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R.J. King et al.
tency is related to a steric effect. Previous work has shown that glycation of proteins involved in coagulation and lysis alters clot structure and/or the efficiency of fibrinolysis. For example, fibrinogen glycation alters fibrin network characteristics and the degree of protein glycation corre- lates with glycemic control measured as HbA1c.20,21 Proteins in the fibrinolytic system are also affected, as plasminogen glycation in diabetes compromises conver- sion to plasmin and modulates enzyme activity.22 There was no clear correlation between the number of lysine residues glycated and the antifibrinolytic effects of C3 but this may be due to the small number of samples analyzed or it may simply indicate that some lysine residues are more important than others and extensive glycation of multiple residues is not required to observe an effect. Overall, however, our data suggest that glycation of C3 increases its antifibrinolytic effect, although it remains unclear which lysine residues are important for the observed effect.
proteins with previous work demonstrating the impor- tance of such interactions between fibrinogen and factor XIII.23
A key finding of our work is a proof of concept for a novel methodology to modulate fibrin clot lysis, and hence thrombosis risk, in individuals with diabetes. We describe a new technique that identified a small binding protein with two variable loops (affimer A6), with one of the loops sharing sequence identity with an exposed por- tion of C3, likely to be involved in protein-protein interac- tions. Interestingly, affimer A6 was able to abolish C3- induced prolongation of lysis with high specificity, regard- less of whether C3 was purified from samples from con- trols or patients with type 1 diabetes. Moreover, affimer A6 was capable of reducing plasma clot lysis in samples from both healthy controls and patients with diabetes. We and others have shown that changes in clot lysis by 6-18% are clinically significant24-26 and therefore the observed 7- 11% reduction in clot lysis by affimer A6 is likely to be clinically meaningful. Although speculative, this targeted effect on clot lysis is unlikely to increase bleeding risk sig- nificantly, making this approach clinically promising.
The predicted binding sites of both loops of affimer A6 were in close proximity within the b chain of fibrinogen and close to the region of fibrinogen that interacts with C3 as determined by microarray screening. Considering the data together, it appears that the N terminus of the b chain of fibrinogen is a binding site for complement C3,
AB
C
Figure 5. In-vivo glycation of C3 and lysine residues involved. Six individual C3 samples from patients with type 1 diabetes and healthy controls were analyzed by matrix-assisted laser desorption/ionization mass-spectrometry (MALDI–MS) for post-translational glycation. (A) Six lysine residues showed additional glycation in samples from the patients with diabetes. (B) The spatial position of glycated residues within C3 are shown. (C) Detailed analysis of the tandem mass spectrometry fragment from C3 with the molecular mass of 1,206 Daltons and the interpretation by the Mascot search engine is shown together with a protein score of 125 for post-translational glycation modification. AU: arbitrary units.
Our binding studies indicate that three areas on the b chain of fibrinogen play a role in C3-fibrinogen interac- tions. In particular, two of these areas, located in the N-ter- minus, were in close proximity and separated by a single amino acid. We should acknowledge that the microarray technique only identifies linear interactions and it is possi- ble that additional conformational interactions take place between the two proteins. However, we should not underestimate the importance of linear binding between
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haematologica | 2021; 106(6)