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H J Ehrlich

Publications and source records attributed to H J Ehrlich.

At least 19 recordsLinked to original sources
PubMed2007

Evaluation of pharmacokinetics, efficacy and safety of Immunate solvent detergent in previously treated patients with severe haemophilia A.

Immunate Solvent Detergent (S/D) is a plasma derived, purified, human factor VIII (FVIII) - von Willebrand factor (VWF) complex subjected to two virus inactivation/removal processes: S/D and vapor heat treatment. This prospective, multicentre, three-part clinical study evaluated the pharmacokinetics (in comparison to the predecessor product Immunate), efficacy and safety of Immunate S/D in 56 previously treated patients with severe haemophilia A. Subjects received Immunate S/D on-demand, as a prophylactic regimen or both. The results of the pharmacokinetic population demonstrate that Immunate and Immunate S/D were equivalent with respect to the FVIII - and to the retrospectively VWF - parameters assessed. A total of 623 bleeding episodes were reported in 47/56 subjects. The duration of prophylaxis ranged from 0.1-5.2 months with a total of 175.6 months. The median number of bleeds per month in subjects on prophylaxis was 0 (range 0-10). Ninety-six percent of bleeding episodes were rated as having an excellent or good response. For most bleeding episodes (89%), subjects required only one infusion with a mean dose of 29.6 IU kg(-1). No FVIII inhibitory antibodies were observed in any subject. No related serious adverse events were reported. Thus, the introduction of S/D treatment did not alter the PK characteristics and function of VWF and FVIII molecules of Immunate S/D which is effective and safe for treatment of bleeding episodes, management of surgical procedures, and prophylaxis.

Detergents↗
PubMed2006

Safety and immunogenicity of the modified adult tick-borne encephalitis vaccine FSME-IMMUN: results of two large phase 3 clinical studies.

A prospective, randomised, multicentre, single-blind phase 3 study was performed to assess the safety of a vaccination schedule consisting of two vaccinations (21-35 days apart) with the tick-borne encephalitis (TBE) vaccine FSME-IMMUN "adults" (five consecutive lots) in comparison to another licensed TBE vaccine (Encepur), with polygeline) (two lots) in healthy volunteers (n=3966) aged 16-65 years. The safety of the third vaccination with FSME-IMMUN "adults" (6 months after the first vaccination) was investigated in a follow-up study on the same population (n=3705) and TBE antibody titres were analysed pre- and post-vaccination in a subgroup of volunteers (n=564). Following the first vaccination, the overall incidence of fever (> or =38.0 degrees C) was 0.8% in the FSME-IMMUN "adults" study group and 5.6% in the comparator study group; fever was mainly mild. The fever rate after the second vaccination was 0.6% and 0.5% in the two study groups, respectively. Local and systemic reactions after the first vaccination occurred with a lower frequency in the FSME-IMMUN "adults" study group than in the comparator group. Upon analysing the tolerability of the third vaccination with FSME-IMMUN "adults", similar results were determined in both study groups of volunteers previously vaccinated with FSME-IMMUN "adults" or with the comparator vaccine. The immunogenicity results demonstrated similar seroconversion rates (as determined by ELISA or neutralization test) before and after the third vaccination in the FSME-IMMUN "adults" group and in the comparator group respectively. The results of both studies demonstrate that: (1) FSME-IMMUN "adults" is safe and highly immunogenic, (2) all five production lots of FSME-IMMUN "adults" were consistent with respect to a low rate of adverse events, (3) FSME-IMMUN "adults" induces considerably lower adverse reaction rates than the comparator vaccine after the first vaccination, and (4) two vaccinations with the comparator vaccine can be successfully followed by a third vaccination with FSME-IMMUN "adults".

Adolescent↗
PubMed2006

Prospective open-label study of pharmacokinetics, efficacy and safety of a new 10% liquid intravenous immunoglobulin in patients with hypo- or agammaglobulinemia.

