Fibrinogen counteracts the antiadhesive effect of fibrin-bound plasminogen by preventing its activation by adherent U937 monocytic cells.

BACKGROUND: Fibrinogen and plasminogen strongly reduce adhesion of leukocytes and platelets to fibrin clots, highlighting a possible role for these plasma proteins in surface-mediated control of thrombus growth and stability. In particular, adsorption of fibrinogen on fibrin clots renders their surfaces non-adhesive, while the conversion of surface-bound plasminogen to plasmin by transiently adherent blood cells results in degradation of a superficial fibrin layer, leading to cell detachment. Although the mechanisms whereby these proteins exert their antiadhesive effects are different, the outcome is the same: the formation of a mechanically unstable surface that does not allow firm cell attachment. OBJECTIVES: Since fibrin clots in circulation are exposed to both fibrinogen and plasminogen, their combined effect on adhesion of monocytic cells was examined. METHODS: Fibrin gels were coated with plasminogen and its activation by adherent U937 monocytic cells in the presence of increasing concentrations of fibrinogen was examined by either measuring (125) I-labeled fibrin degradation products or plasmin amidolytic activity. RESULTS: Unexpectedly, the antiadhesive effects of two fibrin binding proteins were not additive; in fact, in the presence of fibrinogen, the effect of plasminogen was strongly reduced. An investigation of the underlying mechanism revealed that fibrinogen prevented activation of fibrin-bound plasminogen by cells. Confocal microscopy showed that fibrinogen accumulates in a thin superficial layer of a clot, where it exerts its blocking effect on activation of plasminogen. CONCLUSION: The results point to a complex interplay between the fibrinogen- and plasminogen-dependent antiadhesive systems, which may contribute to the mechanisms that control the adhesiveness of a fibrin shell on the surface of hemostatic thrombi.

Plasminogen on the surfaces of fibrin clots prevents adhesion of leukocytes and platelets.

BACKGROUND AND OBJECTIVES: Although leukocytes and platelets adhere to fibrin with alacrity in vitro, these cells do not readily accumulate on the surfaces of fibrin clots in vivo. The difference in the capacity of blood cell integrins to adhere to fibrin in vivo and in vitro is striking and implies the existence of a physiologic antiadhesive mechanism. The surfaces of fibrin clots in the circulation are continually exposed to plasma proteins, several of which can bind fibrin and influence cell adhesion. Recently, we have demonstrated that adsorption of soluble fibrinogen on the surface of a fibrin clot results in its deposition as a soft multilayer matrix, which prevents attachment of blood cells. In the present study, we demonstrate that another plasma protein, plasminogen, which is known to accumulate in the superficial layer of fibrin, exerts an antiadhesive effect. RESULTS: After being coated with plasminogen, the surfaces of fibrin clots became essentially non-adhesive for U937 monocytic cells, blood monocytes, and platelets. The data revealed that activation of fibrin-bound plasminogen by the plasminogen-activating system assembled on adherent cells resulted in the generation of plasmin, which decomposed the superficial fibrin layer, resulting in cell detachment under flow. The surfaces generated after the initial cell adhesion remained non-adhesive for subsequent attachment of leukocytes and platelets. CONCLUSION: We propose that the limited degradation of fibrin by plasmin generated by adherent cells loosens the fibers on the clot surface, producing a mechanically unstable substrate that is unable to support firm integrin-mediated cell adhesion.

The alternatively spliced alpha(E)C domain of human fibrinogen-420 is a novel ligand for leukocyte integrins alpha(M)beta(2) and alpha(X)beta(2).

