• Electron microscopy and hydrogen-deuterium exchange establish the C1 domain as the major binding site for the VWF D′D3 domain on FVIII.

  • Additional sites implicated in the FVIII-VWF interaction are located within the a3 acidic peptide and the A3 and C2 domains of FVIII.

Association with the D′D3 domain of von Willebrand factor (VWF) stabilizes factor VIII (FVIII) in the circulation and maintains it at a level sufficient to prevent spontaneous bleeding. We used negative-stain electron microscopy (EM) to visualize complexes of FVIII with dimeric and monomeric forms of the D′D3 domain. The EM averages show that FVIII interacts with the D′D3 domain primarily through its C1 domain, with the C2 domain providing a secondary attachment site. Hydrogen-deuterium exchange mass spectrometry corroborated the importance of the C1 domain in D′D3 binding and implicates additional surface regions on FVIII in the interaction. Together, our results establish that the C1 domain is the major binding site on FVIII for VWF, reiterate the importance of the a3 acidic peptide in VWF binding, and suggest that the A3 and C2 domains play ancillary roles in this interaction.

The high-affinity, noncovalent association of factor VIII (FVIII) with von Willebrand factor (VWF) in the circulation protects FVIII from otherwise rapid clearance.1,2  Quantitative insufficiency of VWF, characteristic of type 1 and type 3 von Willebrand disease (VWD), as well as impaired FVIII binding by VWF, observed in type 2N VWD, result in a secondary deficiency of FVIII and a bleeding diathesis.1,3,4  During biosynthesis, VWF monomers (∼260 kDa) form disulfide-bonded dimers through their C-terminal cystine knot (CK) domains. Upon formation of homotypic disulfide bonds between juxtaposed N-terminal D′D3 domains, these dimers form mature VWF multimers that range in size from 500 kDa to >20 MDa. The structural determinants for FVIII binding have been localized to the D′D3 domain by proteolytic fragmentation and by analysis of naturally occurring type 2N VWD mutations.5,6  Consistent with these findings, recombinant D′D3 domains have been shown to be sufficient to elevate levels of endogenous FVIII in VWF-deficient mice.7  The reciprocal binding site for VWF on FVIII was initially localized broadly to its light chain.8  Subsequent studies demonstrated that the a3 acidic peptide region (E1649-R1689)9  and a restricted fragment thereof (K1673-R1689),10  as well as sulfation on residue Y1680 within this region, are important for VWF binding.11  In addition, mutation analysis and antibody competition studies have implicated the C1 and C2 domains in VWF binding.9,12-16  To better understand the interaction between FVIII and VWF, we visualized FVIII-D′D3 complexes by electron microscopy (EM) and used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify structural perturbations in FVIII upon D′D3 binding.

B domain–deleted FVIII and FVIII-Fc fusion protein (rFVIIIFc), and monomeric and dimeric forms of VWF D′D3 domain, were expressed in HEK 293 cells and purified as described in supplemental Methods, found on the Blood Web site. Experimental methods for EM and HDX-MS are also described in the supplemental Methods.

Electron microscopy of FVIII in complex with D′D3 domains

Negatively stained FVIII in complex with dimeric D′D3 showed substantial structural variability (Figure 1A), but many particles appeared to consist of 2 peripheral densities connected by a smaller central density (Figure 1A, circled). Class averages (Figure 1B and supplemental Figure 1) revealed the peripheral densities as FVIII, and thus the central, elongated density as the dimeric D′D3 (Figure 1B, arrows). The angle between the 2 FVIII molecules varies dramatically (supplemental Video 1), and the dimeric D′D3 can also be positioned differently with respect to the 2 FVIII molecules. In panels 1 to 4 of Figure 1B, the long axis of the dimeric D′D3 is aligned with the 2 C domains of the FVIII on top (indicated by horizontal arrows), but only in some cases is it also aligned with the C domains of the second FVIII (panels 1 and 2 vs panels 3 and 4). In the remaining panels of Figure 1B, the long axis of the dimeric D′D3 is positioned either diagonally (panels 5-8) or perpendicular (panels 9-12) to the 2 C domains of the FVIII on top (indicated by the direction of the arrows).

