His2315/Gln2316 of the Factor VIII C2 Domain Interact with Phospholipid Membranes and Influence Activity of the Factor Xase Complex.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1727-1727
Author(s):  
Gary E. Gilbert ◽  
Randall J. Kaufman ◽  
Patricia Price ◽  
Hongzhi Mao ◽  
Steven W. Pipe
Keyword(s):  
Amino Acids ◽  
Factor Viii ◽  
Phospholipid Bilayer ◽  
Crystallographic Structure ◽  
Phospholipid Membranes ◽  
C2 Domain ◽  
Wild Type ◽  
Phospholipid Binding ◽  
Hydrophobic Amino Acids ◽  
Type Factor

Abstract A major phospholipid-binding motif of factor VIII is localized in the C2 domain. Two pairs of hydrophobic amino acids in the factor VIII C2 domain (Met2199/Phe2200, Leu2251/Leu2252) have previously been shown to interact with phospholipid membranes. However mutations of hydrophilic amino acids (Gln2213, Arg2215, Asn2217, Arg2220, Lys2249) that were predicted to interact with the membrane surface, based upon the crystallographic structure, have not altered phospholipid binding activity. The established hydrophobic interactions do not account for the demonstrated hydrophilic interactions of factor VIII with the phospholipid bilayer. We hypothesized that His2315 and/or Gln2316, interact with phospholipid membranes, based upon their contribution to the epitope for phospholipid-blocking mAb B02C11 and based upon phosphatidylserine binding by C2 domain peptides that include these residues. Factor VIII His2315Ala/Gln2316Ala (fVIII 2315/16) was prepared by PCR mutagenesis and expressed from COS-1 cells. Secreted factor VIII was purified by immunoaffinity chromatography and evaluated in ELISA assays, aPTT assays with factor VIII deficient plasma, and in defined assays with varying phospholipids. The specific activity of fVIII 2315/16 was 67 ± 9% of wild type factor in a commercial aPTT assay with a large excess of phospholipid. The apparent affinity for extruded phospholipid vesicles (0.1 μm diameter) of composition phosphatidylserine:phosphatidylethanolamine: phosphatidylcholine 4:20:76 was 32 ± 10% of wild type factor VIII in a factor Xase assay with varying phospholipid. When the phospholipid concentration was saturating, the Vmax was 55% of factor Xase with wild type factor VIII. We conclude that His2315 and/or Gln2316 constitute the first identified hydrophilic amino acids of factor VIII that interact with a phospholipid membrane. His2315/Gln2316, combined with the two pairs of phospholipid-interactive hydrophobic amino acids constitute three spatially separated phospholipid-interactive points that define a phospholipid-interactive plane on the factor VIII C2 domain. This is consistent with the hypothesis that the phospholipid-binding plane of the C2 domain overlaps substantially with the epitope for BO2C11 and is nearly coincident with the phospholipid-binding surface predicted from the crystallographic structure.

The Mutations SER2117→LEU, and TRP/TRP2063/64→MET/PHE in the Hydrophobic Spikes of the Factor V C2 Domain Lead to Tenacious Phospholipid Binding and Suggest a Mechanism through Which Phosphatidylserine Activates the Prothrombinase Complex.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2703-2703
Author(s):  
Gary E. Gilbert ◽  
Valerie A. Novakovic ◽  
Randal J. Kaufman ◽  
Hongzhi Miao ◽  
Steven W. Pipe
Keyword(s):  
Specific Activity ◽  
Phospholipid Membranes ◽  
Unexpected Result ◽  
Factor V ◽  
C2 Domain ◽  
Wild Type ◽  
Hydrophobic Residues ◽  
Phospholipid Binding ◽  
Prothrombinase Complex ◽  
Type Factor