BACKGROUND AND OBJECTIVES: The aim of this study was to evaluate the pharmacokinetics, efficacy and safety of a newly developed 10% liquid immunoglobulin preparation in patients with primary immunodeficiency diseases. This new preparation for intravenous use includes three dedicated virus clearance steps in its manufacturing process to ensure a high margin of viral safety. MATERIALS AND METHODS: This was a prospective, open-label, non-controlled, multicentre study. Twenty-two subjects with primary immunodeficiency were treated initially with three infusions of a licensed intravenous immunoglobulin to standardize the immunoglobulin G (IgG) replacement therapy of all subjects to the same intravenous product. A total of nine infusions of the new 10% liquid preparation were subsequently administered. RESULTS: The median terminal half-life of total IgG following administration of the new preparation was 30.1 days. Median terminal half-lives for IgG subclasses IgG(1), IgG(2), IgG(3) and IgG(4) were 28.3, 31.3, 20.9 and 24.2 days, respectively. The median total serum IgG steady-state trough level was 8.51 g/l. No severe infection episodes started after initiation of treatment with the new preparation. The median rate of mild or moderate infection episodes was 0.48 per month. A total of 194 infusions with the new 10% liquid immunoglobulin preparation were administered. The mean dose per infusion was 0.41 g/kg body weight and the maximum infusion rates recorded were 8 ml/kg/h. Adverse experiences were mostly mild and unrelated to the study drugs. Only 4% of infusions with the new product were followed by one or more related adverse experiences. CONCLUSION: The new 10% liquid immunoglobulin preparation was well tolerated and shown to have an excellent pharmacokinetic, efficacy and safety profile. The liquid formulation provides convenience to patients and healthcare professionals.

Adult↗
PubMed2002

Safety of factor VIII inhibitor bypass activity (FEIBA): 10-year compilation of thrombotic adverse events.

Published and unpublished spontaneously reported thrombotic adverse events (AEs) in factor VIII inhibitor bypass activity (FEIBA(R)) recipients were compiled for the most recent 10-year period during which FEIBA(R) units equivalent to 3.95 x 105 typical infusions were distributed worldwide. A total of 16 thrombotic AEs were documented over the 10-year period, corresponding to an incidence of 4.05 per 105 infusions (95% CI, 2.32-6.58 per 105 infusions). Disseminated intravascular coagulation (n=7) and myocardial infarction (n=5) were the most frequent thrombotic AEs. One fatality occurred in an 87-year-old metastatic cancer patient. In 13/16 (81%) patients known risk factors were present, most commonly FEIBA(R) overdose in 8/16 (50%), obesity in 3/16 (19%) and serum lipid abnormalities in 2/16 (12%). These findings indicate that thrombotic AEs in FEIBA(R) recipients are very rare. Recognition of risk factors and avoidance of FEIBA(R) overdosage may avert thrombotic AEs.

Adult↗
PubMed1992

Mapping of binding sites for heparin, plasminogen activator inhibitor-1, and plasminogen to vitronectin's heparin-binding region reveals a novel vitronectin-dependent feedback mechanism for the control of plasmin formation.

