Iren B. Kovacs

High-dose aspirin inhibits shear-induced platelet reaction involving thrombin generation.

BackgroundA unifying concept of explaining all pharmacological actions of aspirin by the irreversible blockage of the enzyme cyclooxygenase and therefore the inhibition of prostaglandin biosynthesis has left many unanswered questions. Methods and ResultsTwo hundred ninety-four patients taking 75 mg/day aspirin were tested 3 months after coronary artery bypass surgery. Platelet thromboxane formation (whole blood aggregation to arachidonate) was completely prevented in 80% of patients. Compared with matched healthy controls (n = 95), a significant platelet hyperreactivity was observed in patients (p<0.0001 versus <0.002). Ninety patients were advised to increase their daily dose of aspirin from 75 mg to 300 mg. Platelet reactivity retested 1 month after increasing the dose has significantly decreased (p = 0.0008; <0.0001), whereas it remained unchanged in those patients (n = 84) who continued with the same dose regimens. In normal subjects, ingestion of a single 600-mg aspirin significantly inhibited shear-induced platelet reaction. ConclusionsIt is concluded that aspirin does affect the platelet response to shear forces, but this requires higher dosage (>300 mg/day), suggesting a mechanism probably different from that of interference with thromboxane formation.

Nifedipine in the treatment of Raynaud's phenomenon in patients with systemic sclerosis

A double‐blind, placebo‐controlled, cross‐over trial of nifedipine 10 mg three times daily for 6 weeks, in 10 patients with Raynauds phenomenon secondary to systemic sclerosis, is reported. A significant reduction in the duration of attacks of Raynauds phenomenon was observed. Nifedipine therapy also reduced the number and severity of attacks of Raynauds phenomenon and the development of new digital ulcers, and increased the digital blood flow, but none of these changes was statistically significant. No alteration in red blood cell deformability or leukocyte chemiluminescence was observed during nifedipine treatment.

Elevated plasma lipid hydroperoxides in patients with coronary artery disease.

Sustained presence of lipid peroxides in the circulation and their plasma carrier is a controversial issue. Particularly, there is no firm evidence for an increased plasma lipid peroxide level in patients with atherosclerosis. In this study, a strong correlation was found between plasma total lipid hydroperoxide and lipid hydroperoxide content of LDL cholesterol (r = 0.882; p < 0.001; n = 16). Lipid hydroperoxides in plasma were carried almost exclusively (89%) in LDL. In 70 patients tested 3 months after coronary artery bypass graft surgery with a specific assay, plasma lipid hydroperoxide levels were significantly increased when compared with matched healthy controls (4.31 +/- 0.23 nmol/ml and 2.34 +/- 0.13 nmol/ml, p < 0.0001, patients vs controls, respectively). These concentrations are 10 times lower than those detected by the nonspecific thiobarbituric acid assay. However, considering the in vitro concentration range in which oxidized LDL exerts important atherogenic effects, the elevated plasma lipid hydroperoxide levels measured in atherosclerotic patients have pathologic significance.

Reduction of heparin binding to and inhibition of platelets by aprotinin

The direct effect of aprotinin on in vitro platelet function was assessed by hemostatometry (n = 10). No significant enhancement was demonstrated. However, aprotinin reduced platelet inhibition secondary to heparin. Hemostatometry demonstrated a significant preservation of in vitro platelet function (n = 25) (p = 0.04), which was particularly marked (p = 0.003) in the subgroup (n = 7) demonstrating a severe inhibition of platelet function with heparin. Aprotinin significantly reduced the binding of tritium-labeled heparin to both nonactivated (n = 25) (p = 0.004) and activated platelets (n = 25) (p < 0.0001). We conclude that interference with heparin-induced inhibition of platelet function by aprotinin may be one of its hemostatic actions in cardiac surgery. This effect is probably secondary to aprotinin reducing binding of heparin to platelets.

Lipid peroxidation by activated platelets: A possible link between thrombosis and atherogenesis

Generation of reactive oxygen species (ROS) by platelets was detected by a cyano-tetrazolium dye (CTC) which forms fluorescent formazan on the cell surface upon reduction. Fluorescence was quantitated by a densitometric device. Resting platelets in plasma produced significant fluorescence (P < 0.0001), and addition of thrombin enhanced the fluorescence of the coagulated platelet mass even further (by 2 h, a 6- and 8-fold increase over fluorescence of platelet-free plasma was measured, respectively). Blood containing CTC was perfused through a glass capillary tubing and the action of shear forces resulted in the formation of an occlusive platelet thrombus. Such thrombi (formed either from whole blood or platelet-rich plasma) were intensely fluorescent, indicating formation of ROS in the platelet mass (a 10- and 8-fold increase in fluorescence over coagulated plasma, respectively). Lipid peroxide content of resting platelets in platelet-rich plasma was doubled over 24 h storage, while addition of thrombin caused a 7.4-fold increase (P < 0.0001) of lipid peroxides in the retracted platelet-rich plasma-clot. Transition metal chelator and antioxidant prevented lipid peroxidation by platelets in response to thrombin. Thrombin activation of (washed) platelets in plasma-free medium caused only 1.4-fold increase in oxidation of added low density lipoprotein (LDL). In contrast, thrombin activation of platelets suspended in de-lipidated autologous plasma resulted in a 5.25-fold increase (P < 0.0001) in LDL oxidation. Generation of ROS and lipid peroxides by platelets can be an important mechanism through which thrombotic events contribute to atherogenesis.