Abstract Factor V (fV) binds to phospholipid (PL) membranes via a motif localized to the C2 domain. We and others have shown that PL binding is mediated by two pairs of hydrophobic residues, each displayed at the tips of β-hairpin turns. The homologous hydrophobic residues in the C2 domain of factor VIII also contribute to PL binding. We hypothesized that the solvent-exposed hydrophobic residues of the fV C2 domain make specific contacts that influence membrane affinity and activity of fV. To test this hypothesis we have prepared fVIII/fV hybrid mutants in which amino acid(s) of the fV C2 domain were changed to the homologous residues of fVIII (Mutants #1 W/W 2063/2064 M/F, #2 L/S 2116/2117 L/L, and #3 W/W/S 2063/2064/2117 M/F/L). Mutants were expressed in COS-1 cells and purified by FPLC. The specific activity of the fV/FVIII hybrids #2 and #3 exceeded those of wild type factor V by approx. 10-fold and approx. 4-fold, respectively, in a prothrombin time assay with factor V deficient plasma. Apparent PL affinites were evaluated in a prothrombinase complex assay with limiting phospholipid. The apparent affinities are 9.8 and 21-fold higher than wild type factor V for mutants #2 and #3 which contain the S→L change in the second hydrophobic spike. An unexpected result was that mutants 1–3 supported prothrombinase activity in the absence of added phospholipid, in contrast to wild type fV. We hypothesized that this activity resulted from phospholipid that was not dissociated from fV during purification. This hypothesis was supported because activity was eliminated by incubation of the factor V mutants with phospholipase A2 or by incubation with lactadherin. The tenacity of the phospholipid binding was further investigated by washing immobilized wild type fV and fV mutants with CHAPS prior to elution from an FPLC column. fV mutants #2 and #3 retained activity after the CHAPS wash, free of added PL and activity remained inhibitable by lactadherin, further illustrating the tenacious phospholipid affinity. We utilized Mutants #1 & 2 in the absence of added PL to evaluate the mechanism through which soluble phosphatidylserine with 6-carbon acyl chains (C6PS) enhances activity of the prothrombinase complex. In the absence of phospholipid vesicles C6PS enhanced activity of Mutant 1 equivalent to wild type fV (> 20 fold). However, enhancement of mutant 2 activity, which presumably retained phospholipid via residues 2116/2117, was < 3-fold. This suggests that C6PS functions to activate wild type fV, in part, by engaging the free LS 2116/2117 hydrophobic spike. Together, these data indicate that the hydrophobic spikes of factor V influence the specific activity of factor V, that the high affinity reversible binding of fV to phospholipid membranes is readily perturbed by mutations, and that activation of the prothrombinase complex by C6PS and phospholipid membranes likely involves engagement of amino acids 2116/2117.

B-Cell Epitope Mapping of Monoclonal Anti-Factor VIII C2 Domain Inhibitors: Identification of Amino Acids That Contribute Significant Antigen-Antibody Binding Affinity.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1211-1211
Author(s):  
Jasper C. Lin ◽  
Jason T. Schuman ◽  
Shannon L. Meeks ◽  
John F. Healey ◽  
Arthur R. Thompson ◽  
...  
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibodies ◽  
Amino Acid ◽  
Factor Viii ◽  
Dissociation Constants ◽  
Antibody Binding ◽  
Amino Acid Substitutions ◽  
C2 Domain ◽  
Wild Type ◽  
Mutant Proteins

