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.

Phase I trial of methotrexate-albumin in a weekly intravenous bolus regimen in cancer patients. Phase I Study Group of the Association for Medical Oncology of the German Cancer Society.

Methotrexate-albumin conjugate (MTX-HSA) is a novel human albumin-based prodrug conjugate of methotrexate (MTX). A low MTX loading rate provided optimal tumor targeting and therapeutic efficacy during preclinical testing. The objectives of this first Phase I study of MTX-HSA were to determine dose-limiting toxicity (DLT) and maximum tolerated dose (MTD) in a weekly regimen. Seventeen cancer patients who were no longer amenable to standard treatment were enrolled and were evaluable for DLT. Up to eight injections were performed in weekly intervals. Dose escalation was as follows: 20, 40, 50, and then 60 mg/m2 MTX-HSA (based on the amount of MTX bound to albumin). Additional MTX-HSA courses were feasible in case of tumor response. DLT (mainly stomatitis, Common Toxicity Criteria grade 3) occurred, beginning at the 50 mg/m2 dose level after repeated administrations; in one case, thrombocytopenia was dose-limiting. Two events of DLT occurred at the 60 mg/m2 dose level within the first two administrations. Mild anemia, transaminitis, and one case of skin toxicity were found. No significant leukopenia, nausea, renal toxicity, or other toxicities were observed. MTX-HSA was well tolerated. Drug accumulation occurred on the weekly schedule. The half-life of the drug was estimated to be up to 3 weeks. Tumor responses were seen in three patients: (a) a partial response was seen in one patient with renal cell carcinoma (response duration, 30 months, ongoing); (b) a minor response was seen in one patient with pleural mesothelioma (response duration, 31 months, ongoing); and (c) a minor response was seen in one patient with renal cell carcinoma (response duration, 14 months until progression). Poststudy treatment was administered at 2-4-week intervals. No signs of toxicity or drug accumulation were seen. Altered pharmacological properties of MTX-HSA such as plasma half-life, tumor targeting, or intracellular metabolism might have contributed to these responses. The MTD for weekly administration was 4 x 50 mg/m2 MTX-HSA during short-term treatment. A regimen with MTX-HSA injections of 50 mg/m2 every 2 weeks was recommended for a further clinical Phase I study.

Separation of fibrinogen degradation products (FDP) by means of adsorption to insolubilized fibrinmonomer (FM-ag).

The phenomenon of complex formation between fibrinmonomer and fibrinogen degradation products was investigated by means of adsorption of FDP to insolubilized thrombin-modified fibrinogen (FM-ag). Since it could be demonstrated that there are different adsorption characteristics for early FDP and late FDP, the possibility of separation of FDP by means of affinity chromatography on FM-ag columns was evaluated using plasmic digests of 3H-Ac-labelled fibrinogen. The identification of FDP was performed by disc-electrophoresis. The results indicate that the adsorption of early FDP is comparable to the behaviour of fibrinogen, whereas late FDP show essential difference in the affinity towards FM-ag, evident by the result that fragment E adsorbs only to a minimal extents. Fragments D and E derived from fibrinogen as well as from non-crosslinked fibrin, revealed identical adsorption characteristics. Under specified conditions the procedure is suitable as a preparative method for the separation of fragments D and E.

A 96-well microfiltration assay for measurement of total clottable fibrinogen.

In clinical routine use, fibrinogen is measured by clotting-time methods, or by clot turbidity in photometric prothrombin time determination. For calibration of these assays measurement of total thrombin-clottable protein has been recommended. We have now developed a microfiltration assay for total thrombin-clottable protein. Plasma samples were mixed with thrombin in a 96-well microfiltration device. After clot formation, the fluid was extracted by vacuum suction, and fibrin adherent to the filter membranes washed with buffer. Membrane segments with adherent fibrin were recovered from the 96-well manifold with a punch and transferred to tubes containing denaturing buffer solution. After dissolution of fibrin, protein concentration was determined by optical absorption at 280 nm. The microfiltration assay displayed a high correlation with the total clottable protein method (R = 0.95), and fibrinogen antigen (r = 0.96). Correlation with clotting time assays, and PT-derived fibrinogen in 150 clinical plasma samples was in the range of r = 0.84 to r = 0.97. Intraassay and day-to-day variability of the assay was comparable to the conventional total clottable fibrinogen assay. The novel microfiltration assay appears to be well suited for measurement of large series of samples for calibration, screening purposes, and clinical trials.

