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Biomedical subjects

D L Heene

Publications and source records attributed to D L Heene.

At least 55 records · Page 3Linked to original sources
PubMed1997

Pharmacokinetics of methotrexate-albumin conjugates in tumor-bearing rats.

Linking chemotherapeutic drugs to a macromolecular carrier system may enhance tumor targeting, reduce toxicity and overcome drug resistance mechanisms. As an elementary model to evaluate the pharmacological properties of macromolecular drug carrier systems we chose rat serum albumin (RSA) for carrier and methotrexate (MTX) as antineoplastic drug. The conjugation procedure yielded conjugates with an approximate 1:1 molar loading rate (MTX(1)-RSA). In the first part of the study a residualizing [111In]DTPA protein label was used for mapping in vivo the catabolic sites of the native carrier protein and of the MTX(1)-RSA drug conjugate in Walker 256 carcinosarcoma bearing rats. The tumor accumulation was about 14% of the injected dose for the RSA and MTX(1)-RSA tracers after 24 h. Tracer entrapment by organs with an active mononuclear phagocyte system was low (liver below 7% and spleen below 1.5% of the injected dose after 24 h). The 1:1 conjugation of MTX to RSA did not decisively alter the pharmacokinetic properties nor the tumor or tissue distribution of the native carrier protein RSA. In the second part of the study the different properties of the MTX(1)-RSA conjugate were compared with MTX in vivo. About 2 mg MTX/kg body weight either of the drug conjugate or of the original drug were injected after being additionally spiked with radiolabeled tracers. Plasma concentrations were simultaneously determined by immunological and radioactive means. After 24 h about 12% MTX(1)-RSA was found in circulation compared to 0.03% MTX. Favorable tumor accumulation rates of about 14% were achieved for MTX(1)-RSA versus 0.04% for MTX. About 45-fold more of the injected dose of [3H]MTX accumulated in the liver as compared to the tumor (1.5 versus 0.03%) after 24 h. Conjugation of MTX to RSA reversed this ratio in favor of the tumor to 1:1.4 (13.6 versus 9.6%). In conclusion, the potential therapeutic benefit of the MTX(1)-RSA conjugate lies in its very long tumor exposure time and its improved tumor accumulation rate compared to conventional MTX. In addition the conjugation to albumin might enhance the therapeutic effects over those achieved by long-term continuous infusion of MTX, as MTX(1)-RSA enters the cells by a different uptake mechanism. This might also help to circumvent MTX resistance mechanisms, such as a reduction in folate receptor numbers or impaired MTX polyglutamylation.

Animals↗
PubMed1997

Continuous measurement of hemodynamic alterations during pharmacologic cardiovascular stress using automated impedance cardiography.

The contribution of computerized impedance cardiography in monitoring and differentiating cardiovascular responses to pharmacologic stress after the administration of dipyridamole (group 1, n = 24) or dobutamine (group 2, n = 26) was investigated during stress echocardiography. Heart rate, stroke volume index, cardiac index and systemic vascular resistance index were evaluated continuously with an automated, computerized, signal-averaged impedance cardiography system. Dipyridamole had little average effect on heart rate, stroke volume index, and cardiac index. The responses were similar in patients with positive (n = 9) or negative (n = 15) stress echocardiography test results (as characterized by echocardiographic wall-motion abnormalities). Dobutamine induced a similar mean increase in heart rate in patients with negative (n = 13) or positive (n = 13) results on stress echocardiography. The mean increase in stroke volume index induced by dobutamine was greater in patients with negative stress echocardiography test results than in patients with stress-induced wall-motion abnormalities. This distinction was also seen in the cardiac index; the mean change in patients with negative stress echocardiography test results was larger than in patients with positive results. It is concluded that automated computerized impedance cardiography not only allows surveying and monitoring hemodynamic changes during pharmacologic stress echocardiography but also contributes to differentiation of pathologic stress responses.

