Alpha-tocopherol, an effective inhibitor of platelet adhesion

Platelet adhesiveness was tested ex vivo in a group of six normal individuals receiving varying doses of alpha-tocopherol. Adhesion to glass slides coated with fibronectin, collagen, fibrinogen, or plasma proteins was studied by perfusing platelet-rich plasma through a flow chamber that allowed time- and space-resolved observations of platelet adhesion. Platelet adherence was measured in an area of parallel flow lines and low shear rate under standardized conditions before and after dietary supplementation with vitamin E at doses of 200 and 400 IU/d. Platelet adherence differed in magnitude depending on the adhesive surface. There was a distinct preference of platelets to adhere to sites that had been previously occupied. A remarkable decrease in platelet adherence was observed after vitamin E supplementation. The average decrease in adhesion after 2 weeks of 200 IU vitamin E was 75%. After 2 weeks of 400 IU vitamin E, platelet adhesion was reduced by 82%. The inhibitory activity of alpha-tocopherol was dose dependent and correlated well with the increase in alpha-tocopherol concentration in platelets after supplementation. Scanning electron microscopy revealed a striking decrease of pseudopodium formation in alpha-tocopherol- enriched platelets. Our results suggest that vitamin E may also be an effective antiadhesive agent in vivo.     

R68070, a combined thromboxane/endoperoxide receptor antagonist and thromboxane synthase inhibitor, inhibits human platelet activation in vitro and in vivo: a comparison with aspirin

We have investigated the effects of R68070 on platelet function in vitro and in vivo. The drug inhibits U46619-induced aggregation (IC50 = 1.2 x 10(-6) mol/L), blocks serum thromboxane formation (IC50 = 1 x 10(-7) mol/L), and increases serum prostaglandin (PG)E2 and 6-keto-PGF1 alpha levels, indicating that it combines thromboxane receptor blocking and thromboxane synthase inhibiting properties. The thromboxane-dependent aggregation of blood platelets is blocked by R68070, whereas no inhibition of thromboxane independent pathways occurs. A double-blind, randomized, cross-over study was performed on nine volunteers, comparing 400 mg placebo, 400 mg aspirin, and 400 mg R68070. Thromboxane-dependent aggregations were significantly inhibited by R68070 and by aspirin, the latter still having the most pronounced action. However, R68070 was clearly more powerful than aspirin (P less than .0005) in prolonging the bleeding time. Serum TxB2 formation was completely inhibited with both treatments, whereas serum 6-keto-PGF1 alpha and PGE2 and intralesional 6-keto-PGF1 alpha were inhibited after aspirin and stimulated after R68070. We conclude that R68070 inhibits platelet thromboxane synthase and its thromboxane receptor both in vitro and in vivo; local reorientation of cyclic endoperoxide metabolism toward prostacyclin induces a stronger inhibition of hemostasis than that produced by aspirin.     

Cumulative inhibitory effect of low-dose aspirin on vascular prostacyclin and platelet thromboxane production in patients with atherosclerosis

The relationship between the antithrombotic and antiplatelet effects of aspirin is complex, since aspirin influences other systems that protect against thrombosis as well as inhibiting platelet function. We investigated possible cumulative effects of low-dose aspirin on vascular production of prostacyclin in patients with documented atherosclerotic cardiovascular disease. Candidates for coronary artery vein graft bypass ingested 20 mg of aspirin daily during the week before surgery, and platelet aggregation, platelet formation of thromboxane A2 (TXA2), aortic and saphenous vein production of prostacyclin (PGI2), and hemostatic status were measured at the time of the bypass surgery. Low-dose aspirin markedly inhibited platelet aggregation responses and reduced TXA2 generation by greater than 90%, effects similar to those observed with much higher doses of aspirin. Both aortic and saphenous vein production of PGI2 were inhibited by 50% compared with PGI2 produced by vascular tissues of control subjects who received no aspirin preoperatively (51 +/- 10 pg 6-keto-PGF1 alpha/mg aortic wet weight [mean +/- SEM] in aspirin-treated subjects vs 130 +/- 16 pg/mg in control subjects, and 71 +/- 8 pg/mg saphenous vein wet weight vs 131 +/- 17 pg/mg). Blood loss at surgery was not significantly increased by preoperative low-dose aspirin as measured by chest tube drainage (754 +/- 229 ml in aspirin-treated subjects vs 645 +/- 271 ml in control subjects), hematocrit nadir (31.2 +/- 1.9% vs 31.8 +/- 1.7%), or transfusions (2.2 +/- 1.3 units of red blood cells vs 2.2 +/- 1.7 units).(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of CV-4151, a selective inhibitor of thromboxane synthetase,on prostanoid formation and platelet aggregation in humans

