Effect of thrombin inhibitors and a glycoprotein IIb/IIIa receptor antagonist in an ex vivo human experimental thrombosis model.

The development of new antithrombotic drugs to inhibit the blood coagulation system and to block receptors of platelets represents an important area of medical research. The results of the efficacy in animal models are of critical importance for the further development of these compounds for human use. The transferability of such data from animal to human species still remains controversial. The antithrombotic effects of direct thrombin inhibitors and a glycoprotein IIb/IIIa (GPIIb/IIIa) receptor antagonist were investigated in an experimental thrombosis model using human blood in order to analyze the feasibility of such a model.

[Prevention of thromboembolism with low-molecular-weight heparin in ambulatory surgery and unoperated surgical and orthopedic patients]. / Thromboembolieprophylaxe mit niedermolekularem Heparin bei ambulanten operierten und nicht-operierten chirurgischen und orthopädischen Patienten.

Prophylaxis of thromboembolism is now well established in orthopaedic outpatients with plaster cast and after elective hip surgery. The present study was undertaken to evaluate the safety of out-of-hospital prevention of venous thromboembolism and to determine the incidence of thromboembolic complications in orthopaedic and surgical patients with or without surgical intervention on an out-patient basis during prophylaxis of thromboembolism with low-molecular-weight heparin and to study the feasibility of this treatment regimen. The treatment period was 1-4 weeks (mean 17 days). Main indications for prophylaxis of thromboembolism were arthroscopy and surgical or non-surgical intervention of bone fractures of the lower leg. The incidence of clinically diagnosed venous thromboembolism was 11/1604 (0.7%) in operated and 8/1017 (0.8%) in non-operated patients. Pulmonary embolism occurred twice in operated patients (0.1%) and in none of the non-operated patients. Minor bleeding complications were rare and major bleeding complications did not occur. Haematomas at the injection site occurred in only 4% of patients. Thrombocytopenia did not occur in any patient. The present study demonstrates the feasibility and safety to prophylaxis of thromboembolism with low-molecular-weight heparin in orthopaedic operated and non-operated out-patients with various orthopaedic or surgical diseases leading to immobilization. The incidence of clinically apparent thromboembolic complications is low and similar to medical bedridden inpatients.

Comparison of the pharmacodynamic and pharmacokinetic profiles of two low-molecular-mass heparins in rats.

The pharmacodynamic and pharmacokinetic properties of certoparin and dalteparin were analyzed after intravenous and subcutaneous administration. The two different LMMHs exhibited different molecular mass and in vitro activities. The aim of the present study was to show the extent to which these in vitro differences influenced the biological activity and pharmacokinetics in vivo. It was possible to measure the plasma concentrations of the LMMHs by using a competitive assay with protamine-coated latex beads. Both LMMHs showed a biphasic aXa and aIIa activity curve after intravenous injection. Certoparin and dalteparin showed comparable aXa pharmacodynamics after IV and SC injection. By measuring the aIIa activities after IV administration of the LMMHs, T1/2, Amax, and AUC were comparable but tPC differed. After SC injection the aIIa activities of the LMMHs exhibited comparable T1/2 and Amax but different AUC and tPC. The plasma concentrations of the LMMHs showed comparable T1/2 but certoparin exhibited a higher Amax and AUC after IV and SC administration. The relative bioavailability of the aXa activity, aIIa activity, and the plasma concentrations ranged between 70 and 100%. The differences in the aIIa pharmacodynamic and pharmacokinetic profiles of certoparin and dalteparin may be caused by the differences in the in vitro activities and the different molecular mass. Clinical relevance of the different pharmacologic profiles is only expected of the aIIa activity-related biological effects of the LMMHs.

Long-term anticoagulation of outpatients with adverse events to oral anticoagulants using low-molecular-weight heparin.

