American Society of Hematology

Figure 5.

$Figure 5. Polyphosphate nanoparticle formation depends on divalent metal ion incorporation and promotes contact system activation. (A) Fractionation of SYTOX-supplemented platelet lysate by ultracentrifugation on sucrose density gradients in the absence or presence of EDTA (250 mM). Data show normalized means ± standard deviation of 3 independent fractionation experiments. (B) Scanning electron microscopy of spreading platelets in the absence or presence of EDTA (250 mM). (C-D) Platelet adhesion and spreading on immobilized VWF under flow. A scale bar is shown in the left panel (10 μm). All images are shown at the same magnification; insets show indicated areas at higher magnification. Times (above) represent the time course, starting from the moment of first stable platelet adhesion in the image field. (C) Polyphosphate exposure was tracked with SYTOX (orange). After 15 minutes, either buffer or EDTA (50 mM) was perfused over spread platelets. (D) Polyphosphate exposure was detected by the binding of Alexa488-conjugated PPX_Δ12 (green) after 25 minutes of platelet spreading in the absence or presence of EDTA (250 mM). Microscopic images were acquired at a magnification of ×1000. A scale bar (10 μm) is shown in the first panel of the series. All images are shown at the same magnification. Times (indicated above) represent the time course, starting from the first moment of stable platelet adhesion in the image field. Insets show indicated areas at higher magnification. Images are representative for experiments that were performed >3 times. (E-F) Plasma contact system activation by 50 µg/mL calcium-preadsorbed synthetic short-chain polyphosphate (PolyP; 70-mers). (E) Kallikrein-like activity was monitored by conversion of a chromogenic substrate and (F) formation of enzyme-C1 inhibitor complexes in plasma (after 15 minutes). Where indicated, contact system activation was triggered in the presence of aprotinin (100 U/mL) or by polyphosphate (PolyP) that was pretreated with EDTA (100 mM) for 10 minutes prior to contact system activation. 100% indicates the amount of complexes that are formed in plasma when exposed to positive control contact activators for 30 minutes. **P < .01, analyzed by unpaired Student t test. (G-H) Kaolin (1.5 µg/mL) or anion exchange–purified platelet polyphosphate (20 µM; expressed as monophosphate units) triggered kallikrein-like activity in the absence or presence of EDTA, monitored by chromogenic substrate conversion.$

Polyphosphate nanoparticle formation depends on divalent metal ion incorporation and promotes contact system activation. (A) Fractionation of SYTOX-supplemented platelet lysate by ultracentrifugation on sucrose density gradients in the absence or presence of EDTA (250 mM). Data show normalized means ± standard deviation of 3 independent fractionation experiments. (B) Scanning electron microscopy of spreading platelets in the absence or presence of EDTA (250 mM). (C-D) Platelet adhesion and spreading on immobilized VWF under flow. A scale bar is shown in the left panel (10 μm). All images are shown at the same magnification; insets show indicated areas at higher magnification. Times (above) represent the time course, starting from the moment of first stable platelet adhesion in the image field. (C) Polyphosphate exposure was tracked with SYTOX (orange). After 15 minutes, either buffer or EDTA (50 mM) was perfused over spread platelets. (D) Polyphosphate exposure was detected by the binding of Alexa488-conjugated PPX_Δ12 (green) after 25 minutes of platelet spreading in the absence or presence of EDTA (250 mM). Microscopic images were acquired at a magnification of ×1000. A scale bar (10 μm) is shown in the first panel of the series. All images are shown at the same magnification. Times (indicated above) represent the time course, starting from the first moment of stable platelet adhesion in the image field. Insets show indicated areas at higher magnification. Images are representative for experiments that were performed >3 times. (E-F) Plasma contact system activation by 50 µg/mL calcium-preadsorbed synthetic short-chain polyphosphate (PolyP; 70-mers). (E) Kallikrein-like activity was monitored by conversion of a chromogenic substrate and (F) formation of enzyme-C1 inhibitor complexes in plasma (after 15 minutes). Where indicated, contact system activation was triggered in the presence of aprotinin (100 U/mL) or by polyphosphate (PolyP) that was pretreated with EDTA (100 mM) for 10 minutes prior to contact system activation. 100% indicates the amount of complexes that are formed in plasma when exposed to positive control contact activators for 30 minutes. **P < .01, analyzed by unpaired Student t test. (G-H) Kaolin (1.5 µg/mL) or anion exchange–purified platelet polyphosphate (20 µM; expressed as monophosphate units) triggered kallikrein-like activity in the absence or presence of EDTA, monitored by chromogenic substrate conversion.

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