Biosynthesis and Apical Secretion of ADAMTS13 Metalloprotease in Vascular Endothelial Cells and in Madin-Darby Canine Kidney Cells.

ADAMTS13, a reprolysin-like metalloprotease, limits platelet aggregation by cleaving von Willebrand factor. Deficiency of plasma ADAMTS13 activity can lead to a life-threatening complication, thrombotic thrombocytopenic purpura (TTP). It has been shown that ADAMTS13 is synthesized in human and mouse hepatic stellate cells. But detection of ADAMTS13 mRNA by a reverse transcriptase polymerase chain reaction (RT-PCR) in many other tissues (brain, heart, lungs, kidneys, pancreas, adrenal gland, prostate, uterus and placenta) suggests that the vascular endothelial cells may also produce low level of ADAMTS13 protease. We showed that full-length ADAMTS13 mRNA and protein were detected in human umbilical cord vein endothelial cells (HUVEC), human aortic endothelial cells (HAEC), and microvascular endothelial cells (ECV304) by RT-PCR, SDS-polyacrylamide gel electrophoresis and Western blot. The ADAMTS13 in the cell lysates and the serum-free conditioned media of HUVEC, HAEC and ECV304 cleaved a peptidyl substrate, FRETS-VWF73, specifically, and such a proteolytic cleavage could be completely abolished by 10 mM EDTA. Immunohistochemistry showed that both endogenous and recombinant ADAMTS13 in HUVEC and HAEC appeared to be co-localized with von Willebrand factor in the endoplasmic reticulum and/or Golgi apparatus, but not in the Weibel-Parade bodies, suggesting that ADAMTS13 is readily secreted rather than stored inside the cells. To determine whether ADAMTS13 is secreted into the lumen of the vessels, we assessed the polarity of ADAMTS13 secretion in ECV304 and Madin-Darby canine kidney (MDCK) cells, two well-characterized polarized endothelial and epithelial cell lines for studying protein sorting. Both endogenous ADAMTS13 and recombinant ADAMTS13-V5-His expressed in ECV304 was predominantly secreted into the apical domain of the cells. Similarly, the recombinant ADAMTS13-V5-His in MDCK cells was also secreted predominantly into the apical domain. Apical secretion of ADAMTS13-V5-His appeared to depend on at least a transient association of ADAMTS13-V5-His with Triton X-100 insoluble glycosphingolipid and cholesterol-enriched microdomains, namely rafts. Deletion of the distal carboxyl terminal CUB (Complement C1r/C1s, Urinary EGF, and Bone morphogenic protein) domains or removal of membrane cholesterol by treatment of MDCK cells expressing recombinant ADAMTS13-V5-His with lovastatin and methyl-β-cyclodextrin completely abolished the raft association and apical secretion of ADAMTS13-V5-His in MDCK cells. Moreover, an ADAMTS13 mutation (4143insA), which naturally occurs in a patient with TTP that deletes the second CUB domain not only significantly impaired the amount of total ADAMTS13 protein secreted into the conditioned medium, but also reversed the polarity of ADAMTS13 secretion in MDCK cells. Our data demonstrate that: 1) active full-length ADAMTS13 protease is produced in human vascular endothelial cells; 2) the CUB domains are critical for apical secretion via their interaction with lipid rafts; 3) mutations in ADAMTS13 gene that disrupt sorting signals may result in congenital ADAMTS13 deficiency, leading to TTP in some cases. Considering a large surface area of the vascular endothelial cells, apically secreted ADAMTS13 from these cells may contribute significantly to plasma pool of ADAMTS13 protease.