Given that this cohort is less diverse in genetic ancestry than the one used for the calcium assay (seven Europeans, one Indian, and two Asians here versus three Europeans, two Asians, two Indians, one Caribbean, one African American, and one African), less donor self-clustering may be a result of donor similarity. To examine this, we calculated the mean of the 10 donor standard deviations (intradonor variation) as well as the standard deviation of the mean value for each donor (interdonor variation) for each of the 45 data points. The ratio of intradonor variation divided by the interdonor variation is shown in Figure 3, where red indicates that variation within the repeats of a donor was larger than variation across donors, while blue indicates variation across the cohort was more significant. For 17 of the 30 PAC-1 or anti-CD62P binding data points, the intradonor variation was dominant, which makes it difficult to reliably distinguish donors and contributes to the low self-clustering. Some self-clustering is achieved, however, because of the relative donor specificity of PS exposure, anti-CD62P binding in the presence of PGE2, and PAC-1 binding in the absence of convulxin. The annexin V positive data for non-CVX conditions can be ignored as there is no PS exposure.
In addition to the genetics of the donors, the range of the experimental space that was explored may contribute to the reduced self-clustering of this assay compared with the previous calcium based assay. Time constraints limited study to only pairwise combinations at EC50 concentrations, as completion of the 135 combinations of a pairwise scan at 0.1, 1, and 10× EC50 would take 4.5 h to complete. Platelets removed from the body, as well as the agonists, lose reactivity over that amount of time, and fixation is not possible because it artificially induces PS exposure and diminishes PAC-1 binding (24). Given this limited experimental space and our cohort of genetically similar, healthy donors, 4 of 10 self-clustering is likely a typical level of patient specificity that can be achieved currently. Increasing the number of experiments per donor to four or six may increase specificity, but the largest gains to be made are in expanding the concentrations of agonists tested. Testing multiple concentrations of each agonist would provide information about donor-specific sensitivity and maximum response that would allow this assay to provide a more complete picture of platelet activation.
In order to further investigate donor specificity, the percentage of cells exposing PS in response to convulxin and SFLLRN costimulation for each donor's two samples was compared (Figure 4). This response shows correlation between samples (R2 = 0.2577), and a wide range of values are seen. This is consistent with previously reported donor specificity of maximal PS exposure between 20%–70% upon treatment with convulxin and thrombin (25). Such high values are not achieved in this assay due to lower agonist concentrations, but the variable potential for PS exposure among donors is still evident. Expansion of the experimental space to include higher agonist concentrations would provide valuable patient-specific information about maximal responses in PS exposure as well as integrin activation and degranulation, but this requires a flow cytometer that can collect and measure samples more quickly.
There is potential for improvement of this assay, which already gives a wealth of data about the effects of multiple simultaneous signals and significant donor specificity. Currently, there are some flow cytometers that are capable of analyzing a full 384-well plate in 15 min. Design of such a machine that also has the necessary liquid handling capabilities to add agonists at well-defined times before analysis would make full pairwise agonist scanning in flow cytometry possible. At such a point, the ability to test all 135 pairwise conditions for integrin activation, degranulation, PS exposure, and possibly even more measures of activation could provide a tremendous amount of donor specific information that could be utilized in models of thrombosis (4). Any response for which a fluorescently labeled probe exists can easily replace or be added to the three in this assay with the inclusion of a sufficient amount to measure the full dynamic range of the agonists. The number of responses that can be measured simultaneously is limited only by the ability of the flow cytometer to record their emission spectra.
This study was supported by NIH R01 HL-103419 to S.L.D. This paper is subject to the NIH Public Access Policy.
The authors declare no competing interests.
Correspondence Address correspondence to Scott L. Diamond, Institute for Medicine and Engineering, Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA. E-mail: [email protected]">[email protected]References
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