Comparative study on the in vitro and in vivo activities of heparinoids derivative investigated on the animal model.

In this study we compared the antithrombotic and anticoagulant properties of sodium and calcium derivatives of pentosan polysulfate (Na-PPS, Ca-PPS), unfractionated heparin (UFH), and low-molecular-weight heparin (Fraxiparin). The antithrombotic effects of these agents have been investigated in an experimental thrombosis model in which rat mesenteric venules with a diameter of 20-30 microm were injured by well-defined argon laser lesions. Furthermore, the in vivo and in vitro anticoagulant activities [activated partial thromboplastin time (aPTT), Heptest] of these agents have been studied. Thrombus formation was significantly inhibited after s.c. injection of Na-PPS and Ca-PPS in doses >10 mg/kg. The duration of the antithrombotic effect lasted 8 h for Na-PPS and 12 h for Ca-PPS. After oral administration of Na-PPS, an antithrombotic effect was not observed. Oral application of Ca-PPS in doses >20 mg/kg significantly inhibited thrombus formation. Na-PPS and Ca-PPS markedly prolonged clotting time in aPTT and Heptest in concentrations ranging from 0.01 to 0.2 mg/ml rat PTT. Two h after s.c. administration of these agents in a dose of 10 mg/kg, the aPTT increased threefold and the Heptest 2.5-fold compared with controls. After oral application of 50 mg/kg Na-PPS and Ca-PPS, no effect on the coagulation test could be measured. Intravenous injection of UFH prolonged the Heptest after 1 min and the aPTT after 30 min. In ex vivo studies of aPTT and Heptest performed in rat plasma between 2 and 24 h after s.c. injection of 0.2 mg/kg Fraxiparin, no inhibition of any coagulation test was measured. The antithrombotic effect of 0.2 mg/kg Fraxiparin after s.c. injection was significant. Intravenous injection of 20 U/kg UFH significantly inhibited thrombus formation. The smallest antithrombotic effect was after i.v. injection of UFH.

Experimental studies on the anticoagulant and antithrombotic effects of sodium and calcium pentosan polysulphate.

In the present study we have compared the antithrombotic and anticoagulant properties of sodium and calcium derivatives of pentosan polysulphate (Na-PPS, Ca-PPS). The antithrombotic effect of these agents have been investigated in an experimental thrombosis model in which rat mesenteric venules diameter of 20-30 microm were injured by well defined Argon laser lesions. Furthermore, the in vivo and in vitro anticoagulant activities (aPTT, Heptest) of these agents have been studied. Thrombus formation was significantly inhibited after s.c. injection of Na-PPS and Ca-PPS in doses above 10 mg/kg. The duration of the antithrombotic effect lasted 8 h for Na-PPS and 12 h for Ca-PPS. After oral administration of Na-PPS an antithrombotic effect was not observed. Oral application of Ca-PPS in doses higher than 20 mg/kg significantly inhibited thrombus formation. Na-PPS and Ca-PPS markedly prolonged clotting time in aPTT and Heptest in concentrations ranging from 0.01 to 0.2 mg/ml rat PTT. Two h after s.c. administration of these agents in a dose 10 mg/kg, the aPTT increased 3-fold and Heptest 2.5-fold compared to controls. After oral application of 50 mg/kg Na-PPS and Ca-PPS no effect on coagulation test could be measured.

Low molecular weight heparin versus acenocoumarol in the secondary prophylaxis of deep vein thrombosis.

The aim of this study was to determine the efficacy and safety of subcutaneous weight-adjusted dose low molecular weight heparin (LMWH) compared with oral anticoagulant (OA) in the prevention of recurrent venous thromboembolism. In a prospective multicenter trial, 202 patients with symptomatic proximal deep vein thrombosis (DVT) were included. As soon as the diagnosis of DVT was confirmed by phlebography, 101 were randomly assigned to receive LMWH (nadroparin) for secondary prophylaxis and 101 to receive OA (acenocoumarol). Patients in both groups were initially treated with nadroparin in a dose of 85 anti-Xa IU/kg s.c. every 12 h. Secondary prophylaxis with either nadroparin, 85 anti-Xa IU/kg s. c. once daily, or acenocoumarol was continued for at least 3 months. Three patients in the LMWH group and 6 in the OA group were excluded from analysis for various reasons. During the one-year combined secondary prophylaxis and surveillance period, 7 of of the 98 evaluable patients (7.1%) in the LMWH group and 9 of the 95 evaluable patients (9.5%) in the OA group had a documented recurrence of venous thromboembolism (Fisher's exact test, p = 0.61). Of these, 2 patients who received LMWH and 7 patients on acenocoumarol had recurrences in the 3-month period of secondary prophylaxis. Four patients (4.1%) in the LMWH group developed bleeding complications during this study period, as compared with 7 (7.4%) in the OA group (Fisher's exact test, p = 0.37). There were two major bleedings, one in the LMWH group and one in the OA group. Eleven patients died, 5 (5.1%) in the LMWH group and 6 (6.3%) in the OA group. It is concluded that nadroparin in a dose of 85 anti-Xa IU/kg s.c. once daily provides an effective and safe alternative to oral anticoagulants in the secondary prophylaxis of DVT.

