The ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) are a family of proteases that include 19 members. ADAMTS are closely related to the ADAM (a disintegrin and metalloproteinase) subfamily of metalloendopeptidases, sharing similar metalloproteinase domains. ADAMTS are secreted proteases that are involved in matrix proteoglycan cleavage, processing of collagen, and regulating processes such as angiogenesis and hemostasis (Porter et al. 2005).
ADAMTS13 is a protein of 1,427 amino acids with a deduced molecular weight of 190 kDa (Levy et al. 2001). ADAMTS13 was originally identified as a von Willebrand factor (vWF)-cleaving protease (vWF-cp), later discovered to be a novel member of the ADAMTS family. Following genome-wide linkage analysis, the ADAMTS13 gene was located on the human chromosome 9q34 (Levy et al. 2001).
Ultrahigh-molecular-weight (HMW) vWF multimers were found to persist in the plasma of patients with thrombotic thrombocytopenic purpura (TTP), a blood disorder caused by absence of ADAMTS13 activity, resulting in increased platelet aggregation and thrombocytopenia (Moake et al. 1982). The known types of TTP include familial deficiencies in ADAMTS13 or the development of antibodies against ADAMTS13 (Fujikawa et al. 2001).
Protein Function and Regulation of the Activity
Before the discovery of ADAMTS13, it was hypothesized that there may be a large “depolymerase” with proteolytic or disulfide-bond-reducing capacity, responsible for cleaving ultra HMW vWF that is deficient in TTP patients. Further, ultra HMW vWF multimers have been shown to be reduced following transfusion of healthy plasma in TTP patients, suggesting that healthy plasma may provide this missing “depolymerase” (Moake et al. 1982, 1985).
The purified ADAMTS13 protein has been found to be a HMW protein with proteolytic activity that cleaves the scissile bond in vWF between Try1605 and Met1606. However, this protease has shown to be different from known matrix metalloproteinases, serine proteases, or calpains. This vWF-cp has been found at significant levels in the plasma, with a concentration of 1–1.6 μg/mL (Gerritsen et al. 2001; Zheng et al. 2003) and a half-life of 2–3 days (Furlan et al. 1996, 1999). Further analysis revealed that its proteolytic function is shear-dependent. Interestingly, the unfolding of vWF multimers during shear enhances the proteolytic activity of vWF-cp, suggesting that the folded confirmation of vWF protects the scissile bond from ADAMTS13 cleavage (Tsai 1996).
The main cause of reduced ultra HMW vWF proteolysis in TTP patients is still unknown. Purification of the vWF-cp revealed a protein with a molecular weight of 190 kDa. Further, the N-terminal sequence of the purified protein showed similarity to the deduced amino acid sequence of a gene on human chromosome 9q34. A search of the human expressed sequence tag (EST) database, with a probe sequence of the aligned region, revealed a novel metalloproteinase of the ADAMTS subfamily (Fujikawa et al. 2001). Similarly, genome-wide linkage analysis of TTP patients confirmed that a gene on chromosome 9q34 region is responsible for encoding ADAMTS13 (Levy et al. 2001; Cal et al. 2002). For the first time, genetic analysis not only identified the full-length cDNA sequence of ADAMTS13 but also provided the evidence that the vWF-cp is in fact ADAMTS13 (Levy et al. 2001). It has further revealed that the proteolytic activity of ADAMTS13 for vWF is regulated by both calcium and zinc ions (Anderson et al. 2006).
ADAMTS13 is found to be constitutively released from human umbilical vascular endothelial cells (HUVECs). However, ADAMTS13 expression, assessed by immunofluorescence staining, has not been detected in mitochondria, lysosomes, or Weibel-Palade bodies of HUVECs, suggesting that ADAMTS13 is transferred directly from the endoplasmic reticulum (ER), via the Golgi, to the plasma membrane without being stored in subcellular compartments (Turner et al. 2009). Interestingly, ADAMTS13 expression is not altered by histamine stimulation, whereas vWF release from HUVECs is increased by up to 15-fold upon stimulation with the same agonist (Turner et al. 2009). Following the release, long vWF strings anchor to the HUVEC cell surface. ADAMTS13 has been observed to attach to the vWF; thus the constitutive release of ADAMTS13 may assist in preventing the buildup of hyperadhesive vWF strings anchored to the cell surface (Turner et al. 2009).
