The role of ß-barrels 1 and 2 in the enzymatic activity of factor XIII A-subunit.

Essentials The roles of ß-barrels 1 and 2 in factor XIII (FXIII) are currently unknown. FXIII truncations lacking ß-barrel 2, both ß-barrels, or full length FXIII, were made. Removing ß-barrel 2 caused total loss of activity, removing both ß-barrels returned 30% activity. ß-barrel 2 is necessary for exposure of the active site cysteine during activation. SUMMARY: Background Factor XIII is composed of an activation peptide segment, a ß-sandwich domain, a catalytic core, and, finally, ß-barrels 1 and 2. FXIII is activated following cleavage of its A-subunits by thrombin. The resultant transglutaminase activity leads to increased resistance of fibrin clots to fibrinolysis. Objectives To assess the functional roles of ß-barrels 1 and 2 in FXIII, we expressed and characterized the full-length FXIII A-subunit (FXIII-A) and variants truncated to residue 628 (truncated to ß-barrel 1 [TB1]), residue 515 (truncated to catalytic core [TCC]), and residue 184 (truncated to ß-sandwich). Methods Proteins were analyzed by gel electrophoresis, circular dichroism, fluorometric assays, and colorimetric activity assays, clot structure was analyzed by turbidity measurements and confocal microscopy, and clot formation was analyzed with a Chandler loop system. Results and Conclusions Circular dichroism spectroscopy and tryptophan fluorometry indicated that full-length FXIII-A and the truncation variants TCC and TB1 retain their secondary and tertiary structure. Removal of ß-barrel 2 (TB1) resulted in total loss of transglutaminase activity, whereas the additional removal of ß-barrel 1 (TCC) restored enzymatic activity to ~ 30% of that of full-length FXIII-A. These activity trends were observed with physiological substrates and smaller model substrates. Our data suggest that the ß-barrel 1 domain protects the active site cysteine in the FXIII protransglutaminase, whereas the ß-barrel 2 domain is necessary for exposure of the active site cysteine during activation. This study demonstrates the importance of individual ß-barrel domains in modulating access to the FXIII active site region.

The Michigan Risk Score to predict peripherally inserted central catheter-associated thrombosis.

Essentials How best to quantify thrombosis risk with peripherally inserted central catheters (PICC) is unknown. Data from a registry were used to develop the Michigan Risk Score (MRS) for PICC thrombosis. Five risk factors were associated with PICC thrombosis and used to develop a risk score. MRS was predictive of the risk of PICC thrombosis and can be useful in clinical practice. SUMMARY: Background Peripherally inserted central catheters (PICCs) are associated with upper extremity deep vein thrombosis (DVT). We developed a score to predict risk of PICC-related thrombosis. Methods Using data from the Michigan Hospital Medicine Safety Consortium, image-confirmed upper-extremity DVT cases were identified. A logistic, mixed-effects model with hospital-specific random intercepts was used to identify factors associated with PICC-DVT. Points were assigned to each predictor, stratifying patients into four classes of risk. Internal validation was performed by bootstrapping with assessment of calibration and discrimination of the model. Results Of 23 010 patients who received PICCs, 475 (2.1%) developed symptomatic PICC-DVT. Risk factors associated with PICC-DVT included: history of DVT; multi-lumen PICC; active cancer; presence of another CVC when the PICC was placed; and white blood cell count greater than 12 000. Four risk classes were created based on thrombosis risk. Thrombosis rates were 0.9% for class I, 1.6% for class II, 2.7% for class III and 4.7% for class IV, with marginal predicted probabilities of 0.9% (0.7, 1.2), 1.5% (1.2, 1.9), 2.6% (2.2, 3.0) and 4.5% (3.7, 5.4) for classes I, II, III, and IV, respectively. The risk classification rule was strongly associated with PICC-DVT, with odds ratios of 1.68 (95% CI, 1.19, 2.37), 2.90 (95% CI, 2.09, 4.01) and 5.20 (95% CI, 3.65, 7.42) for risk classes II, III and IV vs. risk class I, respectively. Conclusion The Michigan PICC-DVT Risk Score offers a novel way to estimate risk of DVT associated with PICCs and can help inform appropriateness of PICC insertion.

Diabetes and cardiovascular disease: pathophysiology of a life-threatening epidemic.

