The Impact of Patient Characteristics and Antiplatelet Regimes on Clot Microstructure in Patients Treated for ST Elevation Myocardial Infarction: Clot Microstructure can Evaluate Therapeutic Efficacy.

BACKGROUND: Unfavourable clot microstructure is associated with adverse outcomes in ST elevation myocardial infarction (STEMI). We investigated the effect of comorbidities and anti-platelet treatment on clot microstructure in STEMI patients using fractal dimension (df), a novel biomarker of clot microstructure derived from the visco-elastic properties of whole blood. METHODS: Patients with STEMI (n = 187) were recruited sequentially receiving aspirin with Clopidogrel (n = 157) then Ticagrelor (n = 30). Patient characteristics and blood for rheological analysis obtained. We quantified df using sequential frequency sweep tests to obtain the phase angle of the Gel Point which is synonymous with the clot microstructure. RESULTS: Higher df was observed in males (1.755 ± 0.068) versus females (1.719 ± 0.061, p = .001), in patients with diabetes (1.786 ± 0.067 vs 1.743 ± 0.046, p < .001), hypertension (1.760 ± 0.065 vs 1.738 ± 0.069, p = .03) and previous MI (1.787 ± 0.073 vs 1.744 ± 0.066, p = .011) compared to without. Patients receiving Ticagrelor had lower df than those receiving Clopidogrel (1.708 ± 0.060 vs 1.755 ± 0.067, p < .001). Significant correlation with df was found with haematocrit (r = 0.331, p < .0001), low-density lipoprotein (LDL) (r = 0.155, p = .046) and fibrinogen (r = 0.182, p = .014). Following multiple regression analysis, diabetes, LDL, fibrinogen and haematocrit remained associated with higher df while treatment with Ticagrelor remained associated with lower df. CONCLUSIONS: The biomarker df uniquely evaluates the effect of interactions between treatment and underlying disease on clot microstructure. STEMI patients with diabetes and elevated LDL had higher df, indicating denser clot. Ticagrelor resulted in a lower df than Clopidogrel signifying a less compact clot.

The efficacy of low molecular weight heparin is reduced in COVID-19.

BACKGROUND: A significant degree of mortality and morbidity in COVID-19 is through thromboembolic complications, only partially mitigated by anticoagulant therapy. Reliable markers of infection severity are not fully established. OBJECTIVES: This study investigated whether visco-elastic biomarkers predict disease severity on presentation to the Emergency Department (ED) and how they measure response to anticoagulationMETHODS:Patients testing positive for COVID-19 at a large University Teaching Hospital ED were recruited at presentation. Multiple blood samples were taken throughout hospital admission to monitor disease progression with end outcome recorded. Visco-elastic markers, fractal dimension (df) and Time to Gel Point (TGP) which measure the properties of the incipient clot were compared in patients with and without anticoagulation by Low Molecular Weight Heparin (LMWH). RESULTS: TGP and df did not predict severity of infection with COVID-19. Although LMWH prolonged TGP, there was no change in df indicating LMWH did not change clot microstructure. CONCLUSIONS: Therapeutic efficacy of LMWH appears blunted in COVID-19 infection. This may be due to the inflammatory state creating a resistance to LMWH activity, which may explain why LMWH appears less effective in COVID-19 compared to other disease states. COVID-19 was not predicted by visco-elastic testing at the time of ED presentation.

High levels of soluble RAGE are associated with a greater risk of mortality in COVID-19 patients treated with dexamethasone.

