A functional assay for heparin in serum using a designed synthetic receptor

Article abstract of DOI:10.1002/anie.200501437

(Graph Presented) Excellent selectivity in highly competitive serum was displayed by a synthetic fluorescent receptor incorporating phenylboronic acid and ammonium groups for the therapeutic anticoagulant heparin. The concentration of heparin at clinicall

Full text of DOI:10.1002/anie.200501437

Angewandte
Chemie
Molecular Recognition
cationic protein protamine is an excellent ligand for heparin
and is often used for the calibration of aPTT reagents. The
highly anionic nature of heparin makes it an ideal target for
synthetic receptors that incorporate functional groups for
molecular recognition which participate in ion-pairing or
other anion-binding interactions.
DOI: 10.1002/anie.200501437
A Functional Assay for Heparin in Serum Using a
Designed Synthetic Receptor**
The design of heparin receptor 1 (Figure 1a) builds upon
the successes of synthetic receptors reported previously. For
Aaron T. Wright, Zhenlin Zhong, and Eric V. Anslyn*
A common objective when developing synthetic receptors is
high binding affinity and specificity for an analyte. This aim is
challenging when targeting a complex analyte in a compet-
itive crude medium such as serum, urine, or saliva.[1] We
report herein the development of a synthetic receptor with
high affinity and selectivity in human and equine serum for
the clinical anticoagulant heparin.[2]
Two forms of heparin are in clinical use: unfractionated
heparin (UFH) with a molecular weight range from 3000–
30000 Da, and low-molecular-weight heparin (LMWH) with
a mean molecular weight of 5000 Da. Heparin concentration
and activity is monitored during surgery, and in post-
operative therapy, to prevent complications such as hemor-
rhaging.[3] Current methods for heparin quantification employ
the activated clotting time (ACT), activated partial thrombo-
plastin time (aPTT), chromogenic antifactor Xa assay,
electrochemical and piezoelectric assays, and complexation
with protamine.[4] Although these are proven methods, they
are often arduous, inaccurate, costly, and not always amenable
to clinical settings.[5] Recently, an engineered GST fusion
protein containing three hyaluronan binding domains from a
heparin binding protein was used to accurately measure
heparin in plasma at clinically relevant values, but it has not
been employed clinically.[6]
Heparin cannot be expressed exactly using a conventional
chemical formula. It is a heterogeneous mixture of diverse
chain lengths consisting of repeating copolymers of 1!4-
linked iduronic acid and glucosamine residues in a semi-
random order (Figure 1b). Heparin has the highest anionic
charge to mass ratio of any biopolymer, as a result of
numerous sulfate and carboxylate functionalities in the
biopolymer chain. Its activity occurs by binding to anti-
thrombin III, a naturally occurring protease inhibitor, which
accelerates the rate of inhibition of coagulation proteases
factor Xa and thrombin by antithrombin III.[7] Clinically
administered heparin binds to its natural substrate anti-
thrombin III primarily through cationic ion-pairing interac-
tions with the sulfate and carboxylate groups.[8] Similarly, the
Figure 1. a) Heparin receptor 1. b) Major unit of heparin.
example, we recently created receptor 2 (Figure 2), which
incorporates phenylboronic acids and ammonium groups, for
the complexation of anionic polysaccharides such as heparin,
hyaluronic acid, and chondroitin-4-sulfate.[9] Phenylboronic
acids with an ortho-aminomethyl group are known to form
boronate esters with alcohols in aqueous media, and have
been used extensively for the molecular recognition of
saccharides.[10] It has been found that these boronate esters
have particularly high affinity for anions with closely
appended hydroxy groups.[11] Furthermore, ion pairing is
anticipated between the ammonium functionalities and the
carboxylate and sulfate groups. Receptor 2 had good selec-
tivity for heparin relative to the other anionic polysaccha-
rides. However, because heparin does not consist of a single
entity, its concentration must be defined by considering
[*] A. T. Wright, Dr. Z. Zhong, Dr. E. V. Anslyn
Department of Chemistry and Biochemistry
The University of Texas at Austin
Austin, TX 78712 (USA)
Fax: (+1)512-471-8696
E-mail: Anslyn@ccwf.cc.utexas.edu
[**] We gratefully acknowledge support for this work from the US NIH
(EB00549).
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Figure 3. 1:1 binding isotherm for 1 and UFH. The Ka value obtained
from the curve was 1.4”108 mꢀ1
.
Figure 2. Previously reported receptor that had insufficient sensitivity
to be active and accurate in a biological medium.
various subunits so that a binding constant can be measured.
A binding affinity of 3.8 ” 104 mꢀ1 was calculated when using a
heparin concentration that is defined by the concentration of
each disaccharide unit. This affinity was insufficient to allow
quantification of heparin in serum at physiologically relevant
concentrations. Moreover, although an indicator-displace-
ment assay with 2 gave a dramatic yellow to purple color
change in response to heparin,[12] we found that the indicator
bound nonspecifically with proteins in crude serum, and
thereby skewed the quantitative results.
From the lessons gleaned from the studies using 2, we
designed a second-generation heparin receptor with two goals
in mind, both of which are embodied in the structure of 1.
First, the cavity was enlarged to allow the arms containing the
boronic acid and ammonium groups to encompass a larger
surface of the oligosaccharide, which we predicted would
raise the affinity by cooperatively increasing the number of
interactions. Second, a fluorescent scaffold was incorporated
into the design of the receptor, thereby avoiding the use of an
indicator-displacement assay. This new signaling technique
should also increase the overall sensitivity of the system. We
used 1,3,5-triphenylethynylbenzene as the core unit to satisfy
both requirements. However, because of the excellent
