Endothelial cell ADAMTS-13 and VWF: production, release, and VWF string cleavage

Abstract

Human umbilical vein endothelial cell (HUVEC)-released ADAMTS-13 (a disintegrin and metalloprotease with thrombospondin repeats) and HUVEC-secreted von Willebrand factor (VWF) strings were investigated under static conditions that allow the accumulation and analysis of ADAMTS-13. The latter was released constitutively from HUVECs and cleaved the secreted and cell-anchored VWF strings progressively during 15 minutes in Ca(2+)/Zn(2+)-containing buffer. HUVEC ADAMTS13 mRNA expression was approximately 1:100 of VWF monomeric subunit expression. In contrast to multimeric VWF stored within Weibel-Palade bodies and secreted rapidly in response to cell stimulation, ADAMTS-13 was released directly from the Golgi to the cell exterior without an organelle storage site. The constitutive release of ADAMTS-13 continued at the same slow rate regardless of the presence or absence of histamine stimulation of HUVECs. Consequently, the percentage of VWF strings cleaved by ADAMTS-13 at VWF Y(1605)-M(1606) decreased as the rate of VWF string secretion was increased by cell stimulation. Blockade of HUVEC ADAMTS-13 activity by antibodies to different ADAMTS-13 domains made it possible to detect the attachment of ADAMTS-13 all along the lengths of HUVEC-secreted VWF strings. Constitutive ADAMTS-13 released from endothelial cells may contribute to the maintenance of cell surfaces free of hyperadhesive VWF multimeric strings.

Figures

Figure 1

Quantification of ADAMTS13 and VWF secreted by unstimulated and histamine-stimulated HUVECs. Culture media was collected from HUVECs grown in 48-well plates at 0, 5, 15, 30, 60, and 180 minutes after the addition of 60 μL of serum-free media with 1% BSA ± 100μM histamine. Samples were collected into 10mM EDTA, and levels of VWF and ADAMTS-13 were measured by immunoassay. (A) There were no significant differences in the ADAMTS-13 levels (pg/mL) measured from the stimulated (•) and unstimulated HUVECs (■) at each time point (n = 8-10). (B) Levels of VWF secreted after histamine stimulation (■) and VWF released in the absence of stimulation (•) were significantly different at each point measured after 0 minutes (n = 5-6).

Figure 2

Cleavage of HUVEC-anchored VWF strings by HUVEC-released ADAMTS13. HUVECs were stimulated for 2 minutes with 100μM histamine, washed, and incubated statically for 0 to 2 minutes (A-B), 5 minutes (C-D), or 10 to 15 minutes (E-F) in Ca2+/Zn2+-containing buffer. The cells were fixed and stained with rabbit anti–VWF plus goat anti–rabbit IgG-488 and DAPI. The lengths of VWF strings were measured from microscope fields (201 μm × 150 μm; ×200) after each incubation time (n = 10), and were categorized as follows: lengths > 150 μm; 101 to 150 μm; 51 to 100 μm; 21 to 50 μm; and <20 μm. Representative images and graphs (means ± SD) are shown for each time interval. The arrows on panels C and E indicate cleaved VWF strings. The panels demonstrate that endogenous ADAMTS-13 released from HUVECs progressively cleaved VWF strings anchored to the same and/or nearby cells into smaller forms.

Figure 3

Specific blockade of VWF string cleavage by antibody to ADAMTS13. HUVECs were stimulated with 100μM histamine in buffer ± 20 μg/mL anti–ADAMTS-13 (BL156) for 2 minutes. After washing, the cells were incubated for 5, 10, and 20 minutes in Ca2+/Zn2+-containing buffer alone or buffer containing 20 μg/mL anti–ADAMTS-13. After incubation, the cells were fixed and stained with rabbit anti–VWF plus goat anti–rabbit IgG-488. The number and lengths of cell-anchored VWF strings were measured from microscope fields (201 μm × 150 μm; ×200) from 3 to 4 experiments at each time point and shown as means ± SD. (A) VWF strings with lengths exceeding 150 μm after 5-, 10-, and 20-minute incubations with Ca2+/Zn2+-containing buffer alone (□) or buffer containing 20 μg/mL anti–ADAMTS-13 (▩). (B) Lengths of long VWF strings (> 150 μm, 101-150 μm, and 51-100 μm) and lengths of cleaved VWF strings (< 20 μm) in the presence and absence of anti–ADAMTS-13 after each incubation time. A greater number of long VWF strings was quantified in the presence of anti–ADAMTS-13. (C) HUVECs were stimulated with 100μM histamine for 2 minutes, washed, and incubated in serum-free media ± 20 μg/mL anti–ADAMTS-13. Levels of VWF released into cell supernatants and collected into 10mM EDTA after 5 minutes (P = .05; n = 10) and 15 minutes (P = .01; n = 8) were significantly lower in supernatants from cells incubated with antibodies to ADAMTS-13.

