Research ArticleBIOCHEMISTRY

Autoregulation of von Willebrand factor function by a disulfide bond switch

See allHide authors and affiliations

Science Advances  28 Feb 2018:
Vol. 4, no. 2, eaaq1477
DOI: 10.1126/sciadv.aaq1477
  • Fig. 1 The reduced A2 domain binds to the A1 domain and masks interaction with platelet GPIb.

    (A) The A1 domain binds the GPIb receptor on platelets, the A2 domain Tyr1605-Met1606 peptide bond is cleaved by ADAMTS13, and the A3 domain binds collagen exposed during vascular damage. The oxidized A2 crystal structure has a Protein Data Bank (PDB) identifier 3GXB. (B) Recombinant A2 (residues M1473 to L1675) produced in Escherichia coli and resolved on SDS–polyacrylamide gel electrophoresis (PAGE). The positions of molecular weight markers are indicated on the left. (C) Differential cysteine alkylation and mass spectrometry method of measuring the redox state of the A2 domain cysteines. Unpaired cysteine thiols in the A2 domain are alkylated with 12C-IPA, and the disulfide bonded cysteine thiols with 13C-IPA after reduction with DTT. Alternatively, unpaired cysteine thiols in the A2 domain were alkylated with 13C-IPA, the protein was digested with Glu-C, and peptides were analyzed by tandem mass spectrometry. A search for peptides labeled with 13C-IPA at Cys1669 and an unknown adduct at Cys1670 was undertaken. The only adduct detected was S-glutathionylation. (D) The recombinant A2 domain exists in oxidized (87%), glutathionylated (11%), and reduced (2%) forms. Treatment of the domain with DTT results in 96% reduced and 4% oxidized protein. (E) Reduced but not oxidized A2 domains bind to the A1 domain immobilized on plastic. A2 binding was corrected for nonspecific binding to bovine serum albumin (BSA)–blocked wells. (F) Biomembrane force probe (BFP) protein functionalization. The probe bead (bottom left) was coated with the A1 domain and streptavidin (SA) for attachment of the bead to a biotinylated red blood cell (RBC). A1 is the focus for interaction with the GPIb on an aspired platelet (bottom right). The soluble A2 domain competes for this interaction. (G) Force-time traces from two representative test cycles. A target was driven to approach a probe, contacted, and retracted. In a no-bond event (black), the cycle ended after the probe-target separation. In a bond event (blue), the target was held (marked by *) at a preset force until dissociation. Lifetime is measured from the point when the clamped force (30 pN) was reached to the point when the bond dissociated, signified by a force drop to zero. (H) The reduced but not oxidized A2 domain competes for binding of platelet GPIb to the A1 domain (left). There were very few interactions of platelets with BSA-coated beads in the absence or presence of A2 (right). The adhesion frequencies (mean ± SEM) were measured on BFP from three independent experiments (n = 3 donors for human platelets) in the absence or presence of the 5 μM A2 domain. For each experiment, three (BSA-coated beads) or five (A1-coated beads) random bead-platelet pairs with 50 touches were analyzed and averaged. *P < 0.05, unpaired, two-tailed Student’s t test. n.s., not significant.

  • Fig. 2 Oxidized VWF is more effective than reduced VWF at engaging platelet GPIb under fluid shear conditions.

    (A) HEK293 cells were transfected with wild-type (wt), C1669A, or C1669A,C1670A mutant VWF plasmids. Conditioned medium was collected after 3 days, and equal weights of protein were resolved by agarose gel electrophoresis and blotted with anti-VWF polyclonal antibodies. (B) Representative images of platelet adhesion on channels coated with wt or double Cys mutant (A2ΔCC) VWF at fluid shear rates of 900, 1800, and 3000 s−1. (C) Surface coverage of whole-blood platelets on channels coated with wt or A2ΔCC VWF at fluid shear rates of 900, 1800, and 3000 s−1. Bars and errors are mean ± SEM of three technical replicates of platelets from four healthy donors (n = 12 measurements). **P < 0.01 and ***P < 0.001. (D) Surface coverage of washed platelets on channels coated with wt or A2ΔCC VWF at a fluid shear rate of 1000 s−1. Bars and errors are mean ± SEM of three technical replicates of platelets from three healthy donors (n = 9 measurements). **P < 0.01.

  • Fig. 3 Interaction of reduced and glutathionylated VWF with GPIb is characterized by low collision frequency and short bond lifetimes.

    (A) BFP bright-field scheme (top) and protein functionalization (bottom). A micropipette-aspirated RBC with a probe bead attached to the apex (top left) was aligned against a human platelet held by an apposing micropipette (top right). The probe bead was covalently linked to a polyclonal antibody (pAb) for the capture of VWF and SA for the attachment of the bead to a biotinylated RBC (bottom left). VWF is the focus for interaction with the GPIb on an aspirated platelet (bottom right). (B) Adhesion frequencies of five bead-platelet pairs with 50 touches for each pair. Errors are mean ± SEM. (C) Lifetime of VWF-platelet GPIb bonds versus clamp force in BFP. Results represent mean ± SEM of >50 measurements per point. (D) Recombinant wt VWF exists in oxidized (52%), glutathionylated (24%), and reduced (24%) forms. (E) Lifetime distributions of VWF-platelet bonds measured by BFP were compared at three representative forces: 25, 32.5, and 40 pN. (F and G) A two-state single-bond dissociation model was fit to the wt VWF bond lifetime distribution to evaluate model parameters at the indicated force bin. The kinetic parameters are the slow (k1) and fast (k2) off rates of dissociation from the two dissociation states (G) and the fractions (w1, w2; w1+ w2 = 1) of bonds associated with the corresponding short-lived (high off rate) and long-lived (low off rate) states. The error bars represent ±95% confidence interval (CI) of the best-fit value. The fraction of oxidized (52%) and glutathionylated plus reduced (48%) forms of wt VWF corresponding to (E) is shown on the right.

