Biomedical Engineering Reference
In-Depth Information
kidney (BHK) or chinese hamster ovary (CHO) cells have been shown to possess a 10 times lower
UFH affi nity
68,104
because of variable glycosylation on Asn155.
105
However, the major AT glyco-
forms that have been shown to have signifi cant carbohydrate-related structure-function differ-
ences are the 4 glycan-containing α-AT and β-AT species that is missing a glycan at Asn135.
76,75
Of these human plasma-derived products, α-AT has reduced heparin affi nity relative to β-AT,
76,106
which is not surprising since the Asn135 glycan is within the region of Lys125, Arg129, and Arg145
UFH-binding site residues. Therefore, it might be thought that covalent ATH preparations may
tend to have a bias toward β-AT since there is no physical obstruction from the Asn135 glycan for
heparin's approach during synthesis. However, steric issues between the Asn135-AT glycan and
UFH with respect to AT binding to heparin are not the only factors. Rapid heparin-binding kinetic
studies revealed that the glycan at Asn135 gives only a small reduction in initial weak binding.
106
It seems that the rate of conformational change to the fi nal high-affi nity complex with heparin is
slower for α-AT, leading to an overall reduction in affi nity relative to the β form.
106
Va r iat ion i in
heparin affi nity between the two AT isoforms has extended to signifi cant
in vivo
effects. Heparin-
like GAGs such as heparan sulfate contain the pentasaccharide AT-binding sequence.
107
Research
has shown that affi nity to these heparinoids translates into regulation of AT pharmacodynamics
since most β-AT is bound to heparan sulfate on vessel surfaces while the vast majority of α-AT
remains in the circulation.
70
These data provide the potential for targeting heparin to areas of
vascular damage by covalent linkage to a low glycan-containing AT that has increased vessel wall
GAG affi nity. Practical examples of such a design will be described later (Section 18.7).
18.3 HEPARIN
18.3.1 C
HEMICAL
S
TRUCTURE
OF
H
EPARIN
UFH is derived from natural heparin in the mucosa of intestine and lung.
108
Heparin itself is part of
the broad GAG classifi cation of molecules
109
present in multi and single-celled organisms.
110
GAGs
are polymers of uronic acid-hexosamine disaccharides that are synthesized in variable length
straight chains.
111
Different subcategories of GAGs are defi ned by the type of uronic acid and/or
the hexosamine makeup as well as type and degree of substituent groups present on the saccharide
residues. For example, heparin and heparan sulfate have glucosamine, while chondroitin sulfates
such as dermatan sulfate have galactosamine.
111
With respect to the uronic acid moiety, heparins
and heparan sulfates are composed of mixtures of glucuronic and iduronic acids, but molecules of
dermatan sulfate have only iduronic acid and other chondroitin species have molecules with only
glucuronic acid residues.
111,112
Further refi nement of structure is gained by groups present on the
saccharide rings. Glucosamines in heparin and heparan sulfate are either N-acetylated or N-sulfated,
while galactosamines of dermatan sulfate and chondroitin sulfates are mainly N-acetylated. Between
heparin and heparan sulfate some statistical differences in hexosamine sulfation occur. Thus,
80% of
heparin glucosamines are N-sulfated but roughly half of heparan sulfate glucosamines are sulfated.
113
Combined with the degree of O-sulfation in heparin (
>
2 O-sulfates/disaccharide)
114
relative to
heparan sulfate (from 0.75 to 0.2 O-sulfates/disaccharide),
113
many features distinguish these GAGs
as distinctly separate biosynthetic groups of molecules.
As stated, UFH is prepared from the native heparin in tissue. Heparin chains are built up as
a posttranslational modifi cation on a core protein within mast cells. Heparin GAGs, ranging from
60,000 to 100,000 Da, can number up to 10 per polypeptide and are attached to serine residues
through O-glycosidic bonds.
115
Biosynthesis of heparin chains initiates by stepwise glycosyl-
transferase addition of monosaccharides to form a xylose-galactose-galactose-glucuronic acid
linkage region sequence, the terminal xylose residue being glycosidically linked to a serine residue
in the core polypeptide.
116
Chain growth continues to make a repeating disaccharide structure
in which uronosyl-β-1
>
4 bonds.
117
As indicated
above, many functional group substituents are simultaneously added as the polysaccharide chain is
→
4 glycosaminosyl units are linked by α-1
→