The role of epidermal growth factor in platelet-endothelium interactions.

The objective of this study was to determine whether endogenous EFG released after submaximal physical exercise, affects platelet-endothelium interactions. Sixteen healthy male volunteers, aged 23-26 years, were submitted to a submaximal bicycle ergometry test. Blood for determination of EGF concentrations, platelet function studies (concentrations of beta-TG, PF4 and TXB2) and endothelium activity (LTC4 and endothelin-1,2 concentrations) was taken via an intravenous catheter before starting exercise and 15, 30 and 60 min after. A similar scheme was followed to investigate changes in the same parameters induced by a slow intravenous infusion of 0.3 mg/kg b.w. phentolamine (an alpha-adrenergic blocker) before exercise. Plasma concentrations of EGF and the markers of platelet function-beta-TG and PF4 as well as LTC4 concentrations increased only 15 min following exercise. The concentrations of TXB2, and endothelium-1,2 were almost unchanged 15 min after the submaximal bicycle ergometry test. Phentolamine markedly decreased the EGF concentrations in plasma (15 min following exercise) while at 30 and 60 min after exercise it had no effect on this parameter. No significant changes in concentrations of beta-TG, PF6, LTC4 and endothelin-1,2 after phentolamine infusion were found. These results show that increase of plasma EGF following exercise was accompanied with increase of beta-TG, PF4 and LTC4 concentrations. Inhibition of alpha-adrenergic receptors with phentolamine abolished the exercise-induced increase in plasma EGF concentration. The findings suggest that endogenous EGF may affect the platelet function and changes the reactivity of the vascular endothelium.

Release of human monocytes protein C and the cofactor protein S in vitro.

Monocytes may contribute to a coagulation process by expression of the tissue factor and by synthesizing factors V, VII, X and XIII. Cultured blood monocytes also express both the tissue plasminogen activator and the plasminogen activator inhibitor I. The present study assesses the ability of human monocytes to secrete protein C and its cofactor protein S, which are potent inhibitors of the clotting cascade. Monocytes derived from the blood of healthy volunteers and prepared according to Boyum were cultured for up to 36 hours with or without lipopolisaccharide from Escherichia coli. After different times of incubation, the concentrations of proteins C and S in the supernatants were measured in order to determine synthesis of proteins, monocytes were cultured in the presence or absence of cycloheximide, the protein synthesis inhibitor. The concentration of protein C was estimated by means of the ELISA Protein C test (Boehringer Mannheim). Protein S concentrations were measured by rocket immunoelectrophoresis according to Laurell, using monospecific antisera (American Diagnostica Inc., N.Y.). The study showed that human monocytes, when stimulated by lipopolisaccharide, release proteins C and S in vitro. The concentration of these proteins in the culture supernatants markedly increased with time during the 36-hour observation. The supernatants obtained from the culture of unstimulated monocytes did not contain detectable quantities of the investigated proteins. The exposure of the cells to cycloheximide did not suppress the release of proteins C and S. In conclusion, our results suggest that monocytes are not able to synthesize proteins C and S. They can only release these factors. Furthermore, monocytes may be responsible for coagulation due to the inactivation of factors Va and VIIIa by protein C.

[Case report presentation for treatment of pure red cell aplasia]. / Opis przypadku leczenia wybiórczej aplazji czerwonokrwinkowej.

