Factor VIII moiety of recombinant Factor VIII Fc fusion protein impacts Fc effector function and CD16+ NK cell activation.

Recombinant Factor VIII-Fc fusion protein (rFVIIIFc) is an enhanced half-life therapeutic protein product used for the management of hemophilia A. Recent studies have demonstrated that rFVIIIFc interacts with Fc gamma receptors (FcγR) resulting in the activation or inhibition of various FcγR-expressing immune cells. We previously demonstrated that rFVIIIFc, unlike recombinant Factor IX-Fc (rFIXFc), activates natural killer (NK) cells via Fc-mediated interactions with FcγRIIIA (CD16). Additionally, we showed that rFVIIIFc activated CD16+ NK cells to lyse a FVIII-specific B cell clone. Here, we used human NK cell lines and primary NK cells enriched from peripheral blood leukocytes to study the role of the FVIII moiety in rFVIIIFc-mediated NK cell activation. Following overnight incubation of NK cells with rFVIIIFc, cellular activation was assessed by measuring secretion of the inflammatory cytokine IFNγ by ELISA or by cellular degranulation. We show that anti-FVIII, anti-Fc, and anti-CD16 all inhibited indicating that these molecules were involved in rFVIIIFc-mediated NK cell activation. To define which domains of FVIII were involved, we used antibodies that are FVIII domain-specific and demonstrated that blocking FVIII C1 or C2 domain-mediated membrane binding potently inhibited rFVIIIFc-mediated CD16+ NK cell activation, while targeting the FVIII heavy chain domains did not. We also show that rFVIIIFc binds CD16 with about five-fold higher affinity than rFIXFc. Based on our results we propose that FVIII light chain-mediated membrane binding results in tethering of the fusion protein to the cell surface, and this, together with increased binding affinity for CD16, allows for Fc-CD16 interactions to proceed, resulting in NK cellular activation. Our working model may explain our previous results where we observed that rFVIIIFc activated NK cells via CD16, whereas rFIXFc did not despite having identical IgG1 Fc domains.

In-depth structure-function profiling of the complex formation between clotting factor VIII and heme.

BACKGROUND AND AIMS: Blood disorders, such as sickle cell disease, and other clinical conditions are often accompanied by intravascular hemolytic events along with the development of severe coagulopathies. Hemolysis, in turn, leads to the accumulation of Fe(II/III)-protoporphyrin IX (heme) in the intravascular compartment, which can trigger a variety of proinflammatory and prothrombotic reactions. As such, heme binding to the blood coagulation proteins factor VIII (FVIII), fibrinogen, and activated protein C with functional consequences has been demonstrated earlier. METHODS: We herein present an in-depth characterization of the FVIII-heme interaction at the molecular level and its (patho-)physiological relevance through the application of biochemical, biophysical, structural biology, bioinformatic, and diagnostic tools. RESULTS: FVIII has a great heme-binding capacity with seven heme molecules associating with the protein. The respective binding sites were identified by investigating heme binding to FVIII-derived peptides in combination with molecular docking and dynamic simulation studies of the complex as well as cryo-electron microscopy, revealing three high-affinity and four moderate heme-binding motifs (HBMs). Furthermore, the relevance of the FVIII-heme complex formation was characterized in physiologically relevant assay systems, revealing a ~ 50 % inhibition of the FVIII cofactor activity even in the protein-rich environment of blood plasma. CONCLUSION: Our study provides not only novel molecular insights into the FVIII-heme interaction and its physiological relevance, but also strongly suggests the reduction of the intrinsic pathway and the accentuation of the final clotting step (by, for example, fibrinogen crosslinking) in hemolytic conditions as well as a future perspective in the context of FVIII substitution therapy of hemorrhagic events in hemophilia A patients.

Lactadherin's Multistate Binding Predicts Stable Membrane-Bound Conformations of Factors V and VIII's C Domains.

