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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
2
a b c
1.5 1.5 1.5
n.s. n.s.
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
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"
OD 650 nm
OD 650 nm
0.10 0.10 0.10
5
kDa$
Vehicle
0.6
70# PACMA-
DTT
55#
OD 650 nm
0.4
35#
25# 0.2
0.0
0 10
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
7
b"
Proteinase*K*
Proteinase)K)
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)
Reduced Fraction
40*
0.6 0.6 Bepristat*2a*
Bepristat
0.6
)
0.2
2A) 38*
* * *
0.4 0.4 0.4
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*
Reduced Fraction
Reduced Fraction
40*
0.6 0.6 Bepristat*2a*
Bepristat) 0.6 0.2
2A) 38*
0.4 0.4 0.4
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
Reduced Fraction
Reduced Fraction
0.1
0.2 0.2 0.2
[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
9
PDI Active-site disulfide Eo', mV
No Addition a (Cys53-Cys56) -206 37
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