The fibrin assay comparison trial (FACT): correlation of soluble fibrin assays with D-dimer.

Soluble fibrin (SF) is regarded as an indicator of acute fibrin formation and a precursor of fibrin thrombi. Using a set of clinical plasma samples, and fibrin derivatives, five assays for measurement of SF, including two chromogenic assays, two ELISA systems, and one latex-enhanced photometric immunoassay were compared. Correlation between SF assays was moderate (Spearman's rho values between 0.344 and 0.805). Re-calibration with serial dilutions of desAABB-fibrin monomer resulted in adjustment of the numerical scale of the assays without improving correlation. All SF assays reacted with purified crosslinked fibrin derivatives. Using clinical plasma samples, Spearman's rho of SF assays with D-dimer consensus values based upon results of 23 quantitative D-dimer assays were between 0.491 and 0.911. Although all SF assays react with desAABB-fibrin monomer complexes, SF assays are heterogeneous concerning reactivity with fibrin compounds observed in clinical plasma samples. The prospect of a common calibrator for SF assays therefore seems to be remote. Since SF assays react with crosslinked fibrin derivatives, it is not possible to clearly distinguish between acute fibrin formation, and fibrin dissolution on the basis of the results of current SF assays.

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.

The Fibrin Assay Comparison Trial (FACT): evaluation of 23 quantitative D-dimer assays as basis for the development of D-dimer calibrators. FACT study group.

Although D-dimer has gained widespread clinical use as a parameter for detection of in vivo fibrin formation, the issue of standardization of D-dimer assays remains to be resolved. The FACT study was performed to generate basic data for development of calibrators and standard preparations. A set of 86 samples, including plasma samples from patients with DIC, DVT. and other clinical conditions, serial dilutions of pooled plasma samples, and plasma samples containing fibrinogen- and fibrin derivatives, were distributed to 12 manufacturers of D-dimer assays. D-dimer assays differ concerning specificity for crosslinked fibrin, and preference for either high molecular weight fibrin complexes, or low molecular weight fibrin degradation products. Terminal plasmin digests of fibrin clots for calibration produce aberrant results in some assays, especially those with preference for high molecular weight crosslinked fibrin derivatives. The best conformity is achieved by the use of pooled plasma samples from patients with high levels of D-dimer antigen in plasma. In vitro preparations containing a comparable composition of fibrin derivatives to clinical plasma samples may also serve as reference material.

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)

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.

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.

Monitoring of heparin and low molecular weight heparin with capillary and venous whole blood.

A method for determination of antifactor Xa-like activity in capillary whole blood obtained from the fingertip is described, which employs the Heptest coagulation assay. Values obtained with capillary whole blood are compared to values of corresponding plasma and venous whole blood samples. Normal values in plasma, venous whole blood, and capillary blood from the fingertip were 17.1 +/- 2.1, 10.0 +/- 1.3 and 10.4 +/- 1.3 sec, respectively. The intraindividual coefficients of variations range from 0.4 to 1.8% in all assays. The day to day coefficient of variation of normal values ranged between 0.8 and 2.0% for all assays. The within assay coefficients of variation ranged from 3.0 to 7.7% for whole blood samples and from 1.5 to 2.2% for plasma samples after addition of no, 0.2 or 1.0 units of normal or LMW heparin to the samples. After administration of heparin or LMW heparin in healthy persons the coagulation values of the different coagulation assay systems displayed coefficients of correlation between r = 0.87 and r = 0.95. Correlation coefficients between the coagulation tests and the S 2222 chromogenic substrate method ranged from r = 0.77 to r = 0.92. In patients, who received LMW heparin for prophylaxis of thromboembolism the coagulation assay correlated between r = 0.78 and 0.92. The coagulation assays and the S 2222 method displayed coefficients of correlation between r = 0.74 and r = 0.83. The data indicate that Heptest sensitively measures antifactor Xa-like activity in capillary whole blood as well as venous whole blood samples containing low quantities of heparin or LMW heparin.

