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Blood, Vol. 113, Issue 24, 6051-6060, June 11, 2009
C-terminal ADAMTS-18 fragment induces oxidative platelet fragmentation, dissolves platelet aggregates, and protects against carotid artery occlusion and cerebral stroke
Blood Li et al.
113: 6051
Supplemental materials for: Li et al
Files in this Data Supplement:
- Figure S1. Activation of gel-filtered platelets with anti–GPIIIa49-66 Ab (JPG, 64.7 KB)
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Measurement of peak P-selectin release at 20 min with FITC Ab by flow cytometry. Ctl refers to buffer control. A20, A10, and A5 refer to uM ADP. CIgG refers to control IgG. PtlgG refers to patient IgG, n=4.
- Figure S2. Effect of PGE1 and dibutyrl cyclic AMP on patient anti–GPIIIa49-66 Ab-induced platelet oxidation and fragmentation (JPG, 72.3 KB)
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(A) Fragmentation in presence of PGE1. 1 × 107 gel filtered platelets, labelled with anti–GPIIIa49-FITC were suspended in 100 ul of filtration buffer with addition of control or patient IgG (25 ug/ml) or thrombin (0.5 u/ml) in the presence and absence of 1 uM PGE1 for 4 hrs at 37°C. Platelets were then analyzed by flow cytometry, n=6. (B) Oxidation in the presence of PGE1. FITC-labelled platelets were loaded with the oxidative fluor, DCFH (10 uM) prior to incubation as in (A), n=5. (C) Fragmentation in presence of dibutyrl cyclic AMP (CMP). Similar experiment as in (A) except for the use of 10 uM dibutyrl cyclic AMP, rather than PGE1, n=3. (D) Oxidation in presence of dibutyrl cyclic AMP. Similar experiment as in (B) in which platelets were loaded with DCFH, n=3.
- Figure S3. Effect of patient Ab on oxidative fragmentation of Gαq −∕− platelets (JPG, 55.9 KB)
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(A and B) Fragmentation and oxidation were measured as in Figure S2, with Gαq −∕− platelets, n=5.
- Figure S4. Purification of ADAMTS-18 C-terminal fragments (JPG, 45 KB)
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SDS-PAGE of purified ADAMTS-18 C-terminal fragments stained with Coomassie Blue. M refers to the M.W. markers. T refers to unpurified bacterial extract. Number above the gel refers to the number of amino acids in the fragments.
- Figure S5. RT-PCR of ADAMTS-18 in HUVEC, ± 0.5 u/ml thrombin for 1 hr at 37°C (JPG, 43.7 KB)
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- Figure S6. Thrombin enhances the secretion and activation of ADAMTS-18 from BMEC (JPG, 83.9 KB)
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HBMEC and HUVEC were incubated with 0.5 u/ml thrombin for 30 min, washed, and then incubated for 2–4 hrs as in Figure 4 prior to testing for platelet particle formation in the presence and absence of oxidative inhibitors, diphenylene iodonium (DPI), and catalase (Cat). CTL refers to control buffer; HUVEC and BMEC refer to effect of conditioned media from these cells on platelet particle formation. DPI 10, 20, and 180 refer to nM DPI incubated platelets plus conditioned media. Cat 500 and 4,000 refer to catalase units/ml incubated with platelets plus media.
- Figure S7. TNFα stimulates the release of ADAMTS-18 from HUVEC (JPG, 53.2 KB)
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Cultured HUVEC were treated with TNFα for 30 min, washed, and then incubated for 2–4 hrs, as in Figure 4. The supernatant was assayed for ADAMTS-18 by ELISA (A) and function (B), n=4.
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