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D L Heene

Publications and source records attributed to D L Heene.

At least 73 records · Page 4Linked to original sources
PubMed1994

Semisynthesis and analysis of lipophilically modified unfractionated and low molecular mass heparins.

Unfractionated heparin and LMMH were substituted with different lipophilic organic compounds. Specifically endpoint attached (LMMH-tyramine and LMMH-tyramine-FITC) and nonspecifically substituted heparins (acylated heparins, and LMMH-biotin and LMMH-cholesterol hemisuccinate) were obtained. The lipophilically substituted heparins were analysed by HPSEC and showed different retention times, high peak purity, different UV/VIS absorbances, and areas under the absorbance time curve. The determination of the average molecular mass Mn, Mm, and Mz and the polydispersity P was performed by PAGE. The substituted heparins showed an increase in their molecular mass Mm, ranging from 2.9 to 129.7% unfractionated heparin and 3.9 to 224.0% (LMMH) compared with the parent compounds (unfractionated heparin and LMMH). The anticoagulant activity was measured by anti-Factor Xa. Lipophilically modified heparin had an aXa activity ranging from 52 to 168 U/mg (unfractionated) and 60 to 108 U/mg (LMMH) and antithrombin activity ranging from 31 to 270 U/mg (unfractionated) and 5 to 15 U/mg (LMMH). The thrombin generation inhibition assay demonstrated an effective anticoagulant potency of the modified compounds. They were neutralized by different amounts (1.1 to 4.1, w/w) of protamin. 1H NMR spectroscopy revealed the specific endpoint attachment of tyramine to LMMH and FITC to LMMH-tyramine. The lipophilically modified heparins showed intact anticoagulant properties and are now used for pharmacokinetic investigations.

Acylation↗
PubMed1994

Comparison of three heparin bovine serum albumin binding methods for production of antiheparin antibodies.

IgG antiheparin antibody secreting hybrid cells have not been successfully raised so far. Therefore, we compared three different conjugating procedures for preparation of LMMH-protein conjugates. Albumin was chosen as carrier protein. Only the conjugate prepared by reductive amination induced antiheparin antibody production in mice. Conjugates prepared by the carbodiimide method and Mannich reaction did not induce heparin immunogenicity and did not induce antibody production in mice. Antiheparin antibody production in mice and by hybrid cells was detected by assaying serum and culture supernatant. LMMH-tyramine, labeled with 125I, was bound by the antibodies and immunoprecipitated by goat anti-mouse IgG antibodies. The tracer was competitively inhibited by nonlabeled heparin for binding to the heparin antibodies. Hybridoma cells producing the specific antiheparin antibody were cloned and subcloned up to monoclonality. Six hybrids were selected and designated H1.18, H1.21, H3.1, H3.17, H3.18, H4.24. In conclusion, six monoclonal antiheparin antibodies were raised using a LMMH-albumin conjugate prepared by reductive amination.

Animals↗
PubMed1994

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.

Animals↗
PubMed1994

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.

Antigen-Antibody Reactions↗
PubMed1993

Protamine neutralization of the release of tissue factor pathway inhibitor activity by heparins.

The present study was designed to investigate the action of protamine on the release of tissue factor pathway inhibitor (TFPI) activity by unfractionated (UF) and low molecular weight (LMW) heparin in healthy individuals. 5000 IU UF-heparin or 5000 IU LMW-heparin were given intravenously followed by saline, 5000 U protamine chloride or 5000 U protamine sulfate intravenously after the 10 min blood sample. Then serial blood samples for the measurement of TFPI activity and anti-factor Xa-activity were taken, in order to detect a possible relation between the remaining anti-factor Xa activity after neutralization of LMW-heparin with protamine and TFPI activity and to establish whether or not a rebound phenomenon of plasmatic TFPI occurs. There was no difference in the release and in the kinetics of TFPI by UF- and LMW-heparin with subsequent administration of saline. After administration of protamine TFPI activity decreased immediately and irreversibly to pretreatment values. There were no differences between protamine chloride and protamine sulfate on the effect of TFPI induced by UF- or LMW-heparin. No rebound phenomenon of TFPI activity occurred. In contrast anti-factor Xa- activity, as measured by the chromogenic S2222-assay, issued the known differences between UF- and LMW-heparin. The half-life of the aXa-effect of LMW-heparin was twice as long as of UF-heparin. Protamine antagonized UF-heparin completely and about 60% of the anti-factor Xa activity of LMW-heparin, using chromogenic S2222-method. No differences could be detected for protamine chloride and sulfate form of protamine. It is assumed that protamine displaces heparins from the binding sites of TFPI.(ABSTRACT TRUNCATED AT 250 WORDS)

Factor Xa Inhibitors↗
PubMed1993

Altered pharmacokinetic properties of a lipophilically derivatized low-molecular-weight heparin in rats.

