Fibrinogen-sepharose interaction with prothrombin, prethrombin 1, prethrombin 2 and thrombin.

Binding of prothrombin, prethrombin 1, prethrombin 2 and thrombin to fibrinogen-Sepharose was studied. Thrombin and prethrombin 2 bound to fibrinogen-Sepharose, while prethrombin 1 and prothrombin did not. Bound thrombin and prethrombin 2 were recovered from the column by eluting with 0.

Highly enriched preparations of cultured myocardial cells for biochemical and physiological analyses.

Cultures of enzyme-dissociated myocardial cells contain myocytes as well as other cell types (e.g. fibroblasts); therefore, cell separation is necessary to interpret accurately biochemical measurements of substances or physiological measurements of processes common to more than one type of cell.

Inhibited thrombins. Interactions with fibrinogen and fibrin.

Fibrin-monomer-Sepharose was used to study thrombin binding to fibrin and the role of the enzyme active centre in this interaction. Binding properties of preformed enzyme-inhibitor complexes, as well as inhibition of thrombin already adsorbed to fibrin monomer, were investigated. No apparent difference was found in binding properties of phenylmethanesulphonyl fluoride-, D-Phe-Pro-Arg-CH2Cl- and dansylarginine NN-(3-ethylpentane-1,5-diyl)amide-inhibited thrombins.

Factor XIII A is synthesized and expressed on the surface of U937 cells and alveolar macrophages.

Factor XIII A subunit was detected in U937 cells and human alveolar macrophages by immunohistology and Western blotting. U937 cells synthesize factor XIII A subunit de novo under serum-free, platelet-free conditions, as indicated by 35S-methionine labeling and immunoprecipitation. Thrombin-dependent activity was demonstrated to account for 98% of the total transglutaminase activity in U937 cells (1.

Characterization of thrombospondin as a substrate for factor XIII transglutaminase.

Thrombin activation of platelets induces the release of a high molecular weight glycoprotein, thrombospondin. On treatment with factor XIII transglutaminase and [3H]putrescine, thrombospondin undergoes specific incorporation of this labeled amine, with 2-3 mol of putrescine being incorporated per mol of thrombospondin. Analysis of plasmin digests of [3H]putrescine-thrombospondin showed that the Mr 53,000-core peptide contains the glutamine site for amine incorporation.

Biosynthesis of factor XIII B subunit by human hepatoma cell lines.

The plasma transglutaminase, factor XIIIa (FXIIIa), circulates as a zymogen containing two proteins, A and B, arranged in a noncovalent tetrameric complex, A2B2. Biosynthesis of plasma FXIII has not previously been demonstrated. In the present study, direct evidence has been obtained that two human hepatoma cell lines, Hep G2 and PLC/PRF/5, synthesize and secrete FXIII B protein.

Detection of malignant tumors. Water-suppressed proton nuclear magnetic resonance spectroscopy of plasma.

A sensitive and specific blood test for cancer has long been sought. The water-suppressed proton nuclear magnetic resonance (NMR) spectrum of plasma is dominated by the resonances of plasma lipoprotein lipids. We measured the mean line widths of the methyl and methylene resonances, which were found to be correlated with the presence or absence of malignant tumors.

Influence of Ca2+ on the structure of reptilase-derived and thrombin-derived fibrin gels.

The effects of Ca2+ ion on the structure of thrombin-derived and reptilase-derived fibrin gels formed at various ionic strengths were studied turbidimetrically. For both enzymes clotting times were shorter, final gel turbidities were higher and fibre mass/length ratios were increased as the ionic strength was lowered. The addition of 5 mM-Ca2+ augmented each of these effects for any given ionic strength.

Binding and covalent cross-linking of purified von Willebrand factor to native monomeric collagen.

We have analyzed the interaction of the adhesive glycoprotein, von Willebrand factor (vWF), with native monomeric collagen monolayers by adsorbing acid soluble Types I and III collagen derived from calf skin to polystyrene microtiter wells and incubating the wells with purified human 125I-vWF. The binding of 125I-vWF was saturable, reversible, specific, and was abolished by heat denaturation of the collagen monomers. Binding was half-maximal at 5 micrograms/ml, and, at saturation, 7.

Factor V is a substrate for the transamidase factor XIIIa.

Coagulation Factor V (Mr = 330,000), upon cleavage by thrombin, produces Factor Va, which is composed of two subunits with Mr values of 94,000 and 74,000, along with two activation fragments possessing no known function. Studies were undertaken to assess the ability of the transamidase Factor XIIIa to covalently incorporate the lysine analogs [3H]putrescine and dansylcadaverine into the thrombin-cleaved (activated) and unactivated forms of human and bovine Factor V. The incorporation of either probe into thrombin-activated Factor V proceeded at an initial rate approximately twice that for unactivated Factor V.

Covalent crosslinking of von Willebrand factor to fibrin.

Factor XIIIa crosslinks a limited number of substrates via epsilon(gamma-glutamyl)-lysyl bond formation. It crosslinks fibrin to itself, alpha 2-plasmin inhibitor and fibronectin to fibrin, and fibronectin to collagen. Results presented here show that plasma von Willebrand factor (vWF) is a substrate for factor XIIIa and can be crosslinked to fibrin during gel formation.

Analysis of elevated fibrin(ogen) degradation product levels in patients with liver disease.

