1. health and fitness
2. disease

Evaluation of Plasma Alpha-2-Macroglobulin and Interactions with
Tumour Necrosis Factor-Alpha in Horses with Endotoxemic Signs
Nathalie Cote, Donald R. Trout, and Anthony M. Hayes
is neither activated nor depleted leur forme native. Peu d'attacheduring endotoxemia, and (3) the ment a ete note entre 1251-FNThr et
The electrophoretic position and binding interactions between equine la Ma2 ou aux autres proteines plasbehavior of the native and activated a2M and TNF-a are too low to matiques equines. Un attachement
forms of equine plasma alpha-2- implicate equine a2M as a regulator faible entre '251-FNThr et les formes
macroglobulin (a2M) were charac- of TNF-a during endotoxemia in native et activee par le methylamine
terized and compared to human horses.
de Ma2 a ete observe, avec un meila2M by nondenaturing polyacryleur attachement a la forme activee.
lamide-gel electrophoresis (PAGE).
Cette etude a permis de demontrer
Plasma a2M was also compared
RESUMI#
que: (1) la Ma2 equine plasmatique
between 6 normal horses and
se comporte de facon similaire a la
6 horses with clinical signs of colic
La migration et le comportement Ma2 humaine lors de l'e'lectroand endotoxemia due to volvulus or de la macroglobuline alpha-2 (Ma2) phoreses; (2) la Ma2 plasmatique
enteritis. Native and activated forms plasmatique equine, sous sa forme equine peut etre activee en une
of a2M were quantified by PAGE native et activee, ont ete caracte- forme plus rapide lors de l'elecand densitometry. Binding of radio- rises et compares a la Ma2 humaine trophorese, mais n'est pas activee
labeled recombinant human tumour suite a une electrophorese sur gel de ou epuisee lors d'une endotoxemie;
necrosis factor-alpha (1251-rhTNF-a) polyacrylamide non-denaturant (3) les reactions d'attachement
to native and activated forms of (PAGE). Nous avons compare entre la Ma2 et le FNT-a sont trop
equine a2M was also evaluated by egalement la Ma2 plasmatique de faibles pour associer la Ma2 comme
autoradiography and densitometry six chevaux normaux et de six un regulateur du FNT-a lors d'une
of PAGE. Equine plasma a2M chevaux avec des signes cliniques de endotoxemie chez les chevaux.
(Traduit par docteur Serge Messier)
migrated as a single band at a posi- colique et d'endotoxemie suite a un
tion equivalent to native human volvulus ou une enterite. Les formes
a2M. Methylamine-reacted equine native et activee de Ma2 ont ete
INTRODUCTION
plasma samples resulted in faster quantifiees par PAGE et densitomigration of a2M in a similar posi- metrie. L'attachement de facteur
Endotoxemia and septic shock are
tion to activated human a2M. How- alpha de necrose tumorale humain
ever, in methylamine-reacted equine recombinant marque (1251-FNThr) important problems in various forms
plasma, an intermediate a2M band aux formes native et activee de Ma2 of acute gastrointestinal disease.
was consistently present between equine a egalement ete evalue par Compared to other species, horses
the bands corresponding to native autoradiographie et densitometrie. have been shown to be relatively senand activated a2M. Amounts of La migration de Ma2 plasmatique sitive to the effects of bacterial endoplasma a2M were similar in equine apparaissait sous une bande toxin (1). Endotoxin is a lipopolysacnormal and endotoxemic horses, unique a une position equivalente a charide, with a toxic component
and remained in the electrophoreti- la Ma2 native humaine. La Ma2 des located in the lipid-A fraction, from
cally slow or unreacted native form. echantillons de plasma equin traites the outer cell wall of various gramThe vast majority of '251-rHuTNF-a au methylamine avait une migra- negative enteric bacteria (2,3). Endodid not bind to a2M or other equine tion plus rapide et se retrouvait a toxin elicits some of its toxic effects
plasma proteins. 1251-rHuTNF-a une position similaire a la Ma2 by activating the release of inflammabound weakly to both native and humaine activee. Toutefois, dans les tory mediators from host cells, espefast methylamine-reacted equine echantillons de plasma traites, une cially macrophages (4). These inflamforms of a2M, although binding was bande intermediaire de Ma2 etait matory mediators include arachidonic
better to the activated form. This presente entre les bandes corres- acid metabolites, such as thromboxstudy indicates that: (1) equine pondant aux formes native et acti- ane and prostaglandins E2 (PGE2) and
plasma a2M behaves similarly to vee de Ma2. Les quantites de Ma2 12 (PGI2) (5,6). Other biologically
human a2M on PAGE, (2) plasma plasmatiques etaient semblables active cytokine mediators, such as
a2M of horses can be activated to chez les chevaux normaux et endo- interleukin-1 (IL-1) and tumour
electrophoretically fast forms, but it toxetmiques, et demeuraient sous necrosis factor-alpha (TNF-a), are
ABSTRACT
Department of Large Animal Surgery (Cote, Trout), Department of Pathology (Hayes), University of Guelph, Guelph, Ontario NI G 2W1.