Blood levels of the soluble receptor for advanced glycation end-products (sRAGE) are acutely elevated during the host inflammatory response to infection and predict mortality in COVID-19. However, the prognostic performance of this biomarker in the context of treatments to reduce inflammation is unclear. In this study we investigated the association between sRAGE and mortality in dexamethasone-treated COVID-19 patients. We studied 89 SARS-CoV-2 positive subjects and 22 controls attending the emergency department of a University Teaching Hospital during the second wave of COVID-19 and measured sRAGE at admission. In positive individuals sRAGE increased with disease severity and correlated with the National Early Warning Score 2 (Pearson's r = 0.56, p < 0.001). Fourteen out of 72 patients treated with dexamethasone died during 28 days of follow-up. Survival rates were significantly lower in patients with high sRAGE (> 3532 pg/mL) than in those with low sRAGE (p = 0.01). Higher sRAGE levels were associated with an increased risk of death after adjustment for relevant covariates. In contrast, IL-6 did not predict mortality in these patients. These results demonstrate that sRAGE remains an independent predictor of mortality among COVID-19 patients treated with dexamethasone. Determination of sRAGE could be useful for the clinical management of this patient population.

Impaired fibrinolysis in severe Covid-19 infection is detectable in early stages of the disease.

BACKGROUND: A significant degree of mortality and morbidity in Covid-19 is due to thromboembolic disease. Coagulopathy has been well described in critically unwell patients on ICU. There is less clear evidence regarding these changes at the time of presentation to the Emergency Department and the progression of disease over time. OBJECTIVE: We sought to investigate whether coagulation markers can predict severity and how they change over the disease course. METHODS: Patients presenting to a single University Teaching Hospital were recruited and followed up if PCR was positive. Alongside routine blood testing, Rotational Thromboelastometry (ROTEM) was performed. Outcome data was recorded for all patients, and ROTEM values were compared across outcome groups. RESULTS: Extem and Intem Maximum Lysis were significantly reduced in those who died or required an ICU admission, indicating a reduced ability to break down clot mass in the most critically unwell patients. CONCLUSION: Comparisons between groups demonstrated that one distinguishing feature between those who require ICU admission or die of Covid-19 compared with those who survive a hospital stay to discharge was the extent to which fibrinolysis could occur. Mortality and morbidity in Covid-19 infection appears in part driven by an inability to break down clot mass.

Candidate Bioinks for Extrusion 3D Bioprinting-A Systematic Review of the Literature.

Purpose: Bioprinting is becoming an increasingly popular platform technology for engineering a variety of tissue types. Our aim was to identify biomaterials that have been found to be suitable for extrusion 3D bioprinting, outline their biomechanical properties and biocompatibility towards their application for bioprinting specific tissue types. This systematic review provides an in-depth overview of current biomaterials suitable for extrusion to aid bioink selection for specific research purposes and facilitate design of novel tailored bioinks. Methods: A systematic search was performed on EMBASE, PubMed, Scopus and Web of Science databases according to the PRISMA guidelines. References of relevant articles, between December 2006 to January 2018, on candidate bioinks used in extrusion 3D bioprinting were reviewed by two independent investigators against standardised inclusion and exclusion criteria. Data was extracted on bioprinter brand and model, printing technique and specifications (speed and resolution), bioink material and class of mechanical assessment, cell type, viability, and target tissue. Also noted were authors, study design (in vitro/in vivo), study duration and year of publication. Results: A total of 9,720 studies were identified, 123 of which met inclusion criteria, consisting of a total of 58 reports using natural biomaterials, 26 using synthetic biomaterials and 39 using a combination of biomaterials as bioinks. Alginate (n = 50) and PCL (n = 33) were the most commonly used bioinks, followed by gelatin (n = 18) and methacrylated gelatin (GelMA) (n = 16). Pneumatic extrusion bioprinting techniques were the most common (n = 78), followed by piston (n = 28). The majority of studies focus on the target tissue, most commonly bone and cartilage, and investigate only one bioink rather than assessing a range to identify those with the most promising printability and biocompatibility characteristics. The Bioscaffolder (GeSiM, Germany), 3D Discovery (regenHU, Switzerland), and Bioplotter (EnvisionTEC, Germany) were the most commonly used commercial bioprinters (n = 35 in total), but groups most often opted to create their own in-house devices (n = 20). Many studies also failed to specify whether the mechanical data reflected pre-, during or post-printing, pre- or post-crosslinking and with or without cells. Conclusions: Despite the continued increase in the variety of biocompatible synthetic materials available, there has been a shift change towards using natural rather than synthetic bioinks for extrusion bioprinting, dominated by alginate either alone or in combination with other biomaterials. On qualitative analysis, no link was demonstrated between the type of bioink or extrusion technique and the target tissue, indicating that bioprinting research is in its infancy with no established tissue specific bioinks or bioprinting techniques. Further research is needed on side-by-side characterisation of bioinks with standardisation of the type and timing of biomechanical assessment.