Bleeding complications are one of the major risks during oral anticoagulation. If further anticoagulation is indicated, low-molecular-weight heparin (LMWH) may offer an alternative treatment in those patients. In a prospective, nonrandomized study, 120 patients have been switched from oral anticoagulants to LMWH because of bleeding complications or other severe side effects during treatment with vitamin K antagonists. Indication for further anticoagulation was prophylaxis of recurrent thromboembolism, artificial heart valve replacement, atrial fibrillation with embolism and cardiomyopathy. The treatment period ranged from 2 months to 10.8 years. No fatal embolism occurred. One major but not severe episode of gastrointestinal bleeding occurred in a patient with an as yet unknown colon carcinoma. The cumulative treatment period amounts to 250 years. No drop in platelet count occurred in any patient. No other side effects were observed. LMWH was injected subcutaneously at doses ranging from 2500 to 15,000 anti-factor Xa units per day by the patient himself. The dose was adjusted on the basis of body weight, bleeding risk and thromboembolic risk. The results indicate that LMWH may be effectively and safely used as alternative anticoagulant regimen in patients with side effects or other complications on oral anticoagulants.

Anticoagulation in patients with heparin-induced thrombocytopenia type II.

Heparin-induced thrombocytopenia (HIT) together with simultaneously occurring thromboembolism is a serious complication of heparin treatment. At present an immunologic cause of this side effect of heparin is equivocally accepted. However, further anticoagulation of these patients is still debated. The present article summarizes the treatment of 20 patients with such complications. Two of these patients did not develop thrombocytopenia but presented cutaneous allergy or necrosis. All patients were treated either with intravenous heparinoid (Orgaran) or with low-molecular-weight heparin without/with simultaneous intravenous high-dose immunoglobulins or with intravenous r-hirudin. Based on these experiences the treatment of choice depends at present on the availability of the anticoagulants and on the local experience with the different anticoagulants. In the future r-hirudins and other nonheparin thrombin inhibitors may become the drugs of choice in this indication. Surgical intervention has to be considered additionally.

Measurement of fluorescent-labeled LMM-heparin in biological fluids using protamine-linked microbeads.

A quantitative assay for fluorescent heparin in a purified system and in plasma was developed (Piazolo et al: Semin Thromb Hemostas 20:227-235, 1994). The protamine microbeads (1.6 microns) showed a broad size distribution and a large standard variation in low concentrations. Our aim was to optimize these protamine microbeads for the measurement of fluorescent heparin. The following results were obtained: Paramagnetic protamine microbeads of different average diameters (0.8, 1.6, 2.8, and 4.5 microns) were synthesized by cyclocarbodiimide and tosyl activation. These microbeads bind heparin and are assayed using flow cytometry. The protamine concentration on the surface of the beads ranged between 2.0 and 61 mg/mL. The protamine microbeads bound fluorescent heparin and were analyzed by flow cytometry. The protamine microbeads bound LMM-heparin-tyramine-FITC dose dependently in saline solution, plasma, and blood. There are substantial differences between the microbeads of different origins with regard to the amount of protamine bound, the sensitivity of the detection, and the reliability for the determination of heparins in plasma and blood. The minimal sensitivity of the final method was 0.001 U/mL LMMH-tyramine-FITC in saline solution and in plasma. Human blood cells were not bound to protamine microbeads. The half-maximal binding of LMMH-tyramine-FITC of the different protamine-coated microbeads ranged from 1.7 to 8.0 micrograms/mL in saline solution, 2.3 to 8.7 micrograms/mL in plasma, and 3.1 to 6.4 micrograms/mL in blood. We conclude that all protamine microbeads can be used to quantify the concentration of LMMH-tyramine. Protamine Dynabeads M-450 (diameter 4.5 microns) have advantages over other microbeads because of their more homogeneous size distribution, a higher selectivity, and they can be measured together with leukocytes. They are currently used to develop a competitive binding assay for heparin in plasma.

Chromatographic and electrophoretic applications for the analysis of heparin and dermatan sulfate.

Heparin and dermatan sulfate are highly sulfated polydisperse glycosaminoglycans. The methods to determine such compounds include chromatographic and electrophoretic techniques. Here we report on the performances of various analytical methods for the characterization and the determination of GAGs. Heparin, low-molecular-mass heparins, dermatan sulfate and low-molecular-mass dermatan sulfate were analyzed. High-performance size exclusion chromatography was used to determine the molecular mass, polydispersity, absorbance and the area under the absorbance-time curve. Polyacrylamide gel electrophoresis was used to determine the average molecular mass and the polydispersity. Heparin and dermatan sulfate preparations were analyzed by capillary electrophoresis using reversed polarity. The results obtained reflect different performances of various analytical methods used to characterize GAGs.

