Post-prandial rise of microvesicles in peripheral blood of healthy human donors.

BACKGROUND: Microvesicles isolated from body fluids are membrane - enclosed fragments of cell interior which carry information on the status of the organism. It is yet unclear how metabolism affects the number and composition of microvesicles in isolates from the peripheral blood. AIM: To study the post - prandial effect on microvesicles in isolates from the peripheral blood of 21 healthy donors, in relation to blood cholesterol and blood glucose concentrations. RESULTS: The average number of microvesicles in the isolates increased 5 hours post - prandially by 52%; the increase was statistically significant (p = 0.01) with the power P = 0.68, while the average total blood cholesterol concentration, average low density lipoprotein cholesterol concentration (LDL-C) and average high density lipoprotein cholesterol concentration (HDL-C) all remained within 2% of their fasting values. We found an 11% increase in triglycerides (p = 0.12) and a 6% decrease in blood glucose (p < 0.01, P = 0.74). The post - prandial number of microvesicles negatively correlated with the post - fasting total cholesterol concentration (r = - 0.46, p = 0.035) while the difference in the number of microvesicles in the isolates between post - prandial and post - fasting states negatively correlated with the respective difference in blood glucose concentration (r = - 0.39, p = 0.05). CONCLUSIONS: In a population of healthy human subjects the number of microvesicles in isolates from peripheral blood increased in the post - prandial state. The increase in the number of microvesicles was affected by the fasting concentration of cholesterol and correlated with the decrease in blood glucose.

Interaction between equally charged membrane surfaces mediated by positively and negatively charged macro-ions.

In biological systems, charged membrane surfaces are surrounded by charged molecules such as electrolyte ions and proteins. Our recent experiments in the systems of giant phospholipid vesicles indicated that some of the blood plasma proteins (macro-ions) may promote adhesion between equally charged membrane surfaces. In this work, theory was put forward to describe an IgG antibody-mediated attractive interaction between negatively charged membrane surfaces which was observed in experiments on giant phospholipid vesicles with cardiolipin-containing membranes. The attractive interactions between negatively charged membrane surfaces in the presence of negatively and positively charged spherical macro-ions are explained using functional density theory and Monte Carlo simulations. Both, the rigorous solution of the variational problem within the functional density theory and the Monte Carlo simulations show that spatial and orientational ordering of macro-ions may give rise to an attractive interaction between negatively charged membrane surfaces. It is also shown that the distinctive spatial distribution of the charge within the macro-ions (proteins) is essential in this process.

Suppression of membrane microvesiculation--a possible anticoagulant and anti-tumor progression effect of heparin.

Heparins (unfractionated and low molecular weight (LMWH) heparins) primarily used as anticoagulants, were found to be effective also in slowing down the development of some types of cancer. On the other hand, the number of microvesicles in the peripheral blood originating from the budding of cell membranes (mostly platelets) is increased in hypercoagulabile states as well as in cancer, indicating a possible common underlying mechanism. It was hypothesized that by mediating an attractive interaction between phospholipid membranes heparin suppresses microvesiculation and thereby acts as an anticoagulant and anti-tumor agent. In this work, the effect of LMWH nadroparin on phospholipid membranes was tested in vitro in a system of giant phospholipid vesicles (GPVs) created by electroformation and observed under the phase contrast microscope. Plasma of different blood donors containing different concentrations of nadroparin was added to the suspension of GPVs to induce adhesion between GPVs. The attractive interaction between membranes was assessed by measuring the average effective angle of contact between the adhered GPVs. It was found in healthy donors, in a donor with gastrointestinal cancer and in a donor with rheumatoid arthritis that adding therapeutic doses of nadroparin to the plasma samples enhanced adhesion of phospholipid membranes in a dose and time-dependent manner while nadroparin alone had no effect within the therapeutic concentration range. The results are in favor of the hypothesis that suppression of microvesiculation underlies both, the anticoagulant and the anti-tumor progression effect of heparin.

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.

Agglutination of like-charged red blood cells induced by binding of beta2-glycoprotein I to outer cell surface.

