Apo A-I binding to platelets detected by flow cytometry.

Lipoprotein-platelet interactions are very important in atherosclerosis and thrombosis. Several studies have been carried out on specific binding of various lipoproteins to platelets. But there is considerable disagreement about the details of these binding sites. Although low-density lipoprotein (LDL) receptors of several cells have been studied extensively, there is little datum about high-density lipoprotein (HDL) receptors. Apolipoprotein (apo) A-I may play a major role in the determination of the specificity of HDL receptors. In this study, binding of apo A-I to platelets was investigated by using a flow cytometric method. Citrated blood samples were obtained from five healthy and seven hypercholesterolemic subjects. Apo A-I antibody was incubated with the citrated whole blood before and after activation with ADP or thrombin receptor agonist peptide (TRAP). Then fluorescein isothiocyanate (FITC)-labeled secondary antibodies were added and analyzed on a Becton-Dickinson FACSort flow cytometer. In the hypercholesterolemic group, apo A-I binding to platelets was found to be significantly decreased after activation with TRAP (P<.05), but not after activation with ADP. In the control group, after platelet activation with ADP or TRAP, the apo A-I MFI values were not found to be significantly different from the values of resting platelets (P>.05). In this study, we demonstrated that apo A-I can bind to platelets, and this supports the hypothesis that apo A-I may play a major role in HDL binding to platelets.

The importance of Lp(a)-fibronectin interaction in atherogenesis.

An elevated concentration of lipoprotein (a) (Lp(a)) in serum has been considered a risk factor for coronary heart disease by various investigators. The apo(a) portion of Lp(a) binds to the carboxyterminal heparin binding domain of fibronectin. Lp(a) bound to fibronectin is internalized through the fibronectin receptor pathway and thereby causes increased accumulation of lipid and foam cell formation. In the present study, fibronectin and Lp(a) concentrations have been assayed in patients with coronary heart disease (CHD) and healthy subjects. Serum total cholesterol, triglyceride, LDL, VLDL and HDL cholesterol levels in patients with coronary heart disease were significantly different from the control group (p < 0.001). Plasma fibronectin (p < 0.05) and serum Lp(a) (p < 0.001) levels were found to be significantly elevated when compared with the control group.

Effect of defibrotide on platelet function.

Defibrotide, a polydeoxyribonucleotide, has been found to modulate endothelial cell function, causing an increase in tissue plasminogen activator (t-PA) levels, a decrease in plasminogen activator inhibitor (PAI) levels, and an increase in prostaglandin I2 (PGI2) formation in humans. Defibrotide has no direct anticoagulant effect but has a synergistic action with heparin. A strong antithrombotic effect has been observed in animal models. Thus, defibrotide has a beneficial effect in cases of deep venous thrombosis (DVT), peripheral obliterative vascular disorder (POVD), stroke, vasculitis, and thromboembolism. Defibrotide also inhibits platelet function and activation. A significant decrease in platelet aggregate formation on the suture line in microarterial anastomosis in rats is one way defibrotide can inhibit platelet function and activation. In humans, a slight prolongation' of the lag period in collagen-induced aggregation has been observed. In addition, a slight decrease in the maximum amplitude of the secondary wave of ADP and adrenalin-induced aggregations was also found. Platelet adhesion is diminished, the platelet differential count on formvar membrane is altered, and platelet aggregate formation is significantly inhibited. With an increase in platelet cyclic AMP (cAMP) content and a decrease in malonyl dialdehyde (MDA) and thromboxane B2 (TXB2) formation, the levels of platelet secretion products such as PF-4 and beta-thromboglobulin (beta-TG) in plasma decreased progressively. It was also demonstrated that the 14C-glucose transport defect of the platelet membrane of atherosclerotic patients was partially corrected with defibrotide treatment.

Persistent vacuoles in leukocytes: familial Jordans anomaly.

Multiple persistent vacuoles were seen in the neutrophils, monocytes and eosinophils of a 9 year old boy and his 10 year old sister. The siblings were both asymptomatic. In the bone marrow, the cytoplasmic vacuoles were also present in the promyelocytes, myelocytes and metamyelocytes, but not in the myeloblasts and they tended to be single and large in immature cells. The cytoplasmic vacuoles did not stain with PAS, Sudan Black or Oil Red O; Sudan III positivity of the vacuoles was found only in a very small number of granulocytes. The vacuoles appeared as round and bright bodies with phase contrast microscopy. By electron microscopy, the vacuoles contained material of low electron density and had no surrounding membrane. Granulocyte functions were unimpaired. Muscle biopsy showed normal morphology. This anomalous vacuolization of the leukocytes is consistent with familial Jordans anomaly.

The effect of endothelin-1 on vena jugularis thrombus model in rabbits.

Endothelins (ET) are the most important vasoconstrictors known, and administration results in contraction of vascular strips in man and experimental animals in vitro. We examined the effects of ET-1 on thrombus formation in rabbits. We used vasoconstrictor and thrombus forming agents and we selected an animal model, the vena jugularis thrombus model. In addition, intravascular endothelium was examined ultrastructurally. The ET-1 level is known to be high in patients with hypertension; if these patients also have atherosclerosis, then intravascular thrombus formation may increase. In the vena jugularis thrombus model, thromboplastin and ET-1 act synergistically to increase intravascular thrombus formation. On injection of ET-1 dose dependent vasoconstriction was shown in the vessel wall. Although similar maximal contraction is achieved, a decrease in vessel diameter is associated with increased potency of ET-1 and thromboplastin. The results suggest that ET-1 may regulate vascular tone through constriction of vessels.

