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Pairwise agonist scanning-flow cytometry (PAS-FC) measures inside-out signaling and patient-specific response to combinatorial platelet agonists
Daniel T. L. Jaeger and Scott L. Diamond
Institute for Medicine and Engineering, Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA
BioTechniques, Vol. 54, No. 5, May 2013, pp. 271–277
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Understanding the response of cells to multiple stimuli is vital for predicting donor specific responses and better understanding the signaling pathways involved. This is of particular importance in platelets because exposure of phosphatidylserine (PS) occurs upon costimulation but not with a single agonist. Here, we describe a multiplexed pairwise agonist scanning-flow cytometry (PAS-FC) method of measuring platelet inside-out responses to all pairs of six platelet agonists (convulxin, SFLLRN, AYPGKF, ADP, U46619, and PGE2) used at their EC50 concentrations. These agonists allowed exploration of platelet signaling downstream of GPVI, PAR-1, PAR-4, P2Y1, P2Y12, TP, and IP receptors. The three-color flow cytometry method simultaneously measured integrin αIIbβ3 activation with PAC-1 antibody, P-selectin exposure (via α granule release) with anti-P-selectin, and PS exposure with annexin V. These responses were consistent across a healthy male donor pool. In duplicate measurements with each donor, 4 of the 10 donors had a sufficiently unique 45-parameter (15 pairs × 3 colors) phenotype to self-cluster (P < 0.001). This method has the potential for efficiently scanning for patient specific responses across a broad agonist-receptor space.

Cells are subject to numerous dose dependent agonist signals at a given time in a biological system, resulting in a complex integrated response. In many instances, signaling pathways use common second messengers that may result in synergistic or antagonistic effects that could not be predicted from study of the agonists in isolation (1). Platelets respond to multiple signals in vivo in a donor specific manner to halt blood loss (2), a process which is also central to the thrombotic risks associated with the 2 million heart attacks and strokes that kill 800,000 people each year in the United States alone (3). Platelets are an ideal cell type for studying the effects of multiple signaling pathways because they are anucleate, easily obtained from donors, and their responses can be used to make donor specific predictions about thrombosis (4).

In vivo, a platelet can encounter numerous different activating and inhibitory signals at the same time, and the integrated response to all of these determines the prothrombotic state of the platelet. In a thrombotic event, the first signal encountered is often activation of glycoprotein VI (GPVI) ITAM tyrosine kinase signaling upon binding collagen in the exposed sub-endothelial matrix (5). This occurs during platelet rolling, enabled by GPIb-IX-V binding vWF, which gives the platelet time for the GPVI signal to induce calcium mobilization that activates αIIbβ3 and α2β1 integrins to firmly adhere the platelet to the site of injury (6). In addition to activation of adhesion molecules, ADP is released from the dense granules early during the activation process and signals via platelet P2 receptors in an autocrine and paracrine fashion (7). After activation dependent synthesis by cyclooxygenase 1 (COX1), thromboxane A2 (TXA2) is also released and binds platelets in the lumen and at the site of injury (8). Somewhat independent of the platelets, tissue factor (TF) exposed at the site of the injury initiates the extrinsic pathway of the coagulation cascade leading to the production of thrombin, which very strongly activates platelets through the PAR receptors. In addition to these and other minor activating signals, the endothelium constitutively releases prostacyclin (PGI2) and nitric oxide (NO) as inhibitory signals to platelet activation.

Method summary

Platelet rich plasma obtained from human blood was stimulated with all pairs of six different agonists. For platelet phenotyping, 96-well plate flow cytometry allowed 3-color detection of integrin activation, granule release, and phosphatidylserine exposure.

Platelets can encounter each of these signals at varying doses during the thrombotic process and combinations of four or more simultaneously would be common in the core of a growing thrombus in vivo. However, most in vitro studies of these signaling pathways are done in isolation. The effects of thrombin (9), ADP (10), and collagen (11) are known in great detail, but the previous studies do not address the conditions of the non-isotropic environment of a thrombus with many signaling molecules (12). To investigate the effects of simultaneous addition of agonist pairs, we have extended the previous Ca2+ assay that found donor specificity in platelet cytosolic calcium levels to utilize flow cytometry (2). Since integrin activation, degranulation, and phosphatidylserine (PS) exposure are all activation markers downstream of the cytosolic calcium, they may also exhibit donor specificity. Binding of the IgM antibody PAC-1 was used to measure the degree of integrin activation as it only binds the active form of the integrin αIIbβ3 (13). Secretion was measured using an IgG antibody against P-selectin (CD62P), which is exposed on the platelet surface when α granules fuse with the plasma membrane. This is not a direct measure of dense granule secretion, but dense granules release before α granules (14), so it is indicative of release of both types. Finally, the exposure of PS to serve as a catalytic surface for the coagulation cascade on the plasma membrane outer leaflet was measured by binding of annexin V (15). These three measures of platelet activation give a fuller representation of platelet behavior upon multiple agonist activation.

Materials and methods

Platelet Preparation

Whole blood was drawn from healthy male volunteers, according to the University of Pennsylvania Institutional Review Board guidelines with informed consent, into Phe-Pro-Arg-chloromethylketone (PPACK; Haematologic Technologies, Essex Junction, VT) with a final anticoagulant concentration of 100 µM. All donors affirmed not taking any medication for 10 days prior to donation and not consuming alcohol for 3 days prior to donation. The whole blood samples were centrifuged at 120× g for 10 min to obtain platelet rich plasma (PRP), which was diluted to 10% v/v in HEPES buffered saline (HBS; 20 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2 at pH 7.4). Calcium was added to the buffer to facilitate proper formation and activation of the αIIbβ3 integrin as well as binding of annexin V to exposed PS.

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