Abstract The most troublesome clinical complication that can afflict hemophilia A patients who receive factor VIII (FVIII) infusions as replacement therapy is the development of an anti-FVIII immune response, in which antibodies bind to functionally important FVIII surfaces, thereby blocking the pro-coagulant function of this important plasma protein cofactor. These antibodies, commonly referred to as “FVIII inhibitors”, bind primarily to the FVIII A2 and C2 domains and to the C-terminal region of the C1 domain, and inhibitors mapping to other regions have also been seen. There are multiple epitopes on the FVIII C2 domain, reflecting both its immunogenicity/antigenicity and its diverse roles in mediating interactions between FVIII and other molecules. For example, the C2 domain is essential for binding of FVIII to its carrier protein von Willebrand factor (VWF). Proteolytic activation to FVIIIa causes its release from VWF and subsequent binding to negatively charged membrane surfaces, e.g. on activated platelets, whereupon a region that overlaps the VWF binding site contacts the membrane. The C2 domain also interacts with thrombin and factor Xa, which both can activate FVIII. To better understand the basis for FVIII inhibition, and to better delineate functionally important FVIII surfaces, a panel of 56 murine anti-C2 monoclonal antibodies was generated. Competition ELISAs and functional assays were used to classify the antibodies into five groups corresponding to distinct regions on the C2 surface, which comprised a larger number of distinct epitopes (Meeks et al., Blood110, 4234–42, 2007). The present study is a high-resolution mapping of the epitopes recognized by six representative antibodies (2-77, 2-117, 3D12, 3E6, I109 and I54) using surface plasmon resonance (SPR). Each antibody was immobilized covalently via amine coupling to a CM5 chip or was captured by a rat anti-mouse IgG attached covalently to a CM5 chip. Referring to the FVIII C2 domain crystal structure (Pratt et al., Nature402, 439–42, 1999), surface-exposed amino acids were selected for mutagenesis using the Stratagene Quik-Change system, and C2 constructs with single substitutions to alanine or amino acids that were structurally similar to the wild-type residues were generated. Forty-five of these proteins were expressed in E. coli and purified; their purity and structural integrity were confirmed by SDS-PAGE and Western blot analysis. The on- and off-rates for binding of these proteins to the six monoclonal antibodies were determined using a Biacore T100 instrument. Mutations that affected binding significantly were analyzed by measuring association and dissociation constants over a temperature gradient (10–40°C), yielding estimates of changes in antibody-binding energy (ΔΔGº) of these mutant proteins compared to wild-type C2. Van’t Hoff analysis was carried out to determine the relative contributions of enthalpy and entropy to the binding energies. Interestingly, C2 binding to each antibody was abrogated by 1–5 of the 45 amino acid substitutions tested. Each of these C2 mutants bound to other antibodies with affinities similar to that of wild-type C2, indicating that this was not an artifact due to protein misfolding. The following substitutions resulted in little or no binding, as evidenced by a completely abated signal (very low Rmax compared to the wild-type C2 protein): L2273A (2-77, 2-117), R2220A (3D12, I109), Q2231A (I54) and T2272A (I109). Additional mutant proteins with reduced binding to inhibitor(s) displayed markedly higher dissociation constants and sometimes less pronounced differences in association constants compared to wild-type C2. Although several FVIII residues contributed to more than one epitope, each antibody had a unique epitope map profile. Our results suggest that a limited number of amino acid substitutions could produce a modified FVIII protein capable of eluding immunodominant inhibitors. This approach could eventually find clinical application as a novel strategy to achieve hemostasis in patients with an established FVIII inhibitor.

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

2021 ◽  
Author(s):  
Yuto Nakajima ◽  
Hiroaki Minami ◽  
Keiji Nogami
Keyword(s):  
Surface Plasmon Resonance ◽  
Factor Viii ◽  
Heavy Chain ◽  
Synthetic Peptides ◽  
C2 Domain ◽  
Wild Type ◽  
Cross Link ◽  
Domain Specific ◽  
Cofactor Activity ◽  
Acidic Region

AbstractFactor 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.

scholarly journals Some factor VIII inhibitor antibodies recognize a common epitope corresponding to C2 domain amino acids 2248 through 2312, which overlap a phospholipid-binding site

Blood ◽  
1995 ◽  
Vol 86 (5) ◽  
pp. 1811-1819 ◽  
Author(s):  
D Scandella ◽  
GE Gilbert ◽  
M Shima ◽  
H Nakai ◽  
C Eagleson ◽  
...  
Keyword(s):  
Amino Acids ◽  
Factor Viii ◽  
Binding Site ◽  
Light Chain ◽  
Intrinsic Factor ◽  
Factor Viii Inhibitor ◽  
C2 Domain ◽  
Amino Acid Residues ◽  
Fviii Inhibitor ◽  
Human Factor Viii

