P J Fay

University of Rochester, Rochester, New York, United States

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Publications (140)738.27 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: An important negative regulator of factor VIIIa (FVIIIa) cofactor activity is A2 subunit dissociation. FVIII molecules with stabilized activity have been generated by elimination of charged residues at the A1-A2 and A2-A3 interfaces. These molecules exhibited reduced decay rates as part of the enzymatic factor Xa generation complex (FXase) and retained their activities under thermal and chemical denaturing conditions. We describe here the potency and efficacy of one such stability variant, D519V/E665V, derived from B-domain-deleted FVIII (BDD-FVIII). The major effect of A2 stabilization was on cofactor activity. D519V/E665V potency was increased 2-fold by the 2-stage chromogenic assay relative to BDD-FVIII. D519V/E665V demonstrated enhanced thrombin generation responses (5-fold by peak thrombin) relative to BDD-FVIII. In vivo consequences of enhanced cofactor activity of D519V/E665V included >4-fold increased maximal platelet-fibrin deposition after laser injury and 2-fold increased protection from bleeding in an acute prolonged vascular injury model in hemophilia A mice. These results demonstrate that noncovalent stabilization of the FVIII A2 subunit can prolong its cofactor activity, leading to differential enhancement in clot formation over protection from blood loss in hemophilia. The FVIII molecule described here is the first molecule with clear efficacy enhancement due to noncovalent stabilization of the A2 domain.
    Blood 11/2014; · 9.78 Impact Factor
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    ABSTRACT: Proteolytic cleavage of factor VIII (FVIII) to activated FVIIIa is required for participation in the coagulation cascade. The A2 domain is no longer covalently bound in the resulting activated heterotrimer and is highly unstable. Aspartic acid (D) 519 and glutamic acid (E) 665 at the A1-A2 and A2-A3 domain interfaces were identified as acidic residues in local hydrophobic pockets. Replacement with hydrophobic valine (V; D519V/E665V) improved the stability and activity of the mutant FVIII over the wild-type (WT) protein in several in vitro assays. In the current study, we examined the impact of mutations on secondary and tertiary structure as well as in vivo stability, pharmacokinetics (PK), efficacy, and immunogenicity in a murine model of Hemophilia A (HA). Biophysical characterization was performed with far-UV circular dichroism (CD) and fluorescence emission studies. PK and efficacy of FVIII was studied following i.v. bolus doses of 4, 10 and 40 IU/kg with chromogenic and tail clip assays. Immunogenicity was measured with the Bethesda assay and ELISA after a series of i.v. injections. Native secondary and tertiary structure was unaltered between variants. PK profiles were similar at higher doses, but at 4 IU/kg plasma survival of D519V/E665V was improved. Hemostasis at low concentrations was improved for the mutant. Immune response was similar between variants. Overall, these results demonstrate that stabilizing mutations in the A2 domain of FVIII can improve HA therapy in vivo.
    The AAPS Journal 06/2014; · 4.39 Impact Factor
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    ABSTRACT: Factor (F)VIIIa, a heterotrimer comprised of A1, A2, and A3C1C2 subunits, is labile due to the tendency of the A2 subunit to dissociate from the A1/A3C1C2 dimer. As dissociation of the A2 subunit inactivates FVIIIa activity, retention of A2 defines FVIIIa stability and thus, FXase activity. Earlier results showed that replacing residues D519, E665, and E1984 at the A2 domain interface with Ala or Val reduced rates of FVIIIa decay, increasing FXa and thrombin generation. We now show the enhanced FVIIIa stability of these variants results from increases in inter-A2 subunit affinity. Using a FVIIIa reconstitution assay to monitor inter-subunit affinity by activity regeneration, the apparent Kd value for the interaction of wild-type (WT) A2 subunit with WT A1/A3C1C2 dimer (43 ± 2 nM) was significantly higher than values observed for the A2 point mutants D519A/V, E665A/V, and E1984A/V which ranged from ~5 to ~19 nM. Val was determined to be the optimal hydrophobic residue at position 665 (apparent Kd = 5.1 ± 0.7 nM) as substitutions with Ile or Leu at this position increased the apparent Kd value by ~3- and ~7-fold, respectively. Furthermore, the double mutant (D519V/E665V) showed an ~47-fold lower apparent Kd value (0.9 ± 0.6 nM) than WT. Thus these hydrophobic mutations at the A2 subunit interfaces result in high binding affinities for the A2 subunit and correlate well with previously observed reductions in rates in FVIIIa decay.
