Leeches, snakes, ticks, and vampire bats in today's cardiovascular drug development.
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Animals↗
Biomedical subjects
N U Bang
Publications and source records attributed to N U Bang.
Gamma-carboxylated isoforms of recombinant human protein S with different biologic properties.
Human protein S (HPS), a regulator of hemostasis, is a vitamin K-dependent plasma protein with potential clinical utility. We have obtained high-level expression of the cDNA for HPS in two mammalian cell lines. Both cell lines secreted single chain recombinant HPS (rHPS) in serum-free medium as determined by Western blot analysis. The ability of the rHPS from both cell lines to act as a cofactor for human protein C (HPC) was determined; the rHPS secreted from the human 293 cell line had an activity six times that of the rHPS from the AV12-664 Syrian hamster cell line. Furthermore, the relative specific cofactor activity of rHPS from the 293 cell line was actually 2.5-fold higher than that of single-chain human plasma-derived HPS. Essentially all of the rHPS secreted from the 293 cell line exhibited a calcium-dependent elution profile on anion exchange chromatography, whereas only 25% to 35% of the hamster cell-derived rHPS exhibited this profile. However, the calcium-eluted rHPS from the AV12 cell line had a high specific cofactor activity, equivalent to that of the 293-derived rHPS. A NaCl-elutable rHPS fraction (calcium nondependent) was isolated from the recombinant AV12-664 cell line, further purified, and found to have reduced activity, only 40% that of the calcium-dependent rHPS. The only observable difference in the calcium-dependent and nondependent rHPS molecules was in the content of gamma-carboxyglutamic acid (Gla); the calcium-dependent material contained approximately 10 mol Gla/mol protein whereas the calcium-nondependent material contained only approximately 8 mol Gla/mol of protein. In addition, the calcium-nondependent rHPS had reduced ability to interact with phospholipid vesicles as evidenced by an eightfold increase in the apparent kd. Our data demonstrate the isolation of rHPS with high specific activity, and show that a reduction in as few as two Gla residues dramatically decreases its functional cofactor activity for HPC, due to a reduction in ability to interact with the phospholipid bilayer.
1-Carboxyglutamic Acid↗
Functional properties of the recombinant kringle-2 domain of tissue plasminogen activator produced in Escherichia coli.
The kringle-2 domain (residues 176-262) of tissue-type plasminogen activator (t-PA) was cloned and expressed in Escherichia coli. The recombinant peptide, which concentrated in cytoplasmic inclusion bodies, was isolated, solubilized, chemically refolded, and purified by affinity chromatography on lysine-Sepharose to apparent homogeneity. [35S]Cysteine-methionine-labeled polypeptide was used to study the interactions of kringle-2 with lysine, fibrin, and plasminogen activator inhibitor-1. The kringle-2 domain bound to lysine-Sepharose and to preformed fibrin with a Kd = 104 +/- 6.2 microM (0.86 +/- 0.012 binding site) and a Kd = 4.2 +/- 1.05 microM (0.80 +/- 0.081 binding site), respectively. Competition experiments and direct binding studies showed that the kringle-2 domain is required for the formation of the ternary t-PA-plasminogen-intact fibrin complex and that the association between the t-PA kringle-2 domain and fibrin does not require plasmin degradation of fibrin and exposure of new COOH-terminal lysine residues. We also observed that kringle-2 forms a complex with highly purified guanidine-activated plasminogen activator inhibitor-1, dissociable by 0.2 M epsilon-aminocaproic acid. The kringle-2 polypeptide significantly inhibited tissue plasminogen activator/plasminogen activator inhibitor-1 interaction. The kringle-2 domain bound to plasminogen activator inhibitor-1 in a specific and saturable manner with a Kd = 0.51 +/- 0.055 microM (0.35 +/- 0.026 binding site). Therefore, the t-PA kringle-2 domain is important for the interaction of t-PA not only with fibrin, but also with plasminogen activator inhibitor-1 and thus represents a key structure in the regulation of fibrinolysis.
Cloning, Molecular↗
Stable expression of a secretable deletion mutant of recombinant human thrombomodulin in mammalian cells.
