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ObjectiveNeprilysin (NEP), a zinc metallo-endopeptidase, has a role in blood pressure control and lipid metabolism. The present study tested the hypothesis that NEP is associated with insulin resistance and features of the metabolic syndrome (MetS) in a study of 318 healthy human subjects and in murine obesity and investigated NEP production by adipocytes in-vitro.Methods and ResultsIn 318 white European males, plasma NEP was elevated in the MetS and increased progressively with increasing MetS components. Plasma NEP activity correlated with insulin, homeostasis model assessment and body mass index in all subjects (p<0.01). Quantitative RT-PCR and Western blotting showed that in human pre-adipocytes NEP expression is upregulated 25-30 fold during differentiation into adipocytes. Microarray analysis of mRNA from differentiated human adipocytes confirmed high NEP expression comparable to adiponectin and plasminogen activator inhibitor-1. In a murine model of diet-induced insulin resistance, plasma NEP levels were significantly higher in high fat diet (HFD)-fed compared with normal chow diet (NCD)-fed animals (1642±529 and 820±487 pg/μl, respectively; p<0.01). Tissue NEP was increased in mesenteric fat in HFD compared with NCD-fed mice (p<0.05). NEP knock out mice did not display any changes in insulin resistance, glucose tolerance or body and epididymal fat pad weight compared to wild type mice.ConclusionsIn humans, NEP activity correlated with body mass index and measures of insulin resistance with increasing levels in subjects with multiple cardiovascular risk factors. NEP protein production in human adipocytes increased during cell differentiation and plasma and adipose tissue levels of NEP were increased in obese insulin resistant mice. Our results indicate that NEP associates with cardio-metabolic risk in the presence of insulin resistance and increases in obesity.

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Background-The fibrinogen A␣ Thr312Ala polymorphism occurs within the ␣C domain of fibrinogen, which is important for lateral aggregation and factor XIII-induced cross-linking of fibrin fibers. We have previously shown an association of Ala312 fibrinogen with poststroke mortality in subjects with atrial fibrillation and with pulmonary embolism in subjects with venous thrombosis. Methods and Results-We studied the properties of clots formed from purified Ala312 and Thr312 fibrinogen and found that Ala312 fibrinogen produces stiffer clots, associated with increased ␣ chain cross-linking, as demonstrated by SDS-Page. On electron microscopy analysis, we found larger fiber diameters in the Ala312 clots and observed a lower number of fibers per square micrometer. The number of branch points per square micrometer was similar between genotypes. Conclusions-Our study shows that Ala312 influences clot structure and properties by increased factor XIII cross-linking and formation of thicker fibrin fibers. These findings may provide a mechanism by which Ala312 fibrinogen could predispose to clot embolization.

Activated coagulation factor XIII (FXIIIa) cross-links the ␥-chains of fibrin early in clot formation. Cross-linking of the ␣-chains occurs more slowly, leading to high molecular weight multimer formations that can also contain ␥-chains. To study the contribution of FXIIIa-induced ␥-chain cross-linking on fibrin structure and function, we created 2 recombinant fibrinogens (␥Q398N/Q399N/K406R and ␥K406R) that modify the ␥-chain crosslinking process. In ␥K406R, ␥-dimer crosslinks were absent, but FXIIIa produced a cross-linking pattern similar to that observed in tissue transglutaminase crosslinked fibrin(ogen) with mainly ␣-␥ crosslinks. In Q398N/Q399N/K406R, cross-links with any ␥-chain involvement were completely absent, and only ␣-chain crosslinking occurred. Upon cross-linking, recombinant normal fibrin yielded a 3.5-fold increase in stiffness, compared with a 2.5-fold increase by ␣-chain cross-linking alone (␥Q398N/Q399N/K406R). ␥K406R fibrin showed a 1.5-fold increase in stiffness after cross-linking. No major differences in clot morphology, polymerization, and lysis rates were observed, although fiber diameter was slightly lower in crosslinked normal fibrin relative to the variants. Our results show that ␥-chain crosslinking contributes significantly to clot stiffness, in particular through ␥-dimer formation; ␣-␥ hybrid cross-links had the smallest impact on clot stiffness. IntroductionAfter cross-linking by FXIIIa, fibrin becomes markedly more resistant to proteolytic and mechanical disruption. The introduction of N⑀-(␥-glutamyl)lysine isopeptide bonds between fibrin molecules within and between clot fibers has a remarkable effect on the rheologic properties of clots. [1][2][3][4] This stabilization of fibrin fibers leads to the formation of a rigid and elastic structure that is capable of stopping leakages in the circulatory system, whereas bleeding is a frequent problem without FXIIIa cross-linking. Of the 3 fibrinogen chains, only the ␣-and ␥-chains, but not the ␤-chains, become cross-linked by FXIIIa. During clot formation, at the early stages of polymerization, cross-linking occurs within emerging protofibrils between ␥K406 and ␥Q398 and/or ␥Q399, resulting in the formation of ␥-dimers. [5][6][7] Multiple cross-linking between fibrin ␣-chains results in the formation of ␣-polymers. 6 Over time, FXIIIa has been shown to generate cross-linked chain structures containing various combinations of ␣-and/or ␥-chains; mainly these are thought to be ␣ n , 8 but ␥ 3 , ␥ 4 , and hybrid ␣ p ␥ q (n, p, and q ϭ 1, 2, 3, respectively, and so forth) are also known to occur. 9,10 The effect of these different cross-linking formations on fibrin clot function has not been fully clarified. Previously, the increase of stiffness in clots has been attributed to ␣-chain cross-linking. 11 On the other hand, a recent study suggested that ␥-chain cross-linking played a major role in the determination of the viscoelastic properties of fibrin. 12 However, in these studies, chain-specific cross-linking was manipulated either by ...

