Zn2+ mediates high affinity binding of heparin to fibrinogen Zn2+ Mediates High Affinity Binding of

21 Pages·2013·0.32 MB·English

JBC Papers in Press. Published on August 29, 2013 as Manuscript M113.469916
The latest version is at http://wwZwn2.+jb mce.odiragt/ecsg hii/gdho ai/f1fi0n.i1ty0 b7i4n/djbincg. Mof1 h1e3p.a4r6in9 9to1 6fi brinogen

Zn2+ Mediates High Affinity Binding of Heparin to the αC Domain of Fibrinogen‡

James C. Fredenburgh,1,2 Beverly A. Leslie, 1,2 Alan R. Stafford, 1,2 Teresa Lim, 1,2
Howard H. Chan, 1,2 Jeffrey I. Weitz*§1,2,3
From the Thrombosis and Atherosclerosis Research Institute1 and Departments of Medicine2 and
Biochemistry and Biomedical Sciences3, McMaster University, Hamilton, Ontario, Canada

Running Title: Zn2+ mediates high affinity binding of heparin to fibrinogen

*Address correspondence to Dr. Jeffrey Weitz, Thrombosis and Atherosclerosis Research Institute, 237
Barton St. E, Hamilton, Ontario L8L 2X2 CANADA Phone [phone] FAX [phone]
E-mail: [email protected]

Keywords: heparin; fibrinogen; zinc

Capsule
Background: The interaction of heparin with fibrinogen compromises its anticoagulant activity.
Results: Zn2+ promotes heparin binding to His545-His546 on the fibrinogen α-chain. D
Conclusion: We identified a novel Zn2+-dependent heparin binding site on fibrinogen. ow
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Significance: Platelet release of Zn2+ at sites of vascular injury may promote heparin binding to loa
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fibrinogen, thereby further attenuating the anticoagulant activity of heparin. ed
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SUMMARY heparin-catalyzed inhibition of factor Xa by h
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The non-specific binding of heparin to plasma antithrombin is compromised by fibrinogen to a ://w
proteins compromises its anticoagulant activity greater extent when Zn2+ is present. These w
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by reducing the amount of heparin available to results reveal the mechanism by which Zn2+ .jb
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bind antithrombin. In addition, interaction of augments the capacity of fibrinogen to impair .org
heparin with fibrin promotes formation of a the anticoagulant activity of heparin. b/
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ternary heparin-thrombin-fibrin complex that Heparin and heparin derivatives are effective gu
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protects fibrin-bound thrombin from inhibition anticoagulants that target thrombin, factor Xa and st o
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by the heparin-antithrombin complex. Previous other upstream enzymes in the coagulation system. A
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studies have shown that heparin binds the E Heparin principally functions as a catalyst by ril 1
domain of fibrinogen. The current investigation binding antithrombin and promoting its interaction , 2
examines the role of Zn2+ in this interaction with coagulation proteases. Two distinct 019
because Zn2+ is released locally by platelets and mechanisms comprise heparin’s catalytic role and
both heparin and fibrinogen bind the cation, both require heparin to bind antithrombin (1). For
resulting in greater protection from inhibition inhibition of factor Xa, the conformational change
by antithrombin. Zn2+ promotes heparin in antithrombin that accompanies heparin binding
binding to fibrinogen, as determined by is essential. In contrast, because this
chromatography, fluorescence, and surface conformational change is without catalytic effect
plasmon resonance. Compared with intact for thrombin, heparin serves as a template onto
fibrinogen, there is reduced heparin binding to which both the protease and inhibitor bind, thereby
fragment X, a clottable plasmin degradation promoting bimolecular interaction.
product of fibrinogen. A monoclonal antibody Despite its efficacy, the anticoagulant response
directed against a portion of the fibrinogen αC to therapeutic doses of heparin is unpredictable.
domain removed by plasmin attenuates binding Consequently, frequent coagulation monitoring is
of heparin to fibrinogen and a peptide analog of necessary to ensure that therapeutic levels of
this region binds heparin in a Zn2+-dependent heparin are achieved (2). This variability is due in
fashion. These results indicate that the αC part to interaction of heparin with numerous
domain of fibrinogen harbors a Zn2+-dependent plasma proteins, some of which are acute phase
heparin binding site. As a consequence, reactants whose levels vary in certain pathological
1

