Fig 1.
RGS10 deficiency does not affect platelet RGS protein levels.
(A) DNA was isolated, and PCR was performed as described in the “Methods” section. DNA was separated on a 1% agarose gel and visualized using a gel documentation system. (B) Platelet extracts (2 x 108/ml) were prepared from wild type (WT) and the Rgs10-/- mice. Proteins were probed using Western Blot.
Table 1.
Peripheral blood cell counts in WT and Rgs10-/- mice.
Fig 2.
Deletion of RGS10 alters agonist-induced platelet activation.
Platelets from Rgs10-/- or WT mice (2 x 108/ml) were exposed to (A) 1 μM ADP, (B) 2.5 μM ADP, (C) 40 μM TRAP4, (D) 80 μM TRAP4, (E) 0.1 U/ml thrombin, (F) 0.2 U/ml thrombin, (G) 0.25 μM U46619, and (H) 0.5 μM U46619. Platelet aggregation was measured using constant stirring. Each experiment was repeated at least 3 times, with blood pooled from a group of eight mice.
Fig 3.
Aggregation in response to TRAP4 in the presence or absence of apyrase.
Platelets from (A) WT or (B) Rgs10-/- mice (2 x 108/ml) were incubated with 0.5 units of apyrase for 3 minutes, then exposed to 40 μM TRAP4. Platelet aggregation was measured using constant stirring. Each experiment was repeated at least 3 times, with blood pooled from a group of eight mice.
Fig 4.
Deletion of RGS10 protein causes enhanced integrin αIIbβ3 activation in stimulated platelets.
Platelets from Rgs10-/- or WT mice (105 platelets/100 μl) were stimulated with (A) 5 μM ADP and (B) 80 μM TRAP4, fixed, and labeled with JonA antibody. Samples were analyzed using a flow cytometer. Average mean fluorescence intensities shown. Experiment was conducted in duplicate, and was repeated at least 3 times, with blood pooled from a group of eight mice. (P<0.05 using Unpaired t-test).
Fig 5.
Deletion of RGS10 causes upregulation of α-granule secretion.
Platelets from Rgs10-/- or WT mice (105 platelets/100 μl) were stimulated with 0.1 U/ml thrombin, fixed, and labeled with CD62P antibody. Samples were analyzed using a flow cytometer. Average mean fluorescence intensities shown. Experiment was conducted in duplicate, and repeated at least 3 times, with blood pooled from at least eight mice. (P<0.05 using Unpaired t test).
Fig 6.
Deletion of RGS10 protein alters physiological hemostasis and development of thrombosis.
(A) Bleeding times were measured in Rgs10-/- (n = 5) or WT (n = 4) mice following venisection, as described in the “Methods” section. Each point represents the bleeding time of a single animal (P < 0.03 Mann-Whitney test). (B) Thrombosis was induced in Rgs10-/- (n = 5) and WT (n = 5) mice using chemical injury (FeCl3), as described in the “Methods” section. Each point represents an occlusion time of a single animal (; P<0.0079 Mann-Whitney test).