    Thrombosis and haemostasis. 06/2014; 112(3).
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    ABSTRACT: The Factor VIII (FVIII) crystal structure suggests a possible bonding interaction of His281 (A1 domain) with Ser524 (A2 domain), although the resolution of the structure (~4 Å) does not firmly establish this bonding. In order to establish that side chains of these residues participate in an inter-domain bond, we prepared and examined the functional properties of a residue swap variant (His281Ser/Ser524His) where His281 and Ser524 residues were exchanged with one-another and disulfide-bridged variant (His281Cys/Ser524Cys) where the two residues were replaced with Cys. The latter variant showed efficient disulfide bonding of the A1 and A2 domains. The swap variant showed WT-like FVIII and FVIIIa stability, which were markedly reduced in for His281Ala and Ser524Ala variants in an earlier study. The disulfide-bridged variant showed ~20% increased FVIII stability and FVIIIa did not decay during the time course measured. This variant also yielded 35% increased thrombin peak values compared with WT in a plasma-based thrombin generation assay. Binding analyses of His281Ser-A1/A3C1C2 dimer with Ser524His-A2 subunit yielded a near WT-like affinity value, whereas combining the variant dimer or A2 subunit with the WT complement yielded ~5 and ~10-fold reductions, respectively, in affinity. Other functional properties including thrombin generation potential, FIXa binding affinity, Km for FX of FXase complexes, thrombin activation efficiency, and down-regulation by activated protein C showed similar results for the two variants compared with WT FVIII. These results indicate that the side chains of His281 and Ser524 are in close proximity and contribute to a bonding interaction in FVIII that is retained in FVIIIa.
    Journal of Biological Chemistry 04/2014; · 4.65 Impact Factor
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    ABSTRACT: FVIIIa is labile due to the dissociation of A2 subunit. Previously, we introduced hydrophobic mutations at select A1/A2/A3 subunit interfaces yielding more stable FVIII(a) variants. Separately we showed that altering the sequence flanking the primary FXa cleavage site in FVIIIa (Arg336) yielded reduced rates of proteolytic inactivation of FVIIIa. In this study we prepared the FXa-cleavage resistant mutant (336(P4-P3')562) combined with mutations of Ala108Ile, Asp519Val/Glu665Val or Ala108Ile/Asp519Val/Glu665Val and examined the effects of these combinations relative to FVIII thermal stability, rates of FVIIIa decay and proteolytic inactivation of FVIIIa by FXa. Thermal decay rates for 336(P4-P3')562/Ala108Ile, 336(P4-P3')562/Asp519Val/Glu665Val, and 336(P4-P3')562/Ala108Ile/Asp519Val/Glu665Val variants were reduced by ~2- to 5-fold as compared with wild-type (WT) primarily reflecting the effects of the A domain interface mutations. FVIIIa decay rates for 336(P4-P3')562/Asp519Val/Glu665Val and 336(P4-P3')562/Ala108Ile/Asp519Val/Glu665Val variants were reduced by ~25 fold, indicating greater stability than the control Asp519Val/Glu665Val variant (~14-fold). Interestingly, 336(P4-P3')562/Asp519Val/Glu665Val and 336(P4-P3')562/Ala108Ile/Asp519Val/Glu665Val variants showed reduced FXa-inactivation rates compared with the 336(P4-P3')562 control (~4-fold), suggesting A2 subunit destabilisation is a component of proteolytic inactivation. Thrombin generation assays using the combination variants were similar to the Asp519Val/Glu665Val control. These results indicate that combining multiple gain-of-function FVIII mutations yields FVIII variants with increased stability relative to a single type of mutation.
