Arhgef1 Plays a Vital Role in Platelet Function and Thrombogenesis

Abstract

Background Platelets are the cellular mediators of hemostasis and thrombosis, and their function is regulated by a number of molecular mediators, such as small GTP ases. These small GTP ases are themselves regulated by guanine nucleotide exchange factors such as Arhgefs, several of which are found in platelets, including the highly expressed Arhgef1. However, the role of Arhgef1 in platelets has not yet been investigated. Methods and Results We employed mice with genetic deletion of Arhgef1 (ie, Arhgef1^-/-) and investigated their platelet phenotype by employing a host of in vivo and in vitro platelet assays. Our results indicate that Arhgef1^-/- mice had prolonged carotid artery occlusion and tail bleeding times. Moreover, platelets from these mice exhibited defective aggregation, dense and α granule secretion, α II bβ3 integrin activation, clot retraction and spreading, in comparison to their wild-type littermates. Finally, we also found that the mechanism by which Arhgef1 regulates platelets is mediated in part by a defect in the activation of the RhoA-Rho-associated kinase axis, but not Rap1b. Conclusions Our data demonstrate, for the first time, that Arhgef1 plays a critical role in platelet function, in vitro and in vivo.

Keywords: Arhgef1; cardiovascular diseases; platelets; small GTPases; thrombosis.

Figure 1

Confirmation of Arhgef1 deletion in mice. A, DNA from Arhgef1 KO and WT mice was isolated and polymerase chain reaction was performed as described in the Methods section. DNA was separated on a 2% agarose gel and visualized using a gel documentation system. B, Arhgef1 protein levels in wild‐type (WT) and Arhgef1^−/− mice as determined by Western blotting using platelet extracts (2×10⁸/mL). C, β3 is the subunit of the αIIbβ3 integrin, whereas G₁₃ is the α subunit of the GTPase‐binding protein G₁₃.

Figure 2

Arhgef1 deletion prolongs thrombus occlusion and bleeding times in mice. (A) Thrombosis was induced in Arhgef1^−/− (n=8) and wild‐type (WT; n=8) mice using ferric chloride, as described in the Methods section. Each point represents the occlusion time of a single animal (***P<0.001). B, Bleeding times were measured in Arhgef1^−/− (n=8) or WT (n=8) mice as described in the Methods section. Each point represents the bleeding time of a single animal (***P<0.001).

Figure 3

Arhgef1 deletion reduces platelets aggregation and dense granule secretion. A through D, Platelets from Arhgef1^−/− and wild‐type (WT) mice were stimulated with thrombin (A and B), U46619 (C), or collagen (D) before their aggregation response was monitored using an aggregometer (inset shows quantification of maximal aggregation). E through H, Platelets from Arhgef1^−/− and WT mice were incubated with luciferase luciferin (12.5 μL) before being stimulated with thrombin (E and F), U46619 (G), or collagen (H). ATP release was detected as luminescence and measured by a lumiaggregometer. Each experiment was repeated at least 3 times, with blood pooled from a group of 8 mice each time (*P<0.05; **P<0.01).

Figure 4

Arhgef1 deletion reduces α‐granule secretion and integrin αIIbβ3 activation. Platelets from Arhgef1^−/− and wild‐type (WT) mice were washed before stimulation with thrombin, U46619, or collagen, and incubation with (A through C) fluorescein isothiocyanate–conjugated CD62P antibody (for α granules), or (D through F) fluorescein isothiocyanate–conjugated JON/A antibody, respectively. The fluorescent intensities were measured by flow cytometry. Average mean fluorescence intensities are shown. Each experiment was repeated at least 3 times, with blood pooled from a group of 8 mice each time.

Figure 5

Arhgef1 deletion impairs clot retraction and platelet spreading. A, Whole blood was collected from both Arhgef1^−/− and wild‐type (WT) mice and washed platelets were isolated as discussed in the Methods section. Clot retraction was initiated by adding thrombin (0.1 U/mL) and photographed every 5 minutes for 30 minutes. B, Arhgef1^−/− and WT platelets were allowed to adhere to fibrinogen‐coated coverslips for 5 minutes after stimulation with thrombin (0.1 U/mL). Tetramethylrhodamine‐conjugated phalloidin in 10% fetal bovine serum/PBS with 0.2% saponin was used to stain for F‐actin and imaged using Leica DMi8 inverted widefield fluorescence microscope with integrated high precision focus drive. Images were processed using LAS X Wizard imaging software. Data are representative of 3 independent experiments. Each experiment was repeated at least 3 times, with blood pooled from a group of 8 mice each time.

Figure 6

Arhgef1 deletion inhibits RhoA activation and phosphorylation of Rho‐associated kinase (ROCK) in platelets. A, Arhgef1^−/− and wild‐type (WT) washed platelets (4×10⁸/mL) were stimulated with thrombin (0.1 U/mL) for 0, 30, 60, 180, and 300 seconds before the RhoA‐GTP pulldown assay was performed. RhoA‐GTP and total RhoA were detected by Western blot using anti‐RhoA antibody. B, Platelets (4×10⁸/mL) from Arhgef1^−/− and WT mice were prepared and stimulated with thrombin (0.1 U/mL) for 3 minutes and proteins were lysed using 5× sample buffer. Proteins were separated by SDS‐PAGE and immunoblotted using antibodies to pROCKIISer1366 and actin. C, Arhgef1^−/− and WT washed platelets (4×10⁸/mL) were stimulated with thrombin (0.1 U/mL) for 0, 30, 60, 180, and 300 seconds before the Rap1b‐GTP pulldown assay was performed. Rap1b‐GTP and total Rap1b was detected by Western blot using anti‐Rap1b antibody. Western blot data represent 3 individual experiments, with blood pooled from a group of 8 mice each time.