Biomedical subjects
Publications and source records attributed to P Gresele.
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Several reports have suggested that the activity of platelet phospholipase A2 is modulated by GTP-binding protein(s) whose nature and properties need to be defined. Fluoroaluminate is known to activate G-proteins and this leads to a number of cellular responses including the activation of phospholipases. This paper demonstrates that human platelets, prelabelled with [3H]arachidonic acid, produce free arachidonic acid when stimulated with fluoroaluminate and this effect is time- and dose-dependent. The production of arachidonic acid is not inhibited by neomycin, a PI-cycle inhibitor, but is completely abolished by mepacrine, an inhibitor of both phospholipase A2 and C. At low concentration of fluoroaluminate (10 mM NaF) phospholipase A2 but not phospholipase C is activated. In addition, fluoroaluminate treatment releases beta-thromboglobulin (beta-TG) and this effect is not inhibited by acetylsalicylic acid. Under identical conditions both neomycin and mepacrine suppress the release of arachidonic acid and beta-TG induced by thrombin. Sodium nitroprusside, which increases cGMP levels in platelets, inhibits arachidonic acid liberation and beta-TG release in thrombin-stimulated platelets but has no effect in fluoroaluminate-treated platelets; cGMP was reported to suppress phospholipase C activation. These results are consistent with the hypothesis that, in thrombin-stimulated platelets, the liberation of arachidonic acid and beta-TG are strictly dependent on the activation of phospholipase C. We have also provided evidence for the existence of a phospholipase A2 activated by a G-protein which is independent from the degradation of phosphoinositides and, contrary to phospholipase C, it is not down regulated by cGMP.
The mechanisms underlying the defective platelet function in cirrhotic patients were investigated. Eleven cirrhotic patients with mild disease (group 1), 20 patients with severe cirrhosis (group 2), and 31 controls were studied. Platelet aggregation was significantly reduced in cirrhotics compared with controls. Compared with controls, cirrhotic patients in group 2 showed a significant reduction in the total content of adenosine triphosphate (57.8 +/- 7.8 vs. 26.1 +/- 6.3 mumol/10(11) platelets; P less than 0.05), 5-hydroxytryptamine (285 +/- 26 vs. 104 +/- 38 nmol/10(11) platelets; P less than 0.05), beta-thromboglobulin (2129 +/- 120 vs. 1223 +/- 161 ng/10(8) platelets; P less than 0.01), and platelet factor 4 (1389 +/- 108 vs. 805 +/- 176 ng/10(8) platelets; P less than 0.05). In patients with severe disease, an increase in plasma beta-thromboglobulin-platelet factor 4 ratio, an index of in vivo platelet activation, was observed (controls, 3.50 +/- 0.50; group 1, 4.02 +/- 0.80; and group 2, 6.59 +/- 1.15). Our data indicate the existence of a platelet storage pool defect, which may favor the bleeding tendency of cirrhotic patients.
Thromboxane A2 (TXA2) plays a pivotal role in platelet activation and is involved in the development of thrombosis. Thromboxane synthase inhibitors suppress TXA2 formation and increase the synthesis of the antiaggregatory prostaglandins PGI2 and PGD2; however, accumulated PGH2 may interact with the platelet and vessel wall TXA2 receptor, thus reducing the antiplatelet effects of this class of drug. TXA2 receptor antagonists block the activity of both TXA2 and PGH2 on platelets and the vessel wall. Very recently, drugs possessing both thromboxane synthase-inhibitory and thromboxane receptor-antagonist properties have been developed. Paolo Gresele and colleagues explain here why these drugs can be more efficacious than traditional antiplatelet agents and review the available experimental studies involving these drugs.