Thrombomodulin is an endothelial cell membrane protein which plays a central regulatory role in the protein C anticoagulant pathway. The human thrombomodulin intronless gene was isolated from a genomic DNA library and used to isolate the coding region. A mammalian expression vector, phd-TMD1, encoding all the extracellular domains of human thrombomodulin but lacking the transmembrane and cytoplasmic domains was constructed. Stable phd-TMD 1 transformants, in both hamster AV12-644 and human 293 cells, expressed functionally active recombinant thrombomodulin as a secreted, soluble product. Soluble thrombomodulin was secreted as two major proteins of 105 kDa and 75 kDa, both of which were purified to homogeneity. The kinetic properties for protein C activation of the two proteins were very different: the Kd for thrombin, Km for protein C, and Ca2+ optima were 3.0 nM, 1.5 microM, and 1-3 mM for the 105-kDa protein and 16 nM, 2.3 microM, and 0.2-0.5 mM for the 75-kDa protein. In clotting and platelet activation assays, the 105-kDa protein was a much more potent anticoagulant than the 75-kDa protein. Both forms of the protein had the amino-terminal sequence Ala19-Pro-Ala-Glu-Pro-Gln. Amino acid composition analysis indicated that both forms of the protein had the same amino acid content which was consistent with the predicted protein comprising residues Ala19 to Ser515. The difference in size appeared to be due to glycosylation as both forms were of similar size following chemical deglycosylation. These studies suggest that (1) secretable thrombomodulin derivatives can be used to study structure-function relationships of the extracellular domains of this important regulatory protein, (2) the extent of glycosylation has profound effects on the kinetic and anticoagulant properties of human thrombomodulin, and (3) soluble recombinant human thrombomodulins may be developed as clinically significant therapeutic anticoagulants.
Amino Acids↗
Decreased thrombin affinity of cell-surface thrombomodulin following treatment of cultured endothelial cells with beta-D-xyloside.
Thrombomodulin, an endothelial cell-surface anticoagulant, has been postulated to contain a glycosaminoglycan. Thrombomodulin function was therefore studied in endothelial cells treated with beta-D-xyloside, an inhibitor of glycosaminoglycan attachment to proteoglycan core proteins. Beta-D-xyloside caused a reproducible 3 to 5-fold increase in the Km of thrombomodulin for thrombin and a 20-30% decrease in the rate of protein C activation by the thrombin-thrombomodulin complex. These results support a role for glycosaminoglycans in thrombomodulin function and suggest that beta-D-xylosides can be used to investigate both the anticoagulant mechanisms and the biosynthesis of cell-surface thrombomodulin.
Animals↗
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↗
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↗
Direct expression of recombinant activated human protein C, a serine protease.
Human protein C, like other serine proteases, is normally secreted as an inactive zymogen. It is converted to its active form extracellularly by limited proteolysis with the thrombin-thrombomodulin complex. This activation results from the removal of a 12-residue activation peptide from the NH2 terminus of the heavy (COOH-terminal) chain. We report here a successful strategy for the activation of human protein C during post-translational cellular processing, resulting in the secretion of activated protein C from transfected mammalian cells. Deletion of the nucleotides encoding the activation peptide resulted in the expression of a protease with less than 5% of the expected activity. However, the replacement of the activation peptide with an 8-residue sequence (Pro-Arg-Pro-Ser-Arg-Lys-Arg-Arg) involved in the proteolytic processing of the human insulin receptor precursor resulted in the direct expression of fully activated protein C. The mutant protein was shown to be correctly processed by NH2-terminal sequence analysis. This strategy for successful expression of an activated form of protein C may apply to the expression of active forms of other proteases which are naturally expressed as zymogens.
Amino Acid Sequence↗
After coronary thrombolysis and reperfusion, what next?
Thrombolytic therapy for the removal of intravascular thrombi was introduced when streptokinase was first given to humans 40 years ago, the same year the American College of Cardiology was founded. Streptokinase was first administered to patients with acute myocardial infarction in 1959. Today, thrombolytic therapy has been established to offer significant benefits to patients with acute myocardial infarction provided they are brought to medical attention early enough after the onset of symptoms. The two major agents, streptokinase and recombinant tissue-type plasminogen activator (rt-PA), have been shown to result in reperfusion of infarct-related arteries, to salvage ischemic myocardium, to improve myocardial performance and to reduce mortality. In spite of these impressive gains, this novel therapy has shortcomings. The interval from the start of thrombolytic treatment to coronary reperfusion varies significantly from patient to patient and may, at times, be too long to produce a real benefit in terms of salvage of ischemic myocardium. The rate of reocclusion lies somewhere between 10% and 20% and appears not to be influenced by concomitant heparin anticoagulation. The rate of bleeding complications even with the "fibrin-specific" rt-PA is higher than anticipated and may range from 10% to 30%. As a consequence, intensive efforts are being directed at the development of improved thrombolytic agents and for adjunctive therapy evaluating better anticoagulants than heparin and better antiplatelet agents than aspirin. This review is a status report summarizing where we are in thrombolytic therapy in acute myocardial infarction, where we need to improve treatment results and what is being done mainly at the preclinical level to bring about such improvements.