The finding that human factor VIII (fVIII) inhibitor antibodies with C2 domain epitopes interfere with the binding of fVIII to phosphatidylserine (PS) suggested that this is the mechanism by which they inactivate fVIII. We constructed a recombinant C2 domain polypeptide and demonstrated that it bound to all six human inhibitors with fVIII light chain specificity. Thus, some antibodies within the polyclonal anti-light chain population require only amino acids within C2 for binding. Recombinant C2 also partially or completely neutralized the inhibitor titer of these plasmas, demonstrating that anti-C2 antibodies inhibit fVIII activity. Immunoblotting of a series of C2 deletion polypeptides, expressed in Escherichia coli, with inhibitor plasmas showed that the epitopes for human inhibitors consist of a common core of amino acid residues 2248 through 2312 with differing extensions for individual inhibitors. The epitope of inhibitory monoclonal antibody (MoAb) ESH8 was localized to residues 2248 through 2285. Three human antibodies and anti-C2 MoAb NMC-VIII/5 bound to a synthetic peptide consisting of amino acids 2303 through 2332, a PS- binding site, but MoAb ESH8 did not. These antibodies also inhibited the binding of fVIII to synthetic phospholipid membranes of PS and phosphatidylcholine, confirming that the blocked epitopes contribute to membrane binding as well as binding to PS. In contrast, MoAb ESH8 did not inhibit binding. As the maximal function of activated fVIII in the intrinsic factor Xase complex requires its binding to a phospholipid membrane, we propose that fVIII inhibition by anti-C2 antibodies is related to the overlap of their epitopes with the PS-binding site. MoAb ESH8 did not inhibit fVIII binding to PS-containing membranes, suggesting the existence of a second mechanism of fVIII inhibition by anti-C2 antibodies.

Thrombin-Catalyzed Cleavages of Factor VIII at Arg372 and Arg1689 Are Facilitated by the Acidic Residues Glu720-Asp725.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2686-2686
Author(s):  
Jennifer Newell ◽  
Qian Zhou ◽  
Philip J. Fay
Keyword(s):  
Factor Viii ◽  
Specific Activity ◽  
Factor Xa ◽  
Factor X ◽  
Wild Type ◽  
Factor Viiia ◽  
N Terminus ◽  
Acidic Residues ◽  
Type Factor ◽  
A2 Domain

Abstract Factor VIIIa acts as an essential cofactor for the serine protease factor IXa, together forming the Xase complex which catalyzes the conversion of factor X to factor Xa. The procofactor, factor VIII circulates as a heterodimeric protein comprised of a heavy chain (A1–A2-B domains) and a light chain (A3-C1-C2 domains) and is activated by proteolytic cleavage by thrombin at Arg372 (A1–A2 junction), Arg740 (A2-B junction), and Arg1689 (near the N-terminus of A3). The regions adjacent to the A1, A2, and A3 domains contain high concentrations of acidic residues and are designated a1 (residues 337–372), a2 (residues 711–740), and a3 (residues 1649–1689). In addition, the N-terminus of the A2 domain (residues 373–395) is rich in acidic residues, and results from a previous study revealed that this region contributes to the rate of thrombin-catalyzed cleavage at Arg740 (Nogami et. al., J. Biol. Chem. 280:18476, 2005). In this study we reveal a role for the acidic region following the A2 domain (a2, residues 717–725) in thrombin-catalyzed cleavage at both Arg372 and Arg1689. The factor VIII mutations Asp717Ala, Glu720Ala, Asp721Ala, Glu724Ala, Asp725Ala, and the double mutations of Glu720Ala/Asp721Ala and Glu724Ala/Asp725Ala were constructed, expressed, and purified from stably-transfected BHK cells as B-domainless protein. Specific activity values for the variants, relative to the wild type value were reduced to 70% for Asp717Ala; ∼50% for Glu720Ala, Asp721Ala, Glu724Ala, and Asp725Ala; and ∼30% for Glu720Ala/Asp721Ala and Glu724Ala/Asp725Ala. SDS-PAGE and western blotting of reactions containing the factor VIII variants and thrombin showed reductions in the rates of thrombin cleavage at both Arg372 and Arg1689 as compared to wild-type factor VIII. The cleavage rates for the single mutations comprising acidic residues 720–724 of factor VIII were reduced from ∼3-5-fold at Arg372, whereas this rate for the Asp717Ala mutant was similar to the wild-type value. The double mutations of Glu720Ala/Asp721Ala and Glu724Ala/Asp725Ala showed rate reductions of ∼7- and ∼27-fold, respectively at Arg372. While the rate for thrombin-catalyzed cleavage at Arg1689 in the Glu720Ala variant was similar to wild-type, rates for cleavage at this site were reduced ∼30-fold compared to wild-type factor VIII for the Asp721Ala, Glu724Ala, Asp725Ala, and Glu720Ala/Asp721Ala mutants, and ∼50-fold for the Glu724Ala/Asp725Ala variant. Furthermore, the generation of factor VIIIa activity following reaction with thrombin as assayed by factor Xa generation showed that all the mutants possessed peak activity values that were ∼2-3-fold reduced compared to wild type factor VIIIa. Moreover, in all the mutants the characteristic peak of activation was replaced with a slower forming, broad plateau of activity, with the double mutants showing the broadest activation profiles. These results suggest that residues Glu720, Asp721, Glu724, and Asp725 following the A2 domain modulate thrombin interactions with factor VIII facilitating cleavage at Arg372 and Arg1689 during procofactor activation.

