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Evgueni L Saenko

Publications and source records attributed to Evgueni L Saenko.

At least 19 recordsLinked to original sources
PubMed2006

Identification of coagulation factor VIII A2 domain residues forming the binding epitope for low-density lipoprotein receptor-related protein.

Regulation of the coagulation factor VIII (fVIII) level in circulation involves a hepatic receptor low-density lipoprotein receptor-related protein (LRP). One of two major LRP binding sites in fVIII is located within the A2 domain (A2), likely exposed within the fVIII complex with von Willebrand factor and contributing to regulation of fVIII via LRP. This work aimed to identify A2 residues forming its LRP-binding site, previously shown to involve residues 484-509. Isolated A2 was subjected to alanine-scanning mutagenesis followed by expression of a set of mutants in a baculovirus system. In competition and surface plasmon resonance assays, affinities of A2 mutants K466A, R471A, R484A, S488A, R489A, R490A, H497A, and K499A for LRP were found to be decreased by 2-4-fold. This correlated with 1.3-1.5-fold decreases in the degree of LRP-mediated internalization of the mutants in cell culture. Combining these mutations into pairs led to cumulative effects, i.e., 7-13-fold decrease in affinity for LRP and 1.6-2.2-fold decrease in the degree of LRP-mediated internalization in cell culture. We conclude that the residues mentioned above play a key role in formation of the A2 binding epitope for LRP. Experiments in mice revealed an approximately 4.5 times shorter half-life for A2 in the circulation in comparison with that of fVIII. The half-lives of A2 mutant R471A/R484A or A2 co-injected with receptor-associated protein, a classical ligand of LRP, were prolonged by approximately 1.9 and approximately 3.5 times, respectively, compared to that of A2. This further confirms the importance of the mutated residues for interaction of A2 with LRP and suggests the existence of an LRP-dependent mechanism for removing A2 as a product of dissociation of activated fVIII from the circulation.

Alanine↗
PubMed2006

Role of the B domain in proteolytic inactivation of activated coagulation factor VIII by activated protein C and activated factor X.

Hereditary deficiency of factor VIII (FVIII), haemophilia A, is treated by plasma-derived FVIII (pd-FVIII) or recombinant FVIII (rFVIII) infusions. B-domain-deleted FVIII (BDD-rFVIII), although generally safe and effective, was less effective than pd-FVIII in prophylaxis -- evidenced by a 2.5-fold higher bleeding incidence. Assessment of BDD-rFVIII activity in chromogenic and one-stage clotting assays gives up to 50% difference in activity values. As earlier studies demonstrated identical activation and cofactor activity of BDD-rFVIII and pd-FVIII, we decided to study susceptibility of thrombin-activated pd-FVIII, full-length rFVIII and BDD-rFVIII to proteolytic inactivation by activated protein C (APC) and activated factor X (FXa) in a purified system. Proteolysis was monitored by Western blot using monoclonal antibodies C5 and R8B12 specific for the A1 and A2 domains, respectively. Inactivation was monitored by measuring the residual cofactor activity of FVIII forms in a one-stage clotting assay. Proteolysis of A1 and A2 domains of activated BDD-rFVIII proceeded 11 or 13 times faster than that of pd-FVIII or full-length rFVIII. Inactivation of activated BDD-rFVIII was two to three times faster by APC and five to six times faster by FXa. We suggest that differences in proteolytic inactivation may contribute to differences between BDD-rFVIII and pd-FVIII in assaying and in clinical use.

Blotting, Western↗
PubMed2006

Factor VIIIa regulates substrate delivery to the intrinsic factor X-activating complex.

