Regulator of G-Protein Signaling 16 Is a Negative Modulator of Platelet Function and Thrombosis

Abstract

Background Members of the regulator of G-protein signaling ( RGS ) family inhibit G-protein coupled receptor signaling by modulating G-protein activity. In platelets, there are 3 different RGS isoforms that are expressed at the protein level, including RGS 16. Recently, we have shown that CXCL 12 regulates platelet function via RGS 16. However, the role of RGS 16 in platelet function and thrombus formation is poorly defined. Methods and Results We used a genetic knockout mouse model approach to examine the role(s) of RGS 16 in platelet activation by using a host of in vitro and in vivo assays. We observed that agonist-induced platelet aggregation, secretion, and integrin activation were much more pronounced in platelets from the RGS 16 knockout ( Rgs16 ^-/-) mice relative to their wild type ( Rgs16 ^+/+) littermates. Furthermore, the Rgs16 ^-/- mice had a markedly shortened bleeding time and were more susceptible to vascular injury-associated thrombus formation than the controls. Conclusions These findings support a critical role for RGS 16 in regulating hemostatic and thrombotic functions of platelets in mice. Hence, RGS 16 represents a potential therapeutic target for modulating platelet function.

Keywords: RGS16; hemostasis; platelet; regulator of G‐protein signaling; signal transduction; thrombosis.

Figure 1

RGS16 is expressed in human platelets. RGS16 expression was analyzed in lysates of human platelets by immunoblotting (2×10⁸/mL sample). RGS16 indicates regulator of G protein signaling 16.

Figure 2

Deletion of RGS16 alters agonist‐induced platelet activation and dense granule secretion. Platelets from Rgs16 ^+/+ and Rgs16 ^−/− mice were prepared and aliquots (2.5×10⁸/mL) were stimulated with (A and B) 80 μmol/L TRAP4, (C and D) 0.05 U/mL thrombin, or (E and F) 1.25 μg/mL collagen. Platelet aggregation (A, C, and E) and ATP release (B, D, and F) were measured with constant stirring in a lumiaggregometer. Each experiment was repeated at least 3 times, each with blood pooled from a group of 8 mice++. RGS16 indicates regulator of G protein signaling 16; TRAP4, protease‐activated receptor 4.

Figure 3

Deletion of RGS16 alters collagen‐induced platelet activation and dense granule secretion in aspirin and apyrase‐treated platelets. Platelets from Rgs16 ^+/+ and Rgs16 ^−/− mice were prepared and aliquots (2.5×10⁸/mL) pretreated with or without aspirin or apyrase were stimulated with 1.25 μg/mL collagen. Platelet aggregation of the Rgs16 ^+/+ (A and E) and Rgs16 ^−/− (C and G) as well as ATP release of the Rgs16 ^+/+ (B and F) and Rgs16 ^−/− (D and H) were measured with constant stirring in a lumiaggregometer. Each experiment was repeated at least 3 times, each with blood pooled from a group of 8 mice. RGS16 indicates regulator of G protein signaling 16.

Figure 4

Deletion of RGS16 protein causes enhanced P‐selectin expression in stimulated platelets. Platelets from Rgs16 ^+/+ or Rgs16 ^−/− mice platelets (10⁵ platelets/100 μL) were stimulated with (A) 80 μmol/L TRAP4 (P<0.00001; ANOVA) and (B) 0.05 U/mL thrombin (P<0.00001; ANOVA) for 3 minutes, fixed, and labeled with CD62P antibody. Samples were analyzed using a flow cytometer. Average mean fluorescence intensities are shown. Experiment was repeated at least 3 times, with blood pooled from a group of 8 mice each time. MFI indicates mean fluorescence intensity; RGS16, regulator of G protein signaling 16; TRAP4, protease‐activated receptor 4.

Figure 5

Deletion of RGS16 protein causes enhanced αIIbβ3 activation in stimulated platelets. Platelets from Rgs16 ^+/+ or Rgs16 ^−/− mice platelets (10⁵ platelets/100 μL) were stimulated with (A) 80 μmol/L TRAP4 (P<0.00001; ANOVA) and (B) 0.05 U/mL thrombin (P<0.00001; ANOVA) for 3 minutes, fixed, and labeled with JON/A antibody. Samples were analyzed using a flow cytometer. Average mean fluorescence intensities are shown. Experiment was repeated at least 3 times, with blood pooled from a group of 8 mice each time. MFI indicates mean fluorescence intensity; RGS16, regulator of G protein signaling 16; TRAP4, protease‐activated receptor 4.

Figure 6

Deletion of RGS16 protein causes enhanced phosphatidylserine expression in stimulated platelets. Platelets from Rgs16 ^+/+ or Rgs16 ^−/− mice platelets (10⁵ platelets/100 μL) were stimulated with (A) 80 μmol/L TRAP4 (P<0.00001; ANOVA) and (B) 0.05 U/mL thrombin (P<0.00001; ANOVA) for 3 minutes, fixed, and labeled with Annexin V antibody. Samples were analyzed using a flow cytometer. Average MFIs are shown. Experiment was repeated at least 3 times, with blood pooled from a group of 8 mice each time. MFI indicates mean fluorescence intensity; PS, phosphatidylserine; RGS16, regulator of G protein signaling 16; TRAP4, protease‐activated receptor 4.

Figure 7

Deletion of RGS16 protein alters physiological hemostasis and development of thrombosis. (A) Bleeding times were measured in Rgs16 ^+/+ (n=8) or Rgs16 ^−/− (n=8) mice following venisection. Each point represents the bleeding time of a single animal (P=0.0002; Mann–Whitney test). (B) Thrombosis was induced in Rgs16 ^+/+ (n=5) or Rgs16 ^−/− (n=5) mice using chemical injury (FeCl₃). Each point represents an occlusion time of a single animal (P=0.0079; Mann–Whitney test). ***P<0.0001, whereas **P<0.001. RGS16 indicates regulator of G protein signaling 16.