S-Homocysteinylated Proteins - Homocysteine in Protein Structure/Function and Human Disease - page 126

S-Hcy-factor Va in the plasma or for the impairment of protein C activation by
thrombin in experimental hyperhomocysteinemia in humans or animals [443].

7.3.2 Annexin A2

Studies of annexin A2, a calcium-regulated and phospholipid-binding protein,
reveal the existence of an antifibrinolytic mechanism involving S-homocystei-
nylation [145]. The annexin A2 complex is the endothelial cell-surface co-receptor
for plasminogen and tissue plasminogen activator (TPA) that accelerates the cata-
lytic activation of plasmin, the major fibrinolytic enzyme in mammals. The binding
of TPA and plasminogen to annexin A2 increases the catalytic efficiency of TPA-
dependent plasmin generation 60-fold [444]. TPA binding is inhibited by the
hexapeptide LCKLSL corresponding to residues 7-12 of annexin A2. Hcy
decreases the ability of annexin A2 to bind to TPA, while cysteine has no effect
[445]. Mass spectroscopic studies have shown that purified annexin A2 incubated
in vitro with Hcy becomes S-homocysteinylated at residue Cys8 [446]. Ex vivo
studies with endothelial cells labeled with [ 35 S]Hcy show a prominent 35 S-labeled
36-kDa band on nonreducing SDS-PAGE gels that corresponds to annexin A2 and
disappears on reducing gels. This suggests that Hcy forms a disulfide bond with the
residue Cys8 of annexin A2 in cultured endothelial cells. As a result of this
posttranslational modification, plasminogen activation on the endothelial cell sur-
face is significantly impaired [446].
Annexin A2 binds Hcy also in vivo in a mouse model of hyperhomocysteinemia
[145]. When mice are fed with a high-Met, low-folate diet, plasma Hcy levels
increase to about 70

M, and annexin A2 becomes S-homocysteinylated, as
demonstrated by the immunostaining of lung sections using a polyclonal antibody
generated against S-homocysteinylated A2 N-terminal peptide. (This antibody is
specific for A2 and fails to react with any other protein.)
Annexin A2 isolated from the lung tissue of hyperhomocysteinemic mice fails to
bind TPA and does not support plasminogen activation, whereas annexin A2
isolated from control mice was fully active in TPA binding and plasminogen
activation. Treatment with 2-mercaptoethanol of annexin A2 isolated from the
hyperhomocysteinemic mice restores its activity, which shows that S-homocystei-
nylation impairs normal annexin A2 function in vivo. The hyperhomocysteinemic
mice exhibit microvascular fibrin accumulation in the kidneys, heart, and lung, as
well as diminished angiogenesis [145]. This phenotype of the hyperhomocys-
teinemic mouse is similar to the phenotype observed in the annexin A2 knockout
mouse [447].
In a chemical FeCl 3 vascular injury model of thrombosis, complete occlusion of
the carotid artery occurs in six of seven mice on high-Met diet but only in one of
seven mice on a control diet. Furthermore, post-injury blood flow in mice on high-
Met diet is reduced to 50 %, whereas post-injury blood flow in control mice is
essentially not affected. This prothrombotic phenotype is reversed by infusion of

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