Development of a perfusion chamber assay to study in real time the kinetics of thrombosis and the antithrombotic characteristics of antiplatelet drugs.

BACKGROUND: Arterial thrombosis triggered by vascular injury is a balance between thrombus growth and thrombus fragmentation (dethrombosis). Unbalance towards thrombus growth can lead to vascular occlusion, downstream ischemia and tissue damage.Here we describe the development of a simple methodology that allows for continuous real time monitoring and quantification of both processes during perfusion of human blood under arterial shear rate conditions. Using this methodology, we have studied the effects of antiplatelet agents targeting COX-1 (aspirin), P2Y12 (2-MeSAMP, clopidogrel), GP IIb-IIIa (eptifibatide) and their combinations on the kinetics of thrombosis over time. RESULTS: Untreated samples of blood perfused over type III collagen at arterial rates of shear promoted the growth of stable thrombi. Modulation by eptifibatide affected thrombus growth, while that mediated by 2-MeSAMP and aspirin affected thrombus stability. Using this technique, we confirmed the primacy of continuous signaling by the ADP autocrine loop acting on P2Y12 in the maintenance of thrombus stability. Analysis of the kinetics of thrombosis revealed that continuous and prolonged analysis of thrombosis is required to capture the role of platelet signaling pathways in their entirety. Furthermore, studies evaluating the thrombotic profiles of 20 healthy volunteers treated with aspirin, clopidogrel or their combination indicated that while three individuals did not benefits from either aspirin or clopidogrel treatments, all individuals displayed marked destabilization profiles when treated with the combination regimen. CONCLUSIONS: These results show the utility of a simple perfusion chamber technology to assess in real time the activity of antiplatelet drugs and their combinations. It offers the opportunity to perform pharmacodynamic monitoring of arterial thrombosis in clinical trials and to investigate novel strategies directed at inhibiting thrombus stability in the management of cardiovascular disease.

Longitudinally quantitative 2-deoxy-2-[18F]fluoro-D-glucose micro positron emission tomography imaging for efficacy of new anticancer drugs: a case study with bortezomib in prostate cancer murine model.

PURPOSE: The aim of this study was to validate quantitative metabolic response of tumors to a treatment measured by longitudinal 2-deoxy-2-[(18)F]fluoro-D-glucose (FDG) micro positron emission tomography (microPET) as a robust tool for preclinical evaluation of new anticancer agents. PROCEDURES: Severe combined immunodeficiency mice with CWR22 xenografts were intravenously treated with bortezomib (Velcade) at 0.8 mg/kg on days 0, 3, 7, 10, and 14 and imaged with FDG microPET before, during and after treatment. Quantitative indices of tumor FDG uptake were developed. RESULTS: FDG microPET images successfully revealed the gradual reduction of tumor FDG uptake on day 4 onward despite no absolute tumor shrinkage. The standardized uptake values of FDG in tumors was reduced to 43% of the baseline values. Using the total tumor FDG uptake as the viable tumor burden, we found 86% tumor inhibition, compared to a 55% tumor growth inhibition in tumor volume measurement. CONCLUSION: FDG microPET imaging can provide an additional dimension of the efficacy of anticancer therapies that may otherwise be underestimated by tumor volume measurement.

Agonist-induced calcium response in single human platelets assayed in a microfluidic device.

To facilitate drug discovery directed toward platelet-specific targets, we developed a platelet isolation and fluorophore-loading method that yields functionally responsive platelets in which we were able to detect agonist-induced calcium flux using a microfluidics-based screening platform. The platelet preparation protocol was designed to minimize preparation-induced platelet activation and to optimize signal strength. Measurement of platelet activation, as monitored by ratiometric determination of agonist-induced calcium flux in fluor-loaded human platelets, was optimized in a macrosample cuvette format in preparation for detection in a microfluidic chip-based assay. For the microfluidic device used in these studies, a cell density of 1 to 2 x 10(6) platelets per milliliter and a nominal flow rate of 5 to 10 nl per second provided optimal event resolution of 5 to 20 platelets traversing the detection volume per unit time. Platelets responded in a dose-dependent manner to adenosine diphosphate and protease-activating peptide (PAR) 1 thrombin receptor-activating peptide (TRAP). The work presented here constitutes proof-of-principle experiments demonstrating the enabling application of a microfluidic device to conduct high-throughput signaling studies and drug discovery screening against human platelet targets.