Figure 1

EM analysis of FVIII in complex with dimeric and monomeric D′D3. (A) Representative raw image of dimeric FVIII-D′D3 in negative stain. Some of the complexes are circled. The scale bar represents 50 nm. (B) Selected class averages of dimeric FVIII-D′D3 complex illustrating that the angle between the 2 FVIII molecules varies greatly. White arrows indicate the density representing the dimeric D′D3 domain. The side length of individual panels is 75.1 nm. (C) Representative raw image of monomeric FVIII-D′D3 in negative stain. The scale bar represents 50 nm. (D) Selected class averages of monomeric FVIII-D′D3 complex illustrating that structural heterogeneity persists in this complex. White arrows indicate the density representing the monomeric D′D3 domain. The side length of individual panels is 33.4 nm.

Figure 1

EM analysis of FVIII in complex with dimeric and monomeric D′D3. (A) Representative raw image of dimeric FVIII-D′D3 in negative stain. Some of the complexes are circled. The scale bar represents 50 nm. (B) Selected class averages of dimeric FVIII-D′D3 complex illustrating that the angle between the 2 FVIII molecules varies greatly. White arrows indicate the density representing the dimeric D′D3 domain. The side length of individual panels is 75.1 nm. (C) Representative raw image of monomeric FVIII-D′D3 in negative stain. The scale bar represents 50 nm. (D) Selected class averages of monomeric FVIII-D′D3 complex illustrating that structural heterogeneity persists in this complex. White arrows indicate the density representing the monomeric D′D3 domain. The side length of individual panels is 33.4 nm.

Close modal

To distinguish whether the observed heterogeneity resulted from structural variability of the 2 D′D3 domains in the dimeric construct or from different ways in which FVIII interacts with a D′D3 domain, we analyzed FVIII in complex with monomeric D′D3. The particles appeared more homogeneous (Figure 1C), but class averages revealed that structural variability persisted (Figure 1D and supplemental Figure 2). In particular, the density representing monomeric D′D3 (indicated by arrows) varies in shape and location relative to FVIII (supplemental Video 2). The difference in shape may be caused by different orientations or by partial loss of density upon averaging. Importantly, although in some averages D′D3 appears to interact with both C domains of FVIII (Figure 1D, panels 1-7), in other averages it appears to interact with only 1 C domain (Figure 1D, panels 8-12). In the conventional orientation used to display FVIII, the molecule has the shape of a reversed “R,” with the C1 domain at the bottom left of the molecule and the C2 domain at the bottom right (supplemental Figure 3). Our averages thus suggested that the C1 domain is the primary binding site for D′D3 and that the C2 domain is a secondary binding site. To confirm this conclusion and to further characterize the interaction between D′D3 and FVIII, we performed HDX-MS.

Hydrogen-deuterium exchange mass spectrometry

Because of its ready availability and an affinity for VWF that is comparable with that of FVIII, pharmaceutical-grade rFVIIIFc was used for HDX-MS.17  HDX-MS was performed with rFVIIIFc alone and in complex with monomeric D′D3. Stringent criteria were used to determine whether differential deuterium exchange was statistically significant: for a given peptide, the mass difference must exceed ±0.5 Da for at least 1 time point, and the sum of mass differences over all time points must exceed ±1.1 Da.18  Based on these criteria, 10 distinct regions of differential deuterium exchange were initially identified within the FVIII component (Figure 2A). Region 1 was identified in the heavy chain of FVIII, within the A1 domain and encompassing residue C248, which forms a disulfide bond with C329. Although differential deuterium uptake at this site cannot be ruled out, given the distance from known VWF interaction sites, the observed mass difference (supplemental Figure 4B, peptide 74) could be more readily rationalized by intramolecular hydrogen transfer upon generation of protein CysS• radicals during disulfide bond photolysis, as has been observed for immunoglobulins.20  The remaining 9 regions were located within the light chain: 2 in A3, 5 in C1, and 2 in C2. Each cluster of overlapping peptides was further analyzed to establish logical consistency of their respective overlap patterns and to narrow down the sequences to which perturbations in deuterium exchange could clearly be attributed (supplemental Figures 5 through 13). Three regions (4, 7, and 10) were eliminated from further consideration on the basis of irreconcilable peptide overlap patterns. Of the remaining 6 regions, 2 could be unambiguously assigned in the A3 domain: one in the a3 acidic peptide (Region 2, V1670-D1678; Figure 2A and supplemental Figure 5) and one in the A3 domain proper (Region 3; V1857-N1861 and E1875: Figure 2A-B and supplemental Figure 6). Three regions were unambiguously assigned in the C1 domain: Region 5 (W2062-S2069; Figure 2A-C and supplemental Figure 8), Region 6 (T2086-S2095; Figure 2A-C and supplemental Figure 9), and Region 8 (S2157-L2166; Figure 2A-C and supplemental Figure 11). One region was assigned in the C2 domain and corresponded to the C2-A1 domain interface (Region 9; Q2235-T2244; Figure 2A-C and supplemental Figure 12).