Abnormal clot microstructure formed in blood containing HIT-like antibodies.

INTRODUCTION: Thrombosis is a severe and frequent complication of heparin-induced thrombocytopenia (HIT). However, there is currently no knowledge of the effects of HIT-like antibodies on the resulting microstructure of the formed clot, despite such information being linked to thrombotic events. We evaluate the effect of the addition of pathogenic HIT-like antibodies to blood on the resulting microstructure of the formed clot. MATERIALS AND METHODS: Pathogenic HIT-like antibodies (KKO) and control antibodies (RTO) were added to samples of whole blood containing Unfractionated Heparin and Platelet Factor 4. The formed clot microstructure was investigated by rheological measurements (fractal dimension; df) and scanning electron microscopy (SEM), and platelet activation was measured by flow cytometry. RESULTS AND CONCLUSIONS: Our results revealed striking effects of KKO on clot microstructure. A significant difference in df was found between samples containing KKO (df = 1.80) versus RTO (df = 1.74; p < 0.0001). This increase in df was often associated with an increase in activated platelets. SEM images of the clots formed with KKO showed a network consisting of a highly branched and compact arrangement of thin fibrin fibres, typically found in thrombotic disease. This is the first study to identify significant changes in clot microstructure formed in blood containing HIT-like antibodies. These observed alterations in clot microstructure can be potentially exploited as a much-needed biomarker for the detection, management and monitoring of HIT-associated thrombosis.

In vitro clot model to evaluate fibrin-thrombin effects on fractal dimension of incipient blood clot.

INTRODUCTION: This aim of this study is to investigate the individual effects of varying concentrations of thrombin and fibrinogen on clot microstructure (characterised through the fractal dimension of the incipient clot network, df) and clot formation time (TGP) using a fibrin-thrombin clot model. df and TGP markers are measured using a haemorheological method that has already been investigated for whole blood. METHODS: This is an in vitro study using three thrombin concentrations (0.1, 0.05 and 0.02 NIH/ml) and two fibrinogen concentrations (8 mg/ml and 12 mg/ml) to investigate a fibrin-thrombin clot model. The haemorheological changes were measured at the gel point using df and TGP. RESULTS: Fractal dimension (df) increased with increasing concentrations of thrombin both at 8 mg/ml (1.60±0.024, 1.67±0.022, 1.74±0.079) and 12 mg/ml fibrinogen concentrations (1.63±0.02, 1.87±0.019, 1.95±0.014). On the other hand, TGP decreased for both 8 mg/ml (1089±265, 637±80, 223±22 seconds) and 12 mg/ml fibrinogen concentrations (2008±247, 776±20, 410±20 seconds). In contrast to previous studies investigating whole blood, TGP increased with higher fibrinogen levels. CONCLUSIONS: The findings from this fibrin-thrombin clot model confirmed that df and TGP can detect changes in the incipient clot following manipulation of fibrinogen and thrombin concentration. df increases (indicating stronger clot) with higher concentrations of thrombin and fibrinogen. On the other hand, TGP decreased as expected with higher thrombin level but not with higher fibrinogen concentrations.

Printability of pulp derived crystal, fibril and blend nanocellulose-alginate bioinks for extrusion 3D bioprinting.

