Biomedical subjects

S D Carson

Publications and source records attributed to S D Carson.

At least 19 recordsLinked to original sources

PubMed1992

Monoclonal antibodies against the C-terminal peptide of human tissue factor for studies of the cytoplasmic domain.

The carboxyl-terminal cytoplasmic domain of human tissue factor (the essential cofactor for coagulation factor VII) is a prospective regulatory domain through which viable cells may control the expression of tissue factor activity. Furthermore, this domain is subject to post-translational modifications of as yet unknown functional significance. Hybridomas producing antibodies against the C-terminal domain of tissue factor were obtained using splenocytes harvested from mice immunized with a synthetic peptide corresponding to the nine terminal residues of the protein sequence. These antibodies, C28 1.1 and C28 2.1, react with purified placental tissue factor, but not with a recombinant soluble tissue factor lacking the cytoplasmic and membrane-spanning domains. This confirms that tissue factor from biological membranes contains the entire cytoplasmic domain predicted from the cDNA sequence, and provides a mechanism to determine whether tissue factor used experimentally retains this proteolytically sensitive epitope. The antibodies have been employed to demonstrate selective proteolytic removal of the carboxyl-terminal peptide from tissue factor. Limited experiments indicate that they will be useful for characterizing the distribution of vesicles with regard to the possible orientations of tissue factor on their surface.

Amino Acid Sequence↗

PubMed1991

Lipoprotein associated coagulation inhibitor, factor VII, antithrombin III, and monocyte tissue factor following surgery.

Fifteen patients undergoing major surgical procedures were evaluated for lipoprotein associated coagulation inhibitor (LACI) antigen, factor VII (F VII), antithrombin III (AT III), and peripheral blood monocyte tissue factor (TF) activity immediately before surgery and on following days. A peak in monocyte TF activity occurred between the first and fifth days after surgery in 10 of the patients, while LACI, F VII, and AT III levels dropped in a qualitatively parallel manner in 8 of these patients. LACI, F VII, and AT III levels decreased after surgery in two additional patients even though TF activity also decreased after surgery in these patients. In the remaining 3 patients who developed infections during the study, TF activity rose within 2 days of the diagnosis of infection in addition to the postoperative peak. In two of these patients, LACI levels increased dramatically near the end of the study period without concomitant changes in F VII and AT III. Overall, the presurgical TF levels in disrupted monocytes varied 52-fold and the maximal TF activity varied 24-fold among patients. The TF response following surgery is therefore heterogenous in both temporal occurrence and magnitude of the postsurgical peak. The patients also varied considerably in the presurgical levels of monocyte TF activity. A possible association between the level of presurgical TF activity and the magnitude of the postsurgical peak was noted.(ABSTRACT TRUNCATED AT 250 WORDS)

Antithrombin III↗

PubMed1991

Tissue factor in chronic villitis of unestablished etiology.

We have previously reported the presence in normal human placentae of coagulation, macrophages and helper T lymphocytes in inflammatory foci known as villitis of unestablished etiology. In order to investigate the link between coagulation and immunity, we have studied fetal stem vessel endothelium for tissue factor, which is made available by cytokines and activates coagulation via the extrinsic pathway. We found that fetal stem vessel endothelial cells of normal chorionic villi did not react with antibody to tissue factor. Normal placentae contain small numbers of villitis areas and endothelium in these areas was reactive with antibody to tissue factor. Endothelial tissue factor reactivity was more prominent in placentae from secondary recurrent spontaneous aborters and these placentae have greatly increased numbers of villitis areas. The tissue factor availability on fetal stem vessel endothelium may result from immunologically mediated cytokine release. The net effect of these reactions is the presence of lymphocytes, macrophages, coagulation, necrosis and vasculitis in villitis.

Abortion, Habitual↗

PubMed1991

Identification of peptides within a known protein sequence using COMSEQ analysis of data containing multiple sequences.

