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Biomedical subjects

M D Smirnov

Publications and source records attributed to M D Smirnov.

At least 19 recordsLinked to original sources
PubMed2001

Factor V Leiden mutation in one family of Chinese origin.

OBJECTIVE: To investigate the factor V Leiden mutation associated with activated protein C resistance (APCR) in Chinese. METHODS: Thirty "normal" individuals and twenty patients with thrombotic disease from Chinese Han Nationality were studied with APTT +/- APC, PCR followed by MnLI restriction enzyme analysis, PCR based direct sequence-specific primers (PCR-SSP) and DNA sequence analysis. RESULTS: In one healthy control, the activated protein C (APC) sensitivity ratio (SR) was found to be significantly lower (0.8) than that in other normal control (> 2.0). This individual was identified to be heterozygous for FV Leiden mutation (Arg506-Gln). His grand-uncle, father, brother and son were also identified to be heterozygous for FV Leiden. The APC resistance was found in 3 other cases of thrombotic diseases, but with no FV Leiden mutation. CONCLUSION: This is the first four generations family case of FV Leiden mutation associated with APCR reported within Chinese ethnic population. It is note-worthy that more FV Leiden or whether other gene defects may be associated with APC resistance and acquired APCR causing thrombosis in Chinese population.

Activated Protein C Resistance↗
PubMed2000

Lipid oxidation enhances the function of activated protein C.

Although lipid oxidation products are usually associated with tissue injury, it is now recognized that they can also contribute to cell activation and elicit anti-inflammatory lipid mediators. In this study, we report that membrane phospholipid oxidation can modulate the hemostatic balance. Oxidation of natural phospholipids results in an increased ability of the membrane surface to support the function of the natural anticoagulant, activated protein C (APC), without significantly altering the ability to support thrombin generation. Lipid oxidation also potentiated the ability of protein S to enhance APC-mediated factor Va inactivation. Phosphatidylethanolamine, phosphatidylserine, and polyunsaturation of the fatty acids were all required for the oxidation-dependent enhancement of APC function. A subgroup of thrombotic patients with anti-phospholipid antibodies specifically blocked the oxidation-dependent enhancement of APC function. Since leukocytes are recruited and activated at the thrombus or sites of vessel injury, our findings suggest that after the initial thrombus formation, lipid oxidation can remodel the membrane surface resulting in increased anticoagulant function, thereby reducing the thrombogenicity of the thrombus or injured vessel surface. Anti-phospholipid antibodies that block this process would therefore be expected to contribute to thrombus growth and disease.

Humans↗
PubMed2000

Mechanisms by which soluble endothelial cell protein C receptor modulates protein C and activated protein C function.

The endothelial cell protein C receptor (EPCR) functions as an important regulator of the protein C anticoagulant pathway by binding protein C and enhancing activation by the thrombin-thrombomodulin complex. EPCR binds to both protein C and activated protein C (APC) with high affinity. A soluble form of EPCR (sEPCR) circulates in plasma and inhibits APC anticoagulant activity. In this study, we investigate the mechanisms by which sEPCR modulates APC function. Soluble EPCR inhibited the inactivation of factor Va by APC only in the presence of phospholipid vesicles. By using flow cytometric analysis in the presence of 3 mM CaCl(2) and 0. 6 mM MgCl(2), sEPCR inhibited the binding of protein C and APC to phospholipid vesicles (K(i) = 40 +/- 7 and 33 +/- 4 nM, respectively). Without MgCl(2), the K(i) values increased approximately 4-fold. Double label flow cytometric analysis using fluorescein-APC and Texas Red-sEPCR indicated that the APC.sEPCR complex does not interact with phospholipid vesicles. By using surface plasmon resonance, we found that sEPCR also inhibited binding of protein C to phospholipid in a dose-dependent fashion (K(i) = 32 nM). To explore the possibility that sEPCR evokes structural changes in APC, fluorescence spectroscopy studies were performed to monitor sEPCR/Fl-APC interactions. sEPCR binds saturably to Fl-APC (K(d) = 27 +/- 13 nM) with a maximum decrease in Fl-APC fluorescence of 10.8 +/- 0.6%. sEPCR also stimulated the amidolytic activity of APC toward synthetic substrates. We conclude that sEPCR binding to APC blocks phospholipid interaction and alters the active site of APC.

Binding Sites↗
PubMed2000

Antiphospholipid antibodies and the protein C pathway.