Plasma protein-mediated attractive interaction between membranes of red blood cells (RBCs) and phospholipid vesicles was studied. It is shown that beta(2)-glycoprotein I (beta(2)-GPI) may induce RBC discocyte-echinocyte-spherocyte shape transformation and subsequent agglutination of RBCs. Based on the observed beta(2)-GPI-induced RBC cell shape transformation it is proposed that the hydrophobic portion of beta(2)-GPI molecule protrudes into the outer lipid layer of the RBC membrane and increases the area of this layer. It is also suggested that the observed agglutination of RBCs is at least partially driven by an attractive force which is of electrostatic origin and depends on the specific molecular shape and internal charge distribution of membrane-bound beta(2)-GPI molecules. The suggested beta(2)-GPI-induced attractive electrostatic interaction between like-charged RBC membrane surfaces is qualitatively explained by using a simple mathematical model within the functional density theory of the electric double layer, where the electrostatic attraction between the positively charged part of the first domains of bound beta(2)-GPI molecules and negatively charged glycocalyx of the adjacent RBC membrane is taken into account.

Antiphospholipid antibodies as non-traditional risk factors in atherosclerosis based cardiovascular diseases without overt autoimmunity. A critical updated review.

The role of antiphospholipid antibodies (aPL) associated with cardiovascular diseases has been extensively studied in autoimmune patients, however it was largely unknown whether and how aPL associate with coronary artery disease (CAD), ishemic stroke (IS) and peripheral artery disease (PAD) in non-autoimmune patients. The current review attempts to prioritize for the first time clinical studies based on cause-outcome and strengths relationships in reference to aPL and CAD/PAD, in addition to supplementing Brey's comprehensive review on IS with other, additional studies. Our overview indicates that all case-control and cross-sectional studies found an aPL association with CAD, PAD and IS, while cohort and nested case-control studies reported a prevailing negative risk association between aPL and IS (confirming Brey), with an unclear/unresolved risk association between aPL and CAD. The only cohort, follow-up study found in PAD reported on positive risk association between aPL and disease. The most frequently associated aPL in all studies reported, irrespective of disease, was aCL, with a less frequent association reported for LA, aß2GPI and other aPL.

Nanoparticles isolated from blood: a reflection of vesiculability of blood cells during the isolation process.

BACKGROUND: Shedding of nanoparticles from the cell membrane is a common process in all cells. These nanoparticles are present in body fluids and can be harvested by isolation. To collect circulating nanoparticles from blood, a standard procedure consisting of repeated centrifugation and washing is applied to the blood samples. Nanoparticles can also be shed from blood cells during the isolation process, so it is unclear whether nanoparticles found in the isolated material are present in blood at sampling or if are they created from the blood cells during the isolation process. We addressed this question by determination of the morphology and identity of nanoparticles harvested from blood. METHODS: The isolates were visualized by scanning electron microscopy, analyzed by flow cytometry, and nanoparticle shapes were determined theoretically. RESULTS: The average size of nanoparticles was about 300 nm, and numerous residual blood cells were found in the isolates. The shapes of nanoparticles corresponded to the theoretical shapes obtained by minimization of the membrane free energy, indicating that these nanoparticles can be identified as vesicles. The concentration and size of nanoparticles in blood isolates was sensitive to the temperature during isolation. We demonstrated that at lower temperatures, the nanoparticle concentration was higher, while the nanoparticles were on average smaller. CONCLUSION: These results indicate that a large pool of nanoparticles is produced after blood sampling. The shapes of deformed blood cells found in the isolates indicate how fragmentation of blood cells may take place. The results show that the contents of isolates reflect the properties of blood cells and their interaction with the surrounding solution (rather than representing only nanoparticles present in blood at sampling) which differ in different diseases and may therefore present a relevant clinical parameter.

Mechanical stability of membrane nanotubular protrusions influenced by attachment of flexible rod-like proteins.