Copyright 2013 by The American Society for Biochemistry and Molecular Biology, Inc.
Zn2+ mediates high affinity binding of heparin to fibrinogen

states. Previously, we have shown that Research Labs, Inc (South Bend, IN). Fibrinogen
displacement of catalytic heparin from plasma and fibrinogen fragment X were prepared and
proteins recovers heparin’s anticoagulant activity characterized as described (13-15). Human
in vitro (3). Thus, interaction with plasma antithrombin was from Affinity Biologicals Inc.
proteins reduces the bioavailability of heparin. (Ancaster, ON). Unfractionated heparin (heparin),
Abundant plasma proteins that bind heparin deaminated heparin (catalog number H7405), and
include fibrinogen, histidine-rich glycoprotein 5-iodoacetamidofluorescein (5-IAF) were from
(HRG)1, and high molecular weight kininogen Sigma. Like unfractionated heparin, which has
(HK) (4-6). equivalent inhibitory activity against factor Xa and
In addition to reducing the concentration of thrombin, deaminated heparin has anti-factor Xa
heparin available to bind to antithrombin, there is and anti-thrombin activities of 101 and 104
another consequence of the high affinity of units/mg, respectively. αC-20, a peptide analog of
fibrinogen for heparin. Because both heparin and fibrinogen Aα529-548 containing an added NH -
2
fibrinogen also bind thrombin, a ternary complex is terminal Cys residue
formed that sequesters thrombin and restricts (CGSESGIFTNTKESSSHHPGI) and αC-20AA, a
accessibility of antithrombin (6). Thus, thrombin variant with Ala residues in place of the two His
bound to heparin and fibrin within this ternary residues, were synthesized by GenScript Corp.
complex retains its catalytic activity and is (Scotch Plains, NJ). A polyclonal antibody
D
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protected from inhibition (6,7). This phenomenon directed against αC-20 was raised in sheep by w
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likely contributes to the prothrombotic nature of Affinity Biologicals Inc. and the IgG fraction was lo
a
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thrombi and to the rethrombosis that can occur then affinity purified using immobilized αC-20. ed
despite heparin therapy (2). Thrombin was radiolabeled at its active site by fro
m
Another factor that may compromise the reaction with 125I-Tyr-Pro-Arg-chloromethyl h
availability of heparin is Zn2+, an essential metal ketone (13). Monoclonal antibodies directed ttp://w
ion that has numerous effects in hemostasis and is against various epitopes on the αC domain, w
w
present in plasma at 10-20 µM (8,9). Recently, we designated F-102 and F-103, 134-B29, and TF 359 .jb
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demonstrated that Zn2+ augments ternary heparin- were generous gifts from Drs. Joan Sobel (16), .o
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thrombin-fibrin-heparin complex formation and Zaverio Ruggeri (17), and Bohdan Kudryk (18), b/
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increases the protection of thrombin from respectively. Chromogenic substrate for factor Xa, g
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inhibition by antithrombin in the presence of fibrin BioPhen CS-11-(65), was from Aniara (Mason, st o
(10). The 4-fold increase in the affinity of heparin OH), whereas FluoZin-1, a fluorescent indicator n A
for fibrin in the presence of Zn2+ leads to a similar for Zn2+, was from Invitrogen (Grand Island, NY). pril 1
elevation in the apparent affinity of thrombin for Heparin-Sepharose chromatography. , 2
fibrin. Zn2+ also has been shown to increase the Fibrinogen and fragment X were subjected to 01
9
affinity of heparin for HRG and HK; interactions chromatography on a 1 ml Hi-Trap heparin-
that reduce the catalytic activity of heparin. HK Sepharose column (GE Healthcare) using a
and HRG bind Zn2+ through their histidine-rich Beckman System Gold chromatography system.
regions and the negatively charged sulfate groups The column was equilibrated with 10 mM Tris-
of heparin then bind Zn2+ (11). Although it is HCl, pH 7.4, 0.005% Tween 20 (Tw), in the
known that fibrinogen binds Zn2+ (12), the effect of absence or presence of 12.5 µM ZnCl , 2 mM
2
Zn2+ on heparin binding has not been investigated. CaCl , or 2 mM EDTA. CaCl and ZnCl
2 2 2
The current study reveals a novel Zn2+-dependent concentrations were chosen to approximate those
heparin binding site localized to the αC domains of in plasma. Protein (1 mg in 1 ml) was applied to
fibrinogen that is distinct from the previously the column in equilibration buffer at a flow rate of
reported heparin binding site on the β-chains. 1 ml/min. After washing, a 40 ml linear gradient
from 0 to 1 M NaCl in equilibration buffer was
Experimental procedures applied, fractions were collected and absorbance
Materials. Human thrombin, plasminogen-free was monitored at 280 nm.
fibrinogen, and factor Xa were from Enzyme Fluorescence studies
2
Zn2+ mediates high affinity binding of heparin to fibrinogen