Among the mechanisms suggested for the prothrombotic activity of lupus anticoagulant and antiphospholipid antibodies is the direct inhibition of the anticoagulant activated protein C (APC) pathway. Although some pathological antibodies may be directed towards the proteins involved, we hypothesize that populations exist which selectively inhibit the APC complex as a result of differences in the phospholipid requirements of this complex as compared to those of the procoagulant complexes. The most prominent feature is the requirement for the presence of phosphatidylethanolamine in the membrane for APC anticoagulant function. This mimics the requirements for inhibitory activity of at least a subset of autoantibodies associated with thrombosis. The role of oxidation of the phospholipid in APC function and antibody reactivity is also discussed.

Antibodies, Antiphospholipid↗
PubMed1999

Inhibition of activated protein C anticoagulant activity by prothrombin.

In this study, we test the hypothesis that prothrombin levels may modulate activated protein C (APC) anticoagulant activity. Prothrombin in purified systems or plasma dramatically inhibited the ability of APC to inactivate factor Va and to anticoagulate plasma. This was not due solely to competition for binding to the membrane surface, as prothrombin also inhibited factor Va inactivation by APC in the absence of a membrane surface. Compared with normal factor Va, inactivation of factor Va Leiden by APC was much less sensitive to prothrombin inhibition. This may account for the observation that the Leiden mutation has less of an effect on plasma-based clotting assays than would be predicted from the purified system. Reduction of protein C levels to 20% of normal constitutes a significant risk of thrombosis, yet these levels are observed in neonates and patients on oral anticoagulant therapy. In both situations, the correspondingly low prothrombin levels would result in an increased effectiveness of the remaining functional APC of approximately 5-fold. Thus, while the protein C activation system is impaired by the reduction in protein C levels, the APC that is formed is a more effective anticoagulant, allowing protein C levels to be reduced without significant thrombotic risk. In situations where prothrombin is high and protein C levels are low, as in early stages of oral anticoagulant therapy, the reduction in protein C would result only in impaired function of the anticoagulant system, possibly explaining the tendency for warfarin-induced skin necrosis.

Anticoagulants↗
PubMed1999

The effect of membrane composition on the hemostatic balance.

The phospholipid composition requirements for optimal prothrombin activation and factor Va inactivation by activated protein C (APC) anticoagulant were examined. Vesicles composed of phosphatidylethanolamine (PE) and phosphatidylcholine (PC) supported factor Va inactivation relatively well. However, optimal factor Va inactivation still required relatively high concentrations of phosphatidylserine (PS). In addition, at a fixed concentration of phospholipid, PS, and APC, vesicles devoid of PE never attained a rate of factor Va inactivation achievable with vesicles containing PE. Polyunsaturation of any vesicle component also contributed significantly to APC inactivation of factor Va. Thus, PE makes an important contribution to factor Va inactivation that cannot be mimicked by PS. In the absence of polyunsaturation in the other membrane constituents, this contribution was dependent upon the presence of both the PE headgroup per se and unsaturation of the 1,2 fatty acids. Although PE did not affect prothrombin activation rates at optimal PS concentrations, PE reduced the requirement for PS approximately 10-fold. The Km(app) for prothrombin and the Kd(app) for factor Xa-factor Va decreased as a function of increasing PS concentration, reaching optimal values at 10-15% PS in the absence of PE but only 1% PS in the presence of PE. Fatty acid polyunsaturation had minimal effects. A lupus anticoagulant immunoglobulin was more inhibitory to both prothrombinase and factor Va inactivation in the presence of PE. The degree of inhibition of APC was significantly greater and much more dependent on the phospholipid composition than that of prothrombinase. Thus, subtle changes in the phospholipid composition of cells may control procoagulant and anticoagulant reactions differentially under both normal and pathological conditions.

Blood Coagulation↗
PubMed1999

Relocating the active site of activated protein C eliminates the need for its protein S cofactor. A fluorescence resonance energy transfer study.

The effect of replacing the gamma-carboxyglutamic acid domain of activated protein C (APC) with that of prothrombin on the topography of the membrane-bound enzyme was examined using fluorescence resonance energy transfer. The average distance of closest approach (assuming kappa2 = 2/3) between a fluorescein in the active site of the chimera and octadecylrhodamine at the membrane surface was 89 A, compared with 94 A for wild-type APC. The gamma-carboxyglutamic acid domain substitution therefore lowered and/or reoriented the active site, repositioning it close to the 84 A observed for the APC. protein S complex. Protein S enhances wild-type APC cleavage of factor Va at Arg306, but the inactivation rate of factor Va Leiden by the chimera alone is essentially equal to that by wild-type APC plus protein S. These data suggest that the activities of the chimera and of the APC.protein S complex are equivalent because the active site of the chimeric protein is already positioned near the optimal location above the membrane surface to cleave Arg306. Thus, one mechanism by which protein S regulates APC activity is by relocating its active site to the proper position above the membrane surface to optimize factor Va cleavage.