Membrane-Interactive Amino Acids in the Factor VIII C1 Domain Are Cooperative with C2 Domain Epitopes for Membrane Binding and Cofactor Function.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 848-848
Author(s):  
Junhong Lu ◽  
Steven W. Pipe ◽  
Hongzhi Miao ◽  
Marc Jacquemin ◽  
Gary E. Gilbert
Keyword(s):  
Amino Acids ◽  
Factor Viii ◽  
Membrane Binding ◽  
Binding Motif ◽  
C2 Domain ◽  
Factor X ◽  
Apparent Affinity ◽  
Factor Ixa ◽  
C1 Domain ◽  
Membrane Interactive

Abstract Abstract 848 Background: Factor VIII functions as a cofactor in blood coagulation. When released from a non-covalent complex with von Willebrand factor (vWf), activated factor VIII assembles with factor IXa on phosphatidylserine (PS)-containing membranes to form the factor Xase complex. Binding to PS-containing membranes amplifies the activation of factor X by several orders of magnitude. Factor VIII is composed of three A domains, one B domain and two C domains (C1 and C2). The role of C2 domain, including the orientation with respect to membrane surface, vWf-binding motif, and protein-protein contact sites among Xase complex, are relatively well-documented. Recently, the position of the C domains in the factor VIII crystal structure suggested a possible role for the C1 domain in membrane binding. We recently confirmed the participation of K2092 and F2093 of the factor VIII C1 domain in membrane binding (Meems et al. Blood 2009 First edition Aug 18). This work explores the participation of additional C1 domain amino acids and the way the corresponding motif(s) cooperate with motifs of the C2 domain for membrane binding. Methods: Four factor VIII C1 domain mutants encompassing the lower surface of the C1 domain (Arg2090/GLy2091, Lys 2092/Phe2093, Gln2042/Tyr2043, and Arg2159) had individual or paired amino acids mutated to alanine. Mutants were produced in COS-1 cells and purified by immunoaffinity chromatography. The specific activities of these mutants were assessed in a commercial PTT assay as well as phospholipid-limiting and phospholipid-saturating factor Xase assay. Their affinities to factor IXa and factor X were measured by titration experiments using different concentrations of factor IXa and factor X, respectively. Binding to plasma vWf was evaluated in a competition, solution phase enzyme-linked immunosorbent assay (ELISA). The cooperative role of C1 and C2 domains in membrane-binding for cofactor activity was carried out using C1 mutants and antibodies against established membrane-interactive C2 domain motifs, ESH4 and BO2C11. Results: In a competition ELISA for vWf, the affinity of Arg2159 was reduced more than 50-fold, while the other mutants were normal. All mutants had reduced specific activity (range 24-61% of wild type) in a commercial PTT assay containing excess phospholipid. All mutants had decreased apparent affinity for vesicles with limiting (4%) PS by 33, 5, 20, and 18-fold for Arg2090/GLy2091, Gln2042/Tyr2043, Arg2159, and Lys 2092/Phe20933, respectively. However, addition of excess vesicles led to near normal activity for Arg2159. Mutants Arg2090/GLy2091 and Gln2042/Tyr2043 both had 4-fold decreased apparent affinity for factor X and 77% and 84% reduction in Vmax even when phospholipid and factor X were in excess. Mutant Lys 2092/Phe2093 had