Diabetes is associated with the development of premature cardiovascular disease (CVD), which relates to the clustering of risk factors such as dyslipidaemia, hypertension, obesity and hyperglycaemia in the presence of insulin resistance. In addition, diabetes is associated with an inflammatory and pro-thrombotic environment, exacerbating the development of atherothrombosis. Insulin resistance and hyperglycaemia both contribute to the development of endothelial cell dysfunction and increased oxidative stress, culminating in accelerated atherosclerosis. Clot formation and function are also directly affected by insulin resistance and hyperglycaemia, with increased levels of coagulation factors and anti-fibrinolytic proteins and a fibrin network that is more resistant to lysis, coupled with increased platelet activation.It is well recognised that the intensification of glycaemic control leads to a reduction in microvascular complications in type 1 and type 2 diabetes; however, the same is less clear with macrovascular disease. Several randomised studies have attempted to address the effect of short-, medium- and long-term glycaemic control on cardiovascular outcomes, with mixed results. The overall interpretation of these trials suggests that intensive glycaemic control in patients with a relatively short duration of diabetes, without very poor control and with no CVD, might be safe and associated with fewer cardiovascular events.This review will summarise the effects of hyperglycaemia on the development of atherothrombosis and examine key cardiovascular outcome trials following intensive glucose control.

(19)F(α,n) thick target yield from 3.5 to 10.0 MeV.

Using a target of PbF2, the thick-target yield from the (19)F(α,n) reaction was measured from E(α)=3.5-10 MeV. From these results, we infer the thick-target neutron yields from targets of F2 and UF6 over this same alpha-particle energy range.

The influence of type 2 diabetes on fibrin clot properties in patients with coronary artery disease.

Type 2 diabetes mellitus (T2DM) increases the risk of coronary thrombosis and both conditions are associated with altered fibrin clot properties. However, the influence of T2DM on fibrin clot properties in patients with coronary artery disease (CAD) remains unclear. We aimed to investigate the influence of T2DM on fibrin clot properties in patients with CAD. Fibrin clot structure and fibrinolysis were investigated in 581 CAD patients (148 with T2DM) using turbidimetric assays, confocal and scanning electron microscopy. Clots made from plasma and plasma-purified fibrinogen were studied, and plasma levels of inflammatory markers were analysed. T2DM patients had increased clot maximum absorbance compared with non-diabetic patients (0.36 ± 0.1 vs 0.33 ± 0.1 au; p=0.01), displayed longer lysis time (804 [618;1002] vs 750 [624;906] seconds; p=0.03) and showed more compact fibrin structure assessed by confocal and electron microscopy. Fibrinogen levels were elevated in T2DM (p< 0.001), but clots made from purified fibrinogen showed no differences in fibrin properties in the two populations. Adjusting for fibrinogen levels, T2DM was associated with C-reactive protein and complement C3 plasma levels, with the former correlating with clot maximum absorbance (r=0.24, p< 0.0001) and the latter with lysis time (r=0.30, p< 0.0001). Independent of fibrinogen levels, females had more compact clots with prolonged lysis time compared with males (all p-values< 0.001). In conclusion, T2DM is associated with prothrombotic changes in fibrin clot properties in patients with CAD. This is related to quantitative rather than qualitative changes in fibrinogen with a possible role for inflammatory proteins.

The effect of blood coagulation factor XIII on fibrin clot structure and fibrinolysis.

BACKGROUND: Factor XIII is a 320 kDa tetramer, comprising two enzymatic A-subunits and two carrier B-subunits (FXIII A2 B2). Activated FXIII (FXIIIa) catalyses the formation of ε-(γ-glutamyl)lysyl covalent bonds between γ-γ, γ-α and α-α chains of adjacent fibrin molecules and also cross-links the major plasmin inhibitor, α2-antiplasmin, to fibrin. OBJECTIVES: We investigated the role of FXIII cross-linking of fibrin directly in clot morphology and its functional effect on clot formation and lysis, in the absence of α2-antiplasmin. RESULTS AND CONCLUSIONS: Our data show that the presence of FXIII during clot formation results in fibrin clots that have a significant 2.1-fold reduction in pore size, as determined by the Darcy constant, Ks, and formed thinner fibers (74.7 ± 1.5 nm) and higher density of fibers compared with those without FXIII (86.0 ± 1.7 nm, P < 0.001), as determined by scanning electron microscopy. Additionally, fibrinolysis showed a significant increase in the time to lysis for clots formed in the presence of FXIII in both static and flow systems. These data demonstrate that independent of α2-antiplasmin, FXIII activity plays a role in increasing the stability of the fibrin clot by altering its structure and increasing the resistance to fibrinolysis.