Summary The pharmacokinetics and pharmacologic effects of a potent, selective inhibitor of thromboxane synthetase, CV-4151 [(E)-7-phenyl-7-(3-pyridyl)-6-heptenoic acid] on prostanoid formation and platelet aggregation were studied in 42 healthy male volunteers. The drug was well tolerated. After oral administration of 10 to 100 mg of CV-4151, peak plasma levels of 1–6 g/mL were reached in a dose-dependent mannor within 1 hour. Elimination followed first-order fashion with elimination half-life of about 1 hour. Serum levels of thromboxane B₂ reduced to 4% to 15% of control at 2 hours after drug ingestion dose-dependently. Serum levels of 6-Keto-prostaglandin F₁ increased to about four to six times basal levels. Platelet aggregation induced by collagen and arachidonate was inhibited in most eases. Such pharmacologic effects outlasted serum drug levels. In repeated administration, stable inhibition of serum thromboxane B₂ production and platelet aggregation in proportion to the enhancement of serum 6-keto-prostaglandin F₁ production was observed although no drug accumulation was found. These results indicate that CV-4151 may be suitable for clinical trials in cardiovascular diseases in which imbalance between thromboxane and prostacyclin may be involved in the pathogenesis.First Department of Medicine, Osaka University School of Medicine     

Mechanisms underlying the inhibitory effects of lipopolysaccharide on human platelet adhesion

Alterations in platelet aggregation in septic conditions are well established. However, little is known about the effects of lipopolysaccharide (LPS) on platelet adhesion. We have therefore investigated the effects of LPS in human platelet adhesion, using an in vitro model of platelet adhesion to fibrinogen-coated wells. Microtiter plates were coated with human fibrinogen, after which washed platelets (6 × 10⁸ platelets/ml) were allowed to adhere. Adherent platelets were quantified through measurement of acid phosphatase activity. Calcium mobilization in Fura2-AM-loaded platelets was monitored with a spectrofluorimeter. Platelet flow cytometry in thrombin-stimulated platelets was performed using monoclonal mouse anti-platelet GPIIb/IIIa antibody (PAC-1). Prior incubation of washed platelets with LPS (0.01–300 µg/ml) for 5 to 60 min concentration- and time-dependently inhibited non-activated platelet adhesion. In thrombin-activated (50 mU/ml) platelets, LPS inhibited the adhesion to a significantly lesser extent than non-activated platelets. Cyclohexamide, superoxide dismutase polyethylene glycol (PEG-SOD) or catalase polyethylene glycol did not affect the LPS responses. No alterations in cyclic GMP levels were seen after platelet incubation with LPS, except with the highest concentration employed (300 µg/ml) where an increase of 36% (P < 0.05) was observed. Thrombin increased by 7.5-fold the internal Ca²⁺ platelet levels, an effect markedly inhibited by LPS. Thrombin induced concentration-dependent platelet GPIIb/IIIa activation, but LPS failed to affect the activation state of this membrane glycoprotein. In conclusion, LPS inhibits human platelet adhesion to fibrinogen by mechanisms involving blockade of external Ca²⁺, independently of cGMP generation and activation of GPIIb/IIIa complex.     

Cathepsin G-dependent platelet stimulation by activated polymorphonuclear leukocytes and its inhibition by antiproteinases: role of P-selectin-mediated cell-cell adhesion

Human PMN stimulated by fMLP are able to activate coincubated, autologous platelets. Cathepsin G, a neutral serine protease stored in the azurophilic granules of PMN, is the major platelet activator in this system. We previously proposed that shear-induced close PMN- platelet contact creates the conditions for which cathepsin G activity on platelets is protected against antiproteinases. The aim of this study was to investigate the adhesive mechanisms, possibly creating between PMN and platelet membranes the microenvironment in which cathepsin G, discharged from stimulated PMN onto adherent platelets, is protected against antiproteinases. Microscopic examination showed that under conditions of high shear, 71.3% +/- 6.1% of PMN were associated to platelets forming small clumps. This percentage decreased to 10% +/- 2% and 13% +/- 4%, respectively, in the presence of an inhibitory antibody to P-selectin or 20 mmol/L mannose-1-phosphate and to 10.8% +/- 3.7% when cells were not stirred. Similarly, PMN pretreatment with neuraminidase abolished PMN binding to platelets. These results indicate that P-selectin mediates PMN-platelet adhesion occurring before PMN stimulation. Prevention of PMN-platelet contact significantly potentiated the inhibitory effect of alpha 1-protease inhibitor on subsequent cathepsin G-induced platelet serotonin release. Because anti-P-selectin antibody, mannose-1-phosphate, and neuraminidase treatment of PMN did not modify PMN-induced platelet activation in the absence of antiproteinases, it is suggested that P- selectin-mediated PMN-platelet adhesion results in the formation of a sequestered microenvironment between cell membranes, in which higher amounts of antiproteinases are required to prevent the activity of released cathepsin G. These data add a new functional role to P- selectin-mediated PMN-platelet adhesion that could be important in vivo because of the presence of antiproteinases in plasma.     

