The antiplatelet activity of rutaecarpine, an alkaloid isolated from Evodia rutaecarpa, is mediated through inhibition of phospholipase C

In this study, the mechanism involved in the antiplatelet activity of rutaecarpine in human platelet suspensions was investigated. In platelet suspensions (4.5 x 10(8)/ml), rutaecarpine (100 and 200 microM) did not influence the binding of FITC-triflavin to platelet glycoprotein IIb/IIIa complex. Additionally, rutaecarpine (200 microM) did not significantly change the fluorescence of platelet membrane labeled with diphenylhexatriene (DPH). On the other hand, rutaecarpine (50 and 100 microM) dose-dependently inhibited the increase in intracellular free Ca2+ of Fura 2-AM loaded platelets stimulated by collagen. Moreover, rutaecarpine (100 and 200 microM) did not significantly affect the thromboxane synthetase activity of aspirin-treated platelet microsomes. Furthermore, retaecarpine (100 and 200 microM) significantly inhibited [3H]arachidonic acid 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. Rutaecarpine (100 and 200 microM) did not significantly affect nitrate production in collagen (10 microg/ml)-induced human platelet aggregation. On the other hand, various concentrations of rutaecarpine (50, 100, and 200 microM) dose-dependently inhibited [3H]inositol monophosphate formation stimulated by collagen (10 microg/ml) in [3H]myoinositol-loaded platelets at different incubation times (1, 2, 3, and 5 minutes). It is concluded that the antiplatelet activity of rutaecarpine may possibly be due to the inhibition of phospholipase C activity, leading to reduce phosphoinositide breakdown, followed by the inhibition of thromboxane A2 formation, and then inhibition of [Ca2+]i mobilization of platelet aggregation stimulated by agonists.     

Reduction by arachidonic acid of prostaglandin I2-induced cyclic AMP formation. Involvement of prostaglandins E2 and F2 alpha

Arachidonic acid reverses the increase in cyclic AMP levels of washed human platelets exposed to prostaglandin (PG)I2, under conditions where the PGH2 analogue U46619 is ineffective. This effect of arachidonic acid was inhibited by aspirin, a cyclooxygenase inhibitor, but not by the thromboxane (Tx) synthase inhibitor Ridogrel, which induces, by inhibiting the conversion of PGH2 into TxA2, an overproduction of PGE2, PGD2 and PGF2 alpha. Addition of PGE2 or PGF2 alpha, which share a receptor with PGI2, to washed human platelets also induced a decrease in cyclic AMP levels, but PGD2, which interacts with a different receptor, had no effect. Thus neither PGD2, PGG2, PGH2, TxA2 nor TxB2 formed from arachidonic acid via the cyclooxygenase pathway is involved in the decrease in cyclic AMP levels. These findings were confirmed using forskolin, a diterpene from the labdane family, which enhanced the formation of cyclic AMP synergistically with the PGs. Also, arachidonic acid, unlike U46619, is able to reverse the inhibition of platelet aggregation by PGI2 after a lag phase of about 4 min. Our data indicate that arachidonic acid decreased cyclic AMP levels through its cyclooxygenase metabolites PGE2 and PGF2 alpha probably interacting competitively with the receptor of PGI2. In addition, intracellular cyclic AMP levels and the degree of aggregation of platelets by arachidonic acid seem to be inversely correlated.     

Isolation of human platelet membrane microparticles from plasma and serum

Methods have been developed to isolate human platelet membrane fragments from plasma and serum. Rabbit antibody produced against the human platelet membrane glycoprotein complex, IIb/IIIa, was utilized in an immunoelectrophoretic assay to evaluate the amount of this antigen in various microparticle preparations. The serum concentration of platelet microparticles was more than tenfold greater than that observed for plasma (65 micrograms/ml versus 4.4 micrograms/ml, respectively). Ultrastructural evaluation of either plasma or serum-derived microparticles disclosed a variety of membrane fragments and membrane-bound vesicles with occasional fragments of red blood cells, white blood cells, and platelets. In contrast, microparticle preparations derived from isolated washed platelets after thrombin stimulation contained a heterogeneous array of membrane fragments, vesicles, and granules but no identifiable red cell, white cell, or platelet fragments. Thus, these studies demonstrate that normal human plasma and serum contain platelet membrane fragments that are produced during cell activation. If a similar loss of platelet membranes occurs in vivo following reversible platelet activation, it is possible that the resulting membrane modifications may be of importance in both the structural and functional changes that develop during platelet senescence.     