Protein binding to negatively charged lipids is essential for maintaining numerous vital cellular processes where its dysfunction can lead to various diseases. One such protein that plays a crucial role in this process is lactadherin, which competes with coagulation factors for membrane binding sites to regulate blood clotting. Despite identifying key binding regions of these proteins through structural and biochemical studies, models incorporating membrane dynamics are still lacking. In this study, we report on the multimodal binding of lactadherin and use it to gain insight into the binding mechanisms of its C domain homologs, factor V and factor VIII. Molecular dynamics simulations enhanced with the highly mobile mimetic model enabled the determination of lactadherin's multimodal binding on membranes that revealed critical interacting residues consistent with prior NMR and mutagenesis data. The binding occurred primarily via two dynamic structural ensembles: an inserted state and an unreported, highly conserved side-lying state driven by a cationic patch. We utilized these findings to analyze the membrane binding domains of coagulation factors V and VIII and identified their preferred membrane-bound conformations. Specifically, factor V's C domains maintained an inserted state, while factor VIII preferred a tilted, side-lying state that permitted antibody binding. Insight into lactadherin's atomistically resolved membrane interactions from a multistate perspective can guide new therapeutic opportunities in treating diseases related to blood coagulation.

Investigation of the effect of pre-analytical factors on particle concentration and size in cryoprecipitate using nanoparticle tracking analysis.

BACKGROUND: Cryoprecipitate is used primarily to replenish fibrinogen levels in patients. Little is known about the presence of micro- or nano-sized particles in cryoprecipitate. Therefore, we aimed to quantify these particles and investigate some pre-analytical considerations. MATERIALS AND METHODS: Particle concentration and size distribution were determined in 10 cryoprecipitate units by nanoparticle tracking analysis (NTA). The effects of freeze-thawing cryoprecipitate and 0.45 µm filtration with either regenerated cellulose (RC) or polytetrafluoroethylene (PTFE) filters before sample analysis were examined. RESULTS: Neither the size nor concentration of particles were affected by two freeze/thaw cycles. PTFE filtration, but not RC filtration, significantly reduced particle mean and mode size compared to RC filtration and mode size compared to unfiltered cryoprecipitate. The 10 cryoprecipitate units had an average particle concentration of 2.50 × 1011 ± 1.10 × 1011 particles/mL, a mean particle size of 133.8 ± 7.5 nm and a mode particle size of 107.9 ± 11.1 nm. CONCLUSION: This study demonstrated that preanalytical filtration of cryoprecipitate units using RC filters was suitable for NTA. An additional freeze/thaw cycle did not impact NTA parameters, suggesting that aliquoting cryoprecipitate units prior to laboratory investigations is suitable for downstream analyses.

Factor VIII A3 domain residues 1793-1795 represent a factor IXa-interactive site in the tenase complex.

BACKGROUND: Factor (F)VIII functions as a cofactor in the tenase complex responsible for conversion of FX to FXa by FIXa. Earlier studies indicated that one of the FIXa-binding sites is located in residues 1811-1818 (crucially F1816) of the FVIII A3 domain. A putative, three-dimensional structure model of the FVIIIa molecule suggested that residues 1790-1798 form a V-shaped loop, and juxtapose residues 1811-1818 on the extended surface of FVIIIa. AIM: To examine FIXa molecular interactions in the clustered acidic sites of FVIII including residues 1790-1798. METHODS AND RESULTS: Specific ELISA's demonstrated that the synthetic peptides, encompassing residues 1790-1798 and 1811-1818, competitively inhibited the binding of FVIII light chain to active-site-blocked Glu-Gly-Arg-FIXa (EGR-FIXa) (IC50; 19.2 and 42.9 µM, respectively), in keeping with a possible role for the 1790-1798 in FIXa interactions. Surface plasmon resonance-based analyses demonstrated that variants of FVIII, in which the clustered acidic residues (E1793/E1794/D1793) or F1816 contained substituted alanine, bound to immobilized biotin labeled-Phe-Pro-Arg-FIXa (bFPR-FIXa) with a 1.5-2.2-fold greater KD compared to wild-type FVIII (WT). Similarly, FXa generation assays indicated that E1793A/E1794A/D1795A and F1816A mutants increased the Km by 1.6-2.8-fold relative to WT. Furthermore, E1793A/E1794A/D1795A/F1816A mutant showed that the Km was increased by 3.4-fold and the Vmax was decreased by 0.75-fold, compared to WT. Molecular dynamics simulation analyses revealed the subtle changes between WT and E1793A/E1794A/D1795A mutant, supportive of the contribution of these residues for FIXa interaction. CONCLUSION: The 1790-1798 region in the A3 domain, especially clustered acidic residues E1793/E1794/D1795, contains a FIXa-interactive site.

