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Isolation From Commercial Aurintricarboxylic Acid of the Most Effective
Polymeric Inhibitors of von Willebrand Factor Interaction With Platelet
Glycoprotein Ib. Comparison With Other Polyanionic and
Polyaromatic Polymers
By Mark Weinstein, Evan Vosburgh, Martin Phillips, Nancy Turner, Leslie Chute-Rose, and Joel Moake
Solutions of commercial aurintricarboxylic acid (ATA) inhibit
ristocetin- or shear stress-induced, von Willebrand factor
(vWF)-mediated platelet aggregation by interacting with
vWF and blocking its attachment to platelet membrane
glycoprotein Ib. ATA has also been shown to prevent cyclic
platelet clumping in a dog model of coronary artery thrombosis. Because these ATA solutions are actually a heterogeneous mixture of polyanionic, polycarboxylic polyaromatic
polymersof molecular weight (Mr) 200 to greater than 6,000,
we separated the most effective inhibitory components of
commercial ATA using exclusion chromatography. ATA polymers larger than Mr 700 inhibited ristocetin-induced, vWFmediated platelet aggregation more effectively than smaller
ATA polymers, whereas shear-induced, vWF-mediated platelet aggregation was optimally inhibited by ATA polymers of
Mr 2 2,500. Platelet aggregation mediated by vWF was not
inhibited by a nonphenolic, polyanionic polymer (polyglutamic acid) or by a polyphenolic ATA-like polymer (aurin)
devoid of carboxyl groups. Polyanionic, polysulfonated aromatic polymers (polystyrene sulfonate) of Mr 35,17.4,8,and
4.6 x lo3 inhibited ristocetin- and shear-induced, vWFmediated aggregation with less potency on a mass/volume
basis than large polymers of ATA. We conclude that a
polyanionic, polycarboxylated, polyphenolic ATA polymer of
Mr 2,500 is optimally potent as an inhibitor of shear- and
ristocetin-induced, vWF-mediated platelet aggregation and
is likely to be more effective than solutions of commercial
ATA as an anti-arterial thrombotic agent.
8 1991 by TheAmerican Society of Hematology.
PLATELET
to ATA but without carboxyl groups, was also evaluated.
The results of this study provide structure-function information about ATA that will be useful in designing compounds
to inhibit platelet aggregation optimally in vivo in animals
and humans.
aggregation can be initiated by high fluid
shear stress, as seen in stenosed, atherosclerotic blood
vessels. The interaction of platelets with von Willebrand
factor (vWF) is essential for the formation of shear-induced
platelet aggregates,’,’ and is a factor in the evolution of
arterial thrombi involving platelet adherence to vessel wall
components.’ Shear stress-induced platelet aggregation is
dependent on metabolizing platelets with intact membrane
glycoproteins (GP) Ib and IIb-IIIa, large multimers of vWF,
adenosine diphosphate (ADP), and calcium. Platelets bind
to vWF, particularly to the largest multimeric forms of the
oligomeric protein, through the interaction of GPIb and the
GPIIb-IIIa complex with specific domains on vWF. Compounds that inhibit this association have the potential of
being useful anti-arterial thrombotic agents.
