Humate p

Wild type full-length VWF (amino acids M₁-K₂₈₁₃)
was expressed in HEK293 cells and cultured as described previously [56 (link), 57 (link)]. P1337L, V1316M and S1285F were inserted into full-length cDNA
of wild-type VWF within the mammalian expression vector pcDNA3 [57 (link)] by site-directed mutagenesis employing the
QuickChange kit (Stratagene). The VWF concentrates Wilate (Octapharma),
Koãte DVI (Bayer), Humate-P (CSL Behring) and Alphanate (Grifols) were
provided by the Mayo Clinic Special Coagulation Laboratory. All concentrates
were excessively dialyzed into PBS prior to usage. An SDS-PAGE gel of all four
concentrates can be found in the HXMS Supporting Information (Fig.S27). VWF was
quantified by fluorescence and the commercial REAADS antigen activity assay
(Diapharma¹) as
described in the LTMS
Supporting Information (Fig.S11).

Mice were prepared for surgery as described recently^{33 (link)}. An AlexaFluor647-labeled antibody against fibrin (2 μg per mouse) (gift from Dr. Rodney Camire) and Humate-P^® (50 u/kg) (CSL Behring) were administered intravenously prior to imaging. The saphenous vein was injured by two maximum-strength 532-nm laser pulses (70 μm; 100 Hz; duration, 7 ns; 10 ms pulse interval) (Ablate! photoablation system; Intelligent Imaging Innovations, Denver, CO, USA). Images were captured on a Zeiss Axio Examiner Z1 microscope equipped with a 20X water immersion objective lens (Zeiss, Jena, Germany) with a numerical aperture of 1 and a working distance of 1.8 mm and a Yokogawa confocal scanning unit, CSU-W (Yokogawa Electric Co.). Fluorescence excitation was achieved with either a multi-color LED light source (Lumencor) for epifluorescence or 488, 561, 640 nm lasers for confocal imaging (Intelligent Imaging Innovations). All images were captured with an Orca Flash 4.0 camera (Hammamatsu). Data was analyzed with SLIDEBOOK 6.0 (Intelligent Imaging Innovations).

VWF concentration was determined using a flow cytometer-bead sandwich assay, using multimeric VWF purified from humate-P (CSL Behring) as a standard [4 (link)]. Human-murine VWF chimera levels in mouse plasma was determined using calibration curves generated by spiking VWF^−/− mouse plasma with human plasma pooled from 5 normal, adult volunteers.
VWF multimer distribution was assayed using 0.6-1.2% agarose gel electrophoresis [21 (link)]. To assay VWF susceptibility to proteolysis by ADAMTS13, 10μg/mL VWF was incubated with 10U/mL ADAMTS13 (WHO standard) and 1.6M urea at 37°C for 24h. Western blotting of cleaved VWF products was assayed under standard reducing conditions using 4%-8% SDS-PAGE followed by detection using anti-VWF Ab (Dako).