American Society of Hematology

Figure 5

$Figure 5. Defective hemostasis and platelet activation in RanBP10-deficient mice. (A) RanBP10 mice have a prolonged bleeding time. Tails were cut at the tip and blood flow was followed by submerging in 37°C saline until flow discontinued for at least 30 seconds. Bleeding stopped within 5 minutes in only 2 of 10 mutant mice but in 8 of 10 wild-type mice. (B) Mutant platelets fail to show full CD62P expression after agonist stimulation. Whole blood of wild-type and mutant mice was stimulated with 1mM PAR4 peptide before adding an fluorescein isothiocyanate-conjugated anti-CD62P specific antibody. Platelets were analyzed by flow cytometry showing a markedly reduced expression of CD62P. (C) Dose-response curve of PAR4p-stimulated platelets from 4 RanBP10-null (gray curve) and 5 wild-type mice (black curve), showing reduced reactivity with suboptimal concentrations. Error bars indicate 1 SD. (D) Platelets lacking RanBP10 fail to secrete the dense-granule marker CD63 after incubation with 1mM PAR4p (gray curve, right panel), compared with wild-type controls (gray curve, left panel). One representative of 3 experiments is shown. Black curves represent unstimulated controls. (E) Flow cytometric analysis from 4 wild-type (♦) or 4 RanBP10-null mice (gray circles) stimulated with 10 or 20 μg/mL collagen reveals slightly reduced CD62P expression in mutant animals. (F) Mutant mice show a reduced release of platelet granules after agonist stimulation. Washed platelets from wild-type (+/+) and knockout (−/−) mice were incubated with 1 or 0.1 U thrombin or 1mM PAR4 peptide as indicated, for 5 minutes before centrifugation. Platelet supernatant (S) was removed before adding 4× sample buffer. Pellets (P) were solubilized with 1× sample buffer overnight, and equal volumes of both fractions were subjected to SDS-PAGE. PF4 distribution was analyzed by immunoblotting. Mutant platelets failed to degranulate after low thrombin or PAR4p stimulation but showed partial response to high thrombin. (G) Normal expression of α-granule proteins in RanBP10-deficient platelets (−/−) compared with wild-type controls (+/+). Total platelet lysates were immunoblotted with antibodies against von Willebrand factor (VWF), P-selectin, and PF4. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (H) Immunofluorescence staining against tubulin (green) showed contraction of the MT coil in most PAR4p-stimulated wild-type platelets (top left, white arrows) while staining for granule marker protein PF4 (red) was weak. In activated RanBP10−/− platelets tubulin staining revealed larger MT coils (bottom left). PF4 staining was stronger implying that granule release was incomplete, which is also shown in the merged panels. Scale bar indicates 2 μm.$

Defective hemostasis and platelet activation in RanBP10-deficient mice. (A) RanBP10 mice have a prolonged bleeding time. Tails were cut at the tip and blood flow was followed by submerging in 37°C saline until flow discontinued for at least 30 seconds. Bleeding stopped within 5 minutes in only 2 of 10 mutant mice but in 8 of 10 wild-type mice. (B) Mutant platelets fail to show full CD62P expression after agonist stimulation. Whole blood of wild-type and mutant mice was stimulated with 1mM PAR4 peptide before adding an fluorescein isothiocyanate-conjugated anti-CD62P specific antibody. Platelets were analyzed by flow cytometry showing a markedly reduced expression of CD62P. (C) Dose-response curve of PAR4p-stimulated platelets from 4 RanBP10-null (gray curve) and 5 wild-type mice (black curve), showing reduced reactivity with suboptimal concentrations. Error bars indicate 1 SD. (D) Platelets lacking RanBP10 fail to secrete the dense-granule marker CD63 after incubation with 1mM PAR4p (gray curve, right panel), compared with wild-type controls (gray curve, left panel). One representative of 3 experiments is shown. Black curves represent unstimulated controls. (E) Flow cytometric analysis from 4 wild-type (♦) or 4 RanBP10-null mice (gray circles) stimulated with 10 or 20 μg/mL collagen reveals slightly reduced CD62P expression in mutant animals. (F) Mutant mice show a reduced release of platelet granules after agonist stimulation. Washed platelets from wild-type (+/+) and knockout (−/−) mice were incubated with 1 or 0.1 U thrombin or 1mM PAR4 peptide as indicated, for 5 minutes before centrifugation. Platelet supernatant (S) was removed before adding 4× sample buffer. Pellets (P) were solubilized with 1× sample buffer overnight, and equal volumes of both fractions were subjected to SDS-PAGE. PF4 distribution was analyzed by immunoblotting. Mutant platelets failed to degranulate after low thrombin or PAR4p stimulation but showed partial response to high thrombin. (G) Normal expression of α-granule proteins in RanBP10-deficient platelets (−/−) compared with wild-type controls (+/+). Total platelet lysates were immunoblotted with antibodies against von Willebrand factor (VWF), P-selectin, and PF4. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (H) Immunofluorescence staining against tubulin (green) showed contraction of the MT coil in most PAR4p-stimulated wild-type platelets (top left, white arrows) while staining for granule marker protein PF4 (red) was weak. In activated RanBP10^−/− platelets tubulin staining revealed larger MT coils (bottom left). PF4 staining was stronger implying that granule release was incomplete, which is also shown in the merged panels. Scale bar indicates 2 μm.

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