    Thrombosis and Haemostasis 03/2014; 112(1). · 5.76 Impact Factor
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    ABSTRACT: Factor (F) Xa proteolytically activates FVIII by cleaving P1 residues Arg372, Arg740 and Arg1689. The Arg372 site represents the rate-limiting step for procofactor activation, whereas cleavage at Arg740 is a fast step. FXa also catalyzes inactivating cleavages that occur on a slower time scale than the activating ones. In order to assess the role of sequences flanking the Arg372 and Arg740 sites, recombinant FVIII variants were prepared where P3-P3´ sequences were swapped individually or in combination. Replacing the Arg372 flanking sequence with that from the Arg740 site increased the rate of cleavage at Arg372, as judged by the ~5-fold increased rate in A1 subunit generation, and reduced the FVIIIa-dependent lag time for in situ FXa generation. The reciprocal swap yielded a nearly 2-fold rate increase in Arg372 cleavage while the combined double swap variant showed a 10-fold rate increase at that site consistent with the individual effects being additive. Although this cleavage represents the slow step for activation, the rate of this reaction appeared ~9-fold greater than the rate of the primary inactivating cleavage at Arg336 to generate the A1336 product. Interestingly, replacement of the Arg372 flanking sequence with the Arg740 sequence combined with an Arg740Gln mutation yielded both more rapid cleavage of the Arg372 site as well as accelerated inactivating cleavages within the A1 subunit. These results indicate that flanking sequences in part modulate the reaction rates required for procofactor activation and influence the capacity for FXa as an initial activator of FVIII rather than inactivator.
    Biochemistry 10/2013; · 3.38 Impact Factor
  • Hironao Wakabayashi, Philip J Fay
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    ABSTRACT: Factor (F)VIII consists of a heavy chain [HC; A1(a1)A2(a2)B domains] and light chain [LC; (a3)A3C1C2 domains]. In order to gain insights into a role of the FVIII C domains we eliminated C1 domain by replacing it with the homologous C2 domain. FVIII stability of the mutant (FVIIIC2C2) as measured by thermal decay at 55°C of FVIII activity was markedly reduced (~11 fold) while the decay rate of FVIIIa due to A2 subunit dissociation was similar to WT FVIIIa. The binding affinity of FVIIIC2C2 for phospholipid membranes as measured by fluorescence resonance energy transfer was modestly lower (~2.8 fold) than that for WT FVIII. Among several anti-FVIII antibodies tested [anti-C1 (GMA8011), anti-C2 (ESH4 and ESH8), and anti-A3 (2D2) antibody], only ESH4 inhibited membrane binding of both WT FVIII and FVIIIC2C2. FVIIIa cofactor activity measured in the presence of each of the above antibodies was examined by FXa generation assays. The activity of WT FVIIIa was inhibited by both GMA8011 and ESH4 while the activity of FVIIIC2C2 was inhibited by both the anti-C2 antibodies, ESH4 and ESH8. Interestingly FIXa binding affinity for WT FVIIIa was significantly reduced in the presence of GMA8011 (~10 fold) while the anti-C2 antibodies reduced FIXa binding affinity of FVIIIC2C2 variant (~4 fold). Together, the reduced stability plus impaired FIXa interaction of FVIIIC2C2 suggest that the C1 domain resides in close proximity to FIXa in the FXase complex and contributes a critical role to FVIII structure and function.
