Figure 1.
Dose-response analysis of thrombin- and collagen-induced platelet aggregation and shape change.
The aggregation of human washed platelets in response to various concentrations of thrombin or collagen was analyzed by turbidimetry under stirring conditions in the absence of exogenous fibrinogen. In parallel, the individual morphological changes induced by the same concentrations of agonists were analyzed by DIC microscopy. A–B. Diagrams showing representative dose-response aggregation tracings obtained for thrombin (A) and collagen (B). CTL corresponds to non-treated resting platelets. C. Bar graph showing the mean maximum platelet aggregation in response to various doses of thrombin or collagen. # indicates a significant statistical difference (p<0.05) from non-stimulated resting human washed platelets (CTL). n = 5 different blood donors. D–E. DIC microscopy images of the morphological aspect of platelets after incubation with or without the indicated concentrations of thrombin (D) or collagen (E). The images shown are representative of 6 independent experiments performed with different blood donors. Scale bars = 5 µm. White arrowheads indicate platelet membrane protrusions, black arrows indicate microvesicles.
Figure 2.
Dose-response analysis of thrombin- and collagen-induced phosphatidylserine exposure in human platelets.
Phosphatidylserine exposure by human washed platelets was quantified by measurement of fluorescent annexin V binding in flow cytometry. A. Representative flow cytometry histograms of annexin V binding to non-stimulated resting (control) platelets or to platelets incubated with various concentration of thrombin or collagen, as indicated above the charts. The histogram obtained for control platelets was superimposed in grey to that of stimulated platelets for better visualization of fluorescence shifts. B. Bar graph representing the mean annexin V binding to non-treated control platelets (CTL) and to thrombin- or collagen-stimulated platelets. Results are expressed as percent relative to the mean fluorescence intensity of CTL platelets. n = 4 different blood donors, # indicates a significant statistical difference (p<0.05) from CTL platelets.
Figure 3.
Dose-response analysis of thrombin- and collagen-induced P-selectin surface expression.
A. Representative flow cytometry histograms of P-selectin surface expression of non-treated (control) and thrombin- or collagen-treated platelets, as indicated. The histogram obtained for control platelets was superimposed in grey to that of stimulated platelets for better visualization of fluorescence shifts. B. Bar graph representing the mean percentage of P-selectin-positive cells of non-treated control (CTL) and thrombin- or collagen-stimulated platelets. n = 8 independent experiments with different blood donors, # indicates a significant statistical difference (p<0.05) from CTL. C. Immunolocalization of P-selectin (green) in permeabilized control and agonist-stimulated platelets. Merged images of DIC and green fluorescence are shown. Bar = 5 µm.
Figure 4.
Dose-response relationship between agonist concentration and secretion of dense and alpha-granule-derived bioactive agents.
The release of dense and alpha-granule components by human platelets in response to various concentrations of thrombin and collagen was assessed and compared by measuring serotonin (A), ATP (B), platelet factor 4 (PF4) (C), TGF-β1 (D), and angiopoietin-1 (E) levels in platelet supernatants. All bar graphs represent the mean levels calculated from 6 independent experiments performed with different blood donors. # indicates a significant statistical difference (p<0.05) from CTL.
Figure 5.
Comparison of soluble factor release and surface activation marker expression by stirred platelets exposed to collagen.
A. ATP levels in suspensions of stirred platelets as determined using a luminescence-based assay. B. PF4 levels in suspensions of stirred platelets as determined using a fluorescent microspheres-based flow cytometric immunoassay. C–E. Surface exposure of P-selectin (C) and phosphatidylserine (D), as well as binding of the PAC-1 antibody to activated GPIIb/IIIa (E) on stirred platelets were measured by flow cytometry. All bar graphs represent means calculated from a minimum of 4 independent experiments performed with different blood donors. # indicates a significant statistical difference (p<0.05) from CTL.
Figure 6.
Low dose collagen-induced platelet secretion requires GPVI signalling.
A. Intracellular calcium levels in collagen-stimulated platelets as assessed by measurement of calcium-dependent fluorescence of Oregon Green 488 BAPTA-1AM-loaded platelets. The calcium curves shown are representative of four independent experiments using different blood donors. B–D. To assess the contribution of GPVI-signalling to low-dose collagen-induced secretion of platelet soluble factors, platelets were stimulated with 0.25 µg/mL collagen in the presence or absence of a GPVI blocking Fab fragment antibody (Fab 9O12, 50 µg/mL) and the levels of serotonin (B), TGF-β1 (C), and angiopoietin-1 (D) were then measured in platelet supernatants. All bar graphs represent the mean levels from a minimum of 4 independent experiments. NS: non-statistically significant.