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Biomedical subjects

E L Saenko

Publications and source records attributed to E L Saenko.

At least 19 recordsLinked to original sources
PubMed2006

Strategies towards a longer acting factor VIII.

The reduced mortality, improved joint outcomes and enhanced quality of life, which have been witnessed in the developed world for patients with haemophilia, have been an outstanding achievement. Advancements in biotechnology contributed significantly through the development of improved pathogen screening, viral inactivation techniques and the development of recombinant clotting factors. These were partnered with enhanced delivery of care through comprehensive haemophilia centres, adoption of home therapy and most recently effective prophylaxis. This came at great costs to governments, medical insurers and patients' families. In addition, barriers persist limiting the adoption and adherence of effective prophylactic therapy. Biotechnology has been successful at overcoming similar barriers in other disease states. Long-acting biological therapeutics are an incremental advance towards overcoming some of these barriers. Strategies that have been successful for other therapeutic proteins are now being applied to factor VIII (FVIII) and include modifications such as the addition of polyethylene glycol (PEG) polymers and polysialic acids and alternative formulation with PEG-modified liposomes. In addition, insight into FVIII structure and function has allowed targeted modifications of the protein to increase the duration of its cofactor activity and reduce its clearance in vivo. The potential advantages and disadvantages of these approaches will be discussed.

Chemistry, Pharmaceutical↗
PubMed2006

Factor V C2 domain contains a major thrombin-binding site responsible for thrombin-catalyzed factor V activation.

Factor (F)V is converted into its active form, FVa, by limited proteolysis. Thrombin-catalyzed activation of FV is essential for its full cofactor activation. Previously, we reported that thrombin was bound to the C2 domain in the light chain of FVIII. As FV has a similar domain structure to FVIII, we focused on the FV C2 domain as a possible binding region for thrombin. Kinetic parameters, measured by surface plasmon resonance, revealed that the K(d) values of anhydro-thrombin for FV, FVa, and the FV C2 domain were 66, 240, and 670 nmol L(-1), respectively. FV activation was increased by approximately 9-fold by the addition of thrombin. In the presence of the FV C2 domain, this increase of the FV activation was inhibited. However, FV activation was not inhibited by the addition of the FVIII C2 domain. FV was cleaved into a 105-kDa heavy chain and a 71/74-kDa light chain by thrombin-catalyzed proteolysis at Arg709, Arg1018 and Arg1545. In the presence of the FV C2 domain, the cleavage was inhibited at all sites. Proteolysis was not affected by the addition of the FVIII C2 domain. These results indicated that the FV C2 domain contains a major binding site for thrombin and that this domain is necessary for the proteolysis at all cleavage sites. Furthermore, the present results also suggested that thrombin has an independent binding site for FV different from that for FVIII.

Binding, Competitive↗
PubMed2005

Initiation and propagation of coagulation from tissue factor-bearing cell monolayers to plasma: initiator cells do not regulate spatial growth rate.

Exposure of tissue factor (TF)-bearing cells to blood is the initial event in coagulation and intravascular thrombus formation. However, the mechanisms which determine thrombus growth remain poorly understood. To explore whether the procoagulant activity of vessel wall-bound cells regulates thrombus expansion, we studied in vitro spatial clot growth initiated by cultured human cells of different types in contact pathway-inhibited, non-flowing human plasma. Human aortic endothelial cells, smooth muscle cells, macrophages and lung fibroblasts differed in their ability to support thrombin generation in microplate assay with peaks of generated thrombin of 60 +/- 53 nmol L(-1), 135 +/- 57 nmol L(-1), 218 +/- 55 nmol L(-1) and 407 +/- 59 nmol L(-1) (mean +/- SD), respectively. Real-time videomicroscopy revealed the initiation and spatial growth phases of clot formation. Different procoagulant activity of cell monolayers was manifested as up to 4-fold difference in the lag times of clot formation. In contrast, the clot growth rate, which characterized propagation of clotting from the cell surface to plasma, was largely independent of cell type (< or = 30% difference). Experiments with factor VII (FVII)-, FVIII-, FX- or FXI-deficient plasmas and annexin V revealed that (i) cell surface-associated extrinsic Xase was critical for initiation of clotting; (ii) intrinsic Xase regulated only the growth phase; and (iii) the contribution of plasma phospholipid surfaces in the growth phase was predominant. We conclude that the role of TF-bearing initiator cells is limited to the initial stage of clot formation. The functioning of intrinsic Xase in plasma provides the primary mechanism of sustained and far-ranging propagation of coagulation leading to the physical expansion of a fibrin clot.

