Dugina Tn

Receptors of the PAR Family as a Link between Blood Coagulation and Inflammation

Blood coagulation plays a key role among numerous mediating systems that are activated in inflammation. Receptors of the PAR family serve as sensors of serine proteinases of the blood clotting system in the target cells involved in inflammation.Activation of PAR-1 by thrombin and of PAR-2 by factor Xa leads to a rapid expression and exposure on the membrane of endothelial cells of both adhesive proteins that mediate an acute inflammatory reaction and of the tissue factor that initiates the blood coagulation cascade. Certain other receptors (EPR-1, thrombomodulin, etc.), which can modulate responses of the cells activated by proteinases through PAR receptors, are also involved in the association of coagulation and inflammation together with the receptors of the PAR family. The presence of PAR receptors on mast cells is responsible for their reactivity to thrombin and factor Xa and defines their contribution to the association of inflammation and blood clotting processes.

Immobilized Thrombin Receptor Agonist Peptide Accelerates Wound Healing in Mice

To accelerate the healing processes in wound repair, attempts have been repeatedly made to use growth factors including thrombin and its peptide fragments. Unfortunately, the employment of thrombin is limited because of its high liability and pro-inflammatory actions at high concentrations. Some cellular effects of thrombin in wound healing are mediated by the activation of protease activated receptor-1 (PAR-1). The thrombin receptor agonist peptide (TRAP:SFLLRN) activates this receptor and mimics the effects of thrombin, but TRAP is a relatively weak agonist. We speculated that the encapsulated peptide may be more effective for PAR-1 activation than nonimmobilized peptide and developed a novel method for TRAP encapsulation in hydrogel films based on natural and synthetic polymers. The effects of an encapsulated TRAP in composite poly(N-vinyl caprolactam)-calcium alginate (PVCL) hydrogel films were investigated in a mouse model of wound healing. On day 7 the wound sizes decreased by about 60% under TRAP-chitosan-containing PVCL films, as compared with control films without TRAP. In the case of TRAP-polylysine-containing films no significant decrease in wound sizes was found. The fibroblast/macrophage ratio increased under TRAP-containing films on day 3 and on day 7. The number of proliferating fibroblasts increased to 150% under TRAP-chitosan films on day 7 as compared with control films. The number of [3H]-thymidine labeled endothelial and epithelial cells in granulation tissues was also enhanced. Thus, the immobilized TRAP to PVCL-chitosan hydrogel films were found to promote wound healing following the stimulation of fibroblast and epithelial cell proliferation and neovascularization. Furthermore, TRAP was shown to inhibit the secretion of the inflammatory mediator PAF from stimulated rat peritoneal mast cells due to augmentation of NO release from the mast cells. The encapsulated TRAP is suggested to accelerate wound healing due to the anti-inflammatory effects and earlier development of the proliferative phase of wound healing.

Stabilization of proteases by entrapment in a new composite hydrogel

A new one-step procedure for entrapping proteases into a polymeric composite calcium alginate-poly(N-vinyl caproladam) hydrogel was developed that provided 75–90% retention of the activity of entrapped enzymes compared to soluble ones. Properties of entrapped carboxypeptidase B, trypsin, and thrombin were investigated. The immobilized enzymes were active within a wide pH range. The temperature optima of entrapped trypsin and carboxypeptidase B were approx 25°C higher than that of the soluble enzymes, and the resistance to heating was also increased. The effects of various polar and nonpolar organic solvents on the entrapped proteases were investigated. The immobilized enzymes retained their activity within a wide concentration range (up to 90%) of organic solvents. Gel-entrapped trypsin and carboxypeptidase (CPB) were successfully used for obtaining human insulin from recombinant proinsulin. The developed stabilization method can be used to catalyze various reactions proceeding within wide pH and temperature ranges.

Activation of mast cells induced by agonists of proteinase-activated receptors under normal conditions and during acute inflammation in rats.

Functions of thrombin as a modulator of inflammation and tissue repair are mediated by the proteinase-activated receptor (PAR) family. Some of these effects may be induced by activation of mast cells. To characterize the degranulation of rat peritoneal mast cells in response to PAR agonists, the effects of thrombin, trypsin and peptide agonists of PARs (PAR-AP, proteinase-activated receptor-activating peptides) on secretion were investigated. The release of beta-hexosaminidase by thrombin (0.01-1 microM) was concentration-dependent and mediated via PAR(1), as evidenced by cathepsin G (100 microM)-induced inactivation of PAR(1) and thrombin-stimulated PAR(1) desensitization. Trypsin (1 microM) accelerated histamine secretion. The PAR(1)-AP, TRAP (SFFLRN, 1-100 microM) and the PAR(2)-AP SLIGRL (5-100 microM) caused the release of histamine, and beta-hexosaminidase from inflammatory mast cells were obtained from a model of acute peritonitis in rats. Relative to the response to compound 48/80, the thrombin- and TRAP-induced release of beta-hexosaminidase was higher in inflammatory mast cells than in the control. This suggests that additional exposure of PAR(1) on mast cells to PAR agonists or an increase in PARs sensitivity to PAR agonists probably occurred during acute inflammation.

