A3 domain residue Glu1829 contributes to A2 subunit retention in factor VIIIa

BACKGROUND: Factor VIII (FVIII) is activated by thrombin to the labile FVIIIa, a heterotrimer of A1, A2 and A3C1C2 subunits, which serves as a cofactor for FIXa. A primary reason for the instability of FVIIIa is the tendency for the A2 subunit to dissociate from FVIIIa leading to an inactive cofactor and consequent loss of FXase activity. OBJECTIVE: Based on our finding of low-specific activity and a fast decay rate for a FVIII point mutation of Glu1829 to Ala (E1829A), we examined whether residue Glu1829 in the A3 subunit is important for A2 subunit retention. RESULTS: The rate of activity decay of E1829A was approximately fourteen fold faster than wild-type (wt) FVIIIa and this rate was reduced in the presence of added A2 subunit. Specific activity values for E1829A measured by one-stage and two-stage assays were approximately 14% and approximately 11%, respectively, compared with wt FVIII. Binding affinity for the A1 subunit to E1829A-A3C1C2 was comparable to wt A3C1C2 (K(d) = 20.1 +/- 3.4 nM for E1829A, 15.3 +/- 3.7 nM for wt), whereas A2 subunit affinity for the A1/A3C1C2 dimer forms was reduced by approximately 3.6-fold as a result of the mutation (K(d) = 526 +/- 107 nM for E1829A, 144 +/- 21 nM for wt). CONCLUSION: As modeling data suggest that Glu1829 is located at the A2-A3 domain interface these results are consistent with Glu1829 contributing to the interactions involved with A2 subunit retention in FVIIIa.     

Epitope mapping factor VIII A2 domain by affinity-directed mass spectrometry: residues 497–510 and 584–593 comprise a discontinuous epitope for the monoclonal antibody R8B12

The murine monoclonal antibody R8B12 recognizes the C-terminal region (residues 563-740) of the A2 subunit of factor VIIIa [J Biol Chem 266: 1991; p. 20139], as judged by Western blotting. However, the location of the epitope within this region is not known. In the present study, we used affinity-directed mass spectrometry to map the epitope. A2 subunit was digested with trypsin or chymotrypsin and then subjected to immunoprecipitation (IP) using R8B12 IgG. Masses of the affinity-selected peptides were determined directly from the immune complexes by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Proteolysis of A2 with the two proteases generated a pre-IP peptide fingerprint that covered approximately 70% of the A2 domain sequence. Analysis of the post-IP tryptic peptide fingerprint showed two masses, 1309 and 1653 Da representing residues 584-593 and 497-510, respectively, determined from a theoretical database search and confirmed by direct sequencing. Results using a chymotryptic digest yielded a single, weakly reactive fragment consistent with residues 577-586, suggesting the importance of residues Ser584-Tyr586 in forming the epitope. A synthetic peptide to residues 584-593 was immunoprecipitated by the IgG and blocked R8B12-directed blotting to A2 subunit. The 497-510 and 584-593 segments were observed to be adjacent and surface exposed in the A2 domain model, and together with the above results suggest that A2 domain residues 497-510 and 584-593 represent a discontinuous epitope for R8B12. Furthermore, based upon blotting specificity, we speculate that residues 584-593 make a substantially greater contribution to the binding energy for this interaction.     

A modified thrombin generation test for the measurement of factor VIII concentrates

Summary. It has been well documented that there is an uncertainty over the true factor (F)VIII level in postinfusion samples due to assay discrepancies. The thrombin generation test (TGT) was used as a potentially more physiological approach to assess and compare FVIII concentrates. FVIII concentrates were added to artificial FVIII‐deficient plasma. Thrombin generation was initiated by the addition of FIXa (14 nm ), phospholipid and CaCl₂. Thrombin was measured by subsampling into fibrinogen, and curves quantified as area under the curve (AUC) and time taken to half‐maximum (t_½max). Addition of one plasma‐derived concentrate to as little as 0.005 IU mL^?1 gave a normal AUC, but prolonged t_½max. Increasing FVIII to 1 IU mL^?1 had little effect on AUC, but did reduce the t_½max to 64 s (normal 114 s). A range of plasma‐derived and recombinant concentrates were tested at 1 IU mL^?1; results were similar, except the B‐domain deleted concentrate, which had the most rapid initial rate of thrombin generation (t_½max 48 s, P < 0.05). Two hemophilic plasmas (< 0.01 IU mL^?1) produced large amounts of thrombin (AUC 65% and 69%), although t_½max was prolonged. Addition of a FVIII antibody abolished thrombin generation, indicating that these plasmas contained low levels of FVIII. Decreasing the FIXa concentration (0.2 nm ) minimized thrombin generation in hemophilic plasma but not in normal plasma. These results indicate that FVIII < 0.01 IU mL^?1 can generate significant quantities of thrombin depending upon the amount of FIXa present. The TGT could prove useful for patient monitoring in gene therapy and prophylaxis.     

