Potent Inhibitor Of Platelet Aggregation Research Articles

Risk of stroke associated with abciximab among patients undergoing percutaneous coronary intervention.

Abciximab, a potent inhibitor of the platelet glycoprotein IIb/IIIa receptor, reduces thrombotic complications in patients undergoing percutaneous coronary intervention (PCI). Because of its potent inhibition of platelet aggregation, the effect of abciximab on risk of stroke is a concern. To determine whether abciximab use among patients undergoing PCI is associated with an increased risk of stroke. Combined analysis of data from 4 double-blind, placebo-controlled, randomized trials (EPIC, CAPTURE, EPILOG, and EPISTENT) conducted between November 1991 and October 1997 at a total of 257 academic and community hospitals in the United States and Europe. A total of 8555 patients undergoing PCI with or without stent deployment for a variety of indications were randomly assigned to receive a bolus and infusion of abciximab (n = 5476) or matching placebo (n = 3079). One treatment group in EPIC received a bolus of abciximab only. Risk of hemorrhagic and nonhemorrhagic stroke within 30 days of treatment among abciximab and placebo groups. No significant difference in stroke rate was observed between patients assigned abciximab (n = 22 [0.40%]) and those assigned placebo (n = 9 [0.29%]; P =.46). Excluding the EPIC abciximab bolus-only group, there were 9 strokes (0.30%) among 3023 patients who received placebo and 15 (0.32%) in 4680 patients treated with abciximab bolus plus infusion, a difference of 0.02% (95% confidence interval [CI], -0.23% to 0.28%). The rate of nonhemorrhagic stroke was 0.17% in patients treated with abciximab and 0.20% in patients treated with placebo (difference, -0.03%; 95% CI, -0.23% to 0.17%), and the rates of hemorrhagic stroke were 0.15% and 0.10%, respectively (difference, 0.05%; 95% CI, -0.11% to 0.21%). Among patients treated with abciximab, the rate of hemorrhagic stroke in patients receiving standard-dose heparin in EPIC, CAPTURE, and EPILOG was higher than in those receiving low-dose heparin in the EPILOG and EPISTENT trials (0.27% vs 0.04%; P =.057). Abciximab in addition to aspirin and heparin does not increase the risk of stroke in patients undergoing PCI. Patients undergoing PCI and treated with abciximab should receive low-dose, weight-adjusted heparin.

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

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

Antiplatelet Therapy for Acute Coronary Syndromes

A common feature of acute coronary syndromes (ACS) is a partial occlusion of major coronary arteries due to atherosclerotic plaque disruption and subsequent thrombus formation. The antiplatelet drug aspirin has been one part of the standard therapy for ACS but has a number of adverse effects such as upper gastrointestinal bleeding and peptic ulceration. New antiplatelet agents, such as the thienopyridine derivatives and glycoprotein IIb/IIIa receptor antagonists, are potent inhibitors of platelet aggregation and thrombus formation. They reduce cardiac mortality and morbidity in patients with ACS. When used in addition to aspirin and/or heparin, they produce further reductions in cardiac death and myocardial infarction.

Synthesis and biological evaluation of novel pyrazoles and indazoles as activators of the nitric oxide receptor, soluble guanylate cyclase.

Database searching and compound screening identified 1-benzyl-3-(3-dimethylaminopropyloxy)indazole (benzydamine, 3) as a potent activator of the nitric oxide receptor, soluble guanylate cyclase. A comprehensive structure-activity relationship study surrounding 3 clearly showed that the indazole C-3 dimethylaminopropyloxy substituent was critical for enzyme activity. However replacement of the indazole ring of 3 by appropriately substituted pyrazoles maintained enzyme activity. Compounds were evaluated for inhibition of platelet aggregation and showed a general lipophilicity requirement. Aryl-substituted pyrazoles 32, 34, and 43 demonstrated potent activation of soluble guanylate cyclase and potent inhibition of platelet aggregation. Pharmacokinetic studies in rats showed that compound 32 exhibits modest oral bioavailability (12%). Furthermore 32 has an excellent selectivity profile notably showing no significant inhibition of phosphodiesterases or nitric oxide synthases.

