Aims/hypothesis Impaired fibrin clot lysis is a key abnormality in diabetes and complement C3 is one protein identified in blood clots. This work investigates the mechanistic pathways linking C3 and hypofibrinolysis in diabetes using ex vivo/in vitro studies. Methods Fibrinolysis and C3 plasma levels were determined in type 1 diabetic patients and healthy controls, and the effects of glycaemia investigated. C3 incorporation into fibrin clots and modulation of fibrinolysis were analysed by ELISA, immunoblotting, turbidimetric assays and electron and confocal microscopy. Results Clot lysis time was longer in diabetic children than in controls (599±18 and 516±12 s respectively; p<0.01), C3 levels were higher in diabetic children (0.55±0.02 and 0.43± 0.02 g/l respectively; p<0.01) and both were affected by improving glycaemia. An interaction between C3 and fibrin was confirmed by the presence of lower protein levels in sera compared with corresponding plasma and C3 detection in plasma clots by immunoblot. In a purified system, C3 was associated with thinner fibrin fibres and more prolongation of lysis time of clots made from fibrinogen from diabetic participants compared with controls (244±64 and 92±23 s respectively; p<0.05). Confocal microscopy showed higher C3 incorporation into diabetic clots compared with controls, and fully formed clot lysis was prolonged by 764±76 and 428±105 s respectively (p<0.05). Differences in lysis, comparing diabetes and controls, were not related to altered plasmin generation or C3-fibrinogen binding assessed by plasmon resonance. Conclusions/interpretation C3 incorporation into clots from diabetic fibrinogen is enhanced and adversely affects fibrinolysis. This may be one novel mechanism for compromised clot lysis in diabetes, potentially offering a new therapeutic target.

An enhanced thrombotic environment and premature atherosclerosis are key factors for the increased cardiovascular risk in diabetes. The occlusive vascular thrombus, formed secondary to interactions between platelets and coagulation proteins, is composed of a skeleton of fibrin fibres with cellular elements embedded in this network. Diabetes is characterised by quantitative and qualitative changes in coagulation proteins, which collectively increase resistance to fibrinolysis, consequently augmenting thrombosis risk. Current long-term therapies to prevent arterial occlusion in diabetes are focussed on anti-platelet agents, a strategy that fails to address the contribution of coagulation proteins to the enhanced thrombotic milieu. Moreover, antiplatelet treatment is associated with bleeding complications, particularly with newer agents and more aggressive combination therapies, questioning the safety of this approach. Therefore, to safely control thrombosis risk in diabetes, an alternative approach is required with the fibrin network representing a credible therapeutic target. In the current review, we address diabetes-specific mechanistic pathways responsible for hypofibrinolysis including the role of clot structure, defects in the fibrinolytic system and increased incorporation of anti-fibrinolytic proteins into the clot. Future anti-thrombotic therapeutic options are discussed with special emphasis on the potential advantages of modulating incorporation of the anti-fibrinolytic proteins into fibrin networks. This latter approach carries theoretical advantages, including specificity for diabetes, ability to target a particular protein with a possible favourable risk of bleeding. The development of alternative treatment strategies to better control residual thrombosis risk in diabetes will help to reduce vascular events, which remain the main cause of mortality in this condition.

