Page 118 - 2021_06-Haematologica-web

R.J. King et al.
prediction for the two loops on the b chain was adjacent to the peptide sequences (peptides B and C) identified by the microarray screening (Figure 4E).
Mechanistic insight into potential enhancement of fibrinolysis prolongation by C3 from patients with diabetes
We have previously shown that pooled C3 purified from patients with diabetes may have enhanced anti-fib- rinolytic effects compared with the protein from healthy controls.7 The current work, using C3 purified from indi- vidual plasma samples, demonstrates that C3 from patients with diabetes has enhanced anti-fibrinolytic activity compared with control protein, at least in some samples. We therefore investigated whether increased protein glycation may be one of the mechanisms involved in the enhanced anti-fibrinolytic effects of C3. Mass spec- trometry identified similar glycation of amino acids serine, threonine, arginine, and asparagine in C3 samples from controls and patients with diabetes. However, additional lysine residues were noted to be glycated in all six dia- betes samples but not in the control group, affecting six separate residues, with a mean of 3±0.9 modifications in each sample. Details of the glycated lysine residues and positions within C3 are illustrated in Figure 5A and B. A
A
characteristic mass-spectrum of a fragment of C3 ana- lyzed by tandem mass spectrometry, with the molecular weight of 1,206 Daltons after tryptic digestion, is shown in Figure 5C together with the protein glycation score. We verified the molecular mass of 1,206 as a characteristic post-translational glycation modification of the fragment with the molecular weight of 882 of C3. This molecular mass 1,206 m/z shows glycation of lysine, highlighted by an asterisk in the amino acid sequence bK*GPLLNK*1209. Although the additional glycation of C3 in patients with diabetes was associated with longer clot lysis time, the Spearman coefficient did not demonstrate a significant correlation between number of glycated lysine residues within C3 and ex vivo plasma clot lysis time (Spearman r=0.19, P=0.73).
Discussion
There are a number of novel findings in this work that can be summarized as follows: (i) C3 purified from indi- vidual patients consistently prolongs clot lysis; (ii) the b chain of fibrinogen in the area of Cys424-Arg445 repre- sents one region of interaction with complement C3; and (iii) affimer proteins provide a tool for targeted modula-
B
Figure 3. Affimer A6 sequence identity with C3 and potential protein-protein interaction sites on C3. (A) Loop 2 of affimer A6 showing sequence identity with the area represented by red spheres on the C3 molecule. (B) Enlargement of the area of potential protein-protein interaction sites on complement C3 (labeled in different colors) identified as the best scoring site using the SiteMap module of Glide (Schrodinger Inc).
1620
haematologica | 2021; 106(6)