficial effects of a new thromboxane synthetase inhibitor in arachidonate-induced sudden death. Thromb. Res. -23:265-273, 1981.

18.

CRESELE, P., DECKMYN, H., ARNOUT, J., LEMMENS, J., JANSSENS, W. and VERMYLEN, J. BM-13,177, a selective blocker of platelet and vessel wall thromboxane receptors, is active in man. The Lancet 1:991-994, 1984.

19.

RIESS, H., HILLER, a new anti-platelet -35:371-378, 1984.

BURKE, S.E., of ATP secretion

F. The measurement of intravascular Bibl. Anat. &229-234, 1973. LEFER, in vivo. --

A.M. and Pharmaceut.

aggregation

FREILICH, A. Continuous Res. 440-43, 1984.

J.B. and LEFER, A.M. Prevention of ATP prostacyclin as assessed by direct on-line N. and Watkins, G.L. teds.) Prostacyclin: New 1985).

W., KOCSIS, J.J., INGERMAN, C.M. and SMITH, sudden death in rabbits. Science 183:1085-1087, 1974. --

E., REINHARDT, drug in patients

by

J.B.

Role of thromboxanes and of eicosanoid induced sudden

8. and BRAUNING, with atherosclerotic

C. Effects of BM-13,177, disease. Thromb. Res.