Biological activity and safety of the subcutaneous administration of high doses of low molecular weight heparin for 8 days in human volunteers.

This study reports on the biological activity and safety of high dose low molecular weight (LMW) heparin therapy administered by two subcutaneous (s.c.) injections daily for 8 days in healthy human volunteers. Group 1 received 2 x 30 aPTT units LMW heparin/kg bodyweight, and group 2 received 2 x 50 aPTT units/kg per day. In group 1, activated partial thromboplastin time (aPTT) and thrombin clotting time (TCT) were uniformly prolonged by 3-5 sec 4 hrs after s.c. administration of heparin. Heptest coagulation time values were prolonged consistently as well by 57 sec on day 1 to 68 sec on day 8. Factor Xa inhibition measured by the S 2222 chromogenic substrate method continuously increased from 0.16 units/ml on day 1 to 0.28 units/ml on day 8. In group 2 prolongation of a aPTT and TCT values increased from 6 sec on day 1 to 15 sec on day 8 and of Heptest time from 70 sec on day 1 to 110 sec on day 8. S 2222 method showed factor Xa inhibitory activity which increased from 0.5 units/ml on day 1 to 0.75 units/ml on day 8. The clinical tolerance of the treatment was good. No changes in clinical chemistry parameters were detected, except for a reversible increase of serum transaminases. The coagulation studies demonstrate accumulation of LMW heparin when high doses are given twice daily. The half life of LMW heparin of factor Xa inhibition increases with increasing doses. Heptest coagulation values were prolonged to 4-6 times the normal values during administration of heparin.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on chemically modified fibrinogen.

Treatment of fibrinogen with maleic acid anhydride renders fibrinogen unclottable depending on the degree of modification of the molecule. According to radioactive studies the release of fibrinopeptides by thrombin or reptilase is undisturbed. The incoagulability is due to inhibition of the polymerization process of fibrinmonomers derived from modified fibronogen, mainly caused by the increase of electronegative charges upon the fibrogen molecule. According to discelectrophoretic analysis modified fibrinogen fails to produce fragments D and E following plasmic digestion, however, may be degraded to high molecular weight products. Modified fibrinogen reveals some similarities to abnormal fibrinogens in congenital dysfibrinogenemia with regard to its functional properties.

Impaired fibrinolytic capacity and tissue plasminogen activator release in patients with restenosis after percutaneous transluminal coronary angioplasty (PTCA).

To assess the role of the fibrinolytic system in the pathogenesis of restenosis after percutaneous transluminal coronary angioplasty (PTCA), we determined the components of this system in a retrospective study, including 16 patients with restenosis (gr. A) and 19 patients with long-term success (gr. B). In both groups at baseline fibrinolytic activity (FA) is unchanged, whereas tissue plasminogen activator antigen (tPA-Ag) is significantly increased (gr. A: 147.0%; gr. B: 139.8%; p less than 0.01). Fibrinolytic capacity (FC) and tPA-Ag release are significantly reduced in the restenosis group (FC: 46.5%, p less than 0.05; tPA-Ag release: 48.3%, p less than 0.01) compared to normal controls as well as to gr. B (FC: 84.3%, p less than 0.05; tPA-Ag release: 79.0%, p less than 0.05). Relating to the contact activation system, F XII (79.5%, p less than 0.05) is significantly, and F XI (82.3%) is clearly reduced in gr. A. Protein C (PC) is significantly elevated in gr. B (117.5%, p less than 0.05). There is a negative correlation between plasminogen activator inhibitor (PAI 1) and HDL-cholesterol (r = 0.37, p less than 0.05). It appears, that there is a typical pattern of defective fibrinolysis in patients with restenosis after PTCA and that this might be a pathogenetic factor in the development of restenosis.

