Novel assay demonstrates that coronary artery disease patients have heightened procoagulant platelet response

Figures

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  Figure 1. Gating strategy for detection of procoagulant platelets in human whole blood using the cytoplasmic cell death marker (GSAO). (A) Platelets are defined as CD41a positive but CD45 negative events. (B) Threshold values for P‐selectin and GSAO‐positive regions are set using a thrombin plus collagen‐stimulated sample stained with IgG isotype control and control GSCA, respectively. GSAO+/P‐selectin+ (Procoag) quadrant (outlined in red) contains procoagulant platelets. GSAO+/P‐selectin− (Apopt) quadrant (outlined in green) contains apoptotic platelets. (C) Very few platelets show the procoagulant phenotype (P‐selectin+/GSAO+) in unstimulated whole blood. (D) Following stimulation with thrombin plus collagen a significant proportion of platelets exhibited the procoagulant phenotype. (E) Characterization of the stimulus‐induced procoagulant platelet response in human whole blood. (F) Synergistic interaction between thrombin and collagen occurred at all levels of individual agonist concentrations. Normalized agonist dose (maximal [100%] for collagen [100 μg mL−1] and thrombin [6.3 U mL−1]) expressed as percent maximal is shown on a logarithmic scale. A representative example is shown. (G)…

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  Figure 2. GSAO‐based assay has advantages over other markers of cell death for identification of procoagulant platelets in human whole blood. (A) Whole blood is the preferred substrate for demonstration of procoagulant platelet potential. Citrated blood samples from individuals were prepared as whole blood (WB), platelet‐rich plasma (PRP) or washed platelet (WP) preparations. Each preparation was treated with thrombin (2 U mL−1), collagen (10 μg mL−1), or a combination of both, then stained with GSAO/P‐selectin. WB demonstrated higher levels of GSAO+/P‐selectin+ platelets compared with PRP or WP from the same draw. (B) Most GSAO+ platelets externalized PS (lactadherin+); however, a significant proportion of lactadherin+ platelets did not take up GSAO. Pseudocolors are used to indicate population density. (C) Thrombin (2 U mL−1)‐treated WB samples were stained either with a standard panel of markers or a modified panel where GSAO was replaced with the amine reactive dye Zombie NIR, fixed and analyzed by flow cytometry. Procoagulant platelets identified by GSAO were not identified by Zombie NIR (n = 3, paired‐t‐test, *P < 0.05). GSAO, [(4‐(N‐(S‐glutathionylacetyl)amino)phenylarsonous acid].…

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  Figure 3. CAD is associated with a heightened procoagulant platelet potential and loss of synergism between thrombin and collagen that is not affected by aspirin. DAPT ameliorates thrombin hypersensitivity in CAD but only partially suppresses thrombin and collagen. (A, B) Blood samples (1–4 per individual) were collected during coronary angiography from 60 patients on either aspirin or no antiplatelet agents. Procoagulant platelets were assayed in whole blood samples treated with thrombin (2 U mL−1) or thrombin (2 U mL−1) plus collagen (10 μg mL−1). Data are shown as the percentage of platelets with the procoagulant phenotype (GSAO+/ P‐selectin+). Linear mixed effects models, two‐tailed tests, *P < 0.05. (C) Platelets from patients with CAD showed increased stimulus‐induced procoagulant potential compared to those without CAD and healthy controls. Two‐way anova analysis with Tukey correction for multiple comparisons. Mean ± SEM, n = 11 (CAD), n = 16 (no CAD) and n = 10 (healthy controls). P < 0.0001. (D) Apoptotic platelet subset (GSAO+/P‐selectin−) did not vary according to the presence of CAD. Two‐way anova analysis with Tukey correction for multiple comparisons. Mean ± SEM, n = 11…

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  Figure 4. Clopidogrel abrogates the abnormal response to ADP and thrombin but not to a thrombin and collagen combination in patients with CAD, and shows a discordant inhibitory effect on platelet aggregation and procoagulant potential. (A) CAD patients without P2Y12 exposure show an increased procoagulant response to ADP 5 μm (2.7% ± 1.8% vs. 7.6% ± 3.2%, *P < 0.05) that is abrogated in patients on DAPT (7.6% ± 3.2% vs. 4.1% ± 2.5%, t‐test **P < 0.01). (B) Platelets from patients with CAD showed heightened procoagulant response to ADP (≥ 0.625 μm) compared with healthy controls. Treatment with clopidogrel restored the pattern of response. n = 3, t‐test *P < 0.05, **P < 0.01. (C) Compared with patients without P2Y12 antagonists, patients on clopidogrel showed significant inhibition of procoagulant platelet formation in response to ADP 5 μm (*P < 0.05) or thrombin 2 U mL−1 (*P < 0.05), but not thrombin 2 U mL−1 and collagen 10 μg mL−1 (t‐test P = 0.99). Patients on prasugrel or ticagrelor demonstrated significant inhibition of thrombin/collagen‐induced procoagulant platelets compared with patients on clopidogrel (27.2 ± 4.5% vs. 12.0 ± 1.8%, n = 8–22, t‐test P < 0.05); however, there was no…