Karl Hawkins

Gel point and fractal microstructure of incipient blood clots are significant new markers of hemostasis for healthy and anticoagulated blood

Here we report the first application of a fractal analysis of the viscoelastic properties of incipient blood clots. We sought to ascertain whether the incipient clots fractal dimension, D(f,) could be used as a functional biomarker of hemostasis. The incipient clot is formed at the gel point (GP) of coagulating blood, the GP demarcating a functional change from viscoelastic liquid to a viscoelastic solid. Incipient clots formed in whole healthy blood show a clearly defined value of D(f) within a narrow range that represents an index of clotting in health, where D(f) = 1.74 (± 0.07). A significant relationship is found between the incipient clot formation time, T(GP), and the activated partial thromboplastin time, whereas the association of D(f) with the microstructural characteristics of the incipient clot is supported by its significant correlation with fibrinogen. Our study reveals that unfractionated heparin not only prolongs the onset of clot formation but has a significant effect on its fractal microstructure. A progressive increase in unfractionated heparin concentration results in a linear decrease in D(f) and a corresponding prolongation in T(GP). The results represent a new, quantitative measure of clot quality derived from measurements on whole blood samples.

Silver nanoparticle based antibacterial methacrylate hydrogels potential for bone graft applications

Infections are frequent and very undesired occurrences after orthopedic procedures; furthermore, the growing concern caused by the rise in antibiotic resistance is progressively dwindling the efficacy of such drugs. Artificial bone graft materials could solve some of the problems associated with the gold standard use of natural bone graft such as limited bone material, pain at the donor site and rejections if donor tissue is used. We have previously described new acrylate base nanocomposite hydrogels as bone graft materials. In the present paper, we describe the integration of silver nanoparticles in the polymeric mineralized biomaterial to provide non-antibiotic antibacterial activity against Staphylococcus epidermidis and Methicillin-resistant Staphylococcus aureus. Two different crosslinking degrees were tested and the silver nanoparticles were integrated into the composite matrix by means of three different methods: entrapment in the polymeric hydrogel before the mineralization; diffusion during the process of calcium phosphate crystallization and adsorption post-mineralization. The latter being generally the most effective method of encapsulation; however, the adsorption of silver nanoparticles inside the pores of the biomaterial led to a decreasing antibacterial activity for adsorption time longer than 2 days.

The effect of alginate oligosaccharides on the mechanical properties of Gram-negative biofilms

The influence of a novel, safe antibiofilm therapy on the mechanical properties of Pseudomonas aeruginosa and Acinetobacter baumannii biofilms in vitro was characterized. A multiscale approach employing atomic force microscopy (AFM) and rheometry was used to quantify the mechanical disruption of the biofilms by a therapeutic polymer based on a low-molecular weight alginate oligosaccharide (OligoG). AFM demonstrated structural alterations in the biofilms exposed to OligoG, with significantly lower Young’s moduli than the untreated biofilms, (149 MPa vs 242 MPa; p < 0.05), a decreased resistance to hydrodynamic shear and an increased surface irregularity (Ra) in the untreated controls (35.2 nm ± 7.6 vs 12.1 nm ± 5.4; p < 0.05). Rheology demonstrated that increasing clinically relevant concentrations of OligoG (<10%) were associated with an increasing phase angle (δ) over a wide range of frequencies (0.1–10 Hz). These results highlight the utility of these techniques for the study of three-dimensional biofilms and for quantifying novel disruption therapies in vitro.

The CentriMag Centrifugal Blood Pump as a Benchmark for In Vitro Testing of Hemocompatibility in Implantable Ventricular Assist Devices