Bacterial Production of Recombinant Coagulation Factor VIII Domains.

Factor VIII (F8) is a blood coagulation protein prearranged in six domains, and its deficiency causes hemophilia A. To fashion functional F8 therapeutics, development of a recombinant F8 (rF8) domain is essential not only for F8 substitution, but also to decipher the F8-related mechanisms. In this study, we generated Glutathione S-transferase (GST)-conjugated recombinant A2 and A3 domains of F8 using Escherichia coli. The high growth rate and economically advantageous protein production system in terms of inexpensive reagents and materials in E. coli cells facilitated the completion of entire process from protein expression to purification in 3-4 days with low production cost. Subsequent assessment of these purified proteins using enzyme-linked immunosorbent assay (ELISA) and antibodies against F8 revealed enhanced detection of rF8-A2 or rF8-A3 in a concentration dependent manner, indicating the presence of the antibody-binding epitopes in these proteins. Furthermore, these proteins are suitable for generating novel antibodies against the F8 domain and F8 domain-capturing affinity columns by enabling their conjugation to GST-capturing beads. Additionally, the recombinant F8 domains produced herein can be used for various studies, which include investigating the explicit roles of the F8 domain in the coagulation process, with domain-specific binding partners, and antibodies.

Mechano-covalent protection of coagulation factor VIII by von Willebrand factor.

von Willebrand factor (VWF) is the protective carrier of procoagulant factor VIII (FVIII) in the shear forces of the circulation, prolonging its half-life and delivering it to the developing thrombus. Using force spectroscopy, VWF-FVIII complex formation is characterized by catch-bond behavior in which force first decelerates then accelerates bond dissociation. Patients with mutations in VWF at the FVIII binding site phenocopies hemophilia A and the most common mutations are of cysteine residues involving multiple disulfide bonds. From differential cysteine alkylation and mass spectrometry experiments, 13 VWF disulfide bonds at the FVIII binding site were found to exist in formed and unformed states, and binding of FVIII results in partial formation of 12 of the VWF bonds. Force spectroscopy studies indicate that the VWF-FVIII bond stiffens in response to force and this feature of the interaction is ablated when VWF disulfide bonds are prevented from forming, resulting in slip-only bond behavior. Exposure of VWF to pathological fluid shear forces ex vivo and in vivo causes partial cleavage of all 13 disulfide bonds, further supporting their malleable nature. These findings demonstrate that FVIII binding to VWF involves dynamic changes in the covalent states of several VWF disulfides that are required for productive interaction in physiological shear forces.

Recombinant factor VIII protein aggregation and adsorption at the liquid-solid interface.