Competitive inhibition of phospholipase A2 activity by the platelet-activating factor antagonist Ro 19-3704 and evidence for a novel suppressive effect on platelet activation

The compound Ro 19-3704 [3-4(R)-2-(methoxycarbonyl) oxy-3-(octadecylcarbamoyl)oxy-propoxy butylthiazolium iodide], initially described as an antagonist of platelet-activating factor, is reported here to directly inhibit rabbit platelet phospholipase (PL) A2 activity, with an IC50 value of 4 to 7 microM. Classical Michaelis-Menten analysis showed that inhibition was reversible and competitive, inasmuch as apparent Km values increased in the presence of Ro 19-3704 (from 0.2-0.4 to 2 microM), whereas Vmax values remained constant (200 +/- 20 nmol/min/10(9) cells). Ro 19-3704 inhibited platelet aggregation, PLA2 release and thromboxane B2 formation induced by thrombin (0.25 U/ml), with IC50 values of 8, 15 and below 5 microM, respectively. Aggregation and PLA2 release by arachidonic acid (100 microM) were also inhibited, but thromboxane B2 formation was unaffected, indicating that Ro 19-3704 does not inhibit cyclooxygenase. Platelet activation by collagen (5 micrograms/ml), the thromboxane mimetic U46619 ([15(S)-hydroxy-11,9(epoxymethano)-prosta-5Z,13E-dienoic acid] 1 microM) and low concentrations of thrombin (0.05-0.1 U/ml) was also inhibited by Ro 19-3704. Inhibition of platelet activation was reversible, suggesting that its suppressive effect was not due to cytotoxicity. Finally, Ro 19-3704 did not stimulate cyclic AMP formation or inhibit phosphodiesterase activity. Ro 19-3704 is a competitive inhibitor of PLA2 activity, and is also endowed with a potent suppressive effect on platelet activation induced by different agonists.     

Thromboxane A2 synthase inhibition and thromboxane A2 receptor blockade by 2-[(4-cyanophenyl)amino]-3-chloro-1,4-naphthalenedione (NQ-Y15) in rat platelets

The effects of 2-[(4-acetylphenyl)amino]-3-chloro-1,4-naphthalenedione (NQ-Y15), a synthetic 1,4-naphthoquinone derivative, on platelet activity and its mechanism of action were investigated. NQ-Y15 caused a concentration-dependent inhibition of the aggregation induced by thrombin, collagen, arachidonic acid (AA), and A23187. The IC50 values of NQ-Y15 on thrombin (0.1 U/mL)-, collagen (10 microg/mL)-, AA (50 microM)-, and A23187 (2 microM)-induced aggregation were 36.2 +/- 1.5, 6.7 +/- 0.7, 35.4 +/- 1.7, and 93.1 +/- 1.4 microM, respectively. NQ-Y15 also inhibited thrombin-, collagen-, AA-, and A23187-stimulated serotonin secretion in a concentration-dependent manner. However, a high concentration (100 microM) of NQ-Y15 showed no significant inhibitory effect on ADP-induced primary aggregation, which is independent of thromboxane A2 (TXA2) production in rat platelets. In fura-2-loaded platelets, the elevation of intracellular free calcium concentration stimulated by AA, thrombin, and 4-bromo-A23187 was inhibited by NQ-Y15 in a concentration-dependent manner. The formation of TXA2 caused by AA, thrombin, and collagen was inhibited significantly by NQ-Y15. NQ-Y15 inhibited TXA2 synthase in intact rat platelets, since this agent reduced the conversion of prostaglandin (PG) H2 to TXA2. Similarly, NQ-Y15 selectively inhibited the TXA2 synthase activity in human platelet microsomes, whereas it had no effect on activity of phospholipase A2, cyclooxygenase, and PGI2 synthase in vitro. NQ-Y15 inhibited platelet aggregation induced by the endoperoxide analogue U46619 in human platelets, indicating TXA2 receptor antagonism, possibly of a competitive nature. These results suggest that the antiplatelet effect of NQ-Y15 is due to a combination of TXA2 synthase inhibition with TXA2 receptor blockade, and that it may be useful as an antithrombotic agent.     