Binding Sites↗
PubMed1999

Lupus anticoagulants, thrombosis and the protein C system.

Although lupus anticoagulants (LAs) are immunoglobulins that inhibit procoagulant reactions in vitro, these molecules are associated with thrombosis in vivo. We and others have hypothesized that this may be due to selective targeting of the activated protein C (APC) anticoagulant pathway. Populations of antibodies that interact with protein C or protein S in ways that inhibit their activity are obvious candidates for such pathological molecules. However, it is less clear how populations that appear to bind to membrane surfaces might target the APC anticoagulant complex selectively. Studies now show that the membrane requirements of the APC anticoagulant complex are significantly different from those of the procoagulant reactions. The most dramatic difference is the requirement for the presence of phosphatidylethanolamine (PE) in the membrane for optimal APC function. The inhibitory activity of at least some LAs is enhanced by the presence of PE, but the anti-APC activity is enhanced even more, resulting in the plasma from these patients clotting faster than normal when APC is present. Structure-function studies have been undertaken to understand the PE dependence of this reaction better. Chimeric proteins in which all or part of the Gla domain of protein C has been replaced by the homologous region of prothrombin have been prepared. Unexpectedly, the PE dependence resides primarily in the C-terminal half of the Gla domain. Using liposomes of various composition, we found both the presence of the PE head group and unsaturation of the fatty acid chains are required for optimal inactivation of factor Va. It is hoped that a better understanding of the biochemistry of these reactions, combined with the use of the chimeric proteins described, will permit us to design better assays for the identification of pathologic LAs.

Humans↗
PubMed1998

A chimeric protein C containing the prothrombin Gla domain exhibits increased anticoagulant activity and altered phospholipid specificity.

To determine the structural basis of phosphatidylethanolamine (PE)-dependent activated protein C (APC) activity, we prepared a chimeric molecule in which the Gla domain and hydrophobic stack of protein C were replaced with the corresponding region of prothrombin. APC inactivation of factor Va was enhanced 10-20-fold by PE. Protein S enhanced inactivation 2-fold and independently of PE. PE and protein S had little effect on the activity of the chimera. Factor Va inactivation by APC was approximately 5-fold less efficient than with the chimera on vesicles lacking PE and slightly more efficient on vesicles containing PE. The cleavage patterns of factor Va by APC and the chimera were similar, and PE enhanced the rate of Arg506 and Arg306 cleavage by APC but not the chimera. APC and the chimera bound to phosphatidylserine:phosphatidylcholine vesicles with similar affinity (Kd approximately 500 nM), and PE increased affinity 2-3-fold. Factor Va and protein S synergistically increased the affinity of APC on vesicles without PE to 140 nM and with PE to 14 nM, but they were less effective in enhancing chimera binding to either vesicle. In a factor Xa one-stage plasma clotting assay, the chimera had approximately 5 times more anticoagulant activity than APC on PE-containing vesicles. Unlike APC, which showed a 10 fold dependence on protein S, the chimera was insensitive to protein S. To map the site of the PE and protein S dependence further, we prepared a chimera in which residues 1-22 were derived from prothrombin and the remainder were derived from protein C. This protein exhibited PE and protein S dependence. Thus, these special properties of the protein C Gla domain are resident outside of the region normally hypothesized to be critical for membrane interaction. We conclude that the protein C Gla domain possesses unique properties allowing synergistic interaction with factor Va and protein S on PE-containing membranes.

Amino Acid Sequence↗
PubMed1997

Thrombogenic mechanisms of antiphospholipid antibodies.

These studies indicate how immunoglobulin populations that react with phospholipid surfaces in the absence of other cofactor molecules can selectively inhibit anticoagulant pathways and lead to a prothrombotic state. These studies combined with those of others indicating the presence of a-PE antibodies (often in isolation) in thrombotic patients illustrate the need to better define the assays to determine patients at risk. Neither the LA assays nor the anti-cardiolipin assays presently in use may be testing for the population(s) of clinical importance. A better understanding of the biochemical requirements of the various reactions involved should help the rational design of such assays. The preliminary studies of Salmon, et al, also show that the genetic context of the patient may contribute to the thrombotic mechanism of any APA present. It is unlikely any single mechanism is responsible for the thrombogenic activity of all APAs associated with thrombosis and this will be a fertile field of investigation for a significant time to come.

Antibodies, Antiphospholipid↗
PubMed1997

[Tomography in diagnosis of sternal osteomyelitis].

The use of transverse direction of smearing in tomography of sternum provides an opportunity of getting the image of the sternum in the direct frontal projection. The displacement of the smearing angle enables increased depth of the imaging layer. This modification provides more complete image of the sternum, improves diagnosis of consolidation disorders and signs of osteomyelitis.

