PubMed Health⌕ Search

Biomedical subjects

G P Samokhin

Publications and source records attributed to G P Samokhin.

At least 19 recordsLinked to original sources
PubMed1998

Coagulation factor XIIIa undergoes a conformational change evoked by glutamine substrate. Studies on kinetics of inhibition and binding of XIIIA by a cross-reacting antifibrinogen antibody.

Coagulation factor XIIIa, plasma transglutaminase (endo-gamma-glutamine:epsilon-lysine transferase EC 2.3.2.13) catalyzes isopeptide bond formation between glutamine and lysine residues and rapidly cross-links fibrin clots. A monoclonal antibody (5A2) directed to a fibrinogen Aalpha-chain segment 529-539 was previously observed from analysis of end-stage plasma clots to block fibrin alpha-chain cross-linking. This prompted the study of its effect on nonfibrinogen substrates, with the prospect that 5A2 was inhibiting XIIIa directly. It inhibited XIIIa-catalyzed incorporation of the amine donor substrate dansylcadaverine into the glutamine acceptor dimethylcasein in an uncompetitive manner with respect to dimethylcasein utilization and competitively with respect to dansylcadaverine. Uncompetitive inhibition was also observed with the synthetic glutamine substrate, LGPGQSKVIG. Theoretically, uncompetitive inhibition arises from preferential interaction of the inhibitor with the enzyme-substrate complex but is also found to inhibit gamma-chain cross-linking. The conjunction of the uncompetitive and competitive modes of inhibition indicates in theory that this bireactant system involves an ordered reaction in which docking of the glutamine substrate precedes the amine exchange. The presence of substrate enhanced binding of 5A2 to XIIIa, an interaction deemed to occur through a C-terminal segment of the XIIIa A-chain (643-658, GSDMTVTVQFTNPLKE), 55% of which comprises sequences occurring in the fibrinogen epitope Aalpha-(529-540) (GSESGIFTNTKE). Removal of the C-terminal domain from XIIIa abolishes the inhibitory effect of 5A2 on activity. Crystallographic studies on recombinant XIIIa place the segment 643-658 in the region of the groove through which glutamine substrates access the active site and have predicted that for catalysis, a conformational change may accompany glutamine-substrate binding. The uncompetitive inhibition and the substrate-dependent binding of 5A2 provide evidence for the conformational change.

Amino Acid Sequence↗
PubMed1997

Thrombin-induced thromboxane synthesis by human platelets. Properties of anion binding exosite I-independent receptor.

These studies have examined the effects of thrombin-related agonists in stimulating thromboxane production by human platelets. The results presented show that (1) the maximal response elicited by thrombin receptor agonist peptide (TRAP) stimulation was 40% to 50% of that seen with thrombin or the thrombin mutant Thrombin Quick I; (2) pretreatment of platelets with prostaglandin E1 or genistein resulted in differential inhibition of thromboxane production in response to TRAP compared with either enzyme agonist; (3) an antibody to the thrombin receptor cleavage site that inhibits increases in intracellular [Ca2+] only partially reduced thromboxane production in response to 5 nmol+L thrombin and 15 nmol/L Thrombin Quick I; (4) preincubation with 20 mumol/L TRAP resulted in desensitization to further stimulation by 100 mumol/L TRAP, but not by 100 nmol/L thrombin; and (5) the response to thrombin after TRAP desensitization was completely inhibited by the tyrosine kinase inhibitor genistein and was independent of an intracellular [Ca2+] flux, The cumulative results may be explained by the existence of two proteolytically activated receptors that result in thromboxane production in response to thrombin. One is the thrombin receptor/substrate, PAR-1. Stimulation through the second receptor/substrate depends on a genistein-sensitive step, is independent of an intracellular Ca2+ flux, and is initiated by a thrombin-activated receptor that does not depend on interaction with anion-binding exosite I, as previously indicated by the relative activity of Thrombin Quick I in stimulating platelet aggregation and thromboxane production. The proposed second thrombin receptor on platelets represents an additional member of the class of proteolytically activated receptors.