Solutions of commercial aurintricarboxylic acid (ATA), a
red dye, inhibit ristocetin- and shear-induced platelet
aggregation in ~ i t r oComponents
.~
of these ATA solutions
bind reversibly to plasma vWF multimers and to endothelial
cell-derived unusually large vWF multimeric forms, but not
to platelets: The components inhibit platelet aggregation
through a mechanism that is independent of ADP and
arachidonic acid. Solutions of commercial ATA also prevent platelet aggregation and thrombosis in an animal
model of coronary arterial thrombosis:
Studies to date have used commercial ATA, which is a
complex mixture of polycarboxylic, polyphenolic polymers
derived from the acid-catalyzed polymerization of salicylic
acid, methylene disalicylic acid, and formaldehyde. With
the goal of identifying the most effective inhibitory components, we have used gel permeation chromatography to
fractionate solutions of commercial ATA. Polymers of
varying size were separated and tested for their capacity to
inhibit ristocetin- and shear-induced, vWF-mediated platelet aggregation. To determine whether the inhibitory properties of ATA are unique, the inhibitory activity of ATA
polymers was compared with that of a polyanion, polyglutamic acid and another aromatic and negatively charged
polymer, polystyrene sulfonate. Aurin, a compound similar
Blood, Vol78, No 9 (November 1). 1991: pp 2291-2298
MATERIALS AND METHODS
Blood was taken from healthy volunteers who had not received
any medication for at least 7 days. All samples were obtained after
receiving informed consent according to the guidelines approved
by the Institutional Review Boards of Boston University School of
Medicine, Baylor College of Medicine, and Rice University. For
studies of shear-induced, vWF-mediated platelet aggregation,
blood was drawn into plastic syringes and anticoagulated with
one-tenth volume of 3.8% sodium citrate. Blood for ristocetinIvWFinduced platelet aggregation studies was anticoagulated with a
one-sixth volume of 1.35% citric acid, 2.5% sodium citrate, and 2%
dextrose (ACD). Platelet-rich plasma (PRP) was made by centrifugation of citrate or ACD-anticoagulated blood at 15% for 10
minutes at 2YC, and was used immediately.
ATA (“85% dye”) and aurin were purchased from Aldrich
(Milwaukee, WI); methylene disalicylic acid from Lancaster Synthesis (Windham, NH);and salicylic acid from Sigma (St Louis, MO).
Polyglutamic acid (Mr 5 to 10 x lo’) was from Bachem (Philadelphia, PA).
From Boston University School of Medicine, Boston MA; and
Baylor College of Medicine, The Methodist Hospital, and Rice
University, Houston, TX.
Submitted Februav 8,1991; accepted June 27, 1991.
Supported in part by National Institutes of Health Grants HL 22355
and HL 35387. M.P. is the recipient of an American HeartAssociation
Clinician-ScientistAward.
Address reprint requests to Mark Weinstein, PhD, Boston University
School of Medicine, S-402,80 E Concord St, Boston, M A 02118.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 I991 by The American Society of Hematology.
0006-4971/91/7809-0007$3.00/0
2291
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2292
Purification and characterization of ATA. For gel filtration
chromatography, 3 g of commercial ATA was mixed with 2 mL 0.01
mol/L Na phosphate, and the pH was corrected to 7.2 with the
dropwise addition of 6 mol/L NaOH. To this mixture was added
1.16 g KSCN and the final volume was adjusted to 6 mL with water
(2 mol/L KSCN). The solution was passed through a 0.2-pm filter
and applied to a 4.5 X 47 cm column of BioRad P-4 gel filtration
media (BioRad, Richmond, CA) in 2 mol/L KSCN/O.Ol mol/L Na
phosphate, pH 7.2. The void volume fractions from three preparations were combined, and the pooled material was separated on a
4.5 X 67 cm column of BioRad P-10,200 to 400 mesh, in 2 mol/L
KSCN/O.Ol mol/L Na phosphate, pH 7.2. ATA polymers in the P-4
and P-10 elution fractions were precipitated by adjusting the pH to
3 to 3.5 with HCI, and concentrated by centrifugation at 6,000s for
15 minutes. The precipitated polymers were washed in 0.15 mol/L
NaCU0.03 mol/L HCI to remove KSCN, and stored at 4°C.
Average Mrs of the ATA polymers were estimated by comparing
their elution volumes with those of polystyrene sulfonate polymers
(PSS) of Mr 1.8,4.6,8.0,and 17.4 X lo) (Polysciences, Warrington,
PA), the peptides GlnTrpGlu and GlyTyr (Bachem), and salicylic
acid (Aldrich). The compounds were separated on a fast protein
liquid chromatography apparatus (FPLC; Pharmacia, Piscataway,
NJ) using a resin of highly cross-linked agarose (Superose 12B)
equilibrated with 2 mol/L KSCN/O.Ol mol/L Na phosphate, pH
7.2. Because PSS is, as ATA, a polyanionic polyaromatic polymer,
it was also tested for its capacity to inhibit ristocetin- and
shear-induced, vWF-mediated platelet aggregation.
ATA polymers of Mr greater than 400 had an Ai: of 80 at pH
7.4.
