Plasma levels of mannan-binding lectin-associated serine proteases are increased in type 1 diabetes patients with insulin resistance.

Activation of the lectin pathway of the complement system, as demonstrated by elevated levels of mannan-binding lectin proteins (MBL), contributes to vascular pathology in type 1 diabetes (T1D). Vascular complications are greatest in T1D individuals with concomitant insulin resistance (IR), however, whether IR amplifies activiation of the lectin pathway in T1D is unknown. We pooled pretreatment data from two RCTs and performed a cross-sectional analysis on 46 T1D individuals. We employed estimated glucose disposal rate (eGDR), a validated IR surrogate with cut-points of: <5.1, 5.1-8.7, and > 8.7 mg/kg/min to determine IR status, with lower eGDR values conferring higher degrees of IR. Plasma levels of MBL-associated proteases (MASP-1, MASP-2, and MASP-3) and their regulatory protein MAp44 were compared among eGDR classifications. In a subset of 14 individuals, we assessed change in MASPs and MAp44 following improvement in IR. We found that MASP-1, MASP-2, MASP-3, and MAp44 levels increased in a stepwise fashion across eGDR thresholds with elevated MASPs and MAp44 levels conferring greater degrees of IR. In a subset of 14 patients, improvement in IR was associated with significant reductions in MASPs, but not MAp44, levels. In conclusion, IR in T1D amplifies levels of MASP-1/2/3 and their regulator MAp44, and improvement of IR normalizes MASP-1/2/3 levels. Given that elevated levels of these proteins contribute to vascular pathology, amplification of the lectin pathway of the complement system may offer mechanistic insight into the relationship between IR and vascular complications in T1D.

Short, frequent, light-intensity walking activity improves postprandial vascular-inflammatory biomarkers in people with type 1 diabetes: The SIT-LESS randomized controlled trial.

AIM: To examine the effect of interrupting prolonged sitting with short, frequent, light-intensity activity on postprandial cardiovascular markers in people with type 1 diabetes (T1D). MATERIALS AND METHODS: In a randomized crossover trial, 32 adults with T1D (mean ± SD age 28 ± 5 years, glycated haemoglobin 67.9 ± 12.6 mmol/mol, 17 women) completed two 7-h laboratory visits separated by >7 days. Participants either remained seated for 7 h (SIT) or interrupted sitting with 3-min bouts of self-paced walking at 30-min intervals commencing 1 h after each meal (SIT-LESS). Physical activity, insulin regimen, experimental start times, and meal consumption were standardized during each arm. Plasma levels of interleukin (IL)-1ß, tumour necrosis factor (TNF)-α, plasminogen activator inhibitor (PAI)-1 and fibrinogen were sampled at baseline, 3.5 and 7 h, and assessed for within- and between-group effects using a repeated measures ANOVA. The estimated glucose disposal rate was used to determine the insulin resistance status. RESULTS: Vascular-inflammatory parameters were comparable between SIT and SIT-LESS at baseline (p > .05). TNF-α, IL-1ß, PAI-1 and fibrinogen increased over time under SIT, whereas these rises were attenuated under SIT-LESS (p < .001). Specifically, over the 7 h under SIT, postprandial increases were detected in TNF-α, IL-1ß, PAI-1 and fibrinogen (+67%, +49%, +49% and +62%, respectively; p < .001 for all). Conversely, the SIT-LESS group showed no change in IL-1ß (-9%; p > .50), whereas reductions were observed in TNF-α, PAI-1 and fibrinogen (-22%, -42% and -44%, respectively; p < .001 for all). The intervention showed enhanced effects in insulin-resistant individuals with T1D. CONCLUSIONS: Interrupting prolonged sitting with light-intensity activity ameliorates postprandial increases in vascular-inflammatory markers in T1D. TRIAL REGISTRATION: The trial was prospectively registered (ISRCTN13641847).

Elimination of fibrin γ-chain cross-linking by FXIIIa increases pulmonary embolism arising from murine inferior vena cava thrombi.