Interaction of fluorescein-heparin with labeled peptides were separated from
fibrinogen. For optimal labeling with fluorescein unincorporated 5-IAF by chromatography on a
or biotin, deaminated heparin (d-heparin) was used Sephadex G-10 column (GE Healthcare)
in some studies. This heparin derivative was equilibrated with water and the concentration of f-
chosen to direct modification to the reducing end αC-20 or f-αC-20AA was quantified by measuring
of the heparin molecule, thereby avoiding random absorbance at 492 nm. HPLC analyses revealed
labeling of internal residues, which may greater than 70% incorporation of 5-IAF into the
compromise the integrity of binding sites (19). D- peptides (not shown). To quantify their interaction
heparin (20 mg) was fluorescein-labeled by with heparin, 900 µl of 100 nM f-αC-20 or f-αC-
reaction with 3 mg fluorescein-5-thiosemicarbizide 20AA in TBS containing 12.5 µM ZnCl was
2
(Invitrogen) in 400 µl phosphate buffer, pH 7.5. placed in a quartz cuvette, stirred with a micro stir
After mixing for 1 h in the dark, the sample was bar and maintained at 23°C using a circulating
passed over a PD-10 column (GE Healthcare) water bath. Fluorescence intensity was monitored
equilibrated with water and 0.5 ml fractions were at excitation and emission wavelengths of 492 and
collected. The heparin-containing fraction was 535 nm, respectively, with an emission filter at 515
lyophilized and resuspended in 500 µl 20 mM nm, before and after the sample was titrated with
Tris-HCl, pH 7.4, 150 mM NaCl (TBS). The aliquots of 66 µM heparin containing 100 nM f-
concentration of fluorescein-d-heparin (f-d- peptide. Fluorescence was allowed to stabilize D
o
heparin) was determined using the extinction before each addition. Intensity values were w
n
coefficient for fluorescein (68,000 M-1•cm-1 at 492 normalized relative to those determined in the loa
d
nm). For fluorescence studies, 900 µl of TBS absence of heparin and plotted versus heparin ed
containing 150 nM f-d-heparin and 2 mM CaCl2, 2 concentration. Data were analyzed by nonlinear from
mM EDTA, or 12.5 µM ZnCl2 was placed in a regression of the binding isotherm to obtain Kd and http
quartz cuvette, stirred with a micro stir bar and values are reported as mean ± standard deviation of ://w
maintained at 23°C with a circulating water bath. three determinations. w
w
Using a LS50B luminescence spectrophotometer Interaction of fibrinogen, fragment X, αC-20, .jb
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(Perkin Elmer, Wellesley, MA) with excitation and and αC-20AA with Zn2+. A cuvette containing 1.0 .org
emission wavelengths of 492 and 535 nm, ml of 0.5 µM FluoZin-1 and 15 µM ZnCl was b/
2 y
respectively, slit widths of 5 and 20 nm, monitored at excitation and emission wavelengths gu
e
respectively, and with a 515 nm cutoff filter, of 495 and 515 nm and slit widths of 10 and 6 nm, st o
fluorescence was monitored before and after respectively. Fluorescence was monitored n A
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titration with 1 to 10 µl aliquots of 25 µM continuously as the sample was titrated with ril 1
fibrinogen or 20 µM fragment X containing the aliquots of fibrinogen, fragment X, αC-20, or αC- , 2
0
appropriate concentration of CaCl2, ZnCl2, or 20AA to 110 µM. Plots of I/Io values versus 19
EDTA. Fluorescence was allowed to stabilize peptide concentration were analyzed by nonlinear
before each addition and after the run, fluorescence regression of a binding isotherm equation to
intensity values (I) obtained from the time drive determine the IC values. These were converted to
50
profile were corrected for dilution, and calculated Kd values using the Cheng-Prusoff equation and
as a ratio of the initial fluorescence (Io). Plots of the affinity of Zn2+ for FluoZin-1, which was
I/Io versus protein concentration were analyzed by determined in a separate experiment. Values are
nonlinear regression analysis of the binding reported as mean ± standard deviation of three
isotherm equation to determine Kd values, which determinations.
are reported as mean ± standard deviation from Binding of Zn2+ to dansyl-fibrinogen. Dansyl-
three determinations (20). fibrinogen was prepared by incubating 3 mg
Interaction of fluorescein-αC-20 and αC-20AA fibrinogen with 0.1 mg dansyl chloride (Sigma) in
with heparin. Fluorescein-labeled αC-20 and αC- the dark for 30 min at 23°C. The reaction mixture
20AA were prepared by incubating 1 mg peptide in was then passed over a PD-10 column equilibrated
1 ml 0.1 M Na phosphate, pH 7.5 with 50 µl of 56 in TBS and the concentration of dansyl-fibrinogen
mg/ml 5-IAF in DMSO for 3 h at 23°C. The was determined by iteratively correcting the A
280
3
Zn2+ mediates high affinity binding of heparin to fibrinogen