Undesired aggregation and adsorption of therapeutic proteins during manufacturing and administration processes can significantly decrease the efficacy of protein drugs, especially when a quantitative treatment is critical. In this study, we investigate molecular interactions of recombinant factor VIII (rFVIII), a therapeutic protein for hemophilia A treatment, at a static liquid-glass interface. We quantitatively analyze the adsorption and aggregation of rFVIII using atomic force microscopy (AFM), dynamic light scattering (DLS) and UV-Vis spectroscopy. We also investigate how PEGylation, temperature, ionic strength and pH affect the rFVIII aggregation and adsorption at the interface over time. The aggregation and adsorption of rFVIII are significantly reduced by decreasing electrostatic attractions in the solution. We observed that the PEGylation endows rFVIII molecules with high stability at the liquid-glass interface in a wide range of temperature, ionic strength and pH. Our studies will help to understand the molecular interactions of how proteins aggregate and adsorb on the solid surface and prevent the undesired events in pharmaceutical applications.

Unveiling the influence of factor VIII physicochemical properties on hemophilia A phenotype through an in silico methodology.

BACKGROUND AND OBJECTIVES: Hemophilia A (HA) is an X-linked blood disorder. It is caused by pathogenic F8 gene variants, among which missense mutations are the most prevalent. The resulting amino acid substitutions may have different impacts on physicochemical properties and, consequently, on protein functionality. Regular prediction tools do not include structural elements and their physiological significance, which hampers our ability to functionally link variants to disease phenotype, opening an ample field for investigation. The present study aims to elucidate how physicochemical changes generated by substitutions in different protein domains relate to HA, and which of these features are more consequential to protein function and its impact on HA phenotype. METHODS: An in silico evaluation of 71 F8 variants found in patients with different HA phenotypes (mild, moderate, severe) was performed to understand protein modifications and functional impact. Homology modeling was used for the structural analysis of physicochemical changes including electrostatic potential, hydrophobicity, solvent-accessible/excluded surface areas, disulfide disruptions, and substitutions indexes. These variants and properties were analyzed by hierarchical clustering analysis (HCA) and principal component analysis (PCA), independently and in combination, to investigate their relative contribution. RESULTS: About 69% of variants show electrostatic changes, and almost all show hydrophobicity and surface area modifications. HCA combining all physicochemical properties analyzed was better in reflecting the impact of different variants in disease severity, more so than the single feature analysis. On the other hand, PCA led to the identification of prominent properties involved in the clustering results for variants of different domains. CONCLUSIONS: The methodology developed here enables the assessment of structural features not available in other prediction tools (e.g., surface distribution of electrostatic potential), evaluating what kind of physicochemical changes are involved in FVIII functional disruption. HCA results allow distinguishing substitutions according to their properties, and yielded clusters which were more homogeneous in phenotype. All evaluated properties are involved in determining disease severity. The nature, as well as the position of the variants in the protein, were shown to be relevant for physicochemical changes, demonstrating that all these aspects must be collectively considered to fine-tune an approach to predict HA severity.

Ectopic Expression of FVIII in HPCs and MSCs Derived from hiPSCs with Site-Specific Integration of ITGA2B Promoter-Driven BDDF8 Gene in Hemophilia A.

Hemophilia A (HA) is caused by mutations in the coagulation factor VIII (FVIII) gene (F8). Gene therapy is a hopeful cure for HA; however, FVIII inhibitors formation hinders its clinical application. Given that platelets promote coagulation via locally releasing α-granule, FVIII ectopically expressed in platelets has been attempted, with promising results for HA treatment. The B-domain-deleted F8 (BDDF8), driven by a truncated ITGA2B promoter, was targeted at the ribosomal DNA (rDNA) locus of HA patient-specific induced pluripotent stem cells (HA-iPSCs). The F8-modified, human induced pluripotent stem cells (2bF8-iPSCs) were differentiated into induced hematopoietic progenitor cells (iHPCs), induced megakaryocytes (iMKs), and mesenchymal stem cells (iMSCs), and the FVIII expression was detected. The ITGA2B promoter-driven BDDF8 was site-specifically integrated into the rDNA locus of HA-iPSCs. The 2bF8-iPSCs were efficiently differentiated into 2bF8-iHPCs, 2bF8-iMKs, and 2bF8-iMSCs. FVIII was 10.31 ng/106 cells in lysates of 2bF8-iHPCs, compared to 1.56 ng/106 cells in HA-iHPCs, and FVIII was 3.64 ng/106 cells in 2bF8-iMSCs lysates, while 1.31 ng/106 cells in iMSCs with CMV-driven BDDF8. Our results demonstrated a high expression of FVIII in iHPCs and iMSCs derived from hiPSCs with site-specific integration of ITGA2B promoter-driven BDDF8, indicating potential clinical prospects of this platelet-targeted strategy for HA gene therapy.