Activation of coagulation factor X (fX) by activated factors IX (fIXa) and VIII (fVIIIa) requires the assembly of the enzyme-cofactor-substrate fIXa-fVIIIa-fX complex on negatively charged phospholipid membranes. Using flow cytometry, we explored formation of the intermediate membrane-bound binary complexes of fIXa, fVIIIa, and fX. Studies of the coordinate binding of coagulation factors to 0.8-microm phospholipid vesicles (25/75 phosphatidylserine/phosphatidylcholine) showed that fVIII (fVIIIa), fIXa, and fX bind to 32 700 +/- 5000 (33 200 +/- 14 100), 20 000 +/- 4500, and 30 500 +/- 1300 binding sites per vesicle with apparent K(d) values of 76 +/- 23 (71 +/- 5), 1510 +/- 430, and 223 +/- 79 nm, respectively. FVIII at 10 nm induced the appearance of additional high-affinity sites for fIXa (1810 +/- 370, 20 +/- 5 nm) and fX (12 630 +/- 690, 14 +/- 4 nm), whereas fX at 100 nm induced high-affinity sites for fIXa (541 +/- 67, 23 +/- 5 nm). The effects of fVIII and fVIIIa on the binding of fIXa or fX were similar. The apparent Michaelis constant of the fX activation by fIXa was a linear function of the fVIIIa concentration with a slope of 1.00 +/- 0.12 and an intrinsic K(m) value of 8.0 +/- 1.5 nm, in agreement with the hypothesis that the reaction rate is limited by the fVIIIa-fX complex formation. In addition, direct correlation was observed between the fX activation rate and formation of the fVIIIa-fX complex. Titration of fX, fVIIIa, phospholipid concentration and phosphatidylserine content suggested that at high fVIIIa concentration the reaction rate is regulated by the concentration of free fX rather than of membrane-bound fX. The obtained results reveal formation of high-affinity fVIIIa-fX complexes on phospholipid membranes and suggest their role in regulating fX activation by anchoring and delivering fX to the enzymatic complex.

Enzyme Activation↗
PubMed2005

Spatial propagation and localization of blood coagulation are regulated by intrinsic and protein C pathways, respectively.

Blood coagulation in vivo is a spatially nonuniform, multistage process: coagulation factors from plasma bind to tissue factor (TF)-expressing cells, become activated, dissociate, and diffuse into plasma to form enzymatic complexes on the membranes of activated platelets. We studied spatial regulation of coagulation using two approaches: 1), an in vitro experimental model of clot formation in a thin layer of plasma activated by a monolayer of TF-expressing cells; and 2), a computer simulation model. Clotting in factor VIII- and factor XI-deficient plasmas was initiated normally, but further clot elongation was impaired in factor VIII- and, at later stages, in factor XI-deficient plasma. The data indicated that clot elongation was regulated by factor Xa formation by intrinsic tenase, whereas factor IXa was formed by extrinsic tenase on activating cells and diffused into plasma, thus sustaining clot growth. Far from the activating cells, additional factor IXa was produced by factor XIa. Exogenously added TF had no effect on the clot growth rate, suggesting that plasma TF does not contribute significantly to the clot propagation process in a reaction-diffusion system without flow. Addition of thrombomodulin at 3-100 nM caused dose-dependent termination of clot elongation with a final clot size of 2-0.2 mm. These results identify roles of specific coagulation pathways at different stages of spatial clot formation (initiation, elongation, and termination) and provide a possible basis for their therapeutic targeting.

Blood Coagulation↗
PubMed2005

The apoE isoform binding properties of the VLDL receptor reveal marked differences from LRP and the LDL receptor.

Apolipoprotein E (apoE) associates with lipoproteins and mediates their interaction with members of the LDL receptor family. ApoE exists as three common isoforms that have important distinct functional and biological properties. Two apoE isoforms, apoE3 and apoE4, are recognized by the LDL receptor, whereas apoE2 binds poorly to this receptor and is associated with type III hyperlipidemia. In addition, the apoE4 isoform is associated with the common late-onset familial and sporadic forms of Alzheimer's disease. Although the interaction of apoE with the LDL receptor is well characterized, the specificity of other members of this receptor family for apoE is poorly understood. In the current investigation, we have characterized the binding of apoE to the VLDL receptor and the LDL receptor-related protein (LRP). Our results indicate that like the LDL receptor, LRP prefers lipid-bound forms of apoE, but in contrast to the LDL receptor, both LRP and the VLDL receptor recognize all apoE isoforms. Interestingly, the VLDL receptor does not require the association of apoE with lipid for optimal recognition and avidly binds lipid-free apoE. It is likely that this receptor-dependent specificity for various apoE isoforms and for lipid-free versus lipid-bound forms of apoE is physiologically significant and is connected to distinct functions for these receptors.