    Journal of Biological Chemistry 09/2013; · 4.65 Impact Factor
  • Hironao Wakabayashi, Philip J Fay
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    ABSTRACT: Factor (F)VIII consists of a heavy chain [A1(a1)A2(a2)B domains] and light chain [(a3)A3C1C2 domains]. Several reports have shown significant changes in FVIII stability and/or activity following selected mutations at the interfaces of the A1-A2, A1-A3, A2-A3, or A1-C2 domains. In this study the remaining inter-FVIII subunit interfaces (A3-C1 and C1-C2) were examined for their contributions to FVIII/FVIIIa stability and activity. We prepared FVIII mutants with a nascent disulfide bridges between A3 and C1 domains (Gly1750Cys/Arg2116Cys and Ala1866Cys/Ser2119Cys) or C1 and C2 domains (Ser2029Cys/Pro2292Cys). We also prepared mutants replacing Arg2116 with hydrophobic residues (Ala and Val) since this C1 domain residue appears to face a pocket of positive electrostatic potential in the A3 domain. Stability was assessed following the rates of loss of FVIII activity at 55ºC and the spontaneous loss of FVIIIa activity from A2 subunit dissociation. FVIII Gly1750Cys/Arg2116Cys showed a marked increase in thermal stability (~3.7 fold) as compared with WT FVIII while the stability of FVIII Ala1866Cys/Ser2119Cys was reduced (~4.7 fold). Although the Ser2029Cys/Pro2292Cys variant showed a modest loss in FVIII stability, the specific activity and thrombin generation potential of this variant were increased (up to 1.2-fold) compared with WT. Furthermore, this variant demonstrated an ~2-fold reduced Km for FX. Mutation of Arg2116 to hydrophobic residues resulted in variable decreases in stability and thrombin generation parameters suggesting a role of this Arg residue contributing to FVIII structure. Taken together, selective modification of the contiguous domain interfaces in FVIII light chain may improve FVIII stability and/or cofactor function.
    Biochemistry 05/2013; · 3.38 Impact Factor
  • Amy E Griffiths, Ivan Rydkin, Philip J Fay
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    ABSTRACT: Factor (F) VIIIa forms a number of contacts with FIXa in assembling the FXase enzyme complex. Surface plasmon resonance was used to examine the interaction between immobilized biotinylated active site-modified FIXa, and FVIII and FVIIIa subunits. The FVIIIa A2 subunit bound FIXa with high affinity (Kd = 3.9 ± 1.6 nM) that was similar to the A3C1C2 subunit (Kd = 3.6 ± 0.6 nM). This approach was used to evaluate a series of baculovirus-expressed, isolated A2 domain (bA2) variants where alanine substitutions were made for individual residues within the sequence 707-714, the C-terminal region of A2 thought to be FIXa-interactive. Three of six bA2 variants examined displayed 2- to 4-fold decreased affinity for FIXa as compared with WT bA2. The variant bA2 proteins were also tested in two reconstitution systems to determine activity and affinity parameters in forming FXase and FVIIIa. Vmax values for all variants were similar to the WT values, indicating that these residues do not affect cofactor function. All variants showed substantially greater increases in apparent Kd relative to WT in reconstituting the FXase complex (8- to 26-fold) compared with reconstituting FVIIIa (1.3- to 6-fold) suggesting that the mutations altered interaction with FIXa. bA2 domain variants with Ala replacing Lys707, Asp712, and Lys713 demonstrated the greatest increases in apparent Kd (17- to 26-fold). These results indicate a high affinity interaction between the FVIIIa A2 subunit and FIXa and show a contribution of several residues within the 707-714 sequence to this binding.
    Journal of Biological Chemistry 04/2013; · 4.65 Impact Factor
  • Hironao Wakabayashi, Philip J Fay
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    ABSTRACT: Factor VIIIa (FVIIIa) binds to phospholipid membranes during formation of the FXase complex. Free thiols of Cys residues of isolated FVIIIa A1 and A2 subunits and the A3 domain of A3C1C2 subunit were labeled with PyMPO or fluorescein (fluorescence donors). Double mutants in the A3 domain (Cys2000Ser/Thr1872Cys and Cys2000Ser/Asp1828Cys) were also produced to utilize Cys1828 and Cys1872 residues for labeling. Labeled subunits were reacted with complementary non-labeled subunits to reconstitute FVIIIa. Octadecylrhodamine incorporated into phospholipid vesicles was used as an acceptor for distance measurements between FVIII residues and membrane surface by fluorescence resonance energy transfer. Results indicated that a FVIII axis on a plane that intersects the approximate center of each domain is oriented with a tilt angle of ~30-50° on the membrane surface. This orientation predicted the existence of contacts mediated by residues 1713-1725 in the A3 domain in addition to a large area of contacts within the C domains. FVIII variants where residues Arg1719 or Arg1721 were mutated to Asp showed a >40-fold reduction in membrane affinity. These results identify possible orientations for FVIIIa bound to the membrane surface and support a new interaction between the A3 domain and membrane likely mediated, in part by Arg1719 and Arg1721.