Vitronectin (VN) has been implicated as a major matrix-associated regulator component of plasminogen activation by serving as a potent stabilizing cofactor of plasminogen activator inhibitor-1 (PAI-1). The direct binding of heparin, plasminogen as well as PAI-1 in its latent and active form to immobilized VN was studied in the absence or presence of competitors. Monoclonal antibodies against the carboxyl-terminal portion of VN inhibited both PAI-1 and plasminogen binding, whereas heparin, heparan sulfate with a high degree of sulfation, or dextran sulfate interfered with PAI-1 binding (KD = 20 nM) only. Utilizing synthetic peptides encompassing overlapping sequences of the heparin-binding domain of VN, adjacent heparin and PAI-1-binding sites were localized within the sequence 348-370 of VN. Although a number of other serine protease inhibitors which do not form binary complexes with VN contain a reactive-site Ser at their P1'-position, a reactive-site P1' mutant of PAI-1 (Met----Ser) showed comparable if not increased binding to VN. Binding of Lys-plasminogen and active-site-blocked plasmin was at least 10-fold higher in affinity (KD = 85-100 nM) compared to Glu-plasminogen (KD approximately 1 microM) and could be inhibited by lysine analogs but not by glycosaminoglycans or PAI-1, indicating that heteropolar plasmin(ogen) binding of VN occurs to an adjacent segment upstream to the heparin and PAI-1-binding sites. This contention was further supported in binding studies with plasmin-modified VN which lost both heparin and PAI-1 binding but exhibited 2-3-fold higher capacity to bind plasminogen. The essential plasmin(ogen)-binding site was mapped by ligand blot analysis to the carboxyl-terminal portion of proteolytically trimmed VN (M(r) = 61,000). Moreover, treatment of the extracellular matrix of human umbilical vein endothelial cells with plasmin resulted in partial degradation of matrix-associated VN and concomitant release of PAI-1, but increased the ability of the matrix by about 2-fold to bind plasminogen. These results are indicative of differential interactions of VN with components of the plasminogen activation system, whereby plasmin itself may provoke the switch of VN from an anti-fibrinolytic into a pro-fibrinolytic cofactor. This process reflects a novel role for the adhesive protein and its degradation product(s) in the possible feedback regulation of localized plasmin formation at extracellular sites.

Amino Acid Sequence↗
PubMed1992

Elucidation of structural requirements on plasminogen activator inhibitor 1 for binding to heparin.

Plasminogen activator inhibitor 1 (PAI-1), a member of the serpin superfamily of proteins, has been demonstrated previously to interact functionally with the glycosaminoglycan heparin (Ehrlich, H.J., Keijer, J., Preissner, K. T., Klein Gebbink, R., and Pannekoek, H. (1991) Biochemistry 30, 1021-1028). Heparin specifically enhances the rate of association between PAI-1 and thrombin about 2 orders of magnitude, whereas no effect is detected with other serine proteases (e.g. factor Xa). For the heparin-dependent serpins antithrombin III and heparin cofactor II, basic amino acid residues in and around the helix D subdomain were proposed to be involved in the binding of glycosaminoglycans. Here we employed site-directed mutagenesis of full-length PAI-1 cDNA to identify the amino acid residues that mediate heparin binding. To that end, 15 single-point mutants of PAI-1, each having individual arginyl, lysyl, or histidyl residues replaced by a neutral (alanyl) residue ("ala-scan"), and one double mutant were constructed, expressed in Escherichia coli, and purified to apparent homogeneity. The purified biologically active proteins were subjected to the following analyses: (i) heparin-dependent inhibition of thrombin; (ii) heparin-dependent formation of sodium dodecyl sulfate-stable complexes with thrombin; and (iii) binding to and elution from heparin-Sepharose. Based on the data presented, we propose that the amino acid residues Lys65, Lys69, Arg76, Lys80, and Lys88 constitute major determinants for heparin binding of PAI-1. These residues are located in and around the helix D domain and are conserved in the other heparin-dependent thrombin inhibitors, antithrombin III and heparin cofactor II.

Base Sequence↗
PubMed1991

On the target specificity of plasminogen activator inhibitor 1: the role of heparin, vitronectin, and the reactive site.

