ORIGINAL RESEARCH Arhgef1 Plays a Vital Role in Platelet Function and Thrombogenesis Hanan Qasim, BS;* Zubair A. Karim, PhD;* Keziah R. Hernandez, MPA; Dante Lozano; Fadi T. Khasawneh, PhD; Fatima Z. Alshbool, PharmD, PhD Background-—Platelets are the cellular mediators of hemostasis and thrombosis, and their function is regulated by a number of molecular mediators, such as small GTPases. These small GTPases 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, Arhgef1À/À) and investigated their platelet phenotype by employing a host of in vivo and in vitro platelet assays. Our results indicate that Arhgef1À/À mice had prolonged carotid artery occlusion and tail bleeding times. Moreover, platelets from these mice exhibited defective aggregation, dense and a granule secretion, aIIbb3 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. ( J Am Heart Assoc. 2019;8:e011712. DOI: 10.1161/JAHA.118.011712.) Key Words: Arhgef1 • cardiovascular diseases • platelets • small GTPases • thrombosis latelets are the cellular mediators of hemostasis and nucleotide–binding proteins. These proteins work as P thrombosis.1–3 Under normal conditions and upon injury intracellular “on-off” signaling switches; coordinating signal to a blood vessel, platelets undergo drastic shape change, transduction and amplification.17,18 Several subfamilies of bind to adhesive protein substrates, and aggregate to form a small GTPases have been identified in platelets19,20 and are plug, ultimately halting bleeding.3–5 Unfortunately, hyperac- cardinal regulators of the actin cytoskeleton dynamics, tivity of platelets serves as a precursor to thrombotic diseases affecting platelet aggregation, secretion, spreading, and such as pulmonary embolism and myocardial infarction.6,7 thrombus formation.4,21 They govern the diverse cellular Platelet activation is regulated by a host of complex processes through cycling between an inactive GDP-bound molecular events that modulate adhesion, aggregation, and formandanactiveGTP-boundformthroughaprocessof thrombus formation, and include phosphorylation,8–10 as well GDP/GTP replacement.22,23 Importantly, the GDP/GTP as calcium-based11 and lipid-based signals.12,13 These events cycling is highly regulated by a group of proteins/protein are coordinated by a number of molecular mediators such domains known as guanine nucleotide exchange factors as small GTPases,14–16 which represent a family of (GEFs) such as Arhgefs.23–25 low-molecular-weight (21–25 kDa) monomeric guanine While the Rho family of GEFs––a few of which have been identified in platelets26––has been thoroughly studied in other cell systems,27–29 their role in platelets has not yet been fully From the Department of Pharmaceutical Sciences, School of Pharmacy, The understood. To this end, it has been reported that: (1) Arhgef3 University of Texas El Paso, El Paso, TX (H.Q., Z.A.K., K.R.H., F.T.K., F.Z.A.); El is not very important for platelet function, at least in mice30; Paso Community College, El Paso, TX (D.L.). (2) Arhgef10 regulates platelet activation in vitro (eg, aggre- *Ms Qasim and Dr Karim contributed equally to this work. gation) and in vivo (eg, hemostasis)31; and (3) Arhegf12 is Correspondence to: Fatima Z. Alshbool, PharmD, PhD, Pharmaceutical “ ” 32 Sciences, School of Pharmacy, The University of Texas at El Paso, TX 79968. essential for normal platelet function. While Arhgef1 is E-mail: [email protected] another member of this family that is highly expressed in 26 Received December 6, 2018; accepted March 25, 2019. platelets, virtually nothing is known regarding its direct role ª 2019 The Authors. Published on behalf of the American Heart Association, in platelet function. Given that RhoA is a crucial regulator of Inc., by Wiley. This is an open access article under the terms of the Creative platelet responses mainly activated downstream of G12/13 Commons Attribution-NonCommercial License, which permits use, distribu- 33 tion and reproduction in any medium, provided the original work is properly and Gq coupled receptors, that these G-proteins are known 34,35 cited and is not used for commercial purposes. to play a vital role in platelet function, and that Arhgef1 DOI: 10.1161/JAHA.118.011712 Journal of the American Heart Association 1 Arhgef1 Regulates Platelet Function Qasim et al ORIGINAL RESEARCH Methods Clinical Perspective All experiments involving animals were performed in compli- What Is New? ance with the institutional guidelines and