  • Fig. 4 The oxidized and reduced forms of the A2 domain have different dynamics and stresses.

    (A) Overall structure of the VWF A2 domain (from the x-ray structure, PDB identifier 3GXB) used as starting conformation for the MD simulations. The structure is shown in cartoon representation, highlighting the Cys1669-Cys1670 disulfide bond in yellow. (B) Dynamics of the oxidized (green) and reduced (orange) VWF A2 domain projected onto the first two eigenvectors (main modes of motion) obtained from principal components analysis (PCA). Each dot represents a conformation observed in the MD simulations. The black dot corresponds to the starting structure for both simulations. (C) Interpolation of the structures along the first PCA eigenvector (main mode of motion), ranging from the conformations sampled in the oxidized state (green) to the reduced state (orange) (going from the extreme left to the extreme right). Ct, C-terminal. (D) Per-residue difference of the root mean square fluctuations (RMSFs) between the reduced (red) and oxidized (oxi) forms of VWF A2. Statistically significant differences are highlighted with the * symbol. The secondary structure of the protein is presented on top. Nt, N-terminal. (E) Force distribution analysis (FDA). (Left) Residue pairs (i,j) with the time-averaged pairwise force of the reduced state minus that of the oxidized state larger than a cutoff value of 90 pN, |〈Fij(red)〉 − 〈Fij(oxi)〉| > 90 pN. Here, | | indicates absolute values. For the dependence on the cutoff value, see fig. S2. Secondary-structure elements of A2 are shown on both axes. To guide the eye, the regions corresponding to the β strands B4, B5, and B6 are shown in gray and the region corresponding to the Cys1669-Cys1670 bond is shown in orange. (Right) The three groups of residue pairs enclosed by circles, showing interactions between the β strands and their surrounding helices, loops, or the C terminus, are explicitly shown as lines connecting points in the A2 structure. (F) The three most favorable structural models of the VWF A1A2 complex predicted by molecular docking and MD simulations (13). A1 (green) and A2 (blue) domains are shown in cartoon representation. The Cys1669-Cys1670 bond (magenta and yellow) is shown in ball-stick representation.

  • Fig. 5 The VWF A2 domain exists predominantly in a reduced dithiol and glutathionylated form in healthy donors.

    (A) Distribution of reduced, glutathionylated, and oxidized VWF A2 in 22 healthy donor plasmas. Mean and range for the two A2 domain cysteine-containing peptides of individual samples are shown on the left, and the mean and SD for the 22 samples are shown on the right. The VWF A2 domain cysteine pair exists as unpaired thiols in ~40%, Cys1669 exists as an unpaired thiol and Cys1670 is glutathionylated in ~36%, and the pair exists in an oxidized disulfide form in ~24%. (B) Posttranslational modifications of the A2 domain cysteines. (C) Distribution of unmodified and oxidized Met1606 in the 22 healthy donor plasmas. The mean and SD for the 22 samples are shown on the right. Only a minor fraction of VWF Met1606 is oxidized in the cohort of healthy donors.

  • Fig. 6 Plasma VWF in ECMO patients is markedly more oxidized than that in heart failure patients not implanted with these devices.

    (A) Results from 9 patients with heart failure not treated with ECMO and (B) results from 11 patients who received ECMO.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/2/eaaq1477/DC1

    Expression and purification of the recombinant A2 domain

    fig. S1. Differential cysteine alkylation and mass spectrometry analysis of the VWF Cys1669-Cys1670 disulfide bond.

    fig. S2. FDA of the VWF A2 domain.

    fig. S3. Concordance between RMSF for the simulated A2 redox states and crystallographic B factors.

    fig. S4. Anticoagulation of blood with calcium chelators (citrate or EDTA) or direct thrombin inhibitors (heparin or hirudin) does not appreciably influence the redox state of the A2 domain cysteines.

    fig. S5. Plasma VWF collected on antibody-coated beads, resolved on reducing SDS-PAGE, and stained with colloidal Coomassie.

  • Supplementary Materials

    This PDF file includes:

    • Expression and purification of the recombinant A2 domain
    • fig. S1. Differential cysteine alkylation and mass spectrometry analysis of the VWF Cys1669-Cys1670 disulfide bond.
    • fig. S2. FDA of the VWF A2 domain.
    • fig. S3. Concordance between RMSF for the simulated A2 redox states and crystallographic B factors.
    • fig. S4. Anticoagulation of blood with calcium chelators (citrate or EDTA) or direct thrombin inhibitors (heparin or hirudin) does not appreciably influence the redox state of the A2 domain cysteines.
    • fig. S5. Plasma VWF collected on antibody-coated beads, resolved on reducing SDS-PAGE, and stained with colloidal Coomassie.

    Download PDF

    Files in this Data Supplement:

Navigate This Article