ADVANCES IN THE PHARMACOLOGY OF ACUTE CORONARY SYNDROME: Platelet Inhibition

The development of potent inhibitors of platelet aggregation has led to significant decreases in morbidity and mortality rates among patients undergoing percutaneous coronary intervention. Clinical trials have demonstrated that agents that block glycoprotein IIb/IIIa receptor-mediated platelet aggregation have an outcome benefit when used acutely in patients with chest pain and ST depression or elevated cardiac enzymes, leading to the integration of these agents into emergency medicine clinical practice. This article provides an overview of the pathophysiology of acute coronary syndrome and the pharmacology of platelet inhibition and reviews the evidence from the clinical trials pertaining to the use of these agents in the emergency department.

Pharmacokinetics and Concentration‐Effect Analysis of Intravenous RGD891, a Platelet GPIIb/IIIa Antagonist, Using Mixed‐Effects Modeling (NONMEM)

RGD891 is a platelet GPIIb/IIIa receptor antagonist and potent inhibitor of platelet aggregation. This compound is biotransformed in vivo to RGD039, which also exhibits high affinity for the GPIIb/IIIa receptor. Pharmacokinetic/pharmacodynamic modeling was employed to describe the concentration-effect relationship of both compounds following the intravenous administration of RGD891 to healthy volunteers. The overall objectives of this work were to support the dose selection process for future intravenous RGD891 safety and efficacy studies. Various intravenous regimens of RGD891 were administered to healthy volunteers enrolled in three Phase I studies. Frequent plasma samples were collected at regular intervals for later measurement of RGD891 and RGD039 concentrations (validated LC/MS/MS methods). The pharmacokinetics of RGD891 and RGD039 were simultaneously analyzed by nonlinear mixed-effect modeling (NONMEM). Pharmacodynamic activity was assessed in all three studies by the degree to which ADP (20 microM)-induced platelet aggregation was inhibited. Population parameters describing the concentration-effect relationship of RGD891 and RGD039 were then generated using a modified competitive Emax-based model. Parent compound is by far the predominant active compound circulating in the plasma following intravenous administration of RGD891. The plasma RGD891 concentration-time data were best fit by a two-compartment structural model. The fit of the basic model was improved when total body weight was introduced as a covariate for RGD891 distribution. Between-subject variability in the RGD891 pharmacokinetic parameters--V1, K10, and K21--was less than 17% (coefficient of variation). Formation of the active metabolite (RGD039; Km) and its elimination (Kem) were assumed to be first-order processes (i.e., one-compartment model). The population pharmacokinetic model could only provide a rough estimate of the plasma concentration-time profile for RGD039 after administration of a given intravenous dosage regimen of RGD891 since metabolite concentrations were relatively low and highly variable. The first-order rate constant describing the formation of RGD039 from RGD891 (Km) was also associated with a substantial degree of between-subject variability (44.9%). The potency of RGD891 toward the inhibition of ADP-induced platelet aggregation was described by the population IC50 value (plasma concentration yielding 50% of maximal inhibition), which ranged from 58.0 to 95.4 ng/mL, depending on the pharmacokinetic-pharmacodynamic (PK-PD) model and the data set used. The relatively low concentrations of the active metabolite achieved following intravenous administration of RGD891 did not permit independent estimation of a population IC50 value for RGD039. Therefore, its potency was fixed at 2.2-fold greater than that of the parent compound (based on previous PK-PD analyses). Intersubject variability in the IC50 values was 30%. Antagonism of the platelet IIb/IIIa receptors by intravenously administered RGD891 was effective in inhibiting ADP-induced platelet aggregation in a reversible and dose-dependent manner. Pharmacodynamic activity was largely attributed to the parent compound and less to the active metabolite based on the relative potencies of both compounds and the plasma concentrations of each achieved following intravenous administration. Intravenous bolus plus maintenance infusion regimens resulted in rapid attainment of steady-state plasma RGD891 concentrations. This combination regimen also provided for a marked and sustained inhibition of platelet aggregation that reached 90% or greater (relative to baseline values) in the higher dose groups. The modified Emax model adequately described inhibition of platelet aggregation following a particular intravenous dosage regimen of RGD891 (within the range of doses administered in the present studies). (ABSTRACT TRUNCATED)

Role of nitric oxide in implantation and menstruation.

