Addition Of Epsilon-aminocaproic Acid Research Articles

Plasminogen binding by oral streptococci from dental plaque and inflammatory lesions

Plasminogen binding by bacteria is a virulence factor important for the entry and dissemination of bacteria in the body. A wide variety of bacteria bind plasminogen, including both organisms causing disease and components of the normal oral flora. The purpose of this study was to examine the characteristics of plasminogen binding by six clinical isolates of oral streptococci from both dental plaque and inflammatory lesions. All the strains bound plasminogen with approximately the same affinity, and binding was specific and lysine-dependent as evidenced by its inhibition by epsilon-aminocaproic acid. All of the test strains were capable of activating bound plasminogen to plasmin without the addition of a plasminogen activator, and subsequent analysis revealed the presence of streptokinase in all strains. However, the streptococci exhibited fibrinolytic activity only in the presence of plasminogen and this could be inhibited by the addition of epsilon-aminocaproic acid. SDS-PAGE and 2D gel electrophoresis coupled with plasminogen ligand blotting showed that only a subset of the total proteins (2-15) were involved in the binding of plasminogen. Partial identification of the binding proteins revealed that four glycolytic enzymes, enolase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate mutase, were predominant in binding plasminogen. The binding of plasminogen by bacteria from pus did not differ from that of the strains from supragingival plaque. The findings illustrate how apparently innocuous commensal bacteria are capable of utilizing a mechanism that is generally regarded as being of importance to pathogenicity and suggest an additional role of plasminogen binding.

Fibrin as an alternative biopolymer to type-I collagen for the fabrication of a media equivalent.

We report here on studies examining the use of fibrin as an alternative to collagen for the entrapment of neonatal aortic rat smooth muscle cells (SMCs) in the fabrication of media equivalents. The studies show increased collagen production by fibroblasts entrapped in fibrin, which suggests that fibrin may be used in the fabrication of tissue equivalents to promote increased protein synthesis and remodeling. However, one of the challenges of working with fibrin is the rapid degradation by SMCs. This degradation was effectively inhibited with the addition of epsilon-aminocaproic acid (EACA) to the culture medium in concentrations ranging from 0.25 to 1 mg/mL. We also present results showing that fibrin stimulates collagen production by SMCs. SMCs in fibrin produced 3.2 and 4.9 times the amount of collagen produced by SMCs in collagen when supplemented with 1 and 0.25 mg/mL EACA, respectively. More than half of the collagen produced appeared in the medium rather than the matrix. The collagen in the medium appeared to be processed beyond the proform and may be in an aggregate form. In addition, the presence of type-III collagen or a type-I trimer was indicated by the results of an analysis of the medium by autoradiography.

The solution phase interaction between apolipoprotein(a) and plasminogen inhibits the binding of plasminogen to a plasmin-modified fibrinogen surface.

In the present study, we assessed the binding of recombinant forms of apolipoprotein(a) [r-apo(a)] to plasminogen. Apo(a)-plasminogen interactions were demonstrated to be lysine-dependent, as they were abolished by the addition of epsilon-aminocaproic acid. Binding of r-apo(a) and plasma-derived Lp(a) to Glu-plasminogen was assessed in solution using a mutant form of recombinant plasminogen [Plg(S741C)] labeled at the active site with 5'-(iodoacetamido)fluorescein. High-affinity binding of apo(a) to plasminogen was observed with the 17-kringle r-apo(a) (Kd = 20.1 +/- 3.3 nM) as well as with plasma-derived Lp(a) (Kd = 5.58 +/- 0.08 nM). Binding studies using various truncated and mutant forms of r-apo(a) demonstrated that sequences within apo(a) kringle IV types 2-9 and the strong lysine binding site (LBS) in apo(a) kringle IV type 10 are not required for high-affinity binding to plasminogen. In all cases, the binding stoichiometry for the apo(a)-plasminogen interaction was determined to be 1:1. Binding data obtained using a 17-kringle r-apo(a) derivative lacking the protease-like domain (17KDeltaP; Kd = 3158 +/- 138 nM) indicate that sequences within the protease-like domain of apo(a) mediate its interaction with LBS in plasminogen. We determined that r-apo(a) and plasminogen bind to distinct sites on plasmin-modified fibrinogen with the concentration of plasminogen binding sites exceeding the concentration of r-apo(a) sites by a factor of 10. Furthermore, r-apo(a) is capable of inhibiting the binding of plasminogen to plasmin-modified fibrinogen surfaces, an effect which we show is attributable to the formation of a solution phase apo(a)/plasminogen complex which exhibits a greatly reduced affinity for plasminogen binding sites on plasmin-modified fibrinogen. The results of this study provide new insights into the mechanism by which apo(a) and Lp(a) may inhibit fibrinolysis, thus contributing to the atherothrombotic risk associated with this lipoprotein.