The electrofocusing of ATA polymers in the P-4 fractions was
performed using a Multiphor I1 electrophoresis chamber (Pharmacia). To make a 22 x 18 cm agarose gel, 0.8 g ISOGEL (FMC,
Rockland, ME) was dissolved in 76 mL of heated H,O. After the
solution was cooled to 60"C, 3.6 mL of Pharmalyte, pH 2.5 to 5, and
0.4 mL of Pharmalyte, pH 4 to 6.5, (Pharmacia) were added. The
gel was cast on gel bond (FMC), with wells located 3 cm from the
cathode. A strip of filter paper soaked in 6.6% Pharmalyte, pH 4 to
6.5, was placed on the surface of one end of the gel, and a strip
soaked in 0.1 mol/L H,SO, was applied at the opposite end. The
respective cathodic and anodic electrodes were placed on top of
these strips. The gels were prefocused for 2.5 hours, beginning at 5
W and reaching a maximum of 15 W (but not exceeding 20 mA).
ATA polymers were prepared for focusing by mixing an aliquot of
0.8 mL of precipitated sample (20 to 25 mg) that had been washed
in 0.01 mol/L HCI and centrifuged at 12,00Qg, with 60 pL of
Pharmalyte, pH 4 to 6.5. The precipitated ATA polymers in each
sample were dissolved by adjusting the pH to 5 to 5.3 with
incremental additions of 10 N NaOH. The samples were focused
for 6.5 hours at a maximum setting of 15 W, 20 mA.
Ristocetin-induced, v WF-mediated platelet aggregation. Studies
of the inhibition of ristocetin-induced platelet aggregation were
performed on a Sienco Dual Sample Aggregation Meter DP-247E
(Sienco, Morrison, CO). To a sample of PRP (160 pL) were added
either 20 pL of veronal-buffered saline (VBS; 0.032 mol/L sodium
barbital, 0.126 mol/L NaCI, pH 7.4) or the test compound dissolved
in VBS, followed by 20 pL of 15 mg/mL ristocetin (BioData,
Hatboro, PA) in VBS. The initial decrease in optical density was
recorded over a period of 4 minutes. Inhibition of ristocetin
cofactor activity (RCof) was calculated by dividing the maximum
rate of decrease in optical density of a mixture containing the test
compound by the maximum rate obtained in the presence of buffer
alone.
Platelet aggregation induced by collagen, ADP, or arachidonic
acid in the presence or absence of PSS was also evaluated in the
WEINSTEIN ET AL
Sienco aggregometer. One hundred sixty microliters of PRP was
mixed for 1 minute either with 20 pL of VBS or 20 FL of VBS
containing 250 pg/mL Mr 17,400 PSS (15 pmol/L final concentration). To this mixture were added either 20 pL of collagen, 1.9
mg/mL; ADP, 20 pmol/L; or arachidonic acid, 5 mg/mL. The
change in optical density was recorded.
Shear-induced, vWF-mediated platelet aggregation. Shear-induced platelet aggregation was performed as previously deusing a Ferranti cone and plate viscometer (Ferranti
Electric, Commack, NY).Samples of PRP (0.6 mL) were added to
the plate in the presence or absence of test compounds that had
been dissolved in 24 pL of phosphate-buffered saline (PBS; 0.011
mol/L KH,PO,, 0.055 mol/L Na,HPO,, 0.15 mol/L NaCI, pH 7.4).
A shear force of 180 dynes/cmzwas applied for 30 seconds. Before
and after shearing, aliquots of PRP were removed for particle
counting (model ZBI and Channelyzer; Coulter Diagnostics, Hialeah, FL).The sample volume for counting was 100 pL, and the
aperture setting was 50 pm. Particles with sizes 520% of the mean
platelet distribution in the unsheared samples were considered as
single platelets. The disappearance of single platelets could be
accounted for by the formation of platelet aggregates. The percentage platelet count remaining in solution after shearing was calculated by dividing the final count of single platelets by the initial
count of single platelets and multiplying by 100. All counts were
performed in duplicate.
A
5
P-4
160
I
1
-
0
ll
Eo
12
120
0
0
C
a
80
g
01
9
40
20
.I
50
40
30
1.
60
70
80
90
100
110
120
Fraction Number
--------_______.