Adult↗
PubMed1997

Fibrinogen heterogeneity in homozygous plasminogen deficiency type I: further evidence that plasmin is not involved in formation of LMW- and LMW'-fibrinogen.

Human plasma fibrinogen is heterogeneous in SDS-polyacrylamide gel electrophoresis and other methods for separation of proteins by molecular size. A high molecular weight fraction (HMW-fibrinogen, 340 kD) contributes approximately 50% of total fibrinogen antigen. Low molecular weight fibrinogen (LMW-fibrinogen, 300 kD) adds another 40%. The residual amount is LMW'-fibrinogen with a molecular weight of 280 kD, and a small amount of very high molecular weight fibrinogen (Fib420), the product of alternative splicing of the A alpha-chain genetic information, resulting in an extended A alpha-chain C-terminus. Fibrinogen was detected by specific immunostaining of nonreduced SDS-PAGE gel immunoblots with antibodies against fibrinopeptide A. Using densitometric scans of the immunoblots we found a ratio of HMW-, LMW- and LMW'-fibrinogen in a patient with homozygous plasminogen deficiency that was similar to the ratio found in immunoblots of plasma from healthy blood donors. Treatment with plasminogen concentrate resulted in a slight decrease of the proportion of HMW-fibrinogen, followed by an increase to 78%. The LMW'-fibrinogen band gained intensity initially, increasing to 11.9% of fibrinogen antigen 6 h after starting plasminogen infusion, but then dropped to levels below detection limit of the immunoblotting assay. LMW-fibrinogen remained constant during the initial 72 h of plasminogen treatment, then dropping to values in the range of 22-25% afterwards. The proportion of HMW-, LMW-, and LMW'-fibrinogen again reached the initial levels two weeks after starting treatment with plasminogen concentrate. We conclude that plasminogen is not involved in the limited proteolysis leading to formation of LMW-fibrinogen and LMW'-fibrinogen in the absence of a generalized fibrinolytic condition. Fibrinolytic activation may lead to the formation of fibrinogen degradation product X, which appears in a similar position as LMW'-fibrinogen in SDS-PAGE.

Alternative Splicing↗
PubMed1997

Fibrin detected in plasma of patients with disseminated intravascular coagulation by fibrin-specific antibodies consists primarily of high molecular weight factor XIIIa-crosslinked and plasmin-modified complexes partially containing fibrinopeptide A.

Pooled plasma from 40 patients with severe disseminated intravascular coagulation (DIC) secondary to septic conditions was subjected to gel permeation chromatography on Sephacryl S-500 HR after sample pretreatment with KSCN for dissociation of non-covalent fibrin complexes. Fibrin antigen in eluates was detected by an array of ELISA tests, using two monoclonal antibodies against fibrin degradation product D-dimer, a monoclonal antibody against an epitope generated by plasmin cleavage of the D-domain, and an antibody against the neo-N-terminus of the alpha-chain of fibrin exposed by cleavage of fibrinopeptide A. Tag antibodies were a polyclonal antibody against the fibrinogen/ fibrin D-domain, a POD-conjugated version of the monoclonal antibody against fibrin alpha-chain neo-N-terminus, and a polyclonal antibody against fibrinopeptide A. Most fibrin-related material present in the pooled DIC plasma was of higher molecular mass than fibrinogen. Fibrin polymers were reactive with antibodies against D-dimer, plasmin cleaved D-domain, and fibrin alpha-chain neo-N-terminus. Part of the polymers reacted with antibodies against fibrinopeptide A, indicating presence of fibrinogen or desA-fibrin monomer within the covalently linked complex. In conclusion, the primary analytes detected by monoclonal antibodies for D-dimer, plasmin-specific epitopes of fibrin degradation products, as well as sites exposed by fibrinopeptide cleavage in plasma from patients with disseminated intravascular coagulation are high molecular weight factor XIIIa-crosslinked fibrin complexes, containing plasmin-cleaved D-domains, intact fibrin monomer units, and fibrinogen or desA-fibrin monomer.

