ELMO1 deficiency enhances platelet function by Akruti Patel, John Kostyak, Carol Dangelmaier, Rachit Badolia, Dheeraj Bhavanasi, Joseph E. Aslan, Salim Merali, Soochong Kim, Johannes A. Eble, Lawrence Goldfinger, and Satya Kunapuli BloodAdv Volume 3(4):575-587 February 26, 2019 © 2019 by The American Society of Hematology

Akruti Patel et al. Blood Adv 2019;3:575-587 © 2019 by The American Society of Hematology

ELMO1 associates with PIP3 in platelets. ELMO1 associates with PIP3in platelets. (A) Resting human platelets (1 × 109 cells/mL) were lysed with NP-40 lysis buffer and incubated with PI(3,4,5)P3 PIP beads or control beads overnight at 4°C. Pull-downs were analyzed by western blot and probed with ELMO1 or Btk antibody. Blots are representative of at least 3 independent experiments. (B) The proteins from resting washed human, WT littermate control, and ELMO1−/− murine platelets were precipitated and analyzed by western blot. The blots were probed for ELMO1 and β-actin as a loading control. Blots are representative of at least 3 independent experiments. MW, molecular weight marker. Akruti Patel et al. Blood Adv 2019;3:575-587 © 2019 by The American Society of Hematology

Enhanced aggregation and granular secretion following GPVI stimulation in ELMO1−/− platelets. Enhanced aggregation and granular secretion following GPVI stimulation in ELMO1−/−platelets. Representative aggregation (A) and dense granule secretion tracings (B) of washed platelets from ELMO1−/− or WT littermate controls activated with the indicated agonists for 4 minutes. Platelet aggregation and dense granule secretion was detected by lumiaggregometry under stirring conditions at 37°C. Quantification of extent of aggregation (C) and dense granule secretion (D) of at least 3 independent experiments from panels A and B, respectively. Data are presented as mean ± SE and were analyzed by Student t test (*P < .05). Flow cytometry analysis of P-selectin exposure (E) and activated integrin αIIbβ3 (F) of washed murine platelets from ELMO1−/− or WT littermate controls activated with the indicated concentrations of CRP for 10 minutes at 37°C. Data are presented as fold increase over basal ± SE from at least 3 independent experiments and were analyzed by Student t test (*P < .05). A.U, arbitrary units. Akruti Patel et al. Blood Adv 2019;3:575-587 © 2019 by The American Society of Hematology

Enhanced thromboxane generation in response to GPVI agonists in ELMO1−/− platelets. Enhanced thromboxane generation in response to GPVI agonists in ELMO1−/−platelets. (A) Washed murine platelets were activated with the indicated concentrations of CRP or collagen for 4 minutes, and the reaction was stopped via flash-freezing. Thromboxane B2 generation was determined using an ELISA kit as per the manufacturer’s instructions. Data are presented as mean ± SE of at least 3 independent experiments and were analyzed by Student t test (*P < .05). (B) Representative aggregation tracings and dense granule secretion of washed platelets from ELMO1−/− or WT littermate controls preincubated with the feedback inhibitors (FBI’s) 10 μM indomethacin, 10 μM MRS-2179, and 100 nM AR-C69931MX for 5 minutes and activated with indicated concentration of CRP for 3 minutes. (C) Quantitation of aggregation and ATP secretion showing the mean ± standard error of the mean and analyzed by Student t test (*P < .05). Akruti Patel et al. Blood Adv 2019;3:575-587 © 2019 by The American Society of Hematology

ELMO1 regulates integrin-mediated platelet spreading. ELMO1 regulates integrin-mediated platelet spreading. (A) Representative images of murine platelets incubated on fibrinogen-coated plates for the times indicated and fixed and stained with FITC-phalloidin. The images were obtained using confocal microscope. Scale bars, 5 μM. (B) Quantification of platelets with a filopodial phenotype expressed as a percentage of adhered platelets. At least 3 fields containing at least 25 platelets were analyzed per replicate (at least 2 replicates per experiment). Data were analyzed via Student t test (*P < .05). WT and ELMO1−/−, n = 4. Akruti Patel et al. Blood Adv 2019;3:575-587 © 2019 by The American Society of Hematology