Submitted May 30, 1995.
150
Can J Vet Res 1996; 60: 150-157
TABLE I. Clinical and laboratory findings from horses with signs of colic and endotoxemia
Mucous Membranes
Refill
(s)
Colour
Horse
4
Congested, cyanotic
I
3
Congested, cyanotic
2
4
Congested, cyanotic
3
3.5
4
Congested, cyanotic
2
Congested
5
4.5
Congested, cyanotic
6
3.5±0.9
Mean±SD
2-4.5
Range
secreted by macrophages in response
to absorbed endotoxin (4,7,8).
TNF-a has been proposed to participate in endotoxin-mediated shock of
various mammals (7,9,10). In vitro
and in vivo, TNF-a produced in
response to endotoxin binds to receptors expressed in various cells and
tissues (7). Several studies have
demonstrated marked tissue damage,
resembling that seen in endotoxemia,
after administration of TNF-ot (11-13).
The tissue damage caused by TNF-ot
in endotoxic shock is partially due to
its ability to stimulate the secretion of
other inflammatory mediators, such as
PGE2 (12,14,15), PGI2 (16), colonystimulating factors (17), IL-1 (18,19)
and fibroblast growth factor (20). It
has also been demonstrated that TNF-ot
inhibits thrombomodulin on endothelial cells (21), increases procoagulant
activity, and enhances both hematopoietic growth factor (22,23) and
platelet activating factor (24,25).
These changes result in a net tendency
for increased thrombosis, which is an
important cause of tissue ischemia in
endotoxemia and septic shock. Other
biological activities of TNF-ot include
induction of nitric oxide secretion by
endothelial cells and macrophages
(26), induction of leukocyte adhesion
molecules on vascular endothelium
(27), and activation of polymorphonuclear neutrophils (28) and platelets
(29).
Because neutralizing antibodies
directed against TNF-a reduce the
detrimental effects of endotoxin if
given prior to endotoxin challenge
(9,30), TNF-a appears to play an
important role in the cascade of many
pathophysiologic aspects of endotoxemia. Accordingly, recent attention
has been directed toward endogenous
or exogenous factors that might counteract TNF-cx or TNF-xa-induced
Extremities
Normal
Normal
Cold
Cold
Cold
Cold
Temp
(OC)
38.1
39.7
40.3
38.7
38.0
Unknown
38.8±1.1
38.0-40.3
Heart rate
/min
104
88
76
100
52
52
77±23
52-104
PCV
(%)
70
73
80
49
47
47
61±15
47-80
inflammatory mediators. Alpha-2macroglobulin (at2M) is a large glycoprotein produced mainly by hepatocytes and is present in high
concentrations in plasma (31). A
major function of ca2M is to act as a
molecular trap for various proteinases
(32); however other roles have been
described. Binding with various proteinases results in conformational
changes in cx2M, along with fast
migration on nondenaturing electrophoretic gel (33,34). Reaction of
a2M with methylamine results in the
same conformational and mobility
change in humans (34). This activated
or fast form of ot2M is rapidly cleared
from the circulation by hepatocytes,
macrophages and other cells that
express appropriate receptors (35,36).
The native or slow form of a2M is not
recognized by a2M receptors and has a
prolonged half-life in the circulation
(36). The a2M has also been shown to
bind to various cytokines, including
TNF-a (37-41), and conformation of
cx2M influences this cytokine binding
(37,39-43). In human, rat, mouse,
swine and bovine plasma, TNF-ot preferentially binds to the activated form
of cx2M which is rapidly cleared from
the circulation (44). However, this
apparent binding affinity of human
fast cx2M to TNF-ax is much lower than
for other cytokines, such as transforming growth factor PI or P2 (45).