    Biochemical Journal 03/2013; · 4.65 Impact Factor
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    ABSTRACT: Basic residues contained in the 39-, 60-, and 70-80-loops of activated protein C (APC) comprise an exosite that contributes to the binding and subsequent proteolytic inactivation of factor (F) VIIIa. Surface plasmon resonance (SPR) showed that WT APC bound to FVIII light chain (LC) and the FVIIIa A1/A3C1C2 dimer with equivalent affinity (Kd = 525 and 546 nM, respectively). These affinity values may reflect binding interactions to the acidic residue-rich a1 and a3 segments adjacent to A1 domain in the A1/A3C1C2 and a3 domain in LC, respectively. Results from SPR, using a panel of APC exosite variants where basic residues were mutated, in binding to immobilized FVIIIa A1/A3C1C2 or LC indicated ~4-10-fold increases in the Kd values relative to WT for several of the variants including Lys39Ala, Lys37-Lys38-Lys39/ Pro-Gln-Glu, and Arg67Ala. On the other hand, a number of APC variants including Lys38Ala, Lys62Ala, and Lys78Ala showed little if any change in binding affinity to the FVIII substrates. FXa generation assays and Western blotting, used to monitor rates of FVIIIa inactivation and proteolysis at the primary cleavage site in the cofactor (Arg336), respectively, showed marked rate reductions relative to WT for the Lys39Ala, Lys37-Lys38-Lys39/ Pro-Gln-Glu, Arg67Ala, and Arg74Ala variants. Furthermore, kinetic analysis monitoring FVIIIa inactivation by APC variants at varying FVIIIa substrate concentration showed ~2.6- to ~4.4-fold increases in Km values relative to WT. These results show a variable contribution of basic residues comprising the APC exosite, with significant contributions from Lys39, Arg67, and Arg74 to forming a FVIIIa-interactive site.
    Biochemistry 03/2013; · 3.38 Impact Factor
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    ABSTRACT: The clinical severity in some patients with haemophilia A appears to be unrelated to the levels of factor (F)VIII activity (FVIII:C), but mechanisms are poorly understood. We have investigated a patient with a FVIII gene mutation at Arg1781 to His (R1781H) presenting with a mild phenotype despite FVIII:C of 0.9 IU/dl. Rotational thromboelastometry using the patient's whole blood demonstrated that the clot time and clot firmness were comparable to those usually observed at FVIII:C 5-10 IU/dl. Thrombin and FXa assays using plasma samples also showed that the peak levels of thrombin formation and the initial rate of FXa generation were comparable to those observed at FVIII:C 5-10 IU/dl. The results suggested a significantly greater haemostatic potential in this individual than in those with severe phenotype. The addition of incremental amounts of FX to control plasma with FVIII:C 0.9 IU/dl in clot waveform analyses suggested that the enhanced functional tenase assembly might have been related to changes in association between FVIII and FX. To further investigate this mechanism, we prepared a stably expressed, recombinant, B-domainless FVIII R1781H mutant. Thrombin generation assays using mixtures of control plasma and FVIII revealed that the coagulation function observed with the R1781H mutant (0.9 IU/dl) was comparable to that seen with wild-type FVIII:C at ~5 IU/dl. In addition, the R1781H mutant demonstrated an ~1.9-fold decrease in Km for FX compared to wild type. These results indicated that relatively enhanced binding affinity of FVIII R1781H for FX appeared to moderate the severity of the haemophilia A phenotype.