were approved by The University of Texas at El Paso Institutional Animal Care • This is the first study to demonstrate that Arhgef1 regulates platelet function, as well as hemostasis and thrombogen- and Use Committee. esis, in mice. The authors declare that all supporting data are available within the article data. Requests for detailed analytic What Are the Clinical Implications? methods will be addressed by the corresponding author. For requests for the Arhgef1 deletion mice, these will be • Arhgef1 represents a potential therapeutic target for referred by the corresponding author to the Mary Lyon managing thrombotic disorders. Centre at MRC Harwell (www.har.mrc.ac.uk), Oxfordshire, United Kingdom, and handled as per our Material Transfer 36 activates G13, we sought to examine the role of Arhgef1 in Agreement. platelet function in vitro and in vivo. Immunoblotting Materials and Methods Immunoblotting was performed as previously described.37,38 Briefly, and to confirm Arhgef1 deletion and that its deletion Reagents and Materials did not impact the expression levels of b3 and G13, platelet Thrombin, collagen, stir bars, and other disposables were from proteins were separated by SDS-PAGE and transferred to Chrono-Log Corporation, whereas U46619 was purchased Immobilon-P PVDF membranes (Millipore Corp). They were from Abcam. Fluorescein isothiocyanate–conjugated anti–P- then probed with anti-Arhgef1, anti-b3, anti-G13, or anti-actin selectin, anti-Arhgef1, anti-actin, anti-RhoA, anti-pROCK, and primary antibodies and visualized with an appropriate alkaline anti-Rap1b antibodies were purchased from Cell Signaling phosphatase–coupled secondary antibody using enhanced fl – Technology, Inc, whereas the anti-b3 and anti-G13 antibodies chemi uorescent substrate (GE Healthcare). For the RhoA were from Sigma Aldrich. The JON/A antibody was from Rho-associated kinase (ROCK) axis, washed platelets (Arhge- À À Emfret analytics. Fibrinogen was purchased from Sigma f1 / and WT) were stimulated with thrombin (0.1 U/mL) for Aldrich. The RhoA and Rap1b Activation Assay Biochem Kits 3 minutes, and processed as described above. Membranes were from Cytoskeleton Inc. Other reagents were of analytical were then probed with anti-RhoA, anti-pROCKIISer1366, and grade. anti-actin antibodies, and visualized with an appropriate horseradish peroxidase–coupled secondary antibody using enhanced chemiluminescence substrate (Thermo Scientific). Animals and Genotyping Images were obtained with ChemiDoc MP Imaging System (Bio-Rad). Arhgef1À/À mice were obtained from the Mary Lyon Centre at MRC Harwell (www.har.mrc.ac.uk), Oxfordshire, United Kingdom, and genotyped using a polymerase chain reac- tion (PCR)–based method. PCR was performed using the Tail Bleeding Time Assay following primers: Arhgef1-F (CCTTTGGGCTCTGACCTCTG), The tail bleeding time assay was performed as previously Arhgef1-R (AAGCCGTCAGTCCTCTGTCC), and CAS1 (TCGTGG- described.39–41 Briefly, mice were anesthetized and placed TATCGTTATGCGCC) with the following PCR condition: denat- on a homeothermic blanket (37°C) before the tail was uration: 94°C for 7 minutes, amplification: 94°C for 1 minute, transected 5 mm from the tip using a sterile scalpel. After 58°C for 1 minute, 72°C for 90 seconds for 34 cycles, and transection, the tail was immediately immersed in saline finally extension at 72°C for 10 minutes. After completion of (37°C constant temperature), and the time to bleeding the PCR cycle, amplified DNA samples were run in 2% agarose cessation was measured. Bleeding stoppage was not con- gel and visualized in the gel documentation system. The sidered complete until bleeding had stopped for 1 minute. expected products per genotype are as follows: wild-type For statistical analysis purposes, if bleeding did not stop in (WT): 520 bp, Het: 362/520 bp, and mutant: 362 bp. 10 minutes, the experiment was halted to limit blood loss Mice were housed in groups of 1 to 5 at 24°C, under 12/ from the mice and 10 minutes was considered the cutoff 12 light/dark cycles, with access to water and food bleeding time. The number of WT and knockout (KO) mice ad libitum. used was 8 each. DOI: 10.1161/JAHA.118.011712 Journal of the American Heart Association 2 Arhgef1 Regulates Platelet Function Qasim et al ORIGINAL RESEARCH In Vivo Ferric Chloride Carotid Artery using the HEMAVET 950FS Multispecies Hematology System Injury–Induced Thrombosis Model and adjusted to the indicated concentrations. This assay