The factor VIII C1 domain contributes to platelet binding

Abstract

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 ...

Structure of the factor VIII C2 domain in a ternary complex with 2 inhibitor antibodies reveals classical and nonclassical epitopes

“…The positioning of the C1 domain relative to the C2 domain supports previous data indicating that the C1 domain contributes to membrane association and the C2 domain is not essential for function. [29][30][31] Lastly, the x-ray crystal structure of the C2 domain/3E6/G99 complex is remarkably similar to the previously modeled ternary complex from low-resolution SAXS data ( Figure 1B). 41 These structural findings suggest that using rapid, low-resolution techniques such as SAXS may be effective tools for characterizing other antibody epitopes for fVIII inhibitor antibodies as well as other antigen/antibody interactions.…”

“…22 In addition to interactions with phospholipid (PL) and VWF, there are previous reports that suggest the C2 domain contributes to the binding of thrombin and factor Xa to full-length fVIII, potentially acting as a docking module that may stabilize and promote the proteolytic activity of factor Xa/thrombin cleavages elsewhere in fVIII during activation. [26][27][28] Recent studies also suggest that the C1 domain contributes to binding both VWF and PS, 29,30 and deletion studies of the C2 domain indicate that it is not essential for PL membrane binding, factor IXa binding, or clotting activity. 31 The C2 domain of fVIII is a major antigenic determinant for recognition by inhibitor antibodies.…”

Platelet binding sites for factor VIII in relation to fibrin and phosphatidylserine

“…Additional work will also be required to indicate whether the C1 domain [15][16][17] or A3 domain 41 also contribute to binding the fibrin-dependent platelet sites.…”

“…14 The C1 and C2 domains mediate membrane binding. [15][16][17][18][19] The fVIII C domains share similar sequence and structure with the C domains of factor V [20][21][22] and lactadherin, 23 a milk fat globule membrane protein. Like fVIII, both factor V and lactadherin bind to PS-containing membranes.…”

High-affinity, noninhibitory pathogenic C1 domain antibodies are present in patients with hemophilia A and inhibitors

“…Several studies have described the involvement of the C1 domain in binding to VWF and phospholipid membranes. 20,21 Recently negativestain electron microscopy and hydrogen-deuterium exchange mass spectrometry (HDX MS) studies have revealed that VWF primarily interacts with the C1 domain of fVIII. 22,23 VWF binding protects fVIII from endocytosis 24 and the human-derived anti-C1 domain antibody KM33 inhibits fVIII uptake by human monocyte-derived dendritic cells (MDDCs).…”