Biomedical subjects

Alexey V Khrenov

Publications and source records attributed to Alexey V Khrenov.

5 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↗

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↗

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↗