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T M Reilly

Publications and source records attributed to T M Reilly.

At least 55 records · Page 3Linked to original sources
PubMed1992

Comparison of recombinant plasminogen activator inhibitor-1 and epsilon amino caproic acid in a hemorrhagic rabbit model.

A rabbit ear model of blood loss was developed to compare the effects of an active form of recombinant plasminogen activator inhibitor-1 (rPAI-1) with epsilon amino caproic acid (EACA) in antagonizing tissue-type plasminogen activator (r-tPA)-induced blood loss. The antagonism of both rebleeding, which occurs as a result of hemostatic plug degradation, and r-tPA-induced hemorrhage, where rabbits lose approximately 30% of their blood volume, was studied. rPAI-1 (1 mg/kg i.v.) or EACA (70 mg/kg i.v.) antagonized the rebleeding induced by r-tPA (10 micrograms kg-1 min-1) to a similar extent. In the hemorrhagic studies, rPAI-1 effectively antagonized the r-tPA-induced hemorrhage with an ED50 of 3 mg/kg i.v., while the ED50 obtained for EACA was 230 mg/kg i.v. rPAI-1 may be of value in reversing r-tPA-induced blood loss during thrombolytic therapy or in clinical situations where excessive fibrinolysis contributes to bleeding.

Amino Acid Sequence↗
PubMed1992

In vivo characterization of a new synthetic thrombin inhibitor.

We have examined the in vivo pharmacology of DuP 714 (Ac-[D]-Phe-Pro-boroArginine), a representative of a new series of synthetic thrombin inhibitors which contain a boronic acid derivative of arginine. Intravenous bolus injections of DuP 714 in anesthetized rats and conscious rabbits produced transient elevations of clotting times. Clinically relevant prolongations of the APTT were also observed in rabbits after i.v. infusion of less than 0.1 mg kg-1 h-1. Efficacy against venous thrombosis was demonstrated in a rabbit model of stasis induced thrombosis. Clots formed in 100% of control animals and only 33% of animals treated with 0.5 mg/kg DuP 714, and were less severe in treated animals. In a rabbit arterial-venous shunt model mimicking arterial thrombosis, occlusion occurred within 30 min in 72% of control animals vs. 11% of animals treated with 0.1 mg kg-1 h-1 DuP 714. Results indicate that DuP 714 is a highly effective anticoagulant which should be useful for the prevention of both venous and arterial thrombotic diseases.

Animals↗
PubMed1992

Effect of thrombin inhibitors on platelet functions: comparative analysis of DuP 714 and hirudin.

Since thrombin plays an important role in platelet-mediated arterial thrombosis, we have examined the antiplatelet activity of a synthetic thrombin inhibitor, DuP 714 (Ac-(D)Phe-Pro-boroArg), in comparison with that of the naturally occurring inhibitor hirudin. Hirudin was slightly more potent than DuP 714 in inhibiting thrombin-induced aggregation in washed human platelets (IC50s of 72 nM and 150 nM, respectively) and in inhibiting the secretion of plasminogen activator inhibitor-I from human platelets (IC50s of 300 nM and 900 nM, respectively). In contrast, DuP 714 was more potent than hirudin in inhibiting thrombin-induced [125I]fibrinogen binding to gel purified platelets, and in inhibiting thrombin-induced intracellular calcium mobilization in washed platelets. These results indicate that the tripeptide DuP 714 has comparable antiplatelet activity to the 65 amino acid hirudin. We conclude that DuP 714 may have clinical utility in the prevention of platelet-dependent, arterial thrombotic processes.

Blood Platelets↗
PubMed1992

The pericyte as a possible osteoblast progenitor cell.

