Matthew Lawrence

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.

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.

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.

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.

Application of ROTEM to assess hypercoagulability in patients with lung cancer

BACKGROUND Venous thromboembolism (VTE) is common in patients with cancer, contributing significantly to morbidity and mortality Currently, no test reliably identifies patients at increased risk of developing VTE who would therefore benefit from prophylactic intervention. The aim of the current study was to evaluate rotational thromboelastometry (ROTEM) in identifying VTE risk in patients with lung cancer. We also compared parameters of ROTEM in patients with limited and extensive disease. METHODS Parameters of ROTEM were measured in 67 patients with lung cancer and 72 age-matched healthy controls and compared with conventional markers of haemostasis. Patients were followed up for 12 months and VTE incidence recorded. RESULTS Lung cancer patients had a reduced clotting time (CT), increased maximum clot firmness (MCF) and increased alpha angle compared with controls. Patients also had significantly higher levels of fibrinogen and PAI-1 than controls and in the former group there was a strong correlation between fibrinogen and both MCF and alpha angle. Six patients developed a VTE during the follow-up period and all had values for MCF at or above the upper limit of normal for EXTEM. CONCLUSIONS This study demonstrates that several ROTEM parameters are significantly different in lung cancer patients compared to healthy age-matched controls, whereas only one of the parameters measured is significantly different between extensive compared to limited disease. No differences were observed between patients who developed a VTE compared to those who did not, highlighting the limitations of ROTEM use in patients with lung cancer.

Effects of unidirectional flow shear stresses on the formation, fractal microstructure and rigidity of incipient whole blood clots and fibrin gels

Abstract Incipient clot formation in whole blood and fibrin gels was studied by the rheometric techniques of controlled stress parallel superposition (CSPS) and small amplitude oscillatory shear (SAOS). The effects of unidirectional shear stress on incipient clot microstructure, formation kinetics and elasticity are reported in terms of the fractal dimension (df) of the fibrin network, the gel network formation time (TGP) and the shear elastic modulus, respectively. The results of this first haemorheological application of CSPS reveal the marked sensitivity of incipient clot microstructure to physiologically relevant levels of shear stress, these being an order of magnitude lower than have previously been studied by SAOS. CSPS tests revealed that exposure of forming clots to increasing levels of shear stress produces a corresponding elevation in df, consistent with the formation of tighter, more compact clot microstructures under unidirectional flow. A corresponding increase in shear elasticity was recorded. The scaling relationship established between shear elasticity and df for fibrin clots and whole blood confirms the fibrin network as the dominant microstructural component of the incipient clot in terms of its response to imposed stress. Supplementary studies of fibrin clot formation by rheometry and microscopy revealed the substantial additional network mass required to increase df and provide evidence to support the hypothesis that microstructural changes in blood clotted under unidirectional shear may be attributed to flow enhanced thrombin generation and activation. CSPS also identified a threshold value of unidirectional shear stress above which no incipient clot formation could be detected. CSPS was shown to be a valuable haemorheological tool for the study of the effects of physiological and pathological levels of shear on clot properties.

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.

Effects of exercise intensity on clot microstructure and mechanical properties in healthy individuals

BACKGROUND Exercise is well established to lead to exercise-induced hypercoagulability, as demonstrated by kinetic coagulation markers. It remains unclear as to whether exercise-induces changes lead in clot development and increased polymerisation. Fractal dimension (df) has been shown to act as a marker of clot microstructure and mechanical properties, and may provide a more meaningful method of determining the relationship between exercise-induced hypercoagulability and potential clot development. METHODS df was measured in 24 healthy individuals prior to, after 5min of submaximal exercise, following maximal exercise, 45min of passive recovery and following 60min of recovery. Results were compared with conventional markers of coagulation, fibrinolysis and SEM images. RESULTS Significantly increased df was observed following exercise, returning to resting values following 60min of recovery. The relationship between df and mature clot microstructure was confirmed by SEM: higher df was associated with dense clots formed of smaller fibrin fibres immediately following exercise compared to at rest. Conventional markers of coagulation confirmed findings of previous studies. CONCLUSION This study demonstrates that df is a sensitive technique which quantifies the structure and properties of blood clots following exercise. In healthy individuals, the haemostatic balance between coagulation and fibrinolysis is maintained in equilibrium following exercise. In individuals with underlying vascular damage who participate in exercise, this equilibrium may be displaced and lead to enhanced clot formation and a prothrombotic state. df may therefore have the potential to not only quantify hypercoagulability, but may also be useful in screening these individuals.

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

Abstract 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.

Cognitive Outcomes following Thrombolysis in Acute Ischemic Stroke: A Systematic Review

BACKGROUND Patients treated with thrombolytic therapy within 4.5 hours after stroke onset appear to have improved survival and functional outcomes. Poststroke cognitive impairment is associated with reduced quality of life and survival and needs to be reviewed in consideration of the administration of thrombolysis. This review aims to systematically evaluate literature exploring the effect of thrombolysis for ischemic stroke on cognition. METHODS An electronic search was conducted to identify articles and gray literature applying broad Medical Subject Heading terms. Literature was reviewed with a 2-step process against predetermined inclusion criteria. All relevant studies were included if they investigated global or individual cognitive domains. RESULTS Three studies satisfied the inclusion criteria but were diverse in outcome measures and duration, their heterogeneity limiting any possible pooled analysis. One study examined long-term treatment effects on global cognition and did not find a positive effect at 6 months. A positive treatment effect was reported in the acute phase in 1 study examining domains of visuoconstructive and perceptive abilities. One study retrospectively analyzed treatment effects on language and found improvement in the acute phase but not in the long term. CONCLUSIONS The limited existing evidence on the effects of thrombolytic therapy on long- and short-term cognition is varied in both outcome measures and diagnostic classifications, making it difficult to extrapolate results to a global stroke population. This review should be used to inform future research in stroke treatment outcomes and highlights the immediate need for larger, more robust studies in this area.