Clinical Biochemistry of Domestic Animals (Sixth Edition) - UMK ...

V. Determinants of RBC Survival
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and Meiselman, 1999 ; Chiu and Lubin, 1989 ). The relative
importance of HzB formation, hemin release, and the generation
of various free radicals remains to be clarified.
The binding of HzB to the inner surface of the RBC
membrane and clustering of band 3 and other components
alter the normal membrane organization. Potential negative
effects include weakening or disruption of the cytoskeleton,
altered distribution of membrane phospholipids, altered cell
surface charges, and formation of abnormal external cell
surface antigens that can be recognized by autologous antibodies
( Low, 1989 ). HzB cause focal membrane rigidity but
do not affect global cellular deformability until they nearly
cover the entire internal surface of RBCs ( Reinhart et al. ,
1986 ).
Hemin released during HzB formation binds to and
causes damage to RBC membranes. Evidence has been
presented that hemin can mediate the dissociation of RBC
cytoskeletal proteins, impair the ability of the RBC membrane
to maintain ionic gradients, oxidize membrane
sulfhydryl proteins, and potentiate peroxide-induced membrane
lipid peroxidation ( Chiu and Lubin, 1989 ; Hebbel
and Eaton, 1989 ; Jarolim et al. , 1990 ). The molecular
mechanism(s) has not been clearly defined, but hemin
may exert its toxic effects via catalysis of the formation of
reactive oxygen species ( Vincent, 1989 ). Iron may also be
released from hemin when oxidative damage results in porphyrin
degradation, and free iron may be a catalyst for the
generation of more oxidants ( Umbreit, 2007 ).
Oxidative injury to RBC membranes is sometimes recognized
by the appearance of eccentrocytes in stained blood
films. Eccentrocytes are formed by the adhesion of opposing
areas of the cytoplasmic face of the RBC membrane
( Fischer, 1986 ). Denatured spectrin is believed to be of primary
importance in the cross bonding of membranes ( Arese
and De Flora, 1990 ; Fischer, 1988 ). These cells have been
reported to occur in horses with red maple toxicity ( Reagan
et al. , 1994 ) and in dogs exposed to acetaminophen and
other nonsteroidal anti-inflammatory drugs, onions, garlic,
naphthalene, propofol, vitamin K, and vitamin K anticoagulant
drugs ( Caldin et al. , 2005 ; Ham et al. , 1973 ; Harvey
et al. , 1986 ; Harvey and Rackear, 1985 ; Lee et al. , 2000 ).
Eccentrocytes have also been reported in the blood of dogs
with ketoacidotic diabetes, inflammation, and neoplasia
(especially lymphoma), secondary to increased endogenous
oxidants ( Caldin et al. , 2005 ). Eccentrocytes occur in RBC
G6PD-deficient and FAD-deficient horses with inadequate
metabolic protection against endogenous oxidants ( Harvey
et al. , 2003 ; Stockham et al. , 1994 ).
4 . RBC Destruction
Following extreme oxidative injury, RBCs may lyse
within the circulation. Hb released to plasma dissociates
into dimers, and is bound to haptoglobin for removal
by macrophages. Free Hb in plasma also scavenges . NO
( Rifkind et al. , 2006 ), which may lead to vasoconstriction,
decreased blood flow, platelet activation, increased
endothelin-1 expression, and organ injury ( Gladwin et al. ,
2004 ; Minneci et al. , 2005 ). Free Hb in plasma spontaneously
oxidizes to MetHb, which activates endothelial cells
by stimulating the production of IL-6 and IL-8 cytokines
and the expression of E-selectin ( Liu and Spolarics, 2003 ).
Hemin released from MetHb is bound by hemopexin for
removal by the liver. Free hemin, like MetHb, promotes
inflammation. It increases vascular permeability, adhesion
molecule expression, and the infiltration of leukocytes
( Wagener et al. , 2001 ). Hemoglobinuria occurs if intravascular
hemolysis is of sufficient magnitude to saturate the
haptoglobin-binding capacity of plasma and exceed the
ability of renal tubules to reabsorb filtered Hb.
In most cases, however, enhanced RBC destruction
results primarily from increased phagocytosis of injured
RBCs by macrophages of the spleen, liver, and bone marrow.
Of the organs of the mononuclear phagocyte system,
the spleen is most adept at recognizing and removing damaged
RBCs. In most species, RBCs must pass through narrow
slits between endothelial cells lining venous sinus
walls of the spleen to reenter the general circulation. As a
consequence, oxidant-induced decreased RBC deformability
tends to result in increased transit time of RBCs in the
splenic reticular meshwork, thereby enhancing the likelihood
of phagocytosis by resident macrophages ( Baerlocher
et al. , 1994 ; Weiss, 1984 ).
When HzB are larger than openings in walls of splenic
venous sinuses, they are retained within the trabecular meshwork
of the spleen. The whole cell may be phagocytized, or
the HzB and closely associated membrane may be removed.
The remainder of the RBC reseals and passes through the
sinus wall. This removal of HzB is the so-called pitting function
of the spleen ( Weiss, 1984 ). Cat spleens have poor pitting
capabilities ( Jain, 1986 ) owing to the presence of large
openings in venous sinus walls ( Blue and Weiss, 1981 ).
Macrophages can recognize damaged RBCs by antibody-dependent
and by antibody-independent mechanisms;
however, the relative importance of the different mechanisms
remains to be defined. Macrophages do not recognize
less-deformable cells per se, but phagocytize them at a
higher rate because of their slower transit time or entrapment
in the spleen as discussed earlier ( Baerlocher et al. , 1994 ).
Oxidative damage to RBCs can induce suicidal death of
RBCs (eryptosis), with reactions similar to some of those
that occur during apoptosis of nucleated cells. Eryptosis is
characterized by Ca 2 entry, RBC shrinkage, membrane
blebbing, activation of proteases, and exposure of phosphatidylserine
in the outer membrane leaflet. The exposed
phosphatidylserine is recognized by receptors on macrophages
that phagocytize and degrade the affected cells
(Lang et al. , 2006 ). Phosphatidylserine exposure has been
reported in RBCs from humans with hemolytic anemia secondary
to PK deficiency and unstable Hbs, but it does not