Determination of modification degree of polysialylated therapeutic proteins using 1H-NMR spectroscopy.

Water soluble polymers and their derivatives bound to proteins can dramatically favor the biological activity of new drugs and vaccines. Quantification of the modification degree of the protein is crucial during the development and licensing phase and later in order to monitor the industrial production process and to match product specification. In this work, we describe an innovative way to measure directly the modification degree of polysialylated proteins using proton NMR (Nuclear Magnetic Resonance) spectroscopy. Following a calibration step, the modification degree can be easily deduced by the integration ratio of a separate signal from the polymer and selected signals from the protein. In fact, the upfield-shifted signals of methyl groups from Valine, Leucine and Isoleucine can be used as an internal calibration reference for the integration. In this paper recombinant factor VIII (rFVIII) and recombinant factor IX (rFIX) proteins modified by polysialic acid (PSA) are used to illustrate the accuracy, reproducibility and ease of the method that may replace or complement wet-chemistry approaches.

Structure of Blood Coagulation Factor VIII in Complex With an Anti-C2 Domain Non-Classical, Pathogenic Antibody Inhibitor.

Factor VIII (fVIII) is a procoagulant protein that binds to activated factor IX (fIXa) on platelet surfaces to form the intrinsic tenase complex. Due to the high immunogenicity of fVIII, generation of antibody inhibitors is a common occurrence in patients during hemophilia A treatment and spontaneously occurs in acquired hemophilia A patients. Non-classical antibody inhibitors, which block fVIII activation by thrombin and formation of the tenase complex, are the most common anti-C2 domain pathogenic inhibitors in hemophilia A murine models and have been identified in patient plasmas. In this study, we report on the X-ray crystal structure of a B domain-deleted bioengineered fVIII bound to the non-classical antibody inhibitor, G99. While binding to G99 does not disrupt the overall domain architecture of fVIII, the C2 domain undergoes an ~8 Å translocation that is concomitant with breaking multiple domain-domain interactions. Analysis of normalized B-factor values revealed several solvent-exposed loops in the C1 and C2 domains which experience a decrease in thermal motion in the presence of inhibitory antibodies. These results enhance our understanding on the structural nature of binding non-classical inhibitors and provide a structural dynamics-based rationale for cooperativity between anti-C1 and anti-C2 domain inhibitors.

Protein residue network analysis reveals fundamental properties of the human coagulation factor VIII.

Hemophilia A is an X-linked inherited blood coagulation disorder caused by the production and circulation of defective coagulation factor VIII protein. People living with this condition receive either prophylaxis or on-demand treatment, and approximately 30% of patients develop inhibitor antibodies, a serious complication that limits treatment options. Although previous studies performed targeted mutations to identify important residues of FVIII, a detailed understanding of the role of each amino acid and their neighboring residues is still lacking. Here, we addressed this issue by creating a residue interaction network (RIN) where the nodes are the FVIII residues, and two nodes are connected if their corresponding residues are in close proximity in the FVIII protein structure. We studied the characteristics of all residues in this network and found important properties related to disease severity, interaction to other proteins and structural stability. Importantly, we found that the RIN-derived properties were in close agreement with in vitro and clinical reports, corroborating the observation that the patterns derived from this detailed map of the FVIII protein architecture accurately capture the biological properties of FVIII.