Plasminogen activator inhibitor 1 (PAI-1) is the fast-acting inhibitor of both tissue-type and urokinase-type plasminogen activators (t-PA, u-PA) and is an essential regulatory protein of the fibrinolytic system. In the presence of either the protein vitronectin or the glycosaminoglycan heparin, PAI-1 is also an efficient inhibitor of thrombin. To assess whether these cofactors turn PAI-1 into a general protease inhibitor or whether their influence is restricted to thrombin, the second-order association rate constants between PAI-1 and the human plasma proteases t-PA, u-PA, plasmin, thrombin, Factor Xa (FXa), and Factor XIIa (FXIIa) in the absence and in the presence of either vitronectin or heparin are determined. In addition, the role of the PAI-1 reactive site P3 to P3' residues for the specificity of inhibition was studied by using PAI-1 reactive site mutants. Our results show that: (1) Heparin exclusively increases the rate of inhibition of thrombin by PAI-1, whereas in the presence of heparin the rate of inhibition of the other proteases is not altered; (2) Vitronectin is an obligatory cofactor for the inhibition of thrombin by PAI-1. In addition, vitronectin moderately increases the rate of inhibition by PAI-1 of u-PA and of plasmin, but does not alter the rate of inhibition of t-PA, FXa, or FXIIa; (3) Apart from the important role of the P1 residue, no consensus can be presented on the nature of other residues within the P3 to P3' region with regard to target protease specificity.

Antithrombin III↗
PubMed1991

The interaction of plasminogen activator inhibitor 1 with plasminogen activators (tissue-type and urokinase-type) and fibrin: localization of interaction sites and physiologic relevance.

Plasminogen activator inhibitor 1 (PAI-1), an essential regulatory protein of the fibrinolytic system, harbors interaction sites for plasminogen activators (tissue-type [t-PA] and urokinase-type [u-PA]) and for fibrin. In this study, anti-PAI-1 monoclonal antibodies (MoAbs) were used to identify interaction sites of PAI-1 with these components. The binding sites of 18 different MoAbs were established and are located on five distinct "linear" areas of PAI-1. MoAbs, binding to two distinct areas of PAI-1, are able to prevent the inhibition of t-PA by PAI-1. In addition, two interaction sites for fibrin were identified on PAI-1. The area located between amino acids 110 and 145 of PAI-1 contains a binding site for both components and its significance is discussed in the context of the t-PA inhibition by fibrin-bound PAI-1. Subsequently, the MoAbs were used to assess the role of platelet-PAI-1 in clot-lysis. An in vitro clot-lysis system was used to demonstrate that clot-lysis resistance is dependent on the presence of activated platelets and that PAI-1 is a major determinant for lysis-resistance. We propose that, upon activation of platelets, PAI-1 is fixed within the clot by binding to fibrin and retains its full capacity to inhibit t-PA and u-PA.

Antibodies, Monoclonal↗
PubMed1991

Vitronectin governs the interaction between plasminogen activator inhibitor 1 and tissue-type plasminogen activator.

The "serpin" plasminogen activator inhibitor 1 (PAI-1) is the fast acting inhibitor of plasminogen activators (tissue-type (t-PA) and urokinase type-PA) and is an essential regulatory protein of the fibrinolytic system. Its P1-P1' reactive center (R346 M347) acts as a "bait" for tight binding to t-PA/urokinase-type PA. In vivo, PAI-1 is encountered in complex with vitronectin, an interaction known to stabilize its activity but not to affect the second-order association rate constant (k1) between PAI-1 and t-PA. Nevertheless, by using PAI-1 reactive site variants (R346M, M347S, and R346M M347S), we show that the binding of vitronectin to the PAI-1 mutant proteins improves plasminogen activator inhibition. In the absence of vitronectin the PAI-1 R346M mutants are virtually inactive toward t-PA (k1 less than 1 x 10(3) M-1 s-1). In contrast, in the presence of vitronectin the rate of association increases about 1,000-fold (k1 of 6-8 x 10(5) M-1 s-1). This inhibition coincides with the formation of serpin-typical, sodium dodecyl sulfide-stable t-PA.PAI-1 R346M (R346M M347S) complexes. As evidenced by amino acid sequence analysis, the newly created M346-M/S347 peptide bond is susceptible to attack by t-PA, similar to the wild-type R346-M347 peptide bond, indicating that in the presence of vitronectin M346 functions as an efficient P1 residue. In addition, we show that the inhibition of t-PA and urokinase-type PA by PAI-1 mutant proteins is accelerated by the presence of the nonprotease A chains of the plasminogen activators.