,') L _ - ------______
B
E
I
P-10
1
20
0
OD
N
16
m
g
a
10
9
6
0
40
60
60
70
80
Fraction Number
Fig 1. Gel filtration chromatography of crude ATA in 2 mol/L
KSCN, 0.01 mol/L Na phosphate, pH 7.2. Crude ATA was first
separated on a BioRad P-4 column (A). The void volume (fraction 11
was then gel filtered on a column of BioRad P-10 (B). Columnfractions
under the bars were pooled and numbered as indicated.
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POLYANIONIC POLYAROMATIC POLYMERS INHIBIT vWF
2293
Table 1. Average MIS ( x l f f ) of ATA Fractions
Column
Fraction
3,P4
5.P4
7.P4
Mr
1.1
0.63
0.30
Column
Fraction
Mr
1,PlO
3,PlO
5,PlO
7,PlO
6.4
3.4
2.5
1.9
Estimated from gel permeation chromatography on Superose 128.
using polymers of polystyrene sulfonates, peptides, and salicylic acid
as Mr standards.
RESULTS
Purification and charactekation of ATA. Components
of crude ATA were separated by gel filtration chromatography using the beaded polyacrylamide resins BioRad P-4
and P-10 (Fig 1). The fractions pooled from these columns
were designated according to their elution position and
column type (eg, 1,PlO refers to the first peak eluted from
the P-10 column). Fractions selected for further study were
rechromatographed on the same resins to narrow the range
of included polymer sizes.
The average Mr of ATA polymers in different fractions
was estimated from elution volumes relative to those of
polymers of PSS, two- and three-residue peptides, and
salicylic acid (Table 1). The separation of peaks extending
from 3,P4 to 7,P4 (Fig 1A) was consistent with a difference
between each of these fractions of Mr 195 k 45.
To characterize further the contents of the P-4 exclusion
column fractions, samples were analyzed by electrofocusing
on agarose gels (Fig 2). Higher Mr ATA polymers, contained in earlier eluting fractions from the P-4 column (Fig
2, lanes 1 through 4), were less acidic than the lower Mr,
later-eluting polymers. Carboxyl groups are the only charged
group present on both large and small polymers; therefore,
the difference in pKa among these polymers is most likely
the result of variability in steric and conformational properties. Fewer bands were visible in gel lanes of later-eluting
fractions (Fig 2, lanes 7 through 9) compared with earliereluting ones. These results indicate that each P-4 column
fraction contains a heterogeneous population of ATA
polymers, and that the lowest Mr polymers are more
homogeneous with respect to charge than larger polymers.
Inhibition of rktocetin-induced, v W-dependent platelet
aggregation by ATA and PSS. P-4 exclusion column fractions of ATA were examined as inhibitors of ristocetininduced, vWF-dependent platelet aggregation in PRP (Fig
3A). Larger ATA polymers were more potent inhibitors of
aggregation than smaller polymers. Polymers less than Mr
300 ( s 7,P4) had minimal inhibitory activity when tested at
a final concentration of 200 pg/mL (Fig 3A). Inhibitory
potency declined slowly over the Mr 1,300 to 700 range (Fig
4A). In contrast, below an Mr of about 700, considerable
increases in ATA polymeric concentrations were required
to produce a 50% inhibition of ristocetin/vWF-aggregation.
On a molar basis (Fig SA), a change in Mr of 400, between
polymers of about Mr 1,300 to those of Mr 900, doubled the
number of micromoles necessary to inhibit ristocetin/vWFplatelet aggregation by 50% (from 26 pmol/L to 52 ymol/
L). A further decrease of Mr 400, from polymers of Mr 900
to 500, however, increased this number of micromoles
almost 10-fold (to 480 pmol/L).
PSS polymers of differing Mrs were also tested as
inhibitors of ristocetin/vWF-aggregation (Fig 3A). As with
ATA, an increase in inhibitor potency was related, in a
P-4 Column Fraction
1
5.0
4.5
Fig 2. P 4 chromatographic
fractions of ATA polymers separated by electrofocusing in agarose. Column fractions correspond to those shown in Fig 1 A
The position of each band is at
the isoelectric pH (pl) of the ATA
polymers in that particular P-4
column fraction.