value with readings determined at A (21). The min at 23°C. Aliquots of 250 µl were injected at a
335
stoichiometry of labeling was 15 dansyl groups per flow rate of 30 µl/min for 4 min prior to injection
fibrinogen and like unlabeled fibrinogen, the of buffer alone. Flow cells were regenerated with
dansyl-fibrinogen was over 95% clottable. To 150 µl of 1.5 M NaCl, 100 µM EDTA, and 0.005%
measure Zn2+ binding, 100 nM dansyl-fibrinogen Tw in HBS, pH 7.4. Using BIAcore software,
in TBS containing 0.4% polyethylene glycol and peak RU values were determined for each run and
0.05% Tw was placed in a cuvette in the corrected for signal from a blank flow cell. The
fluorimeter. Fluorescence was monitored with percentage of fibrinogen bound to b-d-heparin in
excitation at 340 nm and emission at 520 nm in the the presence of each antibody was calculated
presence of a 430 nm cutoff filter. The sample was relative to that in its absence. Studies were
maintained at 23°C and titrated with aliquots of 1 performed two times.
mM ZnCl . Intensity values were obtained from Interaction of Zn2+ with immobilized
2
the time drive profile and plotted as I/Io versus fibrinogen. Using an amine coupling kit,
ZnCl concentration. The data were analyzed by fibrinogen (0.1 mg/ml in 10 mM acetate buffer, pH
2
nonlinear regression of the rectangular hyperbola 5.5) was immobilized on a CM5 sensor chip to
equation to obtain K . Experiments were 10,000 RU in a BIAcore T200 (GE Healthcare).
d
performed three times and data are reported as ZnCl (dissolved in HBS containing 2mM CaCl
2 2
mean ± standard deviation. and 0.05% Tw) was injected into flow cells D
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Surface Plasmon Resonance (SPR) studies containing immobilized fibrinogen for 120 s at a w
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Interaction of fibrinogen with immobilized rate of 30 µl/min to monitor association. Buffer loa
d
heparin. Heparin was biotinylated with biotin- lacking ZnCl was then injected for 300 s to ed
amidohexanoic acid hydrazide (Sigma) in 0.1 M monitor disso2ciation. Between runs, flow cells fro
m
sodium acetate, pH 5.5 (20). Streptavidin (SA) was were regenerated with 30 µl of 3.6% sodium h
attached to a CM5 chip using an amine coupling citrate. Sensorgrams for each Zn2+ concentration ttp://w
kit (GE Healthcare). Biotinylated heparin (b- were background corrected using a blank flow cell. w
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heparin) was adsorbed to the SA flow cell to 640 A plot of background-corrected Req values versus .jb
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RU in 20 mM HEPES, pH 7.4, 150 mM NaCl Zn2+ concentration was analyzed by nonlinear .org
containing 0.005% Tw (HBS-Tw) in a BIAcore regression of a rectangular hyperbola to determine b/
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1000 (GE Healthcare). Aliquots of 250 nM Kd of Zn2+ for fibrinogen. Studies were performed gue
fibrinogen in HBS-Tw containing 0 – 15 µM two times. st o
ZnCl2 were injected over the flow cell at 30 Heparin binding to fibrin clots. d-heparin was n A
µl/min. Between runs, the flow cell was labeled with 125I after modification with 4-OH pril 1
regenerated with 0.5% SDS and 1 mM EDTA. benzhydrazine. Thus, d-heparin (10 mg) was , 2
0
From the sensorgrams, resonance unit (RU) values dissolved in 400 µl acetate buffer, pH 5.5, and 1
9
at equilibrium (Req) were determined and plotted mixed with 100 µl of DMSO containing 2 mg 4-
versus ZnCl concentration. Data were analyzed by OH-benzhydrazine (Sigma). The solution was
2
nonlinear regression of a rectangular hyperbola to mixed overnight at 23°C and then passed over a
determine the K values. Studies were performed Sephadex G10 column equilibrated with water.
d
five times. Fractions containing heparin were pooled,
Effect of antibodies on the interaction of lyophilized, and resuspended in phosphate buffered
fibrinogen with immobilized d-heparin. D-heparin saline. For labeling, 6 mg of modified d-heparin
was biotinylated as described above and adsorbed was reacted with 3 Iodobeads (Pierce) and 2 mCi
to the flow cell of a SA sensor chip (GE Na125I (McMaster University Nuclear Reactor) as
Healthcare) in HBS-Tw in a BIAcore 1000. described (13). The concentration of 125I-d-heparin
Binding was performed in HBS-Tw containing 2 was determined by Azure A assay.
mM EDTA, and 2 mM CaCl or 12.5 µM ZnCl . Binding of 125I-d-heparin to fibrin clots was
2 2
Samples containing 125 nM fibrinogen and 500 performed by measuring the radioactivity in clot
nM antibody were preincubated in HBS, pH 7.4, supernatants. Samples containing 1 µM fibrinogen
containing 12.5 µM ZnCl and 0.005% Tw for 90 and 100 nM 125I-d-heparin in TBS were clotted
2
4
Zn2+ mediates high affinity binding of heparin to fibrinogen

