Hypersensitivity to thromboxane A2 in cholesterol-rich human platelets

The effect of changes in platelet membrane cholesterol content on thromboxane A2 (TXA2)-induced platelet activation was studied. Concentrations of 9,11-epithio-11,12-methano-TXA2 (STA2), a stable analogue of TXA2 which can cause half-maximal aggregation and release of [14C]serotonin in cholesterol-rich platelets were significantly lower than those in cholesterol-normal platelets. STA2-induced increase in cytosolic calcium concentration and [32P]phosphatidic acid formation in cholesterol-rich platelets were significantly greater than those in cholesterol-normal platelets. The maximal concentration of binding site (Bmax) for SQ29548 was significantly increased in cholesterol-rich platelets compared with cholesterol-normal platelets, while the equilibrium dissociation rate constant (Kd) for SQ29548 did not differ between cholesterol-rich and cholesterol-normal platelets. The present study suggested that sensitivity to TXA2 was increased by the incorporation of cholesterol into platelet membrane and that the cause of hypersensitivity to TXA2 in cholesterol-rich platelets may be partly explained by an increase in binding capacity for TXA2.     

Suppression by beta-lactam antibiotics of thromboxane A2 generation and arachidonic acid release in rabbit platelets in vitro

High concentrations of latamoxef and some other beta-lactam antibiotics suppressed thromboxane A2 generation as determined from the thromboxane B2 level in the in vitro aggregation of rabbit platelets in agonist-induced stimulations. In aggregation induced with a low concentration of collagen, the suppression of thromboxane B2 generation correlated well with the suppressions of aggregation and serotonin (5-HT) release; collagen produced thromboxane A2-dependent aggregation and 5-HT release as judged from the inhibitory action of indomethacin. Latamoxef also suppressed thromboxane B2 generation when platelet stimulation was induced by thrombin or platelet activating factor at concentrations at which it did not affect either aggregation or 5-HT release. However, latamoxef did not affect platelet responses including thromboxane B2 generation induced by exogenous arachidonic acid or calcium ionophore, A23187. Beta-lactam antibiotics also interfered with arachidonic acid release from the membrane phospholipids of platelets which had been prelabelled with [3H]arachidonic acid and aggregated with collagen. These results suggest that in the in vitro aggregation of platelets, beta-lactam antibiotics interfere with some of the receptor-stimulated processes which lead to arachidonic acid release from the membranes and this, in turn, suppresses thromboxane B2 generation.     

Two phasic generation of thromboxane A2 by the action of collagen on rat platelets

Two-phase thromboxane A2 (TXA2) generation was observed in washed rat platelets stimulated by collagen. One coincided with shape change and the other with aggregation, and both reactions were inhibited by indomethacin. The collagen-induced first phase was not affected by treatment of the platelets with TXA2 receptor antagonist BM13177, while the shape change, the second phase TXA2 generation and aggregation were completely suppressed. U46619, a stable TXA2 mimetic, induced only platelet shape change and not aggregation or endogenous icosanoid mobilization. However, upon addition of U46619 to platelets previously stimulated by collagen in the presence of indomethacin, icosanoids formation was induced together with aggregation. These results suggest that both the collagen-induced initial TXA2 and the occupation of the receptor by collagen might be a trigger for the second-phase TXA2 formation in concert with platelet aggregation.     

R 68 070: thromboxane A2 synthetase inhibition and thromboxane A2/prostaglandin endoperoxide receptor blockade combined in one molecule--II. Pharmacological effects in vivo and ex vivo