Implantable ventricular assist devices (VADs) have proven efficient in advanced heart failure patients as a bridge-to-transplant or destination therapy. However, VAD usage often leads to infection, bleeding, and thrombosis, side effects attributable to the damage to blood cells and plasma proteins. Measuring hemolysis alone does not provide sufficient information to understand total blood damage, and research exploring the impact of currently available pumps on a wider range of blood cell types and plasma proteins such as von Willebrand factor (vWF) is required to further our understanding of safer pump design. The extracorporeal CentriMag (Thoratec Corporation, Pleasanton, CA, USA) has a hemolysis profile within published standards of normalized index of hemolysis levels of less than 0.01 g/100 L at 100 mm Hg but the effect on leukocytes, vWF multimers, and platelets is unknown. Here, the CentriMag was tested using bovine blood (n = 15) under constant hemodynamic conditions in comparison with a static control for total blood cell counts, hemolysis, leukocyte death, vWF multimers, microparticles, platelet activation, and apoptosis. The CentriMag decreased the levels of healthy leukocytes (P < 0.006), induced leukocyte microparticles (P < 10−5), and the level of high molecular weight of vWF multimers was significantly reduced in the CentriMag (P < 10−5) all compared with the static treatment after 6 h in vitro testing. Despite the leukocyte damage, microparticle formation, and cleavage of vWF multimers, these results show that the CentriMag is a hemocompatible pump which could be used as a standard in blood damage assays to inform the design of new implantable blood pumps.

A new biomarker quantifies differences in clot microstructure in patients with venous thromboembolism

This study compared patients with venous thromboembolism (VTE) to non‐VTE patients using a biomarker of clot microstructure (df) and clot formation time (TGP). df was the only marker that identified a significant difference (P < 0·001) between the VTE (n = 60) and non‐VTE cohorts (n = 69). The ‘abnormal’ clot microstructures in the VTE patients suggests either inadequate response to anticoagulant therapy or the presence of a procoagulant state not detected by other markers of coagulation (i.e., International Normalized Ratio). Furthermore, elevated values of df in first time VTE patients who later develop a secondary event indicates that df may identify those at risk of recurrence.

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.

A study of microstructural templating in fibrin–thrombin gel networks by spectral and viscoelastic analysis

We report a study of the microstructural templating role of incipient fibrin–thrombin gels by analysis of rheological and confocal microscope measurements. Fractal analysis based on the spectral dimension is used, for the first time, to characterise fibrin gel microstructure in terms of the internal connectivity of gel networks. A significant correlation is found between the fractal characteristics of the incipient gel network and its eventual mature form, confirming that incipient gel microstructure templates ensuing gel development. We report an analytical basis for the study of this templating effect which reveals two different regimes of microstructural development. The first involves low thrombin concentration, in which increasing concentration decreases the gel formation time and alters the fractal characteristics of both incipient and mature gels. In the second regime, involving higher thrombin concentrations, the incipient gel formation time and the fractal characteristics of incipient and mature gels show little variation. The network formation is discussed in terms of computer simulations of incipient fractal networks by the activation-limited aggregation of clusters of rod-like particles. The significance of the work is discussed in terms of biomaterials design for applications involving controlled drug release and wound healing, and improved predictions of blood clot susceptibility to lysis.

The Effect of Shear Stress on the Size, Structure, and Function of Human von Willebrand Factor

Clinical outcomes from ventricular assist devices (VADs) have improved significantly during recent decades, but bleeding episodes remain a common complication of long-term VAD usage. Greater understanding of the effect of the shear stress in the VAD on platelet aggregation, which is influenced by the functional activity of high molecular weight (HMW) von Willebrand factor (vWF), could provide insight into these bleeding complications. However, because VAD shear rates are difficult to assess, there is a need for a model that enables controlled shear rates to first establish the relationship between shear rates and vWF damage. Secondly, if such a dependency exists, then it is relevant to establish a rapid and quantitative assay that can be used routinely for the safety assessment of new VADs in development. Therefore, the purpose of this study was to exert vWF to controlled levels of shear using a rheometer, and flow cytometry was used to investigate the shear-dependent effect on the functional activity of vWF. Human platelet-poor plasma (PPP) was subjected to different shear rate levels ranging from 0 to 8000/s for a period of 6 h using a rheometer. A simple and rapid flow cytometric assay was used to determine platelet aggregation in the presence of ristocetin cofactor as a readout for vWF activity. Platelet aggregates were visualized by confocal microscopy. Multimers of vWF were detected using gel electrophoresis and immunoblotting. The longer PPP was exposed to high shear, the greater the loss of HMW vWF multimers, and the lower the functional activity of vWF for platelet aggregation. Confocal microscopy revealed for the first time that platelet aggregates were smaller and more dispersed in postsheared PPP compared with nonsheared PPP. The loss of HMW vWF in postsheared PPP was demonstrated by immunoblotting. Smaller vWF platelet aggregates formed in response to shear stress might be a cause of bleeding in patients implanted with VADs. The methodological approaches used herein could be useful in the design of safer VADs and other blood handling devices. In particular, we have demonstrated a correlation between the loss of HMW vWF, analyzed by immunoblotting, with platelet aggregation, assessed by flow cytometry. This suggests that flow cytometry could replace conventional immunoblotting as a simple and rapid routine test for HMW vWF loss during in vitro testing of devices.

