Ahmed Sabra

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.

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.

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.

Prospective Evaluation of Blood Coagulability and Effect of Treatment in Patients with Stroke Using Rotational Thromboelastometry

BACKGROUND Stroke is the second largest cause of death worldwide. Abnormalities in hemostasis play an important role in the pathophysiology of ischemic stroke (IS). These hemostatic defects can be detected using rotational thromboelastometry (ROTEM) as a global method of measuring coagulation. This study assessed the effects of IS on blood hypercoagulability using ROTEM method, before and subsequent to therapeutic interventions. METHODS In a prospective observational cohort study, whole blood coagulation using ROTEM, along with full blood count and standard coagulation tests, were compared between patients with IS and an age-matched control group of healthy volunteers. Further assessment took place at 2-4 hours and at 24 hours in the stroke group after therapy to assess the effects of therapeutic intervention. RESULTS Seventy-two patients with IS were age-matched to 71 healthy subjects. Clotting time (CT) INTEM (P = .01) and maximum clot firmness (MCF) INTEM (P = .02) were significantly different between stroke patients at baseline and healthy subjects, but this difference disappeared when controlled for by smoking status. There was no association between ROTEM parameters and time from stroke symptom onset or stroke severity as reflected in The National Institute of Health Stroke Scale score. Significant but small changes in the values of MCF-EXTEM, clot formation time (CFT) EXTEM, and alpha-EXTEM CT were observed after therapeutic intervention (thrombolysis or aspirin treatment). CONCLUSIONS ROTEM testing does not seem to detect a hypercoagulable state in patients with IS. Nonetheless, some ROTEM parameters had a small change after antiplatelet therapy or thrombolysis.

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.

10 Real-world evidence of the safety of pharmacologic stress in myocardial perfusion imaging

Introduction Myocardial perfusion imaging (MPI) is increasingly used as recommended by NICE for patients with a 30–60% estimated likelihood of coronary artery disease. We aimed to investigate the safety of drugs used in patients undergoing pharmacologic stress MPI. Methods A 9-month (May 2015–Feb 2016) retrospective study to investigate the incidence of adverse effects (AE) in patients undergoing pharmacologic stress MPI. We employ standardised protocols (Tc-99m) using 4 agents: dipyridamole, regadenoson, adenosine and dobutamine. Clinical data including AEs were collected from cardiobase. Descriptive analysis and frequencies were calculated. AE in MPI were compared to those occurring in 50 patients who underwent conventional exercise tolerance testing (ETT) using chi-squared test. Findings Of the 360 patients (mean age 67 years [SD 11]; 196 men, 164 women), who had pharmacologic stress agent administered, 166 (46%) became symptomatic: dyspnoea (24.7%), chest discomfort (12.8%), flushing (10%), chest pain (3.6%), cough (3.3%), nausea (2.8%), pain elsewhere (2.5%), dizziness (2.5%), vasovagal reaction (2.2%), wheeze (1.9%), headache (1.9%), vomiting (0.8%), abdominal discomfort (0.8%). The commonest symptom was chest discomfort for dobutamine but dyspnoea for all the other agents. Adenosine had the highest incidence of AE (76%). No significant complications were recorded that required hospital admission. When compared with ETT there was no significant difference of adverse effects (MPI 166/360 [44%], ETT 29/50 [58%]; p=0.12). Conclusions This real-world data show that pharmacologic stress MPI is safe when clinical protocols are followed. Technician (physicist)-led testing could be an alternative in busy cardiac units where medical staffing are not always available.

INSIGHTS INTO THE ROLE OF PLATELETS IN FIBRIN CLOT FORMATION AND THE EFFICACY OF ANTI-PLATELET THERAPIES

Platelets and coagulation pathways play important roles in thrombus formation. Configuration of the fibrin network is a vital determinant of clot stability, with dense, less porous clots associated with thrombosis. High on treatment platelet reactivity despite dual antiplatelet therapy (DAPT) is