Apical sorting of ADAMTS13 in vascular endothelial cells and Madin-Darby canine kidney cells depends on the CUB domains and their association with lipid rafts

Abstract

ADAMTS13 biosynthesis appeared to occur mainly in hepatic stellate cells, but detection of ADAMTS13 mRNA in many other tissues suggests that vascular endothelium may also produce ADAMTS13. We showed that ADAMTS13 mRNA and protein were detectable in human umbilical vein endothelial cells, aortic endothelial cells, and endothelium-derived cell line (ECV304). ADAMTS13 in cell lysate or serum-free conditioned medium cleaved von Willebrand factor (VWF) specifically. IntroductionADAMTS13, a member of a disintegrin and metalloprotease with thrombospondin type 1 repeats (ADAMTS) family, limits plateletrich thrombi by cleaving von Willebrand factor (VWF) between the Tyr 1605 and Met 1606 residues. VWF is synthesized in vascular endothelial cells and megakaryocytes or platelets, and normally stored in Weibel-Palade bodies 1 of endothelial cells or the ␣-granules of platelets. 2,3 Upon stimulation, VWF is secreted as unusually large (UL) multimers that may remain associated with the endothelial cell surface. [4][5][6] These UL-VWF multimers may be the preferred substrate for ADAMTS13. 4,5 Inability to cleave these UL-VWF multimers to smaller sizes on the endothelial cell surface may result in an accumulation of UL-VWF, leading to enhanced platelet adhesion and aggregation, and thrombotic thrombocytopenic purpura (TTP), 7,8 characterized by profound thrombocytopenia, microangiopathic hemolytic anemia, and organ failure. 7,8 ADAMTS13 appears to be synthesized in liver, 9-11 particularly in hepatic stellate cells, [12][13][14] which reside in the space of Disse adjacent to the hepatocytes. However, the contribution of hepatic stellate cells in regulation of plasma ADAMTS13 levels remains to be determined. We have recently shown that despite a dramatic increase in ADAMTS13 expression in hepatic stellate cells upon activation by administration of carbon tetrachloride, plasma ADAMTS13 activity is not proportionally increased in rats. 14 In addition, patients with liver cirrhosis or with significant proliferation and activation of hepatic stellate cells exhibit variable or reduced levels of plasma ADAMTS13 activity. 15 Therefore, plasma ADAMTS13 protease may be derived from other sources as well.Recent reports have shown that ADAMTS13 is synthesized and released from human megakaryocytes and platelets. 16,17 ADAMTS13 mRNA is detected in almost every organ tissue by reversetranscriptase-polymerase chain reaction (RT-PCR), [9][10][11]18,19 suggesting that the vascular endothelium may also be the source of plasma ADAMTS13. Considering the enormous surface area of vascular endothelial beds, a small fraction of endothelial cells producing ADAMTS13 at any given time may contribute significantly to plasma levels of ADAMTS13 activity. Consequently, any perturbation of endothelial cell function or ADAMTS13-VWF interactions at the site of their syntheses by inflammatory cytokines or drugs may play an important role in the pathogenesis of TTP 20,21 and perhaps other thrombotic complications. 22 ADAMTS13 consists of signal peptide, propepti...

An IAP retrotransposon in the mouse ADAMTS13 gene creates ADAMTS13 variant proteins that are less effective in cleaving von Willebrand factor multimers

“…It is not clear why mouse ADAMTS13 did not exhibit apical polarity of secretion as previously reported with human ADAMTS13. 7 A similar pattern of substrate-dependent divergence in activity was also observed with human ADAMTS13 and its recombinant variants. Compared with normal human plasma ADAMTS13, truncated proteins AD2 and AD5, but not AD6 or the full-length AD7, exhibited higher activity against VWF73 peptides than against VWF multimers.…”

“…7 To determine whether the IAP-a variant protein was secreted with a different polarity, we transfected MDCK cells grown on Transwell filters with mouse ADAMTS13 cDNA plasmids. The results showed that 48.9% Ϯ 10.1% (n ϭ 5) of recombinant IAP-a and 48.2% Ϯ 9.6% (n ϭ 5) of recombinant mouse full-length ADAMTS13 were secreted apically (P Ͼ .5).…”

“…The mRNA, 4.7 kb in size, is detected primarily in the stellate cells of the liver, 2,3 although it may also be expressed in other cell types. [4][5][6][7] Mutations of the ADAMTS13 gene are detected in patients with congenital thrombotic thrombocytopenic purpura (TTP), whereas autoimmune inhibitors of the enzyme are detected in patients with an acquired form of the disease. 8 ADAMTS13 consists of several homologous domains: a signal peptide, a propeptide, a reprolysin-type metalloprotease domain, a disintegrin domain, a thrombospondin type 1 repeat (TSR), a cysteine-rich domain (Cys), a spacer domain (Spa), 7 additional TSRs, and 2 CUB domains.…”

The role of membrane microdomains in transmembrane signaling through the epithelial glycoprotein Gp140/CDCP1

“…The CUB domain of ADAMTS13 was shown to be important for the constitutive localization of ADAMTS13 to lipid rafts [47]. However, biochemical properties of Gp140 suggest that Gp140 does not always reside in DRMs and may, instead, only transiently associate with these domains upon outside-in signaling or cross-linking (Fig.…”

Biologically active ADAMTS13 is expressed in renal tubular epithelial cells

“…ADAMTS13 antigen is present in the plasma of patients with acquired TTP, where it appears to be in complex with auto-antibodies [12], and is absent in congenital TTP [11,13]. The cellular origin of ADAMTS13 in plasma has not been completely elucidated but the protease has been shown to be synthesized in liver stellate cells and in endothelial cells [14,15]. ADAMTS13 mRNA has been detected in a variety of tissues including the kidneys [4,16,17].…”