Figure 4
Figure 4. Thrombin activation and secretion of ADAMTS-18 in vitro and in vivo. (A) Kinetics of thrombin activation of concentrated HUVEC-conditioned media. HUVEC- conditioned media were treated with thrombin (0.5 units/mL) for various time intervals, followed by neutralization with hirudin. Aliquots were then used to induce platelet fragmentation. Incubation of thrombin and hirudin for 240 minutes prior to incubation had no effect on fragmentation (n = 3). (B) Comparison of anti–GPIIIa49-66 Ab with thrombin-activated HUVEC ADAMTS-18. Thrombin-treated media were neutralized with hirudin prior to incubation with gel-filtered platelets. Hirudin alone had no effect. Note similar kinetic response. (C) In vitro secretion and activation. HUVECs were treated with 0.5 units/mL thrombin for 15 to 30 minutes followed by washing and further incubation for 2 and 4 hours. The supernatant was assayed for ADAMTS-18 by ELISA. (D) Supernatant assay for platelet fragmentation (n = 3-4). (E,F) In vivo secretion and activation. (E) Mice were injected intravenously with various concentrations of thrombin in 100-μL volume and their plasma tested at 1 hour for ADAMTS-18 by ELISA. (F) Plasma from thrombin-stimulated mice was incubated with gel-filtered platelets and platelet fragmentation monitored as described in “Methods.” T-Plasma (plasma from thrombin-stimulated animals at various thrombin concentrations); T-Plasma + Hir (treatment with hirudin to neutralize any residual thrombin); + ADIgG (+ anti-ADAMTS IgG Ab) for 15-minute preincubation prior to platelet incubation with T-Plasma + hirudin; + Ctl IgG (control IgG for + ADIgG). (G) Inhibition of HUVEC-ADAMTS-18 induced platelet fragmentation with anti–ADAMTS-18 Ab or 18-mer peptide. (i) Anti–GPIIIa49-66 Ab was added to gel-filtered platelets in the presence and absence of the 18-mer peptide. (ii) HUVEC-ADAMTS-18 was incubated with control or anti-ADAMTS Ab or 18-mer peptide for 60 minutes at 37°C, prior to incubation with gel-filtered platelets for 4 hours at 37°C (n = 3). (i) Positive control with Buff (buffer), CtIgG (control IgG), PtIgG (patient IgG), 18m (18-mer peptide), or irrel (irrelevant peptide). (ii) AD (HUVEC conditioned media), CtIgG (control IgG), + ADAb (+anti–ADAMTS-18 Ab), 18m, or irrel (n = 6). Where shown, error bars indicate SEM.

Thrombin activation and secretion of ADAMTS-18 in vitro and in vivo. (A) Kinetics of thrombin activation of concentrated HUVEC-conditioned media. HUVEC- conditioned media were treated with thrombin (0.5 units/mL) for various time intervals, followed by neutralization with hirudin. Aliquots were then used to induce platelet fragmentation. Incubation of thrombin and hirudin for 240 minutes prior to incubation had no effect on fragmentation (n = 3). (B) Comparison of anti–GPIIIa49-66 Ab with thrombin-activated HUVEC ADAMTS-18. Thrombin-treated media were neutralized with hirudin prior to incubation with gel-filtered platelets. Hirudin alone had no effect. Note similar kinetic response. (C) In vitro secretion and activation. HUVECs were treated with 0.5 units/mL thrombin for 15 to 30 minutes followed by washing and further incubation for 2 and 4 hours. The supernatant was assayed for ADAMTS-18 by ELISA. (D) Supernatant assay for platelet fragmentation (n = 3-4). (E,F) In vivo secretion and activation. (E) Mice were injected intravenously with various concentrations of thrombin in 100-μL volume and their plasma tested at 1 hour for ADAMTS-18 by ELISA. (F) Plasma from thrombin-stimulated mice was incubated with gel-filtered platelets and platelet fragmentation monitored as described in “Methods.” T-Plasma (plasma from thrombin-stimulated animals at various thrombin concentrations); T-Plasma + Hir (treatment with hirudin to neutralize any residual thrombin); + ADIgG (+ anti-ADAMTS IgG Ab) for 15-minute preincubation prior to platelet incubation with T-Plasma + hirudin; + Ctl IgG (control IgG for + ADIgG). (G) Inhibition of HUVEC-ADAMTS-18 induced platelet fragmentation with anti–ADAMTS-18 Ab or 18-mer peptide. (i) Anti–GPIIIa49-66 Ab was added to gel-filtered platelets in the presence and absence of the 18-mer peptide. (ii) HUVEC-ADAMTS-18 was incubated with control or anti-ADAMTS Ab or 18-mer peptide for 60 minutes at 37°C, prior to incubation with gel-filtered platelets for 4 hours at 37°C (n = 3). (i) Positive control with Buff (buffer), CtIgG (control IgG), PtIgG (patient IgG), 18m (18-mer peptide), or irrel (irrelevant peptide). (ii) AD (HUVEC conditioned media), CtIgG (control IgG), + ADAb (+anti–ADAMTS-18 Ab), 18m, or irrel (n = 6). Where shown, error bars indicate SEM.

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