normal apparent affinity for factor IXa and factor X but > 91% reduction in Vmax. These results indicate that the C1 domain affects interaction with factor X and the Vmax of the factor Xase complex aside from the effect on membrane affinity. To further explore the role of membrane-binding motif in the Xase complex, the activities of mutants were tested with the C2 domain membrane-interactive epitopes blocked by mAb's BO2C11 or ESH4. For WT factor VIII, ESH4 and B02C11 decreased apparent affinity for vesicles of 15% PS by 6-fold and 5-fold, and decreased the Vmax by 0 and 89%, respectively. BO2C11 completely inhibited the activity of Arg2090/GLy2091, Lys 2092/Phe2093, and Arg2159 while ESH4 decreased apparent affinity 2-7-fold for the three mutants. ESH4 decreased the Vmax by 2-5-fold for the mutants. Thus, the intact membrane-binding motif in C1 can independently support Xase activity although the C1 motifs and both C2 membrane-interactive epitopes are required for full activity. Conclusion: Amino acids Arg2090/GLy2091, Lys2092/Phe2093 , Gln2042/Tyr2043, and Arg2159 of the factor VIII C1 domain participate in membrane binding. Our data suggest that engagement of the C1 domain through these residues, together with the ESH4 and the BO2C11 epitopes of the C2 domain, cooperatively influence alignment or an allosteric effect that alters activity for the assembled factor Xase complex. Disclosures: Pipe: Baxter: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees; Wyeth: Speakers Bureau; Inspiration Biopharmaceuticals: Research Funding; CSL Behring: Honoraria.

Contributions of Asn2198, Met2199, and Phe2200 in the factor VIII C2 domain to cofactor activity, phospholipid-binding, and von Willebrand factor-binding

2003 ◽  
Vol 89 (05) ◽  
pp. 795-802 ◽  
Author(s):  
Deborah Lewis ◽  
Mary Pound ◽  
Thomas Ortel
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Hydrophobic Surface ◽  
C2 Domain ◽  
Mutant Proteins ◽  
Phospholipid Binding ◽  
Cofactor Activity ◽  
The Individual ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryThe crystal structure of the factor VIII C2 domain consists of a β-sandwich core from which β-hairpins and loops extend to form a hydrophobic surface. The hydrophobic surface includes M2199 and F2200 at the tip of the 1st β-hairpin. To determine the individual contributions of residues N2198, M2199, and F2200 to phospholipid and von Willebrand factor (vWF) binding properties of factor VIII, we prepared mutant proteins with single alanine substitutions. We found that single mutations at N2198 and M2199 had relatively little impact on cofactor activity, or phospholipid and vWF binding. However the F2200A mutant had slightly lower cofactor activity at subsaturating phospholipid concentrations. Competitive ELISAs suggested that F2200 plays a more important role in both phospholipid-binding and vWF-binding than N2198 and M2199. All mutant proteins were still recognized by a monoclonal antibody and two factor VIII inhibitors that neutralized cofactor activity and blocked factor VIII binding to phospholipids.Presented in part at the XVIII Congress of the International Society on Thrombosis and Haemostasis, Paris, France, 6-12 July 2001, and the 43rd Annual Meeting of the American Society of Hematology, Orlando, Florida, 7-11 December 2001

