Figure 1.
Experimental design and modeling.
Platelets are labeled with DIO and exposed to macrophages (THP-1 cells) via a low speed centrifugation step, then incubated at 37 degrees. The processes of adsorption, phagocytosis, and quenching of the fluorescence of internalized platelets are shown schematically, with reference to the parameters used in numerical modeling. The asterisk denotes that “m” is the functional ratio of platelets to macrophages (see text). Cells are then exposed to PE-anti-CD61, and analyzed by flow cytometry. The expected results (top right) include cells showing variable numbers of internalized platelets, adsorbed platelets, and mixtures of the two. Typical observed results for normal control platelets are also shown (bottom right).
Figure 2.
Observed and modeled fluorescence intensity histograms.
A) Modeled histograms for n = 0 to n = 4. Empirically chosen parameter values (see text) are p = 0.739, alpha = 2.53, m = 2.17. Mean and SD values of the log transformed negative control data are 4.57 and 0.29. B) The sum of the predicted fluorescence intensities for n = 0 to n = 12 is shown in blue. Data for comparison (CD61 negative cells, figure 1) is shown in red.
Figure 3.
Assessment of a series Rt values (p-axis).
Using empirically chosen m and alpha values, and a series of p values, predicted Q1 and Q(2+3) values were compared to observed values for the data shown in figure 1. P value resolution is 0.04 (20 evaluated points). A single Rtmin(j,k) value is identified.
Table 1.
Data and parameter values for figure 3.
Figure 4.
Assessment of a series of Rtmin(j,k) values (alpha axis).
Rt was evaluated for the data shown in figure 1 in an empirically chosen plane defined by M = 2.18. Alpha values assessed ranged from 1 to 4 (resolution 0.15). P values were assessed (for each alpha value) as in figure 3. Left: A single local Rtmin value (Rtmin(k)) is identified in this plane. Right: The graph is rotated 90 degrees on the z axis.
Figure 5.
Assessment of a series of Rtmin(k) values (m axis).
A series of planes (defined by m values) was evaluated as shown in figure 4. P and alpha resolutions were as described in figures 3 and 4. The alpha range was manually adjusted to follow the local minima (LM) evident at lower resolution scans; alpha values for the local minima shown ranged from 4.05 (LM1) to 1.06 (LM7). M resolutions were 0.01 (range m = 1 to m = 2.25) and 0.0625 (m = 2.25 to m = 4).
Figure 6.
Effects of varying key input values on local Rt minima.
A) Normal control platelets were labeled with DIO at the uM concentrations shown, resulting in geometric mean fluorescence intensities of 2796 and 1473, respectively (arbitrary units). Evaluation of parameter space was performed as in figures 3 to 5, at resolutions p = 0.04, alpha = 0.15, and m = 0.0625. Local Rtmin(k) points in parameter space were identified as in figure 5. B) Normal control platelets were labeled at 1.5 uM DIO, and exposed to activated THP-1 cells at the platelet/macrophage ratios shown. Parameter space evaluation was performed as in (A).
Figure 7.
Effect of antibody opsonization on local Rt minima.
Control platelets were exposed to anti-CD61 antibody (50 ng per million platelets) or to an isotype control antibody, then exposed to activated THP-1 cells. Evaluation of parameter space was performed as in figures 3–6.
Figure 8.
Effect of platelet WASP deficiency on phagocytosis.
A) DIO-labeled platelets from an adult control, or from a WAS patient, were exposed to THP-1 cells and analyzed as described in figure 1. B) Parameter space was evaluated as in figures 3 to 5, in comparison to mean values of duplicates of the phagocytosis assays shown in (A). Local Rt minima are shown.
Table 2.
Predicted and observed results for control vs. WAS platelet phagocytosis.