Levosimendan has an inhibitory effect on platelet function

Levosimendan enhances cardiac contractility by increasing myocyte sensitivity to calcium, and induces vasodilatation. Although studies have evaluated the efficacy of levosimendan in heart failure, it is not clear whether it might produce functional influence on platelet response. In this study, the effect of levosimendan on platelet aggregation was investigated. Platelet function tests were performed in 12 healthy male volunteers. Three concentrations of levosimendan solution were prepared that would result in 10, 25, and 45 ng/ml levosimendan concentrations in the blood similar to that observed after clinical therapeutic intravenous application of 0.05-0.1 microg/kg/min. Each concentration of levosimendan solution and a control diluent without levosimendan were incubated with whole blood at 37 degrees C. After incubation for 15 min, aggregation responses were evaluated with adenosine diphosphate (ADP) (5 and 10 microM) and collagen (2 and 5 microg/ml) in platelet-rich plasma. Preincubation with all dilutions of levosimendan inhibited aggregation of platelets induced by ADP and collagen significantly. Levosimendan also inhibited significantly the secondary wave of platelet aggregation induced by ADP. The results showed that there was a relationship between levosimendan concentration and inhibition of platelet aggregation. In conclusion, this study with an in vitro model showed that levosimendan had a significant inhibitory effect on platelets in clinically relevant doses.     

The inhibitory effect of plasma fibronectin on collagen-induced platelet aggregation

Plasma fibronectin (Fn) has been proposed to have an antithrombotic effect, protecting against platelet and fibrinogen consumption after injury. The current study was designed to determine the effect of plasma fibronectin on collagen-induced platelet aggregation. In vitro aggregometry using an isolated homologous rat system, demonstrated a significant (P less than .05) inhibitory effect of 120 micrograms/mL Fn on platelet aggregation as induced by 60 micrograms/mL fibrillar collagen (type I). The inhibition was evidenced by a threefold increase in lag time and a significant decrease in the rate and extent of aggregation. The hypothesis was also tested using an in vivo model of collagen-induced platelet aggregation. The model used was intravenous injection of 2 mg/kg of homologous type I collagen into anesthetized Sprague-Dawley rats. Injection of collagen preincubated with 4 mg/kg Fn resulted in significantly less thrombocytopenia and fibrinogen consumption as compared with injection of collagen alone. The results of both the in vitro and in vivo studies are consistent with the proposed antithrombotic effect of plasma fibronectin.     

Increased thromboxane biosynthesis in a human preparation of platelet activation: biochemical and functional consequences of selective inhibition of thromboxane synthase

Although thromboxane A2 is a potent platelet agonist and vasoconstrictor in vitro, our knowledge of its pathophysiologic importance in human disease is limited. To facilitate the elucidation of its role in vivo, we sought to define a human syndrome in which pharmacologic interventions designed to inhibit the biosynthesis or biologic actions of thromboxane A2 might be appropriately assessed. Patients with severe peripheral vascular disease were selected on the basis of elevated plasma beta-thromboglobulin and circulating platelet aggregates and compared with healthy, age-matched control subjects. In addition to the platelet indexes, their bleeding time was shorter and excretion of 2,3-dinor-thromboxane B2, a noninvasive index of thromboxane formation in vivo, and 2,3-dinor-6-keto-prostaglandin F 1 alpha, the major urinary metabolite of prostacyclin, was markedly increased. A selective inhibitor of thromboxane synthase, imidazo (1,5-2) pyridine-5-hexanoic acid, was administered to these patients under randomized, double-blind, controlled conditions. Platelet aggregation ex vivo, the circulating platelet aggregate ratio, and the bleeding time were all unaltered, despite almost maximal inhibition of platelet thromboxane formation 1 hr after dosing. By contrast, pronounced inhibition of aggregation was observed when platelet cyclooxygenase was inhibited by aspirin. During long-term dosing with the synthetic inhibitor, inhibition of thromboxane biosynthesis was incomplete, which would permit continued thromboxane-dependent platelet aggregation to occur. However, the failure of enzyme blockade to influence platelet function at the time of maximal drug action, despite efficient inhibition of serum thromboxane B2, suggests that accumulation of proaggregatory endoperoxides is also likely to have contributed to the persistence of platelet activation. We have characterized a human preparation in which platelet activation coexists with increased thromboxane biosynthesis. In this setting, platelet activation persists despite long-term administration of a thromboxane synthase inhibitor in a dosing regimen representative of that employed in clinical trials. Prolongation of drug action and combination with antagonists of the shared endoperoxide/thromboxane A2 receptor may be necessary to assess the potential of selective inhibition of thromboxane synthase as a therapeutic strategy in man.     

