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Biomedical subjects

M C Boffa

Publications and source records attributed to M C Boffa.

At least 19 recordsLinked to original sources
PubMed1992

An ultrastructural study of thrombomodulin endocytosis: internalization occurs via clathrin-coated and non-coated pits.

The regulation of thrombomodulin (TM) expression has been reported to occur by several mechanisms. We have examined constitutive internalization of TM using immunofluorescent and electron microscopic (EM) methods. A cell model was developed to study this process by introducing TM DNA into COS-7 cells for expression. The recombinant TM was determined to behave similarly to native TM from human umbilical vein endothelial cells (HUVEC) with respect to M(r) and cell surface functional activity. The transfected cells expressed 8-100-fold more functional TM per cell than HUVEC. Immunofluorescent studies on these cells indicated that anti-TM antibody-TM complex was internalized in a time- and temperature-dependent manner, with internalization detectable within 10 minutes. When the cells were incubated at 4 degrees C with gold-labelled anti-TM antibody, most of the gold particles were surface bound and detected by EM as individual particles or clusters of 2 or 3 particles. Initiation of endocytosis for 10 to 60 minutes resulted in a redistribution of gold particles into small clusters predominantly in non-coated pits and rarely in clathrin-coated pits, subsequently in early endosomes, multivesicular bodies, and lysosomes. Similar studies were performed with gold-conjugated thrombin, demonstrating a similar route of intracellular processing. These studies provide ultrastructural evidence that the process of endocytosis of TM involves the participation of both clathrin-coated and non-coated pits and vesicles, but that the latter process predominates. Further structure/function studies are indicated using our cell model, since defects in the endocytic pathway of this important anticoagulant receptor may contribute to the development of thromboembolic disease.

Animals↗
PubMed1991

Thrombomodulin in the central nervous system.

Thrombomodulin (TM), a thrombin receptor, present on the endothelial surface of blood vessels is a major anticoagulant proteoglycan. In this work the presence of TM antigen in the human brain and in the whole central nervous system of calves was investigated by immunocytochemistry using specific antibodies against human and bovine TM. When TM antigen was well preserved by immediate fixation on frozen tissues, TM was found present in the brain vessels of all human surgical specimens examined as well as in the whole vasculature of the calf central nervous system (CNS), from the medulla to the cortex. Moreover, even without immediate fixation, TM antigen was always found on the surface of the cerebrospinal fluid (CSF) cavity: on the arachnoid and on the inner aspect of the dura mater in humans and in calves. This suggests that the proteolytic enzymes responsible for the cleavage of TM from the endothelium are not present in the CSF and in the meningeal cells. The results reported here emphasize the importance of TM in the CNS as protecting, by its antithrombogenicity, a free circulation of blood and CSF in normal and even more, in pathological situations in which the permeability of the meningeal barrier increases.

Animals↗
PubMed1991

Plasma thrombomodulin as a marker of endothelium damage.

A soluble form of thrombomodulin (TM), an anticoagulant proteoglycan of the endothelial cell membrane, exists in plasma. We recently developed an ELISA assay to measure it. Plasma TM level depends both on endothelium integrity and on the clearance of the molecule. It increases with endothelium damage as seen in different pathological situations.

Biomarkers↗
PubMed1989

In situ hybridization in hematology.

In situ hybridization is used to reveal mRNA presence and amount in a cell. It is of particular interest in Hematology since it allows the study of each individual cell and the investigation of cellular differentiation.

Blotting, Northern↗
PubMed1987

Appraisal of the protein composition of prothrombin complex concentrates of different origins.

The protein composition of 12 different prothrombin complex concentrates, including 2 purified factor IX concentrates and 2 activated fractions, was evaluated. There is a clear difference between ion exchanger-prepared fractions and those obtained by adsorption onto calcium phosphate. The former contain high molecular weight kininogen and complement components, the latter only trace amounts of these proteins but relatively high quantities of the pro- and even partially activated enzymes of the contact phase. Calcium phosphate adsorbed preparations contain less VIII:CAg than the DEAE-Sephadex-prepared fractions. The inorganic adsorbent showed higher affinity for IgG than the ion exchangers.

