Inhibition Of Thromboxane A2 Formation Research Articles

Aspirin resistance: causes, clinical significance, correction

Aspirin is the most frequently prescribed antiplatelet agent today. It exerts its antiplatelet effect by irreversible inactivation of the platelet cyclooxygenase-1, resulting in an irreversible inhibition of thromboxane-A2 formation. The clinical benefit of antiplatelet therapy with acetylsalicylic acid (ASA) in high risk patients has been convincingly demonstrated through the results of multiple placebo-controlled trials. Nevertheless, a large number of patients treated with aspirin suffers an adverse cardiovascular event. This observation led to the concept of ”aspirin resistance“. The mechanisms of aspirin resistance remain to be determined, although different theories are being discussed. Several tests are used to assess resistance to ASA in vitro. Depending on which assay is used and which population is tested, the prevalence of aspirin resistance varies between 5% and 60%. So far, it was not possible to define a clear gold standard for detecting aspirin resistance, which considers both, biochemical data and clinical events, and correlates them in a reproducible way. The clinical implications of aspirin resistance are well-documented through a lot of studies, which conclude that resistance to aspirin in vitro is associated with a significant increased risk for adverse cardiovascular events in cardiovascular patients. Insufficient or excessive antiplatelet effect of acetylsalicylacid may be due not only to changes in the synthesized cyclooxygenase-1, but also to changes in its amount. Literature data on the association of various polymorphic markers of candidate genes with the effectiveness of antiplatelet therapy of ASA are few and contradictory. Therefore, it is currently impossible to identify genetic predictors of the effectiveness of ASA as well as any antiplatelet agent. Continuation of research in this area in the future will predict the patient’s response to a drug and, therefore, individualize the approach to the choice and dosage of antiplatelet drugs, which will reduce the incidence of adverse reactions.

Thrombin-induced lysosomal exocytosis in human platelets is dependent on secondary activation by ADP and regulated by endothelial-derived substances

Exocytosis of lysosomal contents from platelets has been speculated to participate in clearance of thrombi and vessel wall remodelling. The mechanisms that regulate lysosomal exocytosis in platelets are, however, still unclear. The aim of this study was to identify the pathways underlying platelet lysosomal secretion and elucidate how this process is controlled by platelet inhibitors. We found that high concentrations of thrombin induced partial lysosomal exocytosis as assessed by analysis of the activity of released N-acetyl-β-glucosaminidase (NAG) and by identifying the fraction of platelets exposing the lysosomal-associated membrane protein (LAMP)-1 on the cell surface by flow cytometry. Stimulation of thrombin receptors PAR1 or PAR4 with specific peptides was equally effective in inducing LAMP-1 surface expression. Notably, lysosomal exocytosis in response to thrombin was significantly reduced if the secondary activation by ADP was inhibited by the P2Y12 antagonist cangrelor, while inhibition of thromboxane A2 formation by treatment with acetylsalicylic acid was of minor importance in this regard. Moreover, the NO-releasing drug S-nitroso-N-acetyl penicillamine (SNAP) or the cyclic AMP-elevating eicosanoid prostaglandin I2 (PGI2) significantly suppressed lysosomal exocytosis. We conclude that platelet inhibitors that mimic functional endothelium such as PGI2 or NO efficiently counteract lysosomal exocytosis. Furthermore, we suggest that secondary release of ADP and concomitant signaling via PAR1/4- and P2Y12 receptors is important for efficient platelet lysosomal exocytosis by thrombin.

Antiplatelet effects of some aporphine and phenanthrene alkaloids in rabbits and man.

