Biomedical subjects

J Morser

Publications and source records attributed to J Morser.

At least 37 records · Page 2Linked to original sources

PubMed1998

Both cellular and soluble forms of thrombomodulin inhibit fibrinolysis by potentiating the activation of thrombin-activable fibrinolysis inhibitor.

Thrombin-activable fibrinolysis inhibitor (TAFI) is a recently described plasma zymogen that can be activated by thrombin to an enzyme with carboxypeptidase B-like activity. The enzyme, TAFIa, potently attentuates fibrinolysis. TAFI activation, like protein C activation, is augmented about 1250-fold by thrombomodulin (TM). In this work, the effects of both soluble and cellular forms of TM on TAFI activation-dependent suppression of fibrinolysis were investigated. Soluble TM included in clots formed from purified components, barium citrate-adsorbed plasma, or normal human plasma maximally increased the tissue plasminogen activator-induced lysis time 2-3-fold, with saturation occurring at 5, 10, and 1 nM TM in the three respective systems. Soluble TM did not effect lysis in the system of purified components lacking TAFI or in plasmas immunodepleted of TAFI. In addition, the antifibrinolytic effect of TM was negated by monoclonal antibodies against either TAFI or TM. The inhibition of fibrinolysis by cellular TM was assessed by forming clots in dialyzed, barium citrate-adsorbed, or normal plasma over cultured human umbilical vein endothelial cells (HUVECs). Tissue plasminogen activator-induced lysis time was increased 2-fold, with both plasmas, in the presence of HUVECs. The antifibrinolytic effect of HUVECs was abolished 66% by specific anti-TAFI or anti-TM monoclonal antibodies. A newly developed functional assay demonstrated that HUVECs potentiate the thrombin-catalyzed, TM-dependent formation of activated TAFI. Thus, endothelial cell TM, in vitro at least, appears to participate in the regulation of not only coagulation but also fibrinolysis.

Blood Proteins↗

PubMed1998

Generation and characterization of mice deficient in hepsin, a hepatic transmembrane serine protease.

Hepsin is a type II transmembrane serine protease highly expressed on the surface of hepatocytes. The physiological function of hepsin is not known, although in vitro studies indicate that hepsin plays a role in the initiation of blood coagulation and in hepatocyte growth. To determine the functional importance of hepsin, we generated hepsin-deficient mice by homologous recombination. Homozygous hepsin-/- mice were viable and fertile, and grew normally. In functional assays including tail bleeding time, plasma clotting times, and tissue factor- or LPS-induced disseminated intravascular coagulation models, no significant difference was found between hepsin-/- and wild-type litter mates. Liver weight and serum concentrations of liver-derived proteins or enzymes were similar in hepsin-/- and wild-type mice. Interestingly, serum concentrations of bone-derived alkaline phosphatase were approximately twofold higher in hepsin-/- mice of both sexes when compared with wild-type litter mates. No obvious abnormalities were found in major organs in hepsin-/- mice in histological examinations. Our results indicate that hepsin is not essential for embryonic development and normal hemostasis. Hepsin-/- mice will help to evaluate the long-term effects of hepsin deficiency in these animals.

Alkaline Phosphatase↗

PubMed1998

Identification and characterization of two thrombin-activatable fibrinolysis inhibitor isoforms.

Thrombin-activatable fibrinolysis inhibitor (TAFI) is synthesized by the liver and is thought to circulate in plasma as a plasminogen-bound zymogen. When it is activated by the thrombin/thrombomodulin complex, activated TAFI exhibits carboxypeptidase B-like activity. To study the structure-function relationship of TAFI, we expressed recombinant human TAFI in insect cells. During the cloning of TAFI cDNA from several human liver cDNA libraries, we identified a second TAFI cDNA which differed from the published sequence at 2 positions. One of these sequences resulted in a substitution of alanine for threonine at residue 147, the other was a silent mutation. These substitutions were found in several cDNA libraries from different sources. Using Southern blot analysis, we confirmed the existence of this TAFI polymorphism in the population. In order to compare the activation and activity of TAFI isoforms, we expressed both isoforms in the baculovirus expression system, and compared the enzyme kinetics of the purified proteins. The molecular weight of recombinant TAFI is lower than plasma TAFI due to differences in glycosylation. The two recombinant TAFI isoforms had similar activation kinetics and the activated enzymes had similar carboxypeptidase B-like activity towards small molecule substrates. Their ability to retard clot lysis was found to be similar in a plate clot lysis assay.