ELMO1 regulates thrombus formation in vitro. ELMO1 regulates thrombus formation in vitro. Representative light microscopic image of thrombus formation on a collagen-coated surface (A) and percent thrombus area (B). Whole blood from WT littermate controls and ELMO1−/− mice was perfused over a collagen (50 μg/mL)–coated surface at arterial shear rate 1000 s−1 or venous shear rate 200 s−1 for 4 minutes. The direction of flow was right to left. (B) Images were analyzed using ImageJ, and data are presented as percent thrombus area ± SE. Data were analyzed by Student t test (*P < .05). WT and ELMO1−/−, n = 3. Akruti Patel et al. Blood Adv 2019;3:575-587 © 2019 by The American Society of Hematology

ELMO1 regulates thrombus formation and hemostasis in vivo. ELMO1 regulates thrombus formation and hemostasis in vivo. (A) Survival curves of WT and ELMO1−/− mice in a pulmonary thromboembolism model. Time to cessation of respiration was recorded after IV administration of 400 μg/kg collagen and 60 μg/kg epinephrine or phosphate-buffered saline. WT, n = 7; ELMO1−/−, n = 6. (B) Representative carotid artery blood flow tracings (i) and average time to occlusion (ii) from WT and ELMO1−/− mice. A carotid artery was isolated from the indicated mice and was exposed to 7.5% FeCl3 injury for 90 seconds. Data are presented as mean ± SE. WT, n = 12; ELMO1−/−, n = 14. Statistical analysis was performed using a Mann-Whitney U test (*P < .05). (C) Tail bleeding times of WT and ELMO1−/− mice. The distal 3 mm of the tail was cut, and the tail was immersed in 37°C saline. The time it took for bleeding to stop was recorded. WT, n = 38; ELMO1−/−, n = 18. Statistical analysis was performed using a Student t test (*P < .05). Akruti Patel et al. Blood Adv 2019;3:575-587 © 2019 by The American Society of Hematology

ELMO1 regulates RhoG activity in the GPVI pathway. ELMO1 regulates RhoG activity in the GPVI pathway. (A) Rac1 activation in response to CRP in the absence of ELMO1. Washed platelets were stimulated with 1.5 μg/mL CRP for the indicated times and lysed with NP-40 lysis buffer, and active Rac was pulled down using GST-PAK-RBD. Active Rac1 was detected via western blot analysis using a specific antibody to Rac1. (B-C) Time course of ELMO1−/− and WT platelets activated with 1.25 μg/mL CRP at the indicated time points under stirring conditions at 37°C. Proteins were precipitated and analyzed by western blot using indicated antibodies. (D) Surface expression of GPVI in washed murine platelets from WT and ELMO1−/− mice assessed by flow cytometry. GPVI surface expression is a representative of at least 3 independent experiments. (E) RhoG activity in ELMO1−/− and WT murine platelets in response to CRP. Washed murine platelets were activated with 10 μg/mL CRP for 1 minute and lysed with NP-40 lysis buffer. Active RhoG was pulled down using GST-ELMO1 fusion protein, and RhoG activity was detected by western blot. (F) Model of GPVI signaling by ELMO1 in platelets. All western blot images are representative of at least 3 independent experiments. Akruti Patel et al. Blood Adv 2019;3:575-587 © 2019 by The American Society of Hematology

ELMO1 deficiency is dispensable for CLEC-2–mediated platelet reactivity. ELMO1 deficiency is dispensable for CLEC-2–mediated platelet reactivity. Washed platelets isolated from WT, ELMO1−/−, and RhoG−/− mice were diluted to 1.5 × 108 and stimulated with either 5 nM (A) or 10 nM (C) rhodocytin, and subsequent aggregations and secretions were recorded. (B,D) Quantification of ATP secretion expressed as mean ± standard error of the mean following platelet stimulation with either 5 nM or 10 nM rhodocytin. Akruti Patel et al. Blood Adv 2019;3:575-587 © 2019 by The American Society of Hematology