In the present study, we examined
the possibility that a2M may act as a
TNF-binding protein and may be activated by proteinases during endotoxemia/sepsis in horses. The objectives
were: (1) to determine if the amount
of native or activated cx2M was altered
in the plasma of clinically endotoxemic horses, and (2) to evaluate binding between exogenous radiolabeled
rhTNF-a and the different forms of
plasma at2M in horses.
Plasma
protein (g/L)
96
73
55
65
63
54
68±16
54-96
Surgical or
necropsy diagnosis
Enteritis
Volvulus, jejunum
Volvulus, large colon
Volvulus, large colon
Volvulus, large colon
Volvulus, large colon
MATERIALS AND METHODS
Blood samples were initially collected from 5 horses presented to
the Veterinary Teaching Hospital,
University of Guelph with various
abnormalities (1 pleuritis, 1 colitis,
1 colonic volvulus, 1 normal pregnant
mare, and 1 normal horse). These
horses ranged from 2 to 10 y (mean
5.4 ± 3.4 y). Blood samples were subsequently collected from 12 additional horses presented at the Veterinary Teaching Hospital, University of
Guelph; 6 were clinically normal and
6 had signs of colic and endotoxemia.
These 12 horses ranged from 1 to 16 y
(mean 5.9 ± 3.8 y). Signs of endotoxemia included tachycardia, cyanotic
congested mucous membranes, slow
capillary refill time, cold extremities,
and dehydration (Table I). Each sample was collected in a citrate tube,
centrifuged, and the supernatant
frozen in a plastic tube at -70°C until
the time of analysis.
To prepare the activated form of
cx2M, 300 pL of each plasma sample
were dialyzed against 150 mL of 300mM methylamine in 50-mM TrisHCL (pH 8.8) for 24 h at room temperature, followed by 2 dialyses
against phosphate-buffered saline
(PBS pH 7.4) for 18 to 24 h at 4°C
(46). From both the native and methylamine-reacted plasma, aliquots containing 75 ,ug of protein (Protein
Assay, Bio-Rad Laboratories, Toronto,
Ontario) were placed in 50-mM TrisHCI (pH 7.6). One native and one
methylamine-reacted aliquot were
also incubated for 2 h at 37°C with
10 pL (10,000 cpm) of iodine-125labeled recombinant human TNF-ot
('251-rhTNF-ox) (Amersham, Life Science, Oakville, Ontario).
For each plasma sample, PAGE
was performed with and without
151
added '25l-rhTNF-a, on untreated/
native plasma and on plasma reacted
with methylamine. Each aliquot, containing 75 ,ug of protein, was mixed
with an equal volume of sample buffer
(41-mM Tris, 40-mM sodium borate,
20% glycerol, pH 8.6). The proteins
were separated for 6 h at 200 V on
native gradient (4% to 12.5%) PAGE,
with a 4% acrylamide stacking
gel (34). Gels were stained with
Coomassie blue, destained, and dried.
Gels containing samples incubated
with '25I-TNF-a were autoradiographed by exposure to preflashed
(1 ms, to background A540 = 0.15)
X-Omat AR-5 film (Eastman Kodak,
Rochester, New York, USA) at -70°C
for 48 to 72 h, using rare-earth intensifying screens (Lanex Regular, Eastman Kodak, Rochester, New York,
USA). For reference purposes, plasma
ct2M from the initial group of 5 horses
with various abnormalities was also
compared to purified native and activated forms of human a2M on PAGE.
The electrophoretic gels and the
autoradiographs were evaluated subjectively and by quantitative densitometry (Bio Image Whole Band Analysis, Millipore Corp, Ann Arbor,
Massachusetts' USA). The percentage
integral optical density (IOD) of the
native and activated forms of a2M
was calculated from the gels. Percentage IOD is defined as IOD of a specific band divided by total IOD
detected for the sample (lane IOD).
Lane IOD represents total IOD of the
major bands detected by the densitometer for one sample. The IOD of 1251TNF-a was similarly determined
directly from the autoradiograph
films. Quantitative data were statistically analyzed using the general linear
models procedure (GLM) of SAS
(SAS Institute, Cary, North Carolina,
USA) and the student's t-test.