R 68 070 or (E)-5-[[[(3-pyridinyl)[3-(trifluoromethyl)phenyl]- methylen]amino]oxy] pentanoic acid (Janssen Research Foundation, Belgium), a newly developed compound, combining specific thromboxane A2 (TXA2) synthetase inhibition with TXA2/prostaglandin endoperoxide receptor blockade in one molecule, is active in vivo in man and in experimental animals. In man (n = 5), a single oral 400-mg dose of R 68 070 produces deep and protracted inhibition of platelet TXA2 synthetase activity (greater than or equal to 90% for 48 h), increases serum levels of immunoreactive 6-keto-PGF1 alpha, reduces platelet aggregation in P.R.P. induced by U 46619, collagen (greater than 70% for 8 h), arachidonic acid (greater than 90% for 18 h) and prolongs template bleeding times significantly, without affecting plasma coagulation or fibrinolysis. In rats, R 68 070 (1.25 mg/kg orally, -2 h) singly prolongs tail bleeding times as much as a combination of TXA2 synthetase inhibition (dazoxiben 10 mg/kg) and TXA2/prostaglandin endoperoxide receptor blockade (BM 13177 40 mg/kg). In dogs, the compound reduces coronary thrombosis induced by electrical damage (1.25 mg/kg i.v.) and prevents the evolution of occlusion/reperfusion-induced arrhythmias into ventricular fibrillation (2.5 mg/kg i.v.). R 68 070 thus may be an appropriate pharmacological tool to analyze the roles and interactions of agonistic (TXA2, prostaglandin endoperoxides) and antagonistic (PGD2, PGE2, PGI2) metabolites of arachidonic acid in experimental and human pathologies.     

Prostaglandin endoperoxides and thromboxane A2 activate the same receptor isoforms in human platelets

Arachidonic acid (AA) is a potent inducer of platelet aggregation in vitro; this activity is due to its conversion to biologically active metabolites, prostaglandin (PG) endoperoxides and thromboxane A2 (TxA2). PG endoperoxides and TxA, are thought to act on the same receptor; however, at least two isoforms of this receptor have been identified. The aim of our work was to clarify whether endoperoxides and TxA2 activate the same or different receptor subtypes to induce aggregation and calcium movements in human platelets. AA-induced aggregation and calcium rises were still detectable in platelets preincubated with thromboxane synthase inhibitors, which suppress TxA2 formation and induce PGH2 accumulation, suggesting that PG endoperoxides can activate platelets. Exogenously added PGH2 was able to induce aggregation and calcium rises. Pretreatment of platelets with GR32191B or platelet activating factor, which desensitize one of the two receptor subtypes identified in platelets, did not prevent calcium rises induced by endogenously generated or by exogenouly added PGH2, indicating that TxA2 and PG endoperoxides share the same receptor subtype(s) to activate platelets. HEK-293 cells overexpressing either of the two thromboxane receptor isoforms cloned to date (TPalpha and TPbeta) and identified in human platelets, stimulated with PGH2, or with the stable endoperoxide analog U46619, formed inositol phosphates. These data show that endoperoxides and TXA2 mediate their effects on platelets acting on both, and the same, receptor isoform(s).     

Potentiation by adrenaline of Ca2+ influx and mobilization in stimulated human platelets: dissociation from thromboxane generation and aggregation

In a medium containing 1 mM extracellular Ca2+ (Ca2+o), the prior addition of 0.5 microM adrenaline to quin 2-loaded human platelets increased both the rate and amplitude of the rise in cytosolic free Ca2+ (Ca2+i) in response to sub-threshold concentrations of thrombin and PAF and these effects were not prevented by blocking either fibrinogen binding and aggregation or cyclo-oxygenase. In the presence of 2 mM EGTA [( Ca2+o] less than 100 nM), the rate, but not the extent of rise of [Ca2+i] was enhanced by adrenaline, and this was also unaffected by blockade of cyclo-oxygenase. Addition of adrenaline 1 min after the other agonist in the presence of 1 mM Ca2+o resulted in aggregation without further elevation of [Ca2+i]. Adrenaline thus enhances both influx and intracellular mobilization of Ca2+ by a mechanism independent of both fibrinogen binding and thromboxane production, but these effects do not fully explain its potentiation of aggregation by other agonists.     