Apolipoprotein E3↗
PubMed2004

Kinetics of Factor X activation by the membrane-bound complex of Factor IXa and Factor VIIIa.

Intrinsic tenase consists of activated Factors IX (IXa) and VIII (VIIIa) assembled on a negatively charged phospholipid surface. In vivo, this surface is mainly provided by activated platelets. In vitro, phosphatidylcholine/phosphatidylserine vesicles are often used to mimic natural pro-coagulant membranes. In the present study, we developed a quantitative mathematical model of Factor X activation by intrinsic tenase. We considered two situations, when complex assembly occurs on either the membrane of phospholipid vesicles or the surface of activated platelets. On the basis of existing experimental evidence, the following mechanism for the complex assembly on activated platelets was suggested: (i) Factors IXa, VIIIa and X bind to their specific platelet receptors; (ii) bound factors form complexes on the membrane: platelet-bound Factor VIIIa provides a high-affinity site for Factor X and platelet-bound Factor IXa provides a high-affinity site for Factor VIIIa; (iii) the enzyme-cofactor-substrate complex is assembled. This mechanism allowed the explanation of co-operative effects in the binding of Factors IXa, VIIIa and X to platelets. The model was reduced to obtain a single equation for the Factor X activation rate as a function of concentrations of Factors IXa, VIIIa, X and phospholipids (or platelets). The equation had a Michaelis-Menten form, where apparent V(max) and K(m) were functions of the factors' concentrations and the internal kinetic constants of the system. The equation obtained can be used in both experimental studies of intrinsic tenase and mathematical modelling of the coagulation cascade. The approach of the present study can be applied to research of other membrane-dependent enzymic reactions.

Blood Platelets↗
PubMed2004

Inhibitors in hemophilia A: mechanisms of inhibition, management and perspectives.

Factor VIII (FVIII) replacement therapy remains the mainstay in hemophilia A care. The major complication of replacement therapy is formation of antibodies, which inhibit FVIII activity, thus dramatically reducing treatment efficiency. The present review summarizes the accumulated knowledge on epitopes of FVIII inhibitors and mechanisms of their inhibitory effects. FVIII inhibitors most frequently target the A2, C2 and A3 domains of FVIII and interfere with important interactions of FVIII at various stages of its functional pathway; a class of FVIII inhibitors inactivates FVIII by proteolysis. We discuss therapeutic approaches currently used for treatment of hemophilia A patients with inhibitors and analyze the factors that influence the outcome. The choice between options should depend on the level of inhibitors and consideration of efficacy, safety, and availability of particular regimens. Advances of basic science open avenues for alternative targeted, specific and long-lasting treatments, such as the use of peptide decoys for blocking FVIII inhibitors, bypassing them with human/porcine FVIII hybrids, neutralizing FVIII-reactive CD4 T cells with anti-clonotypic antibodies, or inducing immune tolerance to FVIII with the use of universal CD4 epitopes or by genetic approaches.

Animals↗
PubMed2004

Epitope mapping of polyclonal clotting factor VIII-inhibitory antibodies using phage display.

Clotting factor VIII (fVIII)-inhibitory antibodies represent a major problem in the treatment of haemophilia A. To understand the inactivation mechanisms and to pave the way towards modifications of recombinant clotting factors that reduce their immunogenicity, the exact localization of immunodominant epitopes is required. Here, a random peptide phage display library was employed to identify epitopes of polyclonal fVIII antibodies isolated from patient's plasma by affinity chromatography. FVIII-binding specificity and inhibitory activity of the isolated fVIII antibodies were confirmed by ELISA and Bethesda assays. Phage selection on the individual samples yielded several phages which were displaced from binding to the respective antibody preparation by fVIII. Their homology with amino acid motifs of human fVIII and immunoprecipitation results with radioactively labelled fVIII fragments suggested putative epitopes in the A1, A2 and C1 domains of fVIII for one and in the C2 domain for another patient. Synthetic peptides corresponding to the A2, C1 and C2 domain epitopes blocked antibody binding to fVIII and partially neutralized the inhibitory activity of the respective plasma in Bethesda assays. These results provide the proof of principle that random peptide libraries can be used for the mapping of epitopes in a polyclonal antibody preparation.

