Synergy between 15-lipoxygenase and secreted PLA2 promotes inflammation by formation of TLR4 agonists from extracellular vesicles.

Damage-associated endogenous molecules induce innate immune response, thus making sterile inflammation medically relevant. Stress-derived extracellular vesicles (stressEVs) released during oxidative stress conditions were previously found to activate Toll-like receptor 4 (TLR4), resulting in expression of a different pattern of immune response proteins in comparison to lipopolysaccharide (LPS), underlying the differences between pathogen-induced and sterile inflammation. Here we report that synergistic activities of 15-lipoxygenase (15-LO) and secreted phospholipase A2 (sPLA2) are needed for the formation of TLR4 agonists, which were identified as lysophospholipids (lysoPLs) with oxidized unsaturated acyl chain. Hydroxy, hydroperoxy, and keto products of 2-arachidonoyl-lysoPI oxidation by 15-LO were identified by mass spectrometry (MS), and they activated the same gene pattern as stressEVs. Extracellular PLA2 activity was detected in the synovial fluid from rheumatoid arthritis and gout patients. Furthermore, injection of sPLA2 promoted K/BxN serum-induced arthritis in mice, whereby ankle swelling was partially TLR4 dependent. Results confirm the role of oxidized lysoPL of stressEVs in sterile inflammation that promotes chronic diseases. Both 15-LO and sPLA2 enzymes are induced during inflammation, which opens the opportunity for therapy without compromising innate immunity against pathogens.

Disruption of disulfides within RBD of SARS-CoV-2 spike protein prevents fusion and represents a target for viral entry inhibition by registered drugs.

The SARS-CoV-2 pandemic imposed a large burden on health and society. Therapeutics targeting different components and processes of the viral infection replication cycle are being investigated, particularly to repurpose already approved drugs. Spike protein is an important target for both vaccines and therapeutics. Insights into the mechanisms of spike-ACE2 binding and cell fusion could support the identification of compounds with inhibitory effects. Here, we demonstrate that the integrity of disulfide bonds within the receptor-binding domain (RBD) plays an important role in the membrane fusion process although their disruption does not prevent binding of spike protein to ACE2. Several reducing agents and thiol-reactive compounds are able to inhibit viral entry. N-acetyl cysteine amide, L-ascorbic acid, JTT-705, and auranofin prevented syncytia formation, viral entry into cells, and infection in a mouse model, supporting disulfides of the RBD as a therapeutically relevant target.

Extracellular vesicle-mediated transfer of constitutively active MyD88L265P engages MyD88wt and activates signaling.

The link between inflammation and cancer is particularly strong in Waldenström macroglobulinemia (WM), a diffuse large B-cell lymphoma wherein the majority of patients harbor a constitutively active mutation in the innate immune-signaling adaptor myeloid differentiation primary response 88 (MyD88). MyD88Leu265Pro (MyD88L265P) constitutively triggers the myddosome assembly providing a survival signal for cancer cells. Here, we report detection and a functional role of MyD88 in the extracellular vesicles (EVs) shed from WM cells. MyD88L265P was transferred via EVs into the cytoplasm of the recipient mast cells and macrophages, recruiting the endogenous MyD88 that triggered the activation of proinflammatory signaling in the absence of receptor activation. Additionally, internalization of EVs containing MyD88L265P was observed in mice with an effect on the bone marrow microenvironment. MyD88-loaded EVs were detected in the bone marrow aspirates of WM patients thus establishing the physiological role of EVs for MyD88L265P transmission and shaping of the proinflammatory microenvironment. Results establish the mechanism of transmission of signaling complexes via EVs to propagate inflammation as a new mechanism of intercellular communication.

Calcium Ionophore-Induced Extracellular Vesicles Mediate Cytoprotection against Simulated Ischemia/Reperfusion Injury in Cardiomyocyte-Derived Cell Lines by Inducing Heme Oxygenase 1.