The onset of venous thromboembolism, including pulmonary embolism, represents a significant health burden affecting more than 1 million people annually worldwide. Current treatment options are based on anticoagulation, which is suboptimal for preventing further embolic events. In order to develop better treatments for thromboembolism, we sought to understand the structural and mechanical properties of blood clots and how this influences embolism in vivo. We developed a murine model in which fibrin γ-chain cross-linking by activated Factor XIII is eliminated (FGG3X) and applied methods to study thromboembolism at whole-body and organ levels. We show that FGG3X mice have a normal phenotype, with overall coagulation parameters and platelet aggregation and function largely unaffected, except for total inhibition of fibrin γ-chain cross-linking. Elimination of fibrin γ-chain cross-linking resulted in thrombi with reduced strength that were prone to fragmentation. Analysis of embolism in vivo using Xtreme optical imaging and light sheet microscopy demonstrated that the elimination of fibrin γ-chain cross-linking resulted in increased embolization without affecting clot size or lysis. Our findings point to a central previously unrecognized role for fibrin γ-chain cross-linking in clot stability. They also indirectly indicate mechanistic targets for the prevention of thrombosis through selective modulation of fibrin α-chain but not γ-chain cross-linking by activated Factor XIII to reduce thrombus size and burden, while maintaining clot stability and preventing embolism.

Impact of glycaemic technologies on quality of life and related outcomes in adults with type 1 diabetes: A narrative review.

AIMS: To explore the association between the use of glycaemic technologies and person-reported outcomes (PROs) in adults with type 1 diabetes (T1D). METHODS: We included T1D and technology publications reporting on PROs since 2014. Only randomised controlled trials and cohort studies that used validated PRO measures (PROMs) were considered. RESULTS: T1D studies reported on a broad range of validated PROMs, mainly as secondary outcome measures. Most studies examined continuous glucose monitoring (CGM), intermittently scanned CGM (isCGM), and the role of continuous subcutaneous insulin infusion (CSII), including sensor-augmented CSII and closed loop systems. Generally, studies demonstrated a positive impact of technology on hypoglycaemia-specific and diabetes-specific PROs, including reduced fear of hypoglycaemia and diabetes distress, and greater satisfaction with diabetes treatment. In contrast, generic PROMs (including measures of health/functional status, emotional well-being, depressive symptoms, and sleep quality) were less likely to demonstrate improvements associated with the use of glycaemic technologies. Several studies showed contradictory findings, which may relate to study design, population and length of follow-up. Differences in PRO findings were apparent between randomised controlled trials and cohort studies, which may be due to different populations studied and/or disparity between trial and real-world conditions. CONCLUSIONS: PROs are usually assessed as secondary outcomes in glycaemic technology studies. Hypoglycaemia-specific and diabetes-specific, but not generic, PROs show the benefits of glycaemic technologies, and deserve a more central role in future studies as well as routine clinical care.

Interrupting prolonged sitting with frequent short bouts of light-intensity activity in people with type 1 diabetes improves glycaemic control without increasing hypoglycaemia: The SIT-LESS randomised controlled trial.

AIM: To examine the impact of interrupting prolonged sitting with frequent short bouts of light-intensity activity on glycaemic control in people with type 1 diabetes (T1D). MATERIALS AND METHODS: In total, 32 inactive adults with T1D [aged 27.9 ± 4.7 years, 15 men, diabetes duration 16.0 ± 6.9 years and glycated haemoglobin 8.4 ± 1.4% (68 ± 2.3 mmol/mol)] underwent two 7-h experimental conditions in a randomised crossover fashion with >7-day washout consisting of: uninterrupted sitting (SIT), or, interrupted sitting with 3-min bouts of self-paced walking at 30-min intervals (SIT-LESS). Standardised mixed-macronutrient meals were administered 3.5 h apart during each condition. Blinded continuous glucose monitoring captured interstitial glucose responses during the 7-h experimental period and for a further 48-h under free-living conditions. RESULTS: SIT-LESS reduced total mean glucose (SIT 8.2 ± 2.6 vs. SIT-LESS 6.9 ± 1.7 mmol/L, p = .001) and increased time in range (3.9-10.0 mmol/L) by 13.7% (SIT 71.5 ± 9.5 vs. SIT-LESS 85.1 ± 7.1%, p = .002). Hyperglycaemia (>10.0 mmol/L) was reduced by 15.0% under SIT-LESS (SIT 24.2 ± 10.8 vs. SIT-LESS 9.2 ± 6.4%, p = .002), whereas hypoglycaemia exposure (<3.9 mmol/L) (SIT 4.6 ± 3.0 vs. SIT-LESS 6.0 ± 6.0%, p = .583) was comparable across conditions. SIT-LESS reduced glycaemic variability (coefficient of variation %) by 7.8% across the observation window (p = .021). These findings were consistent when assessing discrete time periods, with SIT-LESS improving experimental and free-living postprandial, whole-day and night-time glycaemic outcomes (p < .05). CONCLUSIONS: Interrupting prolonged sitting with frequent short bouts of light-intensity activity improves acute postprandial and 48-h glycaemia in adults with T1D. This pragmatic strategy is an efficacious approach to reducing sedentariness and increasing physical activity levels without increasing risk of hypoglycaemia in T1D.

