Recombinant ADAMTS13 Research Articles

How I treat refractory thrombotic thrombocytopenic purpura

AbstractAcquired thrombotic thrombocytopenic purpura (TTP) is characterized by thrombocytopenia and microangiopathic hemolytic anemia (MAHA) without an obvious cause, and may include fever, mild renal failure, and neurologic deficits. It is characterized by a deficiency of the von Willebrand factor (VWF) cleaving enzyme, ADAMTS13 (a disintegrin and metalloproteinase, with a thrombospondin type 1 motif, member 13), resulting in formation of microthrombi in the high sheer environment of the microvasculature. This causes microvascular occlusion, MAHA, and organ ischemia. Diagnosis is based on the presence of clinical symptoms, laboratory aberrations consistent with MAHA, decreased ADAMTS13 activity, and possibly presence of anti-ADAMTS13 autoantibodies. Upfront treatment of acute TTP includes plasma exchange and corticosteroids. A significant number of patients are refractory to this treatment and will require further interventions. There are limited data and consensus on the management of the refractory TTP patient. Management involves simultaneously ruling out other causes of thrombocytopenia and MAHA, while also considering other treatments. In this article, we describe our management of the patient with refractory TTP, and discuss use of rituximab, increased plasma exchange, splenectomy, and immunosuppressive options, including cyclophosphamide, vincristine, and cyclosporine. We also review recent evidence for the potential roles of bortezomib and N-acetylcysteine, and explore new therapeutic approaches, including recombinant ADAMTS13 and anti-VWF therapy.

ADAMTS13 Endopeptidase Protects against Vascular Endothelial Growth Factor Inhibitor-Induced Thrombotic Microangiopathy.

Thrombotic microangiopathy (TMA) is a life-threatening condition that affects some, but not all, recipients of vascular endothelial growth factor (VEGF) inhibitors given as part of chemotherapy. TMA is also a complication of preeclampsia, a disease characterized by excess production of the VEGF-scavenging soluble VEGF receptor 1 (soluble fms-like tyrosine kinase 1; sFlt-1). Risk factors for VEGF inhibitor-related TMA remain unknown. We hypothesized that deficiency of the VWF-cleaving ADAMTS13 endopeptidase contributes to the development of VEGF inhibitor-related TMA. ADAMTS13(-/-) mice overexpressing sFlt-1 presented all hallmarks of TMA, including thrombocytopenia, schistocytosis, anemia, and VWF-positive microthrombi in multiple organs. Similar to VEGF inhibitor-related TMA in humans, these mice exhibited severely impaired kidney function and hypertension. In contrast, wild-type mice overexpressing sFlt-1 developed modest hypertension but no other features of TMA. Recombinant ADAMTS13 therapy ameliorated all symptoms of TMA in ADAMTS13(-/-) mice overexpressing sFlt-1 and normalized BP in wild-type mice. ADAMTS13 activity may thus be a critical determinant for the development of TMA secondary to VEGF inhibition. Administration of recombinant ADAMTS13 may serve as a therapeutic approach to treat or prevent thrombotic complications of VEGF inhibition.

Platelet-delivered ADAMTS13 inhibits arterial thrombosis and prevents thrombotic thrombocytopenic purpura in murine models

AbstractADAMTS13 metalloprotease cleaves von Willebrand factor (VWF), thereby inhibiting platelet aggregation and arterial thrombosis. An inability to cleave ultralarge VWF resulting from hereditary or acquired deficiency of plasma ADAMTS13 activity leads to a potentially fatal syndrome, thrombotic thrombocytopenic purpura (TTP). Plasma exchange is the most effective initial therapy for TTP to date. Here, we report characterization of transgenic mice expressing recombinant human ADAMTS13 (rADAMTS13) in platelets and its efficacy in inhibiting arterial thrombosis and preventing hereditary and acquired antibody-mediated TTP in murine models. Western blotting and fluorescent resonance energy transfer assay detect full-length rADAMTS13 protein and its proteolytic activity, respectively, in transgenic (Adamts13−/−PltA13), but not in wild-type and Adamts13−/−, platelets. The expressed rADAMTS13 is released on stimulation with thrombin and collagen, but less with 2MesADP. Platelet-delivered rADAMTS13 is able to inhibit arterial thrombosis after vascular injury and prevent the onset and progression of Shigatoxin-2 or recombinant murine VWF-induced TTP syndrome in mice despite a lack of plasma ADAMTS13 activity resulting from the ADAMTS13 gene deletion or the antibody-mediated inhibition of plasma ADAMTS13 activity. These findings provide a proof of concept that platelet-delivered ADAMTS13 may be explored as a novel treatment of arterial thrombotic disorders, including hereditary and acquired TTP, in the presence of anti-ADAMTS13 autoantibodies.

Detection of Plasma Protease Activity Using Microsphere-Cytometry Assays with E. coli Derived Substrates: VWF Proteolysis by ADAMTS13.