4.0
-
2
3
4
5
7
8
9
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2294
c
-
.-c0
.-c
-03
CD
u
e
-e,
0)
WEINSTEIN ET AL
A ,
I
C
.o
e
100
20
-
e
60
c
m
40
c
c
C
C
$
a
-
80
c
h
120
20
0
0
8
a
0
0
50
150
100
-
0
200
0
ATA (Ug/ml)
200
400
600
800
1000
Polystyrene Sulfonate (ug/ml)
80
-
60
-
40
-
20
-
2
o
2
2
P
2
- o
P
5
0
20
40
60
80
100
120
140
ATA (vg/ml)
5
0
100
200
300
400
SO0
Polystyrene Sulfonate (ug/ml)
Fig 3. (A) Inhibition of ristocetin-induced, vWF-mediated platelet aggregation in PRP by ATA (left) and PSS (right). The percent platelet
aggregation was calculated by dividing the initial rate of decrease in optical density of the test mixture by the value obtained with PRP treated
only with buffer. In studies of ATA, P-4 gel filtration peaks 2.P4 through 9.P4 were tested as inhibitors. Higher Mr polymers (lower fraction
numbers) attenuated aggregation better than lower Mr forms (higher number column peaks). The PSS polymer of Mr 1.8 x 101( 0 )was much less
inhibitory than PSS of Mr 4.6 (A), 8.0 (0).17.4 (+),and 35 x lo3(A).(B) Inhibition of shear-induced, vWF-mediated platelet aggregation in PRP.
Commercial ATA and fractionsfrom the BioRad P-4 and P-10 columns (left) as well as PSS polymers (right) were tested. With no inhibitor present,
only 10% of the initial platelets remained unaggregated after a shear stress of 180 dynes/cmzwas applied for 30 seconds.
nonlinear way, to an increase in Mr. On the basis of
concentration as mass per volume, there was little difference in the capacity of PSS polymers between Mr 4,600 and
35,000 to inhibit ristocetin/vWF-induced platelet aggregation by 50% (Fig 4A). In contrast, the Mr 1,800 PSS
polymer was about 10-fold less potent as an inhibitor. If
considered on the basis of molarity, the Mr 1,800 PSS
polymer was 200-fold less effective than the Mr 35,000
polymer (Fig 5A).
The Mr of PSS polymers that attenuated ristocetin/vWFinduced platelet aggregation by 50% was greater than that
for ATA polymers (Fig 4). In terms of mass, ATA was also a
more effective inhibitor than PSS. Only 34 kg/mLof the Mr
1,300 ATA polymer inhibited ristocetin/vWF-induced platelet aggregation by 50%, whereas 100 to 120 pg/mL of any of
the PSS polymers of Mr between 4,600 and 35,000 was
required to produce 50% inhibition.
Other compounds related in structure to ATA were
examined as inhibitors of ristocetin-induced, vWF-mediated platelet aggregation. These compounds included aurin
(100 Fg/mL), salicylic acid (as high as 1 mg/mL), and
methylene disalicylic acid (100 to 400 kg/mL). Polyglutamic acid of Mr 5 to 10 x lo3 (200 pg/mL) was also tested
to determine whether negatively charged polymers i s general have inhibitory activity. None of these sqbstances
inhibited ristocetin/vWF-induced aggregation at the concentrations tested.
Inhibition of shear-induced,v WF-mediatedplatelet aggregation. The effects of ATA polymers on shear-induced,
vWF-dependent platelet aggregation were tested. PRP was
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2295
POLYANIONIC POLYAROMATIC POLYMERS INHIBIT vWF
Z
o
600
1500
1000
10
0
v)
v)
n
ATA Molecular Weight
20
30
40
PSS Molecular Weight (x 1000)
gB
u)
.-0"
5
160
500
140
(d
m
a
g
a
a
.-
s
2 - r
120
400
100
.-c
4-
-0
JCz
-
-z
-'a
0
+.
-0
c
80
,!,
!