Nitric oxide (NO) is a major paracrine mediator of various biological processes, including vascular functions and inflammation. In blood vessels, NO is produced by the low-input constitutive endothelial NO synthase (eNOS) and is a potent vasodilator and platelet aggregation inhibitor. The inducible NOS isoform (iNOS) is capable of producing NO at high concentrations which have pro-inflammatory properties. Immunohistochemical and molecular studies of endometrial NOS expression, as well as animal experiments with NOS inhibitors, indicate that NO plays an important role in endometrial functions such as endometrial receptivity, implantation and menstruation. In rodents, both iNOS and eNOS are highly up-regulated in the implantation sites, and NOS inhibitors show synergistic effects with antiprogestins in inhibiting the establishment of pregnancy. In the human endometrium, eNOS have been localized in the glandular epithelium and in endometrial microvascular endothelium, primarily during the luteal phase. iNOS has been found in the endometrial epithelium during menstruation, in immunocompetent endometrial cells, and in decidualized stromal cells. In primates, NO may be involved in the initiation and maintenance of menstrual bleeding by inducing tissue breakdown and vascular relaxation as well as by inhibiting platelet aggregation. Endometrium-derived NO may also play a role in myometrial relaxation during menstruation. These studies open up new applications for NO-donating and -inhibiting agents in uterine disorders. NO donors may be useful in the treatment of dysmenorrhoea and for promoting fertility. Antiprogestins, progesterone receptor modulators and iNOS inhibitors may find applications in the treatment and prevention of abnormal uterine bleeding.

Brief antecedent ischemia enhances recombinant tissue plasminogen activator-induced coronary thrombolysis by adenosine-mediated mechanism.

Clinical studies have implicated preinfarct angina (brief antecedent ischemia/reperfusion [I/R]) as a predictor of more rapid thrombolysis and lower rates of reocclusion. However, the effects of antecedent ischemia on the efficacy of thrombolysis have not been rigorously assessed. Using a canine model of coronary thrombosis, we aimed to (1) reproduce these clinical findings and (2) determine whether release of adenosine (a potent inhibitor of platelet aggregation via stimulation of platelet A(2) receptors) during brief I/R contributes to this improved patency. To address our first objective, we compared the time required to achieve lysis with recombinant tissue plasminogen activator and patency during the first 2 hours after lysis in dogs in which 1-hour thrombotic occlusion was preceded by brief I/R (10-minute coronary occlusion/10-minute reperfusion) versus 20-minute uninterrupted perfusion (controls). Time to lysis was accelerated in the I/R group versus the control group (11+/-1 versus 35+/-6 minutes, P=0.004). In addition, the duration of subsequent reocclusion was reduced (17+/-12 versus 30+/-11 minutes), and the area of the flow-time profile (normalized to baseline flow x 120 minutes) was increased (64+/-12% versus 35+/-7%, P=0.04) in the I/R cohort. The protocol was then repeated, but all dogs were pretreated with the adenosine A(2)/A(1) antagonist CGS 15943 (CGS, 1.5 mg/kg). Time to lysis (38 versus 39 minutes) and subsequent patency were comparable in the CGS+control group versus the CGS+I/R group. Brief antecedent I/R enhances the efficacy of coronary thrombolysis in this canine model, which is due, at least in part, to an adenosine-mediated mechanism.

Structure–Activity Relationships for Selected Sulfur-Rich Antithrombotic Compounds

We assessed the antithrombotic activity of some simple organosulfur compounds which have some of the functionality found in the disulfide ajoene, a pharmacologically active compound isolated from garlic. The results establish that antithrombotic activity is associated with disulfides directly attached to a phenyl ring and is further enhanced by an α-sulfonyl group. CH3SO2CH2SSPh proved to be a potent inhibitor of platelet aggregation with an IC50 of 5 μM.

