Figure 1.
Figure 1. ADAMTS13 activity in ECs. (A) Confocal image of HUVECs stained with anti-VWF pAb (green, column 1), anti-ADAMTS13 mAb (red, column 2), merged image with 4′,6-diamidino-2-phenylindole (column 3) and green + red colocalization signal (column 4). Top row shows HUVECs stained with both Abs. Second and third rows represent controls with either isotype control anti-l-selectin mAb DREG56 (in place of anti-ADAMTS13, middle) or anti-myosin smooth muscle pAb (in place of anti-VWF, bottom). (B) 2 mg/mL HUVEC or HEK293T cell lysates prepared using detergent lysis was added to 1 μM XS-VWF FRET substrate. Final Triton-X concentration (vehicle control) was 0.2% in all samples. FRET ratio (indicating XS-VWF FRET cleavage) was measured over time. *P < .05 with respect to all other treatments. (C) Western blot showing XS-VWF FRET cleavage at 24 hours in the presence of HUVEC lysate, but not HEK293T lysate or vehicle control. (D) Western blot of crude HUVEC lysates prepared using 3 methods in the presence of 10 mM EDTA: heat denaturation/SDS-boil, detergent lysis/triton, and freeze-thaw. Four bands are noted: band 1 mass corresponds to mature VWF and its glycoforms (230-250 kDa), band 2 (∼200 kDa) origin is unknown, and bands 3 and 4 masses correspond to C-terminal (176 kDa) and N-terminal (140 kDa) cleavage fragments. No bands were present below 140 kDa (data not shown). (E) VWF immunoprecipitated from HUVEC lysates, prepared using different methods, using either anti VWF-A2 mAb 210909 (left gel) or anti-D′D3 mAb DD3.1 (right gel). The same 4 bands are observed. (F) Western blot of HUVEC supernatants collected 2 hours after stimulation with 25 µM histamine, 50 ng/mL TNF-α, 100 ng/mL IL-8, or 10 µM calcium ionophore (left gel) show all 4 bands. Overnight addition of 2 U/mL ADAMTS13 and 1.6M urea in right gel, results in amplification of 176 kDa band. (G) VWF from samples in panel C (left gel, no ADAMTS13) was immunoprecipitated using anti-D′D3 mAb DD3.1 before western blotting. VWF cleavage bands at 176 and 140 kDa are present in all panels. The bottom portion of the panel G gel, containing the 140-kDa fragment, was developed for a longer time. In all cases, % cleavage = cleaved VWF/total VWF = 100× intensity of 176 KDa band/(sum of intensity of 176 KDa + 250 KDa bands). SDS-polyacrylamide gel electrophoresis was performed under reducing conditions and all blots were probed with anti-VWF pAb (Dako). Functionally active ADAMTS13 activity and perinuclear colocalization of VWF and ADAMTS13 was observed in HUVECs.

ADAMTS13 activity in ECs. (A) Confocal image of HUVECs stained with anti-VWF pAb (green, column 1), anti-ADAMTS13 mAb (red, column 2), merged image with 4′,6-diamidino-2-phenylindole (column 3) and green + red colocalization signal (column 4). Top row shows HUVECs stained with both Abs. Second and third rows represent controls with either isotype control anti-l-selectin mAb DREG56 (in place of anti-ADAMTS13, middle) or anti-myosin smooth muscle pAb (in place of anti-VWF, bottom). (B) 2 mg/mL HUVEC or HEK293T cell lysates prepared using detergent lysis was added to 1 μM XS-VWF FRET substrate. Final Triton-X concentration (vehicle control) was 0.2% in all samples. FRET ratio (indicating XS-VWF FRET cleavage) was measured over time. *P < .05 with respect to all other treatments. (C) Western blot showing XS-VWF FRET cleavage at 24 hours in the presence of HUVEC lysate, but not HEK293T lysate or vehicle control. (D) Western blot of crude HUVEC lysates prepared using 3 methods in the presence of 10 mM EDTA: heat denaturation/SDS-boil, detergent lysis/triton, and freeze-thaw. Four bands are noted: band 1 mass corresponds to mature VWF and its glycoforms (230-250 kDa), band 2 (∼200 kDa) origin is unknown, and bands 3 and 4 masses correspond to C-terminal (176 kDa) and N-terminal (140 kDa) cleavage fragments. No bands were present below 140 kDa (data not shown). (E) VWF immunoprecipitated from HUVEC lysates, prepared using different methods, using either anti VWF-A2 mAb 210909 (left gel) or anti-D′D3 mAb DD3.1 (right gel). The same 4 bands are observed. (F) Western blot of HUVEC supernatants collected 2 hours after stimulation with 25 µM histamine, 50 ng/mL TNF-α, 100 ng/mL IL-8, or 10 µM calcium ionophore (left gel) show all 4 bands. Overnight addition of 2 U/mL ADAMTS13 and 1.6M urea in right gel, results in amplification of 176 kDa band. (G) VWF from samples in panel C (left gel, no ADAMTS13) was immunoprecipitated using anti-D′D3 mAb DD3.1 before western blotting. VWF cleavage bands at 176 and 140 kDa are present in all panels. The bottom portion of the panel G gel, containing the 140-kDa fragment, was developed for a longer time. In all cases, % cleavage = cleaved VWF/total VWF = 100× intensity of 176 KDa band/(sum of intensity of 176 KDa + 250 KDa bands). SDS-polyacrylamide gel electrophoresis was performed under reducing conditions and all blots were probed with anti-VWF pAb (Dako). Functionally active ADAMTS13 activity and perinuclear colocalization of VWF and ADAMTS13 was observed in HUVECs.

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