Antibodies, Monoclonal↗
PubMed1997

The loading rate determines tumor targeting properties of methotrexate-albumin conjugates in rats.

Albumin dominates the plasma proteins in man. Following our observation that albumin turnover in rodent tumors is markedly increased, we will present evidence that albumin can be employed as an efficient carrier for targeting cytostatic agents like methotrexate (MTX) into tumors. The considerable discrepancy in the molecular weight of MTX (454 Da) and albumin (about 67,000 Da) tempted researchers to load multiple drug molecules on one carrier molecule. It was supposed that the optimal therapeutic efficacy of MTX protein conjugates could be achieved by increasing the number of the molecules of MTX attached to the carrier. In this paper we will show that only loading rates of close to 1 mol of the cytostatic drug MTX/mol of albumin offer optimal conditions for targeting MTX-albumin conjugates into rodent tumors. Conjugates bearing 5, 7, 10 and 20 molecules of MTX on average showed considerable alterations in the HPLC profiles of the conjugates compared to albumin. Conjugates carrying 5-20 mol MTX, tagged with a residualizing radiolabel, were efficiently trapped by the liver before reaching the tumor. The tumor uptake rates of these conjugates declined dramatically with an increasing molecular load of the cytotoxic drug linked to albumin. Competition experiments with maleylated bovine serum albumin and fucoidan revealed that scavenger receptors present on the cells of the liver monocyte macrophage system were involved in this process. For further preclinical and clinical studies, we chose MTX-albumin conjugates, derivatized at a molar ratio of 1:1. These conjugates enjoy the same favorable tumor targeting properties like albumin, e.g. high tumor uptake rates, low liver uptake rates and a very long biological half-life.

Animals↗
PubMed1996

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.

Calibration↗
PubMed1996

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.

Binding Sites↗
PubMed1996

Effects of low-molecular-weight dermatan sulfate on coagulation, fibrinolysis and tissue factor pathway inhibitor in healthy volunteers.

Low-molecular-weight (LMW)-dermatan sulfate (Desmin) with the mean molecular weight of 5600 Da has been obtained by limited depolymerization of natural dermatan sulfate. The pharmacokinetic and pharmacodynamic data of 100 and 200 mg were analyzed after intravenous injection and of 50, 100 and 200 mg after subcutaneous injection on tissue factor pathway inhibitor (TFPI) antigen and activity, heparin cofactor (HC) II activity, HeptestTM coagulation value, chromogenic S-2222 anti-factor Xa (aXa) assay, activated partial thromboplastin time (APTT), thrombin clotting time (TCT), plasminogen, tissue plasminogen activator activity (t-PA) and plasminogen activator inhibitor (PAI). After i.v.injection of 100 mg and 200 mg Desmin TFPI antigen and activity increased 2.2- and 2.7-fold, and returned to normal values within 60 and 90 min, respectively. Using the HC II assay the elimination half-lives (T1/2 el) increased from 1.9 h to 3.3 h with increasing doses of LMW-dermatan sulfate. T1/2 el were 4.3 and 6.9 h with the Heptest assay and 3.3 and 5.1 with the aXa method, respectively. APTT, TCT and the fibrinolytic parameters were not modified by either dose of i.v. LMW-dermatan sulfate. After s.c. administration of 100 mg or 200 mg LMW-dermatan sulfate no increase of TFPI antigen or activity was detected. T1/2 el was 5.6 h using HC II method, 11.1 h using Heptest and 7.8 h with the aXa activity. The total clearance was about ten-fold higher when determined by the HC II method compared with Heptest and aXa method. The volume of distribution (VD) increased with increasing doses of s.c. LMW-dermatan sulfate and was highest with the HC II method. Intravenous administration of 100 mg protamine chloride 15 min after i.v. dosing of 100 mg LMW-dermatan sulfate did not modify TFPI, coagulation or fibrinolytic parameters. Further analysis of the complex mechanism of action has to include studies which should explain the low release of TFPI in relation to the antithrombotic effects of LMW-dermatan sulfate.