Bovine capillary and microvessel pericytes were grown in monolayer in standard tissue culture medium supplemented with 10% newborn calf serum at various oxygen tensions for up to ten weeks. The pericytes synthesized alkaline phosphatase and formed colonies that mineralized. Energy dispersive X-ray spectrometry revealed the presence of calcium and phosphate, showed positive staining for collagen and glycosaminoglycan, and, most importantly, demonstrated the synthesis of osteocalcin. Cell proliferation, hydroxyproline production, and alkaline phosphatase synthesis were greatest in 3% oxygen, whereas osteocalcin production was least in 3% oxygen. These findings demonstrate that the capillary or microvessel pericyte exhibits phenotypic expressions in vitro that are similar to that of in vitro bone cells, and these expressions may be somewhat oxygen dependent. It is suggested from these findings that the capillary or microvessel pericyte may be an osteoblast precursor cell.

Alkaline Phosphatase↗
PubMed1991

Inhibition of the thrombin-platelet reactions by DuP 714.

The efficacy and specificity of a novel synthetic thrombin inhibitor, DuP 714, on thrombin-induced elevation of cytoplasmic calcium, fibrinogen binding and aggregation in human platelets were examined. Thrombin (0.5 U/ml) stimulated an increase in [125I]fibrinogen binding in gel-filtered platelets which was blocked by DuP 714 with an IC50 value of 2 nM. Thrombin (1 U/ml)-induced elevation of intracellular [Ca2+]i was also blocked by DuP 714 with an IC50 value of 67 nM. A much higher concentration of thrombin (25 U/ml) was used to stimulate aggregation with heparinized platelet-rich plasma. Under these conditions, micromolar concentrations of DuP 714 were needed to inhibit thrombin. In all of these preparations, DuP 714 at concentrations as high as 10(-5) M had no intrinsic effects and did not affect the responses induced by arachidonate, ADP, collagen, epinephrine, vasopressin and serotonin. These data indicate that DuP 714 is a potent and specific thrombin inhibitor capable of arresting the actions of thrombin on human fibrin formation and platelet aggregation and secretion. It may serve as a potential antithrombotic agent for various forms of thrombotic disorders.

Adenosine Diphosphate↗
PubMed1991

A spectroscopic study of the conformations of active and latent forms of recombinant plasminogen activator inhibitor-1.

Recombinant plasminogen activator inhibitor-1 (rPAI-1) purified from Escherichia coli, like its natural counterpart, can exist in either active or latent form. To elucidate the structural basis for these two forms, both active and latent rPAI-1 have been studied using ultra-violet (UV), circular dichroism (CD), and fluorescence spectroscopy. The secondary structures determined by CD show no significant differences and indicate that both the forms are predominantly alpha helical and random. The UV spectra are also very similar with absorption maxima around 278 nm. The structures of the two forms were further characterized by measuring tryptophan fluorescence emissions and their quenching with ionic (iodide) and neutral (acrylamide) quenchers. These data indicate clear differences in the tertiary structures of the two forms with the latent form being more compact and folded in comparison with the active form.

Circular Dichroism↗
PubMed1990

High-level synthesis of biologically active human plasminogen activator inhibitor type 1 (PAI-1) in Escherichia coli.

Segments of a cDNA encoding human plasminogen activator inhibitor type 1 (PAI-1) were subcloned into a highly regulated and inducible Escherichia coli expression system. A plasmid encoding the mature form of human endothelial PAI-1 produced a functional recombinant molecule, as indicated by its ability to inhibit tissue plasminogen activator's enzymatic activity. In contrast to PAI-1 isolated from human fibrosarcoma cells, the biological activity of the recombinant PAI-1 was not dependent on pretreatment with denaturing agents. A construct encoding a polypeptide lacking the first 80 amino acids of PAI-1 also produced elevated levels of the truncated recombinant protein. However, this truncated product was functionally inactive, indicating that an intact N terminus is required for activity.

Amino Acid Sequence↗
PubMed1990

Angiotensin II monoclonal antibody: blood pressure effects in normotensive and spontaneously hypertensive rats.