Blood Coagulation↗
PubMed2005

Two subpopulations of thrombin-activated platelets differ in their binding of the components of the intrinsic factor X-activating complex.

Binding of fluorescein-labeled coagulation factors IXa, VIII, X, and allophycocyanin-labeled annexin V to thrombin-activated platelets was studied using flow cytometry. Upon activation, two platelet subpopulations were detected, which differed by 1-2 orders of magnitude in the binding of the coagulation factors and by 2-3 orders of magnitude in the binding of annexin V. The percentage of the high-binding platelets increased dose dependently of thrombin concentration. At 100 nm of thrombin, platelets with elevated binding capability constituted approximately 4% of total platelets and were responsible for the binding of approximately 50% of the total bound factor. Binding of factors to the high-binding subpopulation was calcium-dependent and specific as evidenced by experiments in the presence of excess unlabeled factor. The percentage of the high-binding platelets was not affected by echistatin, a potent aggregation inhibitor, confirming that the high-binding platelets were not platelet aggregates. Despite the difference in the coagulation factors binding, the subpopulations were indistinguishable by the expression of general platelet marker CD42b and activation markers PAC1 (an epitope of glycoprotein IIb/IIIa) and CD62P (P-selectin). Dual-labeling binding studies involving coagulation factors (IXa, VIII, or X) and annexin V demonstrated that the high-binding platelet subpopulation was identical for all coagulation factors and for annexin V. The high-binding subpopulation had lower mean forward and side scatters compared with the low-binding subpopulation ( approximately 80% and approximately 60%, respectively). In its turn, the high-binding subpopulation was not homogeneous and included two subpopulations with different scatter values. We conclude that activation by thrombin induces the formation of two distinct subpopulations of platelets different in their binding of the components of the intrinsic fX-activating complex, which may have certain physiological or pathological significance.

Annexin A5↗
PubMed2004

Molecular basis of haemophilia A.

Technologies in molecular biology have greatly advanced the knowledge regarding the origin of haemophilia A and the physiology of the factor VIII (FVIII) protein. A variety of different mutations in the FVIII gene have been identified and their effects on the FVIII protein described. It has been shown that the frequency of haemophilia A is due to a high mutation rate predominantly in male germ cells. A significant proportion is originating de novo in early embryogenesis from somatic mutations, a finding that has implications for genetic counselling. The life-cycle of the FVIII protein and its structure-function relationships are continuously clarified. Most recently it has been shown that FVIII clearance from the circulation is mediated by the low-density lipoprotein receptor-related protein (LRP) and cell-surface heparan sulphate proteoglycans (HSPGs). These findings raise hope for novel recombinant FVIII molecules with prolonged half-life that may improve therapies for haemophlia A.

Factor IX↗
PubMed2003

The future of recombinant coagulation factors.

Hemophilias A and B are X chromosome-linked bleeding disorders, which are mainly treated by repeated infusions of factor (F)VIII or FIX, respectively. In the present review, we specify the limitations in expression of recombinant (r)FVIII and summarize the bioengineering strategies that are currently being explored for constructing novel rFVIII molecules characterized by high efficiency expression and improved functional properties. We present the strategy to prolong FVIII lifetime by disrupting FVIII interaction with its clearance receptors and demonstrate how construction of human-porcine FVIII hybrid molecules can reduce their reactivity towards inhibitory antibodies. While the progress in improving rFIX is impeded by low recovery rates, the authors are optimistic that the efforts of basic science may ultimately lead to higher efficiency of replacement therapy of both hemophilias A and B.

