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Hypofibrinolytic effects of complement C3 and its modulation
and reduces the risk of vascular thrombosis without increasing bleeding events.
Complement C3, which is incorporated into fibrin clots, has been shown to modulate fibrin clot lysis,6 an effect that may be exaggerated in patients with higher vascular risk.7 C3 plasma levels have shown correlations with ex-vivo plas- ma clot lysis in individuals with diabetes as well as in healthy controls.7,8 Moreover, plasma levels of this protein were independent predictors of resistance to fibrinolysis in 875 high-vascular-risk patients with type 2 diabetes.9 These data suggest that C3 represents a credible therapeutic target to shorten fibrin clot lysis and ultimately reduce thrombosis risk. However, these functional studies were conducted using pooled C3 and studies assessing consistency of the response in different individuals, crucial to establish the therapeutic potential of C3, are lacking.
We have recently demonstrated that fibrin clot lysis can be modulated, leading to stabilization of the clot, by using small conformational proteins, termed affimers.10,11 Affimer technology was able to correct abnormal lysis of clots made from plasma samples of individuals with hemophilia, indi- cating a potential therapeutic role for these proteins. However, it remains unknown whether affimers can be used to enhance clot lysis, particularly in individuals at high vascular risk.
The aim of this work was to establish the role of C3 as a therapeutic target for enhancing fibrinolysis and reducing thrombosis risk. Therefore, we studied: (i) consistency in the fibrinolytic properties of C3 in healthy controls and patients with high vascular risk; (ii) potential binding sites between fibrinogen and C3; and (iii) modulation of fibrin clot lysis by targeted interference of fibrinogen-C3 interac- tions.
Methods
Only a brief description is provided here; full details can be found in the Online Supplement.
Recruitment of patients
We recruited 12 patients with type 1 diabetes (T1DM) and 12 age- and sex-matched controls to purify fibrinogen and C3. We additionally tested 24 stored plasma samples from individuals with high vascular risk. Ethical approval for the study was provided by the Leeds East, National Research Ethics Service committee and informed consent was obtained from participants in accordance with the Declaration of Helsinki.
Protein purification and turbidimetric analysis
Fibrinogen and complement C3 were purified as described else- where7 and clots were formed from purified fibrinogen in the pres- ence or absence of corresponding C3 from the same individual. Time from full clot formation to 50% lysis was calculated and is presented as clot lysis time throughout this work.
Peptide microarray
A peptide microarray chip containing overlapping 15 amino acid peptides of the fibrinogen molecule (PEPperPRINT, Germany) was synthesized to screen against pooled C3 (0.8 to 8 mg/mL) purified from six healthy individuals.
Non-antibody synthetic proteins (affimers)
Affimers are small proteins composed of a scaffold protein that constrains two variable conformational nine amino acid loops.11,12
Panning and protein production
A phage display library of affimer proteins, comprising 1.3x1010 random variable clones, was screened against human fibrinogen. In order to select specific affimers capable of targeting fibrinogen– C3 interactions, competitive elution with C3 for 20 min was applied in the fourth panning.
Affimers and fibrinolysis
Fibrinogen was incubated with affimer A6 at room temperature for 30 min after which C3 and factor XIII were added and clot for- mation/lysis was triggered as described previously.13
Plasma samples were mixed with increasing concentrations of affimer A6 or control scaffold and then turbidimetric experiments were performed, as described elsewhere.13
Molecular modeling
In order to identify potential sites of ligand binding on C3, the molecular modeling software Autoligand was employed to scan the whole protein.14 Molecular modeling was used to predict bind- ing sites of any peptide sequences identified from the microarray screening and affimer work using the webserver Pepsite2® (http://pepsite2.russelllab.org). Images were viewed and produced using Pymol (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC).15
Mass spectrometry
Purified C3 from six subjects with type 1 diabetes mellitus and six healthy controls was digested by trypsin and analyzed by matrix-assisted laser desorption/ionization -time-of-flight mass spectrometry (MALDI-TOF/MS) as described elsewhere.16 To compare potential differences between in vivo and in vitro C3 gly- cation, we undertook in vitro glycation of the protein as previously described17 and post-translational modifications were assessed as above.
Statistical analysis
Between-group comparisons of normally and non-normally dis- tributed variables were carried out by independent Student t and Mann–Whitney U tests, respectively. Pearson and Spearman coef- ficients were applied to assess correlations between normally and non-normally distributed variables, respectively. Based on previ- ous data7 and assuming a common standard deviation of the vari- able studied of 225 seconds (s) we calculated that the number of samples analyzed (n=24 in total) would be enough to detect a dif- ference of 270 s in C3-induced prolongation of clot lysis time (power 80% at P<0.05).
Results
Patients’ characteristics
Twelve patients with type 1 diabetes and 12 healthy controls were recruited. Their characteristics are shown in Table 1. The patients with diabetes were on no medica- tions other than subcutaneous insulin injections and had no clinically significant microvascular or macrovascular complications of diabetes. The healthy controls were also not taking any prescribed medication.
Effect of C3 on lysis time
One control sample did not lyse within the allotted time and was, therefore, excluded from further analysis. The mean lysis time of all clots made from fibrinogen in the absence and presence of corresponding C3 was 2691±105 and 3057±172 s (P<0.01). The mean lysis time of clots
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