Biomedical Engineering Reference
In-Depth Information
synthesized. These modifi cations involve N-acetylases,
118
N-deacetylases,
118
O-sulfotransferases,
119
N-sulfotransferases,
120
glycosyltransferases,
121
and glucuronosyl-5-epimerases.
119
The order and
degree of action for the various modifying enzymes is intricate and interrelated, which defi nes
the fi nal structures produced. Initially, as the nascent heparin chain grows, freshly synthesized
regions are partially acetylated, sulfated, or unsubstituted on the glycosaminosyl nitrogen groups.
122
Both during and after this period,
122
the evolving chain is increased in N-sulfate by action of an
N-deacetylase and an N-sulfotransferase.
118,122
Further modifi cations proceed following N-sulfation,
with the O-sulfate- and uronic acid-type content within the fi nal product being interdependent. Not
long after the N-sulfotransferase reactions, a glucuronosyl-C5-epimerase converts glucuronosyl
residues at the end of the chain into iduronosyls.
119
Although the epimerase drives the reaction
toward the iduronosyl form, there is a signifi cant reverse reaction that is only blocked if C6 of
the glucosaminosyl residue or C2 of the iduronosyl residue becomes O-sulfated.
119
Within this
O-sulfation pathway, interrelationships also exist whereby glucosaminosyl C6-O-sulfation can
occur with or without 2-O-sulfate groups on the neighboring iduronosyl residue, but C2-O-sulfation
of iduronosyls requires neighboring glucosaminosyl residues to be non-O-sulfated at the C6
position.
123
Furthermore, uronosyl residues are left nonsulfated, if glycosyl transfer of the next
glucosamine occurs before the C2-O-sulfotransferase can act.
123
Apparently, O-sulfation is fairly
effective during heparin GAG synthesis since
∼
78% of uronic acid residues in commercial heparins
are iduronic
117
and
75% of the iduronic acids are 2-O-sulfated.
117,124
By and large, both chain
modifi cation and length of heparins on the core protein are quite variable and partly controlled by
conditions within the synthetic space like substrate availability and cell energetics.
125,126
An important aspect for the type of UFH available for AT-heparin conjugation is the
in vivo
and
in vitro
processing that happens before and during commercial isolation. Heparin chains on
proteoglycans produced by mastocytoma cells undergo partial depolymerization by an endogluc-
uronidase before storage in cytoplasmic granules.
127,128
This degradation within intestinal mucosa
or lung mast cells is responsible for the reduced molecular weight range of 5000-30,000 observed
in the free heparin chains of UFH prepared commercially. More recently, low-molecular weight
heparin (LMWH) has been produced by partial depolymerization of UFH using HNO
2
, base elimi-
nation after partial esterifi cation of uronic acid carboxyls, heparinases, and heparitinases.
129,130
The
reduced molecular weights of LMWHs (from 1800 to 12,000)
129
gives improved pharmacokinetics
and biological characteristics relative to the starting UFH.
∼
18.3.2 F
UNCTIONAL
B
IOCHEMISTRY
OF
H
EPARIN
In vivo
, heparin and other GAGs mainly exist as proteoglycans that function as part of the structural
architecture of the extracellular matrix and as a chemoattractant during processes in tissue.
131,132
In addition, heparin chains provide anticoagulant activity because of the ability to bind to AT or
heparin cofactor II and catalyze their inhibition reactions with coagulation factors.
133
UFH catalysis
of coagulation factor inhibition by AT
in vivo
134
mostly involves the neutralization of thrombin and
FXa.
135,136
Of these two coagulants, it has been shown that enhancement of thrombin's reaction with
AT is the major mode of action for UFH clinical application.
137
In order to assist the
in vivo
inhibition
of thrombin and FXa, heparin molecules must contain a specifi c pentasaccharide sequence
that selectively binds with high affi nity to AT.
138
In native rat skin mast cell proteoglycans, the
pentasaccharide has been shown to occur variously
139
with most proteoglycans having chains with-
out any pentasaccharides, while a minority of proteoglycans had GAG moieties with 1-5 pentasac-
charides per chain.
140
Once fi nal commercial production has been carried out, only one-third of
UFH molecules on the average end up containing the high AT-affi nity pentasaccharide.
141
However,
very small subpopulations within the polydisperse commercial UFH preparations do exhibit two
pentasaccharide sequences per molecule,
142
a fact that can have signifi cant impact in some ATH
synthetic designs. With regard to LMWH, however, the chain cleavages of UFH required to make
these smaller molecules lead to a reduction of intact pentasaccharide.
136,143
In order to determine