Adult↗
PubMed1996

Target-sensitive immunoerythrocytes: interaction of biotinylated red blood cells with immobilized avidin induces their lysis by complement.

Red blood cells (RBC) coated with antibody (immunoerythrocytes) may be useful for drug targeting. Previously we have developed a methodology for avidin (streptavidin)-mediated attachment of biotinylated antibodies (b-Ab) to biotinylated RBC (B-RBC). We have observed that binding of avidin to B-RBC in suspension leads to their complement-mediated lysis by autologous serum. In the present work we have studied the interaction of B-RBC, which are not complement susceptible, with immobilized avidin and their consequent susceptibility to lysis by complement. B-RBC adhered tightly to avidin-coated surfaces and were rendered susceptible to lysis by autologous serum. A long biotin ester provided more effective binding of the B-RBC to immobilized avidin and greater lysis by complement, than a short biotin ester. Based on these results, we have hypothesized that targeting of serum-stable drug-loaded B-RBC attained by step-wise administration of b-Ab and streptavidin may provide target-sensitive lysis of B-RBC. To confirm this hypothesis, we have studied b-Ab and streptavidin mediated targeting of B-RBC to immobilized antigen. Step-wise addition of biotinylated antibody, avidin or streptavidin and b-RBC caused specific binding of B-RBC to immobilized antigen and their subsequent lysis by autologous serum. Therefore, our results obtained in an in vitro model demonstrate that B-RBC might be used for targeting and local release of drug.

Aminocaproates↗
PubMed1996

Monoclonal antibody directed to a fibrinogen A alpha #529-539 epitope inhibits alpha-chain crosslinking by transglutaminases.

A monoclonal antibody (5A2) recognizing a segment near the C-terminus of the fibrin(ogen) A alpha-chain (A alpha #529-539) was found to inhibit alpha-chain crosslinking catalyzed by coagulation factor XIIIa and by tissue-transglutaminase. The rapid gamma-chain cross-linking by factor XIIIa was not affected by the antibody. Results obtained from direct binding and competitive immunoassay established that the antigenic determinant recognized by 5A2 was included within the CNBr fragment referred to as CNBr X (A alpha #518-584), and that it survived trypsin digestion but was destroyed by treatment with Staph V-8 protease or chymotrypsin. Reverse-phase (C-18) high performance liquid chromatography (HPLC) was employed to obtain a CNBr X tryptic fingerprint, which was subsequently characterized by compositional and NH2-terminal analysis. Assay of the HPLC column effluent revealed a single peak of 5A2 immunoreactivity that coincided with elution of the eleven-residue tryptic peptide, A alpha #529-539. When this isolated peptide and its parent CNBr fragment were employed as solution phase competitors in the 5A2 immunoassay, the relative cross-reactivities (18.3%, peptide: fragment) indicated that a significant proportion of the 5A2 epitope was preserved within the small peptide. This is a region that is released from fibrinogen early in its degradation by plasmin. Thus, the antibody can be used as a probe for intact fibrin(ogen) and C-terminal (A) alpha-chain fragments, in addition to assessing roles of the A alpha-chain C-terminus in cross-linking.

Animals↗
PubMed1995

Contact with the N termini in the central E domain enhances the reactivities of the distal D domains of fibrin to factor XIIIa.

