Gillian Stephens

Platelet-derived S100 family member myeloid-related protein-14 regulates thrombosis.

Expression of the gene encoding the S100 calcium-modulated protein family member MRP-14 (also known as S100A9) is elevated in platelets from patients presenting with acute myocardial infarction (MI) compared with those from patients with stable coronary artery disease; however, a causal role for MRP-14 in acute coronary syndromes has not been established. Here, using multiple models of vascular injury, we found that time to arterial thrombotic occlusion was markedly prolonged in Mrp14⁻/⁻ mice. We observed that MRP-14 and MRP-8/MRP-14 heterodimers (S100A8/A9) are expressed in and secreted by platelets from WT mice and that thrombus formation was reduced in whole blood from Mrp14⁻/⁻ mice. Infusion of WT platelets, purified MRP-14, or purified MRP-8/MRP-14 heterodimers into Mrp14⁻/⁻ mice decreased the time to carotid artery occlusion after injury, indicating that platelet-derived MRP-14 directly regulates thrombosis. In contrast, infusion of purified MRP-14 into mice deficient for both MRP-14 and CD36 failed to reduce carotid occlusion times, indicating that CD36 is required for MRP-14-dependent thrombosis. Our data identify a molecular pathway of thrombosis that involves platelet MRP-14 and CD36 and suggest that targeting MRP-14 has potential for treating atherothrombotic disorders, including MI and stroke.

Pharmacokinetic and Pharmacodynamic Effects of Elinogrel Results of the Platelet Function Substudy From the Intravenous and Oral Administration of Elinogrel to Evaluate Tolerability and Efficacy in Nonurgent Percutaneous Coronary Intervention Patients (INNOVATE-PCI) Trial

Background— Elinogrel is the only selective, competitive and reversible platelet P2Y12 inhibitor available in both intravenous (IV) and oral formulations. Methods and Results— This substudy of the Intravenous and Oral Administration of Elinogrel to Evaluate Tolerability and Efficacy in Nonurgent Percutaneous Coronary Intervention patients (INNOVATE-PCI) trial evaluated the pharmacokinetic and pharmacodynamic effects of two dosing regimens of IV followed by oral elinogrel (120 mg IV plus 100 mg oral twice daily; 120 mg IV plus 150 mg oral twice daily) versus standard clopidogrel therapy (300–600 mg oral loading dose plus 75 mg oral maintenance dose) in 56 patients undergoing nonurgent PCI. At time of randomization, 71.4% (40/56) of patients were using maintenance clopidogrel therapy. In the acute phase, an IV bolus of elinogrel achieved more rapid and potent antiplatelet effects compared with clopidogrel, which were sustained during the transition from the IV to the oral formulation in the first 24 hours of the peri-PCI period. During chronic therapy, elinogrel achieved similar levels of platelet reactivity compared with clopidogrel before the next oral dose and, although platelet reactivity was lower with elinogrel up to 6 hours after daily oral maintenance dosing, these differences were not statistically significant. These pharmacodynamic effects matched the pharmacokinetic profile of elinogrel. There were no differences in pharmacodynamic and pharmacokinetic effects between the two elinogrel dosing regimens. Conclusions— Compared with clopidogrel, the combination of IV and oral elinogrel achieves more rapid and enhanced antiplatelet effects that were sustained through the transition to oral elinogrel in the peri-PCI period, but these were not significant during chronic dosing in this pilot investigation. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT00751231.

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

BackgroundArterial 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.ResultsUntreated 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.ConclusionsThese 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.

Sub-optimal inhibition of thrombus formation ex vivo by aspirin in patients with primary thrombocythaemia.

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Tracking flow of leukocytes in blood for drug analysis

Modern microscopy techniques allow imaging of circulating blood components under vascular flow conditions. The resulting video sequences provide unique insights into the behavior of blood cells within the vasculature and can be used as a method to monitor and quantitate the recruitment of inflammatory cells at sites of vascular injury/ inflammation and potentially serve as a pharmacodynamic biomarker, helping screen new therapies and individualize dose and combinations of drugs. However, manual analysis of these video sequences is intractable, requiring hours per 400 second video clip. In this paper, we present an automated technique to analyze the behavior and recruitment of human leukocytes in whole blood under physiological conditions of shear through a simple multi-channel fluorescence microscope in real-time. This technique detects and tracks the recruitment of leukocytes to a bioactive surface coated on a flow chamber. Rolling cells (cells which partially bind to the bioactive matrix) are detected counted, and have their velocity measured and graphed. The challenges here include: high cell density, appearance similarity, and low (1Hz) frame rate. Our approach performs frame differencing based motion segmentation, track initialization and online tracking of individual leukocytes.