Dietary fat intake is associated with insulin resistance and an adverse vascular profile in patients with T1D: a pooled analysis.

BACKGROUND: Insulin resistance (IR) increases vascular risk in individuals with Type 1 Diabetes (T1D). We aimed to investigate the relationship between dietary intake and IR, as well as vascular biomarkers in T1D. METHODS: Baseline data from three randomised controlled trials were pooled. Estimated glucose disposal rate (eGDR) was used as an IR marker. Employing multivariate nutrient density substitution models, we examined the association between macronutrient composition and IR/vascular biomarkers (tumour necrosis factor-α, fibrinogen, tissue factor activity, and plasminogen activator inhibitor-1). RESULTS: Of the 107 patients, 50.5% were male with mean age of 29 ± 6 years. Those with lower eGDR were older with a longer diabetes duration, higher insulin requirements, and an adverse vascular profile (p < 0.05). Patients with higher degrees of IR had higher total energy intake (3192 ± 566 vs. 2772 ± 268 vs. 2626 ± 395 kcal/d for eGDR < 5.1 vs. 5.1-8.6 vs. ≥ 8.7 mg/kg/min, p < 0.001) and consumed a higher absolute and proportional amount of fat (47.6 ± 18.6 vs. 30.4 ± 8.1 vs. 25.8 ± 10.4%, p < 0.001). After adjusting for total energy intake, age, sex, and diabetes duration, increased carbohydrate intake offset by an isoenergetic decrease in fat was associated with higher eGDR (ß = 0.103, 95% CI 0.044-0.163). In contrast, increased dietary fat at the expense of dietary protein intake was associated with lower eGDR (ß = - 0.119, 95% CI - 0.199 to - 0.040). Replacing fat with 5% isoenergetic amount of carbohydrate resulted in decreased vascular biomarkers (p < 0.05). CONCLUSION: Higher fat, but not carbohydrate, intake is associated with increased IR and an adverse vascular profile in patients with T1D.

Total plasma magnesium, zinc, copper and selenium concentrations in obese patients before and after bariatric surgery.

Obesity enhances the risk of type-2 diabetes, cardiovascular disease and inflammatory conditions and often leads to metal dyshomeostasis, which contributes to the negative health aspects associated with the disease. In severe cases, bariatric surgery can be recommended to achieve sustained weight loss and improvement in health. Here, magnesium, zinc, copper and selenium concentrations were examined in 24 obese patients (7 males; 17 females) before and 9 months after undergoing Roux-en-Y gastric bypass surgery. All patients lost weight over this period, with the mean BMI reducing from 51.2±7.1 kg/m2 to 37.2±5.5 kg/m2. Moreover, whole-blood glycated haemoglobin (HbA1c), as a marker of average glycaemia, was also measured and a correlative analysis of this parameter with metal concentrations performed. Significant alterations in the plasma concentrations of magnesium, zinc (both increased by 13.2% and 25.2% respectively) and copper (decreased by 7.9%) were observed over this period (plasma selenium concentration was unchanged), with BMI values correlating with plasma magnesium (p = 0.004) and zinc (p = 0.022) concentrations. At 9 months post-surgery, an increase in mean zinc/copper ratio was observed (0.86±0.29 compared to 0.63±0.14 pre-surgery). Comparison of whole-blood HbA1c concentrations pre- and post-surgery revealed a reduction from 6.50±1.28% pre-surgery to 5.51±0.49% post-surgery. Differences in plasma HbA1c and magnesium at either pre- and post-surgery correlated significantly, as did HbA1c and magnesium levels when pre- and post-surgery values were analysed together. Collectively, this work reveals that bariatric surgery, in conjunction with lifestyle/dietary changes, lead to improvements in the nutritional status of magnesium, zinc and copper. Furthermore, the observed improvements in magnesium and zinc were associated with weight loss and in the case of magnesium, to better glycaemic control.

