![Figure 2. Distinct secretion and surface expression of platelet proangiogenic and antiangiogenic factors. (A-D) Platelet-released SDF-1α (A), VEGF (B), PF4 (C), and endostatin (D) were determined in the supernatants of washed platelets (3 × 108 cells/mL) that had been treated with vehicle, PAR1-AP 10μM, PAR4-AP 100μM, ADP 10μM, ADP 10μM plus the P2Y12 antagonist AZD1283 10μM (AstraZeneca R&D Systems) and CRP 1 μg/mL, using the corresponding DuoSet ELISA kits from R&D Systems. Data are mean ± SEMs. *P < .05, compared with other treatments, except that P2Y1-elevated VEGF level (C) did not differ from PAR4 (P = .46). nd indicates not detectable; n = 3. (E-H) Platelet surface expression or binding of angiogenic factors and P-selectin were monitored by whole blood flow cytometry. Whole blood samples were incubated without or with increasing concentrations of PAR1-AP (E), PAR4-AP (F), ADP (G), or CRP (H) and in the presence of different antibodies (phycoerythrin-conjugated anti-P-selectin monoclonal antibody, BD Biosciences; SDF-1α monoclonal antibody; phycoerythrin-VEGF monoclonal antibody; phycoerythrin-PF4 monoclonal antibody, all from R&D Systems; and antiendostatin monoclonal antibody, Hycult Biotechnology) for 20 minutes at room temperature. The samples incubated with unconjugated antibodies were further labeled with a goat anti–mouse IgG1 fluorescein isothiocyanate-conjugated antibody (AbD Serotec). After washing with 1% bovine serum albumin–PBS and fixed with 0.5% formaldehyde saline, the samples were analyzed using a Beckman Coulter XL-MCL or FC500 flow cytometer. Data plotted are means from 6-9 subjects. (I-K) Signaling mechanisms underlying distinct angiogenic factor release. SDF-1α (I) and endostatin (J) release from washed platelets was induced by 10μM PAR1-AP or 100μM PAR4-AP in the presence of vehicle (0.1% dimethyl sulfoxide in PBS) or signaling inhibitors (−): the tyrosine kinase Src inhibitor < Src (−) > PP2 (final concentration 5μM; Calbiochem), the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (25μM; Cell Signaling Technology), the serine/threonine protein kinase Akt inhibitor SH-6 (30μM; Calbiochem), the PKC inhibitor ro-31-8220 (10μM; Sigma-Aldrich), the mitogen-activated protein kinase kinase inhibitor U0126 (10μM; Cell Signaling Technology), and the p38 mitogen-activated protein kinase inhibitor SB203580 (10μM; Calbiochem). SDF-1α and endostatin levels in the supernatant were determined using corresponding DuoSet ELISA kits. Data are presented as percentage of the control: Control % = ([SDF-1α]PAR+(−) − [SDF-1α]resting)/([SDF-1α]PAR+vehicle − [SDF-1α]resting) × 100%. In parallel with the platelet release assay, platelet surface expression of P-selectin was monitored by flow cytometry. These data are also presented as the percentage of control (K). *P < .05 compared with control (vehicle); n = 5.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/117/14/10.1182_blood-2010-12-327007/4/zh89991169410002.jpeg?Expires=1735834519&Signature=hckGyJhgYUrtiStSd0KLOmGG6hN8Z~dRKsqHTC2RaVEswQ3lxKwNf5Z3ed01fln8Ri3Cbuv5ly-SYb8Vufa~BrjAuT~itQxl3~uOdvyxFQoryyEYaFQRMsDXHpdOxMLaUZLGYuBo8d69ByJ6ouEaEaPuO4oDNuEKGTn28B6Y8WHlU5P9D8v-Rp3qpTHdWk4JnKR~LHG2SW7AkEU-LKvMKtabbVTgHttgEqwn4IobBjXh4e42IJZLLjeP9VcMqme8UdgvbtDvg16bQ0N1T8HEu-E4cY3J4x4vsXaWUq7AotKEnM6dme8gp2PJWotO4YmkjDS1aaBXKGwB9p9ylpfaCA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Distinct secretion and surface expression of platelet proangiogenic and antiangiogenic factors. (A-D) Platelet-released SDF-1α (A), VEGF (B), PF4 (C), and endostatin (D) were determined in the supernatants of washed platelets (3 × 108 cells/mL) that had been treated with vehicle, PAR1-AP 10μM, PAR4-AP 100μM, ADP 10μM, ADP 10μM plus the P2Y12 antagonist AZD1283 10μM (AstraZeneca R&D Systems) and CRP 1 μg/mL, using the corresponding DuoSet ELISA kits from R&D Systems. Data are mean ± SEMs. *P < .05, compared with other treatments, except that P2Y1-elevated VEGF level (C) did not differ from PAR4 (P = .46). nd indicates not detectable; n = 3. (E-H) Platelet surface expression or binding of angiogenic factors and P-selectin were monitored by whole blood flow cytometry. Whole blood samples were incubated without or with increasing concentrations of PAR1-AP (E), PAR4-AP (F), ADP (G), or CRP (H) and in the presence of different antibodies (phycoerythrin-conjugated anti-P-selectin monoclonal antibody, BD Biosciences; SDF-1α monoclonal antibody; phycoerythrin-VEGF monoclonal antibody; phycoerythrin-PF4 monoclonal antibody, all from R&D Systems; and antiendostatin monoclonal antibody, Hycult Biotechnology) for 20 minutes at room temperature. The samples incubated with unconjugated antibodies were further labeled with a goat anti–mouse IgG1 fluorescein isothiocyanate-conjugated antibody (AbD Serotec). After washing with 1% bovine serum albumin–PBS and fixed with 0.5% formaldehyde saline, the samples were analyzed using a Beckman Coulter XL-MCL or FC500 flow cytometer. Data plotted are means from 6-9 subjects. (I-K) Signaling mechanisms underlying distinct angiogenic factor release. SDF-1α (I) and endostatin (J) release from washed platelets was induced by 10μM PAR1-AP or 100μM PAR4-AP in the presence of vehicle (0.1% dimethyl sulfoxide in PBS) or signaling inhibitors (−): the tyrosine kinase Src inhibitor < Src (−) > PP2 (final concentration 5μM; Calbiochem), the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (25μM; Cell Signaling Technology), the serine/threonine protein kinase Akt inhibitor SH-6 (30μM; Calbiochem), the PKC inhibitor ro-31-8220 (10μM; Sigma-Aldrich), the mitogen-activated protein kinase kinase inhibitor U0126 (10μM; Cell Signaling Technology), and the p38 mitogen-activated protein kinase inhibitor SB203580 (10μM; Calbiochem). SDF-1α and endostatin levels in the supernatant were determined using corresponding DuoSet ELISA kits. Data are presented as percentage of the control: Control % = ([SDF-1α]PAR+(−) − [SDF-1α]resting)/([SDF-1α]PAR+vehicle − [SDF-1α]resting) × 100%. In parallel with the platelet release assay, platelet surface expression of P-selectin was monitored by flow cytometry. These data are also presented as the percentage of control (K). *P < .05 compared with control (vehicle); n = 5.