RESULTS
In all normal horse plasma subjected to nondenaturing PAGE, x2M
migrated as a single distinct highmolecular-weight band at a position
equivalent to native human a2M
(Figs. 1 and 2). Methylamine-reacted
equine plasma samples resulted in
faster migration of ct2M, which had a
position similar to methylaminereacted human a2M (Fig. 1). However,
152
B1N
1
MR
2
N
2 3 3 4
MR N MR N
6
N
6
MR
4 5 5 6
MR N MR N
6
MR
Figure 1. Coomassie blue stain (A) and autoradiograph (B) of nondenaturing polyacrylamide gels of human a2M and equine plasma proteins incubated with 1251- rhTNF-a. Lanes
are native plasma (N) or methylamine-reacted (MR) preparations from one horse with pleuritis (1); one horse with colonic volvulus (2); pregnant mare (3); purified human a2M (4);
one normal horse (5) and one horse with colitis (6). a2M bands in native or slow position (S)
are indicated in comparison with the position of reacted intermediate (I) fast (F) forms and
albumin (A).
in the methylamine-reacted equine
plasma, an intermediate a2M band was
consistently present between the bands
corresponding to native and activated
a2M. This intermediate band was not
identified in untreated native samples
(Figs. 1 and 2).
Clinical and laboratory findings
from the 6 horses with signs of colic
and endotoxemia are summarized in
Table I. In these horses, a2M also
migrated as a single major band,
indistinguishable from a2M in native
plasma of normal horses (Fig. 3). In
untreated samples, no differences
were observed in the percentage IOD
of the a2M regions between normal
and endotoxemic horses (Table II).
No activated a2M was present in
native plasma from either group of
horses. However, in both normal
(Fig. 2) and endotoxemic horses
A1
AN
r e:-:ini
1
MR
2
N
2
MR
3
N
3
MR
4
N
4
MR
5 6
MR N
5
N
6
MR
im..,.
-S
F
ML
DISCUSSION
-
B
I
N
I
MR
2
N
2
MR
3
N
3
MR
4
N
4
MR
5
N
5
MR
6
N
6
MR
iII
F
-M-X L
s.t. -...i;.ji
Figure 2. Coomassie blue stain (A) and autoradiograph (B) of nondenaturing polyacrylamide
gels of equine plasma proteins incubated with 1'5I-rhTNF-o. Lanes are native plasma (N) or
methylamine-reacted (MR) preparations from six normal horses. a2M bands in native or slow
position (S) are indicated in comparison with the position of reacted intermediate (I), fast (F)
forms and albumin (A).
(Fig. 3), approximately half (47% ±
13%) of the a2M was converted by
methylamine dialysis to a major activated form, which migrated faster than
the native form of equine a2M. As
described above, a faint intermediate
In all samples, there was minimal
binding between a2M and '251I-rhTNF-a.
Although binding with the intermediate (IOD, 0.04 ± 0.08) and fast activated (IOD, 0.26 ± 0.25) forms of
a2M was greater, it still represented a
minor percentage of total label present (Figs. 2 and 3, Table III). Binding
in the activated-a2M region was significantly greater in methylaminereacted plasma samples (P < 0.5). The
amounts of TNF-a bound to activated
equine a2M in plasma were similar to
those observed for methylaminereacted human ao2M. On one of the
autoradiographs (Fig. 2B), a faint
band of radiolabeled TNF-a binding
was noticed below a2M, in normal
and methylamine-reacted samples.
band of ot2M was consistently visible
on electrophoretic gels of methylamine-reacted samples (Figs. 2 and 3).
However, these intermediate bands
were close to the detection limit set
on the densitometer.
It is known that ot2M is a potential
regulatory factor for proteinases and
cytokine mediators of acute inflammation, endotoxemia and septic shock
(32,37-41). The interaction between
a2M and various proteinases has been
extensively described (32). Following
interaction of a2M with a proteinase,
a2M undergoes a conformational
change that results in the entrapment
of the proteinase (32). The complex
formed, which is often referred to as
the fast form, is characterized by an
increase in mobility on native electrophoretic gels (33,34). In humans,
reaction of a2M with primary amines,
such as methylamine, resulted in the
same conformational and mobility
change (34). Our studies clearly
demonstrate the potential for equine
a2M to be activated to fast electrophoretic forms, similar to a2M of
humans.and other mammals (33). The
native plasma a2M was located in a
similar region on electrophoretic gels
as native human at2M, indicating a
close similarity in molecular size and
function. This finding is consistent
with a previous study that reported
other similarities between equine and
human a2M (47).