Evaluation of systolic performance by automated impedance cardiography.

Impedance cardiography (ICG) is a noninvasive method for evaluating cardiac function. Left ventricular stroke volume (SV) is the basic hemodynamic parameter derived from thoracic bioimpedance curves. Issues of our study were to investigate the diagnostic value of other indices of left ventricular systolic performance, such as ejection fraction (EF), index of contractility (IC), peak flow index (PFI), and acceleration index (ACI), which can also be calculated by ICG. Forty patients (PTS) with suspected coronary artery disease (CAD) were monitored by automated ICG during pharmacologic stress testing with dobutamine. All PTS underwent subsequent cardiac catheterization. In PTS with single vessel disease, the dobutamine-induced changes of SV, EF, IC, PFI, and ACI were comparable to those of PTS without CAD. In PTS with multivessel disease, the impaired systolic performance during dobutamine stimulation could be clearly demonstrated. We conclude that automated ICG is a useful method for monitoring SV and other indices of left ventricular systolic performance for detecting PTS with ischemic left ventricular dysfunction during cardiovascular stress.

Fibrin formation and proteolysis during ancrod treatment. Evidence for des-A-profibrin formation and thrombin independent factor XIII activity.

Ancrod is a purified fraction of venom from the Malayan pit viper Calloselasma rhodostoma, containing a serine protease that cleaves fibrinopeptides A from fibrinogen. We report on a study that involved intravenous and subcutaneous application of ancrod in healthy subjects in which it was shown that ancrod induces the formation of desAA-fibrin complexes that are partially crosslinked by factor XIII proenzyme, and act as cofactor in tPA induced plasminogen activation. The plasmin generated degrades fibrin, as well as fibrinogen, leading to the appearance of large amounts of fibrinogen and fibrin degradation products in the circulation, including fragment D-dimer. At low concentrations of ancrod, formation of desAA-fibrin is preceded by production of desA-profibrin, lacking only one fibrinopeptide A.

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.

Isolation and purification of fibrinogen/fibrin degradation products by chromatography on protamine-agarose.

Protamine-agarose chromatography is introduced as a rapid and efficient method for the isolation of fibrinogen/fibrin degradation products from biological fluids such as human plasma. All fibrinogen/fibrin fragments above a MW of 35000, including fragments D and E are quantitatively adsorbed to insolubilized protamine and can be eluted with a buffer containing 0.2 M sodium citrate/citric acid pH 5.3, following previous elution of non-fibrinogen proteins with a buffer containing 0.8 M NaCl at neutral pH. Fragments D and E are separated by stepwise elution. The efficiency of the method is - evaluated by applying it to plasma samples obtained from healthy donors and from patients with clinical and laboratory evidence of disseminated intravascular coagulation.

Purification of human plasma fibrinogen by chromatography on protamine-agarose.

A study was performed to investigate if protamine covalently attached to CNBr-activated agarose is a useful tool for the purification of fibrinogen from human plasma samples. One chromatography step yielded a preparation with a clottability greater than 90% from platelet-poor plasma with a yield of 65-80% of fibrinogen applied. The preparation is free of plasminogen, immunoglobulins, fibronectin, albumin, antithrombin III, alpha 2-macroglobulin, and alpha 1-antitrypsin, as determined by immunological and functional methods. 6.5 mg of fibrinogen were adsorbed to 1.0 ml of protamine-agarose. Protamine-agarose chromatography can be applied to plasma samples as small as 200 microliters.

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.