It is indicated that nonhomogeneous lateral distribution of membrane attached and flexible rod-like proteins (MRPs) may stabilize nanotubular membrane protrusions. We have shown that curvature induced accumulation of MRPs in the nanotubular membrane protrusion and the corresponding reduction of the membrane free energy are possible if the decrease of the deviatoric free energy of MRPs in the nanotubular protrusions is large enough to overcome the increase of the free energy due to decrease of configurational entropy in the process of lateral sorting of MRPs. The decrease of isotropic curvature energy of MRPs in the region of membrane protrusion is usually not sufficient for substantial MRPs sorting and consequent stabilization of the nanotubular membrane protrusions.

Effects of low-molecular-weight heparin on adhesion and vesiculation of phospholipid membranes: a possible mechanism for the treatment of hypercoagulability in antiphospholipid syndrome.

Heparins represent an efficient treatment of acute thrombosis and obstetric complications in antiphospholipid syndrome (APS). Enhanced microvesiculation of cell membranes, as detected by reduced membrane adhesion, can contribute to hypercoagulability in APS. Healthy donor IgG antibodies significantly increased beta2-glycoprotein I (beta2-GPI)-induced membrane adhesion, indicating that IgG antibodies might supplement the role of beta2-GPI in the regulation of membrane microvesiculation in healthy individuals. Anti-beta2-GPI IgG antibodies significantly reduced beta2-GPI-induced membrane adhesion, suggesting a direct role of anti-beta2-GPI antibodies in enhancing membrane microvesiculation in APS. Therapeutic concentration of nadroparin completely restored beta2-GPI-induced membrane adhesion in the presence of anti-beta2-GPI IgG antibodies. A novel anticoagulant mechanism of nadroparin in APS is suggested that supplements its direct effect on the coagulation cascade. Restoration of adhesion between negatively charged membranes in the presence of nadroparin might decrease shedding of microvesicles into the surrounding solution and could thus contribute to the efficacy of heparin treatment in APS.

Beta2-glycoprotein I and annexin A5 phospholipid interactions: artificial and cell membranes.

The aim of this report was an overview of beta2-glycoprotein I (beta2-GPI)- and annexin A5 (AnxA5)-phospholipid interactions including candidate beta2-GPI receptors, and their relevance to the investigation of physiological/pathological processes. Both beta2-GPI and AnxA5 have thrombomodulatory functions in vivo and their antigenicity is particularly important for thrombotic manifestations and pregnancy complications in antiphospholipid syndrome. Specific elements of beta2-GPI- and AnxA5-phospholipid interactions are different. The crucial elements for beta2-GPI are conformational change and dimerization, both of which are also required and necessary for receptor signaling, leading to the prothombotic state. AnxA5 differs in its ability to crystallize into a protective shield, the disruption of which seems to be the major prothrombotic mechanism. These differences may explain some of the functional consequences of both molecules seen in the pathological conditions. Future alternative therapies of antiphospholipid syndrome are proposed to be based on the expanding knowledge of beta2-GPI- and AnxA5-phospholipid interactions, specifically antagonizing beta2-GPI receptors, as well as inhibiting their signaling pathways.

Attachment of beta 2-glycoprotein I to negatively charged liposomes may prevent the release of daughter vesicles from the parent membrane.

The temperature-induced budding of POPC-cardiolipin-cholesterol, POPC-POPS-cholesterol and POPC-POPG-cholesterol giant lipid vesicles in the presence of beta 2-glycoprotein I (beta 2-GPI) in the outer solution was studied experimentally and theoretically. The observed budding transition of vesicles was continuous which can be explained by taking into account the orientational ordering and direct interactions between oriented lipids. The attachment of positively charged beta 2-GPI to the negatively charged outer surface of POPC-cardiolipin-cholesterol, POPC-POPS-cholesterol and POPC-POPG-cholesterol giant vesicles caused coalescence of the spheroidal membrane bud with the parent vesicle before the bud could detach from the parent vesicle, i.e. vesiculate. Theoretically, the protein-mediated attraction between the membrane of a bud and the parent membrane was described as an interaction between two electric double layers. It was shown that the specific spatial distribution of charge within beta 2-GPI molecules attached to the negatively charged membrane surface may explain the observed attraction between like-charged membrane surfaces.