with 5 nM thrombin in the presence of 12.5 µM The same experiment was then repeated using
ZnCl or 2 mM CaCl and containing 20 µM of fragment X in place of fibrinogen (Fig. 1B).
2 2
either the αC-20 directed IgG or non-immune Fragment X was examined because this high
sheep IgG. After incubation for 1 h, clots were molecular weight, plasmin-derived degradation
compacted by centrifugation. Aliquots of the product of fibrinogen lacks the COOH-terminal
supernatant were counted for radioactivity to half of the Aα chains. In the presence of Ca2+ (or
determine the concentration of free 125I-d-heparin. EDTA), fragment X eluted with a profile similar to
The fraction of 125I-d-heparin bound was that of intact fibrinogen. However, in the presence
normalized and plotted against the IgG of Zn2+, only a small fraction of fragment X bound
concentration. The data were analyzed by heparin with higher affinity than fibrinogen. Based
nonlinear regression of a rectangular hyperbola to on SDS-PAGE analysis (not shown), this minor
determine the IC for inhibition of 125I-d-heparin population may represent a small residual amount
50
binding by the αC-20-directed IgG. of intact fibrinogen in the fragment X preparation.
Factor Xa inhibition. Second order rate The difference in the results obtained with
constants for factor Xa inhibition by antithrombin fragment X and fibrinogen suggests that the region
were determined under pseudo first order of fibrinogen responsible for Zn2+-mediated
conditions (22). Aliquots from incubations binding to heparin resides in the αC domain.
containing 15 nM factor Xa, 100 nM antithrombin, Furthermore, these results reveal that other D
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1 µg/ml heparin and 12.5 µM ZnCl were removed interactions of heparin with fibrinogen also are w
2 n
at fixed time intervals and assayed for residual promoted by Zn2+. lo
a
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chromogenic activity with the factor Xa-directed Affinity of fibrinogen or fragment X for ed
substrate BioPhen CS-11-65. Second order rate fluorescently-labeled heparin. To obtain more fro
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constants were determined in the absence or quantitative data, the affinity of d-heparin for h
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presence of varying concentrations of fibrinogen or fibrinogen or fragment X was determined in the ://w
fragment X. presence of Ca2+ or Zn2+. Binding was determined w
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Statistical analysis. Data are expressed as means ± by monitoring the fluorescence intensity of f-d- .jb
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standard deviation. Unless otherwise stated, heparin as it was titrated with fibrinogen or .o
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significance of differences was examined using fragment X. Titration with fibrinogen in the b/
y
paired t-tests. In all cases, p values less than 0.05 presence of 2 mM CaCl resulted in a g
2 ue
were considered statistically significant. concentration-dependent and saturable decrease in st o
fluorescence, yielding a Kd value of 975 ± 110 nM n A
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Results (Fig. 2A); a value somewhat higher than the Kd of ril 1
Heparin-Sepharose chromatography. To examine 100 nM determined kinetically (23). In the , 2
the potential effect of Zn2+ on the heparin- presence of EDTA, the affinity was 2-fold lower 01
9
fibrinogen interaction, fibrinogen was subjected to and a smaller change in fluorescence was
chromatography on heparin-Sepharose. In the observed. However, in the presence of 12.5 µM
presence of 2 mM CaCl , the bulk of fibrinogen ZnCl there was a greater decrease in fluorescence
2 2
eluted at 200 mM NaCl and a minor fraction eluted at lower concentrations of fibrinogen, such that the
at 400 mM NaCl (Fig. 1A). A similar profile was affinity of d-heparin for fibrinogen in the presence
obtained in the presence of EDTA (not shown). of Zn2+ was 16-fold higher than that determined in
Chromatography was then repeated in the presence its absence (K values of 60 ± 32 and 975 ± 110
d
of 12.5 µM ZnCl . Under these conditions, nM, respectively; p < 0.002).
2
fibrinogen eluted at a higher ionic strength, with The experiment was then repeated using
two peaks at 300 and 400 mM NaCl, respectively, fragment X in place of fibrinogen (Fig. 2B). No
and a trailing edge that eluted with NaCl binding to f-d-heparin was observed in the
concentrations above 500 mM. These findings presence of Ca2+ or EDTA. The lack of binding in
suggest that fibrinogen binds heparin with higher the presence of Ca2+ contrasts with the results
affinity in the presence of Zn2+. obtained using heparin-Sepharose chromatography.
This difference may reflect the high density of
5
Zn2+ mediates high affinity binding of heparin to fibrinogen