ZD1542, a potent thromboxane A2 synthase inhibitor and receptor antagonist in vitro

1. The thromboxane A2 synthase (TXS) inhibitory activity and the thromboxane A2 (TP)-receptor blocking action of ZD1542 (4(Z)-6-[2S,4S,5R)-2-[1-methyl-1-(2-nitro-4-tolyloxy)ethyl]-4-(3- pyridyl)-1,3-dioxan-5-yl]hex-4-enoic acid) has been evaluated in vitro on platelets and whole blood from a range of species including man. Antagonist activity has also been investigated in vascular and pulmonary smooth muscle preparations in vitro. 2. ZD1542 caused concentration-dependent inhibition of human platelet microsomal thromboxane B2 (TXB2) production in vitro (IC50 = 0.016 microM); this inhibition was associated with an increase in prostaglandin E2 (PGE2) and PGF2 alpha formation. 3. ZD1542 also inhibited collagen-stimulated TXS in human, rat and dog whole blood giving IC50 values of 0.018, 0.009 and 0.049 microM respectively. The drug did not modify platelet cyclo-oxygenase activity as inhibition of TXB2 formation was associated with a concomitant increase in the levels of PGD2, PGE2 and PGF2 alpha. ZD1542 had little if any effect against cultured human umbilical vein endothelial cell (HUVEC) cyclo-oxygenase (IC50 > 100 microM) and prostacyclin (PGI2) synthase (IC50 = 18.0 +/- 8.6 microM). 4. ZD1542 caused concentration-dependent inhibition of U46619-induced aggregation responses of human, rat and dog platelets yielding apparent pA2 values of 8.3, 8.5 and 9.1 respectively. The drug was selective as, at concentrations up to 100 microM, it did not modify 5-hydroxytryptamine (5-HT) or the primary phases of adenosine diphosphate (ADP) and adrenaline-induced aggregation. Furthermore, ZD1542 (100 microM) modified only weakly the platelet effects of PGD2, PGE1 and PGI2. 5. ZD1542 also caused concentration-dependent inhibition of U46619-mediated contractions of rat thoracic aorta, guinea-pig trachea and lung parenchyma preparations giving apparent pA2 values of 8.6,8.3 and 8.5 respectively. At concentrations approaching three orders of magnitude greater than those required to block U46619-mediated contractions, the drug did not affect the actions of non-prostanoid agonists or exhibit agonist activity in any of the smooth muscle preparations employed; neither did it interact at EP- or FP-receptors.6. In conclusion, the present study demonstrates that ZD1542 is a drug that exhibits both potent,selective TXS inhibition and TXA2 receptor antagonism.     

Comparative studies of suidatrestin, a specific inhibitor of trehalases

The ability to synthesise prostaglandins and thromboxane from 14C-labelled arachidonic acid was investigated in 11 species of fish from the Arabian Gulf. Cyclooxygenase activity was assessed in washed whole blood cells. Arachidonic acid and its metabolites were extracted and separated on silicic acid columns and thin layer chromatography (silica gel G). Total capacity to convert [14C]arachidonic acid to prostanoids varied from 1 to 35% among the 11 fish species studied. Gray shark (Chiloscyllium griseum) blood cells had the highest capacity (37 +/- 0.4%) to convert arachidonate into prostanoids and two species of catfish (Arius bilineatus and A. thalassinus) exhibited greater than 10% capacity to convert [14C]arachidonate into prostanoids. The major prostanoid synthesised by the two catfish (A. bilineatus and A thalassinus) was 6-keto PGF1 alpha, a stable metabolite of prostacyclin, PGI2. In contrast, A. teunispinis synthesised thromboxane B2, a stable metabolite of thromboxane A2. Thromboxane B2 (TXB2) was the major product synthesised by all three species of shark studied (Chil. griseum, Carcharhinus plumbeus, Carch. melanopterus), with 6-keto PGF1 alpha a minor product. Other fish studied showed a varied pattern of prostanoid synthesis. The synthesis of these prostanoids was almost completely blocked by preincubation of the whole blood cells from catfish and shark with indomethacin (0.5 microM) suggesting the involvement of cyclooxygenase-mediated prostanoid synthesis.     