Cardioprotection against ischemia/reperfusion injury is still an unmet clinical need. The transient activation of Toll-like receptors (TLRs) has been implicated in cardioprotection, which may be achieved by treatment with blood-derived extracellular vesicles (EVs). However, since the isolation of EVs from blood takes considerable effort, the aim of our study was to establish a cellular model from which cardioprotective EVs can be isolated in a well-reproducible manner. EV release was induced in HEK293 cells with calcium ionophore A23187. EVs were characterized and cytoprotection was assessed in H9c2 and AC16 cell lines. Cardioprotection afforded by EVs and its mechanism were investigated after 16 h simulated ischemia and 2 h reperfusion. The induction of HEK293 cells by calcium ionophore resulted in the release of heterogenous populations of EVs. In H9c2 and AC16 cells, stressEVs induced the downstream signaling of TLR4 and heme oxygenase 1 (HO-1) expression in H9c2 cells. StressEVs decreased necrosis due to simulated ischemia/reperfusion injury in H9c2 and AC16 cells, which was independent of TLR4 induction, but not that of HO-1. Calcium ionophore-induced EVs exert cytoprotection by inducing HO-1 in a TLR4-independent manner.

Adamantane Containing Peptidoglycan Fragments Enhance RANTES and IL-6 Production in Lipopolysaccharide-Induced Macrophages.

We report the enhancement of the lipopolysaccharide-induced immune response by adamantane containing peptidoglycan fragments in vitro. The immune stimulation was detected by Il-6 (interleukine 6) and RANTES (regulated on activation, normal T cell expressed and secreted) chemokine expression using cell assays on immortalized mouse bone-marrow derived macrophages. The most active compound was a α-D-mannosyl derivative of an adamantylated tripeptide with L-chirality at the adamantyl group attachment, whereby the mannose moiety assumed to target mannose receptors expressed on macrophage cell surfaces. The immune co-stimulatory effect was also influenced by the configuration of the adamantyl center, revealing the importance of specific molecular recognition event taking place with its receptor. The immunostimulating activities of these compounds were further enhanced upon their incorporation into lipid bilayers, which is likely related to the presence of the adamantyl group that helps anchor the peptidoglycan fragment into lipid nanoparticles. We concluded that the proposed adamantane containing peptidoglycan fragments act as co-stimulatory agents and are also suitable for the preparation of lipid nanoparticle-based delivery of peptidoglycan fragments.

Locked and proteolysis-based transcription activator-like effector (TALE) regulation.

Development of orthogonal, designable and adjustable transcriptional regulators is an important goal of synthetic biology. Their activity has been typically modulated through stimulus-induced oligomerization or interaction between the DNA-binding and activation/repression domain. We exploited a feature of the designable Transcription activator-like effector (TALE) DNA-binding domain that it winds around the DNA which allows to topologically prevent it from binding by intramolecular cyclization. This new approach was investigated through noncovalent ligand-induced cyclization or through a covalent split intein cyclization strategy, where the topological inhibition of DNA binding by cyclization and its restoration by a proteolytic release of the topologic constraint was expected. We show that locked TALEs indeed have diminished DNA binding and regain full transcriptional activity by stimulation with the rapamycin ligand or site-specific proteolysis of the peptide linker, with much higher level of activation than rapamycin-induced heterodimerization. Additionally, we demonstrated reversibility, activation of genomic targets and implemented logic gates based on combinations of protein cyclization, proteolytic cleavage and ligand-induced dimerization, where the strongest fold induction was achieved by the proteolytic cleavage of a repression domain from a linear TALE.

Postulates for validating TLR4 agonists.

TLRs play a central role in the innate immune response, recognizing a variety of molecular structures characteristic of pathogens. Although TLR4, together with its co-receptor myeloid differentiation-2 (MD-2), recognize bacterial LPS and therefore Gram-negative bacterial infections, it also plays a key role in many other pathophysiological processes, including sterile inflammation and viral infection. Specifically, numerous endogenous agonists of TLR4 of notably diverse nature, ranging from proteins to metal ions, have been reported. Direct activation of a single receptor by such a range of molecular signals is very difficult to explain from a structural and mechanistic point of view. It is likely that only a subset of these directly activate the TLR4-MD-2 complex. We propose three postulates aimed at distinguishing the direct agonists of TLR4 from indirect activators. These postulates are as follows: (i) that the agonist requires the TLR4/MD-2 receptor complex; (ii) that agonist formed synthetically or in situ must activate the receptor complex in order to eliminate artifacts of contamination by other agonists; and (iii) that a specific molecular interaction between the agonist and TLR4/MD-2 must be identified. The same type of postulates can be applied to pattern recognition receptors in general.

A role for stefin B (cystatin B) in inflammation and endotoxemia.

