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For many practical applications, monoclonal antibodies must be chemically modified without any significant loss in their immunoreactivity. In some situations, however, the amino acid residue crucial for antibody activity may be highly reactive toward the modifying agent, which results in antibody inactivation. The method to prevent inactivation of a modification-sensitive antinuclear monoclonal antibody by acylating agents was developed. The method is based on the hypothesis that a highly reactive amino group exists within, or in the vicinity of, the binding site of the antibody, providing crucial interaction with negatively charged moieties of DNA. It has been shown that negatively charged polymers, such as dextran sulfate or heparin, may provide temporary protection, presumably interacting noncovalently with this amino group and thus masking it. The protecting molecule can be removed later by chromatography on a protein A column, thus regenerating modified but not inactivated antibody in the free form for use in subsequent applications. In particular, we have modified antibody 2C5 with a chelating agent, diethylenetriaminepentaacetic acid (DTPA) without the loss of activity. Modified antibody was labeled with radioactive isotope, (111)In, via chelation by antibody-attached DTPA. The labeled antibody was shown to demonstrate the same specificity of binding to nucleosomes as the nonmodified antibody, so it may be used in immunoscintigraphy or biodistribution studies. The method might be useful for the modification of other modification-sensitive antibodies with other acylating chemicals, such as crosslinking agents or biotin derivatives., (Copyright 2001 Academic Press.)

We have attempted to simplify the procedure for coupling various ligands to distal ends of liposome-grafted polyethylene glycol (PEG) chains and to make it applicable for single-step binding of a large variety of a primary amino group-containing substances, including proteins and small molecules. With this in mind, we have introduced a new amphiphilic PEG derivative, p-nitrophenylcarbonyl-PEG-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (pNP-PEG-DOPE), synthesized by reaction of DOPE with excess of bis(p-nitrophenylcarbonyl)-PEG in a chloroform/triethylamine mixture. pNP-PEG-DOPE readily incorporates into liposomes via its PE residue, and easily binds primary amino group-containing ligands via its water-exposed pNP groups, forming stable and non-toxic urethane (carbamate) bonds. The reaction between the pNP group and the ligand amino group proceeds easily and quantitatively at pH around 8.0, and remaining free pNP groups are promptly eliminated by spontaneous hydrolysis. Therefore, pNP-PEG-DOPE could serve as a very convenient tool for protein attachment to the distal ends of liposome-grafted PEG chains. To investigate the applicability of the suggested protocol for the preparation of long-circulating targeted liposomes, we have coupled several proteins, such as concanavalin A (ConA), wheat germ agglutinin (WGA), avidin, monoclonal antimyosin antibody 2G4 (mon2G4), and monoclonal antinucleosome antibody 2C5 (mon2C5) to PEG-liposomes via terminal pNP groups and studied whether the specific activity of these immobilized proteins is preserved. The method permits the binding of several dozens protein molecules per single 200 nm liposome. All bound proteins completely preserve their specific activity. Lectin-liposomes are agglutinated by the appropriate polyvalent substrates (mannan for ConA-liposomes and glycophorin for WGA-liposomes); avidin-liposomes specifically bind with biotin-agarose; antibody-liposomes demonstrate high specific binding to the substrate monolayer both in the direct binding assay and in ELISA. A comparison of the suggested method with the method of direct membrane incorporation was made. The effect of the concentration of liposome-grafted PEG on the preservation of specific protein activity in different coupling protocols was also investigated. It was also shown that pNP-PEG-DOPE-liposomes with and without attached ligands demonstrate increased stability in mouse serum.