We presented a case of a 34 year old male patient with pure red cell aplasia. He was treated with antithymocyte globulin (Pasteur Merieux, France) at a dose of 175 mg in 350 ml 0.9% NaCl in intravenous drip infusion--10 drip/min for 5 consecutive days. The therapy resulted in normalization of morphological parameters. Patient was under clinical observation. After 9 years when hemoglobin level decreased monoclonal antibody OKT3 og IgG 2a type (Cilag Ag International, Switzerland) at a dose of 5 mg intravenous for 10 consecutive days was applied. During the first and second therapy course symptoms of serum sickness and "flu like" syndrome was observed. The results of the two treatment appeared positive. We received normalization of morphological parameters.

Effect of nonionic and ionic contrast media on fibrinolysis in patients undergoing angiography.

Radiologic contrast media may influence processes of hemostasis resulting in increased thrombotic or bleeding tendency. A number of clinical case reports suggest that the use of nonionic contrast media is associated with thrombotic complications. In vitro studies have indicated that nonionic contrast media may induce generation of thrombin in blood whereas ionic contrast agents do not show such an effect. Not much is known about the effects of contrast media on the coagulation and fibrinolytic systems in vivo. The aim of this study was to evaluate the systemic effects of markers for activation of fibrinolysis with a nonionic contrast medium (Iopromid/Ultravist-300/Schering AG) and ionic contrast medium (Uropolinum, Polfa) in 82 patients undergoing angiography. We measured tissue plasminogen activator (t-PA) and tissue plasminogen activator inhibitor (PAI), using COA-SET, t-PA and COA-TEST PAI (Chromogenix). Fibrinogen concentration and euglobulin lysis time (ELT) were also estimated. Both contrast agents caused a significant decrease in fibrinogen concentrations. A marked difference was seen for PAI activity. A statistically significant increase was seen in the Iopromid group and no statistically significant rise was seen in the Uropolinum group. t-PA activity remained virtually unchanged in both groups. ELT has been significantly prolonged in patients who received Iopromid but not in those who received Uropolinum. It is likely that nonionic contrast medium could release PAI from platelets and endothelial cells. The changes in fibrinolysis may result from endothelial cell dysfunction.

Interaction between the sulfated lactobionic acid (LW 10082) and other antithrombotic agents in animal thrombosis model.

We investigated the comparative antithrombotic properties of LW 10082, acetylasalicylic acid (Aspisol), low molecular weight heparin (LMWH, CY 216) and sodium heparin using laser-induced thrombosis model. The animal model which has been used is well suited to observe platelet reactions to damaged endothelial cells and also to evaluate the effect of drugs on the formation of platelet thrombi. The rat mesenteric venules with a diameter of 20-30 microns were injured by well defined argon laser lesion. The number of laser injuries which were needed to induce a thrombus intermittently occluded the vessel was counted to quantitate the results. LW 10082 was injected sc 2 h prior to testing. The minimal effective dose of LW 10082 which significantly inhibited thrombus formation was 5 mg/kg. The antithrombotic effect of Aspisol has been investigated after its oral application at doses of 1.5 and 10 mg/kg 2 h before studying the antithrombotic effect. The minimal effective dose for Aspisol was 10 mg/kg. The LMWH has been injected into the rat tail vein in doses of 1, 0.2 and 0.1 mg/kg 30 min before testing. 0.1 mg/kg did not have any antithrombotic effect. The minimal effective dose of the sodium heparin which significantly inhibited thrombus formation was 20 U/kg measured 30 min after its iv injection. The combination of minimal effective dose of LW 10082 and LMWH had a significant additive effect. There was a slight but not significant additive effect between LW 10082, Aspisol and heparin sodium. Our results suggest that combinations of LW 10082 with LMWH may provide a new approach to more effective prophylaxis and treatment of venous or arterial thrombosis.

Effect of troxerutin on laser-induced thrombus formation in rat mesenteric vessels, coagulation parameters and platelet function.

The antithrombotic effect of Troxerutin have been studied in an experimental model of thrombosis in which rat mesenteric vessels (arterioles and venules) 25-30 microns in diameter were injured by well defined argon laser lesions. Furthermore in vitro effect of this agent on coagulation parameters (IIa, Xa inhibition, TT, heptest), and platelet function (platelet adhesion to the siliconised glass and extracellular matrix, platelet spreading) has been investigated 2 h after oral drug administration. Troxerutin at a dose of 10 mg/kg markedly inhibited thrombus formation in venules. Higher dose (50 mg/kg) was needed to obtain the same antithrombotic effect when arterioles were studied. After application of a single dose of Troxerutin (100 mg/kg) antithrombotic effect lasted for 6 h to 7.5 h when venules were studied, and for 4.5 h to 6 h when arterioles were investigated. In in vitro study we did not observe any effect of Troxerutin on coagulation parameters. In concentrations of 100 micrograms/ml in platelet rich plasma Troxerutin significantly inhibited platelet adhesion to the extracellular matrix and siliconised glass as well as platelet spreading. It is likely that this drug possesses antithrombotic effect evaluated by inhibition of platelet function and protection of endothelial cells.

