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High Throughput Platelet Activation Kinetics

Manash S. Chatterjee and Scott L. Diamond. Chemical and Biomolecular Engineering, University of Pennsylvania, 5140 Vagelos Research Laboratories, 3340 Smith Walk, Philadelphia, PA 19104

Several agonists and antagonists acting together regulate the activation state of the platelet in vivo, allowing it to remain in a quiescent state during haemostasis and yet respond efficiently following vascular injury to form a stable thrombus.Using cytosolic Calcium concentration as a metric of platelet activation, we have developed a high throughput kinetic assay which allows us to rapidly study dose responses of agonists, antagonists and drugs as well as combinations of discrete states of these regulators. This assay allows us to probe 96 experimental conditions in replicates of 4 from a mere 15ml human blood draw. Furthermore, we have been able to phenotype 192 mouse platelet conditions using 2ml of pooled mouse blood, thereby greatly increasing throughput per knockout mice.Platelets were stimulated with the Collagen analogue Convulxin, Thrombin receptor agonist peptide (TRAP) and ADP in increasing concentrations to determine the IC50 for each agonist. Concentrations of 0, 0.1, 1 and 10x IC50 were selected, and all the 64 combinatorial combinations of the 4 states of the 3 agonists were studied simultaneously. We found that on short timescales the response is dominated by the characteristics of the GPCR mediated ADP/TRAP signal, whereas on longer timescales it becomes reminiscent of a Convulxin signal. At high concentrations of TRAP, ADP had little distinguishable effect, whereas the effect of Convulxin was apparent at all concentrations studied. Pharmacological treatments using the P2Y1 inhibitor MRS2179, the P2Y12 inhibitor MRS2395 and the COX inhibitor aspirin allowed us to quantify the differential contributions of each of these receptors and autocatalytically produced TxA2 to the overall ADP signal. Using the Calcium chelator EDTA in the medium and controlled recalcification protocols, we were able to distinguish the flux due to store operated extracellular Calcium entry. Using the SERCA pump inhibitor Thapsigargin and the PMCA inhibitor Carboxyeosin, we found evidence that the amplitude and duration of the signal could in part be determined by how quickly Calcium is removed from the cytoplasm by these pumps after agonist induced release from the stores. A Convulxin stimulus was found to produce a slow sustained elevation in cytoplasmic Calcium which was brought down very slowly using only the PMCA pumps. In contrast, a TRAP stimulus produced a rapid Calcium burst which was quickly brought down to baseline using both the PMCA and SERCA pumps.Given the central role of platelets in mediating cardiovascular pathology, this assay shows promise to perform a patient specific scan of ‘Platelet Activation Space' and a control scan of a healthy donor done on the same plate with the objective of diagnosing efficiently undefined platelet defects.

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