selectivity previously found for 2 with heparin, we retained
the side arms containing the boronic acid and ammonium
groups.
repeating unit with which the receptor interacts. The binding
isotherm shown in Figure 3 was achieved by defining the
concentration of heparin to be that of four saccharide units
(an integral number of saccharides is not required to fit the
binding isotherm). The number four supports a stoichiometry
where each receptor on average spans four saccharide units
along the heparin biopolymer. The calculated association
constant between 1 and UFH is 1.4 ” 108 mꢀ1. This value
corresponds to an increase in affinity of nearly 104 for 1 over
2, which was gained by increasing the size of the scaffold. It
should be noted that the glycosaminoglycuronans hyaluronic
acid and chondroitin-4-sulfate did not bind to 1 at low mm
concentrations. This result is further evidence for the high
selectivity of 1 for UFH and LMWH.
As noted above, several clinical methods (such as aPTT)
are calibrated by titration with protamine. Protamine seques-
ters heparin, thereby lowering its bioavailability to bind
antithrombin III. Therefore, if there is a specific binding
interaction between heparin and 1 as postulated above,
protamine should strip heparin from receptor 1, thereby
restoring the fluorescence. Indeed, fluorescence could be fully
reestablished by titration of the complex formed between
receptor 1 and either UFH or LMWH with protamine
(Figure 4). This observation illustrates that the binding
between 1 and heparin is reversible, and acts in an analogous
way to that between heparin and antithrombin III.
An assay for UFH or LMWH in serum requires a binding
interaction between 1 and heparin in the nm range. Titrations
of 1 with UFH and LMWH in water buffered with 10 mm 2-[4-
(2-hydroxyethyl]-1-piperazinyl]ethanesulfonic acid (HEPES)
at pH 7.4 were monitored by fluorescence spectroscopy to
determine the affinity of 1 for UFH and LMWH. The binding
of UFH and LMWH with 1 caused a decrease in the emission
intensity, which resulted in a near complete quenching of the
receptorꢀs emission. Presumably, the interaction of heparin
with 1 leads to conformational restriction of the receptor
“arms”, thereby modulating the fluorescence—a technique
used routinely by Finney and co-workers for creating chemo-
sensors.[13] Titration data at 357 nm was used to generate a
binding isotherm, which was analyzed by using a standard 1:1
binding algorithm (Figure 3). As discussed above, the hetero-
geneous structure of heparin means that one must define a
Our last study targeted the creation of calibration curves
for monitoring UFH and LMWH in serum. Heparin is
administered intravenously or subcutaneously at therapeutic
dosing levels of 2–8 UmLꢀ1 (0.8–3.2 mm) during cardiopul-
monary surgery and emergency deep venous thrombosis
(DVT) conditions to prevent excessive clotting. However,
patients are treated at therapeutic dosing levels of 0.2–
2 UmLꢀ1 (0.08 mm–0.8 mm) in post-operative and long-term
anticoagulant care of DVT. Human and equine serums were
doped with UFH and LMWH at these dosing levels to
simulate monitoring conditions in a clinical setting. A serum
sample (32 mL) doped with UFH or LMWH was added to a
fluorimeter cell containing a total volume of 1.5 mL HEPES
(10 mm) in deionized water. To this was added 2 mL of 1
(2.24 ” 10ꢀ3 mꢀ1). The fluorescence stabilized over a period of
18 minutes, which contrasts the instantaneous response found
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Angewandte
Chemie
Figure 5. Calibration curves for a) LMWH in human serum and
b) UFH in human serum. Conformational changes to 1 upon binding
to heparin result in a diminished fluorescence emission. Greater con-
centrations of UFH and LMWH result in increased fluorescent quench-
ing. The range of detection for both UFH and LMWH was 0–9 UmLꢀ1
(0–3.6 mm).
Figure 4. Reversibility of LMWH:1 binding upon titration with prot-
amine. a) Emission intensity at 357 nm. Dilution was carried out with
aliquots of buffered water (HEPES, pH 7.4). First 11 aliquots of hepa-
~
rin were added ( ), and then 12 aliquots of protamine were added
*
( ). The fluorescent emission was reestablished upon titration.
b) Emission spectrum of 1 upon addition of LMWH. The addition of
protamine reverses the spectrum.
can be created to target very complex bioanalytes, and
function successfully in physiological settings.
Received: April 26, 2005
Published online: August 5, 2005
in buffered water, and indicates that the formation of the
complex with heparin in serum was slow on the laboratory
timescale. This result is potentially a consequence of the
kinetics of release of heparin from natural receptors in the
sera. Emission spectra were recorded after 18 minutes for
each of nine samples with various heparin concentrations to
generate calibration curves (355 nm, Figure 5). Increased
levels of heparin in serum correlated linearly with lower
emission responses for both UFH and LMWH within the
range of clinically relevant concentrations, as was observed
for the fluorimetric titrations using pure heparin in buffered
water. Furthermore, the method worked in both equine and
human samples, thus illustrating that the affinity of the
synthetic receptor for heparin is independent of the mamma-
lian source, and could potentially be used for either human or
veterinary applications.
In summary, we have demonstrated a functional synthetic
fluorescent assay for the clinically administered anticoagulant
heparin. The synthetic receptor highlighted herein showed
remarkable selectivity and affinity for heparin, even in crude
serum. The fluorescence emission was used to generate
calibration curves for UFH and LMWH in serum at clinically
relevant dosing levels (0.2–8.0 UmLꢀ1). These calibration
curves allow the heparin concentration in an unknown sample
to be determined by comparative analysis of the fluorescence
emission. This study demonstrates that synthetic receptors
Keywords: fluorescence · heparin · host–guest systems ·
.
receptors · supramolecular chemistry
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