New Trends in Thromboxane and Prostacyclin Modulators

Thromboxane A2 (TXA2) and prostacyclin (PGI2) are two labile products formed from arachidonic acid by the way of cyclooxygenase. An overproduction of thromboxane A2 has been detected in a series of diseases whereby this prostanoid is assumed to contribute to the underlying pathomechanisms by its potent stimulation of platelet aggregation and smooth muscle contraction. This increased TXA2 biosynthesis is frequently accompanied by a stimulation of prostacyclin formation which is one of the most potent inhibitors of platelet aggregation and smooth muscle contraction. Therefore, TXA2 / prostaglandin endoperoxide H2 receptor antagonists, thromboxane synthase inhibitors and drugs which combine both activities have been developed with the aim to suppress the formation and/or the action of thromboxane A2. Since prostacyclin has been demonstrated to counterbalance the pathological effects of TXA2, several PGI2 agonists have also been developed. This review will highlight the evolution and some of the latest findings in the field of prostacyclin and thromboxane A2 modulators mainly those which are under clinical evaluation or marketed.

Electrospray ionization mass spectrometry of low-molecular-mass S-nitroso compounds and their thiols

Low-molecular-mass S-nitroso compounds (R–S–NO) are potent vasodilators and inhibitors of platelet aggregation. This work describes the electrospray ionization mass spectrometric (ESI-MS) analysis of physiological and synthetic low-molecular-mass S-nitroso compounds and their thiols including S-nitrosoglutathione, S-nitrosocysteine, glutathione and cysteine. Mass spectra of the unlabeled and S- 15N-labeled low-molecular-mass S-nitroso compounds investigated are characterized by abundant cations due to [M+H] +, [M+Na] +, [(M+H)−NO] +, [2 M+H] +, and [(2 M+H)−2NO] +. Mass spectra of low-molecular-mass thiols are characterized by abundant cations due to [M+H] +, [M+Na] + and [2M+H] +. Using off-line electrospray ionization tandem mass spectrometry we unequivocally identified S-[ 15N]nitrosoglutathione in human red blood cells formed after their incubation with S-[ 15N]nitrosocysteine. These results suggest that ESI-MS in combination with an appropriate liquid chromatographic system should be a useful analytical approach for the on-line quantitative determination of low-molecular-mass S-nitroso compounds in biological fluids in the presence of their thiols and nitrite. Considerations were made about on-line ESI-MS and quantitative measurements.

Monophosphoryl lipid A: a novel nitric oxide-mediated therapy to attenuate platelet thrombosis?

Nitric oxide (NO) is a potent inhibitor of platelet aggregation. However, the benefits of NO-based therapies can be confounded by concomitant hypotension. Monophosphoryl lipid A (MLA) is a nontoxic derivative of endotoxin that purportedly increases nitric oxide synthase (NOS) activity and, presumably, NO production, yet has a hemodynamically benign profile. Thus our aims were to determine whether (a) MLA attenuates in vivo platelet aggregation in damaged and stenotic canine coronary arteries by a NO-mediated mechanism but without reductions in arterial pressure; and (b) the platelet inhibitory effects are manifest in vitro. To address the first aim, anesthetized dogs underwent coronary injury + stenosis, resulting in cyclic variations in coronary blood flow (CFVs) caused by the formation/dislodgement of platelet-rich thrombi. In protocol I, dogs received MLA (100 microg/kg + 40 microg/kg/h) or vehicle beginning 15 min before stenosis. Protocol II was identical, except the NOS inhibitor aminoguanidine was coadministered with MLA/vehicle. Coronary patency was assessed throughout the initial 3 h after injury + stenosis. Infusion of MLA did not result in hypotension. However, in protocol I, the median nadir of the CFVs was higher (2.1 vs. 0.8 ml/min; p < 0.05), median duration of total thrombotic occlusion tended to be reduced (0 vs. 10.4 min; p = 0.1), and mean flow-time area, expressed as a percentage of baseline flow, was increased (53 +/- 9% vs. 33 +/- 3%; p < 0.05) in MLA-treated versus vehicle-treated dogs. In contrast, in protocol II, vessel patency was comparable in both groups. Finally, whole blood impedance aggregometry (protocol HI) revealed a significant reduction in the in vitro platelet aggregation in blood samples receiving exogenous MLA, which was blocked by coadministration of exogenous aminoguanidine. Thus MLA attenuates platelet-mediated thrombosis in both damaged and stenotic canine coronary arteries and in vitro, possibly by an NO-mediated mechanism, but without concomitant hypotension.

