Supplementary Figures

a b c
400 150 150
** n.s. n.s.

ERp57 activity in %
ERp5 activity in %
PDI activity in % 300 *
100 100
200
50 50
100

0 0 0
Bepristat 1a
Bepristat 2a

Bepristat 1a
Bepristat 2a

Bepristat 1a
Bepristat 2a
Vehicle

Vehicle

Vehicle
d e
150
n.s.
ERp72 activity in %

100
RFU min-1

50

0
Bepristat 1a
Bepristat 2a
Vehicle

Supplementary Figure 1. Selectivity of the bepristat-mediated augmentation of di-

eosin-GSSG reductase activity. (a) The effect of 30 M bepristats on di-eosin-GSSG
cleavage was evaluated for full-length PDI (n=3) and augmentation was quantified. In
contrast, bepristats did not augment activity of (b) ERp5 (n=3), (c) ERp57 (n=3), (d)
ERp72 (n=3) in the same assay. (e) When no enzyme was added, bepristats failed to
augment the cleavage of di-eosin-GSSG by DTT (n=4). Lack of activity of the bepristats
when used with other thiol isomerases demonstrates the selectively of their augmenting
effect on PDI catalytic activity. Data are mean + SEM from three independent
experiments. One-way ANOVA with Dunnetts post test: *p < 0.05; **p < 0.01; n.s., non
significant.
 Veh NEM Q3R Bp1a Bp2a PAC Bacitr Veh NEM Q3R Bp1a Bp2a PAC Bacitr

Veh NEM Q3R Bp1a Bp2a PAC Bacitr

Veh NEM Q3R Bp1a Bp2a PAC Bacitr

Veh NEM Q3R Bp1a Bp2a PAC Bacitr

Supplementary Figure 2. Original images of representative immunoblot of MPB-binding

(n=3-6) of PDI (a), ERp5 (b), ERp57 (c), TRX (d) and BSA (e) as shown in figure 2C.
Results using vehicle (Veh), N-ethylamine (NEM), quercetin-3-rutinoside (Q3R),
bepristat 1a (Bp1a), bepristat 2a (Bp2a), PACMA-31 (PAC), and bacitracin (Bacitr) are
shown.

2
 a b c
1.5 1.5 1.5
n.s. n.s.

Fold difference ERp57

Fold difference ERp5
Fold difference PDI
n.s.
1.0 1.0 1.0

0.5 0.5 0.5

0.0 0.0 0.0
NEM

NEM

NEM
Q-3-R

Q-3-R

Q-3-R
PACMA-31

PACMA-31

PACMA-31
Bepristat 1a
Bepristat 2a

Bepristat 1a
Bepristat 2a

Bepristat 1a
Bepristat 2a
Bacitracin

Bacitracin

Bacitracin
Vehicle

Vehicle

Vehicle
d e
1.5 n.s. 1.5 n.s.
Fold difference BSA
Fold difference TRX

1.0 1.0


0.5 0.5

0.0 0.0
NEM

NEM
Q-3-R

Q-3-R
PACMA-31

PACMA-31
Bepristat 1a
Bepristat 2a

Bepristat 1a
Bepristat 2a
Bacitracin

Bacitracin
Vehicle

Vehicle

Supplementary Figure 3. Bacitracin and PACMA-31 non-selectively bind to free

sulfhydryls on thiol isomerases. (a) PDI, (b) ERp5, (c) ERp57, (d) thioredoxin, or (e)
BSA were incubated with vehicle, 300 M NEM, 150 M quercetin-3-rutinoside, 150 M
bepristat 1a, 150 M bepristat 2a, 150 M PACMA-31 or 150 M bacitracin prior to
exposure to MPB. Samples were then analyzed by SDS-PAGE and MPB was detected
by Cy5-labeled streptavidin. Fluorescence corresponding to MPB binding was quantified
using ImageQuant 400 analysis software. Values represent mean fluorescence + SEM
(n = 3-6). One-way ANOVA with Bonferronis post test: p < 0.001, NEM vs bepristat
1a, NEM vs bepristat 2a; p < 0.05, PACMA-31 vs bepristat 1a, PACMA-31 vs bepristat
2a; p < 0.05, bacitracin vs bepristat 1a, bacitracin vs bepristat 2a; n.s.,non significant.

3
 40 n.s.

30

MFI
20
10
0
Vehicle

Bep2a
Bep1b
Supplementary Figure 4. Bepristats do not affect P-selectin expression. P-selectin
expression in response to 2 M of the PAR-1 activating peptide, SFLLRN, was
monitored by flow cytometry following exposure of platelets to vehicle control (black) or
45 M bepristat 1b (red) or bepristat 2a (blue). Values represent mean + SEM from three
independent experients. One-way ANOVA was performed, showing no significant
difference between the three conditions (p = 0.84).