Animals↗
Hemostasis and fibrinolysis in renal transplantation.
Results of histologic, biochemical, and immunologic studies suggest that coagulation and fibrinolysis are possibly important in kidney graft rejection, but there is no information about how or why they are involved. The touchstones of their importance are histologic reports of platelet-fibrin thrombi in glomerular vessels of rejecting kidneys. Most of the nonhistologic studies have made use of functional assays aimed at measuring coagulation changes in blood. Little use has been made of immunoassays, and there are almost no studies of recently described anticoagulant systems, such as the protein C pathway. Endothelial cells form the interface between donor and recipient tissues. Antibody or thrombin-stimulated endothelium produces PAF, and thrombin-stimulated endothelium produces both plasminogen activators and plasminogen-activator inhibitors. As important as these reactions might be, information about them has been obtained with the use of either in vitro experiments or artificial systems, and there is no direct evidence that they are relevant to transplantation. However, these observations do show that tissue factor, PAF and fibrinolytic activities can be immunologically triggered. This opens the possibility that allogeneic recognition may initiate the triggering process. The clotting and fibrinolytic phenomena discussed in this overview were consequences of allogeneic recognition. Such recognition reactions cause monocytes and macrophages to produce tissue factor, which is a potent initiator of coagulation. Endothelial cells also can be stimulated to elaborate tissue factor by immune complexes, interleukin-1, or endotoxin. These observations give cause to speculate that the link between immunity and coagulation in kidney transplantation could be products of allogeneic recognition that activate Factor VII and lead to fibrin deposition. If true, this suggests new approaches to the old problems of diagnosis and treatment of rejection reactions in organ transplantation.
Fibrinolysis↗
Thrombolytic therapy in acute myocardial infarction.
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Acute Disease↗
Tissue-type plasminogen activator mutants. Theoretical and clinical considerations.
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Humans↗
Analysis of the plasminogen activator activity of the human glomerulus.
An assay was developed to measure plasminogen activator activity from isolated human glomeruli. Activator was extracted from individual glomeruli with 0.2 M phosphate-buffered saline, pH 7.4 (PBS), containing 0.01% Triton X-100 and quantitated in 125I-fibrin films. Quenching studies using antibodies to tissue plasminogen activator and urokinase revealed that the extracted glomerular plasminogen activator activity contained both tissue plasminogen activator of urokinase. Monoclonal and polyclonal antibodies raised to tissue plasminogen activator demonstrated low-level inhibition of urokinase activity and monoclonal and polyclonal antibodies to urokinase demonstrated low-level inhibition of tissue plasminogen activator activity. The assay should be applicable to the study of glomerular plasminogen activator activity in experimental and human kidney diseases. The detection of antibody cross-reactivity to tissue plasminogen activator and urokinase may be related to the sensitivity of the 125I-fibrin assay and to the structural similarities of these activators.
Humans↗
Effect of insulin immune complexes on human blood monocyte and endothelial cell procoagulant activity.
Insulin antibodies and immune complexes (ICs) are present in most patients with diabetes after initiation of conventional insulin therapy. We studied the ability of bovine insulin, porcine insulin, and human insulin ICs to stimulate the procoagulant activity (PCA) of human blood monocytes (HBMs) and human umbilical vein endothelium (HUVE) in vitro. Polyclonal insulin antibodies from patients with insulin antibody-mediated resistance were isolated by immunoabsorption. PCA expressed by isolated adherent HBMs or cultured HUVE was quantified by radioimmunoassay of fibrinopeptide A with a 1:80 dilution of human plasma. Beef, pork, and human insulin, in quantities determined from titration curves, were added to fixed amounts of antibody to obtain equivalent amounts of ICs. For four different antibodies, the ratios of PCA (expressed by HBMs exposed to ICs or to antibodies alone and normalized for cell numbers) to activity expressed by tissue culture medium were as follows: beef insulin ICs, 2.55 +/- 0.41; por insulin ICs, 1.47 +/- 0.16; human insulin ICs, 1.28 +/- 0.19; and antibody, 1.28 +/- 0.32. The differences between beef and pork insulin ICs and antibody controls were significant. For a fifth antibody, a dose response between beef insulin ICs and PCA expression was demonstrated. For a sixth antibody, HBMs could express PCA when stimulated with antibody of a relatively low binding capacity (0.35 ng bovine insulin per microgram of antibody) only in the presence of lymphocytes. Conditioned medium from these cells significantly stimulated endothelial cell procoagulant activity. These observations raise the possibility that insulin ICs, that is, beef insulin ICs, may generate increased PCA and thereby contribute to the vascular complications of diabetes mellitus.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Anthony P. Fletcher (1919-1987).