Inhibition of human platelet function in vitro and ex vivo by acetaminophen

The effects of acetaminophen (APAP) in vitro, or ex vivo following APAP ingestion, on human platelet aggregation, ¹⁴C-5HT secretion, and thromboxane B₂ (TxB₂) formation were assessed. APAP added in vitro to citrated platelet-rich plasma (PRP) inhibited aggregation, secretion, and TxB₂ formation induced by collagen, epinephrine, arachidonate, and the ionophore A23187, but had no effect on the responses induced by the endoperoxide analog U44069. Arachidonate-induced responses were inhibited by lower concentrations of APAP than were the responses to the other agonists. In PRP obtained 1 hour after ingestion of 650 mg or 1000 mg APAP, arachidonate-induced TxB₂ formation was inhibited by 40–99% in five subjects tested, whereas inhibition of collagen- or epinephrine-induced TxB₂ formation was less consistent. Aggregation and secretion responses were not altered by APAP ingestion m 4 of the 5 subjects, but were inhibited in the remaining subject, who had the highest plasma APAP levels. In contrast to aspirin and indomethacin, APAP-induced inhibition of collagen-stimulated TxB₂ formation could be partially overcome with increasing collagen concentrations. No such partial correction occurred with epinephrine, however. In washed platelet suspensions labeled with ³H-arachidonate, both APAP and aspirin inhibited the formation of labeled PGD₂ and PGE₂, as well as TxB₂. These results suggest that APAP acts in human platelets as a reversible inhibitor of cyclo-oxygenase, as found previously in other tissues, and that recent APAP ingestion can, on occasion, produce inhibition of platelet functional responses measured in vitro.     

Increased thromboxane A2 generation and altered membrane fatty acid composition in platelets from patients with active angina pectoris

Lipid composition of platelet membranes and thromboxane A2 (TxA2) generation by platelets were investigated in eighty-seven anginal patients (forty-two with resting angina in active phase and forty-five with effort stable angina or rest angina in inactive phase) and in forty-five clinically healthy subjects of similar age. All subjects were on the same dietary regimen and the adherence to diet was checked by analysis of red blood cell lipids. Platelets from active angina patients produced more TxA2 than platelets from both inactive patients and controls (p less than 0.001). Moreover patients with active angina had higher arachidonic acid (AA, p less than 0.001) and lower eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) levels in phosphatidylcholine (PC, p less than 0.001), than inactive patients and controls. AA and EPA changes in membrane PC significantly correlated with TxA2 production (p less than 0.001) but not with coronary pathoanatomy. Plasma lipids, content of cholesterol, total phospholipids (and their saturated and unsaturated fatty acids) and the different phospholipid fractions in platelet membrane were not different in the three groups. Present results indicate that in platelets from anginal patients phospholipid fatty acid composition is at least in part independent of plasma composition and that in active angina there are modifications leading to increased TxA2 formation and possibly contributing to the occurrence of ischemic attacks.     

The interrelationship between thromboxane biosynthesis, aggregation and 5-hydroxytryptamine secretion in human platelets in vitro

Platelet aggregation, secretion of 5-hydroxy tryptamine and production of thromboxane B2 were monitored simultaneously in human platelet suspensions in the absence and presence of cyclooxygenase or thromboxane synthetase inhibitors. Aggregation, secretion and thromboxane B2 formation in response to either sodium arachidonate or epinephrine were blocked by aspirin or by 1-N-butyl inidazole suggesting that thromboxane biosynthesis was an essential requirement for platelet activation by these agents. In contrast, thrombin and collagen could apparently induce aggregation and secretion via two pathways: at low doses involving thromboxane production, but at higher doses by a direct mechanism independent of thromboxane biosynthesis. In the case of ADP, inhibition of thromboxane production blocked secretion but had little effect on aggregation, indicating that secretion was probably dependent on thromboxane biosynthesis which probably occurred as a result of aggregation. Thus it appears that although the processes of thromboxane production, release of dense granule constituents and aggregation may often be intimately linked, each process can occur independently of the other, depending upon the stimulus used.     

Low concentrations of sodium azide specifically inhibit a thromboxane A2 pathway in human platelets.

Sodium azide completely inhibits the serotonin release induced by ADP, arachidonic acid and the thromboxane A2 mimetic U46619, but does not have any effect on the activation by PMA. Collagen and thrombin are inhibited when used at low concentrations, but not at high concentration. This pattern of activation suggests that the inhibition by azide is not a metabolic inhibition. The antagonism of U46619-induced secretion was further studied and was shown to be non-competitive. It is selective for certain components of the U46619 stimulus-response coupling: aggregation, serotonin secretion and the activation of protein kinase C are completely or almost completely inhibited by 300 microM sodium azide. Shape change, calcium elevation, cytoplasmic alkalinization and phosphorylation of myosin light chain are only partially modified. This suggests that azide may specifically inhibit one of the different forms of thromboxane A2 receptors present in platelets.     