Figure 4

HUVEC-released ADAMTS13 attached to HUVEC-secreted VWF strings. HUVECs were stimulated with histamine for 2 minutes, washed, and incubated for 20 minutes (without fixative) with polyclonal goat antibodies made against different domains of human ADAMTS-13: (A) BL156 (Tsp1-4 domain); (B) BL152 (metalloprotease domain); and (C) BL153 (distinct peptide within metalloprotease domain); and secondary anti-goat IgG-594 (red). The cells were rewashed before staining with rabbit anti–VWF plus anti-rabbit IgG-488 (green) to visualize VWF strings. Cell nuclei were stained blue with DAPI. The images magnified ×600, represent 6 to 12 experiments, and demonstrate that blocking the activity of HUVEC-released ADAMTS-13 enables the ADAMTS-13 bound only to uncleaved VWF strings to be detected.

Figure 5

Quantification of VWF 176-kD and 140-kD fragments in plasma-derived pVWF. (A) VWF 140-kD fragments (gray) and 176-kD fragments (black) in size-fractionated pVWF were detected with Mab LJ140 and Mab LJ176 by immunoassay, and expressed as percentages of VWF fragments per total quantity of VWF antigen. Results are means ± SD. (B) VWF multimer immunoblot (1% agarose) showing multimer size ranges in pVWF fractions. (C) pVWF (multimer sizes similar to 82) was reduced and separated by SDS-5% PAGE. The immunoblot using polyclonal goat anti–VWF displays intact VWF monomer (225 kD) and faint bands for the 140-kD and 176-kD fragments. (D) Nonreduced samples separated by SDS-0.8% agarose demonstrate the larger plasma VWF multimers present in pVWF and the smaller multimers in CS. (E) Reduced samples of CS and pVWF were separated by SDS-5% PAGE and immunoblotted with Mab LJ140. The VWF 140-kD fragment was undetectable in pVWF samples that contained 1000-fold more VWF antigen (8 μg) than CS samples (8.4 ng). Immunoblots represent similar results obtained in 4 to 5 experiments. (F) Target epitopes of Mab LJ140 and Mab LJ176 that react specifically with either fragment 140-kD or 176-kD flanking the ADAMTS-13 cleavage site within the A2 domain of the VWF monomeric subunit. Cleavage of VWF at peptide bond M1605-Y1606 by ADAMTS-13 results in an increased proportion of 140-kD and 176-kD fragments relative to total VWF protein.

Figure 6

Quantification of ADAMTS13 cleavage sites 1605-6 in VWF A2 domains in VWF strings secreted by stimulated and unstimulated HUVECs. HUVECs grown in 24-well plates were incubated with 200 μL of serum-free media or stimulated with 100μM histamine in the same media at 37°C. Culture media was collected after 60 minutes or 48 hours into 10mM EDTA and analyzed for total VWF antigen and VWF 176-kD fragments by immunoassay. (A) VWF 176-kD fragment levels (pg/mL) were quantified using Mab LJ176 against amino acids M1606-P1620 of VWF monomers, and total VWF antigen levels (ng/mL) were measured using a polyclonal antibody against purified VWF. (B) Ratio of VWF 176-kD fragments per total VWF antigen after 60 minutes and 48 hours from histamine-stimulated and unstimulated HUVECs. Results are means ± SD (n = 12) and demonstrate that there is more ADAMTS-13–mediated cleavage of the few VWF strings secreted at a slow rate from unstimulated HUVECs than the many VWF strings secreted rapidly in response to cell stimulation at 60 minutes. (C) VWF multimer immunoblot representative of soluble VWF collected 1, 24, and 48 hours after HUVEC histamine stimulation in serum-free media. (D) Cartoon illustrating the relationship between rate of VWF string secretion and VWF cleavage multimer length. WPBs, represented by a spool of thread, release longer VWF strings before ADAMTS-13 cleavage under stimulation (rapid release rates) than under unstimulated conditions (basal rate). HUVEC-released ADAMTS-13, represented by the pair of scissors, is slow and continuous and remains constant regardless of the rate of VWF string secretion.