Figure 2

Mapping of the D′D3 domain-binding site on FVIII by HDX-MS. (A) HDX difference plots illustrating localized differential deuterium uptake by the FVIII component of rFVIIIFc in the presence and absence of the monomeric D′D3 construct for the FVIII heavy chain (upper panel) and light chain (lower panel). The x-axis indicates the 463 peptides derived from the B domain–deleted FVIII sequence ordered from the N to C terminus based on their respective midpoints, and the y-axis indicates the calculated mass difference caused by differential deuterium uptake for peptides derived from FVIII in the D′D3-bound and D′D3-free states. FVIII domain boundaries are indicated with arrows. Individually colored curves correspond to the average mass difference values calculated for data acquired at the time points indicated. Vertical gray bars represent the sum of differences across all time points for a given peptide. Positive values for x indicate peptides for which deuterium uptake is reduced in the presence of the D′D3 domain. Peptides and overlapping clusters of peptides exhibiting statistically significant perturbations in deuterium uptake according to criteria defined by Houde et al18  are numbered and indicated with colored horizontal bars or dots, with green indicating those for which noncontradictory peptide overlap patterns could be derived and red indicating those for which this was not possible within the given constraints for statistical significance. (B) Surface representations of the FVIII structure (PDB ID: 2R7E19 ) with individual domains colored as indicated. Numbered areas correspond to sites of differential deuterium uptake identified in (A). (C) Ribbon representations of the FVIII C1 and C2 domains with areas corresponding to differential deuterium uptake numbered as in (B).

Figure 2

Mapping of the D′D3 domain-binding site on FVIII by HDX-MS. (A) HDX difference plots illustrating localized differential deuterium uptake by the FVIII component of rFVIIIFc in the presence and absence of the monomeric D′D3 construct for the FVIII heavy chain (upper panel) and light chain (lower panel). The x-axis indicates the 463 peptides derived from the B domain–deleted FVIII sequence ordered from the N to C terminus based on their respective midpoints, and the y-axis indicates the calculated mass difference caused by differential deuterium uptake for peptides derived from FVIII in the D′D3-bound and D′D3-free states. FVIII domain boundaries are indicated with arrows. Individually colored curves correspond to the average mass difference values calculated for data acquired at the time points indicated. Vertical gray bars represent the sum of differences across all time points for a given peptide. Positive values for x indicate peptides for which deuterium uptake is reduced in the presence of the D′D3 domain. Peptides and overlapping clusters of peptides exhibiting statistically significant perturbations in deuterium uptake according to criteria defined by Houde et al18  are numbered and indicated with colored horizontal bars or dots, with green indicating those for which noncontradictory peptide overlap patterns could be derived and red indicating those for which this was not possible within the given constraints for statistical significance. (B) Surface representations of the FVIII structure (PDB ID: 2R7E19 ) with individual domains colored as indicated. Numbered areas correspond to sites of differential deuterium uptake identified in (A). (C) Ribbon representations of the FVIII C1 and C2 domains with areas corresponding to differential deuterium uptake numbered as in (B).