Adsorption↗
PubMed1987

Preservation of thrombomodulin antigen on vascular and extravascular surfaces.

The protein C anticoagulant system is mediated by thrombin and is highly accelerated by thrombomodulin. We studied the distribution of thrombomodulin antigen (TM Ag) in the rabbit using an affinity-purified antibody raised in a goat against rabbit thrombomodulin. The preservation of TM Ag was highly dependent on immediate fixation of the surface on which it is located. TM Ag was found on the endothelium of the entire vasculature, whereas it was absent from all connective tissue, smooth and striated muscle, secretory epithelia, cartilage, bone, neural tissue, and all parenchyma examined. A new finding was the presence of TM Ag on nonvascular surfaces of body cavities (the mesothelia of pleura, pericardium, and peritoneum, the synovial membrane, and the arachnoid enveloping the central nervous system). By use of a functional assay, TM activity was recovered in buffered saline/detergent solution which was either injected into the intraperitoneal cavity of rabbits in vivo or incubated with the surface of the arachnoid in vitro. These findings extend the importance of anticoagulant mechanisms to the systems of slowly circulating fluids, in which they might be required for maintenance of the flow, and to mesothelial cavities, in which they could be necessary for preventing adherence between the surfaces, in conditions associated with pathological exudation.

Animals↗
PubMed1987

[Different localization of thrombomodulin].

Thrombomodulin (TM) is the endothelial cofactor of the anticoagulant protein C system. The distribution of TM in the organism was studied in the rabbit using a goat anti TM, affinity-purified antibody and a peroxidase-labelled antigoat immunoglobulin. TM antigen was found on the endothelial surface of all blood vessels: capillaries, arteries and veins. The reaction was specific: connective tissue, smooth and striated muscle bone, cartilage, nerve tissue, secretary epithelia and all parenchyma studied were not strained. Moreover TM antigen was present on the surface of serosa: peritoneum, pericardium and pleura as well as on synovial membranes and on arachnoid, all along the central nervous system. It was absent from pia and dura mater. The antigen was found only after formalin fixation on the vessels and body cavities surface on which it lies. This observation shows that the antigen is easily detached from these surfaces and suggest a possible mobility of this endothelial molecule for which it lies. This observation shows that the antigen is easily detached from these surfaces and suggest a possible mobility of this endothelial molecule for which production and function sites might differ.

Animals↗
PubMed1987

Biological and clinical heterogeneity of lupus and lupus-like anticoagulant in fifty-seven patients.

A lupus or lupus-like inhibitor was detected in 57 patients: 24 systemic lupus erythematosus, 9 autoimmune diseases, 10 lymphoproliferative disease, 11 miscellaneous diseases and 3 asymptomatic patients. No hemorrhagic diathesis was observed in spite of major surgery. Thromboembolism occurred in 19 patients. Among them, 5 patients had recurrent abortions. An extensive study of coagulation profile compared different assays to investigate lupus-like inhibitor: the most sensitive assay was the partial thromboplastin time performed without activator. When performed with kaolin, it was the only assay detecting the lupus cofactor. Prothrombin time was prolonged in only 53% of the patients. Factors VIII, IX, XI and XII were in the normal range in 40% of the patients. When decreased, apparent deficiencies were usually not detectable on further dilutions of the test samples. In 7 patients factor XII antigen and activity were both decreased, suggesting an apparent factor XII deficiency. No relationship was observed between thromboembolic events, underlying disease or biological pattern.

Adolescent↗
PubMed1986

Functional domains of rabbit thrombomodulin.