Combining mutations of charged residues at the A2 domain interface enhances factor VIII stability over single point mutations

Summary.  Background:  Factor (F) VIII consists of a heavy chain (A1A2B domains) and light chain (A3C1C2 domains), while the contiguous A1A2 domains are separate subunits in the cofactor, FVIIIa. Recently we reported that procofactor stability at elevated temperature and cofactor stability over an extended time course were increased following replacement of individual charged residues (Asp(D)519, Glu(E)665 or Glu(E)1984) with either Ala (A) or Val (V) (Wakabayashi et al. , Blood, 112, 2761, 2008). Objectives:  In the current study we generated combination mutants at these three sites to examine any additive and/or synergistic effects of these mutations on stability. Methods:  Studies assessing FVIII stability involved monitoring decay rates of FVIII at 55 °C or in guanidinium, decay of FVIIIa following A2 subunit dissociation, and thrombin generation at low (0.3 nmol L⁻¹) FVIII concentration. Results and conclusions:  Similar tendencies were observed within each group of variants. Variants with mutations at D519 and either E665 or E1984 (Group A) generally showed significantly better stability as compared with single mutants. Most variants with mutations at E665 and at E1984 (Group B) did not show significant improvement. Triple mutants with mutations at D519, E665 and E1984 (Group C) showed improvement to a similar degree as the Group A double mutants. Overall, these results indicate that selected combinations of mutations to reduce charge and/or increase hydrophobicity at the A2/A1 and A2/A3 domain interfaces yield FVIII reagents with improved stability parameters.     

Proteolytic cleavage of factor VIII heavy chain is required to expose the binding-site for low-density lipoprotein receptor-related protein within the A2 domain

BACKGROUND: Low-density lipoprotein receptor-related protein (LRP) is an endocytic receptor that contributes to the clearance of coagulation factor (F) VIII from the circulation. Previously, we have demonstrated that region Glu(1811)-Lys(1818) within FVIII light chain constitutes an important binding region for this receptor. We have further found that FVIII light chain and intact FVIII are indistinguishable in their LRP-binding affinities. In apparent contrast to these observations, a second LRP-binding region has been identified within A2 domain region Arg(484)-Phe(509) of FVIII heavy chain. OBJECTIVE: In this study, we addressed the relative contribution of FVIII heavy chain in binding LRP. METHODS AND RESULTS: Surface plasmon resonance analysis unexpectedly showed that FVIII heavy chain poorly associated to the receptor. The binding to LRP was, however, markedly enhanced upon cleavage of the heavy chain by thrombin. The A2 domain, purified from thrombin-activated FVIII, also showed efficient binding to LRP. Competition studies employing a recombinant antibody fragment demonstrated that region Arg(484)-Phe(509) mediates the enhanced LRP binding after thrombin cleavage. CONCLUSIONS: We propose that LRP binding of non-activated FVIII is mediated via the FVIII light chain while in activated FVIII both the heavy and light chain contribute to LRP binding.     

The role of β‐barrels 1 and 2 in the enzymatic activity of factor XIII A‐subunit

Essentials

• The roles of β‐barrels 1 and 2 in factor XIII (FXIII) are currently unknown.
• FXIII truncations lacking β‐barrel 2, both β‐barrels, or full length FXIII, were made.
• Removing β‐barrel 2 caused total loss of activity, removing both β‐barrels returned 30% activity.
• β‐barrel 2 is necessary for exposure of the active site cysteine during activation.

Summary

Background

Factor XIII is composed of an activation peptide segment, a β‐sandwich domain, a catalytic core, and, finally, β‐barrels 1 and 2. FXIII is activated following cleavage of its A‐subunits by thrombin. The resultant transglutaminase activity leads to increased resistance of fibrin clots to fibrinolysis.

Objectives

To assess the functional roles of β‐barrels 1 and 2 in FXIII, we expressed and characterized the full‐length FXIII A‐subunit (FXIII‐A) and variants truncated to residue 628 (truncated to β‐barrel 1 [TB1]), residue 515 (truncated to catalytic core [TCC]), and residue 184 (truncated to β‐sandwich).

Methods

Proteins were analyzed by gel electrophoresis, circular dichroism, fluorometric assays, and colorimetric activity assays, clot structure was analyzed by turbidity measurements and confocal microscopy, and clot formation was analyzed with a Chandler loop system.

Results and Conclusions

Circular dichroism spectroscopy and tryptophan fluorometry indicated that full‐length FXIII‐A and the truncation variants TCC and TB1 retain their secondary and tertiary structure. Removal of β‐barrel 2 (TB1) resulted in total loss of transglutaminase activity, whereas the additional removal of β‐barrel 1 (TCC) restored enzymatic activity to ~ 30% of that of full‐length FXIII‐A. These activity trends were observed with physiological substrates and smaller model substrates. Our data suggest that the β‐barrel 1 domain protects the active site cysteine in the FXIII protransglutaminase, whereas the β‐barrel 2 domain is necessary for exposure of the active site cysteine during activation. This study demonstrates the importance of individual β‐barrel domains in modulating access to the FXIII active site region.     

