Development and validation of a high throughput whole blood thrombolysis plate assay.

The objective of this work was to develop a high throughput assay for testing in vitro the thrombolytic activity using citrated whole blood samples, and to overcome the limitations of currently available techniques. We successfully developed a method that involves forming halo shaped, tissue factor induced, whole blood clots in 96 well plates, and then precisely measuring the thrombolysis process with a spectrophotometer plate reader. We here describe the implementation of this novel method, which we refer to as halo assay, and its validation with plasmin, urokinase and tissue plasminogen activator at different doses. The resulting data is a highly detailed thrombolysis profile, allowing comparison of different fibrinolytic agents. The time point analysis allows kinetic data to be collected and calculated to determine key parameters such as the activation time and the rate of fibrinolysis. We also assessed the capacity of the model to study the effect of clot maturation time on the fibrinolytic rate, an aspect of thrombosis rather unexplored with currently available methods, but of increasing importance in drug development. This novel thrombolysis assay could be an extremely useful research tool; to study the complex process of thrombolysis, and a valuable translational clinical tool; as a screening device to rapidly identify hypo- or hyper-fibrinolysis.

Evaluation of the combined use of adiponectin and C-reactive protein levels as biomarkers for predicting the deterioration in glycaemia after a median of 5.4 years.

AIMS/HYPOTHESIS: Hypoadiponectinaemia and raised C-reactive protein (CRP) level are obesity-related biomarkers associated with glucose dysregulation. We evaluated the combined use of these two biomarkers in predicting the deterioration of glycaemia in a prospective study after a median of 5.4 years. METHODS: In total 1,288 non-diabetic participants from the Hong Kong Cardiovascular Risk Factor Prevalence Study-2, with high-sensitivity CRP (hsCRP) and total adiponectin levels measured were included. OGTT was performed in all participants. Two hundred and six participants had deterioration of glycaemia at follow-up, whereas 1,082 participants did not. RESULTS: Baseline age, hsCRP and adiponectin levels were significant independent predictors of the deterioration of glycaemia in a Cox regression analysis after adjusting for baseline age, sex, BMI, hypertension, triacylglycerols, 2 h post-OGTT glucose and homeostasis model assessment of insulin resistance index (all p < 0.01). The introduction of hsCRP or adiponectin level to a regression model including the other biomarker improved the prediction of glycaemic progression significantly in all participants, especially in women (all p < 0.01). The combined inclusion of the two biomarkers resulted in a modest improvement in model discrimination, compared with the inclusion of either one alone. Among participants with impaired fasting glucose/impaired glucose tolerance (IFG/IGT) at baseline, hsCRP and adiponectin levels were not predictive of progression or improvement of glycaemic status. CONCLUSIONS/INTERPRETATION: Adiponectin and hsCRP levels are independent factors in predicting the deterioration of glycaemia, supporting the role of adiposity-related inflammation in the development of type 2 diabetes. Their combined use as predictive biomarkers is especially useful in women, but not in participants with IFG/IGT.

Serum adipocyte fatty acid-binding protein associated with ischemic stroke and early death.

OBJECTIVE: Adipocyte fatty acid-binding protein (A-FABP) is an adipokine shown to have adverse metabolic and proinflammatory effects, and contributes to atherosclerosis in mice. However, its role in cardiovascular diseases in humans remains to be established. In this case-control study, we investigated the association of serum A-FABP with ischemic stroke, and examined its association with early mortality. METHODS: Serum A-FABP was measured, using ELISA, in 306 subjects with acute ischemic stroke and 306 age-, sex-, and body mass index-matched controls. All controls were free of cardiovascular diseases. Serum A-FABP was also measured in another 60 ischemic stroke subjects who died within 3 months of acute stroke. RESULTS: Serum A-FABP was higher in subjects with ischemic stroke as compared to controls (19.6 ng/mL [14.3-28.4 ng/mL] vs 15.2 ng/mL [10.6-23.6 ng/mL] in men and 32.4 ng/mL [24.5-45.7 ng/mL] vs 22.0 ng/mL [14.3-34.0 ng/mL] in women, stroke vs control, p<0.001). On logistic regression analyses with the model including hypertension, diabetes, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglyceride, lipid-lowering treatment, smoking, and A-FABP, serum A-FABP was independently associated with stroke (odds ratio 2.10, 95% confidence interval 1.50-2.94, p<0.001), and the associations of A-FABP with ischemic stroke were additive to conventional risk factors, as demonstrated on likelihood ratio tests (p<0.001). Furthermore, high serum A-FABP was associated with increased 3-month mortality in ischemic stroke subjects (odds ratio 2.65, 95% confidence interval 1.18-5.96, p=0.018), independent of age and NIH Stroke Scale score. CONCLUSIONS: Serum A-FABP was significantly associated with ischemic stroke in our case-control study, and may serve as a useful prognostic indicator for early mortality.