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Featured researches published by Evgueni L. Saenko.


Journal of Biological Chemistry | 1999

Role of the Low Density Lipoprotein-related Protein Receptor in Mediation of Factor VIII Catabolism

Evgueni L. Saenko; Alexey V. Yakhyaev; Irina Mikhailenko; Dudley K. Strickland; Andrei G. Sarafanov

In the present study, we found that catabolism of coagulation factor VIII (fVIII) is mediated by the low density lipoprotein receptor-related protein (LPR), a liver multiligand endocytic receptor. In a solid phase assay, fVIII was shown to bind to LRP (K d 116 nm). The specificity was confirmed by a complete inhibition of fVIII/LRP binding by 39-kDa receptor-associated protein (RAP), an antagonist of all LRP ligands. The region of fVIII involved in its binding to LRP was localized within the A2 domain residues 484–509, based on the ability of the isolated A2 domain and the synthetic A2 domain peptide 484–509 to prevent fVIII interaction with LRP. Since vWf did not inhibit fVIII binding to LRP, we proposed that LRP receptor may internalize fVIII from its complex with vWf. Consistent with this hypothesis, mouse embryonic fibroblasts that express LRP, but not fibroblasts genetically deficient in LRP, were able to catabolize 125I-fVIII complexed with vWf, which was not internalized by the cells. These processes could be inhibited by RAP and A2 subunit of fVIII, indicating that cellular internalization and degradation were mediated by interaction of the A2 domain of fVIII with LRP. In vivo studies of125I-fVIII·vWf complex clearance in mice demonstrated that RAP completely inhibited the fast phase of the biphasic125I-fVIII clearance that is responsible for removal of 60% of fVIII from circulation. Inhibition of the RAP-sensitive phase prolonged the half-life of 125I-fVIII in circulation by 3.3-fold, indicating that LRP receptor plays an important role in fVIII clearance.


Journal of Biological Chemistry | 1996

Slowed Release of Thrombin-cleaved Factor VIII from von Willebrand Factor by a Monoclonal and a Human Antibody Is a Novel Mechanism for Factor VIII Inhibition

Evgueni L. Saenko; Midori Shima; Gary E. Gilbert; Dorothea Scandella

The anti-factor VIII (fVIII) C2 domain monoclonal antibody ESH8 inhibits fVIII activity only when fVIII is bound to von Willebrand factor (vWf). However, ESH8 binds with similar affinity to fVIII and fVIII·vWf complex, and it does not affect the kinetics of thrombin cleavage at positions 372 and 740 within the fVIII heavy chain and at 1689 within the light chain. The latter is required for fVIII release from vWf. We showed that ESH8 reduced the initial rate of thrombin-activated fVIII (fVIIIa) release from vWf by 4.3-fold compared to that in the absence of antibody. The complex of vWf·fVIII·ESH8 was activated, and the rate constant determined for fVIIIa dissociation from vWf was 4 × 10−3 s−1. We constructed a mathematical model incorporating the measured rates for fVIIIa release from vWf and for inactivation of heterotrimeric fVIIIa due to the spontaneous loss of the A2 subunit and found that the decreased release rate is sufficient to explain our experimentally observed inhibition of fVIII activity by ESH8. We hypothesize that the slowed rate of fVIIIa release from vWf in the presence of ESH8 allows time for inactivation of unstable fVIIIa prior its participation in the formation of the factor Xase complex. The relevance of these findings is illustrated by our observation that reduction of fVIIIa release from vWf represents an additional mechanism of fVIII inhibition by an anti-C2 domain antibody (epitope 2218-2307) from a hemophilia A patient. This rare antibody binds to a more amino-terminal epitope than other human anti-C2 inhibitors, resulting in its lack of inhibition of fVIII binding to vWf but not to phospholipid. These two fVIII ligands therefore bind to C2 sites which do not overlap completely.


Journal of Biological Chemistry | 1997

The acidic region of the factor VIII light chain and the C2 domain together form the high affinity binding site for von willebrand factor.