    Thrombosis and Haemostasis 03/2013; 109(5). · 5.76 Impact Factor
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    ABSTRACT: Although factor (F) VIIIa is inactivated by activated protein C (APC) through cleavages in the FVIII heavy chain-derived A1 (Arg 336) and A2 subunits (Arg 562), the FVIII light chain (LC) contributes to catalysis by binding the enzyme. ELISA-based binding assays showed that FVIII and FVIII LC bound to immobilised active site-modified APC (DEGR-APC) (apparent Kd ~270 nM and 1.0 μM, respectively). Fluid-phase binding studies using fluorescence indicated an estimated K d of ~590 nM for acrylodan-labelled LC binding to DEGR-APC. Furthermore, FVIII LC effectively competed with FVIIIa in blocking APC-catalysed cleavage at Arg 336 ( Ki = 709 nM). A binding site previously identified near the C-terminal end of the A3 domain (residues 2007-2016) of FVIII LC was subjected to Ala-scanning mutagenesis. FXa generation assays and western and dot blotting were employed to assess the contribution of these residues to FVIIIa interactions with APC. Virtually all variants tested showed reductions in the rates of APC-catalysed inactivation of the cofactor and cleavage at the primary inactivation site (Arg 336), with maximal reductions in inactivation rates (~3-fold relative to WT) and cleavage rates (~3 to ~9-fold relative to WT) observed for the Met2010Ala, Ser2011Ala, and Leu2013Ala variants. Titration of FVIIIa substrate concentration monitoring cleavage by a dot blot assay indicated that these variants also showed ~3-fold increases relative to WT while a double mutant (Met2010Ala/Ser2011Ala) showed a>4-fold increase in Km. These results show a contribution of a number of residues within the 2007-2016 sequence, and in particular residues Met2010, Ser2011, and Leu2013 to an APC-interactive site.
    Thrombosis and Haemostasis 12/2012; 109(2). · 5.76 Impact Factor
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    ABSTRACT: Thrombin-catalyzed activation of factor VIII (FVIII) occurs through proteolysis at three P1 Arg residues: Arg(372) and Arg(740) in the FVIII heavy chain and Arg(1689) in the FVIII light chain. Cleavage at the latter two sites is relatively fast compared with cleavage at Arg(372), which appears to be rate-limiting. Examination of the P3-P3' residues flanking each P1 site revealed that those sequences at Arg(740) and Arg(1689) are more optimal for thrombin cleavage than at Arg(372), suggesting these sequences may impact reaction rates. Recombinant FVIII variants were prepared with mutations swapping scissile bond flanking sequences in the heavy chain individually and in combination with a second swap or with a P1 point mutation. Rates of generation of A1 and A3-C1-C2 subunits were determined by Western blotting and correlated with rates of cleavage at Arg(372) and Arg(1689), respectively. Rates of thrombin cleavage at Arg(372) were increased ∼10- and ∼3-fold compared with that of wild-type FVIII when it was replaced with P3-P3' residues flanking Arg(740) and Arg(1689), respectively, and these values paralleled increased rates of A2 subunit generation and procofactor activation. Positioning of more optimal residues flanking Arg(372) abrogated the need for initial cleavage at Arg(740) to facilitate this step. These results show marked changes in cleavage rates correlate with the extent of cleavage-optimal residues flanking the scissile bond and modulate the mechanism for procofactor activation.
    Biochemistry 03/2012; · 3.38 Impact Factor
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    ABSTRACT: Factor (F)VIII can be activated to FVIIIa by FXa following cleavages at Arg(372), Arg(740), and Arg(1689). FXa also cleaves FVIII/FVIIIa at Arg(336) and Arg(562) resulting in inactivation of the cofactor. These inactivating cleavages occur on a slower time scale than the activating ones. We assessed the contributions to cleavage rate and cofactor function of residues flanking Arg(336), the primary site yielding FVIII(a) inactivation, following replacement of these residues with those flanking the faster-reacting Arg(740) and Arg(372) sites and the slower-reacting Arg(562) site. Replacing P4-P3' residues flanking Arg(336) with those from Arg(372) or Arg(740) resulted in ∼4-6-fold increases in rates of FXa-catalyzed inactivation of FVIIIa, which paralleled the rates of proteolysis at Arg(336). Examination of partial sequence replacements showed a predominant contribution of prime residues flanking the scissile bonds to the enhanced rates. Conversely, replacement of this sequence with residues flanking the slow-reacting Arg(562) site yielded inactivation and cleavage rates that were ∼40% that of the WT values. The capacity for FXa to activate FVIII variants where cleavage at Arg(336) was accelerated due to flanking sequence replacement showed marked reductions in peak activity, whereas reducing the cleavage rate at this site enhanced peak activity. Furthermore, plasma-based thrombin generation assays employing the variants revealed significant reductions in multiple parameter values with acceleration of Arg(336) cleavage suggesting increased down-regulation of FXase. Overall, these results are consistent with a model of competition for activating and inactivating cleavages catalyzed by FXa that is modulated in large part by sequences flanking the scissile bonds.
