American Society of Hematology

Figure 1

$Figure 1. Characterization of platelet PN-1. (A) RT-PCR analysis of PN-1 mRNA expression in platelets. (B) Washed platelets were activated by different agonists: 5nM thrombin, 50μM PAR1-AP, 5 μg/mL collagen, 1nM convulxin, and 10μM ADP, for 30 minutes at 37°C, and centrifuged to isolate platelet pellets from supernatants. Platelet pellets (cell extracts) and supernatants (secretion products) were analyzed by Western blot for PN-1 and GAPDH. (C) Washed platelets were pretreated or not with 1 μg/mL prostaglandin E1 before activation by thrombin. Cell extracts were analyzed by Western blot for PN-1 and GPIb. (D) Washed platelets activated by 50μM PAR1-AP were analyzed by flow cytometry with an FITC-coupled anti–PN-1 IgG. (E) Membrane and cytosol fractions of resting platelets analyzed by Western blot for PN-1 and GPIb. (F) Washed platelets treated with 150 μg/mL heparin were analyzed by flow cytometry. The supernatants of heparin-treated platelet were analyzed by Western blot for PN-1 (inset). Data are representative of 3 separate experiments from different donors.$

Characterization of platelet PN-1. (A) RT-PCR analysis of PN-1 mRNA expression in platelets. (B) Washed platelets were activated by different agonists: 5nM thrombin, 50μM PAR1-AP, 5 μg/mL collagen, 1nM convulxin, and 10μM ADP, for 30 minutes at 37°C, and centrifuged to isolate platelet pellets from supernatants. Platelet pellets (cell extracts) and supernatants (secretion products) were analyzed by Western blot for PN-1 and GAPDH. (C) Washed platelets were pretreated or not with 1 μg/mL prostaglandin E₁ before activation by thrombin. Cell extracts were analyzed by Western blot for PN-1 and GPIb. (D) Washed platelets activated by 50μM PAR1-AP were analyzed by flow cytometry with an FITC-coupled anti–PN-1 IgG. (E) Membrane and cytosol fractions of resting platelets analyzed by Western blot for PN-1 and GPIb. (F) Washed platelets treated with 150 μg/mL heparin were analyzed by flow cytometry. The supernatants of heparin-treated platelet were analyzed by Western blot for PN-1 (inset). Data are representative of 3 separate experiments from different donors.

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