Two aporphines (boldine and laurolitsine) and five phenanthrene alkaloids (litebamine, secoboldine, N-cyanosecoboldine, N-methylsecoglaucine and N-methylsecopredicentrine) were evaluated in-vitro for their ability to inhibit platelet aggregation. All seven alkaloids inhibited aggregation of rabbit platelets and inhibited the release of ATP induced by arachidonic acid and collagen in rabbit platelets. Those aggregations induced by platelet-activating factor (PAF), thrombin, U46619 and ADP were inhibited by the three N-substituted secoboldine derivatives only. Thromboxane B2 formation caused by arachidonic acid was also suppressed by these compounds. They did not affect the generation of [3H]inositol monophosphate caused by collagen, PAF and thrombin in the presence of indomethacin. Platelet cyclic AMP level was unaffected by litebamine, but was increased by N-methylsecoglaucine. Litebamine suppressed the secondary aggregation, but not the primary aggregation, induced by ADP and adrenaline in platelet-rich plasma from man, whereas N-methylsecoglaucine inhibited both primary and secondary aggregation. It is concluded that the antiplatelet effect of these seven aporphine and phenanthrene alkaloids is mainly a result of inhibition of thromboxane A2 formation; N-methylsecoglaucine has additional antiplatelet activity as a result of increasing the levels of platelet cyclic AMP.

Incidence of Aspirin 'Resistance' as Determined Using the PFA-100 in Pediatric Patients with Arterial Ischemic Stroke.

A significant benefit of aspirin (ASA) has been demonstrated in the prevention of arterial thrombotic events in high-risk adult patients. Despite ASA therapy, recurrence of thromboembolic events, or treatment failure, has been reported in 10–20% of patients, and this has been termed ASA 'resistance'. This term has also been applied to the failure of ASA to affect ASA-dependent laboratory tests. As there is little information on ASA 'resistance' in children, we examined the efficacy of ASA treatment on platelets from pediatric stroke patients using the PFA-100. Pediatric arterial ischemic stroke afflicts 2–3 children per 100,000 per year, and is associated with recurrent arterial ischemic stroke or transient ischemic attack in 20–40% of cases. Current treatment includes ASA prophylaxis (3 – 5 mg/kg/day) to inhibit platelet function and prevent recurrence, but it is not known whether this ASA dosing regimen is adequate to inhibit platelet function. Our study population consisted of 95 consecutive children with an index arterial ischemic stroke at mean age 5.9 ± 4.8 yrs (range: 0.1 – 17.0 yrs) on active ASA therapy (2.9 ± 1.2 mg/kg/day). Citrated blood samples were obtained at mean age 9.8 ± 4.8 yrs (range: 0.9 – 19 yrs), and were used to measure primary, platelet-related hemostasis in the high-shear PFA-100 system; closure times (CTs) were determined with the collagen/epinephrine cartridge that is sensitive to ASA's inhibition of thromboxane A2 formation via platelet cyclo-oxygenase 1. The mean CT for all 95 patients was 244 ± 68.1 sec, which is greater than 163 sec, the upper limit of the normal range that we have previously determined for healthy children. The majority of patients, 75/95 (79%), had prolonged CTs (172 sec to > 300 sec), indicating inhibition of platelet function by ASA, and they were thus responsive to ASA therapy. 45 of the 75 ASA responders (63%) had CTs > 300 sec, i.e. CTs greater than the maximum test time of 300 sec, demonstrating aperture non-closure. The remaining 20/95 patients (21%) were ASA 'resistant', as they did not respond to ASA therapy, having CTs (93 – 163 sec) within the normal range. Mean ASA dosage did not differ between ASA responders (2.8 ± 1.2 mg/kg/day) and ASA 'resistant' patients (3.0 ± 1.4 mg/kg/day). 6 of the ASA 'resistant' patients had their ASA dosage increased, and on repeat PFA-100 testing, 5/6 showed increased CTs > 163 sec. In conclusion, in children with arterial ischemic stroke, the majority, 79%, demonstrate inhibition of platelet function, as determined using the PFA-100, by ASA therapy at a mean dose of 2.9 mg/kg/day. The reason for the lack of inhibition of platelet function in 21% of pediatric patients, whether increased ASA dosage or alternative anti-platelet agents (e.g. clopidogrel) should be used in ASA 'resistant' patients, and the relationship between ASA 'resistance' and recurrence of arterial thrombotic events in children require further studies.