Antiplatelet and Antithrombotic Effects of Orbofiban, a New Orally Active GPIIb/IIIa Antagonist, in Guinea Pigs

Antiplatelet and antithrombotic effects of orbofiban, a new orally active glycoprotein IIb/IIIa antagonist, were evaluated in guinea pigs. SC-57101A (0.03–1 μM), the hydrochloride salt of the active form of orbofiban, inhibited in vitro ADP- and collagen-induced platelet aggregation in a concentration-dependent manner. Oral administration of orbofiban (3–30 mg/kg) resulted in dose-dependent inhibition of ADP- and collagen-induced platelet aggregation. The inhibition peaked at 1–2 hours postdose and then declined slowly. The agent also showed similar inhibition of platelet aggregation in guinea pigs with dietary-induced hypercholesterolemia. In contrast, the antiaggregatory effects of acetylsalicylic acid differed more widely between normal and hyperlipidemic animals compared to those of orbofiban. Plasma concentration of the active form, measured by a column-switching HPLC method, correlated well with the inhibition of platelet aggregation. Orbofiban (3–100 mg/kg, p.o.) caused dose-dependent inhibition of thrombus formation in an arteriovenous-shunt-thrombosis model. Orbofiban at high doses (⩾30 mg/kg) and acetylsalicylic acid (100 mg/kg) both prolonged cutaneous bleeding time measured by the template method. These results demonstrate that orbofiban is an orally active and potent inhibitor of platelet aggregation with an efficacy that correlates well with the plasma concentration of its active form.

Recombinant decorsin: dynamics of the RGD recognition site.

Decorsin is an antagonist of integrin alphaIIbbeta3 and a potent platelet aggregation inhibitor. A synthetic gene encoding decorsin, originally isolated from the leech Macrobdella decora, was designed, constructed, and expressed in Escherichia coli. The synthetic gene was fused to the stII signal sequence and expressed under the transcriptional control of the E. coli alkaline phosphatase promoter. The protein was purified by size-exclusion filtration of the periplasmic contents followed by reversed-phase high-performance liquid chromatography. Purified recombinant decorsin was found to be indistinguishable from leech-derived decorsin based on amino acid composition, mass spectral analysis, and biological activity assays. Complete sequential assignments of 1H and proton bound 13C resonances were established. Stereospecific assignments of 21 of 25 nondegenerate b-methylene groups were determined. The RGD adhesion site recognized by integrin receptors was found at the apex of a most exposed hairpin loop. The dynamic behavior of decorsin was analyzed using several independent NMR parameters. Although the loop containing the RGD sequence is the most flexible one in decorsin, the conformation of the RGD site itself is more restricted than in other proteins with similar activities.

Effects of COX-2 inhibitors on aortic prostacyclin production in cholesterol-fed rabbits

Abstract Prostacyclin (PGI 2 ) is a potent vasodilator and inhibitor of platelet aggregation that is produced by prostacyclin synthase via the cyclooxygenase (COX) pathway of arachidonic acid metabolism. We investigated the potential role of COX-2 in the production of vasoactive prostanoids by aortic tissue in a rabbit model of dietary cholesterol-induced atherosclerosis. COX-1 was detected as the major isoform by immunoblot analysis in extracts from aortas of normal and 8 week cholesterol-fed animals with COX-2 being induced in atherosclerotic plaques from cholesterol-fed animals. Aortic tissue from cholesterol-fed animals showed decreased levels of basal 6-keto-PGF 1α and PGE 2 production as compared to the normal controls but showed no difference with respect to their ability to synthesize these prostanoids in response to exogenous arachidonic acid. The highly selective COX-2 inhibitors rofecoxib and the furanone DFP at concentrations of up to 10 μmol/l had no effect on the arachidonic acid-dependent production of 6-keto-PGF 1α , in contrast to indomethacin, which caused a complete inhibition at 0.5 μmol/l. Celecoxib caused a significant inhibition of 6-keto-PGF 1α at 10 μmol/l but had little effect when the dose was lowered to 1 μmol/l. Similar effects of these inhibitors were observed with respect to the production of PGE 2 and no major difference was observed between aortic tissues from normal and cholesterol-fed animals with regard to inhibitor sensitivity. These results indicate that in a rabbit model of early stage cardiovascular disease, the basal production of 6-keto-PGF 1α and PGE 2 by aortic tissue is decreased. Furthermore, COX-2 expression is induced in atherosclerotic plaques and may play a role in altering localized synthesis of prostanoids in these lesions but does not appear to significantly impact the arachidonic acid-dependent prostacylin production of aortic tissues, which is largely mediated by COX-1.