Direct inhibition of cyclooxygenase-1 and -2 by the kinase inhibitors SB 203580 and PD 98059. SB 203580 also inhibits thromboxane synthase

The kinase inhibitors SB 203580 and PD 98059 have been reported to be specific inhibitors of the 38- and 42/44-kDa mitogen-activated protein kinase (MAPK) pathways, respectively. In this study, the two inhibitors were found to decrease platelet aggregation induced by low concentrations of arachidonic acid, suggesting that they also interfere with the metabolism of arachidonic acid to thromboxane A2. In support of this, SB 203580 and PD 98059 inhibited the conversion of exogenous [3H]arachidonic acid to [3H]thromboxane in intact platelets. Measurement of platelet cyclooxygenase-1 activity following immunoprecipitation revealed that SB 203580 and PD 98059 are direct inhibitors of this enzyme. Both compounds were shown to inhibit purified cyclooxygenase-1 and -2 by a reversible mechanism. In addition, SB 203580 (but not PD 98059) inhibited platelet aggregation induced by prostaglandin H2 and the conversion of prostaglandin H2 to thromboxane A2 in intact platelets. SB 203580 also inhibited this pathway in platelet microsome preparations, suggesting a direct inhibitory effect on thromboxane synthase. These results demonstrate that direct effects of the two kinase inhibitors on active arachidonic acid metabolites have to be excluded before using these compounds for the investigation of MAPKs in signal transduction pathways. This is of particular relevance to studies on the regulation of cytosolic phospholipase A2 as these two MAPKs are capable of phosphorylating cytosolic phospholipase A2, thereby increasing its intrinsic activity.     

Dual effects of nimesulide, a COX-2 inhibitor, in human platelets

Nimesulide (CAS 51803-78-2) has been shown to exert marked anti-inflammatory effect in several in vivo models of inflammation. Since nimesulide is considered to be a selective inhibitor of COX-2, it has not been studied in detail in relation to its mechanistic effects on platelets, which express COX-1. This study was conducted to investigate the effects of nimesulide in platelet aggregation. We show that nimesulide (1-100 microM) inhibited platelet aggregation induced by adrenaline (20-200 microM). It also inhibited thromboxane A2 (TXA2) formation by platelets at low concentration (IC50; 1 microM). However, much lower concentrations of nimesulide (0.01-0.1 microM) potentiated the aggregatory response of subthreshold concentrations of adrenaline (0.2-2 microM). Such an effect was blocked by Ca2+-channel blockers, verapamil and diltiazem (IC50: 7 and 46 microM, respectively), nitric oxide donor, SNAP (IC50; 2 microM) and cinchonine (10 nM) but not by genistein (up to 10 microM). These results are indicative of the concentration-dependent dual effects of nimesulide on human platelet aggregation. The synergistic effect of low doses of nimesulide and adrenaline seems to be mediated through inhibition of multiple signalling pathways.     

In vitro effects of trapidil on fatty acid and prostaglandin metabolism in platelets

The effects of trapidil, a coronary vasodilator and platelet aggregation inhibitor, on fatty acid metabolism and prostaglandin (PG) formation in platelets were studied using platelet suspensions from six normal subjects. The addition of trapidil to fatty acids in platelet phospholipids decreased palmitoleic acid and arachidonic acid, and increased an unidentified substance, X2 (palmitoleic acid, P < 0.05; arachidonic acid, P < 0.05; X2, P < 0.05). Thrombin stimulation following the addition of trapidil resulted in an increase in stearic acid and a decrease in arachidonic acid, compared with the trapidil-free control samples (stearic acid, P < 0.05; arachidonic acid, P < 0.02). The addition of trapidil tended to increase immunoreactive PGE (iPGE) and iPGF dose-dependently. On the other hand, thrombin stimulation following the addition of trapidil decreased the formation of thromboxane B2 (TXB2) significantly compared with the levels of TXB2 in the trapidil-free samples (10 micrograms/mL trapidil, P < 0.005, 100 micrograms/mL trapidil, P < 0.001). These results show that trapidil increased arachidonic acid mobilization in the platelets.     

