Robert B. Wallis

Mammalian platelet adrenoceptors

1 Mammalian platelets vary widely in their responses to catecholamines in part because these agonists can act via excitatory α‐ and inhibitory β‐adrenoceptors. In the absence of antagonists, adrenaline enhances the response of rabbit platelets to an excitatory agonist, e.g. adenosine‐5′‐O‐(1‐thiodiphosphate) (ADP‐α‐s) acting at another receptor, but has no effect on the response of rat or guinea pig platelets to such an agonist. In the presence of a β‐adrenoceptor antagonist, adrenaline enhances the response of rat, but not guinea‐pig platelets to ADP‐α‐S and the extent of the enhanced effect on rabbit platelets is increased. In the presence of an α‐adrenoceptor antagonist, adrenaline inhibits the response of rabbit and rat platelets to ADP‐α‐S but has no such effect on the response of guinea‐pig platelets. 2 Studies using selective agonists and antagonists demonstrate that the excitatory response of rat platelets to adrenaline is mediated by an α2‐adrenoceptor, and the inhibitory response of rabbit platelets to adrenaline by a β2‐adrenoceptor as is the case in other species which have been examined. 3 The mean α2‐adrenoceptor density on human, rabbit, rat and guinea‐pig platelets as assessed using [3H]‐yohimbine as radioligand is obtained as 258, 270, 42 and < 10 receptors per platelet. 4 The mean β2‐adrenoceptor density on human, rabbit, rat and guinea‐pig platelets as assessed using (‐)‐[125I]‐iodocyanopindolol is obtained as 66, 14, 41 and < 5 receptors per platelet. 5 The nature of the effect of adrenaline on the response of mammalian platelets to other excitatory agonists, e.g. ADP‐α‐S, appears therefore to be largely determined by the absolute number of α2‐adrenoceptors and by the relative content of α2‐ and β2‐adrenoceptors on these cells for the species which have been examined in this analysis.

Particle volume changes associated with light transmittance changes in the platelet aggregometer: dependence upon aggregating agent and effectiveness of stimulus.

The increase in light transmittance of aspirin-treated platelet-rich plasma caused by addition of non-saturating doses of ADP and, at earlier times, of adrenaline is correlated with formation of aggregates having a volume in the range 490-8580 fl. and containing 100-2000 platelets. The disappearance of single platelets and the formation of aggregates having volumes less than 490 fl. makes no significant contribution to the increase in light transmittance. Similar relationships are observed on addition of saturating doses of ADP and adrenaline except that the formation of aggregates larger than 8580 fl. contributes significantly to the initial phase of the increase in light transmittance and is more closely correlated with the overall change in this parameter.

Role of Thromboxane A2

When platelets are stimulated to aggregate and secrete by a sub-maximal concentration of an agonist, the platelets on the outside of an aggregate appear more degranulated than those on the inside (1) (fig. 1). In the example shown, aggregation of rabbit platelet-rich plasma (PRP) was induced by a sub-maximal concentration of collagen.

In vivo redirection of prostaglandin endoperoxides into 6-keto PGF1α formation by thromboxane synthetase inhibitors in the rat

N(7-Carboxyheptyl) imidazole, 4-[2-(1H-imidazol-1-yl) ethoxy] benzoic acid (dazoxiben) and imidazo [1,5-alpha] pyridine-5-hexanoic acid (CGS 13080) are potent selective inhibitors of platelet thromboxane synthetase that have little or no effect on the cyclooxygenase activity. Oral doses of the substances given to rats inhibited platelet thromboxane B2 production induced by intra-venous administration of collagen (100 micrograms/kg). Plasma concentrations of immunoreactive 6-keto PGF1 alpha in treated animals were increased above corresponding concentrations in untreated animals. There were small effects on the thrombocytopenia with CGS 13080 and carboxyheptylimidazole but not with dazoxiben. However these results did not always achieve statistical significance. Confirmation that the immunoreactive prostaglandin measured was actually 6-keto PGF1 alpha was obtained by the facts that indomethacin abolished its appearance in plasma and that the other prostaglandins were not present in sufficient quantities to cross-react with the antiserum to 6-keto PGF1 alpha. Two different antisera to 6-keto PGF1 alpha detected the same changes. Administration of thromboxane synthetase inhibitors to rats causes redirection of prostaglandin production from thromboxane to prostacyclin when platelets are stimulated with collagen in vivo.

CGS 12970: a novel, long acting thromboxane synthetase inhibitor.

1 CGS 12970 is a potent selective inhibitor of human platelet thromboxane synthestase in vitro (IC50 = 12 nM). It is four orders of magnitude less potent as an inhibitor of sheep seminal vesicle cyclo‐oxygenase, bovine aorta prostacyclin synthetase and human leucocyte 15‐lipoxygenase. 2 The compound inhibited collagen‐induced thromboxane B2 production by human platelets in vitro without an effect on the accompanying platelet aggregation induced by collagen, ADP, platelet activating factor, thrombin, arachidonic acid or the prostaglandin mimetic, U 46619. 3 Administration of CGS 12970 to rats inhibited collagen‐induced thromboxane B2 production but had no effect on platelet aggregation ex vivo. It also had no effect on platelet aggregation induced by thrombin or on plasma clotting times. 4 A single oral dose of 1 or 3 mg kg−1 to rabbits inhibited thromboxane B2 production in clotting blood ex vivo for 12 or 24 h respectively. 5 CGS 12970 inhibited thromboxane B2 production in vivo induced by intravenous administration of collagen to rats or calcium ionophore to guinea‐pigs. In both cases there was a concomitant elevation of immunoreactive 6‐keto‐prostaglandin F1α but no effect on the induced thrombocytopenia. 6 As with other thromboxane synthetase inhibitors, CGS 12970 prolonged tail bleeding time in the rat. However, CGS 12970 was not as potent as other thromboxane synthetase inhibitors in this test. 7 In addition to these usual effects of thromboxane synthetase inhibitors, CGS 12970 inhibited the thrombocytopenia induced by the Forssman reaction or ADP administration. In these tests the effect of the compound was of short duration. 8 CGS 12970 had no effect on the thrombocytopenia associated with the Arthus reaction which distinguishes it from cyclo‐oxygenase inhibitors. It also had no effect on thrombus formation on a cotton thread in an arteriovenous shunt in the rat.

