S-Homocysteinylated Proteins - Homocysteine in Protein Structure/Function and Human Disease - page 125

cross-linking to form an insoluble polymeric elastin [438]. Elastin is responsible
for the properties of extensibility and elastic recoil of the extracellular matrix in a
variety of tissues.
Tropoelastin contains two cysteine residues that form an intramolecular disulfide
bond and are located at the conserved C terminus encoded by exon 36 [439].
Studies of coacervation of a recombinant tropoelastin fragment containing the
sole two cysteine residues present in human tropoelastin reveal a statistically
significant increase in the coacervation temperature (1.4 C) after treatment with
0.3-mM Hcy [434]. However, the velocity of coacervation is not affected by Hcy.
Treatment with cysteine does not affect the coacervation temperature of the
tropoelastin fragment. The coacervation of a control fibrillin-1 peptide that does
not contain cysteine residues is not affected by Hcy or cysteine. Although these
findings suggest that Hcy could interfere with the biogenesis of elastic fibers, there
is no direct evidence that S-homocysteinylation is involved.

7.3 Blood Homeostasis Proteins

7.3.1 Factor Va

During blood clotting, the prothrombinase complex, containing factor Va, is
responsible for the conversion of prothrombin to thrombin. Factor V (M wt
330,000 Da) is activated by

-thrombin cleavages at Arg709, Arg1018, and
Arg1545. The product, factor Va, is composed of a heavy chain, residues 1-709
(M wt 105,000 Da), derived from the N terminus of factor V and a noncovalently
associated light chain, residues 1,546-2,196 (M wt 74,000 Da), derived from the
COOH terminus

α

(A3-C1-C2 domains) [440]. Factor Va is proteolytically

inactivated by activated protein C [441].
Treatments with Hcy, cysteine, and Hcy-thiolactone have no effect on factor V
activation by

-thrombin. However, factor Va derived from Hcy-treated, but not
cysteine- or Hcy-thiolactone-treated, factor V is inactivated by activated protein C
at a reduced rate [442]. Incubation with [ 35 S]Hcy results in formation of S-[ 35 S]
Hcy-factor V, which is prevented by the treatment with

α

-mercaptoethanol. After
cleavage of S-[ 35 S]Hcy-factor V with activated protein C, followed by resolution on
nonreducing SDS-PAGE gels, [ 35 S]Hcy is found only in fragments known to
contain free sulfhydryl groups: the light chain (Cys1960, Cys2113), the B region
(Cys1085), and the 26/28-kDa fragment (residues 507-709) (Cys-539, Cys-585).
Treatment with

β

-mercaptoethanol removes all radiolabel [442].
Impaired inactivation of the prothrombinase complex might explain the throm-
botic tendency in CBS-deficient or other hyperhomocysteinemic patients. How-
ever, it is unclear whether the inactivation of the prothrombinase complex by
Hcy occurs in such patients. Furthermore, there is no evidence for the presence of

β