The reaction of Factor XIIIa with fibrin is the last enzyme-catalyzed step on the coagulation cascade, leading to the formation of a normal blood clot. The finding that fibrin is preferred by the cross-linking enzyme about 10-fold over the circulating fibrinogen suggests the operation of a unique substrate-level control for the orderly functioning of the physiological process in the forward direction. An important task is to elucidate the molecular mechanism for the transmission of the signal generated by the thrombin-catalyzed cleavage in the central E domain of fibrin to the distant Factor XIIIa-reactive glutamine residues. By focusing on the substrate sites present in gamma chain remnants of D type domains of fibrinogen and by employing the approach of fragment complementation with the regulatory E domain, which represents the thrombin-modified portion of fibrin, we have now succeeded in reconstructing in solution the phenomenon of kinetic enhancement for the reaction with Factor XIIIa. Two D type preparations (truncated fibrinogen, approximately 250 kDa and D', approximately 105 kDa) were obtained by digestion of human fibrinogen with endo Lys-C. Neither product could be cross-linked by Factor XIIIa, but as shown by the incorporation of dansylcadaverine, both were acceptor substrates for the enzyme. The plasmin-derived D (approximately 105-kDa) product, however, could be cross-linked into DD dimers. In all cases, the admixture of E fragments exerted a remarkable boosting effect on the reactions with Factor XIIIa. Even with native fibrinogen as substrate, cross-linking of gamma chains was enhanced in the presence of E. Nondenaturing electrophoresis was used to demonstrate the complex forming potential of E fragments with fibrinogen, truncated fibrinogen, D', or D. The GPRP tetrapeptide mimic of the GPRV N-terminal sequence of the alpha chains in the E fragments, abolished both complex formation and the kinetic boosting effect of E on the reactions of substrates with Factor XIIIa. Thus, the N-terminal alpha chain sequences seem to act as organizing templates for spatially orienting the D domains, probably during the protofibrillar assembly of the fibrin units, for favorable reaction with Factor XIIIa.

Binding Sites↗
PubMed1994

Substrate specificity of collagenolytic proteases from the king crab Paralithodes camtschatica.

Substrate specificity of two collagenolytic proteases from the king crab Paralithodes camtschatica has been studied. Both proteases are shown to hydrolyze effectively type I and III collagens, gelatin and fibrinogen. The variety of products formed during the enzymatic hydrolysis of the proteins appeared to be different for crab proteases A and C. Studies on peptide hydrolysis demonstrated that protease A cleaves preferably peptide bonds with Arg and Lys as carbonyl components, while protease C prefers hydrophobic amino acids. Kinetic constants of hydrolysis for low molecular weight substrates in the presence of crab proteases have been determined. This allowed us to characterize collagenolytic protease A as a trypsin-like protease. By contrast, collagenolytic protease C was classified as chymotrypsin-like protease although this protease and bovine chymotrypsin are not completely similar. Collagenase substrates Pz-Pro-Leu-Gly-Pro-D-Arg and Z-Gly-Pro-Ala-Gly-Pro-Ala were found to be resistant to both crab proteases.

Amino Acid Sequence↗
PubMed1993

Interaction of avidin-carrying red blood cells with nucleated cells.

In vivo application of red blood cells (RBC) modified with avidin-biotin complex has been suggested recently for various purposes. However, avidin attachment to RBC alters their biocompatibility. Thus, it has been described that avidin-carrying biotinylated RBC were lysed by the complement. In the present work interaction between avidin-carrying RBC and nucleated cells has been examined. It was found that attachment of avidin, but not streptavidin, to RBC led to binding of avidin-carrying RBC to nucleated cells. Adhesiveness of nucleated cells for avidin-carrying RBC varied for different types of nucleated cells. The strongest adhesion was observed with human fibroblasts and rat Kupffer cells, while rat liver endothelial cells were practically non-adhesive for avidin-carrying RBC of corresponding species. In contrast with avidin (streptavidin)-induced lysis by the complement, avidin-induced adhesion was independent of temperature, the presence of divalent ions and mode of avidin attachment. Polyanions (dextran sulphate and heparin) efficiently inhibited the adhesion presumably due to interaction with the membrane-bound avidin. Polyanions to a much lesser extent inhibited lysis of avidin-carrying RBC, which might be a result of their interaction with the complement components. Polycations also blocked adhesion of avidin-carrying RBC to nucleated cells, presumably due to interaction with negatively charged cell-surface components. Therefore, attachment of avidin to RBC alters their biocompatibility, due to both high positive charge of avidin and the cross-linking of biotinylated membrane proteins.