Factor IX↗
PubMed2002

Haemophilia A: effects of inhibitory antibodies on factor VIII functional interactions and approaches to prevent their action.

Factor VIII (FVIII) is an essential component of the intrinsic pathway of blood coagulation. Normal functioning of FVIII requires its interactions with other components of the coagulation cascade. In the circulation, it exists as a complex with von Willebrand factor (vWF). Upon activation by thrombin or activated factor X (FXa), activated FVIII (FVIIIa) functions as a cofactor for the serine protease factor IXa. Their complex assembled on the phospholipid surface activates FX to FXa, which consequently participates in formation of thrombin, the key protease of the coagulation cascade. Genetic deficiency in FVIII results in a coagulation disorder haemophilia A, which is treated by infusions of FVIII products. Approximately 25-30% of patients develop antibodies inhibiting FVIII activity (FVIII inhibitors). The major epitopes of inhibitors are located within the A2, C2 and A3 domains of the FVIII molecule. The inhibitory effects of antibodies are manifested at various stages of the FVIII functional pathway, including FVIII binding to vWF, activation of FVIII by thrombin, and FVIIIa incorporation into the Xase complex. We summarize the current knowledge of the FVIII sites involved in interaction with its physiological ligands and different classes of inhibitory antibodies and describe their inhibitory mechanisms. We outline the strategies aimed to overcome the effects of inhibitory antibodies such as development of human/porcine FVIII molecules, resistant to inhibitors. We also discuss approaches to modulate the antibody response, as well as efforts to develop a long-term immunotolerance to FVIII protein.

Animals↗
PubMed2002

Factor VIII and transmissible spongiform encephalopathy: the case for safety.

Haemophilia A is the most common inherited bleeding disorder, caused by a deficiency in coagulation factor VIII (FVIII). Current treatment of haemophilia A is based on repeated infusions of plasma-derived FVIII concentrate or of recombinant FVIII, which may be exposed to plasma-derived material of human or animal origin used in its tissue culture production process. We review epidemiological and experimental studies relevant to blood infectivity in the transmissible spongiform encephalopathies (TSEs, or 'prion' diseases), and evaluate the hypothetical risk of TSE transmission through treatment with plasma-derived or recombinant FVIII.

Animals↗
PubMed2002

Molecular defects in coagulation Factor VIII and their impact on Factor VIII function.

Molecular defects in Factor VIII (FVIII), such as haemophilia A-related mutations or denaturative conformational changes, may affect the stability of FVIII as well as its interactions with physiological activators, von Willebrand Factor, phospholipid, or conformationally sensitive antibodies. We summarize the contemporary assays which allow identification of impaired functional interactions of FVIII that cause a reduction or loss of its cofactor activity and/or increased immunogenicity. These assays can potentially be used for detection of molecular defects in FVIII and elucidation of the function impaired by these defects.

Blood Coagulation↗
PubMed2001

In hemophilia A and autoantibody inhibitor patients: the factor VIII A2 domain and light chain are most immunogenic.

Factor VIII (fVIII) is a protein cofactor essential for blood coagulation, and it binds in the factor Xase complex to factors IXa, X, and phospholipid. In about 30% of severe hemophilia A patients, treatment with fVIII leads to production of anti-fVIII antibodies. Anti-fVIII autoantibodies also rarely appear in normal individuals. Those antibodies that inactivate fVIII (inhibitors) prevent optimal fVIII therapy. Inhibitor epitopes were previously localized to the fVIII A2, A3, and C2 domains and to an acidic amino acid region between A1 and A2. Such anti-fVIII antibodies interfere with fVIII binding to components of the factor Xase complex and prevent blood coagulation. When total anti-fVIII titers were determined for each fVIII domain in 43 inhibitor plasmas by immunoprecipitation (IP) and inhibitor neutralization assays, the anti-light chain (LCh) antibody titer was highest, anti-A2 was intermediate, and anti-A1 and anti-B were low. The relative immunogenicity of the fVIII domains in hemophilic and autoantibody inhibitor patients was similar.

