During thrombotic or hemostatic episodes, platelets bind collagen and release ADP and thromboxane A 2 , recruiting additional platelets to a growing deposit that distorts the flow field. Prediction of clotting function under hemodynamic conditions for a patient's platelet phenotype remains a challenge. A platelet signaling phenotype was obtained for 3 healthy donors using pairwise agonist scanning, in which calcium dye-loaded platelets were exposed to pairwise combinations of ADP, U46619, and convulxin to activate the P2Y 1 /P2Y 12 , TP, and GPVI receptors, respectively, with and without the prostacyclin receptor agonist iloprost. A neural network model was trained on each donor's pairwise agonist scanning experiment and then embedded into a multiscale Monte Carlo simulation of donor-specific platelet deposition under flow. The simulations were compared directly with microfluidic experiments of whole blood flowing over collagen at 200 and 1000/s wall shear rate. The simulations predicted the ranked order of drug sensitivity for indomethacin, aspirin, MRS-2179 (a P2Y 1 inhibitor), and iloprost. Consistent with measurement and simulation, one donor displayed larger clots and another presented with indomethacin resistance (revealing a novel heterozygote TP-V241G mutation). In silico representations of a subject's platelet phenotype allowed prediction of blood function under flow, essential for identifying patientspecific risks, drug responses, and novel genotypes. IntroductionDuring a clotting event, platelets respond to combinatorial stimuli, including collagen, adenosine diphosphate (ADP), thromboxane A 2 (TXA 2 ), thrombin, epinephrine, and serotonin, as well as endothelialderived inhibitors such as nitric oxide and prostacyclin (PGI 2 ). Excessive platelet buildup at the site of cardiovascular disease and plaque rupture causes more than 1 million heart attacks and strokes in the United States each year. Excessive thrombus formation after plaque rupture remains difficult to predict and can be linked to hyperactive platelet function. 1,2 Therefore, low-dose aspirin to inhibit platelet cyclooxygenase-1 (COX-1) and clopidogrel to inhibit platelet P2Y 12 signaling are used widely in patients with cardiovascular risks, although patient response to these drugs can vary.Interindividual variations in platelet reactivity, even in the healthy population, have been associated with several factors, including female sex, fibrinogen level, ethnicity, inherited variations, and polymorphisms. 3,4 Similarly, platelet dysfunction or antiplatelet therapy can be associated with bleeding risks. [5][6][7] Furthermore, the function of blood is highly dependent on hemodynamic forces; examples include shear-induced platelet activation at Ͼ 5000/s shear rate, 8,9 requirement of VWF in arterial thrombosis, 10-12 shear effects on VWF structure/function and GPIb-VWF A1 domain-bonding dynamics, 13-17 RBC-dependent platelet migration toward the wall, 18,19 and convection-enhanced mass transfer to and from local zones of clotting or bleeding....

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Mass Spectrometry (MS) has been widely reported for measuring the conversion of substrates to products for enzyme assays. These measurements are typically performed by timeconsuming LC-MS to eliminate buffer salts that interfere with electrospray ionization MS. However, matrix-assisted laser desorption ionization, time-of-flight MS (MALDI-TOF MS) offers a label-free and direct readout of substrate and product, a fast sampling rate, and is tolerant of many buffer salts, reagents, and compounds that are typically found in enzyme reaction mixtures. In this report, a demonstration of how MALDI-TOF MS can be used to directly measure ratios of substrates and products to produce IC 50 curves for rapid enzyme assays and compound screening is provided. Typical reproducibility parameters were Ͻ7% RSD-a value comparable to ESI MS quantitative assays and well within the acceptable limits for screening assays. The speed of the MALDI readout is currently about 10 s per sample, thus allowing for over 7500 samples/day. From a simplicity standpoint, the enzymatic reaction mixtures are prepared by liquid handling robots, the reactions are stopped by addition of a 10 times volume of acidic matrix solution, and the samples are simultaneously transferred to MALDI target plate for analysis. Importantly, the ratios of substrate to product are of sufficient reproducibility to eliminate the need for internal standards and, thus, minimize the cost and increasing the speed of assay development. (J Am Soc Mass Spectrom 2006, 17, 815-822)

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