Animals↗
PubMed1993

Tannin-mediated attachment of avidin provides complement-resistant immunoerythrocytes that can be lysed in the presence of activator of complement.

It was shown previously that avidin attachment to biotinylated erythrocytes induces their lysis by homologous complement via the alternative pathway. This phenomenon hinders the use of avidin-coated immunoerythrocytes as carriers for drug targeting. In the present work we demonstrated that attachment of avidin to erythrocytes via the cross-linking agent tannin does not induce their lysis by complement. Tannization allows attachment of about 5 x 10(5) molecules of avidin per erythrocyte, which is comparable to the value obtained after treatment with biotin esters. In contrast to biotinylated avidin-coated erythrocytes, tannized avidin-coated erythrocytes were not lysed by complement. Tannization itself does not reduce the erythrocyte sensitivity to lysis by complement in the presence of activators of the complement (hemolytic antibody or activators of the alternative pathway). Therefore, the avidin-induced lysis by complement depends on the mode of avidin attachment to erythrocyte. Complement-resistant tannized erythrocytes coated with avidin bind biotinylated immunoglobulins (to 7 x 10(4) molecules per cell), suggesting that tannization might be used for the preparation of complement-resistant immunoerythrocytes.

Animals↗
PubMed1993

[Tannin-mediated attachment of avidin to erythrocytes does not cause their lysis by complement].

It was shown previously that avidin attachment to biotinylated erythrocytes induced their lysis by a homologous complement via an alternative pathway. This phenomenon hindered the use of avidin-coated immuno-erythrocytes as carriers for drug targeting. In the present work it has been demonstrated that avidin attachment to erythrocytes via a cross-linking reagent (tannin) does not induce any lysis by the complement. Tannization provides an attachment of up to 5 x 10(5) avidin molecules per erythrocyte which is commensurate with the value obtained after treatment with biotin esters. However, in contrast with biotinylated avidin-coated erythrocytes tannized cells are not lysed by the complement, while tannization itself does not diminish the erythrocyte sensitivity to lysis by the complement in the presence of activators (hemolytic antibody or activators of the alternative pathway). The avidin-induced lysis by the complement depends on the mode of avidin attachment to erythrocytes. Complement-resistant avidin-coated tannized erythrocytes bind biotinylated immunoglobulins and may therefore be used as carriers for drug targeting. The use of hemolytic antibody in biotinylated immunoglobulins attached to avidin-coated erythrocytes provides their controlled lysis by a complement activated via a classical pathway.

Avidin↗
PubMed1992

Avidin-induced lysis of biotinylated erythrocytes by homologous complement via the alternative pathway depends on avidin's ability of multipoint binding with biotinylated membrane.

It was reported that avidin and streptavidin induce lysis of prebiotinylated red blood cells via the alternative pathway of both homologous and heterologous complement. Both of these proteins have four biotin-binding sites, providing a polyvalent interaction with biotinylated components of the erythrocyte membrane. We have compared the effects of mono- and multipoint avidin attachment on the sensitivity of biotinylated erythrocytes to lysis by the complement system. In the presence of anti-avidin antibody, avidin-bearing biotinylated erythrocytes were rapidly lysed by heterologous serum. This lysis was independent from the mode of avidin attachment, implying that complement activation by the classical pathway triggered by interaction between C1 and avidin-bound antibody on the erythrocyte surface is independent from the avidin's ability of polyvalent (multipoint) binding with biotinylated membrane components. In the absence of anti-avidin antibody, biotinylated erythrocytes bearing polyvalently attached avidin were lysed by homologous complement better than cells bearing avidin, which possesses reduced ability for multipoint binding with biotinylated erythrocyte. Two independent approaches to reduce avidin's ability of multipoint binding were used: decrease in surface density of biotin on the erythrocyte membrane and blockage of biotin-binding sites of avidin. Both methods result in reduced lysis of avidin-bearing erythrocytes as compared with erythrocytes bearing an equal amount of polyvalent-bound avidin. Thus the activation of homologous complement via the alternative pathway depends on avidin's ability to 'cross-link' to the biotinylated components of the erythrocyte membrane.