Plasma and serum from patients with liver disease and elevated fibrin(ogen) degradation product (FDP) levels as measured by latex agglutination were analyzed by immunoblotting to characterize the FDP in these patients. An antihuman fibrinogen antibody was used that recognizes fibrinogen, fibrin monomer, soluble high molecular weight fibrinogen and fibrin polymers, as well as high molecular weight cross-linked degradation fragments, and the smaller fragments X, Y, D-dimer, D, and E. The analytic procedures were validated with plasma and serum from patients known to have intravascular fibrinolysis associated either with disseminated intravascular coagulation (DIC) or with thrombolytic therapy.

Macrophage migration in fibrin gel matrices.

Macrophage migration has been extensively studied in vitro on artificial substrates but in vivo macrophages migrate through connective tissue and fibrin gel meshworks that comprise the stroma of many inflammatory reactions and solid tumors. Studies were therefore undertaken to investigate macrophage migration in this more biologically relevant matrix cast either with or without nitrocellulose filter support. Macrophage migration in fibrin gels depended on both fibrin and thrombin concentrations and on the nature of fibrin crosslinking.

Identification of intracellular factor XIII in human monocytes and macrophages.

Factor XIII is a blood protransglutaminase that is distributed in plasma and platelets. The extracellular and intracellular zymogenic forms differ in that the plasma zymogen contains A and B subunits, while the platelet zymogen has A subunits only. Both zymogens form the same enzyme.

Regulation of extravascular coagulation by microvascular permeability.

Extravascular coagulation is a prominent feature of such important pathological processes as cellular immunity and neoplasia and has been thought to result from procoagulants associated with the inflammatory or tumor cells peculiar to these entities. It was found that increased microvascular permeability alone is sufficient to induce equivalent extravascular coagulation in several normal tissues. The results indicate that saturating levels of procoagulant are present even in normal tissues and that microvascular permeability is a rate-limiting step in extravascular coagulation.

Quantitation of fibrinogen influx and fibrin deposition and turnover in line 1 and line 10 guinea pig carcinomas.

Radiolabeled guinea pig fibrinogen (GPF) was used to measure fibrinogen influx and fibrin accumulation in line 1 and line 10 hepato- (bile duct) carcinomas growing in the s.c. space of syngeneic strain 2 guinea pigs over the course of 7 days following transplant, an interval of growth uncomplicated by immunological tumor rejection or by significant tumor necrosis.

Characterization of soluble polymerized fibrin formed in the presence of excess fibrinogen fragment D.

Polymerization of fibrin is inhibited in the presence of excess fibrinogen fragment D. This study was performed in order to test the proposal that these inhibited solutions contain short linear polymers of fibrin (protofibrils) whose further polymerization is prevented as a result of attachment of a molecule of fragment D at each end. Negative-stain electron micrographs, intrinsic viscosities, angular dependence of light scattering intensity, and kinetics of the increase of the scattered intensity with polymerization all were found to support the above model of the inhibited polymer and to reflect the presence of a broad distribution of the lengths of the inhibited fibrin polymers.

Immunocytochemical localization of albumin and factor XIII in thin cryo sections of human blood platelets.

Albumin and factor XIII alpha 2 were localized in human blood platelets by immunofluorescence and protein A-colloidal gold immuno-electron microscopy using thin and ultrathin cryosections of mildly fixed human blood platelets. Albumin was found in alpha-granules, co-distributing with beta-thromboglobulin in double label experiments. In platelets directly fixed in plasma, without previous washing, albumin was also localized on the plasma membrane and in the surface connecting system.

Dependence of the shape of the plasma fibronectin molecule on solvent composition. Ionic strength and glycerol content.

Fibronectin has been purified to apparent homogeneity according to measurements of molecular weight and diffusion constant from light scattering and sedimentation in the analytical ultracentrifuge. This gives two estimates of molecular weight close to 500,000. (The difference with the gel electrophoretic value of 440,000 may indicate the presence of some material of molecular weight higher than that of the monomer.

Studies on the mechanism of thrombin. Interaction with fibrin.

Fibrin monomer Sepharose was used to investigate the interactions of thrombin with fibrin. Thrombin binding was found to be reversible and saturable and to depend on the thrombin: fibrin ratio. Scatchard analysis indicated a single class of binding sites with K alpha = 4.

Hereditary dysfibrinogenemia in a patient with thrombotic disease.

A new case of congenital dysfibrinogenemia, in which the patient has severe thrombotic disease, is reported. The abnormal fibrinogen molecules are characterized by normal fibrinopeptide release with thrombin and defective polymerization in the formation of fibrin. Clotting times with ancrod and reptilase are significantly prolonged.

Thrombin clotting time and fibrinogen concentration in patients treated with coumadin.

Thrombin clotting times and fibrinogen levels were measured in plasma from patients on chronic coumadin therapy who had prolonged prothrombin times. Thrombin clotting times were usually prolonged. Fibrinogen concentrations were generally elevated, but some were normal or even low.

High resolution electrophoretic analysis of human fibrinogen and its crosslinked intermediates.

Heterogeneity in human fibrinogen was examined using an improved two-dimensional isoelectric focusing-SDS polyacrylamide gel electrophoretic procedure. Four different preparations of fibrinogen were compared: single donor fibrinogen prepared from plasma by precipitation with ammonium sulfate or by affinity chromatography on fibrin-monomer Sepharose, fraction 1-4 prepared from Cohn fraction I paste, and Kabi grade L. The subunit A alpha, B beta, and gamma chains in all preparations had marked charge heterogeneity.