Human Prostacyclin Synthase Gene and Hypertension

Prostacyclin (prostaglandin I(2)) is a strong vasodilator that inhibits the growth of vascular smooth muscle cells and is also the most potent endogenous inhibitor of platelet aggregation. Therefore, it has been considered to play an important roles in cardiovascular disease. On the basis of the hypothesis that variations of the prostacyclin synthase gene may also play an important role in human cardiovascular disease, we performed a screening for variations in the human prostacyclin synthase gene. We have detected a repeat polymorphism in the promoter region of the human prostacyclin synthase gene. The number of 9-bp (CCGCCAGCC) repeats in the promoter region, which encodes a tandem repeat of Sp1 transcriptional binding sites, varied between 3 and 7 in Japanese subjects. Luciferase reporter analysis indicated that the alleles of 3 and 4 repeats (R3 and R4, respectively) had less promoter activity in cultured human umbilical vein endothelial cells. We then investigated the possible association of this repeat polymorphism with blood pressure in a large population-based sample (the Suita Study), which consisted of 4971 Japanese participants. Multivariate models indicated that participants with the R3R3, R3R4, or R4R4 genotype (SS genotype, n=80) had significantly higher systolic pressure (P=0.0133) and pulse pressure (P=0.0005). The odds ratio of hypertension (140/90 mm Hg) for the SS genotype was 1.942 (95% confidence interval 3.20 to 1.19, P=0.0084). Repeat polymorphism of the human prostacyclin synthase gene seems to be a risk factor for higher pulse pressure and is consequently a risk factor for systolic hypertension in the Japanese population.

Stimulation of nitric oxide synthesis and protective role of insulin in acute thrombosis in vivo

Administration of physiologic amounts of insulin in mice (200 μunits g body weight) resulted in 9 fold increase of basal nitric oxide level from 0.51 ± 0.1224 nmol ml (mean ± SD, n = 12) to 4.45 ± 0.645 nmol ml after 30min of the injection of the hormone. Since NO is a potent inhibitor of platelet aggregation both in vitro and in vivo, we tested the possibility whether the administration of the hormone would result in the in vivo inhibition of thrombosis through the increase of NO level in the circulation. It was found that administration of insulin (200 μunits g body weight) in mice protected > 90%(p <0.00001, n = 500) of these animals from death due to thrombosis in the coronary arteries induced by ADP injection in the heart. This effect of insulin in vivo was found to be directly related to the hormone induced increase of NO level in the system. The thromboprotective effect of insulin could not be achieved by using either prostacyclin, a well known antithrombotic agent or its stable probe prostaglandin E 1 instead of insulin. The efficacy of insulin was neither related to the blood glucose level nor was the consequence of the hypoglycemic effect of the hormone. In contrast, inhibition of insulin induced increase of NO level resulted in the complete loss of the thromboprotective effect of the hormone. These results suggest that insulin besides being a hypoglycemic hormone could also be a potent antithrombotic humoral factor.

Pharmacological characterization of the human P2Y11 receptor.

1 The human P2Y11 receptor is coupled to both the phosphoinositide and the cyclic AMP pathways. A pharmacological characterization of the recombinant human P2Y11 receptor has been conducted following stable expression in two different cell lines: the 1321N1 astrocytoma cells for inositol trisphosphate measurements and the CHO-K1 cells for cyclic AMP assays. The rank order of potency of a series of nucleotides was almost identical for the two pathways: ATPgammaS approximately BzATP > dATP > ATP > ADPbetaS > 2MeSATP. 2 ADPbetaS, AMPalphaS and A3P5PS behaved as partial agonists of the human P2Y11 receptor. At high concentrations, these three nucleotides were able to partially inhibit the ATP response. 3 Suramin was a more potent antagonist than reactive blue 2, whereas pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid was completely inactive. The P2Y11 receptor proved to be sensitive to suramin in a competitive way with an apparent Ki value of 0.82+/-0. 07 microM. 4 The ATP derivative AR-C67085 (2-propylthio-beta, gamma-dichloromethylene-D-ATP), a potent inhibitor of ADP-induced platelet aggregation, was the most potent agonist of the P2Y11 receptor, among the various nucleotides tested. 5 The pharmacological profile of the recombinant human P2Y11 receptor is closely similar to that of the cyclic AMP-coupled P2 receptor recently described in HL-60 cells, suggesting that it is the same receptor.