Mutagenesis studies revealed that the vWF P3 residue, Leu1603, is essential in determining vWF proteolysis by ADAMTS13. The proteolysis of vWF by ADAMTS13 is significantly reduced when the Leu1603 is substituted with Ala in vWF. Other amino acid substitutions resulted in undetectable levels of proteolysis (Lys and Asn) or complete inhibition of proteolysis (Ser), highlighting the importance of Leu1603 for vWF cleavage by ADAMTS13 (Xiang et al. 2011). Further, close to the active site of ADAMTS13, ten P3 residues have been found to interact with vWF Leu1603. In ADAMTS13, the residue substitutions in two specific clusters, Leu198/Leu273/Leu274 and Val195/Leu151, reduce its vWF cleavage ability, indicating those clusters in ADAMTS13 as the potential sites of interaction with vWF Leu1603. Further analysis revealed that the residues Leu1603, Tyr1605, and Asp1614 in vWF interact with the residues Ley198, Val195, and Arg349, respectively, in the ADAMTS13-vWF complex. These interactions ensure that the Tyr1605-Met1606 scissile bond is positioned into the active site of ADAMTS13 for proteolysis to occur. Interestingly, vWF degradation is influenced by the conformational change in vWF, specifically by the unfolding of the A2 domain under shear stress. vWF then exposes the Tyr1605-Met1606 (P1-P1’) scissile bond for ADAMTS13 cleavage. Exosites on vWF guide the active site of ADAMTS13 specifically to the scissile bond, and then the P1’ residue, vWF Met1606, interacts with Asp252-Pro256 residues at the active site of the metalloprotease domain of ADAMTS13 (Xiang et al. 2011).
Interestingly, vWF has been shown to initiate its own proteolysis via allosteric activation of ADAMTS13. The binding of ADAMTS13 to the D4 domain of vWF causes allosteric activation, enhancing catalytic activity and positioning ADAMTS13 at the scissile bond (Muia et al. 2014). The metalloprotease activity of ADAMTS13 is regulated by its own distal CUB domains. The structural analysis by X-ray crystallography and electron microscopy has revealed that the distal CUB domain and proximal domains of ADAMTS13 make direct contact through folding to inhibit vWF cleavage and unfold following allosteric activation. As such, vWF acts as a cofactor as well as a substrate during the regulation of hemostasis (Muia et al. 2014).
ADAMTS13 may also possess further roles beyond vWF cleavage. Interestingly, increased ADAMTS13 activity was discovered to be a risk factor for prediabetes and type 2 diabetes mellitus in both fasted and non-fasted blood measurements. These results were unaffected by vWF levels, suggesting that ADAMTS13 may be a contributing factor in the development of diabetes. However, further research is required to confirm these biological mechanisms and whether ADAMTS13 antigen levels also play a role (de Vries et al. 2017).
An inhibitor of ADAMTS13 that reduces proteolytic activity, without causing TTP, may prove useful in clinic. Doxycycline is able to inhibit the activity of ADAMTS13 and reduce vWF cleavage. This inhibition is related to the metallic cation-binding properties of doxycycline at the active site of ADAMTS13 (Tsai et al. 1997). In clinical settings, doxycycline may prove useful in controlling ADAMTS13 activity in patients with VWD, a disorder characterized by either a qualitative or quantitative reduction in vWF. Doxycycline is able to protect HMW vWF from degradation and restore vWF binding activity without affecting platelet function (Bartoli et al. 2015).
Major Sites of Expression and Subcellular Localization
Northern blot analysis revealed a strong ADAMTS13 mRNA expression in the liver, suggesting that ADAMTS13 in the circulation may be derived from the liver. Consistent with this, the cDNA of ADAMTS13 has been isolated first from fetal liver tissue. However, reverse-transcription PCR analysis revealed that ADAMTS13 may be expressed in all tissues at low levels, including the prostate and brain (Zheng et al. 2001; Cal et al. 2002). Confirming this, ADAMTS13 expression at mRNA level has also been observed in the kidney, pancreas, spleen, thymus, prostate, ovary, small intestine, and colon (Levy et al. 2001). These observations suggest that highly vascular tissue may not be the primary location for ADAMTS13 expression as initially thought. However, ADAMTS13 expression has not been detected in the brain, heart, or lung (Levy et al. 2001). Furthermore, a full 4.7 kb ADAMTS13 mRNA transcript was discovered in the liver, whereas a smaller 2.4 kb mRNA transcript was identified in the skeletal muscle and placenta, similarly suggesting that the liver may play a role in ADAMTS13 synthesis. ADAMTS13 transcript has also been found relatively at high levels in melanoma and colon carcinoma cells, suggesting a possible role in tumor formation (Cal et al. 2002).
The requirements and location of ADAMTS13 synthesis have been first identified in liver cells using in situ hybridization and immunohistochemistry (Uemura et al. 2005). A strong ADAMTS13 mRNA expression has been detected in perisinusoidal cells, displaying elongated cytoplasmic processes. Furthermore, the ADAMTS13-positive cells in the liver have also been positive for α-smooth muscle actin, a hepatic stellate cell marker expression, revealing that perisinusoidal stellate cells are responsible for the production of ADAMTS13 in the liver (Uemura et al. 2005).
