The Acidic Region of the Factor VIII Light Chain and the C2 Domain Together Form the High Affinity Binding Site for von Willebrand Factor

Saenko, Evgueni L.; Scandella, Dorothea

doi:10.1074/jbc.272.29.18007

Cited by 182 publications

(199 citation statements)

References 44 publications

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“…Alternatively, it is possible that the amino-terminal acidic region of the A3 domain of FVIII may be closely associated with the C2 domain in the threedimensional conformation. vWF-binding sites are known to be located in both the C2 and A3 domains, and FVIII and vWF association is enhanced by the presence of both regions (34). The present anti-C2 antibody, therefore, might have inhibited thrombin cleavage in the amino-terminal A3 region.…”

Section: Competitive Inhibition Of Thrombin Proteolysis Of Lch Bymentioning

confidence: 99%

Factor VIII C2 Domain Contains the Thrombin-binding Site Responsible for Thrombin-catalyzed Cleavage at Arg1689

Nogami¹,

Shima²,

Hosokawa³

et al. 2000

Journal of Biological Chemistry

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Thrombin-catalyzed factor VIII activation is an essential positive feedback mechanism regulating intrinsic blood coagulation. A factor VIII human antibody, A-FF, with C2 epitope, exclusively inhibited factor VIII activation and cleavage at Arg 1689 by thrombin. The results suggested that A-FF prevented the interaction of thrombin with factor VIII and that the C2 domain was involved in the interaction with thrombin. We performed direct binding assays using anhydro-thrombin, a catalytically inactive derivative of thrombin in which the active-site serine is converted to dehydroalanine. Blood clotting factor VIII (FVIII) 1 is a crucial glycoprotein that accelerates the intrinsic coagulation cascade by acting as a cofactor of factor IXa in the tenase complex (1), and a deficiency of FVIII results in the common hereditary bleeding disorder, hemophilia A. FVIII circulates in plasma as a noncovalent complex with von Willebrand factor (vWF) that stabilizes the synthesis and cofactor activity of FVIII (2-4). Mature FVIII is synthesized as a single chain polypeptide of approximately 300 kDa consisting of 2,332 amino acid residues with a mosaic domain structure arranged in the order of A1-A2-B-A3-C1-C2 (5-7) and secreted into plasma as variable series of heterodimers consisting of the heavy chain (HCh) and the light chain (LCh). The HCh is composed of the A1, A2, and parts of the B domain and exhibits size heterogeneity (90 -210 kDa) due to proteolysis within the B domain. The 90-kDa HCh reflects the absence of the B domain, whereas the 210-kDa species includes the intact B domain. The LCh (80 kDa) corresponds to the A3, C1, and C2 domains. The two chains are noncovalently linked by metal ions between the A1 and A3 domains (6, 7).FVIII is transformed into an active form by limited proteolysis by two essential serine proteases, thrombin and FXa (8, 9). This procoagulant activity is more pronounced after thrombin activation than after FXa activation (10). Cleavage at Arg 740 removes the size-heterogeneous B domain from the HCh, producing a 90-kDa HCh fragment, and further cleavage of the 90-kDa HCh fragment (A1-A2) at Arg 372 between the A1 and A2 domains produces 54-(A1) and 44-kDa (A2) species. Cleavage of the 80-kDa LCh fragment (A3-C1-C2) at Arg 1689 between the B and A3 domains removes 40 amino-terminal acidic peptides from the A3 domain and produces a 72-kDa fragment. A unique cleavage by FXa at Arg 1721 produces a 67-kDa LCh fragment. The active form of FVIII is a metal-linked heterotrimer consisting of the A1/A2/A3-C1-C2 domains, lacking the middle B domain (6). Cleavage at Arg 740 between the A2 and B domains does not contribute to the generation of the FVIII activity. In contrast, cleavages at Arg 372 and Arg 1689 are essential for optimal FVIII activity (11-13). Activation of human FVIII at its physiological concentration and at pH 7.4 is followed by a reduction in activity (14). This loss of activity is not caused by further proteolysis but by dissociation of the A2 domain from the active form of FVIII heterotrime...

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Section: Competitive Inhibition Of Thrombin Proteolysis Of Lch Bymentioning

confidence: 99%

Factor VIII C2 Domain Contains the Thrombin-binding Site Responsible for Thrombin-catalyzed Cleavage at Arg1689

Nogami¹,

Shima²,

Hosokawa³

et al. 2000

Journal of Biological Chemistry

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“…The binding of FVIII to vWF is important for the survival of FVIII as the plasma concentration of FVIII is considerably reduced in the absence of vWF (6)(7)(8). FVIII binds to vWF involving A3 and C2 domains of the light chain (9)(10)(11)(12)(13). It has been shown that the A2, A3, and C2 domains of FVIII are involved in the interaction with LRP (14,15).…”

Section: Introductionmentioning

confidence: 99%

Phosphatidylinositol Containing Lipidic Particles Reduces Immunogenicity and Catabolism of Factor VIII in Hemophilia A Mice