Protease levels in human blood are often prognostic indicators of inflammatory, thrombotic or oncogenic disorders. The measurement of such enzyme activities in substrate-based assays is complicated due to the low prevalence of these enzymes and steric hindrance of the substrates by the more abundant blood proteins. To address these limitations, we developed a molecular construct that is suitable for microsphere-cytometer based assays in the milieu of human blood plasma. In this proof of principle study, we demonstrate the utility of this substrate to measure metalloprotease ADAMTS13 activity. The substrate, expressed in E. coli as a fusion protein, contains the partial A2-domain of von Willebrand factor (VWF amino acids 1594–1670) that is mutated to include a single primary amine at the N-terminus and free cysteines at the C-terminus. N-terminus fluorescence conjugation was possible using NHS (N-hydroxysuccinimide) chemistry. Maleimide-PEG(Polyethylene glycol)n-biotin coupling at the C-terminus allowed biotinylation with variable PEG spacer lengths. Once bound to streptavidin-bearing microspheres, the substrate fluorescence signal decreased in proportion with ADAMTS13 concentration. Whereas recombinant ADAMTS13 activity could be quantified using substrates with all PEG repeat-lengths, only the construct with the longer 77 PEG-unit could quantify proteolysis in blood plasma. Using this longer substrate, plasma ADAMTS13 down to 5% of normal levels could be detected within 30 min. Such measurements could also be readily performed under conditions resembling hyperbilirubinemia. Enzyme catalytic activity was tuned by varying buffer calcium, with lower divalent ion concentrations enhancing cleavage. Overall, the study highlights the substrate design features important for the creation of efficient proteolysis assays in the setting of human plasma. In particular, it emphasizes the need to introduce PEG spacers in plasma-based experiments, a design attribute commonly ignored in immobilized peptide-substrate assays.

ADAMTS13 and von Willebrand factor in thrombotic thrombocytopenic purpura.

Pathogenesis of thrombotic thrombocytopenic purpura (TTP) was a mystery for over half a century until the discovery of ADAMTS13. ADAMTS13 is primarily synthesized in the liver, and its main function is to cleave von Willebrand factor (VWF) anchored on the endothelial surface, in circulation, and at the sites of vascular injury. Deficiency of plasma ADAMTS13 activity (<10%) resulting from mutations of the ADAMTS13 gene or autoantibodies against ADAMTS13 causes hereditary or acquired (idiopathic) TTP. ADAMTS13 activity is usually normal or modestly reduced (>20%) in other forms of thrombotic microangiopathy secondary to hematopoietic progenitor cell transplantation, infection, and disseminated malignancy or in hemolytic uremic syndrome. Plasma infusion or exchange remains the initial treatment of choice to date, but novel therapeutics such as recombinant ADAMTS13 and gene therapy are under development. Moreover, ADAMTS13 deficiency has been shown to be a risk factor for the development of myocardial infarction, stroke, cerebral malaria, and preeclampsia.

Bilirubin Directly Inhibits Proteolytic Cleavage of a Fluorogenic VWF73 Peptide and Plasma Von Willebrand Factor

Bilirubin is a yellow breakdown product of heme catabolism. Increased levels of plasma bilirubin are a condition commonly seen in neonates and adults with hemolysis. The unconjugated bilirubin consists of an open chain of four pyrrole-like rings. Previous studies have shown that bilirubin decreases the cleavage of a commercially available FRETS­­­­­-VWF73 assay, resulting in falsely low plasma ADAMTS13 activity. However, the mechanism underlying such a reduction of ­­­­ADAMTS13 activity is not fully understood. Plasma ADAMTS13 activity is critical for differential diagnosis of thrombotic microangiopathy in which low ADAMTS13 activity (less than 5-10%) is more consistent with thrombotic thrombocytopenic purpura (TTP), while normal to mildly reduced ADAMTS13 activity (greater than 10-20%) is the common findings of hemolytic uremic syndrome (HUS). Here, we show that bilirubin directly inhibits the proteolytic cleavage of a fluorogenic VWF73 peptides labeled with fluorescein-5-maleimide (FL485-VWF73) (ext. 485nm/emi.530nm) or DyLight-633-maleimide (DL633-VWF73) (ext.638nm/emi.658 nm) in a concentration-dependent manner (Fig. 1). The IC50 for inhibiting the cleavage of FL485-VWF73 and DL633-VWF73 was estimated to be 13 micro mol/L and 70 micro mol/L, respectively. To confirm the direct inhibitory activity, bilirubin at various concentrations was incubated with plasma-derived multimeric VWF and recombinant ADAMTS13 in the presence of 1.5 M urea, 5 mM Tris-HCl, pH 8.0 for four hours. The cleavage of multimeric VWF was determined by 1% agarose and SDS-polyacrylamide gel electrophoreses, followed by Western blotting with rabbit anti-VWF IgG. The results demonstrated that bilirubin also inhibited recombinant ADAMTS13-mediated cleavage of VWF in a concentration-dependent manner with an IC50 ~0.43 milli mol/L (Fig. 2). The inhibitory activity of bilirubin on the cleavage of a fluorogenic peptide or a multimeric VWF substrate was largely eliminated by addition of bilirubin oxidase that converts bilirubin to biliverdin (not shown). Our results demonstrate for the first time that unconjugated bilirubin directly inhibits plasma and recombinant ADAMTS13 activity in addition to its known interference with certain fluorogenic assays for ADAMTS13 activity. These findings are clinically important concerning diagnosis and differential diagnosis of thrombotic microangiopathy. Additionally, our findings may shed new light on the mechanism of thrombotic complications pertinent to acute hemolysis and liver diseases. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Folded ADAMTS13 Conformation Identified By Small-Angle X-Ray Scattering Can Account for Allosteric Regulation By Distal Thrombospondin-1 and CUB Domains