Animals↗

PubMed1997

Functional characterization of recombinant human meizothrombin and Meizothrombin(desF1). Thrombomodulin-dependent activation of protein C and thrombin-activatable fibrinolysis inhibitor (TAFI), platelet aggregation, antithrombin-III inhibition.

Recombinant human prothrombin (rII) and two mutant forms (R155A, R271A,R284A (rMZ) and R271A,R284A (rMZdesF1)) were expressed in mammalian cells. Following activation and purification, recombinant thrombin (rIIa) and stable analogues of meizothrombin (rMZa) and meizothrombin(desF1) (rMZdesF1a) were obtained. Studies of the activation of protein C in the presence of recombinant soluble thrombomodulin (TM) show TM-dependent stimulation of protein C activation by all three enzymes and, in the presence of phosphatidylserine/phosphatidylcholine phospholipid vesicles, rMZa is 6-fold more potent than rIIa. In the presence of TM, rMZa was also shown to be an effective activator of TAFI (thrombin-activatable fibrinolysis inhibitor) (Bajzar, L., Manuel, R., and Nesheim, M. E. (1995) J. Biol. Chem. 270, 14477-14484). All three enzymes were capable of inducing platelet aggregation, but 60-fold higher concentrations of rMZa and rMZdesF1a were required to achieve the effects obtained with rIIa. Second order rate constants (M-1.min-1) for inhibition by antithrombin III (AT-III) were 2.44 x 10(5) (rIIa), 6.10 x 10(4) (rMZa), and 1.05 x 10(5) (rMZdesF1a). The inhibition of rMZa and rMZdesF1a by AT-III is not affected by heparin. All three enzymes bound similarly to hirudin. The results of this and previous studies imply that full-length meizothrombin has marginal procoagulant properties compared to thrombin. However, meizothrombin has potent anticoagulant properties, expressed through TM-dependent activation of protein C, and can contribute to down-regulation of fibrinolysis through the TM-dependent activation of TAFI.

Antithrombin III↗

PubMed1997

Recombinant advanced glycation end product receptor pharmacokinetics in normal and diabetic rats.

Vascular dysfunction in patients with diabetes mellitus is related to advanced glycation end product (AGE) formation. We previously showed that AGEs produce an increase in vascular permeability and generated an oxidant stress after binding to the receptor (RAGE) present on endothelium. RAGE, a 35-kDa protein that belongs to the immunoglobulin superfamily, has been cloned from a rat lung cDNA library, and recombinant rat soluble RAGE (rR-RAGE) has been produced in insect cells. The sequence of RAGE is highly conserved between human and rat. We studied the biological effect of rR-RAGE and pharmacokinetics of 125I-rR-RAGE after intravenous or intraperitoneal administration in normal and streptozotocin-induced diabetic rats. rR-RAGE prevented albumin or inulin transfer through a bovine aortic endothelial cell monolayer, restored the hyperpermeability observed in diabetic rats or induced in normal rats by diabetic rat red blood cells, and corrected the reactive oxygen intermediate production after intravenous or intraperitoneal administration. After intravenous injection of 125I-rR-RAGE, the distribution half-life was longer (p < or = 0.01) in diabetic (0.15 and 4.01 hr) than in normal (0.02 and 0.21 hr) rats, as was the case for the elimination half-lives (diabetic, 57.17 hr; normal, 26.02 hr; p < or = 0.01). Distribution volume was higher in diabetic than in normal rats (6.94 and 3.24 liter/kg, respectively; p = 0.049). Our study showed that rR-RAGE was biologically active in vivo and slowly cleared, which suggests it could be considered as a potential therapy.

Amino Acid Sequence↗

PubMed1997

Thrombin, thrombomodulin and TAFI in the molecular link between coagulation and fibrinolysis.