The Specific Contribution of Amino Acids 334 and 335 from Factor Va Heavy Chain to the Catalytic Efficiency of Prothrombinase.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1027-1027
Author(s):  
Melissa A. Blum ◽  
Tivadar Orban ◽  
Daniel O. Beck ◽  
Michael Kalafatis
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Heavy Chain ◽  
Factor Xa ◽  
Catalytic Efficiency ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Factor Va ◽  
Cofactor Activity ◽  
Type Factor

Abstract The prothrombinase complex, composed of the enzyme factor Xa, the cofactor factor Va, and the substrate prothrombin associated on a cell surface in the presence of divalent metal ions, catalyzes the activation of prothrombin to thrombin 300,000-fold more effectively than the enzyme, factor Xa, alone. We have demonstrated that amino acids E323, Y324 and E330, V331 are binding sites for factor Xa on the factor Va heavy chain and are required for coordinating the spatial arrangement of enzyme and substrate directing prothrombin cleavage at two spatially distinct sites. We have also demonstrated that amino acid region 332–336 contains residues that are involved in cofactor function. Peptide studies have identified amino acid residues 334DY335 as major participants in factor Va cofactor activity. We have employed site-directed mutagenesis to study the effect of these amino acids on the catalytic efficiency of prothrombinase. Recombinant factor V molecules with the mutations D334K and Y335F, designated factor VKF, and D334A and Y335A, designated factor VAA were produced, transiently transfected, expressed in COS7L cells, and purified. Kinetic studies demonstrate that while factor VaKF has a KD for factor Xa similar to the KD observed for wild type factor Va, the kcat of prothrombinase assembled with factor VaKF has approximately a 1.5-fold decreased value compared to kcat of prothrombinase assembled with the wild type cofactor molecule. On the contrary, prothrombinase assembled with factor VaAA was found to have a nearly 10-fold decrease kcat, compared to prothrombinase assembled with wild type factor Va. This data suggest that not all amino acid substitutions are well tolerated at positions 334–335. Analysis of the sequence 323–340 using the recently published completed model of coagulation factor Va (pdb entry 1Y61) revealed that amino acids 334–335 are located at the end of a beta-sheet. To ascertain the importance of these mutants and their contribution to cofactor activity we have combined the mutations of amino acids 334–335 with mutations at amino acids 323–324 (E323F, Y324F) and 330–331 (E330M, V331I). We thus created quadruple mutants resulting in recombinant factor VFF/KF, factor VFF/AA, factor VMI/KF and factor VMI/AA. These molecules were transiently expressed in COS-7L cells and studied for their ability to be incorporated into prothrombinase. Free energies associated with the catalytic efficiencies of prothrombinase assembled with each mutant were also calculated (ΔΔGint). The ΔΔGint of interaction for the double mutants, factor VaFF/KF and factor VaMI/KF, had positive values indicating that the side chains of amino acids 330EV331, 323EY324 and 334DY335 located in and around the factor Xa binding site interact in a synergistic manner resulting in the destabilization of the transition state complex and a decelerated rate of catalysis. Conversely, combining the factor Xa binding site mutants with recombinant factor VaAA result in ΔΔGint values of approximately zero. In conclusion, the data demonstrate that replacement of amino acids 334–335 by two hydrophilic residues results in decreased cofactor function. In contrast, replacement of these amino acids by two small hydrophobic residues do not appear to be well tolerated by the cofactor resulting in severely impaired cofactor activity. Altogether, these data demonstrate the importance of amino acid residues D334 and Y335 for the rearrangement of enzyme and substrate required for efficient catalysis.