Preferential binding of heparin to granulocytes of various species.

OBJECTIVES: To address the problem of heparin binding to leukocytes of various animal species. DESIGN: Leukocytes of the various species were incubated with fluorescent-labeled, low molecular mass heparin (LMMH). Fluorescence intensity on granulocytes, lymphocytes, and monocytes was analyzed by flow cytometry analysis. SAMPLE POPULATION: Leukocytes were prepared from EDTA-anticoagulated blood of human subjects, rats, rabbits, dogs, pigs, and sheep 3 times. PROCEDURE: The leukocyte populations were identified by their light scatter properties. In addition, phycoerythrin-labeled CD4, CD13, and CD14 antibodies were used to identify human lymphocytes, granulocytes, and monocytes; CD4, GR-1, and CD11b antibodies were used for mouse, and CD45, RP 3, and ED 9 antibodies were used for identification of rat leukocyte subpopulations. RESULTS: Granulocytes, monocytes, and lymphocytes of all species bound LMMH in dose-dependent manner. Binding of LMMH-tyramine (tyr)-fluorescein-5-isothiocyanate (FITC) to granulocytes was higher in human subjects, rats, rabbits, dogs, and pigs, compared with binding to monocytes and lymphocytes. Mouse and sheep granulocytes did not bind more heparin than monocytes or lymphocytes. Binding of LMMH-tyr-FITC was reversible in the presence of unlabeled heparin of LMMH. More then 99% of human, rat, rabbit, dog, and sheep granulocyte populations were distinguished from monocytes and lymphocytes by means of their fluorescence intensity owing to LMMH-tyr-FITC. This separation was not obtained for mouse and pig granulocytes. CONCLUSION: Evidence of specific heparin binding to granulocytes of many species indicates the relevance of fluorescent-labeled LMMH for biological investigations. CLINICAL RELEVANCE: Binding of heparin and LMMH to granulocytes, lymphocytes, and monocytes may have a substantial role in atherosclerosis, inflammation, malignancy, and immunologic diseases.

Analysis of heparin binding to human leukocytes using a fluorescein-5-isothiocyanate labeled heparin fragment.

The binding of LMWH-tyr-FITC to granulocytes, monocytes, and lymphocytes was analyzed by flow cytometry using a low-molecular-weight heparin (LMWH) labeled with fluorescein-5-isothiocyanate (FITC). FITC was covalently bound to tyramine, which was synthesized to LMWH by endpoint-attachment (Malsch et al.: Anal Biochem 217:255-264, 1994). The binding was rapid, specific, dose-dependent, saturable, and reversible. To investigate the molecular weight dependence of heparins, heparin-derived di- to dodecasaccharides were used. With decreasing molecular weight, the amount of oligosaccharides increased; these were bound to granulocytes, monocytes, and lymphocytes (r = -0.77). The degree of sulfation of non-heparin glycosaminoglycans influenced the binding to leukocytes. Decreasing the degree of sulfation decreased the binding. The pentasaccharide did not bind as strongly as the other heparin-derived oligosaccharides, indicating an AT III-independent mechanism. Two classes of heparin binding sites were identified on granulocytes and one class of binding sites on monocytes and lymphocytes. The lowest amount of LMWH-tyr-FITC detected was 1 ng on granulocytes, 0.18 ng on monocytes and 0.01 ng on lymphocytes. The data suggest that heparin and other sulfated polysaccharides may play a role in the physiology of thrombosis, arteriosclerosis, and inflammation by binding to granulocytes, monocytes, and lymphocytes.

N' alkylamine low molecular mass heparins (LMM-heparin-tyramine and LMM-heparin-tyramine-fitc) exhibit long lasting anticoagulant effects.