The thrombin thrombomodulin dependent activation of the plasma protein TAFI (Thrombin Activatable Fibrinolysis Inhibitor) and Subsequent Inhibition of Fibrinolysis by the TAFIa is described. Work to date indicates that TAFIa is a carboxypeptidase B enzyme that suppress fibrinolysis most likely by down regulating the cofactor functions of partially degraded fibrin. The existence of TAFI provides the explanation for the apparent profibrinolytic effect of activated protein C. and implies the existence of an explicit molecular connection between the blood coagulation of fibrinolytic cascades that is expressed through the thrombin thrombomodulin dependent activation of TAFI. Thus, thrombin generation can, in principle, result in the suppression of fibrinolysis.

Amino Acid Sequence↗

PubMed1996

RAGE and amyloid-beta peptide neurotoxicity in Alzheimer's disease.

Amyloid-beta peptide is central to the pathology of Alzheimer's disease, because it is neurotoxic--directly by inducing oxidant stress, and indirectly by activating microglia. A specific cell-surface acceptor site that could focus its effects on target cells has been postulated but not identified. Here we present evidence that the 'receptor for advanced glycation end products' (RAGE) is such a receptor, and that it mediates effects of the peptide on neurons and microglia. Increased expressing of RAGE in Alzheimer's disease brain indicates that it is relevant to the pathogenesis of neuronal dysfunction and death.

Alzheimer Disease↗

PubMed1996

TAFI, or plasma procarboxypeptidase B, couples the coagulation and fibrinolytic cascades through the thrombin-thrombomodulin complex.

TAFI (thrombin-activatable fibrinolysis inhibitor) is a recently discovered plasma protein that can be activated by thrombin-catalyzed proteolysis to a carboxypeptidase B-like enzyme that inhibits fibrinolysis. This work shows that the thrombin-thrombomodulin complex, rather than free thrombin, is the most likely physiologic activator. Thrombomodulin increases the catalytic efficiency of the reaction by a factor of 1250, an effect expressed almost exclusively through an increase in kcat. The kinetics of the reaction conform to a model whereby thrombin can interact with either TAFI (Km = 1.0 microM) or thrombomodulin (Kd = 8.6 nM), and either binary complex so formed can then interact with the third component to form the ternary thrombin-thrombomodulin-TAFI complex from which activated TAFI is produced with kcat = 1.2 s-1. This work also shows that activated TAFI down-regulates tPA-induced fibrinolysis half-maximally at a concentration of 1.0 nM in a system of purified components. This concentration of TAFI is about 2% of the level of the zymogen in plasma, which indicates that ample activated TAFI could be generated to very significantly modulate fibrinolysis in vivo. Therefore, TAFI in vitro and possibly in vivo defines an explicit molecular connection between the coagulation and fibrinolytic cascades, such that expression of activity in the former down-regulates the activity of the latter.

Blood Coagulation↗

PubMed1996

Soluble thrombomodulin antigen in plasma is increased in patients with acute myocardial infarction treated with thrombolytic therapy.

Thrombomodulin (TM) is an integral endothelial cell membrane protein that functions as a cofactor for thrombin mediated activation of protein C. The anticoagulant functions of the protein C system are important in contributing to a hemostatic balance and prevention of thromboembolic disease. It has been suggested that impaired TM cofactor function could also constitute a prothrombotic abnormality leading to thromboembolic diseases. TM exists not only on the surface of endothelial cells but also as soluble fragment(s) circulating in plasma. The concept of a thrombotic occlusion as the critical event in acute myocardial infarction (AMI) forms the rationale for thrombolytic therapy. After successful reperfusion, patients remain at substantial risk for recurrent infarctions due to rethrombosis. The balance between procoagulant and anticoagulant mechanisms in the postthrombolytic phase have not been studied in detail. We have studied whether the plasma levels of soluble TM are influenced by thrombolytic therapy with streptokinase in patients suffering from AMI. Soluble TM concentrations increased significantly by 24 to 48 h after thrombolytic treatment, simultaneously with an increase in C-reactive protein (CRP, a marker of the inflammatory component of the cell damage) and in thio-barbituric acid reactive substances (TBARS, an indirect marker of lipid peroxidation).