A new approach to preparative organic synthesis in aqueous-organic systems is suggested. It is based on the idea that the enzymatic process is carried out in a biphasic system "water-water-immiscible organic solvent." Thereby the enzyme is localized in the aqueous phase-this eliminates the traditional problem of stabilizing the enzymes against inactivation by a nonaqueous solvent. Hence, in contrast to the commonly used combinations "water-water-miscible organic solvent," in the suggested system the content of water may be infinitely low. This allows one to dramatically shift the equilibrium of the reactions forming water as a reaction product (synthesis of esters and amides, polymerization of amino acids, sugars and nucleotides, dehydration reactions, etc.) toward the products. The fact that the system consists of two phases provides another very important sources for an equilibrium shift, i.e., free energies of the transfer of a reagent from one phase to the other. Equations are derived describing the dependence of the equilibrium constant in a biphasic system on the ratio of the volumes of the aqueous and nonaqueous phases and the partition coefficients of the reagents between the phases. The approach has been experimentally verified with the synthesis of N-acetyl-L-tryptophan ethyl ester from the respective alcohol and acid. Porous glass was impregnated with aqueous buffer solution of chymotrypsin and suspended in chloroform containing N-acetyl-L-tryptophan and ethanol. In water (no organic phase) the yield of the ester is about 0.01%, whereas in this biphasic system it is practically 100%. The idea is applicable to a great number of preparative enzymatic reactions.

The purpose of these studies was to identify a possible role for protein kinase C in thromboxane production. The effects of four putative protein kinase C inhibitors were studied with platelet stimulation by thrombin (0.5-150 nM), Thrombin Quick I (1.5-500 nM) or a thrombin receptor (protease activated receptor-1) agonist peptide (TRAP) (5-120 microM). Thromboxane production was increased by the bisindolylmaleimide derivative, 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimi de (GF 109203X), unchanged by the inhibitors 12-(2-cyanoethyl)-6,7, 12,13-tetrahydro-13-methyl-5-oxo-5H-indolo (2,3-a) pyrrolo (3, 4-c)-carbazole (Gö 6976) and 5,21:12,17-dimetheno-18H-dibenzo[i, o]pyrrolo[3,4-l][1,8]diazacyclohexadecine-18,20(19H)-dione, 8-[(dimethylamino)methyl]-6,7,8,9,10,11-hexahydro-, monomethanesulfonate (379196), the latter of which is protein kinase C beta-selective, and decreased by 1-[6-[(3-acetyl-2,4, 6-trihydroxy-5-methylphenyl)methyl]-5,7-dihydroxy-2, 2-dimethyl-2H-1-benzopyran-8-yl]-3-phenyl-2-propen-1-one (rottlerin), an inhibitor selective for protein kinase C delta. These results indicate complex regulation of thromboxane synthesis in human platelets including a probable role for protein kinase C delta. The results taken together further suggest that GF 109203X may suppress negative feedback resulting from an unidentified kinase and that the classical protein kinase C isoforms alpha and beta do not have a significant role in regulating thromboxane production by platelets.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Possibilities of using particles containing ferromagnetic materials and magnetic concentrated colloids for targeted drug transport have been studied. Magnetic particles produced from Sephadex G-25 by ferromagnetic material fixation, polymer-modified iron particles 100 nm in diameter, and concentrated colloid solutions of Fe3O4 are retained in the marginal ear vein of the rabbit by magnetic field at H = 0.5 kHs/cm. The data on magnetic retention of the above-indicated agents in the vein, obtained by measuring 75Se and 131I activity, of labeled particles are presented.

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.

Immunomorphological staining of specimens prepared from human carotid arteries with anti-collagen type I antibodies reveals large amounts of type I collagen in the subendothelium of lipid fibrous plaques. Collagen type I-containing structures, once in direct contact with blood after plaque rupture, can serve as potential targets for selective delivery of liposomes and erythrocytes to these areas. To verify this rationale, [14C]cholesterol oleate-containing liposomes were conjugated with bovine or human anti-collagen type I antibodies or human plasma fibronectin. Biotin derivatives of human anti-collagen type I antibody were coupled to human erythrocytes. Modified liposomes and erythrocytes were perfused in situ through segments of bovine, rabbit, or human arteries partially denuded with a balloon catheter prior to perfusion. After perfusion, the control and denuded areas were excised and subjected to scanning electron microscopic analysis and measurements of associated radioactivity. It was found that conjugates of liposomes or erythrocytes with anti-collagen type I antibodies or fibronectin are selectively bound by endothelium-free zones of arterial segments. Carrier-directed targeting of drug-laden liposomes and erythrocytes to thrombosis-prone areas of arterial lumen is discussed.