Introduction: ADAMTS13 is a multidomain protein with metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C) and spacer (S) domains, followed by 7 T domains and 2 CUB domains. ADAMTS13 cleaves the cryptic Tyr1605-Met1606 bond in the A2 domain of von Willerbrand factor (VWF), which inhibits the growth of platelet-rich thrombi. When subjected to tensile stress in solution, bound to platelets, or on endothelial cell surfaces, VWF interacts with multiple exosites on ADAMTS13, changing the conformation of both proteins. These close contacts enhance the highly specific interaction between ADAMTS13 and VWF in vivo. Interactions between VWF and proximal MDTCS domains of ADAMTS13 have been investigated extensively. ADAMTS13 distal domains T8-CUB2 are required to bind the D4 domain of VWF, and the CUB domains promote the cleavage of platelet-decorated VWF strings. However, the functional relationship between distal and proximal domains is not understood. Using mutagenesis, small-angle X-ray scattering (SAXS) and enzyme kinetics, we now have shown that distal T8-CUB domains interact with and inhibit the proximal MDTCS domains.Methods: Recombinant ADAMTS13, MDTCS, and variants truncated after each distal T domain (MT2, MT3ÉMT8), and VWF D4 domain were produced in T-Rex 293 cell lines and purified to homogeneity. Similar inactive variants of each protein were prepared with the mutation E225Q, which abolishes catalytic activity but does not affect protein folding (MT2Q, MT3QÉMT8Q). SAXS data were collected at the SIBYLS beamline (Lawrence Berkeley National Laboratory) for ADAMTS13 (E225Q) and truncated variants. The radius of gyration (Rg) and maximum particle size (Dmax) were calculated from scattering profiles using DATGNOM. Ab initio envelopes were generated from scattering profiles using DAMMIN. A molecular model of ADAMTS13 was built from crystal structures of ADAMTS4 MD domains (2rjp) and ADAMTS13 DTCS domains (3ghm), and using HHpred to model distal T domains, CUB domains, and linkers after T4 and T8. Plasma VWF was purified from Facteur Willebrand-LFB concentrate on Superdex 200. ADAMTS13 activity assays were performed at pH 6 and pH 7.4 with fluorogenic substrate FRETS-rVWF71.Results: Rg and Dmax progressively increased for variants MDTCS, MT2, MT3, MT4 and MT5, but did not increase proportionately for MT7, MT8 and ADAMTS13 despite a 50% increase in mass (Table 1). This behavior is consistent with a folded conformation. Fitting ADAMTS13 into ab initio envelopes for these variants requires a bend after domain T4, which is followed by a flexible linker that could serve as a hinge. As a consequence, distal T8-CUB2 domains are positioned near the proximal MDTCS domains (Figure 1). At pH 7.4, variants MDTCS through MT7 had similar activity toward FRETS-rVWF71. Full length ADAMTS13 was only ~10% as active at pH 7.4, but was ~7-fold more active at pH 6. MT8 had intermediate activity at pH 7.4 and was not activated by lowering the pH to 6. Adding VWF or recombinant VWF D4 increased the cleavage of FRETS-rVWF71 by full-length ADAMTS13 up to 4-fold at pH 7.4, but had no effect at pH 6 and did not change the activity of MT8 or MDTCS.Conclusions: ADAMTS13 adopts a folded conformation with distal T8-CUB2 domains close to the proximal MDTCS domains and a hinge point between the T4 and T5 domains. The T8-CUB domains inhibit ADAMTS13 activity, and this autoinhibition can be relieved by low pH or by binding to VWF. Thus, VWF functionally serves as both an ADAMTS13 activator and substrate. This allosteric mechanism would localize and concentrate ADAMTS13 activity on VWF multimers at sites of thrombosis.Table 1Properties of ADAMTS13 VariantsVariantMass (kDa)Rg(Angstrom)aDmax(Angstrom)bkcat, pH 6 (min-1)ckcat, pH 7.4 (min-1)cMDTCS6745.2 ± 1.1148.3 ± 2.09.86 ± 0.0410.94 ± 0.01MT27347.8 ± 2.8154.8 ± 6.19.19 ± 0.2410.23 ± 0.07MT37954.3 ± 4.8179.5 ± 10.19.69 ± 0.279.69 ± 0.27MT49058.7 ± 3.7193.1 ± 15.97.85 ± 0.049.80 ± 0.27MT59663.4 ± 3.0210.1 ± 8.97.23 ± 0.239.43 ± 0.22MT710963.8 ± 2.0210.5 ± 11.58.34 ± 0.258.90 ± 0.10MT812164.7 ± 2.5206.2 ± 2.53.01 ± 0.022.44 ± 0.01ADAMTS1314767.2 ± 1.9228.9 ± 6.27.15 ± 0.201.05 ± 0.11aCalculated using the program AutoRg, errors are SD. bCalculated using the program DATGNOM, errors are SD. cErrors are SE [Display omitted] DisclosuresSadler:XO1 Limited: Membership on an entity’s Board of Directors or advisory committees; BioMarin: Consultancy; Band Therapeutics: Consultancy; Baxter HealthCare: Consultancy, Honoraria.