The Cellular and Protein Arms of Coagulation in Diabetes: Established and Potential Targets for the Reduction of Thrombotic Risk.

Diabetes is a metabolic condition with a rising global prevalence and is characterised by abnormally high blood glucose levels. Cardiovascular disease (CVD) accounts for the majority of deaths in diabetes and, despite improvements in therapy, mortality and hospitalisations in this cohort remain disproportionally higher compared to individuals with normal glucose metabolism. One mechanism for increased CVD risk is enhanced thrombosis potential, due to altered function of the cellular and acellular arms of coagulation. Different mechanisms have been identified that mediate disordered blood clot formation and breakdown in diabetes, including dysglycaemia, insulin resistance, and metabolic co-morbidities. Collectively, these induce platelet/endothelial dysfunction and impair the fibrinolytic process, thus creating a prothrombotic milieu. Despite these abnormalities, current antithrombotic therapies are largely similar in diabetes compared to those without this condition, which explains the high proportion of patients experiencing treatment failure while also displaying an increased risk of bleeding events. In this narrative review, we aimed to summarise the physiological functioning of haemostasis followed by the pathological effects of diabetes mellitus on platelets and the fibrin network. Moreover, we carefully reviewed the literature to describe the current and future therapeutic targets to lower the thrombosis risk and improve vascular outcomes in diabetes.

Zn2+ Differentially Influences the Neutralisation of Heparins by HRG, Fibrinogen, and Fibronectin.

For coagulation to be initiated, anticoagulant glycosaminoglycans (GAGs) such as heparins need to be neutralised to allow fibrin clot formation. Platelet activation triggers the release of several proteins that bind GAGs, including histidine-rich glycoprotein (HRG), fibrinogen, and fibronectin. Zn2+ ions are also released and have been shown to enhance the binding of HRG to heparins of a high molecular weight (HMWH) but not to those of low molecular weight (LMWH). The effect of Zn2+ on fibrinogen and fibronectin binding to GAGs is unknown. Here, chromogenic assays were used to measure the anti-factor Xa and anti-thrombin activities of heparins of different molecular weights and to assess the effects of HRG, fibrinogen, fibronectin, and Zn2+. Surface plasmon resonance was also used to examine the influence of Zn2+ on the binding of fibrinogen to heparins of different molecular weights. Zn2+ had no effect on the neutralisation of anti-factor Xa (FXa) or anti-thrombin activities of heparin by fibronectin, whereas it enhanced the neutralisation of unfractionated heparin (UFH) and HMWH by both fibrinogen and HRG. Zn2+ also increased neutralisation of the anti-FXa activity of LMWH by fibrinogen but not HRG. SPR showed that Zn2+ increased fibrinogen binding to both UFH and LMWH in a concentration-dependent manner. The presented results reveal that an increase in Zn2+ concentration has differential effects upon anticoagulant GAG neutralisation by HRG and fibrinogen, with implications for modulating anti-coagulant activity in plasma.

Interindividual variability in average glucose-glycated haemoglobin relationship in type 1 diabetes and implications for clinical practice.