higher molecular weight heparin molecules FluoZin-1. Addition of Zn2+ to FluoZin-1 results
attached to the Sepharose beads. When f-d-heparin in an increase in fluorescence, which is reversed
was titrated with fragment X in the presence of upon titration with a Zn2+-binding ligand (26).
Zn2+, binding was observed and a K value of 539 Titration of the Zn2+/FluoZin-1 mixture with
d
± 185 nM was obtained. Therefore, removal of the fibrinogen resulted in a saturable decrease in
αC domain results in a 9-fold reduction in the fluorescence indicative of near quantitative binding
affinity of fibrinogen for f-d-heparin (from 60 ± 32 of Zn2+ to the peptide (not shown). Accounting for
to 539 ± 185 nM; p=0.03). the affinity of Zn2+ for FluoZin-1, a Kd value of
Zn2+-dependence of fibrinogen binding to 0.67 ± 0.11 µM was obtained. In contrast, Zn2+
immobilized b-heparin. The effect of Zn2+ on bound fragment X with 8.5-fold lower affinity,
fibrinogen binding to immobilized b-heparin was exhibiting a K value of 5.7 ± 0.5 µM.
d
determined using SPR. Increasing concentrations Effect of αC domain-directed antibodies on the
of Zn2+ resulted in a saturable increase in Req heparin-fibrinogen interaction. To validate the
values that yielded half-maximal binding at 4.5 ± existence of a Zn2+-dependent heparin binding site
1.1 µM Zn2+, which is within the physiological in the αC domain of fibrinogen and to begin to
Zn2+ concentration range of about 10 µM (Fig. 3). localize this site, we used SPR to examine the
Identical results were obtained with immobilized effect of various αC domain-directed monoclonal
b-d-heparin, demonstrating that heparin antibodies on the heparin-fibrinogen interaction. D
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deamination does not influence its interaction with The antibodies were directed against the following w
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fibrinogen (not shown). epitopes: Aα 563-578 (F102), Aα 259-276 (F103), lo
a
d
Affinity of Zn2+ for immobilized fibrinogen. The Aα 566-580 (134B-29), and Aα 529-549 (TF359). ed
affinity of Zn2+ for fibrinogen was quantified using Binding of fibrinogen to b-d-heparin was fro
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SPR. Increasing concentrations of ZnCl were monitored in the presence of Zn2+ and in the h
2 ttp
injected into flow cells containing immobilized absence or presence of the antibodies; in all cases ://w
fibrinogen. Sensorgrams revealed a rapid and binding was normalized relative to that observed in w
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concentration-dependent increase in RU values the absence of antibody (data not shown). .jb
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with a subsequent rapid decrease upon injection of Compared with control, F102 and F103 reduced .o
rg
buffer lacking ZnCl2 (Fig. 4). The increase in RU fibrinogen binding by 40 and 30%, respectively, by/
values is attributed to conformational changes in whereas 134B-29 had no effect. TF-359 reduced g
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fibrinogen that occur upon Zn2+ binding that alter fibrinogen binding to heparin by 90%. st o
the refractive index of fibrinogen (24,25). Based Binding of heparin and Zn2+ to αC-20 or αC-20AA. n A
coonn acneanltyrastiiso no,f tZhne2 +p lobti nodfs Rfeibqr ivnaolgueens vweirtshu s aZ nK2d+ Tsuhgeg ersets utlhtsa t wtihthe αmCa jodro mZani2n+--ddierpeecntedde nat nthibeopdarieins pril 1, 2
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value of 9.4 ± 2.2 µM; a value similar to the K of binding site resides in the region of Aα 529-549, 1
d 9
18 µM reported previously (12). near the COOH terminus. The sequence was
A second binding assay was performed to examined for clusters of basic or His residues,
confirm the results obtained using SPR. which could serve as heparin or Zn2+ binding sites,
Fluorescence was monitored as dansyl-fibrinogen respectively. The most likely candidate was the
was titrated with ZnCl . Intensity values increased consecutive His residues at Aα544 and 545.
2
in a concentration-dependent and saturable Consequently, a peptide analog of the Aα529-548
manner, yielding a K of 4.5 ± 0.1 µM (Fig. 4B); a sequence (αC-20) was synthesized and used to
d
value in agreement with that obtained using SPR. raise a polyclonal antibody in sheep. As a control,
Furthermore, the observed change in fluorescence a second peptide with the two His residues
intensity provides independent confirmation that substituted with Ala (αC-20AA) was prepared.
fibrinogen undergoes a conformational change To examine heparin binding, the peptides were
upon Zn2+ binding. labeled at their NH -termini with 5-IAF and
2
Further confirmation of Zn2+ binding to binding to heparin was examined in the absence or
fibrinogen was obtained in a competition presence of Zn2+ or Ca2+. In the presence of EDTA
experiment with the Zn2+-binding fluorophore, or Ca2+, no change in fluorescence intensity was
6
Zn2+ mediates high affinity binding of heparin to fibrinogen