Low concentration of oxidized low density lipoprotein suppresses platelet reactivity in vitro: An intracellular study

The intracellular mechanisms underlying oxidized low density lipoprotein (oxLDL)-signaling pathways in platelets remain obscure and findings have been controversial. Therefore, we examined the influence of oxLDL in washed human platelets. In this study, oxLDL concentration-dependently (20-100 microg/mL) inhibited platelet aggregation in human platelets stimulated by collagen (1 microg/mL) and arachidonic acid (60 microM), but not by thrombin (0.02 U/mL). The activity of oxLDL was greater at 24 h in inhibiting platelet aggregation than at 12 h. At 24 h, oxLDL concentration-dependently inhibited intracellular Ca2+ mobilization and thromboxane B2 formation in human platelets stimulated by collagen. In addition, at 24 h oxLDL (40 and 80 microg/mL) significantly increased the formation of cyclic AMP, but not cyclic GMP or nitrate. In an ESR study, 24 h-oxLDL (40 microg/mL) markedly reduced the ESR signal intensity of hydroxyl radicals (OH(-)) in both collagen (2 microg/mL)-activated platelets and Fenton reaction (H2O2 + Fe2+). The inhibitory effect of oxLDL may induce radical-radical termination reactions by oxLDL-derived lipid radical interactions with free radicals (such as hydroxyl radicals) released from activated platelets, with a resultant lowering of intracellular Ca2+ mobilization, followed by inhibition of thromboxane A2 formation, thereby leading to increased cyclic AMP formation and finally inhibited platelet aggregation. This study provides new insights concerning the effect of oxLDL in platelet aggregation.

The Antiplatelet Activity of Danggijakyaksan by Inhibition of Phospholipase C

We investigated the effects of the traditional Korean prescription, Danggijakyaksan (DJS) on antiplatelet activity in human platelet suspensions. The effect of oriental medicinal prescriptions, Danggijakyaksan consisting of 6 herbes of Paeoniae Radix (2 g), Poria Cocos (1.33 g), Angelicae Sinensis Radix (1 g), Cnidii Rhizoma (1 g), Atractylodis Macrocephalae Rhizoma (1.33 g) and Alismatis Rhizoma (1.66 g), was studied. In this study, the mechanism involved in the antiplatelet activity of DJS in human platelet suspensions was investigated. Danggijakyaksan did not significantly affect the thromboxane synthetase activity of aspirin‐treated platelet microsomes and DJS (20 and 40 µg/mL) significantly inhibited [3H]arachidonic acid (AA) released in collagen‐activated platelets but not in unactivated‐platelets. Nitric oxide (NO) production in human platelets was measured by a chemiluminesence detection method in this study. Danggijakyaksan did not significantly affect nitrate production in collagen (10 µg/mL)‐induced human platelet aggregation. On the other hand, various concentrations of DJS (10, 20, and 40 µg/mL) dose‐dependently inhibited [3H]inositol monophosphate (IP) formation stimulated by collagen (10 µg/mL) in [3H]myoinositol‐loaded platelets at different incubation times (1, 2, 3, and 5 min). It is concluded that the antiplatelet activity of DJS may possibly be due to the inhibition of phospholipase C activity, leading to reduced phosphoinositide breakdown, followed by the inhibition of thromboxane A2 formation, and then inhibition of [Ca2+]i mobilization of platelet aggregation stimulated by agonists.

Inhibition of Platelet Aggregation by Abciximab but Not by Aspirin Can Be Detected by a New Point-of-Care Test, the Hemostatus