The angiotensin II (AII) specific monoclonal antibody KAA8--a selective functional AII antagonist and a hypotensive agent in high renin hypertensive rats--did not lower blood pressure in conscious normotensive rats (NRs) and spontaneously hypertensive rats (SHRs). The non-peptide AII receptor antagonist DuP 753 decreased blood pressure significantly in SHRs but not in NRs. These results suggest the involvement of locally released AII in the control of blood pressure in SHRs but not in NRs.

Angiotensin II↗
PubMed1990

Effects of recombinant plasminogen activator inhibitor type 1 on fibrinolysis in vitro and in vivo.

A biologically active recombinant PAI-1 (rPAI-1) was evaluated for its effects on clot lysis in vitro and in vivo. At concentrations of 0.5 to 10 micrograms/ml, the rPAI-1 significantly prolonged the time to lysis of rabbit euglobulin clots both in the presence and absence of exogenous tissue plasminogen activator. To examine the effects of PAI in vivo, we infused rPAI-1 into conscious rabbits after i.v. injection of homogenized fibrin clots, and assessed fibrinolysis by measuring the appearance of d-dimer fibrin degradation products (FDP). Plasma fibrinolytic activity, PAI activity and antigen were also measured in plasma samples taken during and after infusion of rPAI-1. In control rabbits, endogenous fibrinolytic activity resulted in a significant and continual generation of FDP, reaching 32.6 +/- 13.6 ng/ml 90 minutes after fibrin injection. Infusion of 1, 2, or 5 micrograms/kg/min rPAI-1 led to dose dependent increases in PAI activity and antigen, while FDP levels at 90 minutes were only 8.8 +/- 2.9, 5.7 +/- 2.4, and 0.3 +/- 0.3 ng/ml, respectively. Complete inhibition of fibrinolysis was observed with 10 micrograms/kg/min rPAI-1. These studies directly demonstrate that increases in PAI-1 impair the fibrinolytic system, and support indirect observations of an association between elevated levels of this protein and thromboembolic diseases.

Animals↗
PubMed1990

Purification and characterization of recombinant plasminogen activator inhibitor-1 from Escherichia coli.

A recombinant form of plasminogen activator inhibitor-1 (rPAI-1) has been purified from lysates of pCE1200, a bacterial expression vector containing the full length PAI-1 gene, by utilizing sequential anion exchange and cation exchange chromatography on Q-Sepharose and S-Sepharose columns. Approximately 140 mg of rPAI-1, estimated at 98% purity on the basis of analytical high performance liquid chromatography, could be obtained from 200 g wet weight of cells. The purified protein exhibited a single Coomassie Blue-stainable band at the region of Mr = 42,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an NH2-terminal amino acid sequence consistent with the expected translation product of the pCE1200 PAI-1 insert. The rPAI-1 rapidly inhibited single- and two-chain tissue plasminogen activators, as well as urokinase, with apparent second order rate constants in the range of 2-5 x 10(7) M-1 s-1. A specific activity measurement of 250,000 units/mg was calculated for the rPAI-1 based on its ability to inhibit the enzymatic activity of a single-chain tissue plasminogen activator. Stability studies showed that the activity of the rPAI-1 was very stable when stored at temperatures of 25 degrees C or lower, but decayed within hours when stored at 37 degrees C. Sodium dodecyl sulfate treatment, which partially activates the latent form of natural PAI-1, inactivated rPAI-1. These results show that the purified rPAI-1 produced from pCE1200 displays many of the properties associated with the biologically active form of natural PAI-1.

Amino Acid Sequence↗
PubMed1990

Basic fibroblast growth factor-like activity and receptors are expressed in a human glioma cell line.