Antibodies, Monoclonal↗
PubMed2001

Circulating factor VIII immune complexes in patients with type 2 acquired hemophilia A and protection from activated protein C-mediated proteolysis.

Factor VIII (FVIII) inhibitor antibodies are classified into 2 groups according to the kinetic pattern of FVIII inactivation. Type 2 antibodies are more commonly observed in patients with acquired hemophilia A and do not completely inhibit FVIII activity; in most cases, substantial levels of circulating FVIII are detected. Three type 2 autoantibodies from patients who had normal levels of FVIII antigen despite having low levels of FVIII activity were studied. The antibodies reacted exclusively with the light chain of FVIII but not with the C2 domain, and their epitopes were therefore ascribed to the regions in the A3-C1 domains. Heavy and light chains of FVIII were detected in plasma-derived immune complexes extracted by using protein G Sepharose. Direct binding assays using anhydro-activated protein C (anhydro-APC), a catalytically inactive derivative of activated protein C (APC) in which the active-site serine is converted to dehydroalanine, were used to examine the relation between immune complexes and APC. The intact FVIII, 80-kd light chain, and 72-kd light chain bound in a dose-dependent manner to anhydro-APC, with K(d) values of 580, 540, and 310 nM, respectively, whereas no appreciable binding was detected for the heavy chain. The 3 autoantibodies blocked FVIII binding to anhydro-APC by approximately 80% and consequently inhibited APC-induced FVIII proteolytic inactivation. These antibodies also bound to a synthetic peptide, His2009-Val2018, which contains the APC binding site. The findings suggest that binding of type 2 autoantibodies, recognizing residues His2009 to Val2018, protects FVIII from APC-mediated proteolysis and might contribute to the presence of FVIII immune complexes in the circulation.

Antigen-Antibody Complex↗
PubMed2001

Hemophilia A mutations associated with 1-stage/2-stage activity discrepancy disrupt protein-protein interactions within the triplicated A domains of thrombin-activated factor VIIIa.

Thrombin-activated factor VIII (FVIIIa) is a heterotrimer with the A2 subunit (amino acid residues 373-740) in a weak ionic interaction with the A1 and A3-C1-C2 subunits. Dissociation of the A2 subunit correlates with inactivation of FVIIIa. Patients with hemophilia A have been described whose plasmas display a discrepancy between their FVIII activities, where the 1-stage activity assay displays greater activity than the 2-stage activity assay. The molecular basis for one of these mutations, (ARG)531(HIS), is an increased rate of A2 subunit dissociation. Examination of a homology model of the A domains of FVIII predicted (ARG)531 to lie at the interface of the A1 and A2 subunits and stabilize their interaction. Indeed, patients with mutations either directly contacting (ARG)531 ((ALA)284(GLU), (ALA)284(PRO)) or closely adjacent to the A1-A2 interface in the tightly packed hydrophobic core ((SER)289(LEU)) have the same phenotype of 1-stage/2-stage discrepancy. The (ALA)284(GLU) and (SER)289(LEU) mutations in FVIII were produced by transfection of COS-1 monkey cells. Compared to FVIII wild-type both mutants had reduced specific activity by 1-stage clotting activity and at least a 2-fold lower activity by 2-stage analysis (COAMATIC), similar to the reported clinical data. Analysis of immunoaffinity purified (ALA)284(GLU) and (SER)289(LEU) proteins in an optical biosensor demonstrated that A2 dissociation was 3-fold faster for both FVIIIa mutants compared to FVIIIa wild-type. Therefore, these mutations within the A1 subunit of FVIIIa introduce a similar destabilization of the FVIIIa heterotrimer compared to the (ARG)531(HIS) mutation within the A2 subunit and support that these residues stabilize the A domain interface of FVIIIa.