Accumulation of rhodamine B isothyocyanate-conjugated low density lipoproteins (R-LDL) in cultured endothelial cells from human umbilical cord was studied with a fluorescence activated cell sorter. R-LDL uptake was blocked at 0 degrees C, inhibited by addition of excess of nonlabeled low density lipoprotein, high density lipoprotein-2 or high density lipoprotein-3. High density lipoprotein-2 was about twice as effective in inhibition of R-LDL uptake as high density lipoprotein-3. Endothelial cells that formed a contact-inhibited monolayer lost the ability to incorporate R-LDL via a receptor-mediated pathway. Using R-LDL, it was possible to distinguish cells with different levels of R-LDL incorporation.

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.

The in vivo and in vitro experiments have shown that magnetic erythrocytes (loaded with ferromagnetic material) can be retained in certain sites of the vascular bed. Magnetic erithrocytes could be concentrated in the abdominal part of the dog aorta using a small permanent magnet fixed on the outer wall of the aorta.

A new rapid technique is developed for the determination of complement activity in a large number of samples. Following serial dilution of complement, hemolysis is performed in the same microtiter plate. After the reaction, the degree of hemolysis in wells of the plate is determined spectrophotometrically by measurement of "absorbance" (light scattering) at 630 nm, without additional procedures. This method can find application in clinical and experimental biochemistry for the analysis of a large (up to thousands) number of samples.

The binding of myosin to nylon fiber gives immobilized myosin with a considerable ATPase activity. Treatment of immobilized enzyme with papain results in the entire ATPase activity (known to be concentrated in myosin heads, (fragment HMM S-1)) being replaced from the fiber into the solution; this means that myosin is chemically bound to the fiber via its rod part (fragment LMM+HMM S-2). When nylon fiber is mechanically stretched, the ATPase activity of myosin attached to it sharply decreases; after relaxation of the fiber the enzymatic activity returns to the initial level. The detailed study of this phenomenon has shown that reversible inactivation of myosin upon fiber stretching is not the result of an altered microenvironment of the enzyme. The discovered regulatory effect is ascribed to deformation of myosin molecules induced by support stretching. Thus deformation of the myosin tail (not indispensable for ATPase since its cleaving-off does not alter the enzymatic activity) leads to decrease in the ATPase activity of the enzyme. The possible role of the above phenomenon in the mechanism of muscle contraction is discussed.

Interaction of anti-human collagen types I and III antibodies, as well as human red blood cells conjugated with these antibodies, with the surface of denuded intima of human aorta has been studied. Data on the accessibility of antigenic determinants of collagen types I and III for antibodies and red blood cells conjugated with these antibodies have been obtained in ex vivo experiments in an original model. On the basis of the obtained results it is concluded that antigenic determinants of collagen types I and III exposed as a result of blood vessel wall injury can serve as a target for drug delivery to the injured site(s).

The effect of bloodstream factors (flow rate and blood cellular elements) on the behavior of an experimental system simulating drug targeting to injured sites of vessel walls was studied. The system consisted of red blood cells carrying antibody to type I human collagen (drug carrier) and plastic tube with a collagen-coated inner surface (target). The tube was perfused with a 0.5% (v/v) suspension of 51Cr-labeled red blood cells at different linear flow rates and the red blood cell binding to the tube was determined by gamma-counting. It was demonstrated that an increase in the linear flow rate from 0 to 2 cm/s leads at first to increase of red blood cell binding from 2 X 10(5) to 7 X 10(5) cells/cm2 and then to the decrease of binding back to 2 X 10(5) cells/cm2. In the presence of 50% (v/v) of intact red blood cells the binding continuously increases from 2 X 10(5) to 2.5 X 10(6) cells/cm2 without a subsequent drop. On the basis of the obtained results it is concluded that the behavior of the systems for drug targeting in simple in vitro models can drastically differ from the conditions present in vivo.