Modern methods of automated protein sequence analysis can provide high-quality data with which unambiguous amino-acid sequences can be determined, but analyses are more difficult when the sample is not pure. COMSEQ and auxillary programs were written to facilitate reconciliation of multiple amino-acid sequences potentially contained in noisy data with the known amino-acid sequence of the parent protein. The COMSEQ program prints a matrix in which the first vertical column represents the known amino-acid sequence of a selected protein. Each row of the matrix contains the sequencer yield corresponding to the amino acid in the first column, with each column corresponding to the sequencing reaction cycle. A diagonal which contains net increases of amino acids for each amino acid in the known sequence identifies a peptide potentially contained within the data. The number of matches for each diagonal over the entire known sequence are tabulated and presented as an aid to locating comparisons of greatest interest. The RNDSEQ program conducts multiple analyses using randomized versions of the known amino-acid sequence and tabulates the cumulative frequencies of potential sequence matches irrespective of the true known sequence. TRANSEQ is a utility program that translates edited sequence data from common databases into files that can be used by COMSEQ and RNDSEQ. The programs have been used successfully to identify two co-sequenced peptides from bovine serum albumin, an albumin peptide sequence in the presence of hemoglobin, and to identify two sequences of rat alpha-2u-globulin that differ in their amino termini.

Amino Acid Sequence↗

PubMed1991

A proposed role for silicates and protein in the proliferative effects of saccharin on the male rat urothelium.

High doses of sodium saccharin, a non-genotoxic chemical, lead to the formation of silicate-containing precipitate and microcrystals in urine of male rats. Differences in urinary protein, pH, sodium and other factors affect silicate-containing precipitate and microcrystal formation as well as the bladder effects of sodium saccharin. Total urinary silicon concentration (mostly soluble) in sodium saccharin-fed rats is similar to or lower than the concentration in control rats. Binding of saccharin to male rat urinary proteins was demonstrated by equilibrium-gel filtration. We propose that by binding to urinary proteins under appropriate conditions, saccharin produces a nidus for the formation of silicate-containing precipitate and crystals. These appear to be cytotoxic to the superficial bladder epithelium, with cell death resulting in regenerative hyperplasia. Factors that influence the formation of these silicate-containing materials might provide a rationale for sex, species, dose and dietary differences in response to sodium saccharin.

Animals↗

PubMed1991

Tissue factor antigen in senile plaques of Alzheimer's disease.

Tissue factor (tissue thromboplastin) is the primary initiator of the extrinsic coagulation pathway, triggering a proteolytic cascade when exposed to circulating coagulation factors. In this study, the distribution of tissue factor was examined immunohistochemically in Alzheimer's disease (AD) and control brains. Tissue factor was expressed diffusely in the neocortex, but in AD there was enhanced immunoreactivity in senile plaques. Although tissue factor might potentially contribute to the formation of senile plaques, it could also accumulate in the plaques as a secondary response to other biochemical perturbations.

Adult↗

PubMed1990

Dipyridamole inhibits O2- release and expression of tissue factor activity by peripheral blood monocytes stimulated with lipopolysaccharide.

Monocytes can be induced to synthesize and express tissue factor procoagulant activity. They can also be stimulated to release a broad spectrum of inflammatory agents including superoxide anion (O2-) that are thought to contribute to the pathogenesis of inflammatory diseases. Dipyridamole, an inhibitor of platelet aggregation blocks the lipopolysaccharide (LPS)-induced increase in monocyte-associated tissue factor activity and phorbol myristate acetate (PMA) stimulated O2- release from monocytes and polymorphonuclear leukocytes (PMN). Dipyridamole inhibition of O2- release can be reversed by increased glucose in the culture media, whereas dipyridamole inhibition of tissue factor can not be reversed by increased glucose in the culture media. These results reveal that dipyridamole influences monocytes by at least two distinct mechanisms. Further, it may serve as an anti-thrombotic agent by virtue of its effect on both platelet aggregation and monocyte tissue factor activity.

Biological Transport↗

PubMed1990

The coagulopathy of childhood leukemia. Thrombin activation or primary fibrinolysis?