Effects of antiepileptic drugs on rat platelet aggregation: ex vivo and in vitro study

The influence of conventional antiepileptic drugs (valproate, phenobarbital, diazepam, clonazepam, carbamazepine and diphenylhydantoin) on rat platelet activation induced by arachidonic acid (AA) or adenosine-5'-diphosphate (ADP) was investigated both ex vivo and in vitro on platelet-rich plasma (PRP). It was found that only diazepam, and to a smaller extent clonazepam, impaired rat platelet function. These benzodiazepines did not affect ex vivo platelet aggregation induced by ADP but dose-dependent inhibition of platelet aggregation and malondialdehyde (MDA) synthesis were observed, when the platelets were stimulated with AA (ED(50) of diazepam for aggregation was 2.7 mg/kg and that for MDA synthesis - 3.9 mg/kg). In in vitro study, diazepam was found to be a potent inhibitor of AA-induced platelet aggregation (IC(50) 1.2 microg/ml) and MDA synthesis (IC(50) 4.0 microg/ml). Higher concentrations of diazepam were required to inhibit ADP-induced aggregation (IC(50) 29.0 microg/ml). Clonazepam also exhibited a concentration-dependent inhibitory effect on AA-induced platelet aggregation and MDA synthesis but this effect was weaker when compared to diazepam. The present data demonstrate that diazepam possesed a strong inhibitory effect on rat platelet activation. The correlation between the reduction of platelet aggregation and the synthesis of MDA may suggest that the observed effect of diazepam is due to the inhibition of the cyclooxygenase pathway of the AA metabolism in platelet.     

Effect of picotamide on platelet aggregation and on thromboxane A2 production in vivo

Effects of picotamide (900 mg in 3 oral administrations for 7 days) on ex vivo and in vivo platelet TxA2 production and on platelet aggregation were evaluated in 8 patients with peripheral arteriopathy and in 8 normal subjects. Picotamide significantly reduced ADP-induced platelet aggregation, but had no effect on that induced by arachidonic acid or the thromboxane analogue U46619. Though ex vivo platelet TxA2 production (TxB2 concentration after arachidonic-acid-induced aggregation) was reduced from 946 +/- 141 (mean +/- SD) to 285 +/- 91 ng/ml in controls and from 1515 +/- 673 to 732 +/- 420 ng/ml in patients with arteriopathy, there was no effect on urinary excretion of 2,3-dinor-TXB2 (in vivo indicator of platelet TxA2 production), or on in vivo PGI2 production (urinary excretion of 6-keto-PGF1 alpha and 2,3-dinor-6-keto-PGF1 alpha). In the same subjects, single-dose aspirin reduced ex vivo TxB2 production by at least 98% and 2,3-dinor-TxB2 excretion from 116.7 +/- 61.4 to 32.6 +/- 17.0 ng/g creatinine in control subjects, and from 156.3 +/- 66.1 to 59.1 +/- 19.2 ng/g creatinine in patients with peripheral arteriopathy. Our data suggest that inhibition of platelet TxA2 production in vivo may not be picotamide's main mechanism of action.     

Inhibitors of ex vivo aggregation of human platelets induced by decompression, during reduced barometric pressure

It has been shown experimentally ex vivo that human platelet aggregation is induced by decompression (reduced pressure) produced by various means, i.e., reduced barometric pressure, reduced hydrostatic pressure, and reduced hydrodynamic pressure due to Bernoulli's principle. We report here that the spontaneous platelet aggregation induced by reduced barometric pressure (253 torr for three hours) is inhibited by 1:10(7) diluted Japanese herbal plant oil (JHP) and also by two of its major constituents, menthone and menthol with the median inhibitory concentration (IC50) in the millimolar range. These drugs gave essentially similar results when collagen and ADP were used as aggregating agents. Inhibitor concentrations were determined by microscopic examination of platelets in wet preparations when the aggregating stimulus was reduced pressure and by optical aggregometry when collagen and ADP were the aggregating agents. Potential usefulness of these compounds in the prevention of decompression syndrome (DCS) and acute mountain sickness (AMS) are discussed.     