We have investigated the type of platelet defect that can be detected with the Hemostatus test performed with the Hepcon/HMS instrument (Medtronic) designed to investigate platelet function during and after surgery. This assay is based on the comparison of the activated clotting time of whole blood measured in cartridges containing kaolin or kaolin and platelet-activating factor in different concentrations. Addition of platelet-activating factor shortened the blood activated clotting time when the platelet counts were normal. However, when platelet counts were below 70000/μL, the activated clotting time was prolonged in all channels including those without platelet-activating factor showing the influence of platelets in the formation of the clot under the conditions tested. Inhibition of platelet aggregation with c7E3 (abciximab, ReoPro) also induced a much-prolonged activated clotting time, and a similar finding was seen with blood from a patient with Glanzmann’s thrombasthenia confirming the need for platelet aggregation and/or the glycoprotein (GP) IIb-IIIa complex. In contrast, the interaction of GP Ib with von Willebrand Factor was not of major importance, since inhibition of this interaction with the anti-GP Ib murine monoclonal antibody, ALMA-12, did not modify the activated clotting time. Furthermore, the activated clotting time measured for patients with an acquired defect in von Willebrand Factor activity were unchanged. Finally, inhibition of thromboxane A2 formation by aspirin did not influence the results of this test. Globally, the Hemostatus test was able to detect major abnormalities of GP IIb-IIIa function in the presence or absence of platelet-activating factor.

Inhibitory mechanisms of naloxone on human platelets.

1. In the present study, naloxone was tested for its antiplatelet activities in human platelet-rich plasma (PRP). In human PRP, naloxone (0.1-0.5 mmol/L) inhibited aggregation stimulated by a variety of agonists (i.e. collagen, adenosine diphosphate (ADP), U46619 and adrenaline). 2. Naloxone (0.1-0.5 mmol/L) did not significantly affect cyclic adenosine monophosphate and cGMP levels in human washed platelets, whereas naloxone (0.5 mmol/L) significantly inhibited thromboxane B2 formation stimulated by collagen (5 micrograms/mL) in human washed platelets. 3. Naloxone (0.5 mmol/L) significantly inhibited [3H]-inositol monophosphate formation of [3H]-myoinositol-loaded platelets stimulated by collagen and U46619. Moreover, naloxone did not influence the binding of 125I-triflavin to platelet membranes. Triflavin is an Arg-Gly-Asp-containing specific fibrinogen receptor antagonist. 4. Addition of naloxone (0.5 mmol/L) to platelet preparations tagged with diphenylhexatriene (DPH) resulted in a considerable decrease in relative fluorescence intensity. 5. It is suggested that the anti-platelet effects of naloxone may be caused, at least partly, by the induction of conformational changes in the platelet membrane initially, followed by the inhibition of thromboxane A2 formation and phosphoinositide breakdown of platelets stimulated by agonists.

Enhanced arachidonic acid metabolism in alveolar macrophages from wheezy infants. Modulation by dexamethasone.

To test the hypothesis that alveolar macrophages (AM) from wheezy infants release increased amounts of eicosanoids, as do AM from adults with asthma, we compared eicosanoid release by unstimulated- and ionophore-A23187-stimulated AM from 13 wheezy and six nonwheezy infants and analyzed its regulation by dexamethasone in vitro. Alveolar macrophages from wheezy infants released greater amounts of thromboxane A2 (TxA2) and leukotriene B4 (LTB4) under resting conditions and of TxA2 upon stimulation than did those from control subjects. Dexamethasone induced a dose-dependent inhibition of the spontaneous and A23187-stimulated release of TxA2, but not of the A23187-stimulated release of lipoxygenase products. The inhibition of TxA2 formation was maintained when free arachidonic acid was added during A23187 stimulation, demonstrating that dexamethasone acted mainly at a postphospholipase A2 site. AM exposed to acetylsalicylate and then incubated overnight exhibited de novo cyclooxygenase synthesis, suggesting the presence of the inducible cyclooxygenase as a target for inhibition by dexamethasone. In conclusion, our findings suggest that AM from wheezy infants are activated in vivo to release eicosanoids, as are AM from asthmatic adults, and they support the therapeutic indications of glucocorticoids in severe recurrent wheezing of infancy.

Synthesis and antiplatelet activities of N-arylmethyl-3,4-dimethylpyrano[2,3-c]pyrazol-6-one derivatives.