Amino Acid Sequence↗
PubMed2003

Failure of immunocompetitive capillary electrophoresis assay to detect disease-specific prion protein in buffy coat from humans and chimpanzees with Creutzfeldt-Jakob disease.

The emergence of a new environmentally caused variant of Creutzfeldt-Jakob disease (vCJD), the result of food-born infection by the causative agent of bovine spongiform encephalopathy (BSE), has stimulated research on a practical diagnostic screening test. The immunocompetitive capillary electrophoresis (ICCE) assay has been reported to detect disease-specific, proteinase-resistant prion protein (PrPres) in the blood of scrapie-infected sheep. We have applied this method to blood from CJD-infected chimpanzees and humans. The threshold of detection achieved with our ICCE was 0.6 nM of synthetic peptide corresponding to the prion protein (PrP) C-terminus, and 2 nM of recombinant human PrP at the optimized conditions. However, the test was unable to distinguish between extracts of leucocytes from healthy and CJD-infected chimpanzees, and from healthy human donors and patients affected with various forms of CJD. Thus, the ICCE assay as presently performed is not suitable for use as a screening test in human transmissible spongiform encephalopathies (TSEs).

Amino Acid Sequence↗
PubMed2003

Development of improved factor VIII molecules and new gene transfer approaches for hemophilia A.

Hemophilia A, the most common inherited bleeding disorder, is caused by deficiency or functional defects in coagulation factor VIII (fVIII). Conventional treatment for this disease involves intravenous infusions of plasma-derived or recombinant fVIII products. Although replacement therapy effectively stops the bleeding episodes, it has a risk of transmission of viral blood-borne diseases and development of neutralizing antibodies that inactivate the administered fVIII protein. Hemophilia A is an attractive candidate for application of gene therapy approaches because the therapeutic window is wide and even modest elevation of fVIII levels will correct the hemophilic phenotype. Ongoing preclinical investigations utilize animal models of hemophilia A, including genetically fVIII-deficient mice and naturally fVIII-deficient dogs, to optimize vectors, transgenes and target cell populations for Phase I clinical trials. In this review, we outline the progress in understanding the mechanisms of fVIII turnover, which provides a basis for development of improved fVIII molecules with prolonged half-life in the circulation. We discuss the possibility of incorporating these improved fVIII molecules as transgenes into self-inactivating lentiviral vectors carrying chromatin insulator sequences, representing a new generation of gene delivery vehicle, to target hematopoietic stem cells and endothelial cells. The use of hematopoietic stem cells as the target cell population may prevent inhibitor formation to transduced fVIII by induction of immune tolerance. Alternatively, endothelial cells may support optimal synthesis of fVIII and myeloablative conditioning of patients with radiation or chemotherapy may not be required for efficient engraftment of the engineered cells. Collectively, these proposed advances represent promising prophylactic strategies toward long-term correction of the coagulation defect in this progressively debilitating, life-threatening disease.

Animals↗
PubMed2003

Effect of factor VIII on tissue factor-initiated spatial clot growth.

Using time-lapse videomicroscopy, we studied the role of coagulation factor VIII (fVIII) in tissue factor-initiated spatial clot growth on fibroblast monolayers in a thin layer of non-stirred recalcified plasma from healthy donors or patients with severe Haemophilia A. Analysis of temporal evolution of light-scattering profiles from a growing clot revealed existence of two phases in the clot growth-initiation phase in a narrow (0.2 mm) zone adjacent to activator surface and elongation phase in plasma volume. While the initiation phase did not differ in normal and haemophilic plasmas, the rate of clot growth in the elongation phase in haemophilic plasma constituted only 30% of that in normal plasma. Supplementation of haemophilic plasma with 0.05 U/ml fVIII restored the normal clot growth rate (44.9 +/- 2.5 microm/min) at high but not at low fibroblast density. Our results indicate that the functioning of the intrinsic tenase complex is critical for normal spatial clot growth.