Phosphorylation of the thromboxane receptor alpha, the predominant isoform expressed in human platelets

A single gene encodes the human thromboxane receptor (TP), of which there are two identified splice variants, alpha and beta. Both isoforms are rapidly phosphorylated in response to thromboxane agonists when overexpressed in human embryonic kidney 293 cells; this phenomenon is only slightly altered by inhibitors of protein kinase C. Pharmacological studies have defined two classes of TP in human platelets; sites that bind the agonist I-BOP with high affinity support platelet shape change. Low affinity sites, which irreversibly bind the antagonist GR 32191, transduce platelet activation and aggregation. Isoform-specific antibodies permitted detection of TPalpha, but not TPbeta, from human platelets, although mRNA for both isoforms is present. A broad protein band of 50-60 kDa, reflecting the glycosylated receptor, was phosphorylated upon activation of platelets for 2 min with I-BOP. This was a rapid ( approximately 30 s) and transient (maximum, 2-4 min) event and was inhibited by TP antagonists. Both arachidonic acid and low concentrations of collagen stimulated TPalpha phosphorylation, which was blocked by cyclooxygenase inhibition or TP antagonism. Blockade of the low affinity TP sites with GR 32191 prevented I-BOP-induced TPalpha phosphorylation. This coincided with agonist-induced platelet aggregation and activation but not shape change. Also, activation of these sites with the isoprostane iPF2alpha-III induced platelet shape change but not TPalpha phosphorylation. Heterologous TP phosphorylation was observed in aspirin-treated platelets exposed to thrombin, high concentrations of collagen, and the calcium ionophore A 23187. Both homologous and heterologous agonist-induced phosphorylation of endogenous TPalpha was blocked by protein kinase C inhibitors. TPalpha was the only isoform detectably translated in human platelets. This appeared to correspond to the activation of the low affinity site defined by the antagonist GR 32191 and not activated by the high affinity agonist, iPF2alpha-III. Protein kinase C played a more important role in agonist-induced phosphorylation of native TPalpha in human platelets than in human embryonic kidney 293 cells overexpressing recombinant TPalpha.     

Six primary squamous cell carcinomas of the head and neck

Estrogens have a beneficial effect on atherosclerosis and osteoporosis after menopause, but their exact mechanism of action is still unknown. The aim of the present study was to investigate the effects of estradiol and its metabolites catechol estrogens on arachidonic acid metabolism in vitro. Estradiol had no effect on arachidonic acid metabolism up to 33 microM in A23187-stimulated human whole blood. All catechol estrogens (2-hydroxyestradiol, 2-hydroxyestrone, 4-hydroxyestradiol and 4-hydroxyestrone) had similar kinds of actions on arachidonic acid metabolism, being over ten times more potent inhibitors of leukotriene synthesis (IC50 values 0.044-0.16 microM) than thromboxane (IC50 values 0.99-2.1 microM) and prostaglandin E2 synthesis (IC50 values 0.84-5.5 microM). It is suggested that some of the protective actions of estrogens--e.g., on atherosclerosis and osteoporosis--may be related to the inhibition of leukotriene synthesis by catechol estrogens.     

Inhibition of platelet-vessel wall interactions by thromboxane receptor antagonism in a human in vitro system: potentiation of antiplatelet effects of aspirin

BACKGROUND: Pharmacological inhibition of arachidonic acid metabolism has proven therapeutically useful in the prevention of cardiovascular events. METHODS: We have investigated the ability of Bay u 3405, a synthetic thromboxane antagonist, to interfere with platelet aggregation and arachidonic acid metabolism. The antiplatelet action was also analysed in a perfusion system in which vascular subendothelium was exposed to circulating human blood (10 min; shear rate = 800 s-1). Platelet interactions were morphometrically analysed and results compared with those obtained in studies with blood from donors taking aspirin (acetylsalicylic acid, ASA) (500 mg day-1). The additional effect of Bay u 3405 on the antiplatelet action of ASA was also evaluated. RESULTS: Bay u 3405 caused a dose-dependent inhibition of platelet aggregation induced by U46619 with a maximal effect at concentrations > or = 0.01 microgram mL-1. Higher concentrations (> or = 0.05 micrograms mL-1) also inhibited aggregations induced by ADP or collagen. Bay u 3405 did not interfere with platelet arachidonic acid metabolism. In perfusion studies, Bay u 3405 (0.01 microgram mL-1) significantly decreased the total surface of the vessel covered by platelets (%CS = 18.7 +/- 1.09 vs. 24.4 +/- 1.94; P < 0.05) and the formation of large aggregates %T = 7.5 +/- 0.87 vs. 19.3 +/- 1.61; P < 0.01). ASA treatment reduced platelet aggregate formation (%T = 13.7 +/- 2.06; P < 0.05) but did not affect the total surface covered by platelets. The in vitro addition of Bay u 3405 to blood from ASA-treated donors further reduced the formation of large aggregates (%T = 2.7 +/- 0.79; P < 0.01 vs. ASA). CONCLUSIONS: In vitro effect of Bay u 3405 on platelet function were superior to those observed with ASA. The thromboxane antagonism antagonism provided by Bay u 3405 further enhanced the inhibition of platelet aggregate formation found after ASA treatment.     