Animals↗
PubMed2001

Cell surface heparan sulfate proteoglycans participate in factor VIII catabolism mediated by low density lipoprotein receptor-related protein.

We have demonstrated previously that catabolism of a coagulation factor VIII (fVIII) from its complex with von Willebrand factor (vWf) is mediated by low density lipoprotein receptor-related protein (LRP) (Saenko, E. L., Yakhyaev, A. V., Mikhailenko, I., Strickland, D. K., and Sarafanov, A. G. (1999) J. Biol. Chem. 274, 37685-37692). In the present study, we found that this process is facilitated by cell surface heparan sulfate proteoglycans (HSPGs). This was demonstrated by simultaneous blocking of LRP and HSPGs in model cells, which completely prevented fVIII internalization and degradation from its complex with vWf. In contrast, the selective blocking of either receptor had a lesser effect. In vivo studies of clearance of (125)I-fVIII-vWf complex in mice also demonstrated that the simultaneous blocking of HSPGs and LRP led to a more significant prolongation of fVIII half-life (5.5-fold) than blocking of LRP alone (3.5-fold). The cell culture and in vivo experiments revealed that HSPGs are also involved in another, LRP-independent pathway of fVIII catabolism. In both pathways, HSPGs act as receptors providing the initial binding of fVIII-vWf complex to cells. We demonstrated that this binding occurs via the A2 domain of fVIII, since A2, but not other portions of fVIII or isolated vWf, strongly inhibited cell surface binding of fVIII-vWf complex, and the affinities of A2 and fVIII-vWf complex for the cells were similar. The A2 site involved in binding to heparin was localized to the region 558-565, based on the ability of the corresponding synthetic peptide to inhibit A2 binding to heparin, used as a model for HSPGs.

Animals↗
PubMed2001

Catabolism of the coagulation factor VIII: can we prolong lifetime of f VIII in circulation?

The coagulation factor VIII is required for normal haemostasis, because deficiency or genetic defects in this molecule cause a life-threatening coagulation disorder known as hemophilia A. While the role of f VIII in the intrinsic pathway of blood coagulation has been extensively studied, the mechanisms responsible for f VIII turnover in circulation have not been characterized until recently. This review focuses on the finding that f VIII catabolism in vitro and in vivo is mediated by low-density lipoprotein receptor-related protein (LRP), representing a hepatic clearance receptor. FVIII interaction with LRP involves two distinct sites localized within the C2 and A2 domains of f VIII. We discuss the contribution of the A2 site (residues 484-509) and the C2 site in f VIII catabolism in the presence and absence of vWf. We present the evidence that LRP-mediated f VIII catabolism is facilitated by cell-surface heparan sulfate proteoglycans (HSPGs), which bind to the A2 residues 558-565 of f VIII. Because both LRP- and HSPGs-binding sites within the A2 domain are potentially exposed in the circulating f VIII/vWf complex, we discuss the possibility of prolongation of the f VIII lifetime in circulation by disrupting these sites employing site-directed mutagenesis. In its turn, generation of a novel recombinant f VIII may be prospective for more efficient hemophilia A therapy.

Factor VIII↗
PubMed2001

Stable recombinant expression and characterization of the two haemophilic factor VIII variants C329S (CRM(-)) and G1948D (CRM(r)).