Platelet-free shear flow assay facilitates analysis of shear-dependent functions of VWF and ADAMTS13

IntroductionThe multimeric form of von Willebrand factor (VWF), is the largest soluble protein in mammals and exhibits a multidomain structure resulting in multiple functions. Upon agonist stimulation endothelial cells secrete VWF multimers from Weibel-Palade bodies into the blood stream where VWF plays an essential role in platelet-dependent primary hemostasis. Elongation of VWF strings on the cells’ surface leads to accessibility of VWF binding sites for proteins, such as platelet membrane glycoprotein Ib. The prothrombotic strings are size-regulated by the metalloprotease ADAMTS13 by shear force-activated proteolytic cleavage. Material and MethodsVWF string formation was induced by histamine stimulation of HUVEC cells under unidirectional shear flow and VWF strings were detected employing the VWF binding peptide of platelet glycoprotein Ib coupled to latex beads. VWF strings were then used as substrate for kinetic studies of recombinant and plasma ADAMTS13. ResultsTo investigate specific aspects of the shear-dependent functions of VWF and ADAMTS13, we developed a shear flow assay that allows observation of VWF string formation and their degradation by ADAMTS13 without the need for isolated platelets. Our assay specifically detects VWF strings, can be coupled with fluorescent applications and allows semi-automated, quantitative assessment of recombinant and plasma ADAMTS13 activity. ConclusionsOur assay may serve as a valuable research tool to investigate the biochemical characteristics of VWF and ADAMTS13 under shear flow and could complement diagnostics of von Willebrand Disease and Thrombotic Thrombocytopenic Purpura as it allows detection of shear flow-dependent dysfunction of VWD-associated VWF mutants as well as TTP-associated ADAMTS13 mutants.

ADAMTS13 and its variants promote angiogenesis via upregulation of VEGF and VEGFR2.

Severe plasma ADAMTS13 deficiency results in the clinical disorder thrombotic thrombocytopenic purpura. However, other potential pathophysiological roles of ADAMTS13 in endothelial cell biology remain unexplored. The goals of this study were to understand the angiogenic pathways ADAMTS13 activates and to identify the important structural components of ADAMTS13 that stimulate angiogenesis. Incubation of human umbilical vein endothelial cells (HUVEC) with 150 ng/mL (1 nM) of recombinant human ADAMTS13 induced VEGF expression by 53 % and increased VEGF mRNA by over sixfold, both within 10 min; the measured VEGF levels steadily decreased over 2 h, as shown by Western blot and ELISA. Phosphorylation of VEGFR2 was significantly enhanced in HUVEC after incubation with ADAMTS13 (1 nM). Structure-function analysis showed that an ADAMTS13 variant containing thrombospondin type 1 (TSP1) 2-8 repeats (TSP1 2-8), TSP1 2-8 plus CUB domains (TSP1 2-8 plus CUB), or TSP1 5-8 repeats plus CUB domains (TSP1 5-8 plus CUB) increased HUVEC proliferation by 41-54 % as compared to the EBM-2 controls. Chemotaxis assays further demonstrated that the TSP1 domains of ADAMTS13 increased HUVEC migration by 2.65-fold. Incubation of HUVEC with both ADAMTS13 variants containing TSP1 repeats and anti-VEGF IgG abrogated the enhanced effect of ADAMTS13 on proliferation, migration, and VEGFR2 phosphorylation. In conclusion, ADAMTS13-induced endothelial cell angiogenesis occurs via the upregulation of VEGF and phosphorylation of VEGFR2. This angiogenic activity depends on the C-terminal TSP1 repeats of ADAMTS13.

Effect of recombinant ADAMTS‐13 on microthrombosis and brain injury after experimental subarachnoid hemorrhage

A common complication after aneurysmal subarachnoid hemorrhage (SAH) is delayed cerebral ischemia (DCI), which is associated with vasospasm and other mechanisms such as microthrombosis. ADAMTS-13 activity plays a role in the prevention of thrombus formation in the cerebral microvasculature. Previously, we observed that patients with DCI have lower levels of ADAMTS-13. To examine whether recombinant human ADAMTS-13 (rADAMTS-13) reduces cerebral microthrombus formation and brain injury in an experimental mouse model of SAH including wild-type and ADAMTS-13(-/-) mice. Experimental SAH was induced with the prechiasmatic blood injection model. The following experimental groups were investigated: (i) C57BL/6J mice (n = 10); (ii) C57BL/6J mice (n = 10) treated with rADAMTS-13 20 min after SAH; (iii) ADAMTS-13(-/-) mice (n = 10); and (iv) ADAMTS-13(-/-) mice (n = 10) treated with rADAMTS-13 20 min after SAH. Mice were killed at 48 h. Results are presented as means with standard errors of the mean. Infusion with rADAMTS-13 reduced the extent of microthrombosis by ~ 50% in both wild-type mice (mean fibrinogen area: 0.28% ± 0.09% vs. 0.15% ± 0.04%; P = 0.20) and ADAMTS-13(-/-) mice (mean fibrinogen area: 0.32% ± 0.05% vs. 0.16% ± 0.03%; P = 0.016). In addition, rADAMTS-13 reduced brain injury by > 60% in both wild-type mice (mean microglia area: 0.65% ± 0.18% vs. 0.18% ± 0.04%; P = 0.013) and ADAMTS-13(-/-) mice (mean microglia area: 1.24% ± 0.36% vs. 0.42% ± 0.13%; P = 0.077). Our results support the further study of rADAMTS-13 as a treatment option for the prevention of microthrombosis and brain injury after SAH.