AIM: Glycated haemoglobin (HbA1c) can fail to reflect average glucose levels, potentially compromising management decisions. We analysed variability in the relationship between mean glucose and HbA1c in individuals with diabetes. MATERIALS AND METHODS: Three months of continuous glucose monitoring and HbA1c data were obtained from 216 individuals with type 1 diabetes. Universal red blood cell glucose transporter-1 Michaelis constant KM and individualized apparent glycation ratio (AGR) were calculated and compared across age, racial and gender groups. RESULTS: The mean age (range) was 30 years (8-72) with 94 younger than 19 years, 78 between 19 and 50 years, and 44 were >50 years. The group contained 120 women and 96 men with 106 white and 110 black individuals. The determined KM value was 464 mg/dl and AGR was (mean ± SD) 72.1 ± 7 ml/g. AGR, which correlated with red blood cell lifespan marker, was highest in those aged >50 years at 75.4 ± 6.9 ml/g, decreasing to 73.2 ± 7.8 ml/g in 19-50 years, with a further drop to 71.0 ± 5.8 ml/g in the youngest group (p <0 .05). AGR differed between white and black groups (69.9 ± 5.8 and 74.2 ± 7.1 ml/g, respectively; p < .001). In contrast, AGR values were similar in men and women (71.5 ± 7.5 and 72.5 ± 6.6 ml/g, respectively; p = .27). Interestingly, interindividual AGR variation within each group was at least four-fold higher than average for between-group variation. CONCLUSIONS: In this type 1 diabetes cohort, ethnicity and age, but not gender, alter the HbA1c-glucose relationship with even larger interindividual variations found within each group than between groups. Clinical application of personalized HbA1c-glucose relationships has the potential to optimize glycaemic care in the population with diabetes.

Evaluation of continuous glucose monitoring-derived person-specific HbA1c in the presence and absence of complications in type 1 diabetes.

AIM: To evaluate the accuracy of a novel kinetic model at predicting HbA1c in a real-world setting and to understand and explore the role of diabetes complications in altering the glucose-HbA1c relationship and the mechanisms involved. MATERIALS AND METHODS: Deidentified HbA1c and continuous glucose monitoring values were collected from 93 individuals with type 1 diabetes. Person-specific kinetic variables were used, including red blood cell (RBC) glucose uptake and lifespan, to characterize the relationship between glucose levels and HbA1c. The resulting calculated HbA1c (cHbA1c) was compared with glucose management indicator (GMI) for prospective agreement with laboratory HbA1c. RESULTS: The cohort (42 men and 51 women) had a median age (IQR) of 61 (43, 72) years and a diabetes duration of 21 (10, 33) years. A total of 24 459 days of continuous glucose monitoring (CGM) data were available and 357 laboratory HbA1c were used to assess the average glucose-HbA1c relationship. cHbA1c had a superior correlation with laboratory HbA1c compared with GMI with a mean absolute deviation of 1.7 and 6.7 mmol/mol, r2  = 0.85 and 0.44, respectively. The fraction within 10% of absolute relative deviation from laboratory HbA1c was 93% for cHbA1c and 63% for GMI. Macrovascular disease had no effect on the model's accuracy, whereas microvascular complications resulted in a trend towards higher HbA1c, secondary to increased RBC glucose uptake. CONCLUSIONS: cHbA1c, which takes into account RBC glucose uptake and lifespan, accurately reflects laboratory HbA1c in a real-world setting and can aid in the management of individuals with diabetes.

Diabetes mellitus and perioperative outcomes: a scoping review of the literature.

BACKGROUND: Diabetes mellitus (DM) is frequently encountered in the perioperative period. DM may increase the risk of adverse perioperative outcomes owing to the potential vascular complications of DM. We conducted a scoping review to examine the association between DM and adverse perioperative outcomes. METHODS: A systematic search strategy of the published literature was built and applied in multiple databases. Observational studies examining the association between DM and adverse perioperative outcomes were included. Abstract screening determined full texts suitable for inclusion. Core information was extracted from each of the included studies including study design, definition of DM, type of DM, surgical specialties, and outcomes. Only primary outcomes are reported in this review. RESULTS: The search strategy identified 2363 records. Of those, 61 were included and 28 were excluded with justification. DM was mostly defined by either haemoglobin A1c (HbA1c) or blood glucose values (19 studies each). Other definitions included 'prior diagnosis' or use of medication. In 17 studies the definition was unclear. Type 2 DM was the most frequently studied subtype. Five of seven studies found DM was associated with mortality, 5/13 reported an association with 'complications' (as a composite measure), and 12/17 studies found DM was associated with 'infection'. Overall, 33/61 studies reported that DM was associated with the primary outcome measure. CONCLUSION: Diabetes mellitus is inconsistently defined in the published literature, which limits the potential for pooled analysis. Further research is necessary to determine which cohort of patients with DM are most at risk of adverse postoperative outcomes, and how control influences this association.

Antithrombotic therapy in diabetes: which, when, and for how long?

