Platelet Thromboxane B2 Generation Research Articles

Enantioselective pharmacodynamics of the nonsteroidal antiinflammatory drug ketoprofen: in vitro inhibition of human platelet cyclooxygenase activity.

The pharmacological activity of ketoprofen enantiomers was investigated in humans by an in vitro method. The antiplatelet effect of ketoprofen was assessed by measuring the inhibition of platelet thromboxane B2 (TXB2) generation during the controlled clotting of whole blood obtained from each of four healthy volunteers. Ketoprofen was added separately to whole blood as a range of concentrations of (1) predominantly (S)-ketoprofen, (2) racemic ketoprofen, and (3) predominantly (R)-ketoprofen. (S)-Ketoprofen was found to be solely active at inhibiting human platelet TXB2 production; (R)-ketoprofen was devoid of such activity and did not modify the potency of its optical antipode. A relationship between the percentage inhibition of TXB2 generation and the unbound concentration of (S)-ketoprofen in serum was modelled according to a sigmoidal Emax equation. The mean (+/- SD) serum unbound concentration of (S)-ketoprofen required to inhibit platelet TXB2 generation by 50% (EC50) was 0.320 (+/- 0.062) ng/ml. This value for ketoprofen is considerably lower than previously reported values for (S)-ibuprofen and (S)-naproxen.

Effect of racemic ibuprofen dose on the magnitude and duration of platelet cyclo‐oxygenase inhibition: relationship between inhibition of thromboxane production and the plasma unbound concentration of S(+)‐ ibuprofen.

1. Four healthy male subjects received racemic ibuprofen (200, 400, 800 and 1200 mg), orally, on four occasions, 2 weeks apart, according to a four-way Latin-square design, in order to investigate the influence of increasing dose of ibuprofen on the magnitude and duration of its antiplatelet effect as well as on the relationship between such effect and drug concentration. 2. The antiplatelet effect of ibuprofen was assessed by measuring the inhibition of platelet thromboxane B2 (TXB2) generation during the controlled clotting of whole blood. The plasma unbound concentration of S(+)-ibuprofen, the enantiomer shown in an in vitro study to be responsible for the inhibitory effect of platelet TXB2 generation, was measured using an enantioselective method. 3. The maximum percentage inhibition of TXB2 generation increased significantly with dose from a mean +/- s.d. of 93.4 +/- 1.2% after the 200 mg dose to 98.8 +/- 0.3% after the 1200 mg dose, and there was an increase with dose in the duration of inhibition of TXB2 generation. The effect of ibuprofen on platelet TXB2 generation was transient and mirrored the time-course of unbound S(+)-ibuprofen in plasma; on all but one of the 16 occasions, serum TXB2 concentrations returned to at least within 10% of the pretreatment concentrations within 24 h of ibuprofen administration. 4. For each subject, the relationship between the percentage inhibition of TXB2 generation and the unbound concentration of S(+)-ibuprofen in plasma was modelled according to a sigmoidal Emax equation. The mean plasma unbound concentration of S(+)-ibuprofen required to inhibit platelet TXB2 generation by 50% (EC50) was 9.8 +/- 1.0 micrograms l-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormal Typical Pattern of Platelet Function and Thromboxane Generation in Unstable Angina

Platelet aggregation (PA), platelet thromboxane B2 (TXB2) generation and 14C 5-hydroxytryptamine (5HT) release were studied in 13 patients with unstable angina, and compared to 14 patients with stable angina and 16 healthy controls. A typical pattern, distinct in 4 aspects from stable angina patients or controls, was observed in the unstable angina patients. ADP or collagen induced shape change was 3-4 times greater, the extent of epinephrine induced PA was nil or very low, the extent of collagen induced 14C 5HT release was also reduced while collagen induced platelet TXB2 generation was increased in spite of a reduced extent of PA. The extent of ADP or collagen induced PA was also significantly reduced. These results indicate a platelet membrane abnormality occurring presumably during contact of the circulating platelets with a non-occlusive thrombus observed at sites of ruptured plaques in unstable angina patients. Since also the pattern (20-30% overlap with control values) was distinct from that of stable angina patients, it might indicate an active thrombotic process. Plasma beta-thromboglobulin (beta TG) and TXB2 levels and serum TXB2 generation were also studied in the cardiac patients and controls and in another 10 patients with advanced peripheral occlusive arterial disease (POAD). Plasma beta TG and TXB2 levels were slightly elevated in the unstable angina patients and markedly elevated in the POAD patients. Serum TXB2 generation was, however, elevated in the stable angina patients (p less than 0.002) and more so in the unstable angina patients (p less than 0.001) compared to controls or to POAD patients. This was presumably mediated through enhanced thrombin generation. These results suggest that the measured plasma beta TG variable in the unstable angina patients is not useful in the assessment of in vivo platelet activation. It is presumably reflecting the sum of local enhanced platelet activation (at sites of ruptured plaques) and of reduced function of the "defective" circulating platelets. The ability of the platelets of unstable angina patients to generate large amounts of TXB2 if occurring in vivo might induce an intense coronary vasospasm.

The effect of a thromboxane synthetase inhibitor, OKY-046, on urinary excretion of immunoreactive thromboxane B2 and 6-keto-prostaglandin F1 alpha in patients with ischemic cerebrovascular disease.

