Characterization of Procoagulant COAT Platelets in Patients with Glanzmann Thrombasthenia

Figures

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  Figure 1. Procoagulant collagen-and-thrombin (COAT) platelet formation in patients with Glanzmann thrombasthenia and healthy donors. Absolute percentage of the Annexin-V-positive platelets following activation with convulxin-plus-thrombin in healthy donors (HD) and means of at least two replicates for patients with Glanzmann thrombasthenia (GT). The grey zone (25–55%) indicates the in-house reference ranges (2.5–97.5 percentiles). Statistical significance was determined by Mann–Whitney test, *** p < 0.001.

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  Figure 2. Intracellular ion kinetics following platelet activation with convulxin and thrombin. Platelets from healthy donors (HD) or patients with Glanzmann thrombasthenia (GT) were loaded with ion fluorescent indicator. Fluorescence from ion probes was monitored in FL1 (488:530/30) and median fluorescence intensity (MFI) fold change (relative to the baseline [BSL]) for the whole platelet population was plotted over time. (A) Calcium monitoring with Fluo-3. (B) Sodium monitoring with ION NaTRIUM Green-2 (ING-2). (C) Potassium monitoring with ION Potassium Green-2 (IPG-2). Data from HD represented as mean ± SEM, from experiments with platelets from different donors (n = 6). Data for each GT patient (named PAT_GT1, PAT_GT2, and PAT_GT3) are a mean of individual replicates, n = 3 for PAT_GT1, n = 1 for PAT_GT2, and n = 2 PAT_GT3.

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  Figure 3. Intracellular calcium within first 3 min after activation. Each of the three patients with Glanzmann thrombasthenia (GT) had their first 3 min kinetics after activation plotted with respect to their Fluo-3 median fluorescence intensity (MFI) fold change compared to healthy donors (HD). Co-staining with Cy5 Annexin V allowed differentiating procoagulant COAT platelets (Annexin V positive) from the whole platelet population. (A) PAT_GT1; (B) PAT_GT2; (C) PAT_GT3. HD data are mean ± SEM from experiments with platelets from different donors (n = 6). Data for each GT patient are a mean of individual replicates, n = 3 for PAT_GT1, n = 1 for PAT_GT2, and n = 2 PAT_GT3. Baseline, BSL.

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  Figure 4. GPIIb/IIIa-antagonists effect on the formation of procoagulant COAT platelets. Platelets were pretreated with increasing doses of (A) eptifibatide or (B) tirofiban, and activated with convulxin-plus-thrombin. Relative change of the fraction of Annexin-V-positive platelets compared to control condition (vehicle H2O or DMSO, respectively) is represented as mean ± SEM from experiments with platelets from different donors (n = 3–5).