Thrombomodulin isolated from rabbit lung was separated by ion-exchange chromatography on DEAE-cellulose into a retarded (acidic) and a nonretarded (nonacidic) fraction. Both fractions contained the cofactor required for the activation of protein C. In addition, the acidic fraction (but not the nonacidic fraction) prevented the clotting of fibrinogen by thrombin ("direct" anticoagulant activity) and accelerated the inhibition of thrombin by antithrombin (effect corresponding to 2-10 international units of heparin per mg of protein). Both of these activities were readily neutralized by the synthetic polycation Polybrene, which did not appreciably affect protein C activation. They were also eliminated by digestion of thrombomodulin with bacterial heparinase, which, in addition, converted the acidic form of the protein C activation cofactor to a nonacidic form. Similar conversion observed during storage of thrombomodulin was attributed to endogenous proteinase activity. Density-gradient centrifugation of the acidic form of thrombomodulin in CsCl/4M guanidinium chloride failed to separate either of the direct or antithrombin-dependent anticoagulant activities from the protein C activation cofactor, which showed a buoyant density of 1.31-1.34 g/ml. The nonacidic cofactor had a lower density, 1.26-1.28 g/ml. Unreduced thrombomodulin yielded two major fractions of protein C activation cofactor on NaDodSO4/PAGE, with apparent Mr of approximately 68,000 and 57,000, respectively. The larger component contained essentially all of the direct and antithrombin-dependent anticoagulant activities. We propose that these activities as well as the negative charge and the higher buoyant density of the acidic, Mr 68,000 form of thrombomodulin are due to a heparin-like polysaccharide and, further, that this component can be separated from the major portion of the molecule, which contains the protein C activation site, through the action of a proteinase.

Animals↗
PubMed1984

Studies on prothrombin complex concentrates contact factors, complement components and proteinase inhibitors.

The behaviour of contact factors, complement components and antiproteases during the preparation of prothrombin complex concentrates by adsorption of the clotting components on DEAE-Sephadex has been studied. The pro-enzymes: factors XII, XI and prekallikrein were removed by pre-elution in function of the salt concentration. In contrast, high molecular weight kininogen was considerably enriched in PCC preparations. C4 of the complement system displayed an analogous behaviour. C1s reached a 4-5 fold plasma concentration but C3 only 30% of the normal plasma level. The prothrombin complex concentrate contained no antithrombin III nor alpha 2M nor alpha 2 antiplasmin but a three fold plasma concentration of C1-inactivator and a 15 fold increase of inter-alpha-trypsin inhibitor. NAPTT (Non Activated Partial Thromboplastin Time) ratios did not seem to be in accordance with either the presence or the absence of contact enzymes. Moreover 0.20 M NaCl appeared as the minimal pre-elution molarity necessary to ensure a NAPTT ratio above thrombogenic values. Molecular alteration of high molecular weight kininogen and C4 was observed and its significance discussed. Complex formation between C1-inactivator and proteases was shown to be another sign of undesirable proteolytic events.

Blood Coagulation Factors↗
PubMed1984

Plasma contact activation and decrease of factor V activity on negatively-charged polyelectrolytes.

The effect of negatively-charged polymers, used in some artificial devices, on plasma clotting and kinin systems was studied in vitro using polyelectrolyte complexes. Contact activation was observed as an immediate, transient and surface-dependent phenomenon. After incubation of the plasma with the polymer a small decrease of factor XII activity was noticed, which corresponded to a greater reduction of prekallikrein activity and to a marked kinin release. No significant decrease of factor XII, prekallikrein, HMW kininogen could be detected immunologically. Only the initial contact of the plasma with the polyelectrolyte lead to activation, subsequently the surface became inert. Beside contact activation, factor V activity also decreased in the plasma. The decrease was surface and time-dependent. It was independent of contact factor activation, and appeared to be related to the sulfonated groups of the polymer. If purified factor V was used instead of plasma factor V, inactivation was immediate and not time-dependent suggesting a direct adsorption on the surface. A second incubation of the plasma-contacted polymer with fresh plasma resulted in a further loss of Factor V activity.

Electrolytes↗