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Key Points• Plasminogen is glycated in diabetes, resulting in reduced plasmin generation and impaired protein activity.• Impaired plasminogen function and the consequent hypofibrinolysis in diabetes are reversible by modest improvement in glycemia.Diabetes is associated with hypofibrinolysis by mechanisms that are only partially understood. We investigated the effects of in vivo plasminogen glycation on fibrinolysis, plasmin generation, protein proteolytic activity, and plasminogen-fibrin interactions. Plasma was collected from healthy controls and individuals with type 1 diabetes before and after improving glycemia. Plasma-purified plasmin(ogen) functional activity was evaluated by chromogenic, turbidimetric, and plasmin conversion assays, with surface plasmon resonance employed for fibrin-plasminogen interactions. Plasminogen posttranslational modifications were quantified by mass spectrometry and glycation sites located by peptide mapping. Diabetes was associated with impaired plasma fibrin network lysis, which partly normalized upon improving glycaemia. Purified plasmin (ogen) from diabetic subjects had impaired fibrinolytic activity compared with controls (723 6 16 and 317 6 4 s, respectively; P < .01), mainly related to decreased fibrin-dependent plasmin generation and reduced protease activity (K cat /K M 2.57 6 1.02 3 10 23 and 5.67 6 0.98 3 10 23 M 21 s 21, respectively; P < .05). N«-fructosyl-lysine residue on plasminogen was increased in diabetes compared with controls (6.26 6 3.43 and 1.82 6 0.95%mol, respectively; P < .01) with preferential glycation of lysines 107 and 557, sites involved in fibrin binding and plasmin(ogen) cleavage, respectively. Glycation of plasminogen in diabetes directly affects fibrinolysis by decreasing plasmin generation and reducing protein-specific activity, changes that are reversible with modest improvement in glycemic control. (Blood. 2013;122(1):134-142)

Background and method: Increased plasma clot density and prolonged lysis times are associated with cardiovascular disease. In this study, we employed a functional proteomics approach to identify novel clot components which may influence clot phenotypes. Results: Analysis of perfused, solubilised plasma clots identified inflammatory proteins, including complement C3, as novel clot components. Analysis of paired plasma and serum samples confirmed concentration-dependent incorporation of C3 into clots. Surface plasmon resonance indicated high-affinity binding interactions between C3 and fibrinogen and fibrin. Turbidimetric clotting and lysis assays indicated C3 impaired fibrinolysis in a concentration-dependent manner, both in vitro and ex vivo. Conclusion: These data indicate functional interactions between complement C3 and fibrin leading to prolonged fibrinolysis. These interactions are physiologically relevant in the context of protection following injury and suggest a mechanistic link between increased plasma C3 concentration and acute cardiovascular thrombotic events.

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Endogenous hyperthyroidism is associated with more compact clots and resistance to fibrinolysis ex vivo, related to the degree of hyperthyroidism and C3 plasma levels, and these changes are modulated by achieving euthyroidism. Altered clot structure/lysis may be one mechanism for increased thrombotic risk in hyperthyroidism.

Key Points• FXIIIa exhibits a preference for Q237 in crosslinking reactions within fibrinogen aC (233-425) followed by Q328 and Q366.• None of the reactive glutamines in aC 233-425 (Q237, Q328, and Q366) are required to react first before the others can crosslink.Factor XIIIa (FXIIIa) introduces covalent g-glutamyl-«-lysyl crosslinks into the blood clot network. These crosslinks involve both the g and a chains of fibrin. The C-terminal portion of the fibrin a chain extends into the aC region (210-610). Crosslinks within this region help generate a stiffer clot, which is more resistant to fibrinolysis. Fibrinogen aC (233-425) contains a binding site for FXIIIa and three glutamines Q237, Q328, and Q366 that each participate in physiological crosslinking reactions. Although these glutamines were previously identified, their reactivities toward FXIIIa have not been ranked. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry and nuclear magnetic resonance (NMR) methods were thus used to directly characterize these three glutamines and probe for sources of FXIIIa substrate specificity. Glycine ethyl ester (GEE) and ammonium chloride served as replacements for lysine. Mass spectrometry and 2D heteronuclear single quantum coherence NMR revealed that Q237 is rapidly crosslinked first by FXIIIa followed by Q366 and Q328. Both N-GEE could be crosslinked to the three glutamines in aC (233-425) with a similar order of reactivity as observed with the MALDI-TOF mass spectrometry assay. NMR studies using the single aC mutants Q237N, Q328N, and Q366N demonstrated that no glutamine is dependent on another to react first in the series. Moreover, the remaining two glutamines of each mutant were both still reactive. Further characterization of Q237, Q328, and Q366 is important because they are located in a fibrinogen region susceptible to physiological truncations and mutation. The current results suggest that these glutamines play distinct roles in fibrin crosslinking and clot architecture. (Blood. 2016;127(18):2241-2248