C-terminal Domains Of ADAMTS13 Research Articles

ADAMTS13-/-zebrafish Model Created By Crispr/Cas Reveals a Novel Role of ADAMTS13 in Vascular Development and Prothrombotic Phenotype

Background: Deficiency of plasma ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), a von Willebrand factor (VWF)-cleaving enzyme, in humans results in thrombotic thrombocytopenic purpura (TTP), a potentially fatal syndrome. Previous studies have demonstrated that ADAMTS13, particularly the C-terminal domains of ADAMTS13 may stimulate or inhibit angiogenesis, depending on the growth environment. However, the role of ADAMTS13 in vascular development in vivo is not known.Objective: To identify the novel function of ADAMTS13 beyond the proteolysis of VWF, we generated ADAMTS13 knockdown and knockout zebrafish to determine the vascular development and propensity for development of thrombosis.Methods: Morpholino anti-sense RNA was microinjected to the embryos of a double transgenic Tg (gata1:dsRed; fli1:eGFP)zebrafish to transiently block ADAMTS13 translation. Also, CRSIPR/Cas9 system was employed to generate ADAMTS13 knockout zebrafish. The vascular development and thrombus formation were determined by fluorescent microscopy.Results: Approximately 30% of ADAMTS13 knockdown zebrafish exhibited vascular abnormalities in the dwarf intersegmental vessels and narrow caudal vein plexus (Fig. 1A and B). To confirm the phenotype, ADAMTS13 knockout using CRSIPR/Cas system was performed. Approximately 85% of the F0 fish carried insertion or deletion mutations in the targeted region. The founder fish were outcrossed to wild type fish to generate heterozygous F1 fish. Fourteen different mutation sequences with germ line transmission were identified in 33 F1 mutants. The siblings carrying an 8-bp deletion mutation, which created a premature stop codon in the signal peptide region of ADAMTS13, were then crossed to produce F2 progeny. The genotypes and protein expression were further confirmed by sequencing and by Western blot, respectively. As shown, a significant higher rate of vascular development defects, particularly in intersegmental vessels and caudal vein, was observed in homozygous ADAMTS13 knockout fish compared to the heterozygous and wild type fish (Fig. 1C). Thrombosis was triggered by incubation of larvae with various low concentrations (0.06%-0.25%) of FeCl3 and determined by the real-time change of fluorescence intensity within 10 min in the head region of larvae under a fluorescent microscope. When compared with wild type fish, ADAMTS13-/- fish demonstrated dramatically decreased fluorescence signal (Fig.1D), indicative of the cessation of blood flow elsewhere in the body after FeCl3injury.Conclusion: Our results demonstrate that severe deficiency of ADAMTS13 in zebrafish results in abnormalities of vascular development and increases the propensity of thrombosis after oxidative injury in fish larvae. These results suggest that ADAMTS13 may have other substrates than VWF. The zebrafish model may turn out to be a powerful tool for identifying a novel pro-angiogenic or anti-thrombotic agent. [Display omitted] DisclosuresZheng:Alexion: Research Funding; Ablynx: Consultancy.

Domain Specificity and Mechanism of Action of Anti-ADAMTS13 Antibodies at Presentation and Relapse in Acquired Thrombotic Thrombocytopenic Purpura (TTP)

Acquired thrombotic thrombocytopenic purpura (TTP) is an autoimmune disease due to autoantibodies (Auto-Ab) against ADAMTS13, resulting in a severe deficiency in enzyme activity (<10%). Little is known about the pathogenicity of anti-ADAMTS13 IgG directed against different domains of the enzyme, or how domain specificity alters between presentation, remission or TTP relapse. We developed novel assays to explore domain specificity and inhibitory potential of anti-ADAMTS13 IgG using samples from the UK TTP Registry.Full-length ADAMTS13 and ADAMTS13 fragments (MD, MDTC and MDTCS - N-terminal domains; TSR2-8 and CUB1/2 - C-terminal domains) were expressed in HEK293T cells and purified. Microtitre plates were coated with ADAMTS13 or ADAMTS13 fragments, incubated with TTP plasmas and bound IgG detected. Domain specificity was further analysed by preincubation of patient samples with ADAMTS13 fragments, assessing the ability of fragments to compete for full-length ADAMTS13 binding on the plate. From a cohort of 92 acquired TTP patients at presentation, 100% (92/92) contained auto-Ab that recognised ADAMTS13 N-terminal domains (MDTCS). Of these, 39% (36/92) had auto-Ab recognising the N-terminal domains alone, 22% (20/92) also had anti-TSR2-8, and 39% (36/92) had anti-CUB1/2 auto-Abs. There was no correlation between auto-Ab domain specificity and mortality/disease severity (anti-ADAMTS13 IgG titre, plasma exchanges to remission, platelet count and cardiac involvement).There was no evidence for auto-Ab recognising MD or MDTC domains in 25 patients tested, suggesting immunoreactivity for MDTCS was primarily mediated through the spacer domain. Consistent with this, TTP IgG inhibited proteolysis of VWF115 (a recombinant VWF A2 domain fragment), but failed to inhibit VWF106 proteolysis (VWF A2 domain fragment lacking the spacer domain binding exosite).To examine the inhibitory potency of anti-ADAMTS13 auto-Ab, IgG isolated from TTP patient samples were titrated in reactions measuring MDTCS-mediated proteolysis of FRETS-VWF73. Twenty eight out of 43 presentation samples tested had inhibitory IgG (IC50 ranging from <0.2-5μM total IgG). Addition of excess of MDTC to 19/19 of these samples did not alter the IC50, confirming inhibition in these samples is mediated solely by anti-spacer antibodies. Intriguingly, 15/43 patients had no evidence of inhibitory auto-Ab, suggesting the pathogenic mechanism in these cases may be enhanced clearance of ADAMTS13. We therefore determined ADAMTS13 antigen concentrations by ELISA. All patients at presentation exhibited markedly reduced ADAMTS13 antigen levels (n=91, median 58ng/ml (range 0-450) vs. normal n=67, median 951ng/ml (515-1829); p<0.0001). Interestingly, patients with predominantly C terminal auto-Ab and no evidence of inhibitory auto-Ab had significantly lower ADAMTS13 antigen levels at first presentation than the rest of the cohort (median 2ng/ml (0-141) vs. 57ng/ml (0-356); p=0.006).Cases where rituximab failed to normalise ADAMTS13 activity despite clinical remission were associated with the persistence of inhibitory anti-N-terminal auto-Ab. Two patients with persistently high anti-ADAMTS13 IgG but normal ADAMTS13 activity and antigen levels during remission had non-pathogenic anti-TSR2-8 antibodies that seemingly had no effect upon ADAMTS13 function or clearance. Nine out of 16 patients exhibited altered auto-Ab domain specificity upon relapse, with either loss or development of anti-C-terminal auto-Ab.In conclusion, from our functional studies ADAMTS13 inhibition appears to be mediated exclusively by anti-spacer antibodies, with no evidence for the presence of auto-Ab that recognise/inhibit other N terminal domains. Importantly, we demonstrate ADAMTS13 antigen depletion is a crucial pathogenic mechanism in acquired TTP, accounting for a large proportion of the reduced ADAMTS13 activity detected in patient samples. Our results suggest that auto-Ab directed against the C-terminal domains of ADAMTS13 may be particularly important in mediating this clearance. There is continual development of the autoimmune response during treatment of TTP patients that can result in altered domain specificity at relapse. DisclosuresNo relevant conflicts of interest to declare.