Objectives-The purpose of this study was to investigate the direct effects of aspirin on fibrin structure/function. Methods and Results-Chinese Hamster Ovary cell lines stably transfected with fibrinogen were grown in the absence (0) and presence of increasing concentrations of aspirin. Fibrinogen was purified from the media using affinity chromatography, and clots were made from recombinant protein. Mean final turbidity [OD(ϮSEM)] was 0.083(Ϯ0.03), 0.093(Ϯ0.002), 0.101(Ϯ0.005), and 0.125(Ϯ0.003) in clots made from 0, 1, 10, and 100 mg/L aspirin-treated fibrinogen, respectively (PϽ0.05). Permeability coefficient (Ks cm 2 ϫ10 Ϫ8 ) was 1.68(Ϯ0.29) and 4.13(Ϯ0.33) comparing fibrinogen produced from cells grown with 0 mg/L and 100 mg/L aspirin respectively (PϽ0.05). Scanning electron microscopy confirmed a looser clot structure and increased fiber thickness of clots made from aspirin-treated fibrinogen, whereas rheometer studies showed a significant 30% reduction in clot rigidity. Fibrinolysis was quicker in clots made from aspirin-treated fibrinogen. Ex vivo studies in 3 normal volunteers given 150 mg aspirin daily for 1 week demonstrated similar changes in clot structure/function. Conclusion-Aspirin directly altered clot structure resulting in the formation of clots with thicker fibers and bigger pores, which are easier to lyse. This study clearly demonstrates an alternative mode of action for aspirin, which should be considered in studies evaluating the biochemical efficacy of this agent. Key Words: -fibrinogen Ⅲ aspirin Ⅲ fibrin polymerization Ⅲ fibrin structure Ⅲ acetylation Ⅲ fibrinolysis T he use of antiplatelet agents for prevention of atherothrombotic events is now well established, and aspirin is often the first line agent used in individuals with stable disease. 1 Acetylation of platelet cyclo-oxygenase-1 (COX-1) by aspirin results in irreversible inhibition of thromboxane A 2 (TXA 2 ) production and reduced platelet aggregation potential. This is regarded as the main mechanism for the cardioprotective activity of this agent. However, another mode of action for aspirin, which is not widely considered, is its potential direct effect on clotting factors, including fibrinogen and factor (F) XIII, modulating in the process fibrin clot formation and fibrinolysis. 2 A key event in clot formation is the production of thrombin, which mediates the conversion of fibrinogen into a 3-dimensional network of fibrin fibers, and this is further cross-linked and stabilized by thrombin-activated factor (F)XIII. 3 Clot structure has a role in atherothrombotic disease; clots with thin fibers, small pores, and compact structure are associated with the development of premature and more severe coronary artery disease, which may be related to slower clot lysis of clots. 4,5 Although the ability of aspirin to acetylate fibrinogen has been known for 4 decades, 6 little work has been conducted to study the functional effect of fibrinogen-aspirin interactions, with poor characterization of fibrin gel properties. Limited in vit...

Fibrin fibers, which are ~100 nm in diameter, are the major structural component of a blood clot. The mechanical properties of single fibrin fibers determine the behavior of a blood clot and, thus, have a critical influence on heart attacks, strokes, and embolisms. Cross-linking is thought to fortify blood clots; though, the role of α-α cross-links in fibrin fiber assembly and their effect on the mechanical properties of single fibrin fibers are poorly understood. To address this knowledge gap, we used a combined fluorescence and atomic force microscope technique to determine the stiffness (modulus), extensibility, and elasticity of individual, uncross-linked, exclusively α-α cross-linked (γQ398N/Q399N/K406R fibrinogen variant), and completely cross-linked fibrin fibers. Exclusive α-α cross-linking results in 2.5× stiffer and 1.5× more elastic fibers, whereas full cross-linking results in 3.75× stiffer, 1.2× more elastic, but 1.2× less extensible fibers, as compared to uncross-linked fibers. On the basis of these results and data from the literature, we propose a model in which the α-C region plays a significant role in inter- and intralinking of fibrin molecules and protofibrils, endowing fibrin fibers with increased stiffness and elasticity.

FXIII (Factor XIII) is a Ca²+-dependent enzyme which forms covalent ϵ-(γ-glutamyl)lysine cross-links between the γ-carboxy-amine group of a glutamine residue and the ϵ-amino group of a lysine residue. FXIII was originally identified as a protein involved in fibrin clot stabilization; however, additional extracellular and intracellular roles for FXIII have been identified which influence thrombus resolution and tissue repair. The present review discusses the substrates of FXIIIa (activated FXIII) involved in thrombosis and wound healing with a particular focus on: (i) the influence of plasma FXIIIa on the formation of stable fibrin clots able to withstand mechanical and enzymatic breakdown through fibrin-fibrin cross-linking and cross-linking of fibrinolysis inhibitors, in particular α2-antiplasmin; (ii) the role of intracellular FXIIIa in clot retraction through cross-linking of platelet cytoskeleton proteins, including actin, myosin, filamin and vinculin; (iii) the role of intracellular FXIIIa in cross-linking the cytoplasmic tails of monocyte AT1Rs (angiotensin type 1 receptors) and potential effects on the development of atherosclerosis; and (iv) the role of FXIIIa on matrix deposition and tissue repair, including cross-linking of extracellular matrix proteins, such as fibronectin, collagen and von Willebrand factor, and the effects on matrix deposition and cell-matrix interactions. The review highlights the central role of FXIIIa in the regulation of thrombus stability, thrombus regulation, cell-matrix interactions and wound healing, which is supported by observations in FXIII-deficient humans and animals.