Schematic transgenic construct and characterization of transgenic mice. (A) An expression cassette consists of a murine platelet glycoprotein 1b (mGP1b) proximal promoter sequence (∼2.6 kb), a part of its first exon, an intron, and a full-length human ADAMTS13 cDNA (∼4.3 kb) that is flanked by 5′ and 3′ untranslated regions (UTRs). Primer pairs used for genotyping and RT-PCR, respectively, are indicated in the arrows. (B) Genotyping shows 2 positive (∼0.18 kb) transgenic founders (#10 and #14). (C) RT-PCR demonstrates the platelet-specific expression of transgene mRNA (arrowhead, ∼0.4 kb) in the homozygous transgenic (TG1 and TG2) and heterozygous transgenic (Het) mice, but not in KO, mice. As a positive control, murine PF4 mRNA (open arrowhead, ∼0.3 kb) was amplified in all mice. (D) Western blotting with anti-ADAMTS13-Dis IgG demonstrates a full-length rADAMTS13 protein in duplet (∼195 kDa) (left panel, arrowhead) in transgenic platelets (lane 1), but not in nontransgenic WT platelets (lane 2). Lanes 3 and 4 contain only rADAMTS13 (rA13, arrowhead) and murine VWF (mVWF, arrowheads) for the control. **Fragments of IgG. (E) Western blotting of the same membrane in D with polyclonal anti-VWF IgG; therefore, murine VWF (arrowheads) are detected in lanes 1, 2, and 4, but not in lane 3. (F) ADAMTS13 activity in platelet lysate of KO and TG mice is determined by the cleavage of rF-VWF73 substrate. Normal human plasma is used for calibration, defined as having 1 U/mL activity.