Figure 1.

Effect of rADAMTS-18 on oxidative platelet fragmentation and binding to platelets. (A) Fragmentation with 18-mer peptide. Gel-filtered platelets were treated with biotin-peptide (bp) as well as bp + streptavidin (str) and scrambled biotinylated ADAMTS-18 peptide (scp) to induce integrin clustering (n = 6). Platelet fragmentation was assessed by fluorescence-activated cell sorting (FACS) as described in “Methods.” (B) Platelet oxidation. CT (control IgG), Ab (patient IgG). Eighteen-mer biotin-peptide was incubated with an antibiotin Ab, 1:200 dilution () or irrelevant IgG (□; n = 6). Oxidation was assessed as discussed in “Methods.” (C) ADAMTS-18 or control protein was synthesized by ribosomal translation, using ³⁵S-methionine. ³⁵S-ADAMTS-18 (131 mCi/mg) was added to gel-filtered platelets overnight at 4°C, using doubling concentrations. Ctl refers to ³⁵S-ribosomal translation of luciferase. AD-CTD refers to ³⁵S-ribosomal translation of ADAMTS-18 with absent C-terminal 157 amino acids (13% deleted). Note absence of binding with C-terminal–deficient ADAMTS-18. Eighteen-mer refers to 10 μM nonradioactive 18-mer peptide added with the ³⁵S-ADAMTS-18 incubation. Note inhibition of binding with C-terminal 18-mer peptide. Error bars indicate SEM.