BACKGROUND: One of the main challenges for extrusion 3D bioprinting is the identification of non-synthetic bioinks with suitable rheological properties and biocompatibility. Our aim was to optimize and compare the printability of crystal, fibril and blend formulations of novel pulp derived nanocellulose bioinks and assess biocompatibility with human nasoseptal chondrocytes. METHODS: The printability of crystalline, fibrillated and blend formulations of nanocellulose was determined by assessing resolution (grid-line assay), post-printing shape fidelity and rheology (elasticity, viscosity and shear thinning characteristics) and compared these to pure alginate bioinks. The optimized nanocellulose-alginate bioink was bioprinted with human nasoseptal chondrocytes to determine cytotoxicity, metabolic activity and bioprinted construct topography. RESULTS: All nanocellulose-alginate bioink combinations demonstrated a high degree of shear thinning with reversible stress softening behavior which contributed to post-printing shape fidelity. The unique blend of crystal and fibril nanocellulose bioink exhibited nano- as well as micro-roughness for cellular survival and differentiation, as well as maintaining the most stable construct volume in culture. Human nasoseptal chondrocytes demonstrated high metabolic activity post printing and adopted a rounded chondrogenic phenotype after prolonged culture. CONCLUSIONS: This study highlights the favorable rheological, swelling and biocompatibility properties of nanocellulose-alginate bioinks for extrusion-based bioprinting.

Artificial shear stress effects on leukocytes at a biomaterial interface.

Medical devices, such as ventricular assist devices (VADs), introduce both foreign materials and artificial shear stress to the circulatory system. The effects these have on leukocytes and the immune response are not well understood. Understanding how these two elements combine to affect leukocytes may reveal why some patients are susceptible to recurrent device-related infections and provide insight into the development of pump thrombosis. Biomaterials-DLC: diamond-like carbon-coated stainless steel; Sap: single-crystal sapphire; and Ti: titanium alloy (Ti6 Al4 V) were attached to the parallel plates of a rheometer. Whole human blood was left between the two discs for 5 minutes at +37°C with or without the application of shear stress (0 s-1 or 1000 s-1 ). Blood was removed and used for complete blood cell counts, flow cytometry (leukocyte activation, cell death, microparticle generation, phagocytic ability, and reactive oxygen species [ROS] production), and the production of pro-inflammatory cytokines. L-selectin expression on monocytes was decreased when blood was exposed to the biomaterials both with and without shear. Applying shear stress to blood on a Sap and Ti surface led to activation of neutrophils shown as decreased L-selectin expression. Sap and Ti blunted the LPS-stimulated macrophage migration inhibitory factor (MIF) production, most notably when sheared on Ti. The biomaterials used here have been shown to activate leukocytes in a static environment. The introduction of shear appears to exacerbate this activation. Interestingly, a widely accepted biocompatible material (Ti) utilized in many different types of devices has the capacity for immune cell activation and inhibition of MIF secretion when combined with shear stress. These findings contribute to our understanding of the contribution of biomaterials and shear stress to recurrent infections and vulnerability to sepsis in some VAD patients as well as pump thrombosis.

Characterization of pulp derived nanocellulose hydrogels using AVAP® technology.

Bioinspiration from hierarchical structures found in natural environments has heralded a new age of advanced functional materials. Nanocellulose has received significant attention due to the demand for high-performance materials with tailored mechanical, physical and biological properties. In this study, nanocellulose fibrils, nanocrystals and a novel mixture of fibrils and nanocrystals (blend) were prepared from softwood biomass using the AVAP® biorefinery technology. These materials were characterized using transmission and scanning electron microscopy, and atomic force microscopy. This analysis revealed a nano- and microarchitecture with extensive porosity. Notable differences included the nanocrystals exhibiting a compact packing of nanorods with reduced porosity. The NC blend exhibited porous fibrillar networks with interconnecting compact nanorods. Fourier transform infrared spectroscopy and X-ray diffraction confirmed a pure cellulose I structure. Thermal studies highlighted the excellent stability of all three NC materials with the nanocrystals having the highest decomposition temperature. Surface charge analysis revealed stable colloid suspensions. Rheological studies highlighted a dominance of elasticity in all variants, with the NC blend being more rigid than the NC fibrils and nanocrystals, indicating a double network hydrogel structure. Given these properties, it is thought that these materials show great potential in (bio)nanomaterial applications where careful control of microarchitecture, surface topography and porosity are required.