Inhibition of platelet aggregation by S-nitroso-cysteine via cGMP-independent mechanisms: evidence of inhibition of thromboxane A2 synthesis in human blood platelets

S-Nitroso-cysteine (SNC), a putative endothelium-derived relaxing factor, potently inhibited collagen- and arachidonic acid-induced platelet aggregation (IC50=100 nM) and thromboxane A2 (TxA2) synthesis of human blood platelets. ODQ, a selective inhibitor of the soluble guanylyl cyclase, inhibited SNC-induced formation of cGMP but did not reverse inhibition by SNC of collagen- and arachidonic acid-induced platelet aggregation. Combination of ODQ with SQ-29548, a specific platelet TxA2 receptor antagonist, did not modify the antiaggregatory action of SNC. Our study shows that SNC inhibits platelet aggregation by cGMP-independent mechanisms that may involve inhibition of TxA2 synthesis in human platelets.     

Re-interpreting anaerobic metabolism: an argument for the application of both anaerobic glycolysis and excess post-exercise oxygen comsumption (EPOC) as independent sources of energy expenditure

We studied the effects of porcine factor VIII (P-FVIII; Hyate:C) and other coagulation products employed in the management of patients with hemophilia A, on platelet activation in vitro. Exposure of normal resting platelets to P-FVIII resulted in platelet activation, as manifested by increased expression of the platelet surface activation markers CD62, CD63, and activated-GPIIbIIIa, and by activation-induced modulation of expression of normal platelet membrane glycoproteins CD41, CD42, and CD36. In contrast, platelet activation was not observed after exposure of the platelets to human FVIII, FEIBA, recombinant FVIIa, or cryosupernatant plasma. As with thrombin, exposure of platelets to P-FVIII resulted in the generation of platelet microparticles, an effect not seen not with the other products. In contrast to the characteristic reduction in expression in the number of CD42 molecules detected on thrombin-activated platelets, P-FVIII-stimulated platelets showed a small increase in CD42 expression. In contrast to thrombin, P-FVIII did not cause platelet dense granule release. The results indicate that therapeutic P-FVIII activates platelets, likely in ways that are different from the platelet activation seen with thrombin. The observed platelet activation and microparticle generation may provide a "hypercoagulable" mechanism for hemostasis with P-FVIII therapy separate from, and additional to, that due to increased circulating FVIII levels.     

Increased platelet sensitivity to ADP in mice lacking platelet-type 12-lipoxygenase

Arachidonic acid metabolism is one of several mechanisms culminating in the production of an agonist for platelet activation and recruitment. Although the proaggregatory role of thromboxane A2, a product of the aspirin-inhibitable cyclooxygenase, is well established, relatively little is known regarding the biological importance of arachidonic acid metabolism via the 12-lipoxygenase (P-12LO) pathway to 12-hydro(pero)xyeicosatetraenoic acid. We observed that platelets obtained from mice in which the P-12LO gene has been disrupted by gene targeting (P-12LO-/-) exhibit a selective hypersensitivity to ADP, manifested as a marked increase in slope and percent aggregation in ex vivo assays and increased mortality in an ADP-induced mouse model of thromboembolism. The hyperresponsiveness to ADP is independent of dense granule release, cyclooxygenase-derived eicosanoid synthesis, and protein kinase C activity. The addition of 12-hydroxyeicosatetraenoic acid to P-12LO-/- platelet-rich plasma rescues the hyperresponsive phenotype resulting in a diminished ADP-induced aggregation profile. The enhanced ADP sensitivity of P-12LO-/- mice appears to reveal a mechanism by which a product of the P-12LO pathway suppresses platelet activation by ADP.     