Peng

Straubinger

Balu‐Iyer

2010

AAPS J

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Abstract. Factor VIII (FVIII) is an important cofactor in blood coagulation cascade. It is a multidomain protein that consists of six domains, NH 2 -A1-A2-B-A3-C1-C2-COOH. The deficiency or dysfunction of FVIII causes hemophilia A, a life-threatening bleeding disorder. Replacement therapy using recombinant FVIII (rFVIII) is the first line of therapy, but a major clinical complication is the development of inhibitory antibodies that abrogate the pharmacological activity of the administered protein. FVIII binds to anionic phospholipids (PL), such as phosphatidylinositol (PI), via lipid binding region within the C2 domain of FVIII. This lipid binding site not only consists of immunodominant epitopes but is also involved in von Willebrand factor binding that protects FVIII from degradation in vivo. Thus, we hypothesize that FVIII-PL complex will influence immunogenicity and catabolism of FVIII. The biophysical studies showed that PI binding did not alter conformation of the protein but improved intrinsic stability as measured by thermal denaturation studies. ELISA studies confirmed the involvement of the C2 domain in binding to PI containing lipid particles. PI binding prolonged the in vivo circulation time and reduced catabolism of FVIII in hemophilia A mice. FVIII-PI complex reduced inhibitor development in hemophilia A mice following intravenous and subcutaneous administration. The data suggest that PI binding reduces catabolism and immunogenicity of FVIII and has potential to be a useful therapeutic approach for hemophilia A.

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“…[1][2][3] Upon proteolytic activation, FVIIIa is released from VWF as a heterotrimer composed of the A1 and A2 domains plus the FVIIIa light chain, A3-C1-C2. Activated platelet membranes expose negatively charged phosphatidylserine (PS), which increases from 2% to 10% or more of the surface phospholipids upon activation.…”

Section: Introductionmentioning

confidence: 99%

The factor VIII C1 domain contributes to platelet binding

Hsu

Pratt

Thompson

2008

Blood

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Activated factor VIII (FVIIIa) forms a procoagulant complex with factor IXa on negatively charged membranes, including activated platelet surfaces. Membrane attachment involves the FVIII C2 domain; involvement of the adjacent C1 domain has not been established. Binding of recombinant FVIII C1C2 and C2 proteins to platelets was detected by flow cytometry using (1) anti-C2 monoclonal antibody ESH8 followed by a phycoerythrinlabeled secondary antibody; (2) biotinylated C1C2 detected by phycoerythrinlabeled streptavidin, and (3) C1C2 and C2 site-specifically labeled with fluorescein. Highest binding and lowest background were obtained using fluoresceinconjugated proteins. More than 90% of activated platelets bound C1C2, compared with approximately 50% for equimolar C2. Estimates using fluorescent microbeads indicated approximately 7000 C1C2-binding sites per platelet, approximately 1400 for C2, and approximately 3000 for fluorescein-labeled FVIIIa. Unlike C2 or FVIII(a), C1C2 bound to approximately 700 sites/platelet before activation. C1C2 binding to activated platelets appeared independent of von Willebrand factor and was competed effectively by FVIII(a), but only partially by excess C2. Fluorescein-labeled FVIIIa was competed much more effectively by C1C2 than C2 for binding to activated platelets. Two monoclonal antibodies that inhibit C2 binding to membranes competed platelet binding of C2 more effectively than C1C2. Thus, the C1 domain of FVIII contributes to platelet-binding affinity. IntroductionFactor VIII (FVIII) circulates in plasma in a noncovalent complex with von Willebrand factor (VWF); this interaction is mediated by the FVIII C2 domain and an acidic sequence prior to the A3 domain. [1][2][3] Upon proteolytic activation, FVIIIa is released from VWF as a heterotrimer composed of the A1 and A2 domains plus the FVIIIa light chain, A3-C1-C2. Activated platelet membranes expose negatively charged phosphatidylserine (PS), which increases from 2% to 10% or more of the surface phospholipids upon activation. 4,5 FVIIIa forms a complex with FIXa and calcium on negatively charged phospholipid membranes, enhancing FIXa catalysis 100 000-to 200 000-fold. [6][7][8] Although FVIII can bind to FIXa on phospholipids, 9 or directly to activated platelets, 10 FVIIIa is required for procoagulant activity. 9 A hydrophobic surface on the FVIII(a) C2 domain 11 becomes buried in the phospholipid membrane upon binding, 12,13 and basic C2 residues make favorable charge-charge interactions with negatively charged PS head groups. Although FVIIIa and the light chain bind to PS-containing vesicles and activated platelets with similar affinities, the affinity of the recombinant C2 domain is 5-to 100-fold lower, 10,14,15 suggesting possible roles for the C1 and/or A3 domains.To address the potential role of the C1 domain in FVIII(a) attachment to platelets, a recombinant human FVIII C1C2 protein (residues 2020-2332) was produced in Escherichia coli, refolded, and characterized. The binding of C1C2 to platelet surfaces both before ...

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The Acidic Region of the Factor VIII Light Chain and the C2 Domain Together Form the High Affinity Binding Site for von Willebrand Factor

Cited by 182 publications

References 44 publications

Factor VIII C2 Domain Contains the Thrombin-binding Site Responsible for Thrombin-catalyzed Cleavage at Arg1689

Factor VIII C2 Domain Contains the Thrombin-binding Site Responsible for Thrombin-catalyzed Cleavage at Arg1689

Phosphatidylinositol Containing Lipidic Particles Reduces Immunogenicity and Catabolism of Factor VIII in Hemophilia A Mice

The factor VIII C1 domain contributes to platelet binding

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