Platelet reactivity influences clot structure as assessed by fractal analysis of viscoelastic properties.

Despite the interwoven nature of platelet activation and the coagulation system in thrombosis, few studies relate both analysis of protein and cellular parts of coagulation in the same population. In the present study, we use matched ex vivo samples to determine the influences of standard antiplatelet therapies on platelet function and use advanced rheological analyses to assess clot formation. Healthy volunteers were recruited following fully informed consent then treated for 7 days with single antiplatelet therapy of aspirin (75 mg) or prasugrel (10 mg) or with dual antiplatelet therapy (DAPT) using aspirin (75 mg) plus prasugrel (10 mg) or aspirin (75 mg) plus ticagrelor (90 mg). Blood samples were taken at day 0 before treatment and at day 7 following treatment. We found that aspirin plus prasugrel or aspirin plus ticagrelor inhibited platelet responses to multiple agonists and reduced P-selectin expression. Significant platelet inhibition was coupled with a reduction in fractal dimension corresponding to reductions in mean relative mass both for aspirin plus prasugrel (-35 ± 16% change, p = 0.04) and for aspirin plus ticagrelor (-45 ± 14% change, p = 0.04). Aspirin alone had no effect upon measures of clot structure, whereas prasugrel reduced fractal dimension and mean relative mass. These data demonstrate that platelets are important determinants of clot structure as assessed by fractal dimension (df) and that effective platelet inhibition is associated with a weaker, more permeable fibrin network. This indicates a strong association between the therapeutic benefits of antiplatelet therapies and their abilities to reduce thrombus density that may be useful in individual patients to determine the functional relationship between platelet reactivity, eventual clot quality, and clinical outcome. df could represent a novel risk stratification biomarker useful in individualizing antiplatelet therapies.

Characterisation of clot microstructure properties in stable coronary artery disease.

BACKGROUND: Coronary artery disease (CAD) is associated with an increased prothrombotic tendency and is also linked to unfavourably altered clot microstructure. We have previously described a biomarker of clot microstructure (df) that is unfavourably altered in acute myocardial infarction. The df biomarker assesses whether the blood will form denser or looser microstructures when it clots. In this study we assessed in patients with stable chest pain whether df can differentiate between obstructed and unobstructed CAD. METHODS: A blood sample prior to angiography was obtained from 251 consecutive patients undergoing diagnostic coronary angiography. Patients were categorised based on angiographic findings as presence or absence of obstructive CAD (stenosis ≥50%). The blood sample was assessed using the df biomarker, standard laboratory markers and platelet aggregometry (Multiplate). RESULTS: A significant difference (p=0.028) in df was observed between obstructive CAD (1.748±0.057, n=83) and unobstructive CAD (1.732±0.052, n=168), where patients with significant CAD produce denser, more tightly packed clots. df was also raised in men with obstructive CAD compared with women (1.745±0.055 vs 1.723±0.052, p=0.007). Additionally df significantly correlated with the platelets response to arachidonic acid as measured by the ASPItest area under the curve readings from platelet aggregometry (correlation coefficient=0.166, p=0.008), a low value of the ASPItest indicating effective aspirin use was associated with looser, less dense clots. CONCLUSIONS: For the first time, we characterise clot microstructure, as measured by df, in patients with stable CAD. df can potentially be used to risk-stratify patients with stable CAD and assess the efficacy of therapeutic interventions by measuring changes in clot microstructure.

3D bioprinting for reconstructive surgery: Principles, applications and challenges.

Despite the increasing laboratory research in the growing field of 3D bioprinting, there are few reports of successful translation into surgical practice. This review outlines the principles of 3D bioprinting including software and hardware processes, biocompatible technological platforms and suitable bioinks. The advantages of 3D bioprinting over traditional tissue engineering techniques in assembling cells, biomaterials and biomolecules in a spatially controlled manner to reproduce native tissue macro-, micro- and nanoarchitectures are discussed, together with an overview of current progress in bioprinting tissue types relevant for plastic and reconstructive surgery. If successful, this platform technology has the potential to biomanufacture autologous tissue for reconstruction, obviating the need for donor sites or immunosuppression. The biological, technological and regulatory challenges are highlighted, with strategies to overcome these challenges by using an integrated approach from the fields of engineering, biomaterial science, cell biology and reconstructive microsurgery.