Anticoagulants↗
PubMed1996

Anticoagulant effects and tissue factor pathway inhibitor after intrapulmonary low-molecular-weight heparin.

The present study was designed to investigate the anticoagulant action of inhaled low-molecular-weight (LMW) heparin on the release of tissue factor pathway inhibitor (TFPI) and on antifactor Xa activity, Heptest, activated partial thromboplastin time (APTT) and thrombin clotting time (TCT) in healthy volunteers. 3000 IU (group 1), 9000 IU (group 2), 27,000 IU (group 3) or 54,000 IU (group 4) LMW-heparin were given to 20 healthy volunteers each at 4 weeks intervals by inhalation. For safety reasons a dose escalating design was chosen. APTT and TCT did not change after inhalation of any dose of LMW-heparin as well as all parameters in group 1. In group 2, Heptest coagulation times were prolonged from 18.7 +/- 2.0 s before to 26.1 +/- 5.2 s 6 h and 20.5 +/- 1.9 s 24 h after inhalation and the other parameters remained uneffected. In group 3, S2222 method and the protamine assay increased from 0.01 to about 0.1 IU/ml 6 h after inhalation and returned to normal values after 24 h. TFPI antigen increased from 74.1 +/- 13.9 to 80.5 +/- 14.2 ng/ml 3 h after inhalation. TFPI activity remained unchanged. Heptest coagulation values were prolonged to 42 +/- 7.6 s after 6 h and returned to normal within 72 h after inhalation. In group 4, the following changes were observed: antifactor Xa activity increased to 0.343 +/- 0.196 U/ml after 6 h and normalized after 72 h. The protamine assay detected 0.2 +/- 0.18 U LMWH/ml after 6 h, TFPI antigen increased to 103 +/- 17.9 ng/ml and TFPI activity to 1.14 +/- 0.23 U 3 h after inhalation. All tests were normal after 24 h. Heptest coagulation values increased to 77.5 +/- 11.8 s 6 h after inhalation and normalized after 144 h. The area under the activity time curve of the S2222 method and of the Heptest assay increased with increasing doses (r = 0.677 and r = 0.571), respectively. The calculated elimination half-life of the aXa-effect was 7.5 h using S2222-, Heptest- and protamine assays. The data demonstrate a resorption of LMW-heparin by intrapulmonary route in man. The dose to produce antifactor Xa levels, prolongations of Heptest coagulation values and in releasing TFPI is about ten-fold higher than after subcutaneous administration.

Administration, Inhalation↗
PubMed1996

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.

Animals↗
PubMed1996

[Value of dipyridamole-atropine stress echocardiography for detection of patients with restenosis after PTCA].

UNLABELLED: Stress-Echocardiography has been proven to be a valuable method in the diagnosis of patients with suspected coronary artery disease. It has been demonstrated that the addition of atropine can increase the sensitivity of pharmacological stress-echocardiography-tests. The aim of our study was to evaluate the diagnostic potential of dipyridamole-atropine-echocardiography for the detection of restenosis after coronary angioplasty. We investigated 50 patients 3-6 months after primary successful coronary angioplasty. Restenosis was defined as recurrence of stenosis of > or = 70% at the site of dilatation determined by quantitative coronary angiography. All patients were investigated on antianginal medication and underwent control-coronary angiography within 1-3 days after the echocardiographic study. In 17/23 patients with restenosis, the dipyridamole-atropine-stress-test was pathologic (sensitivity 74%), in 25/27 patients without significant restenosis the echocardiography stress-test was normal (specificity 93%). In 6 patients with restenosis and an unsuspicious stress-test, the percent diameter of restenosis did not exceed > or = 90% lumen narrowing, restenosis of the LAD was correctly identified in all patients (n = 11). The diagnostic accuracy of the dipyridamole-atropine-stress-test for the detection of patients with restenosis after PTCA in our study was 84%, the positive predictive value of a pathologic echocardiography stress-test for the detection of significant restenosis was 89%, the negative predictive value was 81%. CONCLUSIONS: Dipyridamole-atropine-echocardiography has diagnostic potential for non-invasive assessment of patients after coronary angioplasty. In patients with a normal echocardiographic stress-test, the probability of significant restenosis is considerably low, even if restenosis cannot be definitely excluded.