observed (not shown). However, in the presence compare the effects of fibrinogen and fragment X
of 12.5 µM Zn2+, a saturable decrease in f-αC-20 on the catalytic activity of heparin. Previous
fluorescence intensity was observed and yielded a studies have shown that heparin binding to fibrin
K of 691 ± 268 nM (Fig. 5A). In contrast, no reduces the rate of inhibition of thrombin by
d
binding of heparin to αC-20AA was observed. antithrombin (27). It also was observed that
Similar results were obtained in the reciprocal inhibition of factor Xa by antithrombin was
experiment with unlabeled αC-20 and f-d-heparin reduced by fibrin, albeit to a lesser extent because,
(not shown). These results confirm the presence of unlike thrombin, factor Xa does not bind fibrin
a Zn2+-dependent heparin binding site in the αC (23). In the presence of Zn2+, fibrinogen produced
domain. a concentration-dependent reduction in the
Binding of Zn2+ to αC-20 also was compared heparin-catalyzed rate of factor Xa inhibition by
with that to αC-20AA using FluoZin-1. Titration antithrombin, with a 5-fold reduction in rate at a
of the Zn2+/FluoZin-1 mixture with αC-20 resulted fibrinogen concentration of 2 µM (p < 0.05 by
in a saturable decrease in fluorescence (Fig. 5B), two-way ANOVA; Fig. 7). In contrast, the
yielding a K value for the interaction of Zn2+ with heparin-catalyzed rate of factor Xa inhibition by
d
αC-20 of 12.7 ± 3.0 µM. In contrast, when αC- antithrombin decreased less than 2-fold in the
20AA was used in place of αC-20, no binding was presence of increasing concentrations of fibrinogen
detected. These results indicate that the αC-20 when Ca2+ was added in place of Zn2+ (not shown). D
o
peptide contains a Zn2+-binding site that is These results demonstrate that the increased w
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dependent on the His residues at positions 544 and binding of heparin to fibrinogen in the presence of loa
d
545. Zn2+ reduces heparin’s catalytic activity. When ed
Effect of αC-20-directed antibody on the fragment X was substituted for fibrinogen, no fro
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antagonize the binding of heparin to fibrin, confirm the role of the αC domain in the Zn2+- w
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fibrinogen was clotted in the presence of 125I-d- dependent binding of heparin. .jb
c
heparin and increasing concentrations of αC-20- .org
directed or non-immune sheep IgG in the absence Discussion b/
y
or presence of Zn2+ or Ca2+. After 60 min Several groups have identified domains on gu
e
incubation, clots were subjected to centrifugation fibrinogen that contribute to heparin binding. The st o
and the fraction of bound 125I-d-heparin was NH2-terminal region of the Bβ-chain represents n A
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dbelotecrkmedin etdh.e Ibni nthdein pg reosfe n1c2e5I -odf- hCeap2a+,r itnh et oa ntfiibboridny, orenme ocvoanl soefn sfuibsr isnitoep. e pItnidteer aBc,t ieonnd iosw einnhga fnicberdin uwpiotnh ril 1, 2
0
yielding an IC50 value of 0.22 ± 0.06 µM (Fig. 6). a higher affinity for heparin than fibrinogen 19
This confirms that the antibody and heparin bind to (28,29). In contrast to earlier reports (30), these
the same site on fibrin. Non-immune sheep IgG groups failed to observe significant binding of
had no effect on 125I-d-heparin binding to fibrin heparin to the D or αC domains. The heparin
(not shown). However, in the presence of Zn2+, the binding sites on the NH2-termini of the β-chains of
ability of the antibody to block d-heparin binding fibrin are proximal to putative thrombin binding
to fibrin was compromised, yielding a 9-fold sites, consistent with the ability of heparin to
higher IC value of 1.9 ± 0.19 µM (p < 0.002). promote formation of a ternary heparin-thrombin-
50
This finding suggests that the more robust fibrin ternary complex (6). The current work
interaction of heparin with fibrin in the presence of provides evidence for a Zn2+-dependent heparin
Zn2+ impairs binding of the antibody to fibrin. binding site in the αC domain. Although Zn2+ was
Effect of fibrinogen or fragment X on the heparin- not included in previous studies that examined the
catalyzed rate of factor Xa inhibition by heparin-fibrinogen interaction, Zn2+ exists in
antithrombin. As another measure of the plasma at a concentration of ~10 µM (8). In
consequence of Zn2+ promotion of heparin binding addition, Zn2+ is stored in platelet α granules,
to fibrinogen, we used a functional assay to providing a mechanism whereby local
7
Zn2+ mediates high affinity binding of heparin to fibrinogen