Cardiovascular disease remains the main cause of mortality in individuals with diabetes mellitus (DM) and also results in significant morbidity. Premature and more aggressive atherosclerotic disease, coupled with an enhanced thrombotic environment, contributes to the high vascular risk in individuals with DM. This prothrombotic milieu is due to increased platelet activity together with impaired fibrinolysis secondary to quantitative and qualitative changes in coagulation factors. However, management strategies to reduce thrombosis risk remain largely similar in individuals with and without DM. The current review covers the latest in the field of antithrombotic management in DM. The role of primary vascular prevention is discussed together with options for secondary prevention following an ischaemic event in different clinical scenarios including coronary, cerebrovascular, and peripheral artery diseases. Antiplatelet therapy combinations as well as combination of antiplatelet and anticoagulant agents are examined in both the acute phase and long term, including management of individuals with sinus rhythm and those with atrial fibrillation. The difficulties in tailoring therapy according to the variable atherothrombotic risk in different individuals are emphasized, in addition to the varying risk within an individual secondary to DM duration, presence of complications and predisposition to bleeding events. This review provides the reader with an up-to-date guide for antithrombotic management of individuals with DM and highlights gaps in knowledge that represent areas for future research, aiming to improve clinical outcome in this high-risk population.

Non-Traditional Pathways for Platelet Pathophysiology in Diabetes: Implications for Future Therapeutic Targets.

Cardiovascular complications remain the leading cause of morbidity and mortality in individuals with diabetes, driven by interlinked metabolic, inflammatory, and thrombotic changes. Hyperglycaemia, insulin resistance/deficiency, dyslipidaemia, and associated oxidative stress have been linked to abnormal platelet function leading to hyperactivity, and thus increasing vascular thrombotic risk. However, emerging evidence suggests platelets also contribute to low-grade inflammation and additionally possess the ability to interact with circulating immune cells, further driving vascular thrombo-inflammatory pathways. This narrative review highlights the role of platelets in inflammatory and immune processes beyond typical thrombotic effects and the impact these mechanisms have on cardiovascular disease in diabetes. We discuss pathways for platelet-induced inflammation and how platelet reprogramming in diabetes contributes to the high cardiovascular risk that characterises this population. Fully understanding the mechanistic pathways for platelet-induced vascular pathology will allow for the development of more effective management strategies that deal with the causes rather than the consequences of platelet function abnormalities in diabetes.

Strategies for Therapeutic Amelioration of Aberrant Plasma Zn2+ Handling in Thrombotic Disease: Targeting Fatty Acid/Serum Albumin-Mediated Effects.

The initiation, maintenance and regulation of blood coagulation is inexorably linked to the actions of Zn2+ in blood plasma. Zn2+ interacts with a variety of haemostatic proteins in the bloodstream including fibrinogen, histidine-rich glycoprotein (HRG) and high molecular weight kininogen (HMWK) to regulate haemostasis. The availability of Zn2+ to bind such proteins is controlled by human serum albumin (HSA), which binds 70-85% of plasma Zn2+ under basal conditions. HSA also binds and transports non-esterified fatty acids (NEFAs). Upon NEFA binding, there is a change in the structure of HSA which leads to a reduction in its affinity for Zn2+. This enables other plasma proteins to better compete for binding of Zn2+. In diseases where elevated plasma NEFA concentrations are a feature, such as obesity and diabetes, there is a concurrent increase in hypercoagulability. Evidence indicates that NEFA-induced perturbation of Zn2+-binding by HSA may contribute to the thrombotic complications frequently observed in these pathophysiological conditions. This review highlights potential interventions, both pharmaceutical and non-pharmaceutical that may be employed to combat this dysregulation. Lifestyle and dietary changes have been shown to reduce plasma NEFA concentrations. Furthermore, drugs that influence NEFA levels such as statins and fibrates may be useful in this context. In severely obese patients, more invasive therapies such as bariatric surgery may be useful. Finally, other potential treatments such as chelation therapies, use of cholesteryl transfer protein (CETP) inhibitors, lipase inhibitors, fatty acid inhibitors and other treatments are highlighted, which with additional research and appropriate clinical trials, could prove useful in the treatment and management of thrombotic disease through amelioration of plasma Zn2+ dysregulation in high-risk individuals.

Fibrinolysis in Acute and Chronic Cardiovascular Disease.

