TREM-like transcript 1: a more sensitive marker of platelet activation than P-selectin in humans and mice by Christopher W. Smith, Zaher Raslan, Lola Parfitt,

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TREM-like transcript 1: a more sensitive marker of platelet activation than P-selectin in humans and mice by Christopher W. Smith, Zaher Raslan, Lola Parfitt, Abdullah O. Khan, Pushpa Patel, Yotis A. Senis, and Alexandra Mazharian BloodAdv Volume 2(16):2072-2078 August 28, 2018 © 2018 by The American Society of Hematology

TLT-1 expression is greater than P-selectin expression on the surface of activated megakaryocytes and platelets in vitro. TLT-1 expression is greater than P-selectin expression on the surface of activated megakaryocytes and platelets in vitro. (A) Whole-cell lysates of primary BM-derived mouse megakaryocytes 0 to 5 days after thrombopoietin (Tpo) administration were western blotted for TLT-1, P-selectin, and ERK1/2. Representative blots of 2 independent experiments. (B) BM-derived megakaryocyte surface expression of TLT-1 and P-selectin following collagen stimulation (30 µg/mL) for 20 minutes at 37°C. Data are presented as mean fluorescence intensity (MFI) ± SD; n = 7-9. (C-D) Platelet surface expression of TLT-1 and P-selectin was measured by flow cytometry in response to the protease activated receptor agonist thrombin and the glycoprotein VI–specific agonist CRP in murine (C) and human (D) whole blood. (i) Dose-response (thrombin, 0.03-3 U/mL; CRP, 3-30 µg/mL) and (ii) time course (thrombin, 0.1 U/mL; CRP, 10 µg/mL). Data are presented as median fluorescence intensity ± SD; n = 3-6 independent experiments per condition. *P < .05, **P < .01, ***P < .001 vs basal. Christopher W. Smith et al. Blood Adv 2018;2:2072-2078 © 2018 by The American Society of Hematology

TLT-1 appears more rapidly and is detected throughout laser-induced thrombi in vivo. TLT-1 appears more rapidly and is detected throughout laser-induced thrombi in vivo. Mice were injected with anti-GPIbβ, anti-TLT-1, or anti-CD62P antibody. Arterioles of cremaster muscles were subsequently injured by laser. (A) Representative composite bright-field and fluorescence images of platelets (GPIbβ), TLT-1, and P-selectin in thrombi. Scale bars, 10 µm. (B) Quantification of fluorescence area in pixels (data represent mean ± standard error of the mean; n = 25-32 injuries from 4 to 6 mice). (C) Distinct and overlapping staining of TLT-1 and P-selectin in platelets and megakaryocytes. Resting human (top row) and mouse (middle row) platelets seeded on poly-l-lysine and primary mouse BM-derived megakaryocytes (MK) (bottom row) spread on fibrinogen matrix for 15 minutes at 37°C were fixed, permeabilized, and stained with Alexa Fluor 488 anti-TLT-1 and Alexa Fluor 647 anti-P-selectin antibodies. The right column represented an overlay of both images. Scale bars, 5 µm. Images are representative of 3 to 4 independent experiments. (D) Degree of TLT-1 and P-selectin colocalization as determined by the Manders overlap coefficients (M1: TLT-1:P-selectin and M2: P-selectin:TLT-1) in resting human (i) and mouse platelets (ii), and object-based colocalization in mouse BM-derived megakaryocytes (iii) (n = 3-4 independent experiments per condition, 400-600 platelets, and 25-35 megakaryocytes per experiment). Christopher W. Smith et al. Blood Adv 2018;2:2072-2078 © 2018 by The American Society of Hematology

TLT-1 appears more rapidly and is detected throughout laser-induced thrombi in vivo. TLT-1 appears more rapidly and is detected throughout laser-induced thrombi in vivo. Mice were injected with anti-GPIbβ, anti-TLT-1, or anti-CD62P antibody. Arterioles of cremaster muscles were subsequently injured by laser. (A) Representative composite bright-field and fluorescence images of platelets (GPIbβ), TLT-1, and P-selectin in thrombi. Scale bars, 10 µm. (B) Quantification of fluorescence area in pixels (data represent mean ± standard error of the mean; n = 25-32 injuries from 4 to 6 mice). (C) Distinct and overlapping staining of TLT-1 and P-selectin in platelets and megakaryocytes. Resting human (top row) and mouse (middle row) platelets seeded on poly-l-lysine and primary mouse BM-derived megakaryocytes (MK) (bottom row) spread on fibrinogen matrix for 15 minutes at 37°C were fixed, permeabilized, and stained with Alexa Fluor 488 anti-TLT-1 and Alexa Fluor 647 anti-P-selectin antibodies. The right column represented an overlay of both images. Scale bars, 5 µm. Images are representative of 3 to 4 independent experiments. (D) Degree of TLT-1 and P-selectin colocalization as determined by the Manders overlap coefficients (M1: TLT-1:P-selectin and M2: P-selectin:TLT-1) in resting human (i) and mouse platelets (ii), and object-based colocalization in mouse BM-derived megakaryocytes (iii) (n = 3-4 independent experiments per condition, 400-600 platelets, and 25-35 megakaryocytes per experiment). Christopher W. Smith et al. Blood Adv 2018;2:2072-2078 © 2018 by The American Society of Hematology