The pharmacology and clinical pharmacology of defibrotide: a new profibrinolytic, antithrombotic and anti-platelet substance.

Defibrotide, a deoxypolyribonuclide, has been found to modulate endothelial cell function causing increase in t-PA and decrease in PAI levels and also increase in PGI2 production. In addition, it increases platelet c-AMP levels and decreases MDA and TXB2 formation in human. Defibrotide inhibits platelet aggregate formation in vitro experiments as well as end-to-end anostomosis in rats. So, defibrotide inhibits the activation of platelets. Besides an increase of protein C and S levels a synergic action of heparin was observed in animal experiments. A strong antithrombotic effect has been observed in animal models. The drug has a beneficial effect in the cases of DVT, POVD, stroke and thromboembolism. Through its action we may say that the drug acts in a novel fashion in contrast to the other drugs used in this area. Defibrotide is a single-stranded polydeoxyribonucleotide obtained from deoxyribonucleic acid of mammalian lungs by controlled depolimerization. Since 1981 in our laboratory and in the clinical department we have been investigating a newly developed agent defibrotide in vitro experiments, animal experiments, and also its clinical pharmacology and clinical application. Some of our findings are already published and compared with literature (40, 43, 46). Because of the limited space we are not going to review the literature in detail but we are going to summarize our observations on this compound in the following order. I--in vitro experiments, II--Animal experiments, III--clinical pharmacology in human.

Clinical pharmacology and mode of action of a new antithrombotic compound: Defibrotide.

Defibrotide is a derivative of polydeoxyribonucleotide extracted from bovine lung. Defibrotide has been found to modulate endothelial cell function causing increase in t-PA production and release with correction the defect in Cuff test in vascular disorders. Defibrotide causes a significant elevation in the PGI2 formation. In addition increase of platelet c-AMP levels with a decrease of MDA and TXA2 formation has been shown in human subjects. Defibrotide causes an inhibition of platelet activation were demonstrated with surface activation method as well ultrastructurally. Besides, an increase of protein C and FV were observed, a synergic action with heparin was observed. A strong antithrombotic effect has been shown in animal models and unlike most antithrombotic drugs defibrotide did not cause any effect of clotting tests in animals and human subjects. All findings support our earlier suggestion that defibrotide mainly acts via the modulation of endothelial cell function and acts as a novel fashion in contrast to the other drugs used in this area.

Alteration of platelet glucose transport system in atherosclerosis.

Functional and biochemical alterations of platelets in patients suffering from atherosclerosis were studied in our laboratory. One of the most striking alterations observed is in the platelet active glucose transport system. The Na+/K+ gradient dependent active transport system of glucose is found to be absent in the platelets of atherosclerotics. The platelet glucose transport kinetics in these subjects give unsaturable and linear kinetics. Furthermore, the specific glucose binding protein activity detected in the incubation fluid after cold osmotic shock to the platelets of normal subjects is found to be absent in the platelets of atherosclerotics. The platelet active glucose transport system is normal in juvenile onset diabetics, whereas it is impaired in maturity onset diabetics with clinical manifest atherosclerosis. The release inducers like ADP, adrenalin and collagen exert no effect on the platelet active glucose transport system. The specific glucose-binding protein is an unreleasable protein in the platelets of normal subjects. Hence, the absence of active glucose transport system in atherosclerotics is not due to the activated platelets in circulation.

Effects of defibrotide on peripheral obliterative vascular diseases.

Twenty-nine patients with atherosclerotic obliterative vascular disorder and 9 cases of Buerger's disease were treated with 600 mg defibrotide daily for 10 days and then three times weekly for 3 months. The response to therapy was evaluated from hemostatic parameters, venostasis test (cuff test), treadmill testing, and radionuclide arteriography. We observed increased pain-free intervals in daily life and during treadmill testing. There was also a significantly higher response in the cuff test and improved perfusion as seen by radionuclide arteriography.

Effect of defibrotide in electrically induced thrombosis in dogs.

Thrombi were electrically induced in dogs. The alterations of the vessel wall were studied in samples obtained on the 8th day from control and treated animals. Changes in coagulation parameters were monitored daily. Animals were treated with 600 mg defibrotide (Crinos) daily for a week, starting 24 h after the induction of thrombi. In the control dogs there was thickening of intima with endothelial hyperplasia and smooth muscle proliferation. The treated animals did not show any morphological or ultrastructural alterations (SEM). This observation encourages us to examine the role of fibrinolytic and endothelial supportive therapy for prevention of development of vascular changes.

Alteration of prostanoids in atherosclerosis.

We have briefly summarized only the data obtained in our own laboratory without including an extensive literature review. We have evaluated the changes in platelet membrane phospholipids and prostanoid formation in platelets. In atherosclerosis platelets show an activated prostanoid formation, whereas the plasma PGI2 levels are decreased.