Preoperative hemostatic activity and excessive bleeding after cardiopulmonary bypass

The rationale for predicting the risk of excessive postoperative bleeding by assessing the hemostatic status of a patient before cardiopulmonary bypass was investigated. A novel, rapid, overall test (hemostatometry) consisting of a physiologically relevant test of platelet function (shear-induced hemostasis) and coagulation was performed using nonanticoagulated blood and compared with the routine coagulation screen. Two hundred five patients undergoing elective coronary revascularization were studied 3 to 4 days before operation. Forty-nine bled excessively for nonsurgical reasons; none were predicted by the routine coagulation tests. Using a stepwise discriminant analysis, hemostatometry correctly predicted 31 of 49 (63%). Thirty of 156 predicted as bleeders by hemostatometry did not bleed. Thus, preoperative hemostatometry predicted 77% of the true outcome. The false predictions suggest, however, that certain bleeding abnormalities probably acquired during cardiopulmonary bypass cannot be predicted. These findings do not justify the routine use of preoperative tests in assessing the bleeding risk in patients undergoing cardiopulmonary bypass.

Prostacyclin increases filterability of normal and rigidified human red blood cellsin vitro

Red blood cell (RBC) deformability is a critical determinant of capillary flow. In a previous study we found decreased deformability (filterability) of red cells of patients with clinical symptoms of peripheral ischaemia associated with systemic sclerosis and prostacyclin (PGI2) infusion had beneficial effect on both the clinical symptoms and red cell filterability. The present study was therefore designed to investigate the effect of PGI2 on RBC deformabilityin vitro, by measuring the filterability of RBCs suspended in saline. The results demonstrate that PGI2 increases filterabillty of normal RBCs and significantly improves the impaired deformability of cells due to storage or calcium influx.

Inhibition of Vascular Smooth Muscle Cell Migration by Intact Endothelium Is Nitric Oxide-Mediated: Interference by Oxidised Low Density Lipoproteins

Migration of vascular smooth muscle cells (SMCs) leading to neointimal hyperplasia is an early and cardinal feature of atherogenesis. Migration of rat aortic SMCs from an upper chamber towards a lower one has been studied in a microchemotaxis (Boyden) chamber. Spontaneous migration of SMCs was practically prevented by the presence of endothelium in the lower chamber and was reduced if endothelial cells were substituted with endothelial cell-conditioned medium. Endothelial cells which had been treated with either the inhibitor of protein synthesis cycloheximide or nitric oxide synthesis NG-nitro-L-arginine showed no inhibitory effect on SMC migration. Addition of a nitric oxide donor S-nitroso-N-acetylpenicillamine to cell-free medium in the lower chamber prevented SMC migration. Addition of native LDL to endothelial cells had no effect on SMC migration, while (UV light) oxidised LDL completely abolished the inhibitory effect of endothelial cells on SMC migration. It is concluded that via nitric oxide, endothelium exerts a powerful inhibitory effect on SMC migration. This effect of intact endothelium is completely abolished by oxidised LDL applied in a concentration, which is relevant to those measured in plasma of patients with severe coronary artery disease. It is suggested that oxidised LDL may contribute to the pathogenesis of atherogenesis by stimulating migration of SMCs from media to the intima via abolishing the physiological inhibitory effect of normal endothelium.

Infusion of a stable prostacyclin analogue, iloprost, to patients with peripheral vascular disease: lack of antiplatelet effect but risk of thromboembolism

PURPOSE Prostacyclin, a potent inhibitor of platelet function and vasodilator, has been used to treat peripheral vascular disease. The aim of this study was to monitor the thrombotic status of patients treated by infusion of a stable prostacyclin analogue, iloprost. PATIENTS AND METHODS Thirteen patients with peripheral vascular disease underwent iloprost infusion for 3 days (8 hours each day) in a dose ranging from 0.5 to 2 ng/kg/minute. Variable parameters of thrombosis such as platelet reactivity (shear-induced hemostatic plug formation and thrombus formation on a collagen fiber), coagulation, and spontaneous thrombolysis (dislodgment of hemostatic plugs) were measured from non-anticoagulated blood samples by hemostatometry immediately before and 1 hour after the infusion and on the last day, 4 hours after initiation of the infusion. RESULTS Analysis of data from all patients 1 hour after the infusion showed no changes in platelet reactivity and spontaneous thrombolysis, but coagulation was significantly enhanced. In four patients, significant platelet hyperreactivity was observed after the infusion. Four of the five patients tested while undergoing iloprost infusion showed an enhanced thrombotic reaction and markedly enhanced coagulation. Iloprost employed in vitro in a concentration that corresponds to the therapeutic peak blood level caused no inhibition of platelet function but significantly enhanced coagulation. The threshold in vitro iloprost concentration at which anti-platelet effect and increased spontaneous thrombolysis were observed was twice that of the therapeutic blood level. CONCLUSIONS These findings challenge the view that antagonism of platelet function is an important factor of iloprost therapy. Furthermore, platelet hyperreactivity in some patients and markedly enhanced coagulation during and after infusion of iloprost in general, represent a risk of thromboembolism, especially as patients are already in a prethrombotic condition.

Anti-inflammatory effect of sialic acid

Sialic acid was shown to possess anti-inflammatory properties as measured by carrageenan-induced rat paw oedema and pleurisy tests. As the number of leukocytes mobilized was significantly reduced it was concluded that the inhibitory effect of sialic acid on leukocyte accumulation is responsible for the inhibition of exudate/oedema formation.