Once reacted with methylamine,
activated equine ao2M migrated to a
position on gels which was similar to
that of activated human a2M. However, an intermediate band was also
consistently noted in the a2M region.
This intermediate band was located
between the native and activated
153
TABLE II. Percentage integral optical density of a2M in native and methylamine-reacted plasma from normal horses and horses with
endotoxemic signs
Native plasma
Intermediate
Activated
ct2M
aL2M
Native
Horse
Normal horses
Mean ± SD
Range
Ot2M
10.6 ± 1.5
8.8-12.4
Total
a2M
Native
cr2M
Methylamine-reacted plasma
Intermediate
Activated
a2M
C2M
Total
a2M
0
0
0
0
10.6 ± 1.5
8.8-12.4
4.8 ± 1.2
3.7-6.5
*
*
3.4 ± 0.6
3.0-4.6
8.4 ± 0.8
7.3-9.6
0
0
0
0
10.8 ± 2.0
8.8-14.8
3.7 ± 1.3
2.1-5.8
*
*
3.9 ± 1.7
2.7-7.4
7.7 ± 1.3
6.1-9.5
Total
Mean ± SD
10.7 ± 1.7
0
0
Range
8.8-14.8
0
0
* Too faint to be detected by the limit set on the
densitometer
10.7 ± 1.7
8.8-14.8
4.3 ± 1.3
*
*
3.7 ± 1.3
2.7-7.4
8.0 ± 1.1
6.1-9.6
Horses with colic and endotoxemia
Mean ± SD
10.8 ± 2.0
Range
8.8-14.8
TABLE III. Integral optical density of '251-TNFa bound to
with endotoxemic signs
Horse
Normal horses
Mean±SD
Range
Native
TNF/ct2M
Native plasma
Intermediate
Activated
TNF/a2M
TNF/a2M
0
0
3.1-6.5
a2M in native and methylamine-reacted plasma from normal horses and horses
Total
Native
TNF/ct2M
TNF/cr2M
Methylamine-reacted plasma
Intermediate
Activated
TNF/a2M
TNF/a2M
0
0
0
0
0
0
0.01 ±0.01
0-0.4
0±0.01
0-0.01
0.10±0.05
Horses with colic and endotoxemia
Mean ± SD
0
Range
0
0
0
0
0
0
0
0
0
0.08 ± 0.10
0.42 ± 0.27
0-0.24
0.09-0.81
Total
Mean±SD
Range
0
0
0
0
0
0
0±0.01
0.04±0.08
0-0.24
0.26±0.25
0
0
forms of equine plasma ox2M, and was
not present in the native unreacted
samples. Usually, methylamine converts human ox2M to a single fast form,
as demonstrated by control samples in
this study. The human ot2M is probably completely activated by the concentrations of methylamine used.
However, the human-a2M/methylamine reaction has been shown to
occur in a step-wise manner, with
intermediate forms representing partial activation (i.e. reduction of one of
the 2 available thiol-ester linkages
within dimers of a2M subunits) (34).
Therefore, the observed intermediate
band in equine samples may represent
a transitional stage specifically associated with partial activation of
equine a2M by methylamine. It also
suggests that equine a2M is more stable than human ot2M. A similar intermediate form of cx2M was recently
observed in methylamine-reacted
samples of equine synovial fluid
(unpublished observation, Cote et al).
Under the conditions used in our
studies, dialysis of plasma samples
154
0-0.04
with methylamine for 24 h increased
the electrophoretic mobility of equine
a2M. By comparison, Motoshima et al
demonstrated a small change in
equine-ot2M conformation after reaction with methylamine for only
60 min, and the a2M/methylamine
complex failed to migrate faster on
PAGE (47). The shorter dialysis time
could have resulted in incomplete
cleavage of the 4 thiol-ester bonds
within a2M. However, Motoshima
et al also observed the generation of
4 thiol groups, which was consistent
with cleavage of all 4 thiol-ester
bonds (47). Species differences in the
susceptibility of a2M to methylamine
activation have also been documented
(35,48). Although rat and fetal-calf
aL2M reacted with methylamine undergo a conformational change, their
migration on electrophoretic gel is
slower than observed for human a2M
(35,48).