Blood Coagulation↗
PubMed2002

Intrinsic pathway of blood coagulation contributes to thrombogenicity of atherosclerotic plaque.

Thrombosis is the major mechanism underlying acute complications of atherosclerosis. Although thrombogenicity of atherosclerotic plaques has been ascribed to activation of the extrinsic pathway of blood coagulation, in the present study we investigated contribution of the intrinsic factor VIII (fVIII)-dependent pathway. We found that in vitro exposure of human macrophages and smooth muscle cells (SMCs) to atherogenic oxidized low-density lipoprotein (oxLDL) enhances their ability to support activity of 2 major complexes of the intrinsic pathway, Xase and prothrombinase, leading to a 20- and 10-fold increase in thrombin formation, respectively. In contrast, human aortic endothelial cells were less responsive to oxLDL. The increase in the intrinsic procoagulant activity was related to formation of additional fVIII binding sites due to enhanced translocation of phosphatidylserine to the outer surface of oxLDL-treated cells and a 5-fold higher affinity of interaction between components of the Xase complex, activated factors VIII and IX. Processes occurring at early apoptotic stages, including changes in the cell membrane induced by free radicals, may be related to activation of the intrinsic pathway as suggested by effects of inhibitors of early apoptosis on thrombin formation. Immunohistochemical studies on human atherectomy specimens revealed the presence of fVIII in the vicinity of macrophages and SMCs in atheromatous regions with massive deposits of oxLDL, supporting the possible involvement of the intrinsic pathway in thrombus formation in vivo. Our data predict that the intrinsic pathway significantly enhances thrombogenicity of atherosclerotic lesions after removal of the endothelial layer and exposure of SMCs and macrophages to blood flow.

Aorta↗
PubMed2002

A novel mechanism of factor VIII protection by von Willebrand factor from activated protein C-catalyzed inactivation.

The protective effect of von Willebrand factor (VWF) toward activated protein C (APC)-catalyzed inactivation of factor VIII (FVIII) has been attributed mainly to inhibition of FVIII binding to phospholipid. In the present study, we demonstrated that VWF-mediated FVIII protection from APC also results from direct inhibition of FVIII binding to APC. Inhibition of FVIII binding to anhydro-APC by VWF would be consistent with partial or complete overlap of the FVIII binding sites for APC and VWF. We examined, therefore, the inhibitory effects of 6 synthetic peptides spanning residues 1996 to 2028 around the previously localized APC binding region (FVIII residues 2009-2018). Peptide 2009 to 2018 inhibited FVIII binding to anhydro-APC by 83% (50% inhibition, 55 microM). Similarly, peptide 2013 to 2022 inhibited FVIII binding to VWF by 84% (50% inhibition, 25 microM). It was also found that peptides 2009 to 2018 and 2013 to 2022 optimally bound to anhydro-APC and VWF, respectively. A rabbit antipeptide IgG, raised against peptide 2009 to 2022, blocked the binding of both anhydro-APC and VWF to FVIII. This immunoglobulin G inhibited proteolytic cleavage of FVIII by APC. Our results indicate that the essential regions for the binding of APC and VWF to FVIII overlap and that the protective effect of VWF on APC-catalyzed FVIII inactivation includes competitive inhibition of APC binding to FVIII by VWF.

Amino Acid Sequence↗
PubMed2002

Coagulation pathways in atherothrombosis.

Atherosclerosis is a disease recognized as the main cause of death in industrial countries. The current paradigm establishes thrombosis to be the major reason for complications of atherosclerosis, such as myocardial infarction and stroke, and the major factor responsible for atherosclerosis-related mortality. Development of adequate treatment of patients with risk of atherothrombosis requires the comprehensive understanding of mechanisms underlying coagulation processes at the site of atherosclerotic lesion. The present review discusses contribution of the extrinsic and intrinsic pathways of blood coagulation in thrombogenicity of atherosclerotic plaque and factors determining the overall procoagulant/anticoagulant balance.

