Rationale Abdominal aortic aneurysm (AAA) is a complex disease with both genetic and environmental risk factors. Together, 6 previously identified risk loci only explain a small proportion of the heritability of AAA. Objective To identify additional AAA risk loci using data from all available genome-wide association studies (GWAS). Methods and Results Through a meta-analysis of 6 GWAS datasets and a validation study totalling 10,204 cases and 107,766 controls we identified 4 new AAA risk loci: 1q32.3 (SMYD2), 13q12.11 (LINC00540), 20q13.12 (near PCIF1/MMP9/ZNF335), and 21q22.2 (ERG). In various database searches we observed no new associations between the lead AAA SNPs and coronary artery disease, blood pressure, lipids or diabetes. Network analyses identified ERG, IL6R and LDLR as modifiers of MMP9, with a direct interaction between ERG and MMP9. Conclusions The 4 new risk loci for AAA appear to be specific for AAA compared with other cardiovascular diseases and related traits suggesting that traditional cardiovascular risk factor management may only have limited value in preventing the progression of aneurysmal disease.

SummaryFibrinogen is cleaved by thrombin to fibrin, which provides the blood clot with its essential structural backbone. As an acute phase protein, the plasma levels of fibrinogen are increased in response to inflammatory conditions. In addition to fibrinogen levels, fibrin clot structure is altered by a number of factors. These include thrombin levels, treatment with common cardiovascular medications, such as aspirin, anticoagulants, statins and fibrates, as well as metabolic disease states such as diabetes mellitus and hyperhomocysteinaemia. In vitro studies of fibrin clot structure can provide information regarding fibre density, clot porosity, the mechanical strength of fibres and fibrinolysis. A change in fibrin clot structure, to a denser clot with smaller pores which is more resistant to lysis, is strongly associated with cardiovascular disease. This pathological change is present in patients with arterial as well as venous diseases, and is also found in a moderate form in relatives of patients with cardiovascular disease. Pharmacological therapies, aimed at both the treatment and prophylaxis of cardiovascular disease, appear to result in positive changes to the fibrin clot structure. As such, therapies aimed at 'normalising' fibrin clot structure may be of benefit in the prevention and treatment of cardiovascular disease.

Key Points• Thrombin and fibrinogen g9 regulate protofibril packing within fibrin fibers and thereby influence clot stiffness.• Fibrin analysis after dehydration (e.g. electron microscopy) overestimates changes in fiber size due to effects on protofibril packing.Previous studies have shown effects of thrombin and fibrinogen g9 on clot structure. However, structural information was obtained using electron microscopy, which requires sample dehydration. Our aim was to investigate the role of thrombin and fibrinogen g9 in modulating fibrin structure under fully hydrated conditions. Fibrin fibers were studied using turbidimetry, atomic force microscopy, electron microscopy, and magnetic tweezers in purified and plasma solutions. Increased thrombin induced a pronounced decrease in average protofibril content per fiber, with a relatively minor decrease in fiber size, leading to the formation of less compact fiber structures. Atomic force microscopy under fully hydrated conditions confirmed that fiber diameter was only marginally decreased. Decreased protofibril content of the fibers produced by high thrombin resulted in weakened clot architecture as analyzed by magnetic tweezers in purified systems and by thromboelastometry in plasma and whole blood. Fibers produced with fibrinogen g9 showed reduced protofibril packing over a range of thrombin concentrations. High-magnification electron microscopy demonstrated reduced protofibril packing in g9 fibers and unraveling of fibers into separate protofibrils. Decreased protofibril packing was confirmed in plasma for high thrombin concentrations and fibrinogendeficient plasma reconstituted with g9 fibrinogen. These findings demonstrate that, in fully hydrated conditions, thrombin and fibrinogen g9 have dramatic effects on protofibril content and that protein density within fibers correlates with strength of the fibrin network. We conclude that regulation of protofibril content of fibers is an important mechanism by which thrombin and fibrinogen g9 modulate fibrin clot structure and strength. (Blood. 2016;127(4):487-495) IntroductionCoagulation culminates in the production of thrombin, which converts fibrinogen into fibrin, forming the blood clot, to stop bleeding. 1,2 Fibrinogen is a 340-kDa homodimeric plasma protein consisting of 6 polypeptide chains (2Aa, 2Bb, and 2g) linked together by disulphide bonds.3-5 A common variant of fibrinogen, fibrinogen g9 (gA/g9), is produced by alternative splicing of the g-chain mRNA. 6,7 This alternative chain has the final 4 C-terminal residues replaced with 20 different residues, with a high proportion of negatively charged residues. Fibrinogen g9 has an average plasma concentration of 8% to 15%. 6,8 The gA/g9 sequence contains a thrombin binding site, which reduces thrombin inhibition by antithrombin and heparin cofactor II. 9 On the other hand, binding to fibrinogen gA/g9 reduces the availability of thrombin in the circulation, an effect previously described as antithrombin I. 10 We previously found reduced fibrinopeptide (Fp)B but ...

Abdominal aortic aneurysm (AAA) is a silent, progressive disease with a high mortality and an increasing prevalence with aging. Smooth muscle cell (SMC) dysfunction contributes to gradual dilatation and eventual rupture of the aorta. Here we studied phenotypic characteristics in SMC cultured from end-stage human AAA (≥5 cm) and cells cultured from a porcine carotid artery (PCA) model of early and end-stage aneurysm. Human AAA-SMC presented a secretory phenotype and expressed elevated levels of the differentiation marker miR-145 (2.2-fold, p < 0.001) and the senescence marker SIRT-1 (1.3-fold, p < 0.05), features not recapitulated in aneurysmal PCA-SMC. Human and end-stage porcine aneurysmal cells were frequently multi-nucleated (3.9-fold, p < 0.001, and 1.8-fold, p < 0.01, respectively, vs. control cells) and displayed an aberrant nuclear morphology. Human AAA-SMC exhibited higher levels of the DNA damage marker γH2AX (3.9-fold, p < 0.01, vs. control SMC). These features did not correlate with patients' chronological age and are therefore potential markers for pathological premature vascular aging. Early-stage PCA-SMC (control and aneurysmal) were indistinguishable from one another across all parameters. The principal limitation of human studies is tissue availability only at the end stage of the disease. Refinement of a porcine bioreactor model would facilitate the study of temporal modulation of SMC behaviour during aneurysm development and potentially identify therapeutic targets to limit AAA progression.