A series of new 1- and 2-arylmethyl-3,4-dimethylpyrano[2,3-c]pyrazol-6-one derivatives were synthesized and examined for their antiplatelet activities. Some of these compounds showed significant inhibitory activities. Among them, 1-phenylmethyl-3,4-dimethylpyrano[2,3-c]pyrazol-6(1H)-one (4a), 2-(2'-methoxyphenyl)methyl-3,4-dimethylpyrano[2,3-c]pyrazol-6(2H)- one (3e) and 2-(3'-methoxyphenyl)methyl-3,4-dimethylpyrano[2,3-c]pyrazol-6-(2H) - one (3f) were the most effective. These inhibitors acted in a concentration-dependent manner. The antiplatelet effect of compound 3f is due to the inhibition of thromboxane A2 formation and the blockade of thromboxane A2/prostaglandin endoperoxide receptor in washed rabbit platelets.

Potentiation of myocardial salvage by tissue type plasminogen activator in combination with a thromboxane synthetase inhibitor in ischemic cat myocardium.

We studied the effects of a thrombolytic agent (t-PA) and a thromboxane synthetase inhibitor (CGS-13080) in a model of myocardial ischemia and reperfusion. Occlusion of the left anterior descending coronary artery for 2 hours followed by 4 hours of reperfusion in anesthetized cats results in a large washout of creatine kinase into the blood (32 +/- 7 IU/mg protein) and an area of necrotic tissue comprising 52 +/- 5% of the area at risk and 9 +/- 0.6% of the left ventricle. Intravenous administration of t-PA (500 IU/kg.min) for 30 minutes alone at reperfusion or infusion of CGS-13080 (500 micrograms/kg.hr) had no effect on washout of creatine kinase or extent of necrotic tissue development. Administration of the same doses of both t-PA and CGS-13080 together markedly attenuated creatine kinase release to 10 +/- 2 IU/mg protein (p less than 0.01) and reduced the area of necrotic tissue to 9 +/- 2% of the area at risk and only 1.3 +/- 0.3% of the left ventricle (p less than 0.001). No significant sustained effects of these agents were observed on mean arterial blood pressure, heart rate, or the pressure rate index in these experiments. Thus, t-PA and CGS-13080 exert synergistic effects in preserving myocardial integrity in cats subjected to acute myocardial ischemia followed by reperfusion. The mechanism of this beneficial effect does not appear to be via reduced myocardial oxygen demand, increased myocardial oxygen supply, or enhanced inhibition of thromboxane A2 formation. The mechanism of this anti-ischemic effect is not clear but may involve a metabolic or a cytoprotective effect.

Suppression of foam cell lesions in hypercholesterolemic rabbits by inhibition of thromboxane A2 synthesis.

Hypercholesterolemia induces adhesion of blood-borne monocytes to vascular endothelium and their subsequent migration into the intima, where foam cell lesions subsequently develop. The regulating mechanisms for the adhesion and migration are unclear. In this study, a specific thromboxane A2 (TXA2) synthetase inhibitor, UK-38485, was used to treat rabbits fed an atherogenic diet to determine whether inhibition of TXA2, the major metabolite of arachidonic acid in monocytes, affects lesion development. Rabbits were fed a diet supplemented with 2% cholesterol and 8% peanut oil for 12 weeks with or without UK-38485 at a dosage that maintained 80% to 90% inhibition of TXA2 formation in serum. The treatment with UK-38485 had no effect on total serum cholesterol. Both the treated and untreated groups developed subpopulations of high and low responders with respect to the extent of lesion coverage, forming a bimodal distribution. The treatment with UK-38485 significantly (p less than 0.001) reduced the percentage of the thoracic aorta covered by lesions when treated low responders (5.3 +/- 1.0%, n = 12) were compared to untreated low responders (23.6 +/- 2.9%, n = 12). However, UK-38485 had no effect when treated high responders (76.3%, n = 3) were compared to untreated high responders (72.0%, n = 12). Lesion coverage was not correlated with serum cholesterol levels. Stimulation of isolated rabbit monocytes in autologous plasma with 0.66 mM arachidonic acid in the presence of increasing concentrations of UK-38485 caused a dose-dependent inhibition of TXA2 and a concurrent increase in prostaglandin E2 (PGE2).(ABSTRACT TRUNCATED AT 250 WORDS)