C
60
300
c
40
=
20
-
E
\
2
a
24
.*
0
.c
n
"
1
2
3
4
5
6
7
m
v)
n
ATA Molecular Weight ( x 1000)
200
o
10
20
30
40
PSS Molecular Weight (x 1000)
Fig 4. The concentration (pg/mL) of ATA (left) or PSS (right) needed to reduce ristocetin/vWF-mediatedplatelet aggregation by 50% plotted
against polymer Mr. Polymers of ATA of Mr 700 to 1,300 inhibited aggregationto a similar degree as PSS polymersof Mr4.600 to 35,000, and much
more effectively than the smallest PSS polymer tested (Mr 1,800). (B) The concentration (pglmL) of ATA (left) or PSS (right) needed to reduce
shear/vWF-mediated platelet aggregation by 50% plotted against polymer Mr. (*) The Mr 1,800 PSS polymer inhibited ristocetin-induced
aggregation by only 44%. and shear-induced aggregation by 38% at the highest concentrationstested.
incubated with P-4 and P-10 column fractions. A shear
force of 180 dynes/cm2was applied for 30 seconds, and then
the percentage of individual (nonaggregated) platelets
remaining in suspension relative to the initial value was
determined (Fig 3B).
The relationship between ATA polymer size and the
inhibition of shear-induced, vWF-mediated platelet aggregation was analogous to that obtained for ristocetin/vWFinduced aggregation. ATA polymers of Mr 22,500 were
more effective on a molar basis than smaller polymers in
reducing shear-induced, vWF-mediated platelet aggregation (Fig 5B). If considered on the basis of mass/volume
rather than molarity, the Mr 2,500 ATA polymer fraction
inhibited shear-induced, vWF-mediated aggregation better
than fractions of either higher or lower Mr (Fig 4B). The
inhibitory potency of the Mr 2,500 ATA polymer was five
times greater than commercial ATA on the basis of mass.
Polymers of PSS and polyglutamic acid were also tested
for their capacity to inhibit shear-induced, vWF-mediated
platelet aggregation (Fig 3B). On the basis of concentration
expressed as mass/volume, PSS polymers of Mr 4,600 to
35,000were approximately equivalent in inhibitory potency.
The Mr 1,800 polymer did not prevent aggregation by 50%
at the highest concentration tested (480 pg/mL). About 10
times more masdvolume of the most potent PSS polymers
(Mr 35,000) were needed to inhibit aggregation to the same
extent as the ATA polymer of Mr 2,500 (Fig 4B).
Polyglutamic acid and aurin, each at a concentration of
480 pg/mL, did not inhibit shear-induced, vWF-mediated
platelet aggregation.
Effect of PSS onplatelet aggregation induced by arachidonic
acid, AD4 and collagen. In a previous ~ t u d ysolutions
,~
of
commercial ATA were found not to interfere with platelet
aggregation initiated by arachidonic acid or ADP. To
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2296
r
0
0
a
WEINSTEIN ET AL
+
A
0
0
a
5
0
a
c
0
500
1000
I
700
1500
a
ATA Molecular Weight
PSS Molecular Weight (x 1000)
B
v)
I
C
.-0
c
m
CI
:
ID
a
c
.n
.r
-c
0
c
40
30
20
10
a
I<
0
1
2
3
4
5
6
10
7
20
30
40
a
ATA Molecular Weight (x 1000)
PSS Molecular Weight (x 1000)
Fig 5. The molar concentration of ATA (left) or PSS (right) needed to reduce (A) ristocetin- or (B) shear-induced, vWF-mediated platelet
aggregation by 50% plotted against polymer Mr. (*) Results calculatedfor the Mr 1.800 PSS polymer as in Fig 4.
determine whether PSS also lacks inhibitory activity under
these assay conditions, PRP was incubated with arachidonic acid, ADP, or collagen in the presence or absence of
the Mr 17,400 PSS polymer. PSS did not inhibit aggregation
caused by any of these agonists (data not shown).
DISCUSSION
Experiments in this study have identified some of the
properties of ATA polymers that enable them to inhibit the
association of vWF with platelets. Solutions of commercial
ATA, shown in previous ~ t u d i e sto
~ .have
~ arterial antithrombotic properties, were separated into polymers of varying
average Mr. The capacity of these compounds to inhibit
ristocetin- and shear-induced, vWF-mediated platelet aggregation was found to be a function of polymer size, charge,
and aromaticity.