Osteonectin in matrix remodeling. A plasminogen-osteonectin-collagen complex.

Osteonectin is an adhesive glycoprotein synthesized constitutively by osteoblasts, endothelial cells, and megakaryocytes. Bone-derived and platelet-derived osteonectins differ in their electrophoretic mobility and carbohydrate content, and each displays different affinities for collagen matrices. Both types of osteonectin bind to plasminogen (Kd(app), of 4.7 +/- 1.0 x 10(-8) M for bone osteonectin and 1.2 +/- 0.1 x 10(-7) M for platelet osteonectin). The osteonectin-plasminogen interaction is inhibited by alpha 2-antiplasmin and epsilon-aminocaproic acid, suggesting that the interaction is mediated through the kringle 1 region of plasminogen. Both osteonectins enhance the rate of plasmin generation by tissue-type plasminogen activator to approximately the same extent as fibrinogen. Equilibrium binding measurements conducted using total internal reflection fluorescence spectroscopy indicate that plasminogen binds to collagen in the presence of bone osteonectin (Kd = 1.30 +/- 0.1 x 10(-7) M). No binding of plasminogen to collagen matrix was detected in the presence of platelet osteonectin or in the absence of bone osteonectin. Bone osteonectin-dependent binding of plasminogen to collagen matrix is reversed by the addition of epsilon-aminocaproic acid. The ability of both types of osteonectin to bind to and influence plasminogen activation and of bone osteonectin to anchor plasminogen on collagen matrices suggests that osteonectin may play a role in directing extracellular matrix proteolysis.

Role of plasminogen activator and of 92-KDa type IV collagenase in glioblastoma invasion using an in vitro matrigel model.

The invasive nature of human gliomas represents a major factor in preventing their total resection. The exact nature of the underlying mechanisms of tumor cell invasion are still unclear. In this study, we have quantitatively assayed a glioblastoma cell line for its ability to migrate through a polycarbonate filter coated with matrigel which contains a complex of multiple basement membrane components. At 48 h the glioblastoma cell line (U251) showed a rate of invasiveness of 42% and also dependent on the concentration of matrigel. The U251 cell line produced a urokinase type plasminogen activator and a 92-KDa type IV collagenase. Both enzymes were inhibited by the addition of uPA and 92-KDa type IV collagenase antibodies. Those same antibodies reduced the invasion rate of U251 cells from 42% to 12 and 21%, respectively. Similarly, the addition of epsilon-aminocaproic acid (a plasmin inhibitor) or tissue inhibitor of metalloprotease (TIMP2, a collagenase inhibitor) reduced the invasiveness of U251 cells from 42% to 14% and 10%, respectively. Additionally, the other two glioblastoma cell lines (LG11, UWR1) and astrocytes showed a rate of invasiveness at 41%, 61% and 12%, respectively. Finally, the addition of hyaluronic acid to the matrigel, a constituent of brain extracellular matrix, enhanced the rate of invasion. These findings provide evidence for the role of serine proteases and metalloproteases in facilitating the invasion of extracellular matrix components by glioblastoma cell line and suggest a therapeutic role for protease inhibitors in attempting to minimize the invasive propensity of gliomas.

Pepsin fibrinolysis of artificial clots made from fibrinogen concentrate and bovine thrombin: the effect of pH and epsilon aminocaproic acid

Artificial clots made from fibrin glue with and without an inhibitor of fibrinolysis can be used to treat gastrointestinal bleeding. We have been unable to find descriptions of the effects of acid and pepsin upon such artificial clots. Therefore, 10(-2) mol/l epsilon aminocaproic acid was added to fibrin glue in vitro at acid concentrations of pH 1.0 and pH 5.5. Pepsin was added at 9000 U/50 ml, the expected value for fasting human subjects. There was a highly significant reduction in clot survival at pH 1.0. At pH 5.5, clot weight was also significantly decreased with pepsin, compared to control. Thus pepsin and acidity greatly affect survival of artificial clots, but the addition of epsilon aminocaproic acid did not affect clot survival.

Growth characteristics of human esophageal epithelial cells in primary explant and serial culture.