Animals↗
PubMed2002

Anticoagulant effects of a synthetic peptide containing residues Thr-2253-Gln-2270 within factor VIII C2 domain that selectively inhibits factor Xa-catalysed factor VIII activation.

Factor VIII (FVIII), an essential cofactor that accelerates the generation of factor Xa (FXa) in the tenase complex, is activated by proteolytic cleavage by thrombin or FXa. A strong relationship has been reported between high levels of FVIII activity and thrombosis. We have demonstrated previously that an anti-FVIII C2 antibody (ESH8) with a Val-2248-Gly-2285 epitope inhibited FXa-catalysed FVIII activation, and that a synthetic peptide designated EP-2 (residues 2253-2270) blocked C2 domain binding to FXa. We investigated the inhibitory effect of EP-2 on FXa-catalysed FVIII activation and its anticoagulant effect in the blood coagulation system. EP-2 inhibited FXa-catalysed activation in a clotting assay in a dose-dependent manner and reduced FXa generation in a chromogenic assay using FVIII, factor X, factor IXa and phospholipid. The peptide only inhibited FVIII binding to FXa. We also tested the anticoagulant effect of EP-2 in the plasma milieu. The peptide prolonged the activated partial thromboplastin time and activated clotting time in a dose-dependent manner, but not prothrombin time. Our results indicate that EP-2 mediates the anticoagulant effect by specific inhibition of FVIII and FXa interaction in the intrinsic pathway, and that FXa-catalysed FVIII activation plays a significant role in blood clotting. The peptide may provide the basis for the development of novel anticoagulant therapy.

Anticoagulants↗
PubMed2002

Hemophilia A--from basic science to clinical practice.

This article summarizes achievements of basic research and their implementation in clinical treatment of one of the most common inherited bleeding disorders hemophilia A, which is caused by genetic deficiency of coagulation factor VIII (FVIII). We discuss the structure of FVIII, its major interactions in the intrinsic pathway of blood coagulation, and the catabolism of FVIII. We also discuss achievements in the contemporary clinical practice of treatment of hemophilia A. Replacement therapy has substantially improved by development of purification and virus inactivation procedures, allowing preparation of safe and effective therapeutic plasma-derived FVIII concentrates. We give special attention to the principles used in the development of contemporary recombinant FVIII products, which do not inherit a potential risk for viral or prion transmission. Development of FVIII inhibitory antibodies is the major complication of FVIII replacement therapy. We summarize the accumulated knowledge regarding epitopes of FVIII inhibitors and mechanisms by which they inactivate FVIII and discuss approaches to overcome the effects of inhibitors and to prevent their formation by induction of immunotolerance. We also analyze the main concepts and scientific priorities in the gene-therapeutic approach for treatment of hemophilia A.

Animals↗
PubMed2002

Human factor VIII inhibitor alloantibodies with a C2 epitope inhibit factor Xa-catalyzed factor VIII activation: a new anti-factor VIII inhibitory mechanism.

Factor VIII (FVIII) inhibitor alloantibodies react with the A2, C2, or A3-CI domains of FVIII and inactivate FVIII activity. We recently demonstrated that an anti-C2 monoclonal antibody with a Val2248-Gly2285 epitope, inhibited factor Xa (FXa)-catalyzed FVIII activation, and that a FXa binding site for FVIII was located within residues Thr2253-Gln2270. In this study, we investigated whether anti-C2 alloantibodies inhibit FXa-catalyzed FVIII activation. Anti-C2 alloantibodies from four patients inhibited FVIII activation by FXa in one-stage clotting assay. Furthermore, analysis by SDS-PAGE showed that all alloantibodies inhibited FVIII proteolytic cleavage by FXa independently of phospholipid. To confirm direct inhibition of FVIII and FXa interaction, we examined the effect of alloantibodies on FVIII binding to anhydro-FXa, a catalytically inactive FXa, in ELISA. All alloantibodies and C2-affinity purified F(ab)'2 preparations inhibited FVIII binding to anhydro-FXa dose-dependently. Our results revealed a new inhibitory mechanism of FVIII, mediated by inhibition of FXa in the presence of anti-C2 alloantibodies.

Antibody Specificity↗