Little information is available on the prevalence and etiology of the coagulopathy present in some children with acute leukemia at disease presentation. We studied 102 children with newly diagnosed acute leukemia (50 retrospective: Group A; and 52 prospective: Group B) with prothrombin time (PT), partial thromboplastin time (PTT), thrombin time (TT), fibrinogen (FIB), and fibrin degradation products (FDP). All patients in Group B also had assessment of thrombin activation by measurement of the crosslinked fibrin fragment, D-dimer, and of primary fibrinolysis with the B beta 1-42 peptide. Additionally, ten patients from Group B had Factors II, V, VII, and X measured, and eight of these patients had measurement of tissue factor from sonicated bone marrow cells. Thirty-two percent of Group A and 40% of Group B had totally normal coagulation studies, whereas 20% of Group A and 10% of Group B had a severe coagulopathy on disease presentation. A high percentage of both groups had elevated PT (Group A, 52%; Group B, 27%) and increased FDP (Group A, 39%; Group B, 25%). In Group B, 38% of the patients had a positive D-dimer, whereas only 4% of this prospective group had an elevated B beta 1-42 peptide (P less than 0.00001). Nine of ten patients with a positive D-dimer had low levels of one or more of the extrinsic pathway factors. Three of four patients with the highest tissue factor levels were of monocytoid leukemia cell type. These data indicate that the coagulopathy associated with acute leukemia of childhood is usually mediated by thrombin activation.

Adolescent↗

PubMed1990

Consecutive enzyme cascades: complement activation at the cell surface triggers increased tissue factor activity.

Complement activation at the cell surface initiates cell damage through a series of reactions occurring at the cell membrane and, after assembly of the terminal membrane attack complex, produces leakage of cytoplasmic contents from the cell. It has been documented that chemical or physical damage to cell membranes can cause a rapid increase in the expression of tissue factor procoagulant activity. In this study, antibody-mediated complement activation at the cell surface resulted in increased tissue factor activity, which correlated with cytolysis, as measured by 51-chromium release. Therefore, complement fixation on the cell surface can have a direct and immediate stimulatory effect on the coagulation cascade at the point of its initiation, with formation of a fibrin clot requiring only three consecutive proteolytic reactions after immunologically mediated cell damage.

Antibodies↗

PubMed1990

Tissue factor antigen and activity are not expressed on the surface of intact cells isolated from an acute promyelocytic leukemia patient.

Tissue factor activity and antigen were measured in promyelocytes freshly isolated from a patient with acute promyelocytic leukemia (APL), FAB M3. Determination of functional activity revealed that physically disrupted cells expressed considerable tissue factor of which less than two percent was available prior to physical disruption of the cells. No tissue factor antigen was detectable on the cell surface by fluorescence flow cytometry. In contrast, endotoxin-stimulated peripheral blood monocytes and monoblastic cells isolated from a patient with monoblastic leukemia had notable populations of tissue factor-positive cells by flow cytometry, and expressed higher proportions of total tissue factor activity without disruption. While some cell types may express both tissue factor antigen and activity when intact, others, which can be extremely rich in tissue factor, may express neither antigen nor activity without a triggering event such as cell damage.

Antigens↗

PubMed1990

Tissue factor: identification and characterization of cell types in human placentae.

This is an immunohistologic study of tissue factor (TF) in snap frozen, unfixed, human normal-term placentae. Antibodies to TF were a monoclonal to human brain TF purified on a factor VII-agarose affinity column, and a polyclonal to a synthetic polypeptide representing the carboxyl-terminal nine amino acids of human TF. The results detail the localization and distribution of TF and characterize the cells in which it is found. TF was not observed in trophoblast, trophoblastic basement membranes, or noncellular components of connective tissue. TF was identified in some but not all macrophages, most fibroblast-like cells, and occasionally in perivascular cells and endothelium. The most consistent and intense reactions were obtained with vimentin-positive fibroblast-like cells in loose connective tissue. TF usually was not identified in fetal stem vessel endothelial cells, but TF reactivity was found in some of these cells in chorionic villi with histologic evidence of chronic inflammation. Such areas are uncommonly found in normal-term placentae. The vast majority of TF-reactive cells did not react with antibody to factor VII and were not in contact with blood. The biologic purpose of producing relatively great amounts of TF in areas remote from circulating factor VII is not known.