In haemophilia A, the functional defect at the molecular level of most factor VIII (FVIII) missense mutations remains unknown. Site-directed mutagenesis of B domain-deleted FVIII cDNA (FVIIISQ) was used to introduce two mutations associated with severe cross-reacting material (CRM)-negative (FVIII-C329S) or mild/moderate CRM-reduced (FVIII-G1948D) haemophilia A. Wild-type (FVIIISQ-WT) and variant FVIIISQ proteins were successfully expressed after stable transfection in Chinese hamster ovary (CHO) cells, and partially characterized at the intracellular, molecular and functional levels. Reverse transcription polymerase chain reaction analysis confirmed that both transcription and mRNA processing appeared normal in CHO cells transfected with both the wild-type and two variant constructs. In contrast to FVIIISQ-WT, immunofluorescence analysis of both CRM(-) and CRM(r) variants showed intracellular FVIII accumulation within the rough endoplasmic reticulum, suggesting secretion defects in transfected CHO cells. Immunoblot analysis of the FVIIISQ variant proteins that were secreted showed that they were expressed as mixed populations of uncleaved 170 kDa polypeptides, processed 90 kDa heavy chains and 80 kDa light chains, similar to FVIIISQ-WT. Phenotypic analysis of the B domain-deleted FVIIISQ variants expressed in CHO cells correlated well with the patients' reduced FVIII activity and, in addition, surface plasmon resonance studies demonstrated that both missense mutations were associated with increased rates of A2 domain dissociation following thrombin activation. We conclude that the mutations found are responsible for the haemophilia A phenotype, through intracellular retention and decreased stability of the active cofactor FVIIIa.

Animals↗
PubMed2000

Factor VIII C2 domain contains the thrombin-binding site responsible for thrombin-catalyzed cleavage at Arg1689.

Thrombin-catalyzed factor VIII activation is an essential positive feedback mechanism regulating intrinsic blood coagulation. A factor VIII human antibody, A-FF, with C2 epitope, exclusively inhibited factor VIII activation and cleavage at Arg(1689) by thrombin. The results suggested that A-FF prevented the interaction of thrombin with factor VIII and that the C2 domain was involved in the interaction with thrombin. We performed direct binding assays using anhydro-thrombin, a catalytically inactive derivative of thrombin in which the active-site serine is converted to dehydroalanine. Intact factor VIII, 80-kDa light chain, 72-kDa light chain, and heavy chain fragments bound dose-dependently to anhydro-thrombin, and the K(d) values were 48, 150, 106, and 180 nm, respectively. The C2 and A2 domains also dose-dependently bound to anhydro-thrombin, and the K(d) values were 440 and 488 nm, respectively. The A1 domain did not bind to anhydro-thrombin. A-FF completely inhibited C2 domain binding to anhydro-thrombin (IC(50), 18 nm), whereas it did not inhibit A2 domain binding. Furthermore, C2-specific affinity purified F(ab)'(2) of A-FF, and the recombinant C2 domain inhibited thrombin cleavage at Arg(1689). Our results indicate that the C2 domain contains the thrombin-binding site responsible for the cleavage at Arg(1689).

Antibodies, Monoclonal↗
PubMed2000

An alloantibody recognizing the FVIII A1 domain in a patient with CRM reduced haemophilia A due to deletion of a large portion of the A1 domain DNA sequence.

We report the development of a FVIII inhibitor in a patient with severe, cross reacting material reduced (CRM(R)) haemophilia A. The level of Factor VIII antigen (FVIII:Ag) measured by ELISA using anti-C2 monoclonal and alloantibodies was 1.9 U/dl. This baseline FVIII:Ag level was increased to 8.3 U/dl after administration of DDAVP. The anti-FVIII inhibitor titer was 2.9 Bethesda U/ml. DNA analysis showed a large deletion of the FVIII gene from exon 4 to 7, corresponding to amino acid residues 111-317 included within the A1 domain. The size of the gene deletion was approximately 28 kb. 5' and 3' breakpoints were identified by sequencing in intron 3 and intron 7, respectively. FVIII mRNA was detected in the patient's peripheral lymphocytes and the deletion spanning exon 4 to 7 was confirmed at the RNA level. Immunoprecipitation experiments using 125I labeled A1, A2 and light chain demonstrated that the inhibitor reacted only with the 54 kDa A1 domain. The inhibitor activity was more than 95% neutralized by A1 domain polypeptide. Our findings suggest a close relationship between the inhibitor epitope and the specific gene deletion with regard to the pathogenesis of the inhibitor in this patient.

Adult↗