concentrations may be elevated at sites of platelet (4,5). Although these proteins have high His
activation (31). Our results provide further insight content, Zn2+ also binds to peptide sequences
into the mechanism by which heparin interacts containing only 2 or 3 His residues (37,38). Zn2+
with fibrinogen. also promotes heparin binding to other proteins,
The Zn2+-dependence of the interaction of such as heparin cofactor II and endostatin (39,40).
heparin with fibrinogen is consistent with the There are two mechanisms by which Zn2+ may
reported capacity of fibrinogen to bind Zn2+ promote heparin-protein interaction. One is by
(12,32). However, the Zn2+ binding sites on inducing a conformational change in the protein to
fibrinogen have not been identified. In this study, expose a latent heparin binding site (37), while the
we localized the Zn2+-dependent heparin binding other involves Zn2+ serving as a coordinator to
site to a 20-residue segment near the COOH bridge heparin to the protein (41). Although it is
terminus of the α-chain. The location of this site known that Zn2+ binds heparin (42), Zn2+ is unable
was predicted by inspection of the sequence and to promote the interaction of thrombin or
confirmed with the use of antibodies and peptide antithrombin with heparin (39,43). This suggests
analogs. A monoclonal antibody directed against that Zn2+-dependent binding to heparin is protein-
α529-549 reduced binding to heparin by 90%, specific (44). Consistent with this, SPR and
whereas antibodies directed against α563-578 fluorescence analyses suggest that Zn2+ induces
(F102) or α259-276 (F103) reduced binding by less conformational changes in fibrinogen. These D
than 40%. A peptide analog of the α529-548 observations reveal that Zn2+ is a common ow
n
sequence bound f-d-heparin in a Zn2+-dependent mediator of heparin-protein interaction. lo
a
d
fashion. Heparin-binding sites in proteins are One of the consequences of the interaction of ed
typically populated with basic residues (33), and heparin with proteins other than antithrombin is a fro
m
the α-chain COOH terminus has 5 basic residues in reduction in its anticoagulant activity. This has h
ttp
the final 10 amino acids (α601-610). However, been observed with HRG (44,45), HK (5) and ://w
apart from Lys 538, the adjacent His residues at platelet factor 4 (46). Interaction with these w
w
positions 543 and 544 are the only basic residues proteins decreases the anticoagulant activity of .jb
c
in the α529-548 segment. Binding of heparin to heparin by reducing the concentration of heparin .org
His residues is also consistent with the Zn2+ available to bind to antithrombin (3). Fibrinogen is b/
y
dependence of the interaction. His residues a recognized member of this group of heparin- gu
e
represent the principal coordination site for metal binding proteins (28,29). Further compromise of st o
ions in numerous metal-binding proteins (34,35). heparin activity occurs when fibrinogen is clotted n A
Confirming that this segment binds Zn2+, a because heparin promotes thrombin binding to pril 1
fluorescein-labeled analog of the peptide fibrin and induces the formation of a ternary , 2
demonstrated a Zn2+-dependent change in complex that limits inhibition by antithrombin 01
9
fluorescence intensity. Furthermore, substitution (6,27). The importance of the heparin-fibrin
of the two His residues in αC-20 with Ala interaction is highlighted by the fact that higher
abrogated Zn2+ binding. Interestingly, interaction doses of heparin are needed to achieve a
of Zn2+ with amyloid-beta peptide also is mediated therapeutic anticoagulant response in patients with
by adjacent His residues (36). Thus, these results venous thromboembolism than in those with acute
identify the portion of the αC domain responsible coronary syndrome; a phenomenon that has been
for the Zn2+-dependent interaction of heparin with attributed to the larger thrombus burden in patients
fibrin. with venous thrombosis (47). Recently, we
The ability of Zn2+ to promote heparin binding demonstrated that Zn2+ promotes ternary complex
to fibrinogen is not unique. This response is formation, thereby augmenting the protection of
observed with other Zn2+ binding proteins, such as fibrin-bound thrombin from inhibition by
HRG and HK. These two homologous proteins antithrombin (10). The current work shows that
have unique His-rich domains, which bind Zn2+. fibrinogen also compromises heparin-catalyzed
At neutral pH, binding of heparin to HRG or HK is inhibition of factor Xa in the presence of Zn2+.
mediated by His residues and is dependent on Zn2+ Because factor Xa does not bind fibrin, this
8
Zn2+ mediates high affinity binding of heparin to fibrinogen

impairment must result from the inability of fibrin- dependent interaction of heparin with fibrinogen
bound heparin to catalyze antithrombin. This likely adds to the numerous roles that Zn2+ is proposed to
is the result of fibrinogen competitively inhibiting play in hemostasis (9,48), including recent reports
heparin binding to antithrombin. of interaction of Zn2+ with protein S and factor
The current work reveals a novel interaction of VIIa (49,50). Zn2+-mediated promotion of the
heparin with fibrinogen via the αC domain. interaction of heparin with fibrinogen may
Recent work has suggested that the E domain, contribute to the unpredictable anticoagulant
which encompasses the NH -termini of the β- response observed with heparin (2). In addition,
2
chains, is the predominant site of fibrinogen by promoting formation of ternary heparin-
interaction with heparin (28,29). Because Zn2+ thrombin-fibrin complexes (10), Zn2+ may also
was not included, these studies overlooked the αC render fibrin thrombi more thrombogenic. These
interaction. Our findings highlight the potential results illustrate the possibility that many reactions
regulatory role that Zn2+ may have in biological affected by Zn2+ may have been overlooked in
interactions in blood when this metal ion is plasma because of the use of citrate as an
released from activated platelets (4,31). The Zn2+- anticoagulant.

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Zn2+ mediates high affinity binding of heparin to fibrinogen

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10