Base Sequence↗
PubMed1991

Functional interaction of plasminogen activator inhibitor type 1 (PAI-1) and heparin.

Plasminogen activator inhibitor type 1 (PAI-1), the fast-acting inhibitor of tissue-type plasminogen activator (t-PA) and urokinase (u-PA), is a member of the serpin superfamily of proteins. Both in plasma and in the growth substratum of cultured endothelial cells, PAI-1 is associated with its binding protein vitronectin, resulting in a stabilization of active PAI-1. Recently, it has been demonstrated that the PAI-1-binding site on vitronectin is adjacent to a heparin-binding site (Preissner et al., 1990). Furthermore, it can be deduced that the amino acid residues, proposed to mediate heparin binding in the serpins antithrombin III and heparin cofactor II, are conserved in PAI-1. Consequently, here we have investigated whether PAI-1 also interacts with heparin. At pH 7.4, PAI-1 quantitatively binds to heparin-Sepharose and can be eluted with increasing [NaCl]. Binding of PAI-1 to heparin-Sepharose can be efficiently competed with heparin in solution (IC50, 7 microM). In the presence of heparin, the protease specificity of PAI-1 toward thrombin is substantially increased. This is shown by (i) quenching of thrombin activity of PAI-1 in the presence of heparin and (ii) induction of the formation of SDS-stable complexes between thrombin and PAI-1 by heparin. In a dose response curve, both effects reached a maximum at approximately 1 unit/mL and then diminished again upon further increasing the heparin concentration, strongly suggesting a template mechanism as an explanation for the observed effect. In contrast to vitronectin, heparin does not stabilize the active conformation of PAI-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1991

Thrombin neutralizes plasminogen activator inhibitor 1 (PAI-1) that is complexed with vitronectin in the endothelial cell matrix.

Vitronectin endows plasminogen activator inhibitor 1 (PAI-1), the fast-acting inhibitor of both tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), with additional thrombin inhibitory properties. In view of the apparent association between PAI-1 and vitronectin in the endothelial cell matrix (ECM), we analyzed the interaction between PAI-1 and thrombin in this environment. Upon incubating 125I-labeled alpha-thrombin with endothelial cell matrix (ECM), the protease formed SDS-stable complexes exclusively with PAI-1, with subsequent release of these complexes into the supernatant. Vitronectin was required as a cofactor for the association between PAI-1 and thrombin in ECM. Metabolic labeling of endothelial cell proteins, followed by incubation of ECM with t-PA, u-PA, or thrombin, indicated that all three proteases depleted PAI-1 from ECM by complex formation and proteolytic cleavage. Proteolytically inactive thrombin as well as anticoagulant thrombin, i.e., thrombin in complex with its endothelial cell surface receptor thrombomodulin, did not neutralize PAI-1, emphasizing that the procoagulant moiety of thrombin is required for a functional interaction with PAI-1. A physiological implication of our findings may be related to the mutual neutralization of both PAI-1 and thrombin, providing a new link between plasminogen activation and the coagulation system. Evidence is provided that in ECM, procoagulant thrombin may promote plasminogen activator activity by inactivating PAI-1.

Antibodies, Monoclonal↗
PubMed1990

Structural requirements for the extracellular interaction of plasminogen activator inhibitor 1 with endothelial cell matrix-associated vitronectin.

