Christopher G. Skipwith

Multiple domains of ADAMTS13 are targeted by autoantibodies against ADAMTS13 in patients with acquired idiopathic thrombotic thrombocytopenic purpura

Background Type G immunoglobulins against ADAMTS13 are the primary cause of acquired (idiopathic) thrombotic thrombocytopenic purpura. However, the domains of ADAMTS13 which the type G anti-ADAMT13 immunoglobulins target have not been investigated in a large cohort of patients with thrombotic thrombocytopenic purpura. Design and Methods Sixty-seven patients with acquired idiopathic thrombotic thrombocytopenic purpura were prospectively collected from three major U.S. centers. An enzyme-linked immunosorbent assay determined plasma concentrations of anti-ADAMTS13 type G immunoglobulins, whereas immunoprecipitation plus western blotting determined the binding domains of these type G immunoglobulins. Results Plasma anti-ADAMTS13 type G immunoglobulins from 67 patients all bound full-length ADAMTS13 and a variant truncated after the eighth TSP1 repeat (delCUB). Approximately 97% (65/67) of patients harbored type G immunoglobulins targeted against a variant truncated after the spacer domain (MDTCS). However, only 12% of patients’ samples reacted with a variant lacking the Cys-rich and spacer domains (MDT). In addition, approximately 37%, 31%, and 46% of patients’ type G immunoglobulins interacted with the ADAMTS13 fragment containing TSP1 2-8 repeats (T2-8), CUB domains, and TSP1 5-8 repeats plus CUB domains (T5-8CUB), respectively. The presence of type G immunoglobulins targeted against the T2-8 and/or CUB domains was inversely correlated with the patients’ platelet counts on admission. Conclusions This multicenter study further demonstrated that the multiple domains of ADAMTS13, particularly the Cys-rich and spacer domains, are frequently targeted by anti-ADAMTS13 type G immunoglobulins in patients with acquired (idiopathic) thrombotic thrombocytopenic purpura. Our data shed more light on the pathogenesis of acquired thrombotic thrombocytopenic purpura and provide further rationales for adjunctive immunotherapy.

Gain-of-function ADAMTS13 variants that are resistant to autoantibodies against ADAMTS13 in patients with acquired thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) is primarily caused by immunoglobulin G (IgG) autoantibodies against A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats, 13 (ADAMTS13). Nearly all adult idiopathic TTP patients harbor IgGs, which bind the spacer domain of ADAMTS13, a region critical for recognition and proteolysis of von Willebrand factor (VWF). We hypothesize that a modification of an exosite in the spacer domain may generate ADAMTS13 variants with reduced autoantibody binding while preserving or enhancing specific activity. Site-directed mutagenesis was used to generate a series of ADAMTS13 variants, and their functional properties were assessed. Of 24 novel ADAMTS13 variants, 2 (ie, M4, R660K/F592Y/R568K/Y661F and M5, R660K/F592Y/R568K/Y661F/Y665F) exhibited increased specific activity approximately 4- to 5-fold and approximately 10- to 12-fold cleaving a peptide VWF73 substrate and multimeric VWF, respectively. More interestingly, the gain-of-function ADAMTS13 variants were more resistant to inhibition by anti-ADAMTS13 autoantibodies from patients with acquired idiopathic TTP because of reduced binding by anti-ADAMTS13 IgGs. These results shed more light on the critical role of the exosite in the spacer domain in substrate recognition. Our findings also help understand the pathogenesis of acquired autoimmune TTP. The autoantibody-resistant ADAMTS13 variants may be further developed as a novel therapeutic for acquired TTP with inhibitors.

Amino acid residues Arg659, Arg660 and Tyr661 in the spacer domain of ADAMTS13 are critical for cleavage of von Willebrand factor

Previous studies have shown that ADAMTS13 spacer domain is required for cleavage of von Willebrand factor (VWF). However, the exact amino acid residues within this domain critical for substrate recognition are not known. Epitope mapping of anti-ADAMTS13 immunoglobulin G from patients with thrombotic thrombocytopenic purpura and sequence alignment of the ADAMTS13 spacer domains of human, mouse, and zebrafish with these of human and murine ADAMