A New Concept of Action of Hemostatic Proteases on Inflammation, Neurotoxicity, and Tissue Regeneration.

Key hemostatic serine proteases such as thrombin and activated protein C (APC) are signaling molecules controlling blood coagulation and inflammation, tissue regeneration, neurodegeneration, and some other processes. By interacting with protease-activated receptors (PARs), these enzymes cleave a receptor exodomain and liberate new amino acid sequence known as a tethered ligand, which then activates the initial receptor and induces multiple signaling pathways and cell responses. Among four PAR family members, APC and thrombin mainly act via PAR1, and they trigger divergent effects. APC is an anticoagulant with antiinflammatory and cytoprotective activity, whereas thrombin is a protease with procoagulant and proinflammatory effects. Hallmark features of APC-induced effects result from acting via different pathways: limited proteolysis of PAR1 localized in membrane caveolae with coreceptor (endothelial protein C receptor) as well as its targeted proteolytic action at a receptor exodomain site differing from the canonical thrombin cleavage site. Hence, a new noncanonical tethered PAR1 agonist peptide (PAR1-AP) is formed, whose effects are poorly investigated in inflammation, tissue regeneration, and neurotoxicity. In this review, a concept about a role of biased agonism in effects exerted by APC and PAR1-AP via PAR1 on cells involved in inflammation and related processes is developed. New evidence showing a role for a biased agonism in activating PAR1 both by APC and PAR1-AP as well as induction of antiinflammatory and cytoprotective cellular responses in experimental inflammation, wound healing, and excitotoxicity is presented. It seems that synthetic PAR1 peptide-agonists may compete with APC in controlling some inflammatory and neurodegenerative diseases.

Effects of activated protein C on the size of modeled ischemic focus and morphometric parameters of neurons and neuroglia in its perifocal zone.

The effects of activated protein C (APC) on the quantitative parameters of neurons and neuroglia in the perifocal zone of infarction induced in the left hemispheric cortex were studied in two groups of rats. Group 1 animals served as control (control infarction). Group 2 rats were injected with APC (50 µg/kg) in the right lateral cerebral ventricle 3 h after infarction was induced, and after 72 h the infarction size was evaluated and the neurons and neuroglia in the perifocal zone were counted. APC reduced the infarction size 2.5 times in comparison with the control and reduced by 16% the neuronal death in the perifocal zone layer V, causing no appreciable changes in layer III, and did not change the size of neuronal bodies but increased (by 11%) the size of neuronal nuclei in layer III. The protein maintained the sharply increased count of gliocytes in the perifocal zone of infarction and promoted their growth. Hence, APC protected the neurons from death in the ischemic focus by increasing the gliocyte count and stimulating the compensatory reparative processes.

Effect of enteropeptidase on survival of cultured hippocampal neurons under conditions of glutamate toxicity.

The effects of full-size bovine enteropeptidase (BEK) and of human recombinant light chain enteropeptidase (L-HEP) on survival of cultured hippocampal neurons were studied under conditions of glutamate excitotoxicity. Low concentrations of L-HEP or BEK (0.1-1 and 0.1-0.5 nM, respectively) protected hippocampal neurons against the death caused by 100 µM glutamate. Using the PAR1 (proteinase-activated receptor) antagonist SCH 79797, we revealed a PAR1-dependent mechanism of neuroprotective action of low concentrations of enteropeptidase. The protective effect of full-size enteropeptidase was not observed at the concentrations of 1 and 10 nM; moreover, 10 nM of BEK caused death of 88.9% of the neurons, which significantly exceeded the cell death caused by glutamate (31.9%). Under conditions of glutamate cytotoxicity the survival of neurons was 26.8% higher even in the presence of 10 nM of L-HEP than in the presence of 10 nM BEK. Pretreatment of cells with 10 nM of either form of enteropeptidase abolished the protective effect of 10 nM thrombin under glutamate cytotoxicity. High concentrations of BEK and L-HEP caused the death of neurons mainly through necrosis.

[Duodenase activates rat peritoneal mast cells via protease-activated receptors of type 1].

It was found that duodenase, a serine protease from the bovine duodenum, activates rat peritoneal mast cells (PMC) in vitro presumably via protease-activated receptors (PARs). Like thrombin (a serine protease from the blood coagulation system) and the PAR1 agonist peptide (PAR1-AP), duodenase was shown to accelerate the secretion of beta-hexosaminidase (a marker of cell degranulation) by PMC in a dose-dependent manner. The blockage of the proteolytic activity of duodenase toward the substrate Tos-Gly-Pro-Lys-pNA by the soybean Bauman-Birk protease inhibitor substantially reduced (by 40%) the ability of duodenase to stimulate the secretory activity of PMC. Pretreatment of PMC with duodenase decreased the beta-hexosaminidase secretion induced by thrombin and PAR1-AP by 35 and 41.7%, respectively, and abolished the antiinflammatory effect of activated protein C. At the same time, pretreatment of PMC with duodenase did not affect the secretion of beta-hexosaminidase induced by compound 48/80, a nonspecific degranulator of mast cells. Duodenase, unlike PAR1-AP (30-100 microM), in a broad concentration range (10-100 nM) did not induce aggregation of human platelets, but suppressed the platelet aggregation elicited by PAR1-AP.

Role of macromolecular binding site of thrombin molecule in excitation of anticoagulation system.

The reaction of anticoagulation system upon perfusion of humorally isolated (with retained innervation) carotid sinus of a rabbit by alpha-,beta/gamma-, DIP-alpha-thrombin and prethrombin I was studied. DIP-alpha-thrombin without clotting activity was shown to initiate like alpha-thrombin the reflex reaction of anticoagulation system characterized by a sharp increase in non-enzymatic fibrinolysis (by 225%) and total fibrinolytic activity of blood (by 51%). Prethrombin I (thrombin precursor) is also capable of exciting the function of anticoagulation system characterized by an increase in non-enzymatic fibrinolysis (by 82%) and total fibrinolytic activity (by 36%). Furthermore, perfusion of prethrombin I or alpha-thrombin at almost the same molar concentrations resulted in the similar degree of anticoagulation system effector reaction. Reflex response of anticoagulation system was not observed upon perfusion of carotid sinus by beta/gamma-thrombin that has high esterase but little if any clotting activity that appears to be due to molecular changes in the macromolecular binding site region. These data support the suggestion that the effect of anticoagulation system excitation is due to interaction of the macromolecular binding site in the structure of alpha-thrombin with anticoagulation system chemoreceptors.

Protein C activator from the venom of Agkistrodon blomhoffi ussuriensis retards thrombus formation in the arterio-venous shunt in rats.

Protein C (PC) is an anticoagulant protein which, being activated by thrombin, degrades factors V/Va and VIII/VIIIa and releases a tissue-type plasminogen activator. Some Agkistrodon snake venoms contain PC activators which, in experiments, exert an anticoagulant action. An antithrombotic effect of the PC activator from the venom of A. blomhoffi ussuriensis on the model of thrombus formation in the arterio-venous shunt in rats was under investigation. Administration of the PC activator resulted in a dose-dependent prolongation of the thrombus formation time and a decrease in plasma PC activity, which were accompanied by a decrease in factor V activity and APTT prolongation. No reliable changes in the t-PA level, ADP- and epinephrine-induced platelet aggregation were observed. Platelet adhesion to glass beads diminished. We assume that the antithrombotic effect of the PC activator from the A. blomhoffi venom in the platelet-dependent thrombosis model is caused by PC activation and subsequent factor V inactivation as well as by platelet adhesiveness reduction.

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 caprolactam) 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 degrees 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.

[Aptameric DNA--a new type of thrombin inhibitor]. / Aptamernye DNK--ingibitory trombina novogo tipa.

The formation of complexes between various thrombin preparations and a 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 fibrin formation from fibrinogen.

[Thrombin--a regulator of reparative processes in wound healing]. / Trombin--reguliator reparativnikh protsessov pri zazhivlenii ran.

Thrombin, binding to receptors of the protease activated receptor (PAR) family, is involved in wound healing by inducing the reparation processes and regulating the activity of mast cells, which secrete mediators of inflammation. Using thrombin receptor agonist peptide (TRAP-6) for the activation of rat mast cells, effect of several receptors, including PAR-1, on mast cells was demonstrated. It was shown that TRAP increases the concentration of Ca2+ in the cytoplasm of mast cells and regulates cell degranulation, while releasing nitrogen oxide. Thrombin encapsulated in poly(N-vinyl caprolactam)-calcium alginate (PVCL-Ca-Alg) hydrogel films promotes wound healing in rats as demonstrated by the acceleration of fibroblast proliferation and neovascularization.

[Characteristic properties of thrombin neurotropic activity]. / Osobennosti neirotropnogo deistviia trombina.

There are considered the characteristic features of thrombin functional activity in central and peripheral nervous system. A family of specialized membrane receptors--so called PARs (Proteinase Activated Receptors) and their presence in several parts of CNS is described. The concentration- and PAR-dependent neuroprotecting and injuring effects of thrombin in CNS are compared. The literature and original authors data are presented demonstrating the presence of PARs in peripheral nervous system and the ability of endogenous and exogenous thrombin to influence the regeneration of peripheral nerves. The perspectives of experimental approach are discussed, when the exogenous thrombin or peptide-agonists of PARs are used to accelerate the nerve regeneration in vivo.

[Morphologic and molecular aspects of the interaction of platelets with elements of the vascular wall]. / Morfologicheskie i molekuliarnye aspekty vzaimodeistviia trombotsitov s élementami sosudistoi stenki.

The review presents and analyzes the data available on the structure and functions of adhesive proteins, Willebrand's factor, fibrinogen, thrombospondin, and fibronectin, their secretion and interaction with blood cells and subendothelial matrix in the focus of endothelial desquamation. A structural similarity of these modular glycoproteins, their domain structure-related functions, distinctive features of cellular reception are shown. The authors also consider structural changes in platelets in their activation, rearrangement of glycoprotein complexes IIb/IIIa on the membrane for binding adhesive proteins, role of these proteins in the organization of reversible and stabilized cellular aggregates and their location in the area of the damaged endothelium. The evidence is given for one of the most important mechanisms of limiting the platelet formation-activation system of C protein, which is mediated through thrombin binding to endothelial thrombomodulin. Hemostatic abnormalities occur with congenital defects in the structure of adhesive factors or their receptors while protein deficiency in the C protein system results in formation of thromboses.

[Structural and functional bases of hemostasis and its pathology]. / Strukturno-funktsional'nye osnovy gemostaza i ego patologiia.

The multiphase process of blood coagulation occurs owing to the interaction of three links of hemostasis--thrombocyte apparatus, plasma factors, and vascular wall components--and is regulated by the neuro-reflectory mechanisms of the coagulating and anticoagulating systems. At the early stage of hemostasis, a set of reactions begins in the thrombocyte apparatus leading to cell aggregation, secretion of the content of granules, synthesis of prostaglandines and eventually to the formation of thrombocyte thrombus. Disorders in ultrastructures and membranes of thrombocytes, particularly in receptor glycoproteins lead to the development of molecular disease of thrombocyte and to the disturbance of hemostasis. Plasma factors are activated by some proteolysis reactions in the realization of which a great role is played by phospholipids; Ca ions and regulatory proteins. The key reaction of hemostasis is activation of prothrombin into thrombin. Hemostasis disorders at this stage are of congenital (factor deficiency) or acquired (vitamin K deficiency) nature and occur at the molecular level. Hemostasis is terminated by a step-wise formation of fibrin. Disorders of one of the stages of this process due to congenital abnormality of fibrinogen results in coagulation defect due to the molecular disease of fibrinogen. The problem of hemostasis is inseparable from that of the mechanism causing the living host resistance to thrombus formation. The regulation of the liquid state of the blood and its coagulated is performed by the combined functioning of the coagulating and anticoagulating systems.

[Comparative analysis of the effect of alpha-thrombin and trypsin on the state of the anticoagulating system]. / Sravnitel'nyi analiz vliianiia alpha-trombina i tripsina na sostoianie protivosvertyvaiushchei sistemy.

The reflex response of anticoagulating system was studied in perfusion of humorally isolated sinocarotid area with trypsin and alpha-thrombin in rabbits with intact neural connections. Trypsin (1.5 and 3.8 microM) does not induce a reflex release of heparin and plasminogen activator into the blood flow whereas alpha-thrombin (0.5 microM) activates the anticoagulating system which is evident from elongation of the recalcification period, augmentation of total fibrinolytic activity and non-enzyme fibrinolysis of the blood plasma. The level of plasminogen activator sharply rises in the blood with no significant augmentation of the plasmin activity. The data obtained suggest that the specific nature of thrombin in due to the area of high-molecular substrates binding rather than proteolytic activity. Chemoreceptors of the sinocarotid area fail to respond to thrombin after trypsin perfusion which suggests failure of the receptor apparatus after contact with trypsin.

[Activation of the anticoagulation system following intravenous administration of beta-thrombin]. / Aktivatsiia protivosvertyvaiushchei sistemy posle vnutrivennego vvedeniia beta-trombina.

Beta-thrombin possessing high esterase activity and tracing coagulating ability, being product of limited proteolysis of alpha-thrombin in vitro, accelerates recalcification time and thrombin generation in plasma, but not the conversion of prothrombin to enzyme. Thus, beta-thrombin is the activator of early stages of blood coagulation, does not possess fibrinolytic activity and does not activate plasminogen. The i. v. administration of beta-thrombin to rats induces changes in blood coagulability which are accompanied by an increase in plasma recalcification time, total fibrinolytic activity and non-enzymatic fibrinolysis. Nothing of the kind occurs after administration alpha-thrombin, having tracing clotting activity similar to R-thrombin activity. The data obtained suggest the possibility of reflex activation of the anticoagulating system by beta-thrombin or undirectly by alpha-thrombin generated by beta-thrombin activation at early stages of blood coagulation.

[Stimulation of the reflex response of the anticoagulant system by intermediate product-1 of prothrombin proteolysis]. / O vozbuzhdenii reflektornoi reaktsii protivosvertyvaiushchei sistemy promezhutochnym produktom-1 proteoliza protrombina.

Injection of the intermediate 1 of prothrombin proteolysis into the frog's systemic circulation mobilized the anticoagulating and the fibrinolytic potential of the organism. This is not so in frogs with destroyed c.n. s. Electrical activity of the carotid chemoreceptors in considerably higher after perfusion with intermediate 1 of the frog's humorally isolated carotid labyrinth, as well as the total coagulation time, the total fibrinolytic activity, and the non-fermentative fibrinolysis in the systematic circulation. The data obtained suggest that the reflex activation of the physiological anticoagulating system can be induced can be induced by intermediate 1 which has the structure similar to thrombin but no clotting activity.

[Activation of the anticlotting system by perfusion of the carotid sinus with prethrombin 1 in rabbits]. / Vozbuzhdenie protivosvertyvaiushchei sistemy pri perfuzii karotidnogo sinusa krolika pretrombinom 1.

I.v. administration of prethrombin-1 activates the anticoagulating system but not through the thrombin generation. Perfusion of the rabbit humorally isolated carotid sinus with the prethrombin-1 increases the time of plasma recalcification, total fibrinolytic activity and nonenzymatic fibrinolysis, the effect depending on the prethrombin-1 concentration. The data obtained suggest that the prethrombin-1 is able to activate function of the anticoagulating system interacting with chemoreceptors of the isolated carotid sinus. The prethrombin-1 as well as alpha-thrombin induce similar effects in the anticoagulating system. Therefore the activation of the anticoagulating system is due to areas of chemoreceptors binding in the system and not to presence of the enzyme active center.

[Reflex reaction of the anticlotting system to DIP-alpha-thrombin devoid of blood clotting activity]. / Reflektornaia reaktsiia protivosvertyvaiushchei sistemy na DFF-alpha-trombin, lishennyi svertyvaiushchei krov' aktivnosti.

Injection of DFP-alpha-thrombin deprived of the blood coagulating activity into the systemic circulation of rats leads to mobilization of the humoral part of the anticoagulating system. providing high level of the anticoagulating and non-enzymatic fibrinolytic activity. This effect is not followed by increasing of soluble fibrin monomer complexes with fibrinogen or its degradation products concentration, suggesting absence of alpha-thrombin in the circulation. Perfusion of humorally isolated but retaining innervation carotid sinus of rabbit with the DFP-alpha-thrombin changes coagulability in the nonenzymatic fibrinolysis. The data on reflex activation of the anticoagulating system by the DFP-alpha-thrombin suggest the leading role of substrate binding site of the thrombin active in its physiological function.

[Analysis of the mechanisms of effect of beta/gamma-thrombin on the state of the anticoagulating system]. / Issledovanie mekhanizmov vliianiia beta/gamma-trombina na sostoianie protivosvertyvaitshchei sistemy organizma.

A 75-min perfusion of the rabbit humorally isolated carotid sinus with intact innervation with the beta/gamma-thrombin in concentrations of 2.9 X 10(7) M and 8.8 X 10(-7) M did not alter normal values of total and non-enzymatic fibrinolysis or plasma recalcification time in the systemic blood stream. Hence, beta/gamma-thrombin differs from alpha-thrombin in its inability to stimulate chemoreceptors of the carotid sinus which suggests a major part of the additional area for binding macromolecular substrates played in the thrombin interaction with vascular wall's chemoreceptors. I. V. administration of 2.1 X 10(-6) M beta/gamma-thrombin induces in rats a considerable increase of the soluble fibrin: 9.4-fold within the 1st min and 4.5-fold by the 5th min which suggests a generation of endogenous alpha-thrombin in the blood stream. This process maintains the activation of anticoagulating system occurring after the i. v. beta/gamma-thrombin administration.

[Stimulation of the secondary anticlotting system by DFP-alpha-thrombin]. / Vozbuzhdenie vtoroi protivosvertyvaiushchei sistemy DFF-alpha-trombinom.

Inhibition of the alpha--thrombin DFP catalytic portion does not incapacitate it physiologically: i. v. administration of such thrombin stimulates the second anticoagulating system. Aminasin abolishes this effect. These findings suggest that the thrombin preserves the portion of molecule responsible for reflex stimulation of the anticoagulating system. Microstructural changes of the apolar portion in different conditions deprives the alpha--thrombin to its ability to interact with the vascular bed's chemoreceptors. I. v. administration of such thrombin evoked no defense response in the organism.