To examine the relationship between polymer size and
inhibitory potency, gel filtration chromatography was used
to partition solutions of commercial ATA into fractions
containing polymers with relatively narrow distributions of
Mrs. To attain good resolution, it was essential to use a high
ionic strength buffer that impeded the adherence of ATA
polymers to the column resin. Other chromatographic
procedures, such as gel filtration in buffers containing 0.15
mol/L NaCl and 10% to 50% methanol, did not yield
well-resolved fractions (data not shown).
In studies by Gonzales et
in which the size of ATA
polymers was examined, commercial ATA was separated
into high, medium, and low Mr fractions by ultrafiltration
and dialysis. The average Mrs of polymers in these fractions
were determined by vapor-phase osmometry. In the present
study, ATA Mrs were assessed by gel filtration chromatography using polymers of PSS for Mr comparisons. Despite
these differences in technique, the average Mr of the largest
ATA polymers determined in our experiments (Mr 6,400)
was in good agreement with the value of Mr 6,000 5 600
obtained by Gonzales et aL6
The data reported in the present study support the
contention6 that the commonly accepted triphenyl methyl
structure of ATA (Fig 6) is not a major component of the
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2297
POLYANIONIC POLYAROMATIC POLYMERS INHIBIT vWF
4
COOH
/
/
COOH
CCOH
COOH
HypothRtical Polymer of ATA
I
I
~CH-CY-CH-CH,-OI-CY-CH-~
I
I
Polystyrene Sulfonate
INACTIVE
COOH
COOH
COOH
COOH
CHI
CHI
CY
CHz
I
p
I
I
F",
~-NH-~H-~O-NH-CH--CO-NH-CH--CO-NH-CH-~O-
I
I
p
F
i
Polyglutamic Acid
COOH
Salicylic Acid
CWH
COOH
Methylenedisalicylic Acid
Hypothetical Polymer 01 Aurin
Fig 6. Planar representationof compounds tested as inhibitors of
ristocetin- and shear-induced, vWF-mediated platelet aggregation.
The circled segment of the ATA polymer indicates the conventional
representation of ATA. Previous studies' showed that the triphenyl
structure is included in polymers of ATA but does not exist in the free
form. Commercial ATA is, more likely, a heterogeneous mixture of
polymers composed of a number of different protomericcomponents.
solutions of commercial ATA. Most of the polyanionic
constituents of commercial ATA had Mrs greater than that
of the triphenyl methyl unit, Mr 422. Furthermore, the
separation between each of the late eluting gel filtration
fractions (Table 1, Fig 6) was compatible with an Mr
difference between them of approximately 200. Although
the triphenyl methyl structure may be found within ATA
polymers, protomers of the size of salicylic acid (Mr 138),
methyl salicylic acid (Mr 160), or methylene disalicylic acid
(Mr 290) are more in accord with the separation data.
In our previous study of solutions of commercial, unfractionated ATA; the capacity of the dye to bind to vWF and
inhibit shear stress- and ristocetin-induced, vWF-mediated
platelet aggregation was determined. In this report, we
have begun to define the physical and chemical qualities of
ATA that are essential for its inhibitory activity.
The size of ATA polymers directly affected inhibitory
potency. This finding is consistent with the observation' that
a dialysis fraction of commercial ATA containing large
polymeric forms of the compound was more effective than
fractions containing lower Mr polymers as an inhibitor of
cyclic flow reductions in a coronary thrombosis model. The
molar concentration of ATA required to inhibit both
ristocetin- and shear-induced, vWF-mediated platelet aggregation remained constant once a polymer of a critical size
was attained. One possible explanation is that a certain
number of protomers are necessary to form a structure with
the optimal capacity to fit onto vWF monomers in a way
that directly or indirectly alters the structure of the vWF
binding site for GPIb. It would require 16 residues of
methyl salicylic acid, a probable protomeric unit of ATA, to
form a polymer of Mr 2,560 (Mr 2,500 is the Mr of the ATA
polymer that optimally inhibits shear-induced, vWFmediated aggregation). Polymers larger than this would not
be more inhibitory on a molar basis. They would, however,
require more mass per unit volume to attain the same
molarity as the Mr 2,500 polymer. These results suggest that
the dosage of ATA needed to inhibit platelet aggregation in
vivo might be higher for the Mr 6,400 fraction than for the
Mr 2,500 fraction.