Evgueni L. Saenko; Dorothea Scandella

A binding site for von Willebrand factor (vWf) was previously localized to the carboxyl terminus of the C2 domain of the light chain (LCh) of factor VIII (fVIII). The acidic region of the LCh, residues 1649–1689, also controls fVIII·vWf binding by an unknown mechanism. Although anti-acidic region monoclonal antibodies prevent formation of the fVIII·vWf complex, the direct involvement of the acidic region in this binding has not been demonstrated. By limited proteolysis of LCh with Staphylococcus aureus V8 protease, we prepared 14- and 63-kDa LCh fragments, which begin with fVIII residues 1672 and 1795, respectively. Using surface plasmon resonance to measure binding interactions, we demonstrated that the 14-kDa fragment binds to vWf, but its affinity for vWf (K d 72 nm) was 19-fold lower than that of LCh. This was not due to an altered conformation of the acidic region within the 14-kDa fragment, since its affinity for an anti-acidic region monoclonal antibody was similar to that of LCh. All LCh derivatives lacking the acidic region (thrombin-cleaved LCh, recombinant C2, and 63-kDa fragment) had also greatly reduced affinities for vWf (K d 564–660 nm) compared with LCh (K d 3.8 nm). In addition, the similar affinities of these derivatives for vWf indicated that apart from its acidic region, the LCh contains no vWf binding site other than the one within C2. The reduced affinities of the LCh derivatives lacking the acidic region for monoclonal antibody NMC-VIII/5 (epitope, C2 residues 2170–2327) indicated that removal of the acidic region leads to a conformational change within C2. This change is likely to affect the conformation of the vWf binding site in C2, which overlaps the epitope of NMC-VIII/5; therefore, the acidic region also appears to be required to maintain the optimal conformation of this vWf binding site. Our results demonstrate that the acidic region and the C2 domain are both directly involved in forming a high affinity binding site for vWf.


Journal of Biological Chemistry | 1999

Role of factor VIII C2 domain in factor VIII binding to factor Xa

Keiji Nogami; Midori Shima; Kazuya Hosokawa; Toyoaki Suzuki; Takehiko Koide; Evgueni L. Saenko; Dorothea Scandella; Masaru Shibata; Seiki Kamisue; Ichiro Tanaka; Akira Yoshioka

Factor VIII (FVIII) is activated by proteolytic cleavages with thrombin and factor Xa (FXa) in the intrinsic blood coagulation pathway. The anti-C2 monoclonal antibody ESH8, which recognizes residues 2248–2285 and does not inhibit FVIII binding to von Willebrand factor or phospholipid, inhibited FVIII activation by FXa in a clotting assay. Furthermore, analysis by SDS-polyacrylamide gel electrophoresis showed that ESH8 inhibited FXa cleavage in the presence or absence of phospholipid. The light chain (LCh) fragments (both 80 and 72 kDa) and the recombinant C2 domain dose-dependently bound to immobilized anhydro-FXa, a catalytically inactive derivative of FXa in which dehydroalanine replaces the active-site serine. The affinity (K d ) values for the 80- and 72-kDa LCh fragments and the C2 domain were 55, 51, and 560 nm, respectively. The heavy chain of FVIII did not bind to anhydro-FXa. Similarly, competitive assays using overlapping synthetic peptides corresponding to ESH8 epitopes (residues 2248–2285) demonstrated that a peptide designated EP-2 (residues 2253–2270; TSMYVKEFLISSSQDGHQ) inhibited the binding of the C2 domain or the 72-kDa LCh to anhydro-FXa by more than 95 and 84%, respectively. Our results provide the first evidence for a direct role of the C2 domain in the association between FVIII and FXa.


Blood Coagulation & Fibrinolysis | 2004

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

Natalya M. Ananyeva; Sébastien Lacroix-Desmazes; Charlotte A. E. Hauser; Midori Shima; Mikhail V. Ovanesov; Alexey V. Khrenov; Evgueni L. Saenko

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.


Haemophilia | 2006

Strategies towards a longer acting factor VIII

Evgueni L. Saenko; Steven W. Pipe

Summary.  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.