The interaction of human plasma fibrinogen with leukocyte integrins alpha(M)beta(2) (CD11b/CD18, Mac-1) and alpha(X)beta(2) (CD11c/CD18, p150,95) is an important component of the inflammatory response. Previously, it was demonstrated that binding of fibrinogen to these integrins is mediated by gammaC, the globular C-terminal domain of the gamma chain. In this study, evidence was found of another fibrinogen domain that can serve as a ligand for the 2 leukocyte integrins: alpha(E)C, a homologous domain that extends the alpha chains in a recently discovered subclass of fibrinogen known as fibrinogen-420. Recombinant alpha(E)C supported strong adhesion and migration of cells expressing alpha(M)beta(2) and alpha(X)beta(2), including nonactivated and activated U937 and THP-1 monocytoid cells, and neutrophils. Cells transfected with complementary DNA for these integrins also bound alpha(E)C. The specificity of interaction was substantiated by inhibition of cell adhesion with antibodies against alpha(M), alpha(X), and beta(2) subunits. Also, neutrophil inhibitory factor, a specific inhibitor of alpha(M)beta(2) and alpha(X)beta(2) function, efficiently blocked cell adhesion to alpha(E)C. In alpha(M)beta(2) and alpha(X)beta(2), the I domain is the binding site for alpha(E)C, since alpha(E)C bound to recombinant alpha(M) I and alpha(X)I domains in a dose-dependent and saturable manner. Synthetic peptides that duplicated sequences gamma190 to 202 and gamma377 to 395, previously considered putative binding sites in gammaC, effectively inhibited alpha(M)beta(2)- and alpha(X)beta(2)-mediated adhesion to alpha(E)C, suggesting that recognition of alpha(E)C by the I domain involves structural features in common with those of gammaC. These findings identify alpha(E)C as a second domain in fibrinogen-420 that binds alpha(M)beta(2) and alpha(X)beta(2) and can mediate leukocyte adhesion and migration.

Probing the structure-function relationship of alpha-latrotoxin-formed channels with antibodies and pronase.

The major toxic component of black widow spider (Latrodectus mactans tredecimguttatus) venom, alpha-latrotoxin, is known to form ionic channels in different membranes. In order to probe the extramembrane domains of alpha-latrotoxin molecule, alpha-latrotoxin channels in planar lipid membrane were treated with antibodies to latrotoxin or with pronase added to different sides of the membrane. It was found that antibody addition to the same side as the toxin (cis) decreased channel conductance only at positive potentials across the membrane. In contrast, trans side addition of antibodies changed the channel conductance at both positive and negative potentials: at positive potential conductance first slightly increased then decreased by more then 50%; at negative potential it decreased much more quickly, to only about 20% of the initial value. No dependence on membrane potential was found for pronase treatment of incorporated channels. For both cis and trans application of pronase, channel selectivity for Ca2+, Mg2+, Ba2+ and K+, Na+, Li+ ions did not change significantly but Cd2+ block was decreased. Trans pronase treatment also resulted in some rectification of I/V curves and an increase in channel conductance. We interpret these findings as evidence that alpha-latrotoxin channel has protruding parts on both sides of the membrane and that its conformation in the membrane depends on membrane potential.

[Interaction of ribosomes with membranes in vitro]. / Vzaimodeistvie ribosom s membranami in vitro.

Fluorescent "fusion-reporting" probe (R18) was used to study the interaction of ribosomes with membranes in vitro. The latter was incorporated both in the membranes, and ribosomes. The interaction of R18-labeled ribosomes with non-labeled liposomes (of different size and composition) or microsomes increased the fluorescence observed, i.e., the dilution of fluorescent probe took place. The dependence of interaction process on the change of liposomes indicates that the interaction between ribosomes and negatively charged liposomes was more efficient, than that with neutral ones. It is shown that ribosomes can interact with phospholipid membranes even after degradation of protein components accessible for proteins. The interaction of R18-labeled membranes with non-labeled ribosomes results in the increase of fluorescence too. Results obtained indicate to the possibility of direct interaction between ribosomes and membranes.

[Interaction of precursors of secretory proteins and components of a cell-free protein-synthesizing system with phospholipid membranes]. / Vzaimodeistvie predshestvennikov sekretiruemykh belkov i komponentov beskletochnoi beloksinteziruiushchei sistemy s fosfolipidnymi membranami.