The pharmacodynamic and pharmacokinetic properties of endpoint-attached N'alkylamine derivatives of low molecular mass heparin (LMMH), low molecular mass heparin (LMMH), low molecular mass heparin-tyramine (LMMH-tyr) and low molecular mass heparin-tyramine-fluorescein-5-isothiocyanate (LMMH-tyr-fitc) were investigated ex vivo. After intravenous bolus injection of LMMH, LMMH-tyr and LMMH-tyr-fitc (150 aXa U/kg) to Sprague-Dawley rats (n = 8), LMMH-tyr and LMMH-tyr-fitc displayed decreased clearances. The beta-half-life time of the antifactor Xa (aXa) of "endpoint-attached heparins" was significantly prolonged: LMMH-tyr (125 min), LMMH-tyr-fitc (141 min) compared to LMMH (69 min). The pharmacokinetics of LMMH-tyr-fitc were measured with reversed phase high performance liquid chromatography (RP-HPLC). It showed a decreased clearance and a prolonged half-life time (132 min). The selectively tagged LMMH-tyramine and LMMH-tyramine-fitc may be used to investigate the pharmacokinetics, plasma protein and cellular binding of low molecular mass heparins.

Competitive binding of low molecular mass heparin-tyramine fluorescein-5-isothiocyanate and unlabeled glycosaminoglycans to leukocytes.

Antithrombotic, antiarteriosclerotic, and anti-inflammatory actions of heparins may be mediated by binding of heparin to leukocytes. To get the first information on this hypothesis, fluorescein-labeled LMMH-tyramine has been used (LMMH-tyramine-FITC) to analyze the binding of GAGs to lymphocytes, monocytes, or granulocytes. The fluorescence intensity on leukocytes was quantified by flow cytometry analysis. LMMH-tyramine-FITC bound dose dependently to lymphocytes, monocytes, and granulocytes. PE-labeled CD 11c antibodies identified the specificity of the binding of LMMH-tyramine-FITC to granulocytes. UFH and LMMH displaced LMMH-tyramine-FITC dose dependently from leukocytes. Equimolar ratios of LMMH and LMMH-tyramine-FITC revealed a 50% displacement of the labeled compound, indicating the specific binding by the polysaccharide chain. The synthetic pentasaccharide was 10-fold and dermatan sulfate 100-fold less effective than UFH in displacing LMMH-tyramine-FITC from leukocytes. The data indicate that negatively charged GAGs bind to the surface of lymphocytes, monocytes, and granulocytes. By decreasing the number of negatively charged groups of GAGs, the binding to the surface of leukocytes is decreased. The data obtained with the synthetic pentasaccharide indicate a binding of GAGs to the surface of leukocytes, independently of AT III. The cellular binding of heparins may significantly contribute to its antithrombotic and other biologic activities.

Binding of fluorescent-labeled low molecular mass heparin to latex microspheres and to rat and human leukocytes.

Fluorescent-labeled LMMH may be used to investigate the pharmacokinetics of heparins. FITC has been specifically bound by endpoint attachment to LMMH-tyramine. In the present article two in vitro methods are described with the intention of developing new ex vivo test systems. To determine the concentration of heparin, protamine has been bound to the surface of magnetic latex particles. LMMH-tyramine-FITC bound dose dependently in a linear range from 0.1 to 30 micrograms/mL to the protamine-coated latex particles. The fluorescence intensity of the latex particles was analyzed using flow cytometry. No differences in the dilution curves were observed between buffer system, rat and human plasma. This indicated that no interference of heparin binding took place with plasma proteins with respect to the binding to protamine. In the second method the binding of LMMH-tyramine-FITC on rat and human leukocytes was analyzed using flow cytometry. LMMH-tryamine-FITC bound dose dependently to rat and human granulocyes, monocytes, and lymphocytes. A linear relationship of binding was obtained for all three cell lines; the sensitivity was 0.1 microgram/mL for monocytes and lymphocytes and 0.03 microgram/mL for granulocytes without differences between rat and human cells. The data demonstrate a dose-dependent binding of LMMH-tyramine-FITC to protamine coated latex beads and to rat and human leukocytes.