Stefin B (cystatin B) is an endogenous cysteine cathepsin inhibitor, and the loss-of-function mutations in the stefin B gene were reported in patients with Unverricht-Lundborg disease (EPM1). In this study we demonstrated that stefin B-deficient (StB KO) mice were significantly more sensitive to the lethal LPS-induced sepsis and secreted higher amounts of pro-inflammatory cytokines IL-1ß and IL-18 in the serum. We further showed that increased caspase-11 gene expression and better pro-inflammatory caspase-1 and -11 activation determined in StB KO bone marrow-derived macrophages resulted in enhanced IL-1ß processing. Pretreatment of macrophages with the cathepsin inhibitor E-64d did not affect secretion of IL-1ß, suggesting that the increased cathepsin activity determined in StB KO bone marrow-derived macrophages is not essential for inflammasome activation. Upon LPS stimulation, stefin B was targeted into the mitochondria, and the lack of stefin B resulted in the increased destabilization of mitochondrial membrane potential and mitochondrial superoxide generation. Collectively, our study demonstrates that the LPS-induced sepsis in StB KO mice is dependent on caspase-11 and mitochondrial reactive oxygen species but is not associated with the lysosomal destabilization and increased cathepsin activity in the cytosol.

The role of UNC93B1 protein in surface localization of TLR3 receptor and in cell priming to nucleic acid agonists.

Translocation of nucleic acid-sensing (NAS) Toll-like receptors (TLRs) to endosomes is essential for response to microbial nucleic acids as well as for prevention of the autoimmune response. The accessory protein UNC93B1 is indispensable for activation of NAS TLRs because it regulates their response through trafficking to endosomes. We observed that poly(I:C) up-regulates transcription of UNC93B1 and promotes trafficking of TLR3 to the plasma membrane in human epithelial cell line. Up-regulation of UNC93B1 is triggered through TLR3 activation by poly(I:C). Further studies revealed that expression of UNC93B1 promotes trafficking of differentially glycosylated TLR3, but not other NAS TLRs, to the plasma membrane. UNC93B1 promoter region contains binding sites for poly(I:C)- and type I interferon-inducible regulatory elements. UNC93B1 also increases the protein lifetime of TLR3 and TLR9 and augments signaling of all NAS TLRs. Furthermore, we discovered that poly(I:C) pretreatment primes B-cells to the activation by ssDNA via up-regulation of UNC93B1. Our findings identified TLR3 as the important regulator of UNC93B1 that in turn governs the responsiveness of all NAS TLRs.

The lipopolysaccharide core of Brucella abortus acts as a shield against innate immunity recognition.

Innate immunity recognizes bacterial molecules bearing pathogen-associated molecular patterns to launch inflammatory responses leading to the activation of adaptive immunity. However, the lipopolysaccharide (LPS) of the gram-negative bacterium Brucella lacks a marked pathogen-associated molecular pattern, and it has been postulated that this delays the development of immunity, creating a gap that is critical for the bacterium to reach the intracellular replicative niche. We found that a B. abortus mutant in the wadC gene displayed a disrupted LPS core while keeping both the LPS O-polysaccharide and lipid A. In mice, the wadC mutant induced proinflammatory responses and was attenuated. In addition, it was sensitive to killing by non-immune serum and bactericidal peptides and did not multiply in dendritic cells being targeted to lysosomal compartments. In contrast to wild type B. abortus, the wadC mutant induced dendritic cell maturation and secretion of pro-inflammatory cytokines. All these properties were reproduced by the wadC mutant purified LPS in a TLR4-dependent manner. Moreover, the core-mutated LPS displayed an increased binding to MD-2, the TLR4 co-receptor leading to subsequent increase in intracellular signaling. Here we show that Brucella escapes recognition in early stages of infection by expressing a shield against recognition by innate immunity in its LPS core and identify a novel virulence mechanism in intracellular pathogenic gram-negative bacteria. These results also encourage for an improvement in the generation of novel bacterial vaccines.

MARCKS as a negative regulator of lipopolysaccharide signaling.