STUDY OBJECTIVE: Vasodilator prostaglandins have been claimed to be responsible for the coronary haemodynamic and venodilator effects of glyceryl trinitrate, although conflicting results have been reported. The aim of this study was to evaluate whether vasodilator prostaglandins play a role in the effect of glyceryl trinitrate on the distensibility of peripheral muscular arteries in healthy humans. DESIGN: A non-invasive technique, impedance plethysmography, was applied to the assessment of the effects of sublingual glyceryl trinitrate on the compliance of the forearm and digital arteries. The subjects studied received placebo (on two separate occasions), indomethacin (100 mg orally), or ibuprofen (800 mg orally) 1 h before glyceryl trinitrate (0.3 mg sublingual) on four occasions separated from each other by at least 48 h. Blood pressure and heart rate were measured by standard techniques; changes in peripheral arterial compliance were evaluated by impedance plethysmography of the forearm and finger. The study was double blind, cross over, placebo controlled, and randomised. SUBJECTS: 12 healthy male volunteers were enrolled in the study. All subjects were fasting for at least 10 h and had abstained from smoking and from methylxanthine or alcohol containing beverages. MEASUREMENTS AND MAIN RESULTS: Glyceryl trinitrate increased heart rate by 11.6(SEM 1.6) beats.min-1 (p less than 0.0005) and diastolic blood pressure by 8.7(1) mm Hg (p less than 0.01), and decreased systolic blood pressure by 8.7(1.5) mm Hg (p less than 0.01) in placebo treated volunteers; the amplitude of the plethysmograph c wave in the forearm and in the finger was also augmented, by 120(11)% and 78(13)% respectively, indicating an increase in arterial compliance. The results obtained in the two placebo sessions did not differ, indicating good reproducibility of the system. Pretreatment with either indomethacin or ibuprofen did not modify the effects of glyceryl trinitrate on heart rate, blood pressure, and arterial compliance in the forearm and the finger. Both indomethacin and ibuprofen suppressed prostaglandin synthesis, as shown by the striking inhibition of serum TxA2 concentration, by 97.2(1.5)% and 93.7(3.0)%, respectively. CONCLUSIONS: Sublingual glyceryl trinitrate, in doses used clinically, induces a reproducible increase in peripheral arterial compliance in healthy volunteers; prostaglandins do not play any significant role in this effect.
We have evaluated the arachidonic acid (AA) metabolism in patients with myeloproliferative disorders (MPD). In essential thrombocythemia (ET), the generation of thromboxane B2 was found significantly reduced and inversely correlated with platelet count. Polycythemia vera (PV) patients showed an increased formation of this metabolite of AA. Prostaglandin E2 and 6-keto-PGF1 alpha generation were markedly reduced in patients with chronic myelogenous leukemia. Our study confirms that the arachidonate metabolism is frequently deranged in patients with MPD. The opposite changes in thromboxane formation in ET and PV could be one of the factors responsible for the different incidences of thrombotic and hemorrhagic complications in these diseases.
Red cell deformability of 30 nonpregnant volunteers and 20 normal pregnant women in the 3rd trimester was assessed by determination of filterability of red cells suspended either in autologous plasma or in buffer by St. George's filtrometer method. Total red cell deformability was decreased in normal pregnancy [transit time (Tc) = 7.79 +/- 0.86 vs. 6.99 +/- 0.65, p less than 0.01] as compared to nonpregnant women when erythrocytes were studied in buffer suspensions, while the number of profoundly rigid red cells was lower in pregnant patients [clogging particles (CP) = 0.864 +/- 0.225 vs. 1.103 +/- 0.246, p less than 0.01]. Total red cell deformability was no longer reduced in pregnant women when erythrocytes were suspended in autologous plasma (Tc = 7.25 +/- 0.66 vs. 7.13 +/- 0.69, p = NS) while the number of rigid erythrocytes was still lower (CP = 0.802 +/- 0.157 vs. 1.055 +/- 0.210, p less than 0.01). Our data suggest that by the end of normal pregnancy, red cell deformability decreases with a consequent accelerated turnover and a fast elimination of very poorly deformable erythrocytes; plasma alterations in late pregnancy partly counteract the intrinsic loss of deformability of red blood cells.
Picotamide, a new antiplatelet drug which has only slight effects on bleeding time, could be useful, in combination with oral anticoagulants, for the prevention of thromboembolic complications in patients with heart valve prostheses. We have evaluated in a randomized, controlled, double-blind, cross-over study, the effect of picotamide on the anticoagulant activity of warfarin. Administration of 300 mg t.i.d. for 10 days to 10 patients with aortic or mitral valve prostheses did not modify significantly either the level of anticoagulation or the mean daily dosage of warfarin. We observed a trend towards a reduction of plasma levels of beta-thromboglobulin. In conclusion the results of this study show that picotamide does not interfere with the anticoagulant activity of warfarin.