Factor VIII C1 Domain Residues 2092–2093 Participate in Membrane Binding

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 587-587
Author(s):  
Henriet Meems ◽  
Alexander B Meijer ◽  
Dave Cullinan ◽  
Koen Mertens ◽  
Gary E. Gilbert
Keyword(s):  
Factor Viii ◽  
Membrane Binding ◽  
Phospholipid Membranes ◽  
Ionophore A23187 ◽  
C2 Domain ◽  
Microtiter Plate Assay ◽  
Surface Loop ◽  
Lower Affinity ◽  
C1 Domain

Abstract Background: Activated factor VIII (FVIIIa) assembles with factor IXa (FIXa) on the membranes of activated platelets and on synthetic phosphatidylserine(PS)-containing membranes. This membrane-bound complex catalyses the conversion of the zymogen, factor X, to factor Xa. Established membrane-binding amino acids of FVIII are in the C2 domain. However, the C2 domain alone binds with a lower affinity to membranes than intact FVIII or the FVIII light chain, suggesting a role of the A3 and/or C1 domains in membrane binding. Moreover, a possible role for the C1 domain in platelet binding has been recently reported. The position of the C domains in the FVIII crystal structure suggests a possible role for residues from surface loop K2092-S2094 of the C1 domain in membrane binding. The present study addresses the role of this loop in membrane binding. Methods: The role of the C1 surface loop K2092-S2094 was assessed by competition studies using KM33. This is a scFv fragment cloned from the antibody repertoire of a hemophilia A patient, with an epitope that comprises residues 2092–2094. In addition, FVIII mutants incorporating yellow fluorescent protein in place of the B domain and with K2092/F2093 changed to alanine (FVIIIYFP and FVIIIYFP K2092A/F2093A) were expressed and purified. Binding of recombinant FVIII labelled with fluorescein-maleimide (FVIIIfl), FVIIIYFP and FVIIIYFP K2092A/F2093A to phospholipid membranes (4% or 15% PS/20% PE/PC as balance) supported by glass microspheres and purified platelets was measured by flow cytometry. Lower affinity, non-equilibrium binding of sonicated vesicles to immobilized factor VIII was measured in a microtiter plate assay. The cofactor function of FVIII was measured in a factor Xase assay with limiting phospholipid. Results: KM33 inhibited &gt;95% of FVIII binding to phospholipid membranes containing 15% PS, indicating that the C1 domain epitope is important for membrane binding. The affinity of FVIIIYFP K2092A/F2093A for the same membranes was reduced 3-fold compared with FVIIIYFP (Kd’s of 91 ± 6 vs. 31 ± 2 nM). KM33 decreased the overall activity for the factor Xase complex by 95% on vesicles with 15% PS and &gt;99% on vesicles with 4% PS. The implied membrane affinity for FVIIIYFP K2092A/F2093A in the factor Xase complex was decreased 3-fold for vesicles with 15% PS but the Vmax was equivalent to FVIIIYFP. The implied affinity of FVIIIYFP K2092A/F2093A was reduced approximately 40-fold for vesicles with 4% PS confirming the importance of the C1 domain epitope for full factor VIII function. In the microtiter assay, mAb BO2C11, against the C2 domain, blocked approx. 80% of binding to vesicles containing 15% PS, KM33 blocked 5% of binding and both antibodies together blocked ~95% of binding. Binding to 4% PS vesicles was inhibited 70% by KM33 alone and B02C11 alone blocked all binding. Thus, the two membrane-binding motifs are required for detectable binding to membranes with 4% PS but can independently support some binding to membranes with 15% PS. KM33 inhibited approx 90% of FVIII binding to platelets. The binding of FVIIIYFP K2092A/F2093A to platelets stimulated with calcium ionophore A23187 was reduced 50% compared to FVIIIYFP; however binding to platelets stimulated with thrombin receptor activating peptide (TRAP) was comparable to FVIIIYFP. The cofactor function of FVIIIYFP K2092A/F2093A was reduced approximately 80% on platelets stimulated with either TRAP or A23187. Conclusion: The present study demonstrates that the FVIII C1 domain contributes to membrane binding and residues K2092 and/or F2093 participate in this interaction. The relative importance of these residues for membrane binding is dependent on the amount of PS present in synthetic membranes. On platelets K2092 and/or F2093 are necessary for full cofactor function of FVIII.

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