Responses of rabbit platelets to adrenaline induced by other agonists

Abstract ADP, thrombin, 5HT, arachidonate, collagen and stable prostaglandin endoperoxide/thromboxane A 2 analogues all induce rabbit platelets to aggregate in response to subsequent addition of adrenaline as a result of occupancy of an α 2 -adrenoceptor. The maximal rate and extent of the induced aggregatory response to adrenaline varies with the inducing agonist. The aggregatory response to adrenaline induced by collagen, arachidonate or U-46619 is associated with secretion of both 5HT and ADP. Indomethacin or N(1-carboxyheptyl-) imidazole inhibit the response to adrenaline induced by collagen or arachidonate. No secretion accompanies the aggregatory response to adrenaline induced by ADP or thrombin. These results demonstrate for the first time that occupancy of the rabbit platelet α 2 -adrenoceptor can give rise to a secretory response. They also explain the inconsistent reports of direct aggregatory responses to adrenaline observed in rabbit platelet-rich plasma or washed rabbit platelets.

Synergistic responses and receptor occupancy in rabbit blood platelets

Several observations indicate that simultaneous receptor occupancy is necessary for generation of a synergistic response of rabbit platelets to two excitatory agonists. First, an aggregatory response to adrenaline induced by prior addition of 5HT reverses rapidly unless stabilised by inhibition of 5HT uptake. The extent of response correlates with the extracellular 5HT concentration. Second, addition of an ADPase following adrenaline enhances the rate of disaggregation if the response to this agonist has been induced by prior addition of ADP. Third, disaggregation is induced, or enhanced, if an antagonist selective to the inducing agonist is added following completion of the induced aggregatory response. These data suggest marked lability in the mechanisms responsible for generation of synergistic responses.

The Effect of Sulphinpyrazone and Its Metabolites on Platelet Function in vitro and ex vivo

The thioether metabolite of sulphinpyrazone is between 8 and 13 times more potent than the parent compound as a competitive inhibitor of human, guinea pig and rabbit platelet aggregation induced by sodium arachidonate. Of the other known metabolites, the sulphone is approximately equipotent and the p-hydroxy compounds are much less potent that sulphinpyrazone itself. Malondialdehyde biosynthesis from sodium arachidonate by washed human platelets and collagen-induced aggregation of all three species is also inhibited by the thioether. It is 10 times more potent than sulphinpyrazone. ADP-induced aggregation is not affected by sulphinpyrazone, its thioether metabolite, nor the other metabolites. After intravenous administration of the thioether metabolite to groups of guinea pigs the inhibitory effect on sodium arachidonate-induced platelet aggregation ex vivo was long lasting (up to 24 h). In view of the recent information about the metabolism of sulphinpyrazone to its thioether in guinea pigs, we conclude that the thioether metabolite is the substance responsible for the prolonged effect of sulphinpyrazone on platelet fuction in this species and in man.

The effects of recombinant desulphatohirudin on arterial thrombosis in rats.

The role of thrombin in the formation of arterial thrombi is poorly understood. With the new availability of the specific thrombin inhibitor, recombinant desulphatohirudin, in large quantities this is now under investigation. A new model of arterial thrombosis in rats is described where a thrombus is formed on a mechanically injured vessel in vivo. Both platelet and fibrin deposition is inhibited by a recombinant hirudin (CGP 39393) at doses which prolong the activated partial thromboplastin time (APTT) to no more than 3-4 times the control level. In contrast, both unfractionated heparin and Fragmin only inhibit thrombosis when the APTT is excessively prolonged (i.e. to greater than 15 times the control value). It is concluded that CGP 39393 is an effective antithrombotic in arterial thrombosis at lower levels of anticoagulation than either heparin or Fragmin.

EXPRESSION OF THE PLATELET PROCOAGULANT ACTIVITY IN VIVO IN THROMBUS FORMATION IN AN EXTRACORPOREAL SHUNT IN THE RAT

The interaction of platelets and the coagulation system has been investigated in vivo on a cotton thread implanted in an arteriovenous shunt in the rat. Depletion of the circulating platelet count with anti-platelet antibody reduces both thrombus deposition and 125I-labelled fibrinogen incorporation demonstrating the platelet behaviour with either anagrelide or prostacyclin decreases thrombus deposition by a greater extent than that expected from the platelet content of the thrombus. Treatment with warfarin also dramatically decreases both formation and 51Cr-labelled platelet deposition demonstrating the important role of the coagulation system in platelet aggregation in this model. Treatment with Arvin or with heparin also reduced the thrombus weight. It is concluded that this is a model of bidirectional platelet interaction with the coagulation system and may have similarities with thrombosis which occurs in extracorporeal shunts in man.