Aged↗
PubMed1995

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.

Animals↗
PubMed1995

Magnetic bead protamine-linked microtiter assay for detection of heparin using iodinated low-molecular-mass heparin-tyramine.

We have developed a competitive heparin binding assay employing protamine-coated magnetic beads for detection and measurement of heparin. The assay utilizes 125-iodine specifically bound to newly synthesized low-molecular-mass (LMM) heparin-tyramine. The tracer was stable over a period of 3 weeks, as demonstrated by gel filtration chromatography. The protamine-coated beads were found to be stable over at least two months. The heparin-tyramine bead assay had in buffer a lower detection limit of 0.04 microgram/ml and in plasma of 0.23 microgram heparin/ml. 50% binding was obtained at 0.7 microgram/ml and 20% binding at 4 micrograms/ml in plasma. The within assay coefficient of variation ranged from 9 to 28% for unfractionated, high molecular mass (HMM) heparin and from 12 to 15% for LMM-heparins in buffer system and in plasma. Various heparin fractions displaced the tracer from the protamine-coated magnetic beads to different extents. The validity of the assay was proven after intravenous administration of unfractionated and LMM-heparin in man. The elimination rate was similar using the heparin-tyramine bead assay compared with the anti-factor Xa coagulation assay. After intravenous dosing of LMM-heparin the maximal concentration was lower using the heparin-tyramine bead assay compared with the anti-factor Xa coagulation assay. The bead assay was found to be reproducible, valid, and rapid for measurement of the concentration of heparin preparations in purified systems and for HMM-heparin in plasma. Measurement of the concentration of LMM-heparin in plasma has a high coefficient of variation using the binding assay.

Binding, Competitive↗
PubMed1995

Complexes of a modified low-molecular-weight heparin with protamine are predominantly cleared by a macrophage scavenger receptor-mediated process in rats.

Neutralization of heparin with its antidote protamine is associated with side effects such as pulmonary hypertension. The pharmacodynamic effects of protamine treatment are well documented. However, little is known about metabolic fate of heparin-protamine complexes. Twenty Wistar rats received a 131I-labeled low-molecular-weight heparin tracer intravenously. Four groups of animals were formed: a control group receiving the tracer, a second group receiving protamine after tracer application, a third group receiving maleylated bovine serum albumin (mal-BSA) prior to tracer and protamine injections to inhibit scavenger receptors of the reticuloendothelial system, and the last group receiving preformed heparin-protamine aggregates. All animals were examined by scintigraphy. Blood and tissue samples were analyzed for radioactivity. Protamine injection 2 min after heparin tracer application lead to a rapid decline in blood radioactivity. The radioactivity in the liver increased from 17% for the control to 43% after protamine application. Injection of mal-BSA prior to protamine prevented tracer accumulation in the liver and increased urine excretion (34% versus 20%). In vitro preformed heparin-protamine precipitates were rapidly trapped in the liver. We present evidence that, like the polyanionic heparin, polyanionic heparin-protamine complexes are phagocytosed by a scavenger receptor-mediated mechanism by macrophages, predominantly in the liver. The amount, the size, and the charge density of the complexes might trigger a mediator release from macrophages leading to phenomena such as pulmonary hypertension.

Animals↗
PubMed1995

Tissue factor pathway inhibitor: proposed heparin recognition region.