Animals↗
PubMed1991

Avidin attachment to biotinylated erythrocytes induces homologous lysis via the alternative pathway of complement.

Noncovalent attachment of avidin to the membrane of prebiotinylated red blood cells (RBCs) induces lysis via the alternative pathway of complement (APC). Lysis is not species-dependent; RBCs from humans, rabbits, rats, and sheep were lysed with both autologous and all heterologous sera. Both biotinylated and native cells were not lysed. Lysis was observed at an avidin surface density of about 10(5) molecules per cell. Acylation of avidin prevents lysis and decreases the positive charge of the avidin. Lysis depends on the length of the cross-linking agent used for the biotin attachment to the membrane. An increase in the length of the cross-linking agent was accompanied by an enhancement of the lysis and the agglutination titer of biotinylated RBCs in a solution of avidin. It is suggested that avidin attachment induces some transformations of the cell membrane that lead to the conversion from "APC nonactivator" cells to "APC activator" cells. The interaction of avidin with membrane APC-restrictors (decay-accelerating factors, type 1 receptor for complement, homologous restriction factor, and others), the charge of avidin, and its cross-linking ability in lysis are discussed. It is proposed that membrane rearrangement induced by multipoint avidin attachment to biotinylated membrane is the main reason for avidin-induced elimination of APC restriction.

Animals↗
PubMed1991

Streptavidin-induced lysis of homologous biotinylated erythrocytes. Evidence against the key role of the avidin charge in complement activation via the alternative pathway.

It is shown that non-covalent attachment of streptavidin, as well as of avidin, to biotinylated human erythrocytes induces homologous hemolysis by complement. Rabbit antiserum against human C3 is found to inhibit the lysis specifically as compared with non-immune rabbit serum. Efficiency of lysis inhibition is greater for avidin- and streptavidin-induced lysis of biotinylated human erythrocytes than for antibody-sensitized sheep erythrocytes. In contrast to positively charged avidin (pI 11), streptavidin is a neutral protein. Hence, hemolysis of streptavidin-carrying erythrocytes is inconsistent with the suggestion on the crucial role of avidin charge in lysis. Membrane alterations (cross-linking and clusterization of biotinylated components) induced by avidin (streptavidin) seem to be a more plausible explanation for the lysis.

Avidin↗
PubMed1991

Avidin acylation prevents the complement-dependent lysis of avidin-carrying erythrocytes.

Non-covalent binding of avidin to biotinylated erythrocytes results in complement-dependent haemolysis. Biotinylated erythrocytes, as well as native cells, are not lysed by complement. Complement activation requires a tight contact between avidin and the erythrocyte membrane, since avidin does not in itself activate complement and does not inhibit lysis of sensitized sheep erythrocytes. The efficiency of haemolysis depends on avidin's surface density. When the avidin concentration in the reaction mixture is less than 15 micrograms/ml, erythrocyte lysis is not induced. However, the attachment of biotinylated antibodies to avidin-carrying erythrocytes decreases dramatically. Acylation of avidin with succinic anhydride strongly decreases its ability to induce complement-dependent haemolysis. However, the ability of avidin to cross-link the biotin-containing structures decreases after acylation. A 50% modification of avidin by succinic anhydride (pI about 7.0) allows preparation of 'immunoerythrocytes', which retain their affinity to antigen and stability in the presence of complement.

Acylation↗
PubMed1990

Visualization of apo B, fibrinogen/fibrin, and fibronectin in the intima of normal human aorta and large arteries and during atherosclerosis.