The formation of an obstructive thrombus within an artery remains a major cause of mortality and morbidity worldwide. Despite effective inhibition of platelet function by modern antiplatelet therapies, these agents fail to fully eliminate atherothrombotic risk. This may well be related to extensive vascular disease, beyond the protective abilities of the treatment agents used. However, recent evidence suggests that residual vascular risk in those treated with modern antiplatelet therapies is related, at least in part, to impaired fibrin clot lysis. In this review, we attempt to shed more light on the role of hypofibrinolysis in predisposition to arterial vascular events. We provide a brief overview of the coagulation system followed by addressing the role of impaired fibrin clot lysis in acute and chronic vascular conditions, including coronary artery, cerebrovascular, and peripheral vascular disease. We also discuss the role of combined anticoagulant and antiplatelet therapies to reduce the risk of arterial thrombotic events, addressing both efficacy and safety of such an approach. We conclude that impaired fibrin clot lysis appears to contribute to residual thrombosis risk in individuals with arterial disease on antiplatelet therapy, and targeting proteins in the fibrinolytic system represents a viable strategy to improve outcome in this population. Future work is required to refine the antithrombotic approach by modulating pathological abnormalities in the fibrinolytic system and tailoring therapy according to the need of each individual.

Affimer proteins as a tool to modulate fibrinolysis, stabilize the blood clot, and reduce bleeding complications.

Bleeding complications secondary to surgery, trauma, or coagulation disorders are important causes of morbidity and mortality. Although fibrin sealants are considered to minimize blood loss, this is not widely adopted because of its high cost and/or risk for infection. We present a novel methodology employing nonantibody fibrinogen-binding proteins, termed Affimers, to stabilize fibrin networks with the potential to control excessive bleeding. Two fibrinogen-specific Affimer proteins, F5 and G2, were identified and characterized for their effects on clot structure/fibrinolysis, using turbidimetric and permeation analyses and confocal and electron microscopy. Binding studies and molecular modeling identified interaction sites, whereas plasmin generation assays determined effects on plasminogen activation. In human plasma, F5 and G2 prolonged clot lysis time from 9.8 ± 1.1 minutes in the absence of Affimers to 172.6 ± 7.4 and more than 180 minutes (P < .0001), respectively, and from 7.6 ± 0.2 to 28.7 ± 5.8 (P < .05) and 149.3 ± 9.7 (P < .0001) minutes in clots made from purified fibrinogen. Prolongation in fibrinolysis was consistent across plasma samples from healthy control patients and individuals at high bleeding risk. F5 and G2 had a differential effect on clot structure and G2 profoundly altered fibrin fiber arrangement, whereas F5 maintained physiological clot structure. Affimer F5 reduced fibrin-dependent plasmin generation and was predicted to bind fibrinogen D fragment close to tissue plasminogen activator (tPA; residues γ312-324) and plasminogen (α148-160) binding sites, thus interfering with tPA-plasminogen interaction and representing 1 potential mechanism for modulation of fibrinolysis. Our Affimer proteins provide a novel methodology for stabilizing fibrin networks with potential future clinical implications to reduce bleeding risk.

Reduction in cardiovascular mortality following severe hypoglycemia in individuals with type 2 diabetes: the role of a pragmatic and structured intervention : Structured intervention for community hypoglycemia.

BACKGROUND: Mortality in individuals with diabetes with severe hypoglycemia requiring ambulance services intervention is high and it is unclear whether this is modifiable. Our aim was to characterise this high-risk group and assess the impact of nurse-led intervention on mortality. METHODS: In this single centre study, patients with diabetes and hypoglycemia requiring ambulance call out were randomized to nurse led support (intensive arm) or managed using existing pathways (standard arm). A third group agreed to have their data collected longitudinally (observational arm). The primary outcome was all-cause mortality comparing intensive with combined standard and observational arms as well as standard arm alone. RESULTS: Of 828 individuals identified, 323 agreed to participate with 132 assigned to intensive, 130 to standard and 61 to observational arms. Mean follow up period was 42.6 ± 15.6 months. Mortality in type 1 diabetes (n = 158) was similar across study arms but in type 2 diabetes (n = 160) this was reduced to 33% in the intensive arm compared with 51% in the combined arm (p = 0.025) and 50% in the standard arm (p = 0.06). Cardiovascular deaths, the leading cause of mortality, was lower in the intensive arm compared with combined and standard study arms (p < 0.01). CONCLUSIONS: Medium-term mortality following severe hypoglycemia requiring the assistance of emergency services is high in those with type 2 diabetes. In individuals with type 2 diabetes, nurse-led individualized intervention reduces cardiovascular mortality compared with standard care. Large-scale multicentre studies are warranted to further investigate this approach. Trial registration The trial was retrospectively registered on http://www.clinicaltrials.gov with reference NCT04422145.