Basic fibroblast growth factor (bFGF), a potent mitogen and angiogenic peptide, has been examined as an autocrine regulator of glioma cell growth. The addition of purified bovine pituitary bFGF to an established human glioma cell line, SNB-19, doubled the density of these cells in chemically defined medium. Half-maximal stimulation occurred at 8.2 ng/ml (480 pM). Also, human recombinant bFGF (hr-bFGF) significantly enhanced the growth of SNB-19 cells in soft agar. SNB-19 cells expressed both high and low affinity binding sites for hr-bFGF. These cells expressed approximately 13,000 high affinity sites/cell (Kd = 16.6 +/- 1.7 pM) and 9.5 x 10(6) low affinity sites/cell (Kd = 61.2 +/- 4.1 nM). The results of cross-linking experiments with iodinated hr-bFGF demonstrated the presence of two bands with molecular masses of 145 and 130 kDa. High affinity receptors were also demonstrated in SNB-19 tumors grown in nude mice. SNB-19 cell extracts contained mitogenic activity that eluted from heparin-agarose with high salt (1.2-2 M NaCl) and exhibited many properties normally associated with authentic bFGF. This material cross-reacted with a monoclonal antibody to hr-bFGF, comigrated with hr-bFGF by Western blot analysis, competed with 125I-hr-bFGF in a radioreceptor assay, and stimulated SNB-19 cell growth. These results indicate that a human glioma cell line both expresses and utilizes a bFGF-like growth factor. Such a factor may be an important autocrine regulator of glioma cell growth and may also facilitate its neoplastic progression.

Cell Division↗
PubMed1989

Monoclonal antibodies directed against basic fibroblast growth factor which inhibit its biological activity in vitro and in vivo.

A panel of four murine monoclonal IgG1 antibodies (mAbs) to a recombinant form of basic fibroblast growth factor (bFGF) was produced using somatic cell fusion techniques. Non-linear regression analysis of radioimmunoassay data for each mAb yielded the following dissociation constants (nM) for their interactions with bFGF: DE6 (0.822); AF11 (2.0); FE8 (2.31); and DG2 (20.0). One of the mAbs, DG2, was identified as a bFGF neutralizing antibody on the basis of its ability to inhibit, in vitro, the binding of [125I]-bFGF to high and low affinity bFGF sites on cultured baby hamster kidney cells and bFGF-induced [3H]-thymidine incorporation in cultured 3T3 cells, and in vivo, the angiogenic response to bFGF in a rat kidney capsule model of angiogenesis. The other mAbs displayed varying inhibitory activities in these assays. These mAbs, particularly DG2, may be well suited for a number of applications in bFGF research including immunoassays, immunohistochemical studies, and as functional antagonists of bFGF for examining its role in physiological processes such as reproduction, growth, and development.

Animals↗
PubMed1989

Tissue plasminogen activator-induced secretion of type-1 plasminogen activator inhibitor in cultured human fibroblasts.

Tissue plasminogen activator (t-PA) regulation of type-1 plasminogen activator inhibitor (PAI-1) secretion by WI-38 human fibroblasts was investigated utilizing a specific ELISA to measure PAI-1 antigen levels. Incubation of confluent fibroblasts with t-PA in serum-free medium resulted in a concentration and time-dependent increase in the secretion of PAI-1, with the maximum response (a two-fold increase) observed following a 4 hour incubation with 0.1 microM t-PA. This t-PA effect was reduced by cycloheximide, suggesting that the involvement of specific protein synthesis, and by inhibitors of t-PA's enzymatic activity, suggesting a requirement for a free catalytic site. The t-PA effect could not be attributed to endotoxin contamination. Increased levels in cell-associated PAI-1 were also detected following incubation of fibroblasts with t-PA. This previously undescribed effect, although seen at relatively high concentrations of t-PA, may have important biological implications.

Cells, Cultured↗
PubMed1989

Binding of tissue plasminogen activator to cultured human fibroblasts.