Beneficial effect of a thromboxane synthetase inhibitor in traumatic shock

Traumatic shock was induced in anesthetized rats using the Noble-Collip method. This resulted in an abrupt decline in mean arterial blood pressure (MABP) and heart rate. Plasma cathepsin D activity increased sixfold, plasma thromboxane B2 (TxB2) concentration increased 2.5-fold, plasma myocardial depressant factor (MDF) activity increased 3.5 fold, and the mean survival time was 1.4 +/- 0.2 hours. Administration of the selective thromboxane synthetase inhibitor 5-(3-pyridinylmethyl) benzofuran-2-carboxylate (U-63,557A) (4 mg/kg) resulted in a significant improvement in survival time, 3.3 +/- 0.5, p less than 0.01. Plasma cathepsin D activity was not affected by U-63,557A (7.4 +/- 0.8 vs. 8.5 +/- 1.1 U/ml). However, both plasma and peritoneal fluid TxB2 concentration were significantly reduced and accumulation of the toxic peptide, MDF, was significantly blunted (69 +/- 6 vs. 40 +/- 5 U/ml, p less than 0.01). Our data indicate that blockade of thromboxane A2 (TxA2) production by selective synthetase inhibition is beneficial in trauma and support a role for TxA2 in the pathogenesis of circulatory shock.     

Intracoronary administration of a thromboxane A2 synthase inhibitor relieves acetylcholine-induced coronary spasm.

This study sought to clarify the effectiveness of intracoronary administration of a thromboxane (TX) A2 synthase inhibitor, Ozagrel Na, to relieve coronary spasms induced by intracoronary injection of acetylcholine (ACh). An ACh spasm provocation test was performed in 92 consecutive patients with coronary spastic angina using incremental doses of 20, 50, and 80 microg into the right coronary artery, and 20, 50, and 100 microg into the left coronary artery within 20s. A coronary spasm was defined as TIMI 0 or 1 flow and an intracoronary injection of 20 mg Ozagrel Na was administered when it was provoked. Within 2 min of the administration of the TXA2 synthase inhibitor, ACh-induced coronary spasms were relieved (TIMI 3 flow) in 88.1% of procedures without complications. In only 4 cases (4.3%), it took more than 3 min to relieve the coronary spasms. Intracoronary administration of 20mg Ozagrel Na when ACh-induced spasms occurred, shortened the spasm relief time in all 7 patients (200 +/- 59s vs 111 +/- 23s, p < 0.01), improved the maximal ST segment elevation in 5 of them (3.9 +/- 3.7 mm vs 0.7 +/- 1.5 mm, p < 0.05), and stopped chest pain in 4 patients. In 4 patients who had ACh-induced coronary spasm of the left anterior descending artery, the TXB2 concentration in the coronary sinus decreased after intracoronary administration of Ozagrel Na into the left coronary artery (463 +/- 562 vs 96 +/- 45, p < 0.01). In conclusion, intracoronary administration of a TXA2 synthase inhibitor can relieve ACh-induced coronary spasms by inhibiting TXA2 synthesis in the local coronary circulation.     