Carboxyl groups of succinylated bovine serum albumin were activated by isobutylchloroformate in dimethylformamide solution. Subsequent reaction of the mixed anhydride with amino groups of the added peptide provided rapid and efficient coupling of peptide to protein. For different peptides the yield of coupling was equal to 40-100%. These values corresponded to 20-50 mol peptide bound/mol protein. Immunization of rabbits with these conjugates produced antisera to peptides with titers of 1:1000-1:3000 (estimated by enzyme-linked immunosorbent assay).

A new mechanochemical method for enzyme immobilization has been elaborated. The principle of this method consists of the following precepts. Partially hydrolyzed nylon fiber, the surface of which is known to be strewn with microcracks, is reversibly stretched (approximately 25%) and placed into an enzyme solution. Then, in the same solution, the fiber is made to relax and is taken out. The fiber retains considerable enzymatic activity even after numerous thorough washings (in a similar procedure without fiber stretching, equivalent washing removed all the enzymatic activity from the fiber). Immobilization on the fiber proceeds due to trapping of enzyme molecules by the microcavities on the surface of the support. The catalytic activity of mechanochemically immobilized chymotrypsin and trypsin is commensurable with their activity on covalent immobilization on nylon (calculated per unit of the macrosurface). A wide range of commercial polymers may be made of use as supports in the mechanochemical method of immobilization.

1. Covalent binding of model enzymes, chymotrypsin and trypsin, to elastic polymer supports, nylon and viscose (cellulose) fibers, human hair, methacrylate rubber, has been effectuated. On mechanical stretching of the fibers, the catalytic activity of the enzymes bound to them decreases, and when they relax, it increases to the initial level. The data obtained by us fit the concept that the effect is due to reversible deformation of the bound enzyme molecules induced by fiber stretching. 2. Analysis of the dependence of the catalytic activity of the enzymes chemically bound to the fiber on the degree of fiber deformation shows that the reversible inactivation of the enzymes induced by support stretching occurs even if the deformation of the enzymes' molecules is as small as 0.5 A. 3. The deformation of the enzyme molecules induced by fiber stretching entails a change in the substrate specificity of the biocatalysts, i.e. the activity towards "good" substrates decreases, and towards "poor" substrates increases. 4. The deformation of the enzyme molecules induced by fiber stretching results in a decrease of the specific catalytic activity of the biocatalyst, whereas its thermal stability increases. 5. The results obtained allowed a new, mechanochemical, approach to be suggested for studying major problems of enzymatic catalysis.

It is suggested to use 'enzyme(s)-erythrocyte-antibody' complex for modulation of the microenvironment in definite compartments of blood circulation. A model system including peroxidase, human erythrocytes and anti-collagen antibodies was chosen to illustrate the principle. Peroxidase was conjugated to the erythrocyte surface via periodate-oxidized enzyme carbohydrate moiety; biotinylated antibodies were linked by avidin to the biotinylated erythrocytes. The properties of the immunocomplexes obtained have been investigated in an artificial system simulating an injured blood vessel wall. The advantages in using erythrocyte-mediated immunoenzyme complexes for enzyme (drug) targeting are discussed.

The interaction of human red blood cells carrying antihuman collagen antibody with collagen-coated surfaces was studied. Avidin was used as bifunctional crosslinking agent for the attachment of antibody to the red blood cell surface. Antibody-carrying red blood cells efficiently and specifically bound to collagen-coated surface covering a significant part of the surface. The components of normal blood had an insignificant effect on red blood cell binding. A model of drug targeting to the injured sites(s) of blood vessel wall is proposed.