A novel mechanism for hypofibrinolysis in diabetes: the role of complement C3.

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.

Neprilysin, obesity and the metabolic syndrome.

OBJECTIVE: Neprilysin (NEP), a zinc metalloendopeptidase, 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 RESULTS: In 318 white European males, plasma NEP was elevated in the MetS and increased progressively with increasing MetS components. Plasma NEP activity correlated with insulin, homoeostasis model assessment and body mass index (BMI) in all subjects (P<0.01). Quantitative reverse transcriptase PCR (RT-PCR) and western blotting showed that in human pre-adipocytes NEP expression is upregulated 25- to 30-fold during differentiation into adipocytes. Microarray analysis of mRNA from differentiated human adipocytes confirmed high-NEP expression comparable with 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(-1), respectively; P<0.01). Tissue NEP was increased in mesenteric fat in HFD compared with NCD-fed mice (P<0.05). NEP knockout mice did not display any changes in insulin resistance, glucose tolerance, or body and epididymal fat pad weight compared with wild-type mice. CONCLUSION: In humans, NEP activity correlated with BMI 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 cardiometabolic risk in the presence of insulin resistance and increases with obesity.

Effect of the small molecule plasminogen activator inhibitor-1 (PAI-1) inhibitor, PAI-749, in clinical models of fibrinolysis.

BACKGROUND: The principal inhibitor of fibrinolysis in vivo is plasminogen activator inhibitor-1 (PAI-1). PAI-749 is a small molecule inhibitor of PAI-1 with proven antithrombotic efficacy in several preclinical models. OBJECTIVE: To assess the effect of PAI-749, by using an established ex vivo clinical model of thrombosis and a range of complementary in vitro human plasma-based and whole blood-based models of fibrinolysis. METHODS: In a double-blind, randomized, crossover study, ex vivo thrombus formation was assessed using the Badimon chamber in 12 healthy volunteers during extracorporeal administration of tissue-type plasminogen activator (t-PA) in the presence of PAI-749 or control. t-PA-mediated lysis of plasma clots and of whole blood model thrombi were assessed in vitro. The role of vitronectin was examined by assessing lysis of fibrin clots generated from purified plasma proteins. RESULTS: There was a dose-dependent reduction in ex vivo thrombus formation by t-PA (P < 0.0001). PAI-749 had no effect on in vitro or ex vivo thrombus formation or fibrinolysis in the presence or absence of t-PA. Inhibition of PAI-1 with a blocking antibody enhanced fibrinolysis in vitro (P < 0.05). CONCLUSIONS: Despite its efficacy in a purified human system and in preclinical models of thrombosis, the current study suggests that PAI-749 does not affect thrombus formation or fibrinolysis in a range of established human plasma and whole blood-based systems.

Effects of aspirin on clot structure and fibrinolysis using a novel in vitro cellular system.

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(+/-SEM)] was 0.083(+/-0.03), 0.093(+/-0.002), 0.101(+/-0.005), and 0.125(+/-0.003) in clots made from 0, 1, 10, and 100 mg/L aspirin-treated fibrinogen, respectively (P<0.05). Permeability coefficient (Ks cm2 x 10(-8)) was 1.68(+/-0.29) and 4.13(+/-0.33) comparing fibrinogen produced from cells grown with 0 mg/L and 100 mg/L aspirin respectively (P<0.05). 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.

Functional expression of glucose-dependent insulinotropic polypeptide receptors is coupled to differentiation in a human adipocyte model.