Phenotypes and Splice Variants
The gene for ADAMTS13 spans 37,000 bases across 29 exons, encoding a protein of 1,427 amino acids. The DNA sequence analysis has predicted that the exon 17 may undergo alternative splicing, resulting in a frameshift that produces a truncated protein with 842 amino acids and lacks six TSP1 repeats. This suggests the possibility of differential gene expression, producing isoforms with diverse roles (Levy et al. 2001). Further analysis has revealed 12 mutations within the ADAMTS13 gene. These mutations include frameshift mutations (exon 19), single splice mutations (exon 13), and nonconservative amino acid substitutions (exons 3, 6, 10, 13, 17, 19, 22, 24, 26, and 27) responsible for TTP. ADAMTS13 contains a propeptide convertase cleavage site at amino acids 71–74; therefore, proteolytic processing may be required for the activation. Further, the presence of an RGD sequence suggests likely integrin interactions. These results indicate that a complete lack of ADAMTS13 may in fact be lethal (Levy et al. 2001).
Further mutations (Q449stop nonsense and R268P, C508Y, and P475S missense) in the ADAMTS13 gene, which result in congenital TTP, have been identified (Kokame et al. 2002). Further expression analysis has revealed that the R268P and C508Y mutations prevent ADAMTS13 secretion from cells, whereas the Q449stop and P475S mutations reduce the proteolytic activity of ADAMTS13 (Kokame et al. 2002).
Various anti-ADAMTS13 antibodies are commercially available. A rabbit unconjugated polyclonal antibody against human residues 250–350 of ADAMTS13 is available from Novus Biologicals (NB100-584). This antibody is suitable for use in Western blotting. Similarly, a rabbit polyclonal antibody to human ADAMTS13 is available from MyBioSource (MBS630150) and is suitable for Western blot and ELISA. A rabbit unconjugated polyclonal antibody against human ADAMTS13 is available from Biorbyt (orb37949), suitable for use in Western blot and immunohistochemistry paraffin. LifeSpan Biosciences, Inc. offers a wide range of anti-ADAMTS13 antibodies, including a mouse unconjugated monoclonal antibody to human ADAMTS13 (aa34-688) for use in Western blot, immunoprecipitation, flow cytometry, and ELISA (LS-C124984); a sheep biotin-conjugated polyclonal antibody to human ADAMTS13 (aa34-688) for use in Western blot (LS-C124986); and a rabbit unconjugated polyclonal antibody to human ADAMTS13 (aa829-858) for use in immunohistochemistry paraffin and Western blot (LS-C100639). Further, an inhibitory polyclonal goat antibody raised against the peptide sequence CVPGADGLEAPVTEGPGSVDEKLPAPE targets the fourth TSP1 domain of ADAMTS13. This antibody is called BL156 and is available from Bethyl Laboratories (Turner et al. 2009).
ADAMTS13 is a secreted metalloproteinase that is involved in regulating hemostasis, by specifically binding to and cleaving HMW vWF at the A2 domain, and its activity is dependent on optimal concentrations of calcium and zinc ions. The gene for ADAMTS13 spans 37,000 bases across 29 exons, encoding a protein of 1,427 amino acids with a molecular weight of 190 kDa. ADAMTS13 contains an N-terminal signal peptide, a propeptide sequence, a reprolysin-like metalloprotease domain, a nonfunctional disintegrin-like domain, a thrombospondin-1 (TSP1) repeat, a cysteine-rich domain, a spacer domain, seven further TSP1 repeats, and two CUB domains. ADAMTS13 is constitutively excreted from cells, primarily in the liver. The development of TTP is characterized by a lack of ADAMTS13, caused either by familial deficiency of ADAMTS13 expression or the development of antibodies against ADAMTS13. Several mutations have been discovered that result in ADAMTS13 variants responsible for TTP. It has been revealed that vWF works with ADAMTS13 to regulate proteolysis, as well as positioning the scissile bond at the active site for vWF cleavage. Interestingly, the ADAMTS13 molecule adopts a folder configuration to inhibit its own metalloproteinase domain through direct contact with its CUB domains. Once unfolded, the binding of ADAMTS13 to vWF induces the allosteric activation and enhances proteolysis. Furthermore, increased ADAMTS13 activity has been found to be a risk factor for type 2 diabetes mellitus. ADAMTS13 expression is upregulated in tumor cells, suggesting that ADAMTS13 may possess further roles beyond the cleavage of vWF.
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