Apolipoprotein B (apo B), fibrinogen/fibrin, blood platelets, factor VIII-related antigen of the blood coagulation system, and smooth muscle cells (SMC) were identified in the intima of normal and atherosclerotic human aorta and large arteries by the indirect immunofluorescence technique. Fibrinogen/fibrin was revealed by a monoclonal antibody (monAb) against the C-terminal region of human fibrinogen A alpha-chain. Fibronectin was visualized by monAb to the cellular form and against an epitope shared by different fibronectin subunit variants. In normal intima, fatty streaks, small amounts of fibrinogen/fibrin together with large amounts of apo B were observed. Fibronectin detected by two types of monAb was not found in extracellular matrix (ECM), whereas cellular fibronectin encircled SMC. According to the data obtained, fibrinogen/fibrin accumulates in plaques as a result of intramural thrombus incorporation, blood insudation, intramural haemorrhage, and in or around cells, apparently macrophages.

Aged↗
PubMed1990

[Apo B, fibrinogen-fibrin and fibronectin in the intima of the normal human aorta and large arteries and in atherosclerosis].

Apo B, fibrinogen/fibrin, fibronectin, thrombocytes, factor VIII of the blood coagulation and smooth muscle cells (SMC) were identified by the immunofluorescence method in the intima of aorta and big arteries under normal conditions and in atherosclerosis. Monoclonal antibodies (MCA) against C-end fragments of A alpha-fibrinogen chain were used in the study of fibrinogen/fibrin. MCA reacting with plasma fibronectin and those reacting with A-area of the polypeptide chain specific for the cell fibronectin were used for the identification of fibronectin. Small amount of fibrinogen/fibrin, no fibronectin in the extracellular matrix and the cell fibronectin around SMC were observed on the normal intima and lipid strip in spite of the presence of Apo B. The results indicate that fibrinogen/fibrin is accumulated in the plaques due to the incorporation of the wall thrombi, insudation from the blood plasma, intramural haemorrhages as well as around cells, presumably macrophages.

Adult↗
PubMed1987

[Local prevention of thrombosis in the dog carotid artery using magnetically concentrated erythrocytes loaded with aspirin].

Thrombosis was induced in both canine carotid arteries by means of vascular wall flap inversion into their lumens. A red, completely occluding thrombus was formed inside the vessel 4 to 5 hours later. SmCo5 magnet was secured externally to one of the arteries. The constant magnetic field produced by the magnet had no influence on the clot formation. Autologous red cells loaded with ferromagnetic colloid compound and aspirin were administered intravenously through the hind paw route; total aspirin pool was 20 micrograms. Circulating magnetically-charged red cells have been earlier shown to concentrate in a canine artery in the constant magnet area. The administration of magnetically-charged red cells loaded with aspirin completely prevented arterial thrombosis in the magnet-supplied artery, having no detrimental effect on clot formation in the control artery.

Animals↗
PubMed1986

Immunotargeting of erythrocyte-bound streptokinase provides local lysis of a fibrin clot.

The creation of an anticollagen antibody-erythrocyte-streptokinase complex has been described. Immobilization of both proteins on erythrocyte membrane has been performed using an avidin-biotin interaction. Modification of streptokinase with (6-biotinylamido)hexanoic acid N-hydroxysuccinimide ester at the concentration of 1.1 mM (20% modification of protein amino groups) provides effective (up to 90%) attachment of streptokinase to an avidin-carrying erythrocyte surface. The loss of streptokinase activity due to modification under these conditions is not significant. The maximal attachment of streptokinase was equal to about 50 ng per 10(6) erythrocytes, i.e., about 5 X 10(5) molecules of streptokinase per erythrocyte. The presence of streptokinase in the incubation mixture inhibited the attachment of antibodies by about 50%. Nevertheless, co-immobilization of anticollagen antibody (1.0 X 10(5) molecules per cell) and streptokinase (2.8 X 10(5) molecules per cell) on the erythrocyte surface provided firm and specific binding of such erythrocytes to a collagen-coated surface (1.6 X 10(6) bound cells per 1 cm2 on a collagen-coated surface against 0.006 X 10(6) bound cells on a bovine serum albumin-coated surface). Targeting of such erythrocytes led to local lysis of a fibrin clot in the target zone. The properties described offer in principle the possibility of the application of this or a similar system of fibrinolytic agent targeting for the preventive therapy of rethrombosis during surgical manipulations on vessels.

Antibodies↗