We have previously characterized the new antiplatelet agent picotamide as a dual thromboxane synthase inhibitor/thromboxane A2 receptor antagonist in human platelets. We have now studied the antithrombotic activity of this drug in a simple animal model of lung platelet thromboembolism in the mouse. Picotamide, given i.p. 1 hr before the thrombotic challenge, protected mice from death caused by the i.v. injection of collagen plus epinephrine in a dose-dependent way; the dose reducing mortality by 50% was 277 mg/kg while for aspirin it was 300 mg/kg. Picotamide was also able to reduce the mortality provoked by the i.v. injection of the stable TxA2 mimetic U46619; BM 13.505, a pure TxA2-receptor blocker, was also effective while aspirin was totally inactive. Picotamide, finally, reduced the lethal consequences of the i.v. injection of a 12.5% suspension of hardened rat red blood cells, a model in which platelets are not involved; aspirin was totally ineffective in this model while nicardipine, a calcium channel blocker, was active. Picotamide did not inhibit the formation of TxB2 in serum at any of the doses tested (100 to 750 mg/kg i.p.) while it did enhance significantly PGI2-synthesis from mice aortae and, even more, from mice lungs. The i.v. administration of picotamide (250 mg/kg 2 min before the thrombotic challenge) lead to a strong inhibition of serum TxB2 (-84.6%) and was associated with a higher antithrombotic effect.(ABSTRACT TRUNCATED AT 250 WORDS)
Red blood cell filterability as an appropriate method for evaluating red blood cell deformability was studied with different suspending media of erythrocytes. Comparison of autologous plasma and buffer suspensions of healthy subjects' erythrocytes showed no significant difference in filterability. Albumin alone resulted in a dose-dependent increase, while fibrinogen caused a decrease in red cell filterability. In the presence of fibrinogen, albumin showed controversial effects. The results suggest that measurements of red blood cells in their original surroundings give more accurate information about the microcirculation because plasma components may have a crucial influence on erythrocyte deformability.
Picotamide (G137 or N,N'-bis[3-picolyl]-4-methoxy-isophtalamide), a drug which has shown platelet inhibitory effects in vitro and ex vivo, was investigated for its mechanism of action on human platelets in vitro. This compound suppresses the aggregation of human platelets induced by arachidonic acid (IC50: 1.8 x 10(-5) M), low-dose collagen (IC50: 3.5 x 10(-4) M), U46619 (IC50: 1 1.4 x 10(-4) M) and by authentic TxA2 (IC50: 1 x 10(-4) M), without affecting the aggregation induced by A23187 or primary aggregation by ADP. Picotamide inhibits dose-dependently TxA2 synthesis by platelets (IC50: 1.5 x 10(-4) M) and enhances the formation of PGE2. Picotamide-treated platelets also favour the formation of PGI2 by aspirinated endothelial cells; in addition, the drug appears to exert a direct stimulatory effect on PGI2-synthesis, at least at high concentrations. Finally, in platelet-rich plasma stimulated with arachidonic acid, picotamide increases intraplatelet cAMP while no effects on cAMP are detected in unstimulated platelets. In conclusion, picotamide is a dual thromboxane-synthase inhibitor/thromboxane-receptor antagonist in human platelets and introduces a new class of agents potentially useful in antithrombotic therapy.
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The effect of five thromboxane-synthase inhibitors (UK-37248, UK-38485, UK-34787, CGS-13080 and OKY-1581) on arachidonic acid-induced platelet aggregation has been studied in vitro on platelets from 30 different healthy volunteers. The sensitivity of their platelets to adenylate cyclase stimulators or to dibutyryl cyclic AMP has been evaluated contemporarily. In 4 of the 30 volunteers tested no inhibition of platelet aggregation was obtained with any of the five thromboxane synthase inhibitors: these subjects were defined nonresponders; in 13 volunteers inhibition was observed with all the five drugs (responders). Significantly higher amounts of prostaglandin (PG)D2, prostacyclin and adenosine were required to suppress arachidonic acid-induced aggregation of platelets from nonresponders in vitro. No differences were instead observed between responders and nonresponders concerning platelet sensitivity to forskolin or dibutyryl cyclic AMP. The cyclic AMP rise obtained with exogenous prostacyclin was lower in platelets from nonresponders than in those from responders. PGE2 added in vitro to platelets from nonresponders exerted always a proaggregatory effect whereas this PG was antiaggregatory in most of the nonresponders. PGE2 blunted the antiaggregatory activity of PGD2 and limited the cyclic AMP increase induced by PGD2 in all the subjects tested. These data indicate that the unequal functional response of platelets from different subjects to thromboxane synthase inhibition depends essentially on adenylate cyclase function: a relative insensitivity of this enzyme to activating stimuli and the accumulation of substances (PGE2, PG endoperoxides, etc.) reducing the activity of adenylate cyclase may lead to continued platelet activation in some subjects despite the suppression of the synthesis of thromboxane A2.
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We have studied the effect of indobufen, a cyclo-oxygenase blocking agent which has proved useful in patients with obstructive vascular disease, on red blood cell (RBC) filterability in vitro and in a pilot study ex vivo. The addition of indobufen in vitro to blood samples from 10 healthy volunteers did not significantly modify RBC deformability. We evaluated the ex vivo effect of indobufen (200 mg bd) in 14 patients with obstructive vascular disease. A significant improvement in RBC deformability was noted on the 5th, 14th, and 28th days of treatment, 2 h after the morning dose. Acetylsalicylic acid given to 6 similar patients had no effect suggesting that the positive haemorheological effect of indobufen is probably not linked to its cyclooxygenase blocking effect.