特异性血栓素合成酶抑制剂对急性坏死性胰腺炎犬胰腺及肺损伤的影响

目的观察特异性血栓素合成酶抑制剂对急性坏死性胰腺炎（ANP）犬胰腺及肺损伤的影响,探讨其作用机制。方法20只健康雄性杂种犬按数字表法随机分为对照组（4只）、ANP组（8只）及特异性血栓素合成酶抑制剂（商品名：丹奥）治疗组（治疗组,8只）。采用胰管内逆行注射5％牛黄胆酸钠及胰蛋白酶混合液的方法制备ANP模型,治疗组于制模成功后2h起静脉输注2mg／kg体质量的丹奥,每天2次,连用7d。术后动态监测各组血清钙、超敏C反应蛋白（HCRP）、血栓素A2（TXA2）、前列环素（PGI2）水平。7d后处死动物,取胰腺及肺组织行病理学检查,并评分。结果对照组胰腺及肺组织无明显病理改变;ANP组胰腺及肺损伤严重;治疗组胰腺及肺组织损伤较ANP组减轻。对照组、ANP组、治疗组的胰腺病理评分分别为（1．25±0．96）、（7．00±2．39）、（4．63±1．19）分;肺组织病理评分分别为（0．75±0．50）、（7．13±1．55）、（4．88±0．83）分,ANP组、治疗组的胰腺、肺组织病理评分均显著高于对照组,而治疗组的评分又较ANP组显著降低（P值均〈0．05）。对照组术后1d的血清钙、HCRP、TXB2、keto—PGFla水平及TXB2／keto—PGFla比值分别为（2．45±0．07）mmol／L、（30．36±4．29）mg／L、（345．8±46．8）pg／ml、（187．8±18．6）pg／ml、1．85±0．16;ANP组为（2．21±0．08）mmol／L、（72．04±10．22）mg／L、（1227．3±118．2）pg／ml、（368．8±64．4）pg／ml、3．33±0．19;治疗组为（2．32±0．08）mmol／L、（66．51±4,28）mg／L、（1179．5±116．3）pg／ml、（371．8±65．2）pg／ml、3．17±0．18。ANP组与治疗组术后血清钙水平较对照组显著下降,而其他指标显著升高,差异均具有统计学意义（P值均〈0．01）。治疗组血清钙水平显著高于ANP组,血HCRP水平显著低于ANP组,差异有统计学意义（P〈0．05或〈0．01）。结论应用特异性血栓素合成酶抑制剂治疗后ANP犬的胰腺及肺脏损伤程度减轻,其机制与阻断TXA2合成,纠正TXA2／PGI2的比例失衡,改善胰腺及肺组织微循环有关。     

Zileuton,a 5-lipoxygenase inhibitor,increases production of thromboxane A2 and platelet aggregation in patients with asthma

Leukotrienes, generated from arachidonic acid via the lipoxygenase pathway, play an important role in the pathophysiology of asthma. Therefore, leukotriene inhibitors, such as Zileuton, are used in the treatment of asthma. However, thromboxanes, generated from arachidonic acid via the cyclooxygenase pathway, play an important role in platelet aggregation and thrombosis. Therefore, we studied whether Zileuton, by shifting arachidonic acid to the cyclooxygenase pathway, enhances thromboxane production and, hence, platelet aggregation. Blood samples were collected from 10 asthmatic patients before and 2 weeks after standard Zileuton treatment. Spontaneous platelet aggregation was measured in platelet-rich plasma. Platelet-rich plasma was also used to determine thromboxane B(2), a stable metabolite of thromboxane A(2), as the indirect measure of thromboxane A(2) because thromboxane A(2) is too unstable for assay. Baseline thromboxane B(2) and platelet aggregation values in the 10 asthmatic patients were normal. Treatment with Zileuton for 2 weeks significantly increased thromboxane B(2) levels from baseline levels of 267 +/- 54 microg/l to 389 +/- 62 microg/l after 2 weeks of treatment (P < 0.0002). Spontaneous platelet aggregation also increased significantly from baseline values of 4.2 +/- 2.4% to 6.8 +/- 2.8% after 2 weeks of treatment (P < 0.0001). These results establish that Zileuton, an effective drug for asthma, adversely affects in vitro platelet function. The findings suggest that this drug, and perhaps related agents also, may pose a thrombotic risk; clinical attention will be needed to address this possibility.     

Calin--a platelet adhesion inhibitor from the saliva of the medicinal leech.