Translocation of eucaryotic secretory proteins across the phospholipid membrane containing, no protein components has been studied on the model system. The level of translocation was found to depend critically on physical and chemical properties of membranes. It was found that the cell-free system components can exhibit membrane activity.

[Department of neurochemistry]. / Viddil neirokhimiï.

By the beginning of 1980s the questions concerning the mechanism of neurosecretion, the mode of neurotoxin action on this process and the mechanism of protein insertion into biological membranes were the principal directions of studies. Two views on the process of insertion of membrane proteins into, and transport across the biological membranes have been proposed. One hypothesis on membrane biogenesis describes the mechanism of incorporation of protein molecules into phospholipid matrix as a co-translational process. It starts with the synthesis of a hydrophobic N-terminal signal sequence of the protein destined to span the bilayer. In this case the energy of elongation was supposed to be utilized for insertion of the protein into hydrophobic core of the bilayer. Using the model system of co-translational translocation that consisted of liposomes and cell-free translational system including wheat germ extract and poly(A) RNA obtained from the mammary gland it has been studied whether the liposomes could serve as acceptor of synthesized secretory proteins. It is shown that in the presence of casein mRNA the 14-C-labeled product of translation is accumulated inside liposomes. When mRNA for globin, a nonsecretory protein, is translated in this cell-free system, 14-C-label is not found in the internal volume of liposomes. The polypeptides extracted from liposomes after their incubation in the system of casein mRNA translation interact specifically with anti-casein antibodies (D. I. Balkov, A. V. El'skaya, V. K. Lishko et al., 1988). These data provide the evidence that the transfer of synthesizing casein through bilayer lipid membrane does not require a specific receptor. Insertion and transfer of this protein occur co-translationally, due to the interaction of "signal peptide" with membrane lipids. The second model of the assembly of protein into membrane supposes an ability of hydrophilic proteins to cooperate with the phospholipid bilayer. As a result of such cooperation the polypeptide changes its conformation adequately to the hydrophilic environment and is integrated with the bilayer. This second model suggests the existence of water soluble precursors of membrane proteins localized in the cell cytoplasm. The last idea was studied in experiment with a specific hydrophilic protein from the cytoplasmic fraction of excitable tissues. Tetrodotoxin-sensitive (TTX-sensitive) structures have been found in soluble fractions of the brain, heart and skeletal muscle homogenates (V. K. Lishko, M. K. Malysheva, A. V. Stefanov, 1977). Initially we prepared proteoliposomes by sonication of a phospholipid suspension with the supernatants of tissue homogenates.(ABSTRACT TRUNCATED AT 250 WORDS)

[Binding of secreted protein mRNA translation products with artificial phospholipid vesicles]. / Sviazyvanie produktov transliatsii mRNK sekretiruemykh belkov s iskusstvennymi fosfolipidnymi vezikulami.

Translocation of eucaryotic secretory proteins across the phospholipid membrane containing no protein components has been studied. The level of translocation was found to depend critically on the physico-chemical properties of the membranes. It was found also that eucaryotic secretory proteins can pass through the phospholipid bilayer by both co- and post-translational interactions.

The preparation of endotoxin-free L-methionine-alpha-deamino-gamma-mercaptomethane-lyase (L-methioninase) from Pseudomonas putida.

Many types of human and animal tumors have an absolute requirement for methionine. This requirement can be satisfied by homocysteine only in normal cells and tissues. Therefore, methionine may be an important target in cancer therapy. To attack this target we have purified endotoxin-free methioninase from Pseudomonas putida by a novel and simple procedure. This procedure involves (1) a heat step of the cell extract at 60 degrees C for 8 min, (2) DEAE-Toyopearl ion-exchange chromatography, (3) DEAE-Sephadex A50 ion-exchange gel filtration chromatography, and (4) affinity chromatography on Acticlean to remove the endotoxin bound to the enzyme. The yield for this purification was up to 80%. The methioninase has four subunits of approximate molecular weight 43 kDa. This is the first methodology for methioninase that allows rapid purification with high yield and separation from endotoxin suitable for in vivo efficacy testing against methionine-dependent tumors in animal models.