The role of whole blood impedance aggregometry and its utilisation in the diagnosis and prognosis of patients with systemic inflammatory response syndrome and sepsis in acute critical illness

Objective To assess the prognostic and diagnostic value of whole blood impedance aggregometry in patients with sepsis and SIRS and to compare with whole blood parameters (platelet count, haemoglobin, haematocrit and white cell count). Methods We performed an observational, prospective study in the acute setting. Platelet function was determined using whole blood impedance aggregometry (multiplate) on admission to the Emergency Department or Intensive Care Unit and at 6 and 24 hours post admission. Platelet count, haemoglobin, haematocrit and white cell count were also determined. Results 106 adult patients that met SIRS and sepsis criteria were included. Platelet aggregation was significantly reduced in patients with severe sepsis/septic shock when compared to SIRS/uncomplicated sepsis (ADP: 90.7±37.6 vs 61.4±40.6; p<0.001, Arachadonic Acid 99.9±48.3 vs 66.3±50.2; p = 0.001, Collagen 102.6±33.0 vs 79.1±38.8; p = 0.001; SD ± mean)). Furthermore platelet aggregation was significantly reduced in the 28 day mortality group when compared with the survival group (Arachadonic Acid 58.8±47.7 vs 91.1±50.9; p<0.05, Collagen 36.6±36.6 vs 98.0±35.1; p = 0.001; SD ± mean)). However haemoglobin, haematocrit and platelet count were more effective at distinguishing between subgroups and were equally effective indicators of prognosis. Significant positive correlations were observed between whole blood impedance aggregometry and platelet count (ADP 0.588 p<0.0001, Arachadonic Acid 0.611 p<0.0001, Collagen 0.599 p<0.0001 (Pearson correlation)). Conclusions Reduced platelet aggregometry responses were not only significantly associated with morbidity and mortality in sepsis and SIRS patients, but also correlated with the different pathological groups. Whole blood aggregometry significantly correlated with platelet count, however, when we adjust for the different groups we investigated, the effect of platelet count appears to be non-significant.

The evaluation of leukocytes in response to the in vitro testing of ventricular assist devices.

Infection is a clinically relevant adverse event in patients with ventricular assist device (VAD) support. The risk of infection could be linked to a reduced immune response resulting from damage to leukocytes during VAD support. The purpose of this study was to develop an understanding of leukocyte responses during the in vitro testing of VADs by analyzing the changes to their morphology and biochemistry. The VentrAssist implantable rotary blood pump (IRBP) and RotaFlow centrifugal pump (CP) were tested in vitro under constant hemodynamic conditions. Automated hematology analysis of samples collected regularly over 25-h tests was undertaken. A new flow cytometric assay was employed to measure biochemical alteration, necrosis (7-AAD) and morphological alteration (CD45 expression) of the circulating leukocytes during the pumping process. The results of hematology analysis show the total leukocyte number and subset counts decreased over the period of in vitro tests dependent on different blood pumps. The percentage of leukocytes damaged during 6-h tests was 40.8 ± 5.7% for the VentrAssist IRBP, 17.6 ± 5.4% for the RotaFlow CP, and 2.7 ± 1.8% for the static control (all n=5). Flow cytometric monitoring of CD45 expression and forward/side scatter characteristics revealed leukocytes that were fragmented into smaller pieces (microparticles). Scanning electron microscopy and imaging flow cytometry were used to confirm this. Device developers could use these robust cellular assays to gain a better understanding of leukocyte-specific VAD performance.