Modest reductions in plasma concentrations of ot2M have been described
in severely ill patients (49,50). This is
probably secondary to inflammatory
0.03-0.16
0.03-0.81
Total
TNF/ot2M
0.10±0.04
0.60-0.16
0.50 ± 0.32
0.09-0.92
0.30±0.30
0.06-0.92
proteinases, which activate a2M
to
forms that are rapidly cleared from
the circulation (35,46,51). An increase
in the generation of circulating activated a2M has also been reported in
patients with severe pancreatitis, in
which other exocrine proteinases are
released. However, the proportion of
activated ct2M was generally less than
10% (51). In the present study, no significant change in the total amount of
a2M was noted between the normal
and endotoxemic groups, and no circulating activated forms were seen.
This suggests that systemic release of
inflammatory proteinases during
endotoxemia in horses does not cause
cx2M activation and consumption at
the time when clinical signs are
advanced.
TNF-a production has been demonstrated following the administration
of endotoxin in many species (52-57).
In horses, an increase in TNF-a was
noted approximately 1 h after endotoxin administration, and remained
increased for 4 h (55,56). However,
the clinical signs of endotoxemia
A 1
N
2
N
3
N
4
N
1
N
2
N
3
N
" Ig llilll"m l ------- .-----~~--.llrri lw
4
N
6
5
4
3
2
1
MR MR MR MR MR MR
6
N
5
N
5
N
6
N
2 3 4 5 6
1
MR MR MR MR MR MR
.. -~~ -~--~~- .ErIln -----~-~~---~~-------- --- -- ----m . - ---- -------
Figure 3. Coomassie blue stain (A) and autoradiograph (B) of nondenaturing polyacrylamide
gels of equine plasma proteins incubated with 125I-rhTNF-a. Lanes are native plasma (N) or
methylamine-reacted (MR) preparations from six horses with endotoxemic signs. a2M bands
in native or slow position (S) are indicated in comparison with the position of reacted intermediate (I), fast (F) forms and albumin (A).
persist after TNF-a disappears from
the circulation. This suggests that
TNF-a acts more as an intermediate
signal for other cellular and vascular
responses, rather than as the ultimate
effector of endotoxin (58). For example, TNF-a. has been shown to stimulate the production of nitric oxide by
macrophages, endothelium and hepatocytes (59,60). Nitric oxide, an arginine-derived product, stimulates the
production of cyclic guanosine monophosphate (cGMP), which results in
relaxation of smooth muscle (61),
vessel dilatation and hypotension
(62).
Alpha-2-macroglobulin has been
shown to bind to several inflammatory cytokine (37-41). When bound
to a2M, there is varying behavior
amongst cytokines with respect to
receptor interaction and biological
activity (37,39-43). It was recently
hypothesized that a2M may be an
important regulator of TNF-a activity
and distribution, with a2M participating
in a TNF-a rapid-clearance pathway
rather than having any direct neutralizing effect (44). In the present study,
it was demonstrated that the binding
of 125I-rhTNF-a to native or activated
forms of equine a2M was present but
weak. The dense binding noticed on
the figure lB can be explained by
overexposure of the autoradiographs.
The presence of a weak 251I-rhTNF-a
band below the position of a2M on
one gel (Fig. 2B) may be due to binding of '251I-rhTNF-a to another protein
or to a2M residues. A recent study on
human-a2M methylamine/cytokine
interactions also demonstrated that
TNF-a had a low binding affinity
compared to other cytokines (45). The
release of endogenous TNF-a in the
endotoxemic horse might be expected
to compete for binding with a2M, and
consequently result in an underestimation of TNF-a binding to a2M.
However, this seems unlikely since no
significant difference in TNF-a binding affinity was observed between
normal and endotoxemic horses in
this study. The weak binding interactions observed between '251I-rhTNF-a
and equine plasma a2M might be
explained by structural differences
between equine and human TNF-a,
such that equine a2M does not recognize the human form of TNF-a. However, comparison in the amino acid
sequences of human and equine TNFa demonstrated an 85% homology
(63,64). Similarities in the TNF-a
sequence have also been described
among other species (65), and rhTNF-ot
has been demonstrated to bind to
other mammalian a2M (44). Accordingly, while these studies show that
the majority of TNF-a does bind preferentially to an activated form of
equine a2M, the amounts are too
small to implicate a2M as a major carrier or clearance pathway for the elimination of TNF-a during endotoxemia
in horses.
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