Effects of Long-term Infusion of Prostacyclin on Exercise Performance in Patients With Primary Pulmonary Hypertension

To determine whether long-term IV prostacyclin (PGI(2)) use improves exercise capacity in patients with primary pulmonary hypertension (PPH). Cycle ergometry and the 6-min walk was used to evaluate the exercise performance of patients with PPH. The patients underwent serial exercise testing after starting continuous IV PGI(2) and were followed up for 19.5 +/- 7.5 months. Peak work, peak oxygen consumption (f1.gif" BORDER="0">O(2)), peak O(2) pulse, and distance walked in 6 min were used to evaluate performance. PPH is characterized by medial hypertrophy and intimal proliferation of the pulmonary arterioles, leading to elevation of pulmonary artery pressure, right ventricular failure, and death. Palliative treatment consists of vasodilators, anticoagulants, cardiac glycosides, diuretics, and transplantation. PGI(2), a potent vasodilator and inhibitor of platelet aggregation, has been used for long-term treatment when conventional therapy has been unsuccessful. Sixteen patients with PPH (10 women, 6 men; mean age, 24 years). At the initiation of PGI(2), peak work (+/- SD) was 35.5 +/- 11% of predicted; peak f1.gif" BORDER="0">O(2), 39 +/- 10.4%; peak O(2) pulse, 5.0 +/- 1.7 mL/min; and distance on the 6-min walk, 428 +/- 78 feet. At 18 to 27 months, peak work increased to 58.8 +/- 23% of predicted (p = 0.001), peak f1.gif" BORDER="0">O(2) increased to 52 +/- 15% of predicted (p = 0. 02), peak O(2) pulse increased to 7.1 +/- 3.0 mL/beat (p = 0.004), and performance on the 6-min walk increased to 526 +/- 62 feet (p = 0.001). There was a positive correlation between peak f1.gif" BORDER="0">O(2) and peak 6-min walk of 0.6 (p < 0.005) and between peak work and peak 6-min walk of 0.6 (p < 0.005). Exercise capacity improved in our patients at up to 27 months of follow-up. Exercise testing is helpful in assessing the functional capacity of patients with PPH and may be useful in guiding therapy. Patients who deteriorate while receiving optimal conventional therapy should be considered for IV PGI(2) therapy.

Effects of Jarastatin, a Novel Snake Venom Disintegrin, on Neutrophil Migration and Actin Cytoskeleton Dynamics

A new disintegrin, an RGD-containing peptide of 6 kDa called jarastatin, was purified from Bothrops jararaca venom. It is a potent inhibitor of platelet aggregation induced by ADP, collagen, and thrombin. The effect of jarastatin on neutrophil migration in vivo and in vitro and on the actin cytoskeleton dynamics of these cells was investigated. Incubation in vitro with jarastatin significantly inhibited, in a concentration-dependent manner, the chemotaxis of human neutrophils toward fMLP, IL-8, and jarastatin itself. Despite this inhibitory effect, jarastatin induced neutrophil chemotaxis. A significant increase of F-actin content was observed in jarastatin-treated neutrophils. Furthermore, as demonstrated by confocal microscopy after FITC–phalloidin labeling, these cells accumulated F-actin at the plasmalemma, a distribution similar to that observed in fMLP-stimulated cells. Pretreatment of mice with jarastatin inhibited neutrophil migration into peritoneal cavities induced by carrageenan injection. The results suggest that binding of jarastatin to neutrophil integrins promotes cellular activation and triggers a dynamic alteration of the actin filament system and that this is one of the first event in integrin-mediated signaling.