After intravenous and subcutaneous injection, heparins and low-molecular-weight heparins release tissue factor pathway inhibitor (TFPI) into the blood stream. Protamine immediately reverses the plasma levels of TFPI to pretreatment values. A low-molecular-weight dermatan sulphate releases only very small amounts of TFPI after intravenous injection without a clear dose-dependent effect. Together with other data from literature, it seems likely that TFPI is rather specifically released by acidic glycosaminoglycans. In the present paper a binding region for heparins is reported to be located in the C-terminal end. A basic amino acid cluster occurs between 256-lysine and 261-lysine. This proposed heparin recognition region in TFPI is similar to the recognition region in antithrombin III and other proteins. The binding between the basic region at the C-terminal end of TFPI and the negatively charged sulphate or carboxyl-groups of glycosaminoclycans may occur in a linear manner. A comparison of a helical wheel diagram of antithrombin III and tissue factor pathway inhibitor support also the proposal of this form of a heparin recognition region in TFPI. Further studies are now required to analyse these interactions.

Amino Acid Sequence↗
PubMed1995

Monitoring of heparins in haemodialysis using an anti-factor-Xa-specific whole-blood clotting assay.

In haemodialysis low-molecular-weight (LMW) heparin is increasingly used for anticoagulation. The advantages over unfractionated (UF) heparin are the lower bleeding risk and the lack of influence on lipid metabolism. However, no reliable and rapid method is available so far to control the efficacy and safety of LMW heparin during haemodialysis. The specific anti-factor Xa (aXa) chromogenic substrate assays are laborious and time consuming. In the present study we have compared chromogenic assay with a coagulation assay (heptest), which was performed from plasma and whole-blood samples. The effects were compared with unfractionated heparin during haemodialysis. The aXa activity in the chromogenic S2222 assay ranged between 0.2 and 0.5 U/ml with UF heparin and between 0.4 and 0.8 U/ml with LMW heparin during haemodialysis. The coagulometric aXa activity in heptest assay from plasma was 0.6-1.0 U/ml with UF heparin and 1.2-2.0 U/ml with LMW heparin. The Heptest coagulation values from citrated whole blood samples ranged from 0.4 to 0.8 U/ml with UF heparin and from 0.8 to 1.4 U/ml with LMW heparin. The prolongation of the heptest clotting times with UF and LMW heparin was in the range of 4.4 for UF heparin and 5.2 for LMW heparin using the whole-blood assay. Heptest assay from plasma samples yielded prolongations of 6.1 with UF heparin and 6.6 with LMW heparin. The data show that the coagulation assay is rapid and reproducible using plasma or uncentrifuged whole-blood samples and is valid to monitor UF heparin or LMW heparin in patients undergoing chronic intermittent haemodialysis.

Adult↗
PubMed1995

Comparison of immunological and functional assays for measurement of soluble fibrin.

Various assays have been developed for quantitation of soluble fibrin or fibrin monomer in clinical plasma samples, since this parameter directly reflects in vivo thrombin action on fibrinogen. Using plasma samples from healthy blood donors, patients with cerebral ischemic insult, patients with septicemia, and patients with venous thrombosis, we compared two immunologic tests using monoclonal antibodies against fibrin-specific neo-epitopes, and two functional tests based on the cofactor activity of soluble fibrin complexes in tPA-induced plasminogen activation. Test A (Enzymun-Test FM) showed the best discriminating power among normal range and pathological samples. Test B (Fibrinostika soluble fibrin) clearly separated normal range from pathological samples, but failed to discriminate among samples from patients with low grade coagulation activation in septicemia, and massive activation in venous thrombosis. Functional test C (Fibrin monomer test Behring) displayed good discriminating power between normal and pathological range samples, and correlated with test A (r = 0.61), whereas assay D (Coa-Set Fibrin monomer) showed little discriminating power at values below 10 micrograms/ml and little correlation with other assays. Standardization of assays will require further characterization of analytes detected.

Antibodies, Monoclonal↗