The saliva of the medicinal leech, Hirudo medicinalis, contains a potent, hitherto unsuspected, inhibitor of collagen-mediated platelet adhesion/aggregation. Calin, of molecular size approximately 65,000 (reduced), has a rapid (1-10 min) effect on collagen which is reflected in its ability to suppress collagen-induced platelet aggregation, as well as adhesion of platelets to collagen-coated microcarrier beads. It also causes flocculation of Type I collagen fibril suspensions. Calin is differentiated from leech collagenase in two ways: (1) by demonstrating, by SDS-PAGE analysis of the products of incubations of Calin with Type I collagen at 37 degrees C, that Calin binds to but does not cleave collagen; and (2) by showing that Calin cannot be purified using the methods used to isolate leech collagenase. Calin's rapid and unusual interaction with collagen makes it a prime candidate for one of the agents that are the causative factors of the prolonged bleeding phenomenon seen after leech bites.     

Production and characterization of saratin, an inhibitor of von Willebrand factor-dependent platelet adhesion to collagen

Platelets tether to collagen in both a von Willebrand factor (vWF)-dependent and a vWF-independent manner. We have recently characterized a recombinant protein, saratin, isolated from the saliva of the leech Hirudo medicinalis, expressed it in Hansenula polymorpha, and studied its effect on direct and indirect platelet-collagen interactions. Saratin dose dependently inhibited the binding of purified human vWF to human type I and III collagens (IC(50)= 0.23 +/- 0.004 and 0.81 +/- 0.04 microg mL(-1), respectively) and to calf skin collagen (IC(50)= 0.44 +/- 0.008 microg mL(-1)). Furthermore, saratin showed a similar inhibitory potency against the binding of human, rodent, and porcine plasma vWF to these collagens. In a flow chamber under conditions of elevated shear (2700 s(-1)), saratin dose dependently and potently inhibited platelet aggregate formation on a collagen-coated surface (IC(50)= 0.96 +/- 0.25 microg mL(-1)), but at reduced shear (1300 s(-1)) a rightward shift in the dose-response curve was noted (IC(50)= 5.2 +/- 1.4 microg mL(-1)). Surface plasmon resonance analysis revealed both high and low affinity binding sites for saratin on human collagen type III (K(d) 5 x 10(-8) M and 2 x 10(-6) M, respectively). Although low concentrations of saratin, which inhibited platelet adhesion under increased shear (i.e., saturation of high-affinity binding sites), had no effect on vWF-independent collagen-induced platelet aggregation, high concentrations (i.e., saturation of low-affinity binding sites) were found to inhibit platelet aggregation. These data demonstrate that saratin is a potent inhibitor of vWF-dependent platelet adhesion to collagen and hence may have therapeutic potential as an antithrombotic agent.     

Inhibition of thromboxane formation in vivo and ex vivo: implications for therapy with platelet inhibitory drugs

The capacity of platelets to generate thromboxane A2, reflected by measurement of serum thromboxane B2 (TxB2), greatly exceeds the systemic production of thromboxane in vivo. Thus, it is possible that substantial but incomplete inhibition of thromboxane formation ex vivo would still allow marked augmentation of thromboxane production in vivo. To address this hypothesis, we administered aspirin 120 mg, a selective inhibitor of thromboxane synthase (TxSl), 3-(1H-imidazol-1-yl- methyl)-2-methyl-1H-indole-1-propanoic acid (UK-38, 485) 200 mg, and a combination of both drugs to 12 healthy volunteers and measured the effects on serum TxB2 and urinary 2,3-dinor-thromboxane B2 (Tx-M), an index of endogenous thromboxane biosynthesis. Although serum TxB2 was maximally inhibited by 94 +/- 1% after aspirin and 96 +/- 2% after the TxSl, maximal depression of Tx-M was only 28 +/- 8% and 37 +/- 9%, respectively. Combination of aspirin with the TxSl resulted in a small but significant increase in inhibition of thromboxane generation ex vivo (98 +/- 1% v 94 +/- 1%; P less than 0.05), but a disproportionately greater fall in thromboxane synthesis in vivo (58 +/- 7%; P less than 0.01). Consistent with further inhibition of platelet thromboxane synthesis, addition of the TxSl abolished the transient decline in prostacyclin formation after aspirin alone. Administration of a lower dose of aspirin (20 mg) to 6 healthy subjects caused a small reduction in Tx-M (12 +/- 4%; P less than 0.05) and inhibited serum TxB2 by 48 +/- 2%. The relationship between inhibition of platelet capacity to form thromboxane ex vivo (serum TxB2) and synthesis in vivo (Tx-M) departed markedly from the line of identity. When total blockade of the capacity of platelets to generate thromboxane is approached, minor decrements in capacity result in a disproportionate depression of actual thromboxane biosynthesis. These results imply that pharmacologic inhibition of serum TxB2 must be virtually complete before thromboxane- dependent platelet activation is influenced in vivo.