Another property required for inhibition of ristocetinand shear-induced, vWF-mediated platelet aggregation is
the negative charges on the aromatic residues of ATA (Fig
6). Aurin, a mixture of polymers similar to ATA, but lacking
carboxyl groups, did not prevent aggregation in either of the
systems tested. Our experiments with ATA, PSS, and
polyglutamic acid polymers indicate that certain types of
negatively charged aromatic polymers can inhibit ristocetinand shear-induced, vWF-mediated platelet aggregation,
but that inhibitory capacity is not a property of anionic
nonaromatic polymers.
ATA does not prevent ristocetin-induced, vWF-mediated platelet aggregation solely by neutralizing the positive
charge of the antibi~tic.~
Although charge neutralization
might reduce ristocetin activity to some extent,' this cannot
explain completely the inhibitory potency of ATA polymers. Small ATA polymers that are more negatively charged
than larger forms of ATA, as well as the negatively charged
compounds salicylic acid, methylene disalicylic, and polyglutamic acid, were all ineffective inhibitors of ristocetin/vWFinduced aggregation (Fig 6). Even more convincing evidence is provided by the inhibition by ATA of the shear
stress-induced, vWF-mediated platelet aggregation that
occurs in the absence of ristocetin.
Polymers of ATA may bind to one or more positively
charged region(s) in the vWF monomers that comprise the
large vWF multimers, and sterically hinder the adhesion of
vWF to platelet GPIb receptors in both the ristocetin and
shear systems. It is likely that the binding site for ATA
polymers on vWF monomers is in the region between
residues 449 to 728. This region is enriched in positively
charged amino acids, and includes the two discontinuous 15
amino acid sequences in each vWF monomer that may
contribute to the binding site for the platelet membrane
GPlb receptors.' Attachment of a large ATA polymer to
this region between amino acids 449 to 728 may interfere
with the adherence of vWF multimers to platelet GPIb
molecules. Similar conclusions have been derived in a study
of the capacity of ATA analogues to inhibit vWF binding to
heparin."
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2298
WEINSTEIN ET AL
Much higher Mr anionic aromatic PSS polymers were
necessary to effect the same extent of inhibition on a molar
basis as that produced by anionic aromatic ATA polymers.
Given that the backbone of an ATA polymer is comprised,
in part, of phenyl, methyl, and methylene groups,6 in
contrast to the methyl groups of PSS, it is likely that
polymers of ATA are less flexible and form more extended
structures than PSS polymers of similar Mr.
ATA and PSS polymers do not prevent platelet aggregation stimulated by ADP, arachidonic acid, or collagen.
These results indicate that these polymers do not destroy
platelet metabolic function or cover platelet membrane
proteins and render them inaccessible to various agonists.
ATA and PSS polymers do not prevent ADP-induced
aggregation, providing indirect evidence that they do not
adversely affect GPIIb-IIIa complexes to prevent fibrinogen
binding to platelet surfaces. ATA and PSS polymers inhibit
ristocetin-induced, vWF-mediated aggregation of metabo-
lizing and formalin-fixed (data not shown) platelets, implying that their inhibitory effect is the result of blockade of
the interaction of large vWF multimers with platelet GPIb
receptors.
Currently, there is little data available about the toxicity
of ATA. No acute toxicity has been detected in more than
25 dogs administered ATA and ATA polymers5" (unpublished observations). The LD50 in mice is 340 mg/kg."
Chronic toxicity studies have not yet been reported in any
animal model.
In summary, this report describes the isolation of the
most inhibitory ATA polymers from commercial ATA
solutions for use in animal models of arterial thrombosis
and, ultimately, perhaps in humans. The data presented
will also be useful in designing polymers comprised of
protomers with defined size, charge, and aromatic characteristics that will optimally inhibit vWF-platelet interactions and thrombus formation.
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1991 78: 2291-2298
Isolation from commercial aurintricarboxylic acid of the most effective
polymeric inhibitors of von Willebrand factor interaction with platelet
glycoprotein Ib. Comparison with other polyanionic and polyaromatic
polymers
M Weinstein, E Vosburgh, M Phillips, N Turner, L Chute-Rose and J Moake
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