Inherited Thrombotic Thrombocytopenic Purpura in Children

Congenital thrombotic thrombocytopenic purpura (TTP) or Upshaw-Schulman syndrome is caused by homozygous or compound heterozygous mutations in the ADAMTS-13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) gene. We investigated 30 patients with congenital TTP and analyzed clinical data and underlying ADAMTS-13 mutations. All patients showed virtually no ADAMTS-13 activity in plasma. Individual disease burden ranged from mild courses with rare episodes of mild thrombocytopenia to severe courses with chronic kidney disease and central nervous system (CNS) lesions. Two patients died due to complications of TTP. If initiated in a timely manner, plasma transfusions offer a reliable treatment to prevent organ damage. We identified 30 different causative mutations in the ADAMTS-13 gene. Our data do not support the idea of a tight correlation between ADAMTS-13 genotype and severity of disease. The type and magnitude of exogenous triggers for acute bouts of TTP as well as endogenous individual factors participating in the inflammatory response likely represent the foremost determinants of individual clinical courses. Future developments should aim at improving early diagnosis of TTP. To improve feasibility of prophylaxis and treatment of congenital TTP, recombinant ADAMTS-13 therapeutics are highly anticipated.

Identification of N‐linked glycosylation and putative O‐fucosylation, C‐mannosylation sites in plasma derived ADAMTS13

Acquired deficiency of ADAMTS13 causes a rare and life-threatening disorder called thrombotic thrombocytopenic purpura (TTP). Several studies have shown that aberrant glycosylation can play an important role in the pathogenesis of autoimmune diseases.N-linked glycosylation and putative O-fucosylation sites have been predicted or identified in recombinant ADAMTS13. However, it is not known which of these sites are glycosylated in plasma derived ADAMTS13. Here we investigated the presence of putative O-fucosylation, C-mannosylation and N-linked glycosylation sites on plasma derived ADAMTS13. Sites of N-linked glycosylation were determined by the use of peptide N-glycosidase-F (PNGase F), which removes the entire carbohydrate from the side chain of asparagines. Nine of the 10 predicted N-linked glycosylation sites were identified in or near the metalloproteinase,spacer, thrombospondin type 1 repeat (TSR1) and the CUB domain of plasma ADAMTS13. Moreover, six putative O-fucosylated sites were identified in the TSR domains of plasma ADAMTS13 by performing searches of the tandem mass spectrometry (MS/MS) data for loss of hexose (162 Da), deoxyhexose (146 Da), or hexose deoxyhexose(308 Da). The use of electron transfer dissociation (ETD) allowed for unambiguous identification of the modified sites. In addition to putative O-fucosylation and N-linked glycosylation, two putative C-mannosylation sites were identified within the TSR1 and TSR4 domains of ADAMTS13. Our data identify several glycosylation sites on plasma derived ADAMTS13. We anticipate that our findings may be relevant for the initiation of autoimmune reactivity against ADAMTS13 in patients with acquired TTP.

Von Willebrand factor and ADAMTS13 in arterial thrombosis: a systematic review and meta-analysis

Von Willebrand Factor (VWF) plays an important role in hemostasis by mediating platelet adhesion and aggregation. Ultralarge VWF multimers are cleaved by ADAMTS13 in smaller, less procoagulant forms. An association between high VWF levels and cardiovascular disease has frequently been reported, and more recently also an association has been observed between low ADAMTS13 levels and arterial thrombosis. We reviewed the current literature and performed meta-analyses on the relationship between both VWF and ADAMTS13 with arterial thrombosis. Most studies showed an association between high VWF levels and arterial thrombosis. It remains unclear whether ADAMTS13 is a causal independent risk factor because the association between low ADAMTS13 and arterial thrombosis is so far only shown in case-control studies. Prospective studies are awaited. A causal role for ADAMTS13 is supported by mice studies of cerebral infarction where the infusion of recombinant human ADAMTS13 reduced the infarct size.

VWF-mediated leukocyte recruitment with chromatin decondensation by PAD4 increases myocardial ischemia/reperfusion injury in mice

AbstractInnate immune cells play a major role in the early response to myocardial ischemia/reperfusion (MI/R) injury. Recombinant human ADAMTS13 (rhADAMTS13), cleaving von Willebrand factor (VWF), reduces leukocyte recruitment in mice. Death of cardiomyocytes and the possible formation of neutrophil extracellular traps (NETs) may result in chromatin release that is prothrombotic and cytotoxic. We investigated the pathophysiological role of extracellular chromatin during MI/R to evaluate the therapeutic potential of targeting extracellular DNA and VWF by using DNase I with/without rhADAMTS13. Finally, we examined the impact of histone citrullination and NETosis by peptidylarginine deiminase 4 (PAD4) on MI/R. We used a 24-hour MI/R mouse surgical model. MI/R injury caused an increase in plasma nucleosomes, abundant neutrophil infiltration, and the presence of citrullinated histone H3 at the site of injury. Both monotherapies and coadministration of DNase I and rhADAMTS13 revealed a cardioprotective effect, resulting in subsequent improvement of cardiac contractile function. PAD4−/− mice, which do not produce NETs, were also significantly protected from MI/R and DNase I treatment had no further beneficial effect. We demonstrate that extracellular chromatin released through NETosis exacerbates MI/R injury. Targeting both VWF-mediated leukocyte recruitment and chromatin removal may be a new therapeutic strategy to reduce ischemia-related cardiac damage.

Thrombotic thrombocytopenic purpura: basic pathophysiology and therapeutic strategies.