Pro-Con Debate: Fibrinogen Concentrate or Cryoprecipitate for Treatment of Acquired Hypofibrinogenemia in Cardiac Surgical Patients.

Cryoprecipitate has been the gold standard for treating acquired hypofibrinogenemia in cardiac surgery for nearly 50 years. More recently, fibrinogen concentrate has been used off-label in the United States and is the standard in European countries and Canada to treat the acquired hypofibrinogenemia during cardiac surgery. Fibrinogen concentrate has multiple potential advantages including rapid reconstitution, greater dose predictability, viral inactivation during processing, and reduced transfusion-related adverse events. However, because fibrinogen concentrate lacks the other components contained in the cryoprecipitate, it may not be the "ideal" product for replacing fibrinogen in all cardiac surgical patients, particularly those with longer cardiopulmonary bypass duration. In this Pro-Con commentary article, we discuss the advantages and disadvantages of using fibrinogen concentrate and cryoprecipitate to treat acquired hypofibrinogenemia in cardiac surgical patients.

Characterisation and application of recombinant FVIII-neutralising antibodies from haemophilia A inhibitor patients.

The development of anti-drug antibodies (ADAs) is a serious outcome of treatment strategies involving biological medicines. Coagulation factor VIII (FVIII) is used to treat haemophilia A patients, but its immunogenicity precludes a third of severe haemophiliac patients from receiving this treatment. The availability of patient-derived anti-drug antibodies can help us better understand drug immunogenicity and identify ways to overcome it. Thus, there were two aims to this work: (i) to develop and characterise a panel of recombinant, patient-derived, monoclonal antibodies covering a range of FVIII epitopes with varying potencies, kinetics and mechanism of action, and (ii) to demonstrate their applicability to assay development, evaluation of FVIII molecules and basic research. For the first objective we used recombinant antibodies to develop a rapid, sensitive, flexible and reproducible ex vivo assay that recapitulates inhibitor patient blood using blood from healthy volunteers. We also demonstrate how the panel can provide important information about the efficacy of FVIII products and reagents without the need for patient or animal material. These materials can be used as experimental exemplars or controls, as well as tools for rational, hypothesis-driven research and assay development in relation to FVIII immunogenicity and FVIII-related products.

Extending the post-thaw viability of cryoprecipitate.

BACKGROUND: Cryoprecipitate has a short post-thaw expiry time of 6 h. The aim of this study was to assess the stability and function of cryoprecipitate components (FVIII, fibrinogen, vWF, and FXIII) and cryoprecipitate sterility up to 120 h post-thawing when stored at two temperatures (2-6°C and room temperature [20-24°C]). METHODS AND MATERIALS: Twenty batches (110 individual units) of time-expired, thawed cryoprecipitate were collected. Units were sampled at the 6-h expiration mark and then stored at 2-6°C or room temperature (RT). They were resampled every 24 h for 120 h. One unit from each batch was sent for sterility testing at 120 h. Samples had FVIII (one stage and chromogenic), fibrinogen, FXIII, vWFag, and vWF:RCo assays performed in batches. Rotational thromboelastometry (ROTEM) was also performed. RESULTS: FVIII levels declined significantly at 120 h post-thawing at both RT and 2-6°C, but still met international standards for FVIII content. Fibrinogen, vWF antigen, and FXIII levels reduced minimally over 120 h and always met international standard requirements when stored at either temperature. ROTEM analysis demonstrated that fibrinogen function was not compromised at 120 h post-thawing under both storage conditions. vWF:RCo levels declined significantly over 120 h at both storage temperatures. No bacterial contamination was detected in 20 units of cryoprecipitate following storage for 120 h post-thawing. CONCLUSION: These results demonstrate that extension of the storage time of thawed cryoprecipitate to 120 h, stored at either 2-6°C or RT, is feasible while still maintaining required FVIII, fibrinogen, and vWFag levels. Storage at 2-6°C has the advantage of reduced risk of potential bacterial contamination.