O-104 - DP-1904 reduces ischemic myocardial injury with a complete inhibition of thromboxane A2 formation in a ligation-reperfusion model in rabbits

O-104 - DP-1904 reduces ischemic myocardial injury with a complete inhibition of thromboxane A2 formation in a ligation-reperfusion model in rabbits

Effects of platelet-modifying drugs on arterial thromboembolism in baboons. Aspirin potentiates the antithrombotic actions of dipyridamole and sulfinpyrazone by mechanism(s) independent of platelet cyclooxygenase inhibition.

To resolve questions of drug actions, efficacy, and interactions for platelet-modifying agents used clinically, we have compared the relative capacities and mechanisms of aspirin, dipyridamole, sulfinpyrazone, and dazoxiben to prevent arterial thromboembolism in a baboon model. In 136 studies the agents were given twice daily by oral administration both singly and in combination. The antithrombotic efficacy of a given therapy was determined by its capacity to interrupt steady-state platelet utilization induced by thrombogenic arteriovenous cannulae. When given alone, dipyridamole and sulfinpyrazone reduced the rate at which platelets were utilized by thrombus formation in a dose-dependent manner with essentially complete interruption by dipyridamole at 10 mg/kg per d. In contrast, neither aspirin (2-100 mg/kg per d) nor dazoxiben (20-100 mg/kg per d) decreased cannula platelet consumption detectably despite the striking reduction in the capacity of platelets to produce thromboxane B2. However, aspirin, but not dazoxiben, potentiated the antithrombotic effects of dipyridamole and sulfinpyrazone in a dose-dependent fashion without changing the pharmacokinetics for any of the agents. Complete potentiation required aspirin at 20 mg/kg per d to be given with each dose of dipyridamole. Because dazoxiben's blockade of platelet thromboxane A2 production was not associated with antithrombotic potentiation, and because complete potentiation by aspirin required a dose that fully inhibited vascular production of prostaglandin I2 (PGI2), we conclude that aspirin's potentiating effect on dipyridamole is independent of PGI2 production or inhibition of thromboxane A2 formation. In addition, because frequent repeated and synchronous dosing of aspirin was necessary, aspirin's potentiating effects appear to be produced by mechanism(s) unrelated to its potent, irreversible inhibition of platelet cyclooxygenase.

Dose-related kinetics of aspirin. Presystemic acetylation of platelet cyclooxygenase.

When aspirin is administered by mouth in low doses, poor systemic bioavailability may contribute to its apparent dose-related "selective inhibition" of thromboxane A2 formation. Systemic bioavailability of orally administered aspirin is necessary to inhibit prostacyclin synthesis by systemic vascular endothelium, whereas cumulative inhibition of thromboxane A2 formation by platelets may occur in the presystemic (portal) circulation. We simultaneously administered unlabeled aspirin orally and deuterium-labeled aspirin intravenously in five healthy volunteers. This permitted an estimation of the bioavailability of an oral dose from the ratio of plasma drug concentration-time curves for the labeled and the unlabeled species. Systemic bioavailability ranged from 46 to 51 per cent of single oral doses of 20, 40, 325, and 1300 mg of aspirin. Bioavailability was similar after single-dose and long-term oral administration of 325 mg. Thromboxane B2 formation in serum ex vivo after oral administration of 20 mg of unlabeled aspirin was reduced 39 per cent before aspirin was detected in the systemic circulation. Furthermore, incubation of simulated peak plasma aspirin concentrations in whole blood in vitro underestimated the inhibition of thromboxane B2 ex vivo after oral administration of 20 or 40 mg of unlabeled aspirin. These data are consistent with presystemic inhibition of platelets by aspirin and suggest that biochemical "selectivity" might be enhanced by slow administration of very low doses of aspirin, thereby optimizing conditions for cumulative, presystemic acetylation of platelet cyclooxygenase and inhibition of thromboxane formation.