Child↗
PubMed1996

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

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

Aminocaproates↗
PubMed1995

On the role of phosphatidylethanolamine in the inhibition of activated protein C activity by antiphospholipid antibodies.

Phosphatidylethanolamine (PE) is an important membrane component for supporting activated protein C anticoagulant activity but has little influence on prothrombin activation. This difference constitutes a potential mechanism for selective inhibition of the protein C anticoagulant pathway by lupus anticoagulants and/or antiphospholipid antibodies. In this study, we demonstrate that the presence of PE augments lupus anticoagulant activity. In the plasma of some patients with lupus anticoagulants, activated protein C anticoagulant activity is more potently inhibited than prothrombin activation. As a result, in the presence of activated protein C and PE, these patient plasmas clot faster than normal plasma. Patients with minimal lupus anticoagulant activity are identified whose plasma potently inhibits activated protein C anticoagulant activity. This process is also PE dependent. In three patient plasmas, these phenomena are shown to be due to immunoglobulins. The PE requirement in the expression of activated protein C anticoagulant activity and the PE dependence of some antiphospholipid antibodies provide a mechanistic basis for the selective inhibition of the protein C pathway. Inhibition of activated protein C function may be a common mechanism contributing to increased thrombotic risk in certain patients with antiphospholipid antibodies.

Anticoagulants↗
PubMed1994

Phosphatidylethanolamine incorporation into vesicles selectively enhances factor Va inactivation by activated protein C.

Membrane surfaces accelerate the proteolytic inactivation of factor Va by activated protein C. In most coagulation complexes, the most active membrane phospholipid is believed to be phosphatidylserine. In this study, we demonstrate that with phosphatidylserine-containing vesicles, incorporation of phosphatidylethanolamine increased the rate of factor Va inactivation approximately 10-fold at all concentrations of factor Va studied and at all vesicle concentrations at or below the optimum for prothrombin activation. In contrast, phosphatidylethanolamine had very little influence on prothrombin activation. Phosphatidylethanolamine was a critical component of vesicles for the vesicles to support activated protein C anticoagulant activity optimally in plasma. These results demonstrate that the membrane requirements for the different coagulation/anticoagulation complexes differ much more than previously appreciated.

Animals↗
PubMed1993

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

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

Animals↗
PubMed1993

Avidin attachment to biotinylated human neutrophils induces generation of superoxide anion.

The influence of biotinylation and subsequent attachment of avidin on generation of superoxide anion by human neutrophils was studied. Biotinylation of human neutrophils with succinimide ester of biotin does not reduce superoxide generation in response to activation with phorbol myristate acetate (PMA) and formyl peptide (FMLP). Addition of avidin to biotinylated, but not native, leukocytes induces generation of superoxide anion. The kinetics and level of superoxide generation by biotinylated neutrophils in response to addition of avidin were quite similar to those in response to activation with FMLP. The avidin sugar moiety and charge were not involved in superoxide generation, since streptavidin was also active. Both avidin- and PMA-induced superoxide generation were independent of the extracellular calcium, while FMLP-induced superoxide generation was dependent on the presence of calcium in solution. Therefore, interaction of avidin with biotinylated components of the neutrophil membrane alters functional activity of this cell and might induce 'activation-like' reaction of leukocytes.

1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium ↗
PubMed1993

Avidin attachment to biotinylated amino groups of the erythrocyte membrane eliminates homologous restriction of both classical and alternative pathways of the complement.

Lysis of avidin-coated biotinylated sheep red blood cells (RBC) via the classical pathway of homologous (sheep) and heterologous (guinea pig) complement has been studied. The minimal surface density of avidin inducing antibody-dependent lysis via the classical pathway is smaller than that inducing antibody-independent lysis via the alternative pathway. Heterologous lysis via the classical pathway does not depend on the mode of avidin attachment: both biotinylation of membrane amino groups and insertion of biotinyl-lipid into the membrane provide the same lysis of avidin-coated RBCs by guinea pig serum in the presence of anti-avidin antibody. Avidin-free sheep RBC sensitized with hemolytic anti-RBC antibody were lysed by guinea pig, but not by sheep serum, confirming high efficiency of homologous restriction of the complement. However, avidin-coated RBCs were lysed by homologous serum in the presence of anti-avidin antibody at low surface density of avidin attached. The elimination of the homologous restriction depends on the mode of avidin attachment: biotinylation of membrane amino groups provides antibody-mediated lysis via the classical pathway of homologous complement, while insertion of biotinyl-lipid does not provide lysis.

Amines↗