The binding of 125I-labeled, one-chain tissue plasminogen activator (t-PA) by WI-38 cultured human lung fibroblasts was investigated. Binding of t-PA to WI-38 monolayers was specific, saturable and temperature dependent. One and two-chain t-PAs were comparable in their ability to compete with 125I-labeled, one-chain t-PA for binding to fibroblasts, while no inhibition of binding was observed with a 500-fold molar excess of urokinase. Studies with various compounds suggest that neither the catalytic site, the fibrin binding site, nor the carbohydrate moieties on t-PA are involved in its binding to WI-38 cells. At higher temperatures, the amount of cell-bound 125I-t-PA that was removed by either incubation in binding buffer containing an excess of unlabeled t-PA, or by brief treatment with acidic buffer, was small (approximately 20%) suggesting that much of the t-PA is internalized. Electrophoretic analysis of extracts prepared from cells that had been incubated with 125I-t-PA revealed the presence of a major band of 70,000 Mr, which corresponds to intact t-PA. Our results suggest that WI-38 fibroblasts are capable of binding and internalizing t-PA, and that these processes involve a receptor site specific for t-PA.

Binding, Competitive↗
PubMed1989

Monoclonal antibodies to tissue-type plasminogen activator which prolong its clearance in vivo.

Three murine monoclonal antibodies to tissue-type plasminogen activator (t-PA) were evaluated for their effects on the binding of iodinated t-PA to cultured human hepatoma cells (Hep G2), and on extending the half-life of t-PA injected into rabbits. Two of the antibodies, AE5 and EG2, significantly inhibited t-PA binding in vitro, and extended the in vivo half-life of t-PA four to five-fold. A third antibody, BA10, which had a much smaller inhibitory effect on t-PA binding, had no influence in extending t-PA's half-life. MOPC-21, a control antibody not directed to t-PA, had no effect on either test. Our results are the first to correlate different compounds' effects on t-PA binding with their ability to retard t-PA clearance in vivo, and provide additional evidence for the importance of a liver cell receptor in the t-PA clearance process.

Antibodies, Monoclonal↗
PubMed1989

The Mitrofanoff principle for continent urinary diversion.

A continent urinary diversion was formed for 16 patients using the Mitrofanoff principle for continence. As originally described, this system used the appendix tunnelled into the bladder to form a continent catheterisable vesicostomy. We have expanded the technique and have used all available narrow tubes as continent conduits (ureter 10 cases, appendix 5, Fallopian tube 1). The urine container was made of large and small intestine and bladder in several combinations. The system has been very satisfactory: 14 patients were continent and able to catheterise; 1 required a revision to achieve continence and 1 awaits revision; 3 patients required revision procedures for stricture.

Adolescent↗
PubMed1989

Effect of a monoclonal antibody to angiotensin II on hemodynamic responses to noradrenergic stimulation in pithed rats.

A specific angiotensin II monoclonal antibody, KAA8, was used to examine the interaction between sympathetic function and angiotensin II in pithed rats. KAA8, at 5 or 50 mg/kg i.v., did not alter the mean blood pressure, cardiac output, total peripheral resistance, or heart rate responses to sympathetic neural stimulation (0.25-4.0 Hz) or to norepinephrine (0.3-3 micrograms/kg i.v.) but blocked significantly the hemodynamic responses to angiotensin II (0.03-1.0 microgram/kg i.v.) and to angiotensin III (0.3-10 micrograms/kg i.v.). KAA8 treatment also reduced the plasma immunoreactive angiotensin II from 2,880 +/- 475 pg/ml to an undetectable level. In contrast, captopril (5 mg/kg i.v.) and saralasin (10 or 50 micrograms/kg/min i.v.) inhibited the mean blood pressure and total peripheral resistance responses, but not the cardiac output and heart rate responses, to sympathetic neural stimulation and to norepinephrine. These results, which confirm previous findings by Kaufman and Vollmer (Kaufman LJ, Vollmer RR: Endogenous angiotensin II facilitates sympathetically mediated hemodynamic responses in pithed rats. J Pharmacol Exp Ther 1985;235:128-134), demonstrate that angiotensin II selectively potentiates the sympathetic vascular function in the pithed rat. However, our results suggest that circulating angiotensin II does not appear to interact with the sympathetic vascular function. It is speculated that in the pithed rat the sympathetic vascular response is enhanced by vascular-formed angiotensin II.

Angiotensin I↗