Thromboxane synthetase activity is selectively inhibited by (E)-3-[4-(1-imidazolylmethyl)phenyl]-2-propenoic acid hydrochloride monohydrate (OKY-046). A single dose of 100 mg OKY-046 was orally administered to patients with ischemic cerebrovascular disease and healthy volunteers. Platelet aggregation and thromboxane B2 (TXB2) generation of intact and homogenised platelets induced by 1.0 mM sodium arachidonate were measured before and at 1, 4, 6 and 8 h after dosing. OKY-046 inhibited arachidonate-induced aggregation in platelet rich plasma from some, but not all, individuals, whereas platelet TXB2 generation was almost completely inhibited by a single dose of 100 mg OKY-046, in all of the patients and healthy volunteers. Endogenous TXA2 and prostacyclin (PGI2) biosynthesis were assessed by measurement of urinary immunoreactive TXB2 (i-TXB2) and 6-keto-PGF1 alpha (i-6-keto-PGF1 alpha) before and at 0-3, 3-6, 6-9 h after dosing. OKY-046 increased the urinary i-6-keto-PGF1 alpha coincidently with a decrease of urinary i-TXB2, both in patients and healthy volunteers. These effects of a selective thromboxane synthetase inhibitor will improve a disturbed balance between TXA2 and PGI2, associated with the development of ischemic cerebrovascular disease.

Equal Antiplatelet Effects of Aspirin 50 or 324 mg/Day in Patients After Acute Myocardial Infarction

This study explores the effects on some hematological parameters of a low-dose aspirin regimen (50 mg/day) versus a conventional aspirin treatment with reported antithrombotic efficacy (324 mg/day), in patients with acute myocardial infarction. Fifteen patients were randomized into 3 equal groups receiving 50 mg or 324 mg aspirin or placebo, daily for 21 days. Compared with placebo, bleeding time was significantly and similarly prolonged with both aspirin doses (+ 71 +/- 22% and + 69 +/- 20%, mean +/- S.D.). Aspirin 50 mg/day suppressed arachidonate-induced platelet aggregation and secondary phase aggregation after ADP and adrenaline. Collagen aggregation was inhibited by 44 +/- 15%. In no case were differences in the antiplatelet effects of the two doses observed. The effects of 50 mg/day persisted without attenuation during the observation period. Platelet thromboxane B2 generation during arachidonate-induced aggregation was inhibited by 95 +/- 2 and 99 +/- 1% compared to placebo group after 50 and 324 mg/day, respectively (P between doses less than 0.05). No change was observed with any treatment in coagulation time, prothrombin time or plasma thromboplastin time. Thus, in patients with acute myocardial infarction, the antiplatelet effects of aspirin 50 mg/day are stable over time and superimposable on those of 324 mg/day. The antithrombotic efficacy of aspirin 50 mg/day remains to be tested clinically.

Thromboxane B2 production by fetal and neonatal platelets: effect of idiopathic respiratory distress syndrome and birth asphyxia.

To study the production of proaggregatory thromboxane A2 (TxA2) by fetal and neonatal platelets, blood specimens were collected from umbilical cords immediately after delivery at term (n = 22), from newborn infants during the first 10 days of life (n = 85), from infants between 1 and 3 months of age (n = 14), and from healthy adults (n = 18). The blood samples were allowed to clot spontaneously at +37 degrees C for 60 min, and the concentrations of thromboxane B2 (TxB2), a stable metabolite of TxA2, in the sera were measured by radioimmunoassay and expressed as nanograms of TxB2/10(6) platelets. Platelet TxB2 generation in term infants at the age of 1 day (1.344 +/- 0.253 ng/10(6) platelets, mean +/- SE, n = 9) was higher than that in cord blood (0.634 +/- 0.042 ng/10(6) platelets, n = 22), or in infants of 1-3 months of age (0.881 +/- 0.099 ng/10(6) platelets, n = 14), or in adults (0.869 +/- 0.062 ng/10(6) platelets, n = 18). Increase in TxB2 generation following birth was seen already at the age of 1 h (1.076 +/- 0.114 ng/10(6) platelets, n = 9). TxB2 synthesis in preterm infants (1.032 +/- 0.136 ng/10(6) platelets, n = 10) did not differ from that in term infants on the 1st day of life, and idiopathic respiratory distress syndrome had no effect on it (1.029 +/- 0.079 ng/10(6) platelets, n = 19). Severe birth asphyxia was accompanied by reduced TxB2 formation (0.564 +/- 0.201 ng/10(6) platelets, n = 7).(ABSTRACT TRUNCATED AT 250 WORDS)

837 INCREASED PLASMA THROMBOXANE LEVELS IN SICKLE CELL DISEASE

Platelet function abnormalities, i.e., increased circulating platelet aggregates and decreased platelet aggregation, have been observed in sickle cell disease patients. To determine the mechanism of these abnormalities, we studied 13 patients with sickle cell disease and 10 normal subjects. Peripheral venous blood samples were collected for plasma thromboxane B2 (TXB2) (stable metabolite of TXA2) determinations by radioimmunoassay. In addition, platelets were stimulated with sodium arachidonate (1 mM) and thrombin (10 units/m1), and platelet TXB2 generation quantitated. In 11 patients in steady state, plasma TXB2 levels were increased compared to normal subjects (480 ± 41 vs 252 ± 37 pg/ml, P<0.01). In contrast, platelet TXB2 generation in response to sodium arachidonate was lower in patients with sickle cell disease (44 ± 10 vs 103 ± 32 pg/108 platelets, P<0.01). Similarly, thrombin-induced platelet TXB2 generation was decreased (186 ± 45 vs 801 ± 320 pg/108 platelets, P<0.01). In 2 patients with acute vaso-occlusion, plasma TXB2 levels were extremely high (1650 and 1350 pg/m1), but platelet TXB2 generation was similar to those in steady state. Increased plasma TXB2 levels in sickle cell disease patients imply continuous platelet activation and may be a mechanism of increase in circulating platelet aggregates. Decrease in platelet TXB2 generation suggests either substrate or enzyme deficiency due to persistent platelet activation, and may be the cause of decreased platelet aggregation.