4
 a" b" c"

0.15 0.15 0.15

OD 650 nm

OD 650 nm

OD 650 nm
0.10 0.10 0.10

0.05 0.05 0.05

0.00 0.00 0.00

0 10 20 30 0 10 20 30 0 10 20 30
Time (sec) Time (sec) Time (sec)

Vehicle Vehicle Vehicle

0.03 M 0.06 M 3 M
3 M 6 M 300 M
Diluted to 0.03 M Diluted to 0.06 M Diluted to 3 M
DTT DTT DTT

Supplementary Figure 5. Reversibility of bepristats. Reversibility of inhibition of

reductase activity of recombinant PDI by either (a) bepristat 1a, (b) bepristat 2a, or (c)
PACMA-31 was evaluated by 100-fold dilution of a mixture of 3 M bepristat 1a, 6 M
bepristat 2a or 300 M PACMA-31 and monitoring in the insulin turbidimetric assay.
Values represent mean data + SEM.

5
 kDa$

Vehicle
0.6
70# PACMA-
DTT
55#

OD 650 nm
0.4
35#
25# 0.2

0.0
0 10

Supplementary Figure 6. PDI fragments. SDS-PAGE analysis of PDI and PDI

fragments used in these studies.

ab
0.10 0.3

0.08

OD 650 nm
0.2
OD 650 nm

0.06

0.04
0.1
0.02

0.00 0.0
0 10 20 30 40 0 10
Time (min) T

6
 8000
Vehicle
b'x
6000
b'x I272A
RFU
4000

2000

0
400 500 600
Wavelength in nm

Supplementary Figure 7. ANS fluorescence is blocked in the I272A mutant. ANS

was incubated with either vehicle alone, wild-type b'x domain, or a b'x domain mutant in
which isoleucine 272 was mutated to alanine. The I272A mutation inhibited ANS
fluorescence.

7
 b"
Proteinase*K*
Proteinase)K)

a 1*m* 3*m* 5*m* 10*m* 20*m* kDa*

1)min) 2*m* 3)min)
2)min) 5)min) 10)min) 20)min)
Vehicle* 40*
Vehicle*
Vehicle)
Bepristat*1a* 38*
Bepristat*2a*
40*
Bepristat*1a*
Bepristat)
1A) 38*
40*
Bepristat*2a*
Bepristat)
2A) 38*

PDI * bxa * abb *

d" 10
Bepristat 1b

10
Bepristat 2b

55
P (r)

P (r)
5 5
35
25
0 0
0 25 50 75 100 125 0 25 50 75 100 125
Wavelength*(nm)*
Wavelength*(nm)*

bb"
Rg Rg
Proteinase*K*
Proteinase)K)

a" Vehicle* Vehicle*

Vehicle)
1*m*
1)min) 2)min) 3*m*
2*m* 3)min) f" 5*m*
5)min)
0.4
10*m* 20*m*
10)min) 20)min)
kDa*
40*
Vehicle Bepristat*1a* Bepristat 1a Bepristat 2b
Vehicle 38*
1.0 C53-C56 Bepristat*2a*
1.0 C53-C56 1.0 C53-C56 Bepristat 1a
C397-C400 C397-C400 Bepristat*1a*
Bepristat) C397-C400 Reduced Fraction 0.3 Bepristat 2b 40*
0.8 0.8 1A)0.8 38*
Reduced Fraction

Reduced Fraction

40*
0.6 0.6 Bepristat*2a*
Bepristat
0.6
)
0.2
2A) 38*

* * *
0.4 0.4 0.4

PDI bxa abb 0.1

c"
0.2

0.0
0.2

0.0
d"55
0.2

0.0
Bepristat 1b Bepristat 2b

10 -5 10 -4 10 -3 10 -2 10 -1 10 0 10 -5 10 -4 10 -3 10 -2 10 -1 10 0 10 10 -5 10 -4 10 -3 10 -2 10 -1 10 010 0.0
[GSH]2/[GSSG], M [GSH]2/[GSSG], M 35 [GSH]2/[GSSG], M
a a'
Fluorescence*

25
P (r)

P (r)

5 5
15

0 0
0 25 50 75 100 125 0 25 50 75 100 125
Wavelength*(nm)*
Wavelength*(nm)*
Rg Rg

cb"
Proteinase*K*
Proteinase)K)

a"
e" Vehicle* Vehicle*
Vehicle)
1*m* 2)min)
1)min) 3*m*
2*m* 3)min) f" 5*m*
5)min)
0.4
10*m* 20*m*
10)min) 20)min)
40*
kDa*