Therapeutic tumor-specific cell cycle block induced by methionine starvation in vivo.

The ability to induce a specific cell cycle block selectively in the tumor could have many uses in chemotherapy. In the present study we have achieved this goal of inducing a tumor-specific cell cycle block in vivo by depriving Yoshida sarcoma-bearing nude mice of dietary methionine. Further, we demonstrate that methionine depletion also causes the tumor to eventually regress. The antitumor effect of methionine depletion resulted in the extended survival of the tumor-bearing mice. The mice on the methionine-deprived diets maintained their body weight for the time period studied, indicating that tumor regression was not a function of body weight loss. The data reported here support future experiments utilizing methionine depletion as a target for tumor-selective cell cycle-dependent therapy.

Depletion of serum methionine by methioninase in mice.

Methionine dependence is a tumor-specific metabolic defect found in human cancer cell lines as well as in fresh human tumor specimens. Methionine dependent tumors cease growing when deprived of methionine, unlike normal cells which can substitute homocysteine for methionine for their growth requirement. We have previously purified a stable, endotoxin-free methioninase from the bacterium, Pseudomonas putida. We demonstrate in this report that purified methioninase can lower the serum levels of methionine in normal and nude mice from 60 microM to approximately 5 microM within 1 hour. The circulating half-life of methioninase is approximately 100 minutes in mice after i.v. injection. The enzyme therefore seems to be a good candidate as an antitumor agent for methionine-dependent tumors.

Potential-dependent alpha-latrotoxin interaction with black lipid membranes.

The influence of membrane potential on alpha-latrotoxin insertion into bilayer lipid membranes (BLM) has been investigated. It was found that positive potentials cis to toxin application stimulated the formation of channels in the bilayer. A two-step model of latrotoxin/membrane interaction is put forward to explain these data. In the first step, latrotoxin irreversibly binds to the bilayer without forming conductive structures. The second step of the process represents rapid insertion of the protein molecule into the bilayer with the formation of the conducting channel. We imagine the driving force for this process to be the interaction of charged groups in the toxin molecule with the electric field applied across the BLM. Our results are compared with known data on the interaction of LTX with synaptosomal membranes.

[Prevention of postischemic lesions of the myocardium using liposomes]. / Preduprezhdenie postishemicheskikh povrezhdenii miokarda s pomoshch'iu liposom.

The experiments on dogs showed that 60-min blood flow restriction in the left coronary artery branch resulted in pumping and contractile heart dysfunctions. The removal of the blood flow barrier caused reinforcement of the above dysfunctions. The administration of 50 mg/kg liposome prior to reperfusion improved pumping and contractile heart functions and allowed maintenance of stable hemodynamics during the reperfusion.

[Functioning of latrotoxin channels during pH changes]. / Funktsionirovanie latrotoksinovykh kanalov pri izmenenii pH.

Using the fluorescent probe BCECP, the pH dependence of Ca2+ transport in synaptosomes along alpha-latrotoxin-formed channels, was studied. It was found that the pH value in synaptosomes is equivalent to 7.16 +/- 0.09. Acidification or alkalinization of the intracellular medium by 0.1-0.3 pH units had no appreciable influence on the Ca2+ influx along latrotoxin-formed channels. Alteration of external pH caused a parallel shift in the cytoplasmic pH in the synaptosomes. The pH decrease in the external medium down to 6.0 caused the inhibition of Ca2+ fluxes along latrotoxin-formed channels. Dissipation of the proton gradient by high concentrations of KCl in the presence of nigericin decreased the latrotoxin ability to form ionic channels without any loss in the activity of the preformed channels. The influx of bivalent cations along latrotoxin-formed channels led to alkalinization of the synaptosomal cytoplasm to pH of the external medium. This pH change did not depend on the presence of Na+ in the external medium and was blocked by cadmium.