Myristoylated alanine-rich C kinase substrate (MARCKS) is an intrinsically unfolded protein with a conserved cationic effector domain, which mediates the cross-talk between several signal transduction pathways. Transcription of MARCKS is increased by stimulation with bacterial LPS. We determined that MARCKS and MARCKS-related protein specifically bind to LPS and that the addition of the MARCKS effector peptide inhibited LPS-induced production of TNF-α in mononuclear cells. The LPS binding site within the effector domain of MARCKS was narrowed down to a heptapeptide that binds to LPS in an extended conformation as determined by nuclear magnetic resonance spectroscopy. After LPS stimulation, MARCKS moved from the plasma membrane to FYVE-positive endosomes, where it colocalized with LPS. MARCKS-deficient mouse embryonic fibroblasts (MEFs) responded to LPS with increased IL-6 production compared with the matched wild-type MEFs. Similarly, small interfering RNA knockdown of MARCKS also increased LPS signaling, whereas overexpression of MARCKS inhibited LPS signaling. TLR4 signaling was enhanced by the ablation of MARCKS, which had no effect on stimulation by TLR2, TLR3, and TLR5 agonists. These findings demonstrate that MARCKS contributes to the negative regulation of the cellular response to LPS.

TXM peptides inhibit SARS-CoV-2 infection, syncytia formation, and lower inflammatory consequences.

After three years of the SARS-CoV-2 pandemic, the search and availability of relatively low-cost benchtop therapeutics for people not at high risk for a severe disease are still ongoing. Although vaccines and new SARS-CoV-2 variants reduce the death toll, the long COVID-19 along with neurologic symptoms can develop and persist even after a mild initial infection. Reinfections, which further increase the risk of sequelae in multiple organ systems as well as the risk of death, continue to require caution. The spike protein of SARS-CoV-2 is an important target for both vaccines and therapeutics. The presence of disulfide bonds in the receptor binding domain (RBD) of the spike protein is essential for its binding to the human ACE2 receptor and cell entry. Here, we demonstrate that thiol-reducing peptides based on the active site of oxidoreductase thioredoxin 1, called thioredoxin mimetic (TXM) peptides, can prevent syncytia formation, SARS-CoV-2 entry into cells, and infection in a mouse model. We also show that TXM peptides inhibit the redox-sensitive HIV pseudotyped viral cell entry. These results support disulfide targeting as a common therapeutic strategy for treating infections caused by viruses using redox-sensitive fusion. Furthermore, TXM peptides exert anti-inflammatory properties by lowering the activation of NF-κB and IRF signaling pathways, mitogen-activated protein kinases (MAPKs) and lipopolysaccharide (LPS)-induced cytokines in mice. The antioxidant and anti-inflammatory effects of the TXM peptides, which also cross the blood-brain barrier, in combination with prevention of viral infections, may provide a beneficial clinical strategy to lower viral infections and mitigate severe consequences of COVID-19.

Interaction of the HIV-1 gp120 viral protein V3 loop with bacterial lipopolysaccharide: a pattern recognition inhibition.

HIV-1 represents an elusive target for therapeutic compounds due to its high rate of mutation. Targeting structural patterns instead of a constantly changing specific three-dimensional structure may represent an approach that is less sensitive to viral mutations. The V3 loop of gp120 of HIV-1, which is responsible for binding of viral gp120 to CCR5 or CXCR4 coreceptors, has already been identified as an effective target for the inhibition of viral entry. The peptide derived from the V3 loop of gp120 specifically interacts with the lipid A moiety of LPS, as does the full gp120 protein. NMR analysis of V3 in complex with LPS shows formation of an amphipathic turn. The interaction between LPS and V3 relies on the structural pattern, comprising a combination of hydrophobic and charge interactions, similar to the interaction between antimicrobial peptides and LPS. LPS inhibited binding of gp120 to the surface of target T cells. Nonendotoxic LPS antagonists inhibited viral infection, demonstrating the possibility for the development of an inhibitor of HIV-1 attachment to T cells based on the recognition of a conserved structural pattern.

Globular domain of the prion protein needs to be unlocked by domain swapping to support prion protein conversion.

Prion diseases are fatal transmissible neurodegenerative diseases affecting many mammalian species. The normal prion protein (PrP) converts into a pathological aggregated form, PrPSc, which is enriched in the ß-sheet structure. Although the high resolution structure of the normal PrP was determined, the structure of the converted form of PrP remains inaccessible to high resolution techniques. To map the PrP conversion process we introduced disulfide bridges into different positions within the globular domain of PrP, tethering selected secondary structure elements. The majority of tethered PrP mutants exhibited increased thermodynamic stability, nevertheless, they converted efficiently. Only the disulfides that tether subdomain B1-H1-B2 to subdomain H2-H3 prevented PrP conversion in vitro and in prion-infected cell cultures. Reduction of disulfides recovered the ability of these mutants to convert, demonstrating that the separation of subdomains is an essential step in conversion. Formation of disulfide-linked proteinase K-resistant dimers in fibrils composed of a pair of single cysteine mutants supports the model based on domain-swapped dimers as the building blocks of prion fibrils. In contrast to previously proposed structural models of PrPSc suggesting conversion of large secondary structural segments, we provide evidence for the conservation of secondary structural elements of the globular domain upon PrP conversion. Previous studies already showed that dimerization is the rate-limiting step in PrP conversion. We show that separation and swapping of subdomains of the globular domain is necessary for conversion. Therefore, we propose that the domain-swapped dimer of PrP precedes amyloid formation and represents a potential target for therapeutic intervention.