In-situ synthesis of magnetic iron-oxide nanoparticle-nanofibre composites using electrospinning.

We demonstrate a facile, one-step process to form polymer scaffolds composed of magnetic iron oxide nanoparticles (MNPs) contained within electrospun nano- and micro-fibres of two biocompatible polymers, Poly(ethylene oxide) (PEO) and Poly(vinyl pyrrolidone) (PVP). This was achieved with both needle and free-surface electrospinning systems demonstrating the scalability of the composite fibre manufacture; a 228 fold increase in fibre fabrication was observed for the free-surface system. In all cases the nanoparticle-nanofibre composite scaffolds displayed morphological properties as good as or better than those previously described and fabricated using complex multi-stage techniques. Fibres produced had an average diameter (Needle-spun: 125±18nm (PEO) and 1.58±0.28µm (PVP); Free-surface electrospun: 155±31nm (PEO)) similar to that reported previously, were smooth with no bead defects. Nanoparticle-nanofibre composites were characterised using scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), dynamic light scattering (DLS) (Nanoparticle average diameter ranging from 8±3nm to 27±5nm), XRD (Phase of iron oxide nanoparticles identified as magnetite) and nuclear magnetic resonance relaxation measurements (NMR) (T1/T2: 32.44 for PEO fibres containing MNPs) were used to verify the magnetic behaviour of MNPs. This study represents a significant step forward for production rates of magnetic nanoparticle-nanofibre composite scaffolds by the electrospinning technique.

An Investigation Into the Effects of In Vitro Dilution With Different Colloid Resuscitation Fluids on Clot Microstructure Formation.

BACKGROUND: Balancing the beneficial effects of resuscitation fluids against their detrimental effect on hemostasis is an important clinical issue. We aim to compare the in vitro effects of 3 different colloid resuscitation fluids (4.5% albumin, hydroxyethyl starch [Voluven 6%], and gelatin [Geloplasma]) on clot microstructure formation using a novel viscoelastic technique, the gel point. This novel hemorheologic technique measures the biophysical properties of the clot and provides an assessment of clot microstructure from its viscoelastic properties. Importantly, in contrast to many assays in routine clinical use, the measurement is performed using unadulterated whole blood in a near-patient setting and provides rapid assessment of coagulation. We hypothesized that different colloids will have a lesser or greater detrimental effect on clot microstructure formation when compared against each other. METHODS: Healthy volunteers were recruited into the study (n = 104), and a 20-mL sample of whole blood was obtained. Each volunteer was assigned to 1 of the 3 fluids, and the sample was diluted to 1 of 5 different dilutions (baseline, 10%, 20%, 40%, and 60%). The blood was tested using the gel point technique, which measures clot mechanical strength and quantifies clot microstructure (df) at the incipient stages of fibrin formation. RESULTS: df and clot mechanical strength decrease with progressive dilution for all 3 fluids. A significant reduction in df from baseline was recorded at dilutions of 20% for albumin (P < .0001), 40% for starch (P < .0001), and 60% for gelatin (P < .0001). We also observed significant differences, in terms of df, when comparing the different types of colloid (P < .0001). We found that albumin dilution produced the largest changes in clot microstructure, providing the lowest values of df (= 1.41 ± 0.061 at 60% dilution) compared with starch (1.52 ± 0.081) and gelatin (1.58 ± 0.063). CONCLUSIONS: We show that dilution with all 3 fluids has a significant effect on coagulation at even relatively low dilution volumes (20% and 40%). Furthermore, we quantify, using a novel viscoelastic technique, how the physiochemical properties of the 3 colloids exert individual changes on clot microstructure.