VWF is a multimeric plasma glycoprotein that specifically recruits platelets to sites of vessel injury. VWF multimeric size is central to this function, with larger multimers being more hemostatically active. Regulation of VWF multimeric size is mediated by the plasma metalloprotease ADAMTS13 (A Disintegrin And Metalloproteinase with ThromboSpondin type 1 motifs, member 13). This enzyme can only recognize and cleave VWF when it is unraveled by rheological shear forces of the flowing blood. After the exposure of cryptic exosites, VWF recognition by ADAMTS13 involves multiple interactions that enable the protease to cleave VWF. Loss of VWF multimer size regulation caused by severe ADAMTS13 deficiency (either inherited or acquired) is associated with the microvascular thrombotic disorder thrombotic thrombocytopenic purpura (TTP). The sequelae associated with TTP are widely thought to be linked to hyperreactive circulating VWF that cause unwanted platelet aggregation in the high shear environment of the microvasculature. Diagnosis of TTP is primarily made through a combination of symptoms, analysis of plasma ADAMTS13 activity, and detection of inhibitory anti-ADAMTS13 antibodies. Current frontline treatments for TTP include plasma exchange, which serves to remove inhibitory antibodies (in acquired TTP) and provide a source of functional ADAMTS13, and steroids to treat the autoimmune component of acquired TTP. The use of anti-CD20 therapy has also exhibited encouraging results in the treatment of acquired TTP. Newer therapeutic strategies that are currently being explored or are in development include recombinant ADAMTS13, a hyperreactive ADAMTS13 variant, and anti-VWF therapy. This review discusses the basic biochemistry of VWF and ADAMTS13, their dysfunction in TTP, and therapeutic approaches for the amelioration of TTP.

Carboxyl Terminal Domains Of ADAMTS13 Inhibit The Formation Of Ultra Large VWF Strings and Platelet/Leukocyte Aggregates On Endothelial Cells In Murine Mesenteric Venules Via a Disulfide Bond Reduction Mechanism

von Willebrand factor (VWF) undergoes self-association mediated by free cysteine residues between two apposed VWF multimers. This process occurs in solution or between soluble and ultra large (UL) VWF on endothelial cells or matrix under flow. ADAMTS13 is a key regulator for the ULVWF string formation and adhesive function, primarily through its proteolytic cleavage at the Tyr1605-Met1606 bond in the central A2 domain of VWF. The proteolytic cleavage of VWF by ADAMTS13 rapidly removes newly secreted ULVWF on activated endothelial cell surface, thereby preventing excessive platelet aggregation and thrombus formation under physiological conditions. Previous studies have shown that the proteolytic cleavage of both cell-bound and soluble ULVWF by ADAMTS13 depends on its amino terminal MDTCS domains. However, the role of more distal carboxyl-terminal domains is not fully established. Here, we show by N-ethylmaleimide (NEM) labeling and mass spectrometry that TSP1 2-8 repeats and CUB domains of human recombinant ADAMTS13 contain multiple free cysteine residues (-SH). We hypothesize that these free thiols may directly interact with those on the surface of VWF multimers, thereby preventing their lateral association, thereby inhibiting thrombus formation under flow. Using an intravital microscopic imaging technique we demonstrate that even in the absence of circulating ADAMTS13 (Adamts13-/-) the formation and elongation of ULVWF strings on endothelial cells of mesenteric venules after FeCl3 injury are highly dynamic over time (180 seconds), suggesting other proteases or reductases in blood are able cleave or reduce ULVWF strings under flow. However, the average length of ULVWF strings remains relatively constant. Recombinant ADAMTS13 (rA13) at physiological concentration (10 nM), when infused, rapidly cleaves or eliminates preformed ULVWF strings and platelet/leukocyte aggregates on endothelial cells. The efficacy of rA13 in reducing the average length of ULVWF strings and platelet/leukocyte aggregation decreases by ∼50% if rA13 is pretreated with NEM. Moreover, a C-terminal fragment of ADAMTS13 comprising 2-8 TSP1 repeats and two CUB domains (T2C) or containing 5-8 TSP1 repeats and two CUB domains (T5C), when infused, also significantly attenuates the formation and/or elongation of ULVWF strings/bundles and platelet/leukocyte aggregation on endothelial cells under the same conditions. The inhibitory activity of T2C and T5C is dose-dependent. At the concentration of 0.5 µM, the average length of the ULVWF strings and the surface coverage of platelets/leukocytes decrease by ∼50% and ∼40% (p&lt;0.001), respectively. Also, treatment with NEM abolishes the inhibitory effect of T2C and T5C on the ULVWF string formation and platelet/leukocyte aggregation on activated endothelial cells surface (Fig. 1). In conclusion, our results demonstrate for the first time that the distal C-terminal fragments of ADAMTS13 lacking the protease domain are of direct anti-thrombotic activity, which is mediated through its disulfide-bond reduction mechanism. Furthermore, the carboxyl-terminal disulfide bond reducing activity may contribute to the overall anti-thrombotic activity of ADAMTS13 in vivo. Disclosures: No relevant conflicts of interest to declare.