Antibodies, Monoclonal↗

PubMed1989

BASIC program for non-parametric fitting of user-defined functions to experimental data with plotting of results.

Graphic displays of experimental results facilitate decisions regarding mathematical models which relate experimental results to controlled parameters. Experiments frequently produce response variables which are non-linearly related to the controlled variable. Computer programs can be useful to fit model functions to experimental data. SIMPLOT7, written in Microsoft QuickBASIC, provides graphic display of experimental data and non-parametric fitting of user-defined functions to experimental data. The algorithm utilizes simplex-based non-parametric minimization of residuals, and the user-defined function may contain up to seven parameters to be determined for best fit. Once optimum fit values have been determined, both function and data can be graphed for visual inspection of the result.

Computer Simulation↗

PubMed1989

Human fibroblast tissue factor is inhibited by lipoprotein-associated coagulation inhibitor and placental anticoagulant protein but not by apolipoprotein A-II.

Studies of proteins that inhibit tissue factor activity have generally been conducted using either an extracted tissue homogenate ("thromboplastin") or tissue factor protein reconstituted into phospholipid vesicles rather than with tissue factor expressed in cell membranes (its physiological environment). In the present study, a human fibroblast cell strain was used to evaluate the effects of lipoprotein associated coagulation inhibitor (LACI), placental anticoagulant protein (PAP), and apolipoprotein A-II (apo A-II) on human tissue factor in cell membranes. LACI was tested from 7.8 to 500 pmol/L on fibroblasts cultured at cell densities ranging from 3,500 to 9,925 cells/well, and caused a progressive inhibition of tissue factor activity. PAP was tested from 3.9 nmol/L to 1 mumol/L at cell densities ranging from 4,500 to 15,400 cells/well and caused up to 83% inhibition of tissue factor activity. Inhibition by these proteins appeared to be influenced by cell density as well as whether the cells were intact or disrupted. Apo A-II, up to 1 mumol/L, did not inhibit the tissue factor activity of intact or disrupted fibroblasts at any cell density examined even though it did inhibit the activity of tissue factor in phospholipid vesicles. Of these inhibitors of tissue factor-dependent activation of factor X, LACI was the most effective in suppressing the generation of factor Xa activity. The effects obtained with apo A-II are clearly dependent on the nature of the tissue factor preparation with which it is tested. The disparity between the inhibitory effect of apo A-II on the activity of tissue factor reconstituted into lipid vesicles and the absence of effect on the activity of tissue factor remaining in cell membranes serves to reemphasize the necessity of reexamining results obtained with model systems using as nearly physiological reagents as possible.

Annexins↗

PubMed1988

Participation of cell-mediated immunity in deposition of fibrin in glomerulonephritis.

Fibrin deposition is prominent in delayed-type hypersensitivity (DTH) reactions and is initiated by antigen-specific, T-lymphocyte-directed macrophage expression of human tissue factor (HTF). To examine the role of DTH in glomerular fibrin deposition, 10 fibrin-positive and 24 fibrin-negative biopsy specimens from patients with glomerulonephritis (GN) and samples from normal controls were studied with monoclonal antibodies against T cells, macrophages, and HTF. Fibrin-positive sections showed intense glomerular staining for HTF and significantly more T cells and macrophages than fibrin-negative specimens. All the essential elements of DTH reactions can therefore be simultaneously demonstrated within glomeruli from patients with fibrin-related GN. These findings suggest a role for cell mediated immunity in GN.

Adolescent↗

PubMed1988

Effects of lipid-binding proteins apo A-I, apo A-IL, beta 2-glycoprotein I, and C-reactive protein on activation of factor X by tissue factor--factor VIIa.