Vehicle Bepristat 1a Bepristat 2b

Bepristat*1a* Vehicle 38*
1.0 C53-C56 1.0
Bepristat*2a* C53-C56 1.0 C53-C56 Bepristat 1a
Reduced Fraction

C397-C400 C397-C400 Bepristat*1a*

Bepristat) C397-C400 0.3 Bepristat 40*
2b
0.8 0.8 1A) 0.8 38*
Reduced Fraction

Reduced Fraction

Reduced Fraction

40*
0.6 0.6 Bepristat*2a*
Bepristat) 0.6 0.2
2A) 38*
0.4 0.4 0.4

PDI * bxa * abb * 0.1

c" 0.2

0.0
0.2

0.0 d"35
55 0.2

0.0
Bepristat 1b Bepristat 2b
0.0
10 -5 10 -4 10 -3 10 -2 10 -1 10 0 10 -5 10 -4 10 -3 10 -2 10 -1 10 0 10 10 -5 10 -4 10 -3 10 -2 10 -1 10 10 0
2 2 a a'
[GSH] /[GSSG], M [GSH] /[GSSG], M [GSH]2/[GSSG], M
Fluorescence*

25
P (r)

P (r)

15 5 5

0 0
0 25 50 75 100 125 0 25 50 75 100 125
Wavelength*(nm)*
Wavelength*(nm)*
Rg Rg
Supplementary Figure 8. Original images of f"
e" Vehicle following proteinase
Bepristat 1a K digestion (n=3
Bepristat
representative silver stained gels of abbx
for2b all conditions)
0.4
inVehicle
the presence of vehicle (a),
1.0 C53-C56 bepristat 1a
1.0
(b) or bepristat 2a (c).
C53-C56 1.0 C53-C56 Bepristat 1a
Reduced Fraction

C397-C400 C397-C400 C397-C400 Bepristat 2b 0.3

0.8 0.8 0.8
Reduced Fraction

Reduced Fraction

Reduced Fraction

0.6 0.6 0.6 0.2

0.4 0.4 0.4

0.1
0.2 0.2 0.2

0.0 0.0 0.0 0.0

10 -5 10 -4 10 -3

[GSH]2/[GSSG], M
10 -2 10 -1

10 0 10 -5 10 -4 10 -3

[GSH]2/[GSSG], M
10 -2 10 -1 10 0 10 -5 10 -4 10 -3

[GSH]2/[GSSG], M
10 -2 10 -1 10 0
a a' 8
 a b

Supplementary Figure 9. Differential cysteine alkylation and mass spectrometry of

PDI. (a) Reduced disulfide bond cysteines in PDI were alkylated with 12C-IPA and the
oxidized cysteine thiols with 13C-IPA following reduction with DTT. One or both of the
reduced disulfide bond cysteines can be labeled with either alkylator. The ratio of 12C-
IPA to 13C-IPA alkylation represents the proportion of the disulfide bonds in the
population that are in the reduced state. (b) Tandem mass spectrum of the WT PDI
CGHCKALAPEY peptide containing the Cys53 and Cys56 catalytic dithiol/disulfide. The
top panel is an example of the 12C-IPA-alkylated peptide and the bottom panel the 13C-
IPA-alkylated peptide. Both Cys53 and Cys56 are alkylated in these examples. The
accurate mass spectrum of the peptide is shown in the inset (observed [M+2H]2+ =
729.3219 m/z and expected [M+2H]2+ = 729.3207 m/z; observed [M+2H]3+ = 490.5636
m/z and expected [M+2H]2+ = 490.5630 m/z).

9
 PDI Active-site disulfide Eo', mV
No Addition a (Cys53-Cys56) -206 37

a' (Cys397-Cys400) -206 36

+ bepristat 1a a (Cys53-Cys56) -209 60

a' (Cys397-Cys400) -210 72

+ bepristat 2b a (Cys53-Cys56) -201 48

a' (Cys397-Cys400) -200 58

SupplementaryTable 1. Redox potential of PDI in the presence of bepristats.The

calculated equilibrium constants based on results shown in Fig. 5 were used to
determine the standard redox potentials from equation 2 in Methods.

10
Supplementary Methods

P-Selectin expression
P-selectin expression, a marker of platelet activation, was monitored by flow cytometry.
Washed human platelets (2.5 x 108 platelets / mL), prepared as described above, were
incubated with 45 M of bepristat 1b, 45 M of bepristat 2a, or vehicle control. After
exposure to 2 M of SFLLRN, 5 L of PE-conjugated CD62P (BD Biosciences) was
added to 25 L of the platelet sample. Fluorescence and forward scatter measurements
were performed using a Gallios Flow Cytometer (Beckman Coulter). The data was
analyzed using Kaluza software and Graphpad Prism 5.0.

12