Half is enough: Oxidized lysophospholipids as novel bioactive molecules.

Studies in the last decade have established the roles of oxidized phospholipids as modulators of various cellular processes, from inflammation and immunity to cell death. Oxidized lysophospholipids, formed through the activity of phospholipases and oxidative enzymes and lacking an acyl chain in comparison with parent phospholipids, are now emerging as novel bioactive lipid mediators. Their detection and structural characterization have been limited in the past due to low amounts and the complexity of their biosynthetic and removal pathways, but recent studies have unequivocally demonstrated their formation under inflammatory conditions. The involvement of oxidized lysophospholipids in immune regulation classifies them as damage-associated molecular patterns (DAMPs), which can promote sterile inflammation and contribute to autoimmune and chronic diseases as well as aging-related diseases. Their signaling pathways are just beginning to be revealed. As the first publications indicate that oxidized lysophospholipids use the same receptors as pathogen-associated molecular patterns (PAMPs), it is likely that the inhibition of signaling pathways activated by oxidized lysophospholipids would affect innate immunity per se. On the other hand, inhibition or modulation of their enzymatic formation, which would not interfere with the response to pathogens, might be beneficial and is potentially a promising new field of research.

Cleavage-Mediated Regulation of Myd88 Signaling by Inflammasome-Activated Caspase-1.

Coordination among multiple signaling pathways ensures an appropriate immune response, where a signaling pathway may impair or augment another signaling pathway. Here, we report a negative feedback regulation of signaling through the key innate immune mediator MyD88 by inflammasome-activated caspase-1. NLRP3 inflammasome activation impaired agonist- or infection-induced TLR signaling and cytokine production through the proteolytic cleavage of MyD88 by caspase-1. Site-specific mutagenesis was used to identify caspase-1 cleavage site within MyD88 intermediary segment. Different cleavage site location within MyD88 defined the functional consequences of MyD88 cleavage between mouse and human cells. LPS/monosodium urate-induced mouse inflammation model corroborated the physiological role of this mechanism of regulation, that could be reversed by chemical inhibition of NLRP3. While Toll/interleukin-1 receptor (TIR) domain released by MyD88 cleavage additionally contributed to the inhibition of signaling, Waldenström's macroglobulinemia associated MyD88L265P mutation is able to evade the caspase-1-mediated inhibition of MyD88 signaling through the ability of its TIRL265P domain to recruit full length MyD88 and facilitate signaling. The characterization of this mechanism reveals an additional layer of innate immunity regulation.

A Nanoscaffolded Spike-RBD Vaccine Provides Protection against SARS-CoV-2 with Minimal Anti-Scaffold Response.

The response of the adaptive immune system is augmented by multimeric presentation of a specific antigen, resembling viral particles. Several vaccines have been designed based on natural or designed protein scaffolds, which exhibited a potent adaptive immune response to antigens; however, antibodies are also generated against the scaffold, which may impair subsequent vaccination. In order to compare polypeptide scaffolds of different size and oligomerization state with respect to their efficiency, including anti-scaffold immunity, we compared several strategies of presentation of the RBD domain of the SARS-CoV-2 spike protein, an antigen aiming to generate neutralizing antibodies. A comparison of several genetic fusions of RBD to different nanoscaffolding domains (foldon, ferritin, lumazine synthase, and ß-annulus peptide) delivered as DNA plasmids demonstrated a strongly augmented immune response, with high titers of neutralizing antibodies and a robust T-cell response in mice. Antibody titers and virus neutralization were most potently enhanced by fusion to the small ß-annulus peptide scaffold, which itself triggered a minimal response in contrast to larger scaffolds. The ß-annulus fused RBD protein increased residence in lymph nodes and triggered the most potent viral neutralization in immunization by a recombinant protein. Results of the study support the use of a nanoscaffolding platform using the ß-annulus peptide for vaccine design.