ADAMTS13 Expressed In Platelets Offers Systemic Protection Against Arterial Thrombosis and Therapeutic Benefits In Murine Models Of Thrombotic Thrombocytopenic Purpura

ADAMTS13, a plasma metalloprotease, cleaves von Willebrand factor (VWF) and inhibits arterial thrombosis and inflammatory response. Deficiency of plasma ADAMTS13, due hereditary mutations in ADAMTS13 gene or autoantibodies against ADAMTS13 protease, results in a potentially fatal syndrome, thrombotic thrombocytopenic purpura (TTP). Plasma infusion or exchange is the only effective therapy available to date. To develop novel therapeutics against TTP, we tested a hypothesis that recombinant ADAMTS13, expressed specifically in platelets, may offer systemic protection against arterial thrombosis and therefore provide therapeutic benefit for TTP. To test this hypothesis, we first generated transgenic mice (JAX B6SJL) carrying a human full-length ADAMTS13 gene under a murine platelet glycoprotein 1b (CD41) promoter. The transgenic mice were then bred with Adamts13-/- (CAST/Ei) mice for &gt;4 generations. By Western blotting and activity assay, we show that a full-length human ADAMTS13 protein (∼195 kDa) and its proteolytic activity toward a FRETS-hVWF73 peptide are detected in platelet lysate obtained from transgenic (rA13-PltTG) mice but not in Adamts13-/- mice or wild-type mice. Little to no ADAMTS13 activity was detected in plasma in transgenic mice, suggesting the expressed human ADAMTS13 is stored inside platelets. ADAMTS13 was releasable upon stimulation with thrombin (1 U/ml), collagen (10 µg/ml), and AYPGKF (0.5 mM). More significantly, rA13-PltTG mice had higher baseline platelet count than Adamts13-/- mice, but exhibited a dramatically reduced rate of thrombus formation in mesenteric arterioles after oxidative injury with 10% ferric chloride as compared with Adamts13-/-mice and wild-type mice. Finally, rA13-PltTG mice were protected from Shigatoxin-2 (Stx-2) or murine recombinant VWF (mVWF)-induced “TTP-like” syndrome, as demonstrated by fewer rA13-PltTG mice having thrombocytopenia (defined by a 40% drop in platelet count from the baseline after challenge with Stx-2 or mVWF), faster and more complete recovery of thrombocytopenia, and significantly higher survival rate than Adamts13-/- mice. In summary, we have generated transgenic mice expressing human ADAMTS13 in platelets. Platelet ADAMTS13 is releasable upon stimulation by agonists and biologically functional in proteolytic cleavage of VWF in vitro. The platelet-derived ADAMTS13 offers systemic protection against arterial thrombosis after oxidative injury and provide a therapeutic benefit to murine models of TTP, resulting from hereditary ADAMTS13 deficiency. We are now in the process testing the efficacy of this strategy as a novel therapeutic for acquired TTP with inhibitors. Disclosures: No relevant conflicts of interest to declare.

ADAM28 Metalloprotease Domain Is Sufficient For Proteolytic Cleavage Of Von Willlebrand Factor

ADAM28, a member of ADAM (A Disintegrin and Metalloprotease) family, consists of two isoforms: a membrane-type form and a secreted form. Both ADAM28 isoforms are expressed in normal T and B lymphocytes of peripheral blood and lymphatic organs and certain malignant tumor cells. ADAM28 exhibits catalytic activity toward a few substrates including insulin-like growth factor binding protein-3 (IGFBP-3) and von Willebrand factor (VWF). ADAM28 is also overexpressed in human non-small cell lung carcinomas and breast carcinomas, predominantly by maligant cells, and the expression levels correlate with malignant cell proliferation and lymph node metastasis, presumably mediated by the cleavage of VWF. However, the structural components of ADAM28 required for proteolytic cleavage of VWF are not known. In this study, we constructed and expressed a soluble murine ADAM28 lacking the transmembrane domain and cytoplasmic tail (ΔTMA28) and various other carboxyl-terminal truncated ADAM28 variants containing a metalloprotease domain (M), plus a disintegrin domain (MD), and a Cys-rich domain (MDC) in COS-7 cells. All constructs were tagged at their carboxyl termini with a 3xflag epitope. Conditioned medium from transfected cells was collected and concentrated by a filtration technique. Protein concentrations of each construct in the concentrated conditioned medium were determined by a semi-quantitative Western blotting using a monoclonal anti-Flag IgG. A carboxyl-terminal truncated ADAMTS13 fragment (MDTCS) containing a 3xFlag epitope was used for a calibration. The proteolytic cleavage of a murine recombinant VWF (mVWF) by murine recombinant ADAM28 variants was carried out at 37 °C in a buffer containing 1 µM ZnCl2 and 5 mM CaCl2 in the presence or in the absence of guanidine-HCl at neutral pH 7.5. A full-length murine recombinant ADAMTS13 (mFL-A13) was used as a positive control and the conditioned medium derived from untransfected cells was used for a negative control. We show that mVWF that is pre-denatured with 1.0 M guanidine-HCl (at 37 °C for 2 hours) was efficiently cleaved by the constructs M, MD, MDC, and ΔTMA28 at the concentration of 50-100 nM after 18 hours of incubation. The proteolytic cleavage of VWF by ADAMTS28 variants results in dramatically reduced immune reactivity with rabbit anti-VWF IgG and multimer size as demonstrated by SDS-agarose gel (1%) electrophoresis and Western blotting. Paradoxically, this cleavage significantly increases the VWF-collagen binding activity despite of smaller VWF multimer size, suggesting that ADAM28-mediated proteolysis of VWF exposes its high-affinity collagen binding site. The cleavage efficiency of mVWF by all ADAM28 variants is comparable to that by mFL-A13 under the same conditions. The cleavage of native mVWF by ADAM28 is much less than that of pre-denatured VWF. EDTA (10 mM) added to the reaction completely abolishes the proteolytic cleavage of mVWF by all mADAM28 variants. We conclude that a metalloprotease domain of mADAM28 is sufficient for proteolytic cleavage of mVWF. The carboxyl terminal ancillary domains of mADAM28 appear to be dispensable for recognition and cleavage of mVWF. Our findings suggest a different mechanism underlying the proteolysis of VWF by a ADAM family protease. Further investigation into the biological activity of ADAM28 variants under more physiological conditions may shed new light on the mechanism of ADAM and ADAMTS proteolysis. The findings may provide a potential novel therapy for arterial thrombosis and acquired thrombotic thrombocytopenic purpura due to autoantibody inhibition because ADAM28 activity is not expected to be inhibited by ADAMTS13-specific autoantibodies. Disclosures:No relevant conflicts of interest to declare.