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Bibliographies as Topic↗
Vitamin K-dependent carboxylase: possible role of the substrate "propeptide" as an intracellular recognition site.
The liver microsomal vitamin K-dependent carboxylase catalyzes the posttranslational conversion of specific glutamate residues to gamma-carboxyglutamate residues in a limited number of proteins. A number of these proteins have been shown to contain a homologous basic amino acid-rich "propeptide" between the leader sequence and the amino terminus of the mature protein. Plasmids encoding protein C, a vitamin K-dependent protein, containing or lacking a propeptide region were constructed and the protein was expressed in Escherichia coli. The protein products were assayed as substrates in an in vitro vitamin K-dependent carboxylase system. Only proteins containing a propeptide region were substrates for the enzyme. These data support the hypothesis that this sequence of the primary gene product is an important recognition site for this processing enzyme.
Animals↗
Endotoxin enhances tissue factor and suppresses thrombomodulin expression of human vascular endothelium in vitro.
Endotoxemia is frequently associated clinically with disseminated intravascular coagulation (DIC); however, the mechanism of endotoxin action in vivo is unclear. Modulation of tissue factor (TF) and thrombomodulin (TM) expression on the endothelial surface may be relevant pathophysiologic mechanisms. Stimulation of human umbilical vein endothelial cells with endotoxin (1 microgram/ml) increased surface TF activity from 1.52 +/- 0.84 to 11.89 +/- 8.12 mU/ml-10(6) cells at 6 h (n = 11) which returned to baseline by 24 h. Repeated stimulation at 24 h resulted in renewed TF expression. Endotoxin (1 microgram/ml) also caused a decrease in TM expression to 55.0 +/- 6.4% of control levels at 24 h (n = 10) that remained depressed at 48 h. Both effects were dose and serum dependent. A temporary rise in TF expression accompanied by a sustained fall in TM expression comprise a shift in the hemostatic properties of the endothelium that would favor intravascular coagulation and may contribute to the pathogenesis of DIC in gram-negative septicemia.
Cell Membrane↗
Effects of a vitamin K-deficient diet and antibiotics in normal human volunteers.
Decreased concentrations of vitamin K-dependent plasma clotting factors are a well-documented response of vitamin K-deprived patients administered broad-spectrum antibiotics. It has recently been claimed that antibiotics containing a N-methylthiotetrazole (NMTT) side chain cause this response through a direct effect of NMTT on the vitamin K-dependent posttranslational carboxylation of these clotting factors. To further study these relationships, 11 groups of three volunteers were fed a synthetic vitamin K-free diet for 2 weeks. During the last 10 days of vitamin K restriction, seven of the volunteer groups received a therapeutic dose of antibiotics not containing NMTT: ampicillin, sulfamethoxazole-trimethoprim (Bactrim), cefoxitin, cefotaxime, ceftazidime, clindamycin, and piperacillin, and three groups received NMTT-containing antibiotics: moxalactam, cefamandole, and cefoperazone. Serum phylloquinone (vitamin K1) concentrations reflected dietary intake and fell from 1.4 +/- 0.9 ng/ml after 3 days of hospital diet to 0.4 +/- 0.3 ng/ml after 13 days of vitamin K-free diet. Median stool excretion of phylloquinone was 19 micrograms/day while subjects consumed the hospital diet, and fell to 3 micrograms/day by day 6 on vitamin K-free diet. Prothrombin times remained within the normal range throughout the study. Suppression of vitamin K-dependent clotting factor biosynthesis was evident by decreased factor VII levels in seven of the volunteers and by an increased concentration of des-gamma-carboxy (abnormal) prothrombin in 21 of the volunteers. The changes occurred in the control subjects and in subjects receiving all nine of the 10 antibiotics with no consistent pattern.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