Tissue factor is the membrane-associated protein which mediates activation of factors IX and X by factor VII. In a purified, reconstituted bovine system, factor X activation by the tissue factor-factor VIIa complex is inhibited by the mixed apoproteins from human high density lipoprotein (HDL) and by isolated apolipo-protein A-II (apo A-II). Other proteins found associated with plasma lipoproteins, apolipoprotein A-I (apo A-I), C-reactive protein (CRP), and beta 2-glycoprotein I (beta 2 GPI), have been examined for effects on the activation of factor X by tissue factor-factor VIIa. In these experiments, bovine tissue factor, reconstituted into phosphatidylserine-phosphatidylcholine (PS/PC; 30/70) vesicles, was used at a single concentration while factor X (the substrate), factor VIIa (the enzyme), and the potentially inhibitory proteins were varied in a continuous chromogenic assay. Apo A-II and CRP clearly inhibit tissue factor-factor VIIa activation of factor X, while apo A-I and beta 2 GPI have little or no effect. These results demonstrate that different lipid binding proteins vary in their effects on tissue factor activity.

Apolipoprotein A-I↗

PubMed1988

Regional assignment of human tissue factor gene (F3) to chromosome 1p21-p22.

Tissue factor, or coagulation factor III, is a membrane-bound glycoprotein and acts as a cofactor for factor VII-dependent initiation of blood coagulation. The tissue factor gene (F3) was previously assigned to human chromosome 1, region p21-pter. The present report has further refined the mapping position to 1p21-p22 using a cDNA probe for the tissue factor gene and in situ hybridization to metaphase chromosomes.

Chromosome Mapping↗

PubMed1988

Monocyte-associated tissue factor is suppressed by phorbol myristate acetate.

The monocyte is the only normal circulating cell type capable of initiating blood coagulation through the expression of tissue factor. Recently isolated peripheral blood monocytes that contain no demonstrable tissue factor activity can be induced to express tissue factor activity by a number of stimulatory agents. Monocyte-associated tissue factor activity transiently increases in response to adherence to tissue culture plates and, consistent with other reports, markedly increases after the isolated monocytes are treated with endotoxin. Phorbol myristate acetate (PMA) induced an increase in tissue factor activity at low doses (10(-11) to 10(-12) mol/L). Conversely, concentrations of PMA that stimulate release of oxygen metabolites or that cause the cytosol-to-membrane translocation of protein kinase C (PKC) (10(-9) to 10(-7) mol/L) resulted in a rapid decrease in both adherence-induced and endotoxin-induced monocyte tissue factor activity. The effects of PMA on monocytes were time- and dose-dependent with respect to PKC translocation, release of oxygen metabolites, and changes in tissue factor activity. Immunofluorescent staining of monocytes with monoclonal antibody (MoAb) HTF1-7B8, directed against human tissue factor, revealed that tissue factor antigen was induced concurrently with tissue factor activity by adherence and endotoxin and that tissue factor antigen decreased after PMA stimulation.

Cells, Cultured↗

PubMed1988

Protein co-isolated with human tissue factor impairs recovery of activity.

Preparations of human tissue factor isolated by immunoaffinity chromatography contain variable amounts of 47,000 mol wt, 55,000 mol wt, and multimeric tissue factor when analyzed without reduction on polyacrylamide gels in sodium dodecyl sulfate (SDS). When analyzed after reduction, the 47,000 mol wt tissue factor apoprotein and a protein of about 12,000 mol wt are observed. Elution of tissue factor from polyacrylamide gel slices, followed by reassociation with lipids, restored proportionately much greater tissue factor activity with the 47,000-mol wt protein than with the 55,000-mol wt form. Cyanogen bromide cleavage at the single tissue factor methionine revealed that the 12,000-mol wt protein is associated with the carboxyl-terminal peptide derived from the 47,000-mol wt protein. These results reveal that association of the 12,000-mol wt protein with the cytoplasmic domain of tissue factor can modulate its activity in vitro.

Disulfides↗