OBJECTIVE: To establish that human adipocytes express functional glucose-dependent insulinotropic peptide (GIP) receptors and in particular the regulation of GIP receptor (GIPR) expression in the context of the dynamic process of adipocyte differentiation. DESIGN: A combination of semiquantitative real-time PCR and measurement of GIP-stimulated cAMP accumulation was used to establish the expression and functional coupling of GIPRs during in vitro differentiation of human Simpson-Golabi-Behmel syndrome (SGBS) preadipocytes. RESULTS: Semiquantitative real-time PCR revealed that GIPR expression was substantially increased by day 4 of differentiation, reaching a maximum around 6-8 days (approximately 200-fold increase above undifferentiated cells, n=2). We also analysed the expression of the adipocyte fatty acid binding protein (FABP4) to relate GIPR expression to a molecular differentiation marker of adipogenesis. FABP4 expression was barely detectable in undifferentiated cells. However, following exposure to adipogenic medium, FABP4 expression gradually increased, with a maximal expression level around 10 days (approximately 1,600,000-fold increase above undifferentiated cells, n=2). Thus, the increases in GIPR mRNA during adipogenesis occur earlier than FABP4, suggesting that it might represent a gene expressed early in terminal differentiation and thus plays a role in fat droplet formation. A unit of 1 microM GIP failed to raise intracellular cAMP levels above basal levels in undifferentiated cells (n=3). In stark contrast, the 9-day differentiated cells produced a robust concentration-dependent increase in cAMP accumulation following stimulation with GIP, with an EC(50) value of 2.3 nM (n=3). The maximal response represented a 9-34-fold increase in cAMP accumulation above basal levels. CONCLUSIONS: This study demonstrates that GIPRs are expressed by human adipocytes, both GIPR mRNA and functional receptor expression being present in differentiated adipocytes but not in preadipocytes. Further investigation into the functional effects of GIP on differentiated SGBS cells could help towards understanding exactly how GIP regulates fat accumulation in human adipocytes.

Alanine aminotransferase is associated with atherothrombotic risk factors in a British South Asian population.

BACKGROUND: Alanine aminotransferase (ALT) predicts the development of Type 2 diabetes mellitus and cardiovascular disease in Caucasian subjects. OBJECTIVES: This study aimed to determine the incidence of an elevated ALT and its relationship to metabolic and atherothrombotic risk factors in a healthy British South Asian population. PATIENTS/METHODS: One hundred and forty-three participants from the West Yorkshire community were recruited randomly from general practice registers and were grouped according to whether their ALT was above or within the normal range (cut-off 35 IU L(-1)) and examined for differences in metabolic and atherothrombotic risk factors. All participants were originally from South Asia, with their grandparents being born in India, Pakistan, or Bangladesh. RESULTS: The incidence of a raised ALT was 24%. Those with a raised ALT had a more adverse metabolic profile, with significantly higher body mass index, waist/hip ratio, fasting insulin, glucose, homeostasis model assessment homeostasis model assessment insulin resistance (HOMA-IR), and triglycerides, and lower high-density lipoprotein (HDL) cholesterol. Fifty per cent had the metabolic syndrome [International Diabetes Federation (IDF) criteria]. They also had a more adverse atherothrombotic profile, with higher tissue-type plasminogen activator and plasminogen activator inhibitor-1 (PAI-1) antigen. In accordance, the group as a whole showed a positive correlation of ALT (age-adjusted) with waist/hip ratio, insulin, glucose, triglycerides, PAI-1 antigen, factor XIII B subunit, and FXII, and a negative correlation with HDL cholesterol. CONCLUSION: Raised ALT is common in apparently healthy British South Asians, and is significantly associated with an adverse metabolic and atherothrombotic risk profile.

Association between polymorphisms in the Clock gene, obesity and the metabolic syndrome in man.

OBJECTIVE: Accumulating evidence raises the hypothesis that dysregulation of intrinsic clock mechanisms are involved in the development of the metabolic syndrome, type 2 diabetes mellitus and cardiovascular disease. The aim of the present study was to investigate the relationship between three known common polymorphisms in the Clock gene and features of the metabolic syndrome in man. METHODS: Genotype and haplotype analysis was carried out in a cohort of 537 individuals from 89 families characterized for inflammatory, atherothrombotic and metabolic risk associated with insulin resistance. RESULTS: Heritability of the metabolic syndrome, defined according to International Diabetes Federation criteria, was 0.40. Haplotype analysis indicated three common haplotypes: CAT, TGT and CGC (rs4864548-rs3736544-rs1801260) with frequencies of 31, 33 and 28%, respectively. The CGC haplotype was less prevalent in subjects with the metabolic syndrome (P=0.0015) and was associated with lower waist circumference (P=0.007), lower hip circumference (P=0.023), lower body mass index (P=0.043) and lower leptin levels (P=0.028). The CAT haplotype was significantly associated with the presence of the metabolic syndrome (P=0.020). CONCLUSIONS: These findings suggest that the Clock gene CGC haplotype may be protective for the development of obesity and support the hypothesis that genetic variation in the Clock gene may play a role in the development of the metabolic syndrome, type 2 diabetes and cardiovascular disease.