Antithrombotic and thrombolytic activity of heparins in a rat model of laser-induced thrombosis.

The mode of action by which heparins exert their antithrombotic effect is not fully understood. Antithrombotic action persists much longer than the inhibition of even sensitive coagulation parameters. We therefore have investigated antithrombotic and thrombolytic activity of two low molecular weight heparins Fraxiparin (Sanofi) and Clexane (Rhone-Poulenc) in a rat model of laser-induced thrombosis. The antithrombotic effect was measured by the number of laser injuries required to induce a defined venous thrombus which was at least as broad and as high as the vessel diameter. Thrombolytic activity of low molecular weight heparins was investigated by measuring the time from venous occlusion to recanalisation of the vessel. Fraxiparin in minimal dose of 0.001 mg/kg after iv injection showed antithrombotic effect in the laser thrombosis model. After single iv injection of this dose antithrombotic effect of Fraxiparin lasted longer than 6 h but less than 8 h. In higher dose of 0.01 mg/kg after single iv injection thrombolytic activity of Fraxiparin was observed. Also Clexane showed dose dependent antithrombotic and thrombolytic activity in this model. In dose of 0.005 mg/kg after single iv injection of Clexane significant inhibition of thrombus formation was observed. This antithrombotic effect lasted longer than 6 h but less than 8 h. In dose of 0.05 mg/kg after iv injection Clexane showed significant thrombolytic activity. In conclusion, further investigations are needed to optimize prophylactic and therapeutic use of low molecular heparins.

[Examination of interleukin levels and growth factor from blood platelets in leukemia]. / Badania stezen interleukin oraz czynnika wzrostowego z plytek krwi w bialaczkach.

Plasma concentrations of interleukines 1, 3, 6, 8 and platelet derived growth factor (PDGF) were estimated in 45 patients with leukaemias. Among patients were 12 with acute myeloblastic leukaemia-AML (type M1 according to FAB classification), 9 with chronic granulocytic leukaemia-CGL, 10 with blastic crisis of CGL (CGL-BC) and 14 with chronic lymphocytic leukaemia-CLL (in 1 and 2 stage according to Rai classification). Additionally the concentration of IL-3 was measured in 10 patients with exacerbation of CLL. Control group consisted of 12 health volunteers. The concentrations of interleukines and PDGF were determined by means of radioimmunoassay or immunoradiometric assay. In the patients with AML, CGL-BC and CLL significant increase of concentrations of IL-1B, IL-6 were found. The patients suffers from CGL and CGL-BC had increase concentrations of IL-3, but patients with CLL-interleukin 8. The concentrations of PDGF were significantly decreased in the patients with AML, CGL-BC, and CGL. The differences of concentrations of studied interleukines can confirm their great significance in proliferation of leukaemic cells. The decrease of concentrations of PDGF in myelocytic leukaemias may reflect thrombopoietic disturbances in these illnesses.

Prothrombin activation fragment 1 + 2 and thrombin-antithrombin-III complexes in plasma of patients with essential arterial hypertension.

Prothrombin activation fragment 1 + 2 (F1 + 2) and thrombin-antithrombin-III complexes (TAT) were measured in plasma of 34 patients with newly diagnosed essential hypertension. The patients with hypertension showed an increase in both F1 + 2 and TAT concentrations. The obtained results point to the intravascular thrombin generation in very early stages of essential hypertension.

Immunohistochemical demonstration of fibrin II in gastric cancer tissue.

Immunohistochemistry was applied to AMeX-fixed sections of twelve cases of gastric carcinoma obtained at surgical resection to explore the occurrence and distribution of fibrin deposits in situ. Fibrinogen was distributed in abundance throughout perivascular zones and in the connective tissue of the tumor stroma. Fibrin II (des-fibrinopeptide B-type fibrin) was easily identified in a direct apposition to the surface membranes of viable carcinoma cells, predominantly at the host-tumor interface and in the regions immediately adjacent to the zones of angiogenesis. Further studies are required to identify the triggers of the coagulation reactions as well as fibrinolytic system components in the gastric cancer tissue.