Pharmacological analysis of cyclooxygenase-1 in inflammation

The enzymes cyclooxygenase-1 and cyclooxygenase-2 (COX-1 and COX-2) catalyze the conversion of arachidonic acid to prostaglandin (PG) H2, the precursor of PGs and thromboxane. These lipid mediators play important roles in inflammation and pain and in normal physiological functions. While there are abundant data indicating that the inducible isoform, COX-2, is important in inflammation and pain, the constitutively expressed isoform, COX-1, has also been suggested to play a role in inflammatory processes. To address the latter question pharmacologically, we used a highly selective COX-1 inhibitor, SC-560 (COX-1 IC50 = 0.009 microM; COX-2 IC50 = 6.3 microM). SC-560 inhibited COX-1-derived platelet thromboxane B2, gastric PGE2, and dermal PGE2 production, indicating that it was orally active, but did not inhibit COX-2-derived PGs in the lipopolysaccharide-induced rat air pouch. Therapeutic or prophylactic administration of SC-560 in the rat carrageenan footpad model did not affect acute inflammation or hyperalgesia at doses that markedly inhibited in vivo COX-1 activity. By contrast, celecoxib, a selective COX-2 inhibitor, was anti-inflammatory and analgesic in this model. Paradoxically, both SC-560 and celecoxib reduced paw PGs to equivalent levels. Increased levels of PGs were found in the cerebrospinal fluid after carrageenan injection and were markedly reduced by celecoxib, but were not affected by SC-560. These results suggest that, in addition to the role of peripherally produced PGs, there is a critical, centrally mediated neurological component to inflammatory pain that is mediated at least in part by COX-2.     

Contribution of platelet microparticle formation and granule secretion to the transmembrane migration of phosphatidylserine

Activation of human platelets by complement proteins, C5b-9, thrombin plus collagen, or a Ca2+ ionophore results in surface exposure of phosphatidylserine (PS), accompanied by the expression of membrane catalytic activity for the tenase (VIIaIXa) and prothrombinase (VaXa) coagulation enzyme complexes. The mechanism underlying this surface exposure of PS upon platelet activation remains unresolved. Using fluorescent derivatives of PS (NBD-PS), we have investigated how the transmembrane migration of PS is related to microvesiculation of the platelet plasma membrane and to fusion of storage granules with the plasma membrane. Gel-filtered platelets were incubated with NBD-PS, allowing 90 +/- 10% of the incorporated NBD-PS to accumulate into the inner leaflet of the plasma membrane. Migration of NBD-PS from the inner leaflet to the plasma membrane surface was monitored by time-based flow cytometry, and correlated with the appearance of platelet microparticles and alpha-granule secretion. Platelet activation by C5b-9 or the Ca2+ ionophore, A23187, increased surface exposure of NBD-PS, due to acceleration of the apparent rate of migration from inner to outer plasma membrane leaflets. The onset of this accelerated migration of NBD-PS to the surface coincided with the onset of plasma membrane vesiculation, and the NBD-PS that partitioned into the membrane of the shed microparticle was also rapidly exposed to the surface (t1/2 < 2 min). In addition to a temporal correlation, microparticle formation and the surface exposure of inner leaflet NBD-PS showed a similar requirement for Ca2+. These results demonstrate that agonist-induced microvesiculation of the platelet plasma membrane is accompanied by accelerated migration of a PS analogue from the inner leaflet to the surface of the shed microparticle membrane, suggesting the mechanism by which induction of platelet microparticle formation exposes catalytic surface for tenase and prothrombinase assembly.     

The effect of thrombin on the uptake and transformation of arachidonic acid by human platelets

Washed human platelets take up arachidonic acid from plasma and incorporate the fatty acid into the major classes of complex lipids. Thrombin impairs net incorporation. It activates endogenous phospholipases which liberate arachidonic acid from phospholipids. As a consequence of thrombin induced aggregation platelets release arachidonic acid intermediates formed by the action of platelet fatty acid cyclooxygenase and by platelet fatty acid lipoxygenase. Cyclooxygenase, but not lipoxygenase, is inhibited by aspirin and indomethicin. Analysis of the pathways of arachidonic acid metabolism may furnish new insight into platelet function and into disorders of primary hemostasis.