IMMOBILIZED ENZYMES AND CELLS IN POLY(N-VINYL CAPROLACTAM)-BASED HYDROGELS: PREPARATION, PROPERTIES, AND APPLICATIONS IN BIOTECHNOLOGY AND MEDICINE

A one-step mild method for entrapping animal cells and enzymes in macroporous composite poly (N-vinyl caprolactam)-calcium alginate (PVCL-CaAlg) hydrogels is described. Some properties of immobilized enzymes, such as thermal and storage stabilities and stability in water/organic media were investigated. Composite PVCL-CaAlg gels were successfully applied to immobilize a number of proteases, namely, trypsin, α-chymotrypsin, carboxypeptidase B, and thrombin. Thermal stability of the immobilized preparations obtained by entrapment in hydrogel beads allowed us to use them at 65–80†C, while the native enzymes were completely inactivated at 50–55°C. Various applications of enzymes and cells immobilized in beads weredemonstrated. Immobilized trypsin and carboxypeptidase B were applied to prepare human insulin from recombinant proinsulin. The hydrogel beads with entrapped α-chymotrypsin were used in enantioselective hydrolysis of Shiffs base of D,L-phenylalanine ethyl ester (SBPH) in acetonitrile/water medium. Thrombin immobilized in PVCL-based hydrogel films was shown to be a promising compound for wound treatment. To prepare pure preparations of monoclonal antibodies (MAb) several hybridoma cell lines, including hybridoma cell lines producing MAb to interleukin-2, were successfully cultivated in the hydrogel beads.

Activation of rat mast cells upon stimulation of protease-activated receptor (PAR-1)

In vivo experiments on the model of wound healing showed that thrombin and thrombin receptor agonist TRAP-6 stimulated heparin secretion by mast cells in rat subcutaneous fat: the saturation of mast cells with heparin decreased, while degranulation and granulolysis increased.In vitro studies showed that TRAP-6 caused a dose-dependent release of β-hexosaminidase from peritoneal mast cells. TRAP-6 also induced heparin release from these cells and inhibition of amidase activity of thrombin. Heparin released from mast cells had low anticoagulant activity. These data suggest that activation of mast cells with thrombin is mediated by PAR-1.

Study of neurotrophic activity of thrombin on the model of regenerating mouse nerve.

Experiments demonstrated a dose-dependent facilitating effect of thrombin and peptide thrombin receptor agonist PAR1 (TRAP6) on regeneration of mouse peripheral nerve after its crushing. The maximum neurotrophic effect was observed at low concentrations of thrombin (10 nM) and TRAP6 (10 µM).

Effect of synthetic peptide thrombin receptor agonist encapsulated in microparticles based on lactic and glycolic acid copolymer on healing of experimental skin wounds in mice.

PAR1 peptide thrombin receptor agonist (PAR1-AP) was encapsulated in microcorpuscles based on lactic and glycolic acid copolymer. The desorption profile of the preparation was studied in vitro and its wound-healing effects were studied on a model of cut skin wound in mice. The study showed that 90% PAR1-AP was desorbed over 6 h, but the peptide was detected in eluates from the microparticle surface after 23 h. The desorbed peptide retained its physiological activity and was capable of activating PAR1 receptors on human platelets. The study of the dynamics of experimental skin wound healing in mice showed lower number of macrophages in the wounds treated with PAR1-AP microparticles compared to the control (open wounds and wounds covered with microparticles) and higher number of fibroblasts on day 3 of tissue reparation. Hence, PAR1-AP desorbed from microparticles shortened the inflammation phase in the wound. On day 7 the best healing parameters were also observed in wounds treated with PAR1-AP microparticles, which attests to shortening of the proliferation phase and acceleration of wound healing.

[Aptameric DNA--a new type of thrombin inhibitor]

The formation of complexes between various thrombin preparations and 30-mer aptamer DNA was comparatively studied, and a correlation between the complex formation and the fibrinogen-hydrolyzing activity of thrombin was found. The aptamer DNA was shown to inhibit the formation of fibrin from fibrinogen.

Synthetic analog of the thymosinα1 fragment 24–28 alters the coagulating and aggregating activities of α-thrombin

A pentapeptide (EEAEN), which is the 24–28 fragment of the COOH-terminal sequence in the thymosinα1 molecule highly homologous with the 54–58 region of hirudin (with the complementary anion-binding exosite in the thrombin molecule), was synthesized by a solid-phase method. Preincubation of α-thrombin with EEAEN in concentrations of 0.1 pM to 1 nM reduced its clotting activity while preincubation of this enzyme with EEAEN in concentrations of 0.01 to 1 nM reduced its platelet-aggregating activity. The reaction of EEAEN with thrombin is shown to be similar to the reaction of the entire thymosinα1 molecule. It is concluded that the COOH-terminal thymosinα1 peptide EEAEN may be the reactive site responsible for the antithrombin activity of thymosinα1.