The role of cytoplasmic free calcium in the responses of quin2-loaded human platelets to vasopressin.

Responses to vasopressin were studied in human platelets loaded with the fluorescent Ca2+ indicator, quin2. In the presence of 1 mM external Ca2+, vasopressin caused a transient rise in [Ca2+]i from the basal level near 100nM to about 700 nM; peak [Ca2+]i was reached in a few seconds and the level then declined towards resting over several minutes. In the absence of external Ca2+ there was a much smaller rise of similar time-course, suggesting that vasopressin increases [Ca2+]i mainly by stimulated-influx across the plasma membrane but also by partly releasing internal Ca2+. Inhibition of thromboxane A2 formation somewhat reduced the peak [Ca2+]i in the presence of external Ca2+, but had no effect on the response attributed to release of internal Ca2+. With external Ca2+, vasopressin stimulated shape-change, secretion and aggregation. Secretion and aggregation were decreased by about half following blockage of thromboxane production. The ability of vasopressin to induce shape-change and secretion even at near basal [Ca2+]i suggests that activators other than Ca2+ are involved.

Inhibition of thromboxane A2 synthesis in human platelets by coagulation factor Xa.

Factor Xa binds to platelets provided that factor Va is present on the platelet surface, an interaction that results in a striking acceleration of the conversion of prothrombin to thrombin. Thrombin then initiates fibrin formation, induces platelet aggregation, and stimulates the intraplatelet synthesis of thromboxane A2 (TXA2). Addition of thrombin (2.4-14.4 nM) to platelet-rich plasma increased the basal level of TXA2, measured as thromboxane B2, from less than 0.5 pmol per 10(8) platelets to (mean +/- SEM) 100 +/- 22 and 250 +/- 10 pmol per 10(8) platelets, respectively. Treatment of platelet-rich plasma with increasing concentrations of factor Xa (1-12 nM) prior to the addition of thrombin progressively inhibited the production of TXA2. Thrombin (9.6 nM), which produced 93% of the maximal formation of TXA2, was inhibited 70% by factor Xa (10 nM). To identify which of these steps in thromboxane synthesis was inhibited by factor Xa, platelets labeled with [14C]arachidonic acid were exposed to thrombin and products of prostaglandin synthesis were separated by thin-layer chromatography. In contrast to the inhibition of TXA2 synthesis, prostaglandin E2 and prostaglandin F2 alpha synthesis were not inhibited suggesting that neither phospholipase(s) nor cycloxygenase was involved. The inhibition of TXA2 formation by factor Xa could be reversed by increasing the molar ratio of thrombin to factor Xa to 5.5. Incubation of platelets with an IgG fraction of a human monoclonal antifactor V antibody, previously shown to inhibit factor Xa binding, was found to block factor Xa inhibition of TXA2 synthesis. The inhibition of TXA2 synthesis requires the presence of the active site serine of factor Xa and is not specific for TXA2 formation induced by thrombin because it is also demonstrable when the agonist is ADP. Further, factor Xa does not require additional plasma components for its action because its inhibitory effects are detected in gel-filtered platelets. The effect of factor Xa was evident at physiological (1.3 mM) calcium concentrations. These results indicate that factor Xa binding to platelets through factor Va not only stimulates thrombin formation but also has a countervailing effect by inhibiting TXA2 formation.

A Pharmacological Approach to the Inhibition of Platelet Adhesion and Platelet Aggregation

A first level of pharmacological interference with platelet function is located at the site of the agonist-receptor interaction (receptors for collagen, adenosine diphosphate, serotonin, fibrinogen). A second level of interaction is to prevent the mobilization of intracellular calcium ions which can be obtained by inhibition of phosphodiesterase, activation of cAMP (e.g. with prostacyclin analogues), inhibition of thromboxane A2 formation or blocking of its receptors. Compounds preventing the platelet contractile process or platelet secretion could theoretically also prevent some platelet functions.