N-Homocysteinyl-Proteins - Homocysteine in Protein Structure/Function and Human Disease - page 80

formation of multimeric forms of N-Hcy-cytochrome c (Fig. 5.10 ) that are observed
on nonreducing SDS-PAGE gels [78]. Multimers of N-Hcy-cytochrome c can also
be separated by capillary electrophoresis [325]. Furthermore, N-Hcy-cytochrome c
becomes more resistant than the native cytochrome c to proteolytic degradation.
Thus, N-homocysteinylation of susceptible lysine residues in cytochrome c has
important structural and functional consequences, manifested by increased resis-
tance to proteolysis and change in iron redox state. A thiol of the N-linked Hcy
introduced by N-homocysteinylation changes the redox state of the heme ligand of
cytochrome c by rendering it reduced (Fig. 5.10 ) [298].

5.4.2 N-Hcy-Fibrinogen and Fibrin Clot Properties

In vitro studies show that the modification by Hcy-thiolactone interferes with the
function of fibrinogen, a major blood clotting protein. Fibrinogen is a dimer of three
polypeptides, A

, linked by 29 disulfide bonds. During coagulation fibrin-
ogen is converted to an insoluble fibrin by thrombin-catalyzed removal of
fibrinopeptides from the A

α ,B β ,C γ

chains. Although fibrinogen does not have a
free thiol, and thus cannot bind Hcy by a disulfide linkage, the protein is known to
be susceptible to N-homocysteinylation by Hcy-thiolactone in vitro (Table 3.3 ) [78,
139] and, like other circulating proteins, carry N-linked Hcy in vivo in the human
blood (Tables 5.4 and 5.5 ) [79].
Because lysine residues are important for the binding of fibrinolytic enzymes to
fibrin, their modification by Hcy-thiolactone is likely to impair fibrinolysis and lead
to increased thrombogenesis. Indeed, fibrin clots formed from in vitro-prepared N-
Hcy-fibrinogen have more compact structure and lyse slower than clots from
unmodified control fibrinogen [175]. For example, the half lysis time of the clots
formed from N-Hcy-fibrinogen is significantly increased, compared to the clots
formed from the control fibrinogen (15.5

α and B β

3.5 min vs. 11.7 2.1 min,

P < 0.001). The decreased susceptibility to lysis is caused by less efficient activa-

tion of plasminogen on clots formed from N-Hcy-fibrinogen. Although tPA binding
is increased, the binding of plasminogen to N-Hcy-fibrin is not affected relative to
control fibrin [175]. In addition, N-Hcy-fibrinogen acquires the ability to form
disulfide-linked complexes with albumin. However, fibrin clots generated from
albumin-containing complexes of N-Hcy-fibrinogen have half lysis time not
different from those for clots from N-Hcy-fibrinogen [326].
The in vitro prothrombotic effects of N-Hcy-fibrinogen are similar to the
prothrombotic effects of fibrinogen mutations in humans, which introduce a cyste-
ine thiol group, e.g., A

α Arg16 ! Cys, Arg554 ! Cys, Ser532 ! Cys; B β Arg14

! Cys, Arg44 ! Cys, Arg255 ! Cys; and C γ Arg275 ! Cys, Tyr354 ! Cys

[327-330] (human fibrinogen database is available at http://www.geht.org ).
Confocal microscopy of fibrin clots formed from in vitro-prepared N-Hcy-
fibrinogen demonstrates a denser structure with increased branching compared to
control [331]. Consistent with their denser clot structure, lysis of fibrin clots formed