The interaction of plasminogen activator inhibitor-1 (PAI-1) with its binding protein vitronectin (VN) (Declerck, P. J., De Mol, M., Alessi, M.-C., Baudner, S., Paques, E.-P., Preissner, K. T., Müller-Berghaus, G., and Collen, D. (1988) J. Biol. Chem. 263, 15454-15461) in the extracellular matrix (ECM) of cultured human endothelial cells (HUVEC) was studied. Like PAI-1, VN was found associated with the ECM as evidenced by direct antibody binding, by Western blot analysis as well as by diffuse immunofluorescence staining in permeabilized HUVEC. The specific interaction of VN with confluent monolayers of HUVEC was found to be saturable within 2-4 h at 37 degrees C only with respect to binding to the cells, while no saturable binding to the underlying ECM was observed, indicating that the majority if not all ECM-associated VN was derived from the culture medium. In contrast to PAI-1, ECM-associated VN was resistant toward glycine (pH 2.3), guanidine or urokinase treatment, suggesting that VN was tightly associated with the ECM network. Binding of recombinant PAI-1 (rPAI-1) was largely blocked by anti-VN IgG and only partly by anti-collagen IgG but not by antibodies against other ECM components, indicating that VN constitutes the primary binding protein for ECM-associated PAI-1. This contention was supported by ligand blotting experiments in which rPAI-1 was reacted with nitrocellulose replicas of electrophoretically separated ECM components. Protein band(s) (Mr = 63,000-67,000), comigrating with bovine VN (i.e. medium-derived VN) rather than with human VN were identified as major binding component(s). Moreover, binding studies with purified components revealed that PAI-1 did not show any affinity for collagen (type I/III) alone, whereas VN collagen coating was a much better template for PAI-1 binding than VN alone and that conformationally extended VN provides maximal PAI-1 binding capacity. Binding of rPAI-1 to surface-coated VN was saturable and revealed that (unlike urokinase) heparin or the synthetic peptide Gly-Arg-Gly-Asp-Ser did not inhibit PAI-1 binding. Ligand binding of rPAI-1 to nitrocellulose replicas from sodium dodecyl sulfate-polyacrylamide gels containing electrophoretically separated peptides from VN digests documented the association of PAI-1 with Mr = 10,000-20,000 fragments originating from the heparin-binding domain of VN. These results indicate that the exposure of the glycosaminoglycan-binding domain in VN may allow the concomitant binding of PAI-1 and heparin-like molecules to this region of the VN molecule.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1990

Alteration of serpin specificity by a protein cofactor. Vitronectin endows plasminogen activator inhibitor 1 with thrombin inhibitory properties.

Serine protease inhibitors ("serpins") are highly homologous proteins which inhibit selected "target" serine proteases by acting as a pseudo-substrate. Their specificity is primarily determined by the amino acid sequence around the carboxyl-terminally located reactive center (P1-P1'). In addition, the association rate constant between a serpin and a serine protease can be dramatically increased by non-protein cofactors, such as heparin in the case of thrombin inhibition by antithrombin III. In an attempt to alter the specificity of PAI-1 from an inhibitor of the fibrinolytic system to an inhibitor of coagulation, we replaced P1-P1' or P3 through P3' of the reactive center of PAI-1 by the corresponding residues of antithrombin III and assessed whether the mutant proteins, purified from lysates of transformed Escherichia coli cells, had acquired thrombin inhibitory properties. The experiments were performed in the presence and absence of vitronectin, a multifunctional protein which has been shown to bind PAI-1 in plasma and in the matrix of endothelial cells. The second-order rate constants for t-PA inhibition of "wild-type" PAI-1 and PAI P1-P1'ATIII, irrespective of the presence of vitronectin, were similar, whereas replacing P3-P3' resulted in a 40-fold decrease of the second-order rate constant towards t-PA, again independent of vitronectin. In the absence of vitronectin, reactivity of PAI-1 and its "antithrombin III-like" variants towards thrombin was slow; however, PAI-1 P3-P3' ATIII had a 10-fold higher k1 than wild-type PAI-1 (1.3 x 10(4) M-1 s-1 versus 1.1 x 10(3) M-1 s-1). In contrast, in the presence of vitronectin, PAI-1 and even more rapidly PAI-1 P3-P3'ATIII were found to be effective thrombin inhibitors, with k1 values of 2.2 x 10(5) M-1s-1 and 1.8 x 10(6) M-1 s-1, respectively. Thus, in the presence of vitronectin, PAI-1 P3-P3'ATIII displays a 3-fold higher k1 with thrombin than with t-PA. It is shown that vitronectin enhances, in a dose-dependent manner, the formation of sodium dodecyl sulfate-resistant complexes between PAI-1 or mutants thereof and thrombin. Therefore, vitronectin is the first protein described to function as a cofactor for serpin specificity. PAI-1 is proposed to be a versatile inhibitor which, in the presence of vitronectin, can modulate both coagulation and fibrinolysis.