The role of monocytes in the platelet release of von Willebrand factor.

Circulating blood monocytes, which are a source of foam cells contribute to the vascular lesions. Von Willebrand factor (vWF) is an adhesive multimeric glycoprotein that is synthesized only by endothelial cells and megakaryocytes. In the present study we assessed the ability of human platelets to secrete vWF after incubation with blood monocytes in vitro. Monocytes derived from blood of healthy adults were cultured and next incubated with washed platelets. von Willebrand factor antigen (vWF:Ag) was measured in the supernatant by Laurell immunoelectrophoresis. The study showed that Triton X-100 treated platelets released 82-112% of vWF:Ag. The levels of vWF:Ag in the supernatant with untreated platelets and incubated with monocytes without LPS were respectively 36-48% and 45-72%. After incubation washed platelets with LPS stimulated monocytes a significant decrease in vWF:Ag level was observed (7-11%).

[Tissue plasminogen activator and plasminogen activator inhibitor in aqueous humor]. / Stezenia tkankowego aktywatora plazminogenu i dzialanie inhibitora tkankowego aktywatora plazminogenu w plynie komorowym galki ocznej.

BACKGROUND: In the anterior segment of the eye, fibrin clots must be rapidly resorbed to prevent fibrosis. Tissue-type plasminogen activator (t-PA), a serine protease that catalyzes the conversion from plasminogen to plasmin, plays an important role in the fibrinolytic system and has therefore in recent years attracted attention in the field of ophthalmology. MATERIAL AND METHODS: The aqueous humor of patients undergoing cataract surgery was analyzed for the presence of components of the fibrinolytic cascade. The quantities of t-PA and plasminogen-activator inhibitor (PAI) were determined using enzyme-linked immunosorbent assays. We determined t-PA and PAI in the aqueous humor of 64 patients between 59 and 82 years of age. RESULTS: The t-PA levels ranged from 0.65 to 1.75 ng/ml and PAI activity from 3.24 to 5.1 AU/ml. Association between t-PA levels and PAI activity and accompanying diseases or metabolic disorders was noted. The highest concentration of t-PA and the lowest activity of PAI has been observed in aqueous humor of patients with senile cataract. The knowledge about the presence of t-PA in aqueous humor is significant for the recognition of pathological events following intraocular fibrin formation and may be an important basis for therapeutic use of t-PA.

Effects of 0-/-B-hydroxyethyl/rutoside on platelet function and thrombus formation in rat mesenteric vessels.

0-/-B-hydroxyethyl/rutoside has been investigated for its effect on laser-induced thrombus formation in rat mesenteric venules and arterioles. The in vitro effect of this agent on platelet adhesion to bovine subendothelial extracellular matrix (ECM) and glass, on spreading and on platelet aggregation induced by ADP, collagen and epinephrine have also been studied. The animal investigations of 0-/-B-hydroxyethyl/rutoside showed an antithrombotic effect in doses between 5 and 50 mg/kg after i.v. injection. This effect was similar for both arterioles and venules damaged. After i.v. injection of minimum effective doses of 0-/-B-hydroxyethyl/rutoside, the antithrombotic effect lasted longer than 6 hrs but less than 12 hrs. In vitro, 0-/-B-hydroxyethyl/rutoside significantly inhibited platelet adhesion to bovine ECM in concentrations of 20 micrograms/ml PRP, and with 30 micrograms/ml the PRP adhesion to glass and spreading were inhibited. Epinephrine and ADP induced aggregation was inhibited in concentrations higher than 30 micrograms/ml PRP.

Stimulated monocytes release factor XIII subunit A and fibronectin in vitro.

Factor XIII catalyses the formation of covalent bonds between several proteins involved in wound healing. Subunit A of factor XIII has been found inside and on the surface of monocytes and macrophages. In our study we investigated the ability of cultured monocytes to release factor XIII A subunit and its substrate-fibronectin. Human monocytes were cultured for up to 36 hours with lipopolysaccharide (LPS) from Escherichia coli. In supernatants of LPS stimulated monocytes we found 22 +/- 4 nM/ml and 40 +/- 6 nM/ml of factor XIII subunit A after 24 and 36 hours of incubation respectively. We also observed an increase of fibronectin concentration in supernatants of stimulated monocytes. The obtained results suggest that activated monocytes might be a source of factor XIII and fibronectin, which are critical for tissue repair processes.