Journal of Biological Chemistry | 1998

Activation of Factor VIII by Thrombin Increases Its Affinity for Binding to Synthetic Phospholipid Membranes and Activated Platelets

Evgueni L. Saenko; Dorothea Scandella; Alexey V. Yakhyaev; Nicholas J. Greco

Membrane-bound thrombin-activated factor VIII (fVIIIa) functions as a cofactor for factor IXa in the factor Xase complex. We found that binding of heterotrimeric fVIIIa (A1·A2·A3-C1-C2) to synthetic vesicles with a physiologic content of 4% phosphatidylserine (PS), 76% phosphatidylcholine, and 20% phosphatidylethanolamine occurs with a 10-fold higher affinity than that of factor VIII (fVIII). The increased affinity of fVIIIa for PS-containing membranes resulted from the reduced rate of fVIIIa dissociation from the vesicles compared with that of fVIII. Similar affinities of A3-C1-C2, A1·A2·A3-C1-C2, and A3-C1-C2·heavy chain for interaction with PS-containing membranes demonstrate that removal of the light chain (LCh) acidic region by thrombin is responsible for these increased affinities of fVIIIa and its derivatives. Similar kinetic parameters of fVIII and its LCh and C2 domain for binding to PS-containing membranes and to activated platelets indicated that the C2 domain is entirely responsible for the interaction of fVIII with membranes. We conclude that the increased fVIIIa affinity for PS-containing membranes is a result of conformational change(s) within the C2 domain upon removal of the acidic region of the LCh. This conclusion is based on the finding that binding of the monoclonal antibody ESH8 to the C2 domain, which is known to prevent this conformational transition, resulted in fVIIIa binding to PS/phosphatidylcholine/phosphatidylethanolamine vesicles (4/76/20) with a lower affinity similar to that of fVIII. In addition, stabilization of the low affinity binding conformation of the C2 domain of fVIIIa by this antibody led to an inhibition of the fVIIIa activity in the factor X activation complex.


Journal of Biological Chemistry | 2000

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

Keiji Nogami; Midori Shima; Kazuya Hosokawa; Masanori Nagata; Takehiko Koide; Evgueni L. Saenko; Ichiro Tanaka; Masaru Shibata; Akira Yoshioka

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 Arg1689 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 theK 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 (IC50, 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 Arg1689. Our results indicate that the C2 domain contains the thrombin-binding site responsible for the cleavage at Arg1689.


Journal of Thrombosis and Haemostasis | 2005

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

Mikhail V. Ovanesov; Natalya M. Ananyeva; Mikhail A. Panteleev; Fazoil I. Ataullakhanov; Evgueni L. Saenko

Summary.  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 (≤ 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.


Trends in Cardiovascular Medicine | 1999

Role of Activation of the Coagulation Factor VIII in Interaction with vWf, Phospholipid, and Functioning within the Factor Xase Complex

Evgueni L. Saenko; Midori Shima; Andrey Sarafanov

Blood coagulation factor VIII (fVIII) in its nonactivated form circulates in plasma in a complex with von Willebrand factor (vWf). Upon activation by thrombin- or factor Xa-mediated site-specific proteolysis, activated fVIII (fVIIIa) serves as a cofactor for factor IXa. This protein complex assembled on a phospholipid surface (factor Xase) activates factor X. This complex plays the key role in the intrinsic pathway of blood coagulation. We reviewed the molecular events triggered by fVIII activation, which are required for the assembly and functioning of the Xase complex, including fVIIIa dissociation from vWf and a significant increase of fVIII affinity for binding to the phospholipid surface. Both events are mediated by activation-related cleavage within fVIII light chain (LCh), releasing the 40 amino-acid N-terminal LCh peptide, which is followed by a conformational change within the C2 domain. The conformational change within LCh is also required for the optimal fVIII cofactor functioning within the factor Xase complex, exerted via fVIIIa interactions with phospholipid, factor IXa, and factor X. Since factor IXa not only stabilizes but also proteolytically inactivates fVIIIa within the factor Xase complex, the stability of the membrane-bound fVIIIa in the presence and absence of factor IXa is discussed. In conclusion, we outline some new possible directions of the research. One of them arises from the recently demonstrated ability of plasma lipoproteins to provide a phospholipid surface for the assembly of the factor Xase complex in vitro. This finding raises a possibility that lipoproteins participate in factor Xase functioning in vivo and suggests a direct link between elevated levels of lipoproteins associated with atherosclerosis and increased thrombogenicity associated with this disease.

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Midori Shima

Nara Medical University

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Keiji Nogami

Nara Medical University

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Alexey V. Khrenov

Scripps Research Institute

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