Amino Acid Sequence↗
PubMed1990

Recombinant human protein C derivatives: altered response to calcium resulting in enhanced activation by thrombin.

Calcium plays a dual role in the activation of protein C: it inhibits protein C activation by alpha-thrombin, whereas it is required for protein C activation by the thrombomodulin-thrombin complex. Available information suggests that these calcium effects are mediated through calcium induced structural changes in protein C. In this paper, we demonstrate that substitution of Asp167 (located in the activation peptide of human protein C, occupying position P3 relative to the peptide bond Arg169-Leu170 which is susceptible to hydrolysis by thrombin) by either Gly or Phe results in protein C derivatives which are characterized by an altered response to calcium. At 3 mM calcium, alpha-thrombin activated the derivatives 5- to 8-fold faster compared with the wild-type, an effect which was shown to be caused by a decreased inhibitory effect of calcium on the reaction. These same single amino acid substitutions enhanced the affinity of the thrombomodulin-thrombin complex for the substrate at 3 mM calcium 3-(Gly-substitution) to 6-(Phe-substitution) fold, either without influencing kcat (Gly-substitution) or with a 2.5-fold decrease of kcat. For both derivatives, the calcium concentrations resulting in half maximal inhibition of activation by alpha-thrombin and in half maximal stimulation of activation by the thrombomodulin-thrombin complex increased from 0.3 mM to 0.6 mM. It is concluded that Asp167 is involved in the calcium induced inhibition of protein C activation by thrombin. Moreover, our studies demonstrate that it is feasible to enhance the efficiency of enzymatic reactions by introducing point mutations in the substrate.

Amino Acid Sequence↗
PubMed1990

In vivo behavior of detergent-solubilized purified rabbit thrombomodulin on intravenous injection into rabbits.

Thrombomodulin is a thrombin endothelial cell membrane receptor. The thrombomodulin-thrombin complex rapidly activates protein C resulting in anticoagulant activity. We investigated the anticoagulant effects and pharmacokinetic behavior of detergent-solubilized purified rabbit thrombomodulin labeled with iodine 125 when intravenously injected into rabbits. Thrombomodulin half-life (t1/2) was determined by tracking the 125I-radiolabeled protein and the biologic activity as determined by the prolongation of the activated partial thromboplastin time (APTT) and thrombin clotting time (TCT). When 200 micrograms/kg 125I-thrombomodulin was injected into rabbits, the APTT and TCT were immediately prolonged, whereas no effect on the prothrombin time was seen. In vitro calibration curves enabled us to convert the prolongations of the clotting times into micrograms per milliliter thrombomodulin equivalents. The best fit (r greater than 0.99) for the disappearance curves was provided by a two-compartment model with mean t1/2 alpha (distribution phase) of 18 minutes for 125I, 12 minutes for APTT, and 20 minutes for TCT, and mean t1/2 beta (elimination phase) of 385 minutes for 125I, 460 for APTT, and 179 for TCT. The administration of two doses of endotoxin (50 micrograms/kg) 24 hours apart did not accelerate the turnover rate of 125I-thrombomodulin as measured by the disappearance of 125I from the circulation. Thus, detergent-solubilized purified thrombomodulin administered intravenously circulates in a biologically active form for appreciable time periods.

Animals↗