    Journal of Biological Chemistry 03/2012; 287(19):15409-17. · 4.65 Impact Factor
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    ABSTRACT: Factor (F) VIII functions as a cofactor in FXase, markedly accelerating the rate of FIXa-catalyzed activation of FX. Earlier work identified a FX-binding site having μM affinity within the COOH-terminal region of the FVIIIa A1 subunit. In the present study, surface plasmon resonance (SPR), ELISA-based binding assays, and chemical cross-linking were employed to assess an interaction between FX and the FVIII light chain (A3C1C2 domains). SPR and ELISA-based assays showed that FVIII LC bound to immobilized FX (K(d) = 165 and 370 nM, respectively). Furthermore, active site-modified activated protein C (DEGR-APC) effectively competed with FX in binding FVIII LC (apparent K(i) = 82.7 nM). Western blotting revealed that the APC-catalyzed cleavage rate at Arg(336) was inhibited by FX in a concentration-dependent manner. A synthetic peptide comprising FVIII residues 2007-2016 representing a portion of an APC-binding site blocked the interaction of FX and FVIII LC (apparent K(i) = 152 μM) and directly bound to FX (K(d) = 7.7 μM) as judged by SPR and chemical cross-linking. Ala-scanning mutagenesis of this sequence revealed that the A3C1C2 subunit derived from FVIII variants Thr2012Ala and Phe2014Ala showed 1.5- and 1.8-fold increases in K(d) for FX, whereas this value using the A3C1C2 subunit from a Thr2012Ala/Leu2013Ala/Phe2014Ala triple mutant was increased >4-fold. FXase formed using this LC triple mutant demonstrated an ~4-fold increase in the K(m) for FX. These results identify a relatively high affinity and functional FX site within the FVIIIa A3C1C2 subunit and show a contribution of residues Thr2012 and Phe2014 to this interaction.
    Biochemistry 01/2012; 51(3):820-8. · 3.38 Impact Factor
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    Journal of Thrombosis and Haemostasis 12/2011; 10(3):492-5. · 6.08 Impact Factor
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    ABSTRACT: Neutralizing factor (F) VIII antibodies develop in approximately 30% of individuals with hemophilia A and show specificity to multiple sites in the FVIII protein. Reactive epitopes to an immobilized IgG fraction prepared from a high-titer, FVIII inhibitor plasma were determined after immuno-precipitation (IP) of tryptic and chymotryptic peptides derived from digests of the A1 and A2 subunits of FVIIIa and FVIII light chain. Peptides were detected and identified using highly sensitive liquid chromatography-mass spectrometry (LC-MS). Coverage maps of the A1 subunit, A2 subunit and light chain represented 79%, 69% and 90%, respectively, of the protein sequences. Dot blots indicated that the inhibitor IgG reacted with epitopes contained within each subunit of FVIIIa. IP coupled with LC-MS identified 19 peptides representing epitopes from all FVIII A and C domains. The majority of peptides (10) were derived from the A2 domain. Three peptides mapped to the C2 domain, while two mapped to the A1 and A3 domains, and single peptides mapped to the a1 segment and C1 domain. Epitopes were typically defined by peptide sequences of < 12 residues. IP coupled with LC-MS identified extensive antibody reactivity at high resolution over the entire functional FVIII molecule and yielded sequence lengths of < 15 residues. A number of the peptides identified mapped to known sequences involved in functionally important protein-protein and protein-membrane interactions.