Lyophilised reconstituted human platelets increase thrombus formation in a clinical ex vivo model of deep arterial injury

Platelets are the principal component of the innate haemostatic system that protect from traumatic bleeding. We investigated whether lyophilised human platelets (LHPs) could enhance clot formation within platelet-free and whole blood environments using an ex vivo model of deep arterial injury. Lyophilised human platelets were produced from stored human platelets and characterised using conventional, fluorescent and electron microscopic techniques. LHPs were resuspended in platelet-free plasma (PFP) obtained from citrated whole human blood to form final concentrations of 0, 20 and 200 x 10? LHPs/L. LHPs with recalcified PFP or whole blood were perfused through the chamber at low (212 s?1) and high (1,690 s?1) shear rates with porcine aortic tunica media as thrombogenic substrate. LHPs shared morphological characteristics with native human platelets and were incorporated into clot generated from PFP or whole blood. Histomorphometrically measured mean thrombus area increased in a dose-dependent manner following the addition of LHPs to PFP under conditions of high shear [704 μm2 ± 186 μm2 (mean ± SEM), 1,511 μm2 ± 320 μm2 and 2,378 μm2 ± 315 μm2, for LHPs at 0, 20 and 200 x 10? /l, respectively (p= 0.012)]. Lyophilised human platelets retain haemostatic properties when reconstituted in both PFP and whole blood, and enhance thrombus formation in a model of deep arterial injury. These data suggest that LHPs have the potential to serve as a therapeutic intervention during haemorrhage under circumstances of trauma, and platelet depletion or dysfunction.     

In vitro thromboxane synthesis of depleted blood platelets following renal transplantation

Renal transplant rejection is associated with platelet activation in vivo which may lead to partially alpha- and delta-granule-depleted platelets that continue to circulate. These "exhausted" platelets are hemostatically defective. To quantitate the extent of platelet granule depletion following kidney transplantation, we determined intraplatelet levels of beta-thromboglobulin (beta TG), platelet factor 4 (PF4), and serotonin (5-hydroxytryptamine, 5-HT) ex vivo in Triton X-100-treated platelet lysates. To explore biochemical alterations of partially depleted platelets, we studied platelet thromboxane A2 (TXA2) synthesis in citrated platelet-rich plasma (PRP) upon stimulation with thrombin or collagen in 45 recipients of renal allografts and 10 healthy volunteers. The patients were divided into subjects with acute and chronic allograft rejection (N = 15), those with compensated renal failure after kidney transplantation but without evidence of allograft rejection (N = 15), and those with functioning renal transplant (N = 15). The mean intraplatelet content of beta TG (38.6 +/- 4.2 micrograms/10(9) platelets), PF4 (11.8 +/- 1.8 micrograms/10(9) platelets), and 5-HT (274 +/- 31 ng/10(9) platelets) in patients with acute or chronic renal allograft rejection was significantly lower than in other recipients of kidney transplants or healthy volunteers (beta TG: 59.9 +/- 4.7 micrograms/10(9) platelets; PF4: 20.4 +/- 2.3 micrograms/10(9) platelets; 5-HT: 461 +/- 48 ng/10(9) platelets; p less than 0.005 in all cases).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of bortezomib on platelet aggregation and ATP release in human platelets, in vitro

INTRODUCTION: Proteasome inhibitor bortezomib (PS-341) has been the first proteasome inhibitor that has entered clinical trials with its antiproliferative and proapoptotic effects in patients with multiple myeloma. Recent studies indicate that proteasome inhibitors can be useful in prevention of experimental arterial thrombosis in renovascular hypertensive rat models. The aim of the present study is to investigate the effect of bortezomib on in vitro platelet aggregation and adenosine triphosphate (ATP) release of human platelets. MATERIALS AND METHODS: For this purpose, platelet aggregation was induced in the platelet-rich plasma (PRP) using 3 microg ml(-1) collagen, 5 microM adenosine diphosphate (ADP), 10 microM epinephrine and 1 U ml(-1) thrombin and ATP release was induced by collagen. RESULTS AND CONCLUSIONS: Bortezomib showed an inhibitory effect on platelet aggregation induced by ADP in human PRP in a dose- and time-dependent manner, whereas it had no effect on collagen-, epinephrin and thrombin-induced aggregation. ATP-release reaction induced by collagen was inhibited dose- and time-dependently by bortezomib, even though collagen-induced platelet aggregation was apparently not affected in human PRP. These findings indicate that bortezomib may be an antiaggregating agent and its' effects may be related to adenine nucleotide receptor dependent regulatory proteins which are important for physiological and pathophysiological cellular processes. However, our in vitro studies suggest that this hypothesis is inadequate to explain the observations completely. This phenomenon and its clinical implication justify further clinical investigations.