Diabetes mellitus as a prothrombotic condition.

Diabetes mellitus (DM) is characterized by fasting hyperglycaemia and a high risk of atherothrombotic disorders affecting the coronary, cerebral and peripheral arterial trees. The risk of myocardial infarction (MI) is 3-5 fold higher in Type 2 DM and a DM subject with no history of MI has the same risk as a non-DM subject with a past history of MI. In total around 70% of deaths are vascular with poorer outcomes to both acute events and cardiological interventions. It was proposed that clustering of vascular risk factors (hyperinsulinaemia, dysglycaemia, dyslipidaemia and hypertension) around insulin resistance (IR) accounted for the increase in risk with Type 2 DM. The importance of this became apparent with the recognition that risk clustering occurs in normoglycaemic and impaired glucose tolerance (IGT) subjects with IR, in total around 25% of the population in addition to long-standing Type 1 subjects with renal disease. Evidence indicates that thrombotic risk clustering also occurs in association with IR, suppression of fibrinolysis due to elevated concentrations of the fibrinolytic inhibitor, plasminogen activator inhibitor-1 (PAI-1) is invariable with IR and there is evidence that this is regulated by the effects of triglyceride on the PAI-1 gene promoter. Other studies indicated that prothrombotic risk (coagulation factors VII, XII and fibrinogen) also associates with the IR syndrome. The development of endothelial cell dysfunction with suppression of nitric oxide and prostacyclin synthesis, combined with platelet resistance to the anti-aggregatory effects of these hormones leads to loss of control over platelet activation. In addition, hyperglycaemia and glycation have marked effects on fibrin structure function, generating a clot which has a denser structure, resistant to fibrinolysis. The combination of increased circulating coagulation zymogens, inhibition of fibrinolysis, changes in fibrin structure/function and alterations in platelet reactivity creates a thrombotic risk clustering which underpins the development of cardiovascular disease.

Ethnic differences in cardiovascular risk factors in healthy Caucasian and South Asian individuals with the metabolic syndrome.

BACKGROUND: The metabolic syndrome is a cluster of atherothrombotic risk factors that are commonly associated with insulin resistance. OBJECTIVES: The aim of this study was to investigate ethnic differences in insulin resistance and non-traditional cardiovascular risk factors in relation to the International Diabetes Federation (IDF) definition of the metabolic syndrome. PATIENTS AND METHODS: A total of 245 healthy South Asians and 245 age- and sex-matched Caucasians were studied. C-reactive protein (CRP), complement C3, plasminogen activator inhibitor-1 (PAI-1) and tissue plasminogen activator (t-PA) were measured and homeostasis model assessment-insulin resistance (HOMA-IR) was calculated from fasting plasma glucose and insulin levels. RESULTS: Fifty Caucasian (20%) and 95 (39%) South Asian subjects had the metabolic syndrome as defined by the IDF. In South Asian subjects, HOMA-IR, CRP, C3, PAI-1 and t-PA were significantly higher in subjects with the metabolic syndrome. In contrast, in Caucasian individuals there was no difference in HOMA-IR or C3 levels and only CRP, PAI-1 and t-PA were higher in subjects with the metabolic syndrome. In a logistic regression model, plasma levels of CRP and PAI-1 were independent predictors of the metabolic syndrome in Caucasians, whereas plasma levels of C3 and t-PA as well as HOMA-IR were independent predictors of the metabolic syndrome in South Asian subjects. CONCLUSIONS: In the cohort of individuals studied, the IDF definition of the metabolic syndrome was associated with insulin resistance in the South Asian but not the Caucasian population. This work also showed ethnic differences in non-traditional cardiovascular risk factors in the presence of the metabolic syndrome.