Growth characteristics of human esophageal epithelial cells have been determined in primary explant and serial culture. Normal human esophagus was obtained from donor patients in a heart/lung transplantation program; tissue obtained at autopsy (6 to 22 h after death) was not viable. When mucosal specimens (1.5 mm2) were explanted on a plastic surface and attached with a plasma clot, 35% of explants detached from the surface within 48 h. The addition of epsilon amino caproic acid (EACA) to the culture medium increased explant attachment to 93% (P less than 0.001). Outgrowth kinetics were similar in both the presence and absence of EACA. No advantage of human serum over nonhuman sera was observed in primary culture. Esophageal epithelium could be frozen in 10% dimethyl sulfoxide without affecting growth kinetics. Addition of dexamethasone (DEX) significantly altered esophageal cell morphology in primary culture and increased viability on serial culture. Studies of pH revealed an optimum at pH 7.4 with significantly decreased growth occurring at 6.8 and no growth at 6.2. Esophageal cells in primary explant cultures could be released by trypsin and passaged two additional times with an eightfold increase in total number. An increased rate of attachment and multiplication was observed for cells plated on a collagen substrate compared to plastic. The addition of EACA and DEX to the culture media and the subculture on a collagen substrate provide a method for the isolation and serial cultivation of human esophageal cells from biopsy-sized specimens of normal esophageal epithelium.

Properties of a Factor Increasing Monocytopoiesis (FIM) Occurring in Serum During the Early Phase of an Inflammatory Reaction

A factor increasing monocytopoiesis (FIM) has been demonstrated during the onset of an acute inflammatory reaction caused by an intraperitoneal injection of polystyrene latex particles. It is protein in nature, does not contain a carbohydrate moiety essential for its function, and is very probably not a glycoprotein. The molecular weight of FIM lies between 18,000 and 24,000 daltons (determined with both ultrafiltration membranes and gel filtration on Sephadex G100). The monocytosis induced by FIM is dose dependent. FIM is thermolabile, having a half-time of about 20 min at 37 degrees C in serum; temperature inactivation can be delayed by the addition of epsilon-aminocaproic acid, the half-time at 37 degrees C then being about 45 min. In vitro treatment of normal murine blood with the inducers of the inflammatory reaction does not result in FIM activity in the serum. FIM dose not have chemotactic activity toward macrophages, is not a clotting factor, is not a biologically active fragment of the complement system, and has no colony-stimulating or-enhancing activity in the vitro bone marrow colony assay. On the basis of these results, a mechanism is postulated for the humoral regulation of monocytopoiesis.

Altered morphology and increased cell adhesiveness of chinese hamster ovary cells cultured on fibrin.

Chinese hamster ovary cells cultivated on fibrin exhibited different characteristic from cells growing on plastic. While sparsely plated cells on plastic dishes had an epithelioid morphology, cells on fibrin assumed a round shape and then converted to a stretched form with protruded processes that increased with cell density. Within a few days, cells fibrinolysed adjacent fibrin and returned to the morphology seen in plastic dishes. When fibrinolysis was inhibited by epsilon-aminocaproic acid (EACA), cells continued to grow on the fibrin for a longer period and showed dense, criss-crossed fibroblast-type congestion. Whereas, cells on plastic maintained pavement-like epitheloid appearance when they grew to a confluent monolayer. The other altered characteristics on fibrin was increased accumulation of cells in multilayers. Normally as Chinese hamster cells on plastic proliferate, many cells float into the medium instead of piling up after they form a monolayer. On the other hand, cells on fibrin, being maintained by the addition of EACA, remained adherent, piling up multilayers instead of floating into the medium. A possible explanation of these findings is that the surface properties of the stretched cells on fibrin are altered to make them more adhesive. A possible link of these characteristics of the cells on fibrin to tumor cell behavior in vivo is dicussed.

Effect of Fibrinolysin on the Coagulability of Fibrinogen

SummaryStreptokinase or glycerol activated human fibrinolysin was added to human plasma and the enzyme action was stopped by the addition of epsilon amino-caproic acid at different time intervals. The clottable fibrinogen content of the mixtures was determined by diluting the plasma with either physiological saline or distilled water, both containing thrombin or thrombin plus calcium chloride. It was found that before the addition of fibrinolysin the values in distilled water containing calcium chloride were higher than the respective values in plain saline or in saline containing calcium chloride. The drop in clottable fibrinogen after the addition of fibrinolysin was more pronounced when the determinations were performed in plain distilled water. Thus, a crossover between the values obtained in plain distilled water and those in saline gradually occurred, the values in distilled water without calcium chloride becoming the lowest. This is at variance with the results obtained in the presence of added AFIF or fibrinolysed fibrinogen where the values in distilled water were always higher than the ones obtained in saline both in the presence and in the absence of calcium chloride. Since the reversal of coagulability of fibrinogen in physiological saline versus distilled water was reproduced only when we mixed partially lysed human fibrinogen with completely lysed human plasma, the conclusion was drawn that three prerequisites are necessary for this phenomenon to occur: a) the fibrinogen must have been acted upon by fibrinolysin; b) the fibrinogen must be of human origin; c) a substance produced by the action of fibrinolysin on a plasmatic factor other than fibrinogen or, possibly, an intact plasmatic factor, must be present.