Biomedical Engineering Reference
In-Depth Information
Finally, signifi cant yields of AT-heparin conjugate were achieved by Ceustermans et al.
231
Preliminary attempts involved reaction of heparin with tolylene-2,4-diisocyanate according to the
method of Clyne et al.
239-241
using basic conditions to increase amino (or hydroxyl) reactivity with
the isocyanates. A very modest yield (
5%) was realized that was much reduced in ice-cold pH
9.5 borate because of accelerated hydrolysis of the isocyanates on the substituted heparin, prior to
addition of AT. However, heparin modifi cation with the more stable tolylene-2,4-diisothiocyanate
(TDTC) according to Edman and Henschen,
242
prior to reaction with AT in pH 8.5 bicarbonate,
gave a much improved 30% yield.
231
Further optimization of the process followed with the introduc-
tion of amino groups into the starting heparin. This was accomplished either by partial desulfation
of glucosamine aminos or by the attachment of amino-terminating spacer arms to uronic acid
carboxyls.
231
UFH was fi rst chromatographed on immobilized AT to obtain the high-affi nity frac-
tion for modifi cation. The pyridinium salt of high-affi nity heparin, prepared by pyridine titration
of the heparinic acid form, obtained by passage through Dowex 50, was partially N-desulfated by
incubation in 95% dimethylsulfoxide at 23°C for 0.5 h.
243,244
Alternatively, high-affi nity heparin
was conjugated through the uronic carboxyl groups to 1,6-diamino-hexane using a carbodiimide.
231
Both amino-modifi ed heparins were reacted with TDTC (in excess to the amino groups present) to
give products with isothiocyanate functions for linkage with AT. Incorporation of isothiocyanate
groups was improved 2-fold by partial N-desulfation and 2.9-fold by hexyl-amino modifi cation.
231
However, ATH yield from either N-desulfated or hexyl-amino heparins remained at approximately
30% and up to 25% of the product molecules contained two ATs per heparin chain.
231
Table 18.1
gives activities from analyses of materials in such studies. N-desulfation decreased activated par-
tial thromboplastin time (APTT), clotting time by 35% and anti-FXa activity (measuring ability to
catalyze reaction of FXa with added AT) by 24%, compared to the starting high-affi nity heparin
(Table 18.1). Addition of hexyl-amino groups disrupted heparin function to an even greater degree
causing 44% loss in APTT and 39% loss in anti-FXa (Table 18.1). Major functional reduction in
both partially N-desulfated heparin and carboxyl-linked diamino-hexyl heparin would be expected
since N-sulfates and carboxyls are key structures responsible for heparin anticoagulant activity.
245
Noncatalytic direct inhibition of FXa by hexyl-amino ATH was measured to have a second order
rate constant of 2.1
≤
10
6
M
-
1
s
-
1
(Table 18.1), one-third of that value was for noncovalent mixtures
of high-affi nity heparin and saturating amounts of AT.
231
It is possible that not all AT was being
activated by the conjugated heparin since supplementation with additional heparin signifi cantly
increased the rate of FXa inhibition by the hexyl-amino ATH.
231
To eliminate inactive complexes
(some of which may contain two AT perheparin chain), purifi cation of hexyl-amino ATH product
was performed on sepharose-conconavalin A,
246
in addition to chromatographic separations by ion
exchange, gel fi ltration, and sepharose-AT.
231
Test s on t he d fi re ct fi n h ibit ion of t h rombi n by t h is h ig h ly
purifi ed hexyl-amino ATH gave a second order rate constant of 6.7
×
10
8
M
-
1
s
-
1
and a biomolecular
×
10
8
M
-
1
s
-
1
, which was similar to the bimolecular thrombin inhibition rate of
rate constant of 2.5
×
10
8
M
-
1
s
-
1
for high-affi nity heparin and saturating AT.
246
ATH was prepared by Hoylaerts
et al. from high-AT-affi nity hexyl-amino-derivatized molecular weight fractions (
M
r
2.2
×
4300 or
3200) of HNO
2
-reacted UFH but complexes gave reduced second order rate constants for direct
thrombin inhibition (2
=
10
5
M
-
1
s
-
1
for 4300 and 3200
M
r
fractions, respec-
tively) relative to ATH from full-length heparin.
246
This is consistent with a loss in the required
heparin-binding of thrombin with short LMWH chains during inhibition by AT.
155
Work by Björk et al., around the same time period as Ceustermans et al., resulted in ATH that
was attached to the end groups of partially depolymerized heparin fragments.
247
High-AT-affi nity
fractions of HNO
2
-treated UFH (having MW ranging from 3700
247
to 10,000
248
using this partial
depolymerization protocol) were incubated with AT
10
7
M
-
1
s
-
1
and 3
×
×
NaBH
3
CN.
247
In this system, the aldehydes
on anhydromannose terminal residues of the heparin fragment form metastable Schiff bases with
AT lysyl ε-amino groups, that are fi xed by reduction.
247
ATH was separated from unreacted LMWH
by gel fi ltration and fi nally isolated from unreacted AT on a heparin agarose column, to give an
yield of 40% ATH from the starting AT. Since the coupling reaction occurs only at one active group
+