Acute Disease↗

PubMed1995

The structure of a 19-residue fragment from the C-loop of the fourth epidermal growth factor-like domain of thrombomodulin.

The solution structure has been determined for a 19-residue peptide that is fully folded at room temperature. The sequence of this peptide is based on the C-loop, residues 371-389, of the fourth epidermal growth factor-like domain of thrombomodulin, a protein that acts as a cofactor for the thrombin activation of protein C. Despite its small size, the peptide forms a compact structure with almost no repeating secondary structure. The results indicate the structure is held together by hydrophobic interactions, which in turn stabilize the two beta-turns in the structure. The first beta-turn in the C-loop represents a conserved motif that is found in the published structures of five other epidermal growth factor-like proteins. The critical role of Phe376 in the stabilization of the first beta-turn is consistent with mutagenesis data with soluble thrombomodulin. The results also show that a small subdomain of a larger protein can fold independently, and therefore it could act as an initiation site for further folding.

Amino Acid Sequence↗

PubMed1994

Modulation of glycosaminoglycan addition in naturally expressed and recombinant human thrombomodulin.

The two major glycoforms of full-length human thrombomodulin (TM), one with (TM(CS+)) and one without (TM(CS-)) chondroitin sulfate (CS) were analyzed on Western blots of primary and transformed cells and in cells expressing recombinant TM. TM on the surface of Chinese hamster ovary and COS-7 cells is solely TM(CS-). Primary arterial endothelial cells (HAEC and HPAEC) express a greater fraction of TM with CS attached than venous cells (HUVEC). Human lung carcinoma cells (A549) express more TM(CS+) than primary cells and recombinant TM on human melanoma cells (CHL-1) occurs in two very high molecular weight forms of TM(CS+). We explored this variation in TM(CS+) with soluble recombinant TM in several cell lines and analyzed the ambiguous CS addition site in human TM by site-directed mutagenesis. Mutation of Ser474 to Ala blocks CS addition in Chinese hamster ovary and COS-7 cells but not CHL-1 cells which add CS to Ser472 and Ser474. Structure of the O-link domain affects partitioning into TM(CS+) since substituting with the decorin CS addition sequence, substituting all Ser and Thr except Ser474 with Ala, and deleting around the potential beta-turn all increase the ratio of TM(CS+) to TM(CS-). A combination of the decorin substitution and deletion of the remaining O-link domain yields the most TM(CS+).

Amino Acid Sequence↗

PubMed1994

Expression efficiency of the human thrombomodulin-encoding gene in various vector and host systems.

Expression systems were developed for evaluating recombinant human thrombomodulin (TM) production in different host cell lines by investigating the performance of five mammalian expression vectors. The expression vectors were constructed so that they contain multiple monocistronic gene cassettes which include a gene encoding a dominant selectable marker, HyR (hygromycin B phosphotransferase), under the regulation of the thymidine kinase promoter, the target gene which encodes a truncated human re-TM under the regulation of various promoters, an amplifiable gene (Dhfr) encoding murine dihydrofolate reductase under the regulation of either the SV40 early or late promoter along with the SV40 enhancer and the SV40 ori. We tested the performance of the five expression vectors in human embryonic kidney cells (HEK293), baby hamster kidney cells (BHK), human melanoma cells (CHL-1) and Dhfr- Chinese hamster ovary cells (CHO/Dhfr-). We found that the efficiency of DNA uptake, transient expression and stable expression of the different expression vectors were all cell-line dependent. However, the myeloproliferative sarcoma virus (MPSV) LTR promoter consistently showed higher expression levels in all cell lines, particularly in HEK293 cells. These results were confirmed by the distribution curves of the level of expression of individual clones. Furthermore, by amplifying Dhfr in transfected CHO/Dhfr- cells with 100 nM methotrexate, we achieved a 20-fold increase in re-TM production using the SV40 late promoter to control murine Dhfr expression. Our data from DNA and mRNA analysis reveal that pMPSV-TM has a high transcription efficiency.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Recombinant soluble human thrombomodulin: a randomized, blinded assessment of prevention of venous thrombosis and effects on hemostatic parameters in a rat model.