A randomised controlled trial to assess the antithrombotic effects of aspirin in type 1 diabetes: role of dosing and glycaemic control.

BACKGROUND: The enhanced thrombotic milieu in diabetes contributes to increased risk of vascular events. Aspirin, a key antiplatelet agent, has inconsistent effects on outcomes in diabetes and the best dosing regimen remains unclear. This work investigated effects of aspirin dose and interaction with glycaemia on both the cellular and protein components of thrombosis. METHODS: A total of 48 participants with type 1 diabetes and 48 healthy controls were randomised to receive aspirin 75 or 300 mg once-daily (OD) in an open-label crossover study. Light transmittance aggregometry and fibrin clot studies were performed before and at the end of each treatment period. RESULTS: Aspirin demonstrated reduced inhibition of collagen-induced platelet aggregation (PA) in participants with diabetes compared with controls, although the higher dose showed better efficacy. Higher aspirin dose facilitated clot lysis in controls but not individuals with diabetes. Collagen-induced PA correlated with glycaemic control, those in the top HbA1c tertile having a lesser inhibitory effect of aspirin. Threshold analysis suggested HbA1c levels of > 65 mmol/mol and > 70 mmol/mol were associated with poor aspirin response to 75 and 300 mg daily doses, respectively. Higher HbA1c was also associated with longer fibrin clot lysis time. CONCLUSIONS: Patients with diabetes respond differently to the antiplatelet and profibrinolytic effects of aspirin compared with controls. In particular, those with elevated HbA1c have reduced inhibition of PA with aspirin. Our findings indicate that reducing glucose levels improves the anti-thrombotic action of aspirin in diabetes, which may have future clinical implications. TRIAL REGISTRATION: EudraCT, 2008-007875-26, https://www.clinicaltrialsregister.eu/ctr-search/search?query=2008-007875-26 .

Fibrinogen interaction with complement C3: a potential therapeutic target to reduce thrombosis risk.

Complement C3 binds fibrinogen and compromises fibrin clot lysis thereby enhancing thrombosis risk. We investigated the role of fibrinogen-C3 interaction as a novel therapeutic target to reduce thrombosis risk by analysing: i) consistency in the fibrinolytic properties of C3, ii) binding sites between fibrinogen and C3 and iii) modulation of fibrin clot lysis by manipulating fibrinogen-C3 interactions. Purified fibrinogen and C3 from the same individuals (n=24) were used to assess inter-individual variability in the anti-fibrinolytic effects of C3. Microarray screening and molecular modelling evaluated C3 and fibrinogen interaction sites. Novel synthetic conformational proteins, termed Affimers, were used to modulate C3-fibrinogen interaction and fibrinolysis. C3 purified from patients with type 1 diabetes showed enhanced prolongation of fibrinolysis compared with healthy control protein [195±105 and 522±166 seconds, respectively (p=0.04)], with consistent effects but a wider range (5-51% and 5-18% lysis prolongation, respectively). Peptide microarray screening identified 2 potential C3-fibrinogen interactions sites within fibrinogen ß chain (residues 424-433, 435-445). One fibrinogen-binding Affimer was isolated that displayed sequence identity with C3 in an exposed area of the protein. This Affimer abolished C3-induced prolongation of fibrinolysis (728±25.1 seconds to 632±23.7 seconds, p=0.005) and showed binding to fibrinogen in the same region that is involved in C3-fibrinogen interactions. Moreover, it shortened plasma clot lysis of patients with diabetes, cardiovascular disease or controls by 7-11%. C3 binds fibrinogen ß-chain and disruption of fibrinogen-C3 interaction using Affimer proteins enhances fibrinolysis, which represents a potential novel target tool to reduce thrombosis in high risk individuals.