A novel binding site for ADAMTS13 constitutively exposed on the surface of globular VWF

AbstractADAMTS13 metalloprotease regulates the multimeric size of von Willebrand factor (VWF) by cleaving the Tyr1605-Met1606 bond in the VWF A2 domain. The mechanisms of VWF recognition by ADAMTS13 have yet to be fully resolved. Most studies have focused on the role of exosites within the VWF A2 domain, involved in interaction with the ADAMTS13 spacer domain. In the present study, we expressed different C-terminal domain VWF fragments and evaluated their binding to ADAMTS13 and its truncated mutants, MDTCS and del(TSP5-CUB). Using plate binding assay and surface plasmon resonance, we identified a novel ADAMTS13 binding site (KD ∼ 86 nM) in the region of VWF spanning residues 1874 to 2813, which includes the VWF D4 domain and that interacts with the C-terminal domains of ADAMTS13. We show that the interaction occurs even when VWF is in static conditions, assumed to be globular and where the VWF A2 domain is hidden. We demonstrate that C-terminal VWF fragments, as well as an antibody specifically directed toward the VWF D4 domain, inhibit VWF proteolysis by ADAMTS13 under shear conditions. We propose that this novel VWF C-terminal binding site may participate as the initial step of a multistep interaction ultimately leading to proteolysis of VWF by ADAMTS13.

ADAMTS13 Binds VWF Via its C-Terminal CUB2 Domain.

Thrombotic thrombocytopenic purpura (TTP) is a life threatening illness due to a deficiency of the VWF-cleaving protease, ADAMTS13. The ADAMTS13 protein is composed of a propeptide, followed by a typical zinc metalloprotease domain. The C-terminal 2/3 of the molecule contains disintegrin-like, cystine-rich, and spacer domains, as well as a total of eight TSP1 motifs and two CUB domains. The function of this C-terminal portion of the molecule and its composite motifs is unknown, though TSP1 and CUB domains of other proteins have been shown to mediate protein-protein interactions. To further explore the interaction between ADAMTS13 and VWF, we cloned full length human cDNAs for both ADAMTS13 and VWF into the mammalian expression vector pcDNA3.1. These constructs were transiently transfected into 293T cells and COS cells respectively, and conditioned media collected for analysis. Using an anti-myc antibody, myc-tagged VWF co-immunoprecipitated (co-IP) with ADAMTS13, as demonstrated by western blot analysis using antisera raised against a C-terminal peptide derived from the predicted ADAMTS13 sequence. This direct interaction required partial denaturation of VWF in 1M urea, with no co-IP observed in the absence of urea. To map the segment within ADAMTS13 responsible for VWF binding, we cloned a series of overlapping ADAMTS13 fragments into the bacterial expression vector, Pet44b. Fusion proteins were purified by binding of the included His-tag to Ni-NTA beads and incubated with recombinant myc-VWF in the presence of 1M urea. Association with VWF was analyzed by co-IP with anti-myc followed by western blot analysis using an antibody to the C-terminal HSV-tag present in each fusion protein. The CUB2 (Glu1298- Thr1427) fusion protein co-IP'd with full-length VWF and also demonstrated concentration-dependent competition with full-length ADAMTS13 for VWF binding. In summary, we have demonstrated a direct protein-protein interaction between VWF and ADAMTS13. Binding requires partial denaturation of VWF and appears to be mediated primarily through contacts with the ADAMTS13 CUB2 domain. This interaction may account for the previously observed co-purification of VWF and ADAMTS13 from human plasma. Furthermore, the requirement for 1M urea suggests that this interaction may only occur physiologically under conditions of high shear. Though others have shown that the C-terminal domains of ADAMTS13, including CUB2, are not required for VWF cleavage in vitro, our data, together with several C-terminal mutations previously reported in TTP patients, suggest that interactions between VWF and the ADAMTS13 CUB2 domain may be important in vivo.