UNLABELLED: Thrombomodulin is an endothelial surface receptor that binds thrombin and accelerates the activation of protein C. We compared the effects of a recombinant thrombomodulin analog (TME), recombinant hirudin (r-HIR), heparin sodium (HEP), and normal saline (Control) on thrombus formation, activated partial thromboplastin time (APTT), thrombin time (TT), platelet aggregation and tail transection bleeding time (BT) in a rat model of vena cava thrombosis. RESULTS: TME, r-HIR and HEP prevented venous thrombosis in this model in a dose-dependent manner. At the dose required to reduce vena cava thrombosis by 50% (ED50), TME did not prolong the APTT or TT as did HEP and r-HIR. Platelet aggregation in response to thrombin was not effected by TME but was inhibited by both r-HIR and HEP. BT did not differentiate the agents tested. CONCLUSION: TME inhibited venous thrombosis in a rat vena cava model with less effect on hemostatic variables than HEP or r-HIR.

Animals↗

PubMed1994

A new animal model of venous thrombosis in rats with low flow conditions in the venous blood stream.

The aims of the present investigation were to develop a new venous thrombosis animal model with low flow conditions in the venous blood stream and then evaluate this model for testing new anticoagulants. In this model, the vena cava of rats was narrowed with a Doppler flow probe, blood flow velocity continuously recorded and thrombus formation initiated by thromboplastin infusion. Sixty-five minutes following thromboplastin infusion the animals were sacrificed and the following parameters measured: thrombus wet weight, fibrinopeptide A (FpA), activated partial thromboplastin time and platelet number. The new model was evaluated with aspirin, a PGI2 mimetic, heparin and a soluble thrombomodulin analogue. Without thromboplastin infusion no thrombus formation or reduction of blood flow was observed. Controls receiving thromboplastin infusion developed a thrombus, blood flow was arrested, platelet number decreased and FpA was elevated. In contrast, animals pretreated with anticoagulants maintained a residual flow, while thrombus weight, thrombocytopenia and FpA elevation were reduced. The antiplatelet agents were not effective. This study demonstrates that, under low flow conditions, only a combination of blood flow reduction with a hypercoagulable state results in venous thrombus formation. This improved model of venous thrombosis more closely resembles the clinical situation and is applicable for testing anticoagulants.

Animals↗

PubMed1993

The short loop between epidermal growth factor-like domains 4 and 5 is critical for human thrombomodulin function.

Thrombomodulin (TM) is a cofactor for activation of protein C by thrombin. We showed that 80-90% of this cofactor activity is lost by oxidation of Met388, located within the short interdomain loop between epidermal growth factor-like domains 4 and 5 (Glaser, C. B., Morser, J., Clarke, J. H., Blasko, E., McLean, K., Kuhn, I., Chang, R.-J., Lin, J.-H., Vilander, L., Andrews, W. H., and Light, D. R. (1992) J. Clin. Invest. 90, 2565-2573). For each of the 3 amino acids of the loop, site-specific mutants are described in which, 1) all possible single amino acid substitutions are made, 2) deletions are made, or 3) alanine is inserted adjacent to each residue of the loop. Most substitutions within the loop (38/57) result in a > 50% decrease in cofactor activity, while changes in the length of this region result in > 90% loss of activity. Only the Met388-->Leu mutant has higher cofactor activity (2-fold) than wild-type TM. A number of soluble and full-length TM analogs with the Met388-->Leu substitution are improved thrombin cofactors, whether produced in bacteria, insect, or mammalian cells. Detailed kinetic analysis of a soluble TM analog consisting of the six EGF-like domains secreted from insect cells shows that the enhanced activity of the Met388-->Leu mutant results from an increased catalytic efficiency (kcat/Km). This enhancement is maximal at physiological concentrations of calcium. The loss of activity following Met388 oxidation in the wild-type protein is the result of both decreased binding to thrombin (Kd effect) and a decreased interaction of the TM.thrombin complex with protein C (Km effect). We demonstrate the critical role of this interdomain loop in the biological anticoagulant properties of TM.

Amino Acid Sequence↗

PubMed1993

Alanine-scanning mutagenesis of the epidermal growth factor-like domains of human thrombomodulin identifies critical residues for its cofactor activity.