    Journal of Thrombosis and Haemostasis 06/2011; 9(8):1534-40. · 6.08 Impact Factor
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    ABSTRACT: Factor VIII (FVIII) consists of a heavy (A1A2B domains) and light chain (A3C1C2 domains), whereas the contiguous A1A2 domains are separate subunits in the cofactor, FVIIIa. FVIII x-ray structures show close contacts between A1 and C2 domains. To explore the role of this region in FVIII(a) stability, we generated a variant containing a disulfide bond between A1 and C2 domains by mutating Arg-121 and Leu-2302 to Cys (R121C/L2302C) and a second variant with a bulkier hydrophobic group (A108I) to better occupy a cavity between A1 and C2 domains. Disulfide bonding in the R121C/L2302C variant was >90% efficient as judged by Western blots. Binding affinity between the A108I A1 and A3C1C2 subunits was increased ∼3.7-fold in the variant as compared with WT as judged by changes in fluorescence of acrylodan-labeled A1 subunits. FVIII thermal and chemical stability were monitored following rates of loss of FVIII activity at 57 °C or in guanidinium by factor Xa generation assays. The rate of decay of FVIIIa activity was monitored at 23 °C following activation by thrombin. Both R121C/L2302C and A108I variants showed up to ∼4-fold increases in thermal stability but minimal improvements in chemical stability. The purified A1 subunit of A108I reconstituted with the A3C1C2 subunit showed an ∼4.6-fold increase in thermal stability, whereas reconstitution of the variant A1 with a truncated A3C1 subunit showed similar stability values as compared with WT A1. Together, these results suggest that altering contacts at this A1-C2 junction by covalent modification or increasing hydrophobicity increases inter-chain affinity and functionally enhances FVIII stability.
    Journal of Biological Chemistry 05/2011; 286(29):25748-55. · 4.65 Impact Factor
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    ABSTRACT: Activated protein C (APC) inactivates factor VIIIa (FVIIIa) through cleavages at Arg336 in the A1 subunit and Arg562 in the A2 subunit. Proteolysis at Arg336 occurs 25-fold faster than at Arg562. Replacing residues flanking Arg336 en bloc with the corresponding residues surrounding Arg562 markedly reduced the rate of cleavage at Arg336, indicating a role for these residues in the catalysis mechanism. To assess the contributions of individual P4-P3' residues flanking the Arg336 site to cleavage efficiency, point mutations were made based upon those flanking Arg562 of FVIIIa (Pro333Val, Gln334Asp, Leu335Gln, Met337Gly, Lys338Asn, Asn339Gln) and selected residues flanking Arg506 of FVa (Leu335Arg, and Lys338Ile). APC-catalyzed inactivation of the FVIII variants and cleavage of FVIIIa subunits were monitored by FXa generation assays and Western blotting. Specific activity values of the variants were 60-135% of the wild type (WT) value. APC-catalyzed rates of cleavage at Arg336 remained similar to WT for the Pro333Val and Lys338Ile variants and was modestly increased for the Asn339Gln variant; while rates were reduced ~2-3-fold for the Gln334Asp, Leu335Gln, Leu335Arg, and Lys338Asn variants, and 5-fold for the Met337Gly variant. Rates for cofactor inactivation paralleled cleavage at the A1 site. APC slowly cleaves Arg372 in FVIII, a site responsible for procofactor activation. Using FVIII as substrate for APC, the Met337Gly variant yielded significantly greater activation compared with WT FVIII. These results show that individual P4-P3' residues surrounding Arg336 are in general more favorable to cleavage than those surrounding the Arg562 site.
    Thrombosis Research 04/2011; 128(5):470-6. · 3.13 Impact Factor

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3k Citations
738.27 Total Impact Points

Institutions

  • 1984–2014
    • University of Rochester
      • • Department of Biochemistry and Biophysics
      • • Division of Hospital Medicine
      • • School of Medicine and Dentistry
      • • Department of Medicine
      Rochester, New York, United States
  • 2001–2005
    • University Center Rochester
      • • Department of Biochemistry
      • • Department of Medicine
      Rochester, Minnesota, United States
    • Washington University in St. Louis
      • Department of Medicine
      Saint Louis, MO, United States
  • 1994
    • University of Maryland, College Park
      • Department of Microbiology and Immunology
      College Park, MD, United States
  • 1990
    • Red Cross
      Washington, Washington, D.C., United States
  • 1986
    • Rochester General Hospital
      Rochester, New York, United States