Molecular determinants of the factor VIII/von Willebrand factor complex revealed by BIVV001 cryo-electron microscopy.

Interaction of factor VIII (FVIII) with von Willebrand factor (VWF) is mediated by the VWF D'D3 domains and thrombin-mediated release is essential for hemostasis after vascular injury. VWF-D'D3 mutations resulting in loss of FVIII binding are the underlying cause of von Willebrand disease (VWD) type 2N. Furthermore, the FVIII-VWF interaction has significant implications for the development of therapeutics for bleeding disorders, particularly hemophilia A, in which endogenous VWF clearance imposes a half-life ceiling on replacement FVIII therapy. To understand the structural basis of FVIII engagement by VWF, we solved the structure of BIVV001 by cryo-electron microscopy to 2.9 Å resolution. BIVV001 is a bioengineered clinical-stage FVIII molecule for the treatment of hemophilia A. In BIVV001, VWF-D'D3 is covalently linked to an Fc domain of a B domain-deleted recombinant FVIII (rFVIII) Fc fusion protein, resulting in a stabilized rFVIII/VWF-D'D3 complex. Our rFVIII/VWF structure resolves BIVV001 architecture and provides a detailed spatial understanding of previous biochemical and clinical observations related to FVIII-VWF engagement. Notably, the FVIII acidic a3 peptide region (FVIII-a3), established as a critical determinant of FVIII/VWF complex formation, inserts into a basic groove formed at the VWF-D'/rFVIII interface. Our structure shows direct interaction of sulfated Y1680 in FVIII-a3 and VWF-R816 that, when mutated, leads to severe hemophilia A or VWD type 2N, respectively. These results provide insight on this key coagulation complex, explain the structural basis of many hemophilia A and VWD type 2N mutations, and inform studies to further elucidate how VWF dissociates rapidly from FVIII upon activation.

Acidic Region Residues 1680-1684 in the A3 Domain of Factor VIII Contain a Thrombin-Interactive Site Responsible for Proteolytic Cleavage at Arg1689.

Factor VIII (FVIII) is activated by thrombin-catalyzed cleavage at Arg372, Arg740, and Arg1689. Our previous studies suggested that thrombin interacted with the FVIII C2 domain specific for cleavage at Arg1689. An alternative report demonstrated, however, that a recombinant (r)FVIII mutant lacking the C2 domain retained >50% cofactor activity, indicating the presence of other thrombin-interactive site(s) associated with cleavage at Arg1689. We have focused, therefore, on the A3 acidic region of FVIII, similar to the hirugen sequence specific for thrombin interaction (54-65 residues). Two synthetic peptides, spanning residues 1659-1669 with sulfated Tyr1664 and residues 1675-1685 with sulfated Try1680, inhibited thrombin-catalyzed FVIII activation and cleavage at Arg1689. Treatment with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide to cross-link thrombin with either peptide showed possible contributions of both 1664-1666 and 1683-1684 residues for thrombin interaction. Thrombin-catalyzed activation and cleavage at Arg1689 in the alanine-substituted rFVIII mutants within 1663-1666 residues were similar to those of wild type (WT). Similar studies of 1680-1684 residues, however, demonstrated that activation and cleavage by thrombin of the FVIII mutant with Y1680A or D1683A/E1684A, in particular, were severely or moderately reduced to 20 to 30% or 60 to 70% of WT, respectively. Surface plasmon resonance-based analysis revealed that thrombin interacted with both Y1680A and D1683A/E1684A mutants with approximately sixfold weaker affinities of WT. Cleavage at Arg1689 in the isolated light-chain fragments from both mutants was similarly depressed, independently of the heavy-chain subunit. In conclusion, the 1680-1684 residues containing sulfated Tyr1680 in the A3 acidic region also contribute to a thrombin-interactive site responsible for FVIII activation through cleavage at Arg1689.