A dose‐ranging study evaluating the oral factor Xa inhibitor edoxaban for the prevention of venous thromboembolism in patients undergoing total knee arthroplasty

Summary. Background: Edoxaban (the free base of DU‐176b) is an oral, direct factor (F)Xa inhibitor in clinical development for the prevention and treatment of thromboembolic events. Objectives: The aim of the present study was to evaluate the efficacy and safety of edoxaban for the prevention of venous thromboembolism (VTE) in patients undergoing total knee arthroplasty (TKA). Patients/methods: This was a randomized, double‐blind, placebo‐controlled, multicenter study conducted in Japan. A total of 523 Japanese patients were assigned to receive edoxaban 5, 15, 30 or 60 mg once daily or placebo for 11–14 days. A placebo control was used as neither low‐molecular‐weight heparin (LMWH) nor fondaparinux had been approved for thromboprophylaxis at the time of the study in Japan. The primary efficacy outcome was the incidence of VTE (lower‐extremity deep vein thrombosis, symptomatic pulmonary embolism or symptomatic deep vein thrombosis). The primary safety outcome was the incidence of major and clinically relevant non‐major bleeding. Results: Edoxaban produced a significant dose‐related reduction in VTE: the incidence of VTE was 29.5%, 26.1%, 12.5% and 9.1% in the edoxaban 5‐, 15‐, 30‐ and 60‐mg treatment groups vs. 48.3% in the placebo group. The incidence of major and clinically relevant non‐major bleeding was similar across all groups without any significant differences among edoxaban doses or between edoxaban and placebo. Conclusions: Edoxaban demonstrated significant dose‐dependent reductions in VTE in patients undergoing TKA with a bleeding incidence similar to placebo. [This trial is registered with JAPIC, JapicCTI‐060283 (ja).]     

A tripeptide mimetic of von Willebrand factor residues 981–983 enhances platelet adhesion to fibrinogen by signaling through integrin βIIbβ3

BACKGROUND: RGD is a major recognition sequence for ligands of platelet alpha(IIb)beta3. OBJECTIVE AND METHODS: To identify potential binding sites for alpha(IIb)beta3 apart from RGD, we screened phage display libraries by blocking the enrichment of RGD-containing phages with a GRGDS peptide and identified a novel integrin recognition tripeptide sequence, VPW. RESULTS: Platelets adhered to an immobilized cyclic VPW containing peptide in a alpha(IIb)beta3-dependent manner; platelets and alpha(IIb)beta3-expressing CHO cells adhered faster to immobilized alpha(IIb)beta3-ligands in the presence of soluble VPW. In platelets adhering to fibrinogen, VPW accelerated the activation of the tyrosine kinase Syk which controls cytoskeletal rearrangements. In alpha(IIb)beta3-expressing CHO cells, VPW induced a faster formation of stress fibers. Sequence alignment positioned VPW to V980-P981-W982 in the von Willebrand factor (vWf) A-3 domain. In blood from a vWf-deficient individual, VPW increased platelet adhesion to fibrinogen but not to collagen under flow and rescued the impaired adhesion to vWf deficient in A-3. CONCLUSION: These data reveal a VPW sequence that contributes to alpha(IIb)beta3 activation in in vitro experiments. Whether the V980-P981-W982 sequence in vWf shows similar properties under in vivo conditions remains to be established.     

Purified A2 domain of von Willebrand factor binds to the active conformation of von Willebrand factor and blocks the interaction with platelet glycoprotein Ibα

BACKGROUND: von Willebrand factor (VWF) does not interact with circulating platelets unless it is induced to expose the binding site for platelet glycoprotein (GP)Ibalpha in the A1 domain by high shear stress, immobilization, and/or a modulator. Previous studies have implied indirectly that the A2 domain may be involved in regulating A1-GPIbalpha binding. OBJECTIVE AND METHODS: Because the relationship between the A1 and A2 domains has not been defined, we have investigated the effect of the A2 domain on the binding activity of the A1 domain using recombinant A domain polypeptides, multimeric VWF, and monoclonal antibodies (mAb). RESULTS: The A2 domain polypeptide bound specifically to the immobilized A1 domain polypeptide or full-length VWF, with half-maximal binding being obtained at 60 or 168 nm, respectively. This A1-A2 interaction was inhibited by mAb against the A2 or A1 domain and by the A1 domain polypeptide. The A2 domain polypeptide effectively blocked GPIbalpha-mediated platelet adhesion under high flow conditions. The A2 domain polypeptide specifically recognizes the GPIbalpha-binding conformation in the A1 domain, as it only interacted with VWF activated by the modulator ristocetin or immobilized VWF. Furthermore, in contrast to plasma VWF, the ultra-large (UL)VWF multimers or a recombinant VWF-A1A2A3 polypeptide containing a gain-of-function mutation (R1308 L) of type 2B von Willebrand disease bound to the A2 domain polypeptide without the need for ristocetin. CONCLUSIONS: The recombinant A2 domain polypeptide specifically binds to the active conformation of the A1 domain in VWF and effectively blocks the interaction with platelet GPIbalpha under high-flow conditions.