Targeted Delivery of Adamantylated Peptidoglycan Immunomodulators in Lipid Nanocarriers: NMR Shows That Cargo Fragments Are Available on the Surface.

We present an in-depth investigation of the membrane interactions of peptidoglycan (PGN)-based immune adjuvants designed for lipid-based delivery systems using NMR spectroscopy. The derivatives contain a cargo peptidoglycan (PGN) dipeptide fragment and an adamantyl group, which serves as an anchor to the lipid bilayer. Furthermore, derivatives with a mannose group that can actively target cell surface receptors on immune cells are also studied. We showed that the targeting mannose group and the cargo PGN fragment are both available on the lipid bilayer surface, thereby enabling interactions with cognate receptors. We found that the nonmannosylated compounds are incorporated stronger into the lipid assemblies than the mannosylated ones, but the latter compounds penetrate deeper in the bilayer. This might be explained by stronger electrostatic interactions available for zwitterionic nonmannosylated derivatives as opposed to the compounds in which the charged N-terminus is capped by mannose groups. The higher incorporation efficiency of the nonmannosylated compounds correlated with a larger relative enhancement in immune stimulation activities upon lipid incorporation compared to that of the derivatives with the mannose group. The chirality of the adamantyl group also influenced the incorporation efficiency, which in turn correlated with membrane-associated conformations that affect possible intermolecular interactions with lipid molecules. These findings will help in improving the development of PGN-based immune adjuvants suitable for delivery in lipid nanoparticles.

Prevention of microvesiculation by adhesion of buds to the mother cell membrane--a possible anticoagulant effect of healthy donor plasma.

Microvesicles (MVs) found in peripheral blood are derived from the budding of cell membranes and are associated with a higher risk of thrombosis. Recently, a hypothesis has been suggested that certain plasma proteins could suppress microvesiculation by mediating adhesion of the buds to the mother cell membrane. In a pilot study, we have tested this hypothesis by considering the relation between the amount of MVs in peripheral blood and the ability of plasma to induce adhesion between giant phospholipid vesicles (GPVs). MVs were isolated from human plasma and counted by flow cytometry. The adhesion between GPVs was measured by assessing the average angle of contact between the adhered vesicles. It was found that greater ability of plasma to induce adhesion relates to smaller concentration of MVs in plasma. The ratio between the concentration of MVs and the concentration of platelets proved the most efficient parameter to predict the propensity of the membrane to shed vesicles. Our results indicate that a stronger attractive interaction between GPVs mediated by plasma is associated with a smaller amount of MVs per platelets. Plasma that mediates stronger attractive interaction between GPVs might potentially be associated with a smaller risk of thrombosis.

Number of microvesicles in peripheral blood and ability of plasma to induce adhesion between phospholipid membranes in 19 patients with gastrointestinal diseases.

It was recently shown that the plasma protein-mediated attractive interaction between phospholipid membranes could in the budding process cause adhesion of the bud to the mother membrane [J. Urbanija, N. Tomsic, M. Lokar, A. Ambrozic, S. Cucnik, M. Kanduser, B. Rozman, A. Iglic, V. Kralj-Iglic, Coalescence of phospholipid membranes as a possible origin of anticoagulant effect of serum proteins, Chem. Phys. Lipids 150 (2007) 49-57]. Since in the in vivo conditions the budding of cell membranes leads to the release of microvesicles into the circulation, a hypothesis was put forward that the ability of plasma to cause adhesion between membranes supresses the microvesiculation process. In the present work, this hypothesis was tested in a population of 19 patients with gastrointestinal diseases. The number of microvesicles in peripheral blood of patients was determined by flow cytometry while the ability of plasma to cause adhesion between membranes was determined by adding patient's plasma to the suspension of giant phospholipid vesicles created by electroformation method, and measuring the average effective angle of contact between the adhered vesicles. Statistically significant negative correlations between the number of microvesicles and the average effective angle of contact (Pearson coefficient -0.50, p=0.031) and between the number of microvesicles per number of platelets and the average effective angle of contact (Pearson coefficient -0.64, p=0.003) were found, which is in favor of the above hypothesis. Patients with gastrointestinal cancer had larger number of microvesicles (difference 140%, statistical significance 0.033) and smaller average effective angle of contact (difference 20%, statistical significance 0.013) compared to patients with other gastrointestinal diseases.