ADAMTS13 Expressed In Glioblastoma and Astrocytoma Cells Can Modulate Endothelial Cell Migration

ADAMTS13, the von Willebrand factor (vWF) cleaving protease, regulates platelet aggregation and microthrombi formation by cleaving high-molecular weight vWF multimers. It is expressed primarily in hepatic stellate cells, but is also found in endothelial cells. Recently, ADAMTS13 was reported to be expressed and regulated in astrocytes, microglial, neuroblastoma, and adult human brain endothelial cells. Previous in vitro studies by our group with human umbilical vein endothelial cells (HUVEC) showed that ADAMTS13 can promote angiogenesis via upregulating the secretion of VEGF and phosphorylation of VEGFR2, suggesting that ADAMTS13 may also be involved in physiological processes unrelated to vWF cleavage (Lee, M., et al. Microvasc Res. 2012, 84, 109-115). Herein, we report an additional possible role of ADAMTS13 secreted by brain tumor cells to modulate tumor cell angiogenesis.Using a human ADAMTS13 immunoassay, we detected ADAMTS13 in U-87 and LN-229 glioblastoma cell lysates, SW-1088 astrocytoma cell lysates, as well as in the supernatants of all cell lines (> 2.0 ng/mL). Co-incubation of U-87, LN-229, and SW-1088 tumor cell conditioned media with recombinant vWF indicated that brain tumor-secreted ADAMTS13 is biologically active in cleaving vWF multimers (measured by ELISA). Secretion of VEGF was upregulated in LN-229 and SW-1088 cell lines by ADAMTS13. 939 pg/mL and 674 pg/mL of VEGF were measured in LN-229 and SW-1088 cell lysates, respectively, after incubation with 100 ng/mL ADAMTS13. Incubation of LN-229 glioblastoma cells with 10 – 500 ng/mL rh-ADAMTS13 or 50 ng/mL VEGF165 did not affect tumor cell proliferation. No change in tumor cell proliferation was observed when LN-229 cells were incubated with a polyclonal antibody against ADAMTS13 in serum free media supplemented with 10 ng/mL ADAMTS13 or media supplemented with 5% FBS, suggesting that ADAMTS13 secreted by brain tumor cells may be involved in extracellular signaling of endothelial cells.Brain tumor cell secreted-ADAMTS13 induced HUVEC migration in a Matrigel invasion assay. Using a modified Boyden chamber fitted with a Matrigel-coated polycarbonate membrane, LN-229 glioblastoma cells increased HUVEC migration by 83%. LN-229 cells supplemented with 10 ng/mL ADAMTS13 further increased HUVEC migration by 190%, suggesting that tumor cell-secreted ADAMTS13 may modulate EC migration (Fig. 1). Combined with our previous findings suggesting that recombinant ADAMTS13 modulates EC angiogenesis, brain tumor-secreted ADAMTS13 may also be a regulator for tumor vasculature angiogenesis. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Anti-idiotypic small molecules neutralize pathogenic anti-ADAMTS13 autoantibodies in autoimmune Thrombotic Thrombocytopenic Purpura in vitro - new treatment tools in vivo? (P5173)

Abstract A therapy aiming at neutralization of inhibitory autoantibodies (Abs) causing autoimmune thrombotic thrombocytopenic purpura (aTTP) would be highly desirable. We screened a large combinatorial small protein library of Designed-Ankryin-Repeat-Protein (DARPins, Molecular Partners AG, Switzerland; library size 1015-23) for anti-idiotypic binders to an equimolar pool of three spleen-derived inhibitory anti-ADAMTS13 monoclonal Abs of two aTTP patients containing 1 of 4 shared CDR3 motifs. Four unique anti-idiotypic DARPins were purified and their neutralization potential tested against 8 spleen-derived anti-ADAMTS13 Abs (200 nM), belonging to the same, shared CDR3 motif used for selection. Anti-idiotypic DARPins restored ADAMTS13 activity up to 100% (2000 nM). By ELISA we found Abs in plasma of 27 of 37 randomly selected aTTP patients that bound specifically to the anti-idiotypic DARPins. Pre-incubation with anti-idiotypic DARPins (960 nM) prevented binding of plasma anti-ADAMTS13 Ab to recombinant ADAMTS13 in a competition ELISA (n=5). Our findings of anti-idiotypic DARPins able to neutralize several, although similar (belonging to the same CDR3 motif) pathogenic anti-ADAMTS13 Abs and the fact that the DARPins are also specifically recognized by plasma-derived anti-ADAMTS13 Abs of different aTTP patients are promising and suggest that possibly only a limited number of the selected DARPins are required to develop a general treatment for aTTP.