A new generation of lipophilic heparins has been developed that show longer-lasting inhibitory effects on the coagulation system. We have studied the radiopharmacokinetics of a derivatized low-molecular-weight heparin (LMWH) with a residualizing lipophilic tyramine-deoxysorbitol label in comparison with conventional LMWH after intravenous application into Wistar rats. Whole body scintigraphy and analysis of the blood and organ distribution of different tracer preparations revealed that the lipophilically derivatized LMWH substance was predominantly trapped in the liver RES by a scavenger receptor-mediated mechanism. After the saturable uptake mechanism was blocked by maleylated bovine serum albumin, 41.4% of the lipophilic LMWH tracer circulated in blood, as compared with 18.4% of the control and 1% of conventional LMWH. The same results were attained by a competition experiment with an excess of unfractionated heparin. Urinary excretion of the lipophilic tracer among the rats in this competition experiment was considerably lower (13.7%) as compared with conventional LMWH (53.0%). Experiments with lipophilic LMWH tracer bound nonspecifically to rat serum albumin confirmed that the prolonged half-life might in part be due to an increased affinity for albumin. About 59% of the activity of the lipophilic tracer bound to albumin was found in the liver reticuloendothelial system, and only 3.3% were excreted to urine 3 hours after injection. Further studies are necessary to evaluate the accumulation rates and the metabolic fate of lipophilically derivatized heparins in the case of an impeded reticuloendothelial system uptake before attempts are made to therapeutically apply these compounds.

Albumins↗
PubMed1993

Binding of a new monoclonal antibody against N-terminal heptapeptide of fibrin alpha-chain to fibrin polymerization site 'A': effects of fibrinogen and fibrinogen derivatives, and pretreatment of samples with NaSCN.

A novel murine monoclonal antibody against the fibrin alpha-chain N-terminus is presented, which reacts with desAA- and desAABB-fibrin. In immunoblot procedures, the antibody reacted with fibrin degradation products X and Y of non-crosslinked fibrin, and fragment E. No binding was observed to the fibrin fragment D-dimer, and fibrinogen fragments D and E. Minor binding to fibrinogen fragments X, and Y, and desBB-fibrin were presumably due to minor contamination with (desAA)-fibrin. A prerequisite for binding was release of fibrinopeptides A (FpA), the binding site being a fibrin-specific neo-epitope. No binding was observed to fibrinogen or to thrombin-treated dysfibrinogen MANNHEIM III (A alpha 16 Arg-->Cys) molecules, which do not release FpA. The antibody bound to abnormal fibrin molecules prepared from dysfibrinogen MANNHEIM I (A alpha 19 Arg-->Gly), albeit to a lesser extent than to normal fibrin. Binding of the antibody to the fibrin epitope was greatly enhanced by denaturation, e.g. by heat, or by treatment with chaotropic ions. Soluble fibrin in clinical samples is generally found as a complex with fibrinogen, since polymerization sites 'A' exposed by release of FpA react with complementary binding sites 'a' on the D-domains of other fibrin and fibrinogen molecules. Treatment of samples with NaSCN caused dissociation of fibrin monomer complexes. Reassociation was prevented by denaturation of both polymerization sites 'A' and 'a'. The antibody in combination with NaSCN-treatment of samples was useful for specific detection of fibrin monomer in plasma samples. Measurement was not influenced by fibrinogen degradation products, whereas fibrin degradation products at very high concentration caused some underestimation of fibrin monomer concentration.

Animals↗
PubMed1992

[Patent foramen ovale and platypnea after pneumonectomy].

Six months after a pneumonectomy for myeloma, which had preoperatively been indistinguishable from bronchial carcinoma, a 50-year-old man presented with shortage of breath, cyanosis and episodes of syncope on standing or walking, symptoms which improved on lying down (platypnea). On one occasion these symptoms necessitated controlled artificial ventilation, but even at an inspiratory oxygen saturation of 100%, blood gases only partially improved (pCO2 27 mm Hg, pO2 67 mm Hg, O2 saturation 93%). Right heart catheterization in recumbency revealed a right to left shunt at atrial level of 37% of systemic flow. Contrast medium injection into the inferior vena cava near the heart demonstrated cardiac displacement and rotation. Part of the inferior vena cava flow passed into the left atrium via a patent foramen ovale: it is likely that this shunt increased in the upright position. After surgical closure of the patent foramen ovale and partial relocation of the heart (with a vicryl net) the patient has now remained free of symptoms for 5 years.

Dyspnea↗
PubMed1992

Heparin Study in Internal Medicine (HESIM): design and preliminary results.