Thrombomodulin (TM) is an endothelial cell surface-bound cofactor in thrombin-dependent formation of activated protein C, a potent anticoagulant. Cofactor activity has been localized to the carboxyl-terminal half of the six epidermal growth factor-like (EGF) domains of TM (TME). To identify residues in TME that are critical for activity, 77 alanine point mutants were made between Cys-333 and Cys-462 by site-directed mutagenesis (all residues except Ala, Cys, Gly, and Pro). Mutants were expressed in Escherichia coli and cofactor activity measured directly in periplasmic extracts obtained by osmotic shock. Critical residues were defined as those which when mutated had less than 25% cofactor activity of a reference TME. Western blots of non-reduced samples confirmed that alanine substitutions did not significantly decrease expression levels or result in the formation of multimers. In EGF4, which is essential for protein C activation by the thrombin-TM complex, critical residues were: Asp-349, Glu-357, Tyr-358, and Phe-376. In EGF5-EGF6, critical residues within a proposed acidic thrombin-binding region were: Glu-408, Tyr-413, Ile-414, Leu-415, Asp-416, Asp-417, Asp-423, Ile-424, Asp-425, and Glu-426. A potential Ca(2+)-binding site, which is comprised of residues Asp-423, Asp-425, Glu-426, Asn-439, Leu-440, and Phe-444, was also identified and overlaps the thrombin-binding region. Asp-461, in the C-loop of EGF6 previously shown to be critical for thrombin binding, was also critical. Asp-398, Asp-400, Asn-402, and Asn-429 in EGF5 were also critical. Thus, rapid alanine-scanning mutagenesis of TME has identified 22 critical residues in the region comprising EGF4-6, which is essential for thrombin binding and protein C activation by the thrombin-TM complex.

Alanine↗

PubMed1993

The E. coli envY gene encodes a high affinity opioid binding site.

In an attempt to isolate a cDNA encoding an opioid receptor, a cDNA library was constructed in the lambda ZAP vector using NG108-15 mRNA as template. Using an in vitro transcription-translation assay and a sib selection strategy, a single phage was isolated. An RNA transcribed from this cDNA was able to direct in vitro translation of opioid binding sites. The insert was sequenced and comparison with data banks showed a 100% homology with the E. coli envY gene. We assume that the presence of the envY sequence in the NG108-15 cDNA library was due to a contamination of the lambda ZAP vector with E. coli DNA. A search for opioid binding sites on E. coli strains showed that envY+ strains, but not envY- mutants were able to bind opiates. On envY+ cells, the sites are stereospecific, saturable and of high affinity for the opiate ligands. These sites bind opiate agonists and antagonists but neither mu nor delta opioid peptides. In contrast, rabbit reticulocyte lysate primed with RNA transcribed in vitro from the envY sequence elicited the synthesis of an opioid binding site with mixed mu and delta properties. In addition, transfection of the envY sequence into mammalian cells resulted in the expression of opioid binding sites. Depending on the type of cells transfected, these sites were selective for either the mu or delta ligands.

Animals↗

PubMed1993

Thrombomodulin activity and localization.

An overview on the properties, actions and localization of thrombomodulin (TM) in situations of tissue injury and in selected tumors is presented. The localization and activity of TM after injury to vascular endothelium shows that following balloon catheter denudation of the endothelium of the rabbit aorta, the activity and immunohistochemical staining is markedly reduced. The functional and antigenic levels approach the control levels approximately one week after the initial injury. The results suggest that the neointimal smooth muscle cells express TM. This phenotypic plasticity of the neointimal smooth muscle cells may be important in conferring thrombo-resistance to the lumenal lining cells of vessels after injury. Studies are also reviewed on the use of soluble recombinant TM to prevent thrombosis after ligature of vessels in an experimental model. Further characterization on the immunohistochemical distribution of TM in normal tissues and tumors shows that staining with a monoclonal anti TM antibody can be very useful in separating mesotheliomas from pulmonary adenocarcinomas. These studies may lead to insights concerning the role of TM in tissue-injury-repair and tissue differentiation.

Adenocarcinoma↗