Close modal

Despite the structural variability observed by EM among complexes of FVIII with both monomeric and dimeric D′D3, involvement of the C1 domain was a common feature of all complexes. Furthermore, HDX-MS analysis revealed multiple sites of structural perturbation within the C1 domain upon binding of monomeric D′D3. In general, FVIII missense mutations known to impair VWF binding, and thereby giving rise to mild or moderate hemophilia A, cluster within the C1 domain, and approximately half of these are located within or near regions identified by HDX-MS (supplemental Figure 14). Less pronounced perturbations were observed in the a3 acidic peptide, consistent with its established role in VWF binding, as well as in the A3 domain proper and C2 domain, suggesting a secondary role for these sites in the FVIII-D′D3 interaction.

The online version of this article contains a data supplement.

There is an Inside Blood Commentary on this article in this issue.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

The authors thank Drs Damian Houde, Steven Berkowitz, and Andrew Weiskopf for helpful discussions. T.W. is an investigator with the Howard Hughes Medical Institute.

This research was funded in part by Biogen Idec.

Contribution: P.-L.C., G.M.B.-A., R.T.P., J.D.K., and T.W. designed the experiments; P.-L.C., G.M.B.-A., E.S.C., M.G.C., J.D.K., and T.W. performed research and analyzed data; and P.-L.C., G.M.B.-A., J.D.K., and T.W. wrote the manuscript.

Conflict-of-interest disclosure: G.M.B.-A., E.S.C., R.T.P., and J.D.K. are employees of and hold equity interest in Biogen Idec.

Correspondence: Thomas Walz, Department of Cell Biology, Harvard Medical School, 240 Longwood Ave, Boston, MA 02115; e-mail: [email protected]; John D. Kulman, Biogen, 250 Binney St, Cambridge, MA 02142; e-mail: [email protected].

1
Weiss
 
HJ
Sussman
 
II
Hoyer
 
LW
Stabilization of factor VIII in plasma by the von Willebrand factor. Studies on posttransfusion and dissociated factor VIII and in patients with von Willebrand’s disease.
J Clin Invest
1977
, vol. 
60
 
2
(pg. 
390
-
404
)
2
Vlot
 
AJ
Koppelman
 
SJ
Meijers
 
JC
, et al. 
Kinetics of factor VIII-von Willebrand factor association.
Blood
1996
, vol. 
87
 
5
(pg. 
1809
-
1816
)
3
Sadler
 
JE
For the Subcommittee on von Willebrand Factor of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis
A revised classification of von Willebrand disease.
Thromb Haemost
1994
, vol. 
71
 
4
(pg. 
520
-
525
)
4
Nishino
 
M
Girma
 
JP
Rothschild
 
C
Fressinaud
 
E
Meyer
 
D
New variant of von Willebrand disease with defective binding to factor VIII.
Blood
1989
, vol. 
74
 
5
(pg. 
1591
-
1599
)
5
Hampshire
 
DJ
Goodeve
 
AC
The international society on thrombosis and haematosis von Willebrand disease database: an update.
Semin Thromb Hemost
2011
, vol. 
37
 
5
(pg. 
470
-
479
)
6
Foster
 
PA
Fulcher
 
CA
Marti
 
T
Titani
 
K
Zimmerman
 
TS
A major factor VIII binding domain resides within the amino-terminal 272 amino acid residues of von Willebrand factor.
J Biol Chem
1987
, vol. 
262
 
18
(pg. 
8443
-
8446
)
7
Yee
 
A
Gildersleeve
 
RD
Gu
 
S
, et al. 
A von Willebrand factor fragment containing the D’D3 domains is sufficient to stabilize coagulation factor VIII in mice.
Blood
2014
, vol. 
124
 
3
(pg. 
445
-
452
)
8
Hamer
 
RJ
Koedam
 
JA
Beeser-Visser
 
NH
Bertina
 
RM
Van Mourik
 
JA
Sixma
 
JJ
Factor VIII binds to von Willebrand factor via its Mr-80,000 light chain.
Eur J Biochem
1987
, vol. 
166
 