Altered clot microstructure detected in obstructive sleep apnoea hypopnoea syndrome.

Abnormal clot microstructure plays a pivotal role in the pathophysiology of thromboembolic diseases. Assessing the viscoelastic properties of clot microstructure using novel parameters, Time to Gel Point (T GP ), Fractal Dimension (d f ) and clot elasticity (G׳ GP ) could explain the increased cardiovascular and thromboembolic events in patients with Obstructive Sleep Apnoea Hypopnea Syndrome (OSAHS). We wanted to compare T GP , d f , and G׳ GP and their diurnal variation in OSAHS and symptomatic comparators. thirty six patients attending a sleep disturbed breathing clinic with symptoms of OSAHS were recruited. T GP , d f and G׳ GP were measured alongside standard coagulation screening, thrombin generation assays, and platelet aggregometry at 16:00 h and immediately after an in-patient sleep study at 07:30 h. OSAHS group had significantly lower afternoon d f than comparators (1.705±0.033 vs. 1.731±0.031, p<0.05). d f showed diurnal variation and only in the OSAHS group, being significantly lower in the afternoon than morning (p<0.05). Diurnal changes in d f correlated with 4% DR, even after controlling for BMI (r=0.37, p=0.02). The lower d f in the afternoon in OSAHS suggests a partial compensatory change that may make up for other pro-clotting abnormalities/hypertension during the night. The change to the thrombotic tendency in the afternoon is biggest in severe OSAHS. d f Shows promise as a new microstructural indicator for abnormal haemostasis in OSAHS.

A New Class of Safe Oligosaccharide Polymer Therapy To Modify the Mucus Barrier of Chronic Respiratory Disease.

The host- and bacteria-derived extracellular polysaccharide coating of the lung is a considerable challenge in chronic respiratory disease and is a powerful barrier to effective drug delivery. A low molecular weight 12-15-mer alginate oligosaccharide (OligoG CF-5/20), derived from plant biopolymers, was shown to modulate the polyanionic components of this coating. Molecular modeling and Fourier transform infrared spectroscopy demonstrated binding between OligoG CF-5/20 and respiratory mucins. Ex vivo studies showed binding induced alterations in mucin surface charge and porosity of the three-dimensional mucin networks in cystic fibrosis (CF) sputum. Human studies showed that OligoG CF-5/20 is safe for inhalation in CF patients with effective lung deposition and modifies the viscoelasticity of CF-sputum. OligoG CF-5/20 is the first inhaled polymer therapy, represents a novel mechanism of action and therapeutic approach for the treatment of chronic respiratory disease, and is currently in Phase IIb clinical trials for the treatment of CF.

The Effects of Temperature on Clot Microstructure and Strength in Healthy Volunteers.

BACKGROUND: Anesthesia, critical illness, and trauma are known to alter thermoregulation, which can potentially affect coagulation and clinical outcome. This in vitro preclinical study explores the relationship between temperature change and hemostasis using a recently validated viscoelastic technique. We hypothesize that temperature change will cause significant alterations in the microstructural properties of clot. METHODS: We used a novel viscoelastic technique to identify the gel point of the blood. The gel point identifies the transition of the blood from a viscoelastic liquid to a viscoelastic solid state. Furthermore, identification of the gel point provides 3 related biomarkers: the elastic modulus at the gel point, which is a measure of clot elasticity; the time to the gel point (TGP), which is a measure of the time required to form the clot; and the fractal dimension of the clot at the gel point, df, which quantifies the microstructure of the clot. The gel point measurements were performed in vitro on whole blood samples from 136 healthy volunteers over a temperature range of 27°C to 43°C. RESULTS: There was a significant negative correlation between increases in temperature, from 27°C to 43°C, and TGP (r = -0.641, P < 0.0005). Conversely, significant positive correlations were observed for both the elastic modulus at the gel point (r = 0.513, P = 0.0008) and df (r = 0.777, P < 0.0005) across the range of 27°C to 43°C. When temperature was reduced below 37°C, significant reductions in df and TGP occurred at ≤32°C (Bonferroni-corrected P = 0.0093) and ≤29°C (Bonferroni-corrected P = 0.0317), respectively. No significant changes were observed when temperature was increased to >37°C. CONCLUSIONS: This study demonstrates that the gel point technique can identify alterations in clot microstructure because of changes in temperature. This was demonstrated in slower-forming clots with less structural complexity as temperature is decreased. We also found that significant changes in clot microstructure occurred when the temperature was ≤32°C.