Latrotoxin-like properties of a protein from brain.

In bovine brain cortex cytoplasm we have identified a soluble protein (L-protein) of M(r) approximately 90 kDa interacting with polyclonal antibodies to alpha-latrotoxin. The L-protein forms potential-dependent and cation-selective ion channels in BLM, which are blocked by Cd2+. The fusogenic activity of the L-protein was demonstrated on liposomes. We have arrived at the conclusion that the action mechanisms of the L-protein and alpha-latrotoxin are similar.

[A high level of cytoplasmic calcium inactivates ion channels, formed by alpha-latrotoxin in the rat brain synaptosomes]. / Vysokii uroven' tsitoplazmaticheskogo kal'tsiia inaktiviruet ionnye kanaly, formiruemye alpha-latrotoksinom v sinaptosomakh mozga krys.

Effect of alpha-latrotoxin on the concentration level of free calcium [( Ca2+]in) in the rat brain synaptosomes and dependence of the activity of "latrotoxin" channels on [Ca2+]in were studied using fluorescent calcium probe quin-2. It is shown that alpha-latrotoxin exerts effect on calcium permeability of plasmalemma and does not induce calcium ejection from the intracellular compartments. A lag-period is characteristic of alpha-latrotoxin action. A degree of the [Ca2+]in increase in synaptosomes depends on the toxin concentration. When [Ca2+]in increases as a result of preliminary potassium depolarization of plasmalemma of synaptosomes, the amount of incoming calcium ions followed by the toxin effect as well as the calcium input rate considerably decrease. Inactivation of calcium-transferring channels induced by alpha-latrotoxin is not a result of a change in the potential on the membrane, as during the blockage of potential-depending calcium channels by D-600, an increase of KCl in the incubation medium does not influence the alpha-latrotoxin action. Differences in the properties of alpha-latrotoxin channels are discussed in synaptosomes and BLM.

[Latrotoxin channels. Permeability for divalent cations]. / Latrotoksinovye kanaly. Pronitsaemost' dlia dvukhvalentnykh kationov.

The dependence of Ca2+ transport in synaptosomes along the channels formed by alpha-latrotoxin on [Ca2+]in and the feasibility of transport along these channels of other bivalent cations were studied. It was found that the concentration dependence of Ca2+ influx is nonlinear and is described by the Michaelis-Menten kinetics (Km = 1.07 +/- 0.19 mM). Mg2+, Ba2+, Sr2+, Mn2+ and Co2+ competitively inhibited the Ca2+ influx via latrotoxin channels. Studies with the use of the fluorescent Ca2+ probes, Quin-2 and Fura-2, revealed that these cations can also penetrate inside synaptosomes via latrotoxin channels. The bivalent cation influx via latrotoxin channels caused a decrease of the membrane potential of synaptosomes. The similarity of properties of latrotoxin and endogenous Ca2+ channels is discussed.

[Internalization and degradation of latrotoxin bound to rat brain synaptosomes]. / Internalizatsiia i degradatsiia latrotoksina, sviazannogo s sinaptosomami golovnogo mozga krys.

The accessibility to trypsin of 125I-labeled latrotoxin bound to rat brain synaptosomes was investigated. It was shown that latrotoxin bound to synaptosomes in the cold can be practically completely removed by trypsin treatment. The resistance of latrotoxin to proteolysis increases during its incubation with synaptosomes (37 degrees C). Concanavalin A (10(-6) M) decreases toxin binding by 30%, but fully prevents internalization (incorporation). Moreover, latrotoxin is not incorporated into synaptosomal membrane fragments irrespective of duration and temperature of incubation. Latrotoxin incorporated into synaptosomal membranes undergoes degradation by endogenous proteases resulting in the formation of TCA-soluble products.