1
(pg. 
37
-
43
)
9
Saenko
 
EL
Scandella
 
D
The acidic region of the factor VIII light chain and the C2 domain together form the high affinity binding site for von Willebrand factor.
J Biol Chem
1997
, vol. 
272
 
29
(pg. 
18007
-
18014
)
10
Leyte
 
A
Verbeet
 
MP
Brodniewicz-Proba
 
T
Van Mourik
 
JA
Mertens
 
K
The interaction between human blood-coagulation factor VIII and von Willebrand factor. Characterization of a high-affinity binding site on factor VIII.
Biochem J
1989
, vol. 
257
 
3
(pg. 
679
-
683
)
11
Leyte
 
A
van Schijndel
 
HB
Niehrs
 
C
, et al. 
Sulfation of Tyr1680 of human blood coagulation factor VIII is essential for the interaction of factor VIII with von Willebrand factor.
J Biol Chem
1991
, vol. 
266
 
2
(pg. 
740
-
746
)
12
Saenko
 
EL
Shima
 
M
Rajalakshmi
 
KJ
Scandella
 
D
A role for the C2 domain of factor VIII in binding to von Willebrand factor.
J Biol Chem
1994
, vol. 
269
 
15
(pg. 
11601
-
11605
)
13
Shima
 
M
Scandella
 
D
Yoshioka
 
A
, et al. 
A factor VIII neutralizing monoclonal antibody and a human inhibitor alloantibody recognizing epitopes in the C2 domain inhibit factor VIII binding to von Willebrand factor and to phosphatidylserine.
Thromb Haemost
1993
, vol. 
69
 
3
(pg. 
240
-
246
)
14
Bloem
 
E
van den Biggelaar
 
M
Wroblewska
 
A
, et al. 
Factor VIII C1 domain spikes 2092-2093 and 2158-2159 comprise regions that modulate cofactor function and cellular uptake.
J Biol Chem
2013
, vol. 
288
 
41
(pg. 
29670
-
29679
)
15
Jacquemin
 
M
Lavend’homme
 
R
Benhida
 
A
, et al. 
A novel cause of mild/moderate hemophilia A: mutations scattered in the factor VIII C1 domain reduce factor VIII binding to von Willebrand factor.
Blood
2000
, vol. 
96
 
3
(pg. 
958
-
965
)
16
Jacquemin
 
M
Benhida
 
A
Peerlinck
 
K
, et al. 
A human antibody directed to the factor VIII C1 domain inhibits factor VIII cofactor activity and binding to von Willebrand factor.
Blood
2000
, vol. 
95
 
1
(pg. 
156
-
163
)
17
Peters
 
RT
Toby
 
G
Lu
 
Q
, et al. 
Biochemical and functional characterization of a recombinant monomeric factor VIII-Fc fusion protein.
J Thromb Haemost
2013
, vol. 
11
 
1
(pg. 
132
-
141
)
18
Houde
 
D
Berkowitz
 
SA
Engen
 
JR
The utility of hydrogen/deuterium exchange mass spectrometry in biopharmaceutical comparability studies.
J Pharm Sci
2011
, vol. 
100
 
6
(pg. 
2071
-
2086
)
19
Shen
 
BW
Spiegel
 
PC
Chang
 
CH
, et al. 
The tertiary structure and domain organization of coagulation factor VIII.
Blood
2008
, vol. 
111
 
3
(pg. 
1240
-
1247
)
20
Zhou
 
S
Mozziconacci
 
O
Kerwin
 
BA
Schöneich
 
C
The photolysis of disulfide bonds in IgG1 and IgG2 leads to selective intramolecular hydrogen transfer reactions of cysteine Thiyl radicals, probed by covalent H/D exchange and RPLC-MS/MS analysis.
Pharm Res
2013
, vol. 
30
 
5
(pg. 
1291
-
1299
)

Author notes

P.-L.C. and G.M.B.-A. contributed equally to this study.

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