The Heparin Study in Internal Medicine (HESIM) compares the efficacy and safety of an unfractionated (UF) heparin with a low molecular weight (LMW) heparin (CY 216 D) for prevention of proximal deep vein thrombosis (DVT) and pulmonary embolism (PE) in medical inpatients with a high risk for development of thromboembolism. Patients are randomized and receive three times daily 5000 IU UF heparin or once daily 3100 IU LMW heparin and two placebo injections subcutaneously for 10 days. All patients are screened for the presence of proximal DVT at day 1 and 10 by real-time B-mode compression sonography and for PE by repeated clinical examinations. Perfusion scintigraphy is used for confirmation of the clinical diagnosis of PE. The study protocol includes a stratified randomization of patients on admission to the hospital according to one of the following main diagnoses: malignant disease, cardiovascular disease, bronchopulmonary disease, neurologic disease, and other diseases. The present study may serve as a model for further clinical trials in medical inpatients using the biometric approach of statistical analysis for proving equivalence of drug efficacy, and to adopt less sensitive but noninvasive methods for the detection of primary endpoints.

Aged↗
PubMed1992

Haemodilution in acute ischaemic stroke comparison of two haemodilution regimen.

In a prospective study we randomly allocated 50 patients with acute ischaemic stroke in the area of the middle cerebral artery within 12 hours after onset to two moderate hypervolemic haemodilution regimen consisting of 500 ml of 10% hydroxyethyl starch per day for 10 days. In the high haematocrit group the target haematocrit of 41-42% was achieved by 0-3 phlebotomies and additional replacement of that volume with the colloid in 3 days. In the low haematocrit group with 1-4 phlebotomies a target haematocrit of 37-38% was reached in 4 days. The groups did not differ regarding age, risk factors, haematocrit and neurological score. The improvement of the disturbed blood rheology was more pronounced in the low haematocrit group. One death occurred in each group. The neurological score showed a significantly greater increase in the low haematocrit group with +59% at day 5 and +125% at day 11; the data for the high haematocrit group were +34% and +89% respectively. We calculated a correlation (r = 0.36, p less than 0.02) between the rise in neurological score and the reduction of haematocrit. Our data suggest but not do prove that an early start on moderate hypervolemic haemodilution is beneficial in patients with acute ischaemic stroke and disturbed blood rheology.

Aged↗
PubMed1992

Hepatic uptake of a modified low molecular weight heparin in rats.

Fractionated and unfractionated heparins are widely used as antithrombotic agents. Because of their heterogeneous composition, it is difficult to study the pharmacokinetics of these drugs. We now report on a new method for labeling low molecular weight heparins with 131I by binding tyramine to the anhydromannose end of the molecules. We examined the pharmacokinetics of the compound by intravenous injection of 131I-tyramine-heparin into Wistar rats. About 18% of the activity was found in the liver, whereas 33% was detected in urine. Biological activity in terms of Factor Xa inhibition was measurable. Since evidence from cell culture experiments implies that reticuloendothelial cell system receptors might be involved in heparin metabolism, maleylated BSA, a substance known to block scavenger receptors, was injected before the radiolabeled heparin compound. The liver uptake was reduced from 17.4 to 4.8%. Injection of unfractionated heparin before tracer application caused a considerable increase in urine excretion of the tracer substance. To our knowledge, this is the first report that liver uptake of heparins is linked to scavenger receptor mediated mechanisms in vivo. This interaction of heparins with scavenger receptors might play an important role in the biology of the vessel wall.

Animals↗
PubMed1992

Microtiter assay for measurement of factor XIII activity in plasma.

A sensitive, reproducible and rapid quantitative microtiter assay for factor XIII activity is described. The assay is based on incorporation of a soluble amine substrate into casein attached to polystyrol microtiter plates and detection of bound substrate with a monoclonal antibody-enzyme conjugate. Defibrination of plasma samples is not required. The assay shows strong correlation with the immunologic assay for factor XIII catalytic subunit a (r = 0.94), the factor XIII dansylcadaverine assay (r = 0.83), and the factor XIII clot solubility test. Normal values detected in healthy blood donors were in the range of 74% to 117%, with a mean value of 96.9% and a median value of 97.8%; pooled normal plasma was used as a reference. Since the assay is performed in a single microtiter plate and requires few reagents and short incubation periods, it can be easily adapted for clinical use.

Caseins↗
PubMed1992

Risk factor patterns for coronary heart disease in China, Japan and Germany.

The risk factor patterns for coronary heart disease in China, Japan and Germany were studied. 6,025 Germans, 7,580 Japanese and 2,047 Chinese aged 30-59 were investigated following the protocol of the Göttingen Risk, Incidence, and Prevalence Study carried out in West Germany in 1982. It is concluded that in China, the risk factor intervention focuses mainly on smoking and hypertension; smoking also remains the most important risk factor in Japan; while in Germany the major targets are obesity and hyperlipidemia. However, about 38% of the participants from West Germany showed 3 or more risk factors accumulated per person. Thus the multifactorial risk factor reduction might be necessary in Germany.

Adult↗