Fractal dimension (df) as a new structural biomarker of clot microstructure in different stages of lung cancer.

Venous thromboembolism (VTE) is common in cancer patients, and is the second commonest cause of death associated with the disease. Patients with chronic inflammation, such as cancer, have been shown to have pathological clot structures with modulated mechanical properties. Fractal dimension (df) is a new technique which has been shown to act as a marker of the microstructure and mechanical properties of blood clots, and can be performed more readily than current methods such as scanning electron microscopy (SEM). We measured df in 87 consecutive patients with newly diagnosed lung cancer prior to treatment and 47 matched-controls. Mean group values were compared for all patients with lung cancer vs controls and for limited disease vs extensive disease. Results were compared with conventional markers of coagulation, fibrinolysis and SEM images. Significantly higher values of df were observed in lung cancer patients compared with controls and patients with extensive disease had higher values than those with limited disease (p< 0.05), whilst conventional markers failed to distinguish between these groups. The relationship between df of the incipient clot and mature clot microstructure was confirmed by SEM and computational modelling: higher df was associated with highly dense clots formed of smaller fibrin fibres in lung cancer patients compared to controls. This study demonstrates that df is a sensitive technique which quantifies the structure and mechanical properties of blood clots in patients with lung cancer. Our data suggests that df has the potential to identify patients with an abnormal clot microstructure and greatest VTE risk.

The changes in clot microstructure in patients with ischaemic stroke and the effects of therapeutic intervention: a prospective observational study.

BACKGROUND: Stroke is the second largest cause of death worldwide. Hypercoagulability is a key feature in ischaemic stroke due to the development of an abnormally dense clot structure but techniques assessing the mechanics and quality of clot microstructure have limited clinical use. We have previously validated a new haemorheological technique using three parameters to reflect clot microstructure (Fractal Dimension (d f )) ex-vivo, real-time clot formation time (T GP ) and blood clot strength (elasticity at the gel point (G'GP)). We aimed to evaluate these novel clotting biomarkers in ischaemic stroke and changes of clot structure following therapeutic intervention. METHODS: In a prospective cohort study clot microstructure was compared in ischaemic stroke patients and a control group of healthy volunteers. Further assessment took place at 2-4 hours and at 24 hours after therapeutic intervention in the stroke group to assess the effects of thrombolysis and anti-platelet therapy. RESULTS: 75 patients (mean age 72.8 years [SD 13.1]; 47 male, 28 female) with ischaemic stroke were recruited. Of the 75 patients, 32 were thrombolysed with t-PA and 43 were loaded with 300 mg aspirin. The following parameters were significantly different between patients with stroke and the 74 healthy subjects: d f (1.760 ± .053 versus 1.735 ± 0.048, p = 0.003), TGP (208 ± 67 versus 231 ± 75, p = 0.05), G'GP (0.056 ± 0.017 versus 0.045 ± 0.014, p < 0.0001) and fibrinogen (3.7 ± 0.8 versus 3.2 ± 0.5, p < 0.00001). There was a significant decrease in d f (p = 0.02), G'GP (p = 0.01) and fibrinogen (p = 0.01) following the administration of aspirin and for d f (p = 0.003) and fibrinogen (p < 0.001) following thrombolysis as compared to baseline values. CONCLUSION: Patients with ischaemic stroke have denser and stronger clot structure as detected by d f and G'GP. The effect of thrombolysis on clot microstructure (d f ) was more prominent than antiplatelet therapy. Further work is needed to assess the clinical and therapeutic implications of these novel biomarkers.