N-Homocysteinyl-Proteins - Homocysteine in Protein Structure/Function and Human Disease - page 81

Table 5.10 Plasma N-Hcy-fibrinogen, total fibrinogen, and the ratio of N-Hcy-fibrinogen/total
fibrinogen in CBS-deficient patients and unaffected individuals (Data from [115])

N-Hcy-fibrinogen
(nM)

Total fibrinogen
(

N-Hcy-fibrinogen/total
fibrinogen (%) Genotype (n) μ M)

CBS +/+ (7) 35.8 14.3 6.56 1.98 0.61 0.21

CBS /a (27) 58.1 * 1.65 # 0.64 *

72.5 6.95 1.00

CBS /a , noncompliant (1) 314.8 8.30 3.78

a

CBS / patients were on an Hcy-lowering therapy
* P

0.01
# P = 0.32 for CBS / vs. CBS +/+

¼

from N-Hcy-fibrinogen is 1.3 times slower compared to control fibrin. Fibrinogen
purified from human plasma obtained from patients with hyperhomocysteinemia
has higher content of N-linked Hcy compared to fibrinogen from control subjects.
The purified fibrinogen with high in vivo N-linked Hcy content (2.38 M) produces

fibrin clots with a denser structure and a 1.2-fold longer lysis time, compared with
fibrin clots with low in vivo N-linked Hcy content (0.34

μ

M) [331].
Plasma levels of the prothrombotic N-Hcy-fibrinogen are elevated up to tenfold in
CBS-deficient patients (Table 5.10 ) [115] who are known to be prone to
atherothrombosis [46]. Mass spectrometric analyses identify three lysine residues
that carry N-linked Hcy in fibrinogen isolated from CBS-deficient patients, one in
each subunit: Lys562 in

μ

-subunit
(Fig. 5.3 )[215].ThesethreeinvivoN-homocysteinylation sites are also predominant
sites for fibrinogen N-homocysteinylation in vitro. The

α -subunit, Lys344 in β -subunit, and Lys385 in γ

-subunit Lys562 site of
N-homocysteinylation is located in an unstructured region of the

α

α C domain known

to be involved in tPA and plasminogen binding ( α 392-610), which can explain

abnormal characteristics of clots formed from N-Hcy-fibrinogen [175].

N-Hcy-Lys562 is close to the sites of two mutations Ser532- Cys and Arg554-Cys

that are associated with thrombosis [328, 329]. Thus, it is likely that at least
N-Hcy-Lys562 contributes to prothrombotic properties of N-Hcy-fibrinogen in
CBS-deficient patients. Taken together, these findings suggest that fibrinogen
N-homocysteinylation contributes to the pro-coagulant phenotype observed in
hyperhomocysteinemic patients.

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5.4.3 N-Homocysteinylation and LDL Function

Low-density lipoprotein, the major cholesterol transport protein in plasma, enters
cells by binding to specific surface receptors that mediate its cellular uptake and
transport to lysosomes [332]. Lysine residues are involved in the LDL receptor
interaction [333]. LDL is susceptible to N-homocysteinylation by Hcy-thiolactone
in vitro [78, 139] and native LDL carries small amounts of N-linked Hcy in the
circulation (Tables 5.4 and 5.5 ) [79].
Chemical modification of 15-20 % of the lysine residues of LDL by
carbamylation with cyanate or by acetoacetylation with diketene prevents the
LDL from competitively displacing unmodified 125 I-LDL from the high-affinity