Biomedical Engineering Reference
In-Depth Information
on the terminus of each LMWH molecule, the chains are all connected to the AT moiety in the same
way. The anti-FXa activity reported for the LMWH used was slightly lower than that of starting
UFH (Table 18.1) and analyses showed that conjugates contain an average of 0.7 chains per AT.
247
Experiments with mixtures of Björk et al. ATH
+
FXa indicated that 98% of the complex was
AT, reactions with Björk et al. ATH were only mildly reduced
by 1 M NaCl or polybrene (suggesting that the covalent linkage prevented disruption of AT activa-
tion by the LMWH).
247
Lack of polybrene neutralizability would prevent the capability of reversing
anticoagulation by the agent if there were hemorrhagic complications. Assays of the LMWH used
for Björk et al. ATH gave no detectable anti-thrombin catalytic activity (Table 18.1),
249
making the
complex ineffective against thrombin. Thus, the Björk et al. complex could not address thrombin
feedback of the coagulation cascade.
Mitra and Jordan, who were able to overcome previous failures employing CNBr as an activat-
ing reagent, have reported another active ATH product.
250
UFH
active and, unlike starting LMWH
+
57-fold molar excess of CNBr
were reacted at pH 10.7 for 40 min at 23°C. After dialysis under basic conditions to remove unre-
acted CNBr, the activated heparin was reacted with AT (1 mol heparin per 0.02 mol AT) at pH 9.4
for 18 h at 5°C. During incubation, cyanate/iminocarbonate/N-nitrile active groups on the modi-
fi ed heparin reacted with lysyl amino groups on the AT to form amidine ester (
+
-
O
-
CNH
-
NH
-
),
urethane (
) linkages.
Removal of free heparin using immobilized Concanavalin A and removal of unreacted AT by heparin-
sepharose chromatography gave a 40% yield of ATH with respect to starting AT.
250
Although no
data was reported on the number of activated groups initially incorporated into the heparin by
CNBr,
250
use of glycine to block excess active cyanate/iminocarbonate/N-nitrile groups after AT
coupling
250,251
suggests that there was more than one reactive group per heparin and likely
-
O
-
CO
-
NH
-
), guanidine (
-
NH
-
CNH
-
NH
-
), or urea (
-
NH
-
CO
-
NH
-
1 GAG
per AT in the fi nal conjugate. Thrombin and FXa titrations of Mitra and Jordan ATH indicated
that almost the entire product had potent inhibitory activity. Furthermore, the Mitra et al. ATH
exhibited elevated fl uorescence (relative to free AT) that was not enhanced further by addition
of UFH, indicative of the fact that the AT moiety in all ATH molecules was fully activated by
the covalently bonded heparin. Experiments studying inhibition of thrombin and FXa by Mitra
and Jordan ATH in plasma suggested that inhibitory capacity was solely from direct reaction, with
no extra catalytic activity evident.
250,251
Rate experiments were performed with Mitra and Jordan
ATH
≥
thrombin at equimolar concentrations, which allow for calculation of a biomolecular rate
constant.
250
Results for the ATH gave a biomolecular rate of 6.7
+
10
7
M
-
1
s
-
1
(Table 18.1), while
×
similar experiments with noncovalent AT
+
high-AT-affi nity heparin gave a biomolecular thrombin
10
7
M
-
1
s
-
1
, 3.3-fold slower than for Mitra and Jordan ATH. However, due
to the fact that measurements were not made with inhibitor concentrations at 5 to 10 times greater
than the enzyme (pseudofi rst order conditions), comparison of the absolute reaction rates between
covalent and noncovalent complexes is not possible.
Recent work by Chan et al.
141
has provided a new direction in ATH development. The
fundamental difference in Chan et al.'s ATH construction is that no prior modifi cation of either
the AT or UFH reactants used to make the conjugate is required. Preparation of conjugate relies
on novel observations on natural spontaneous reactions
in vivo
and the presence of a subpopula-
tion of molecules within many commercial UFHs. Investigations in diabetic patients have estab-
lished that hemoglobin and several plasma proteins undergo spontaneous glycation. Schiff base
formation occurs between the C1 aldehyde of the open chain form of plasma glucose and the
ε-amino of blood protein lysyl residues.
252
Bonding between sugar and polypeptide is stabilized
by tautomeric Amadori rearrangement of protons on the glucosyl C1 and C2 to an ene-ol-amine
and, eventually, a stable keto-amine.
252
The formation of these long-lived products in diabetics
is signifi cantly affected by glucose concentration, availability of amino groups, pH, and tem-
perature. Nonenzymatic glycation of proteins has been shown to have measurable effects on
the function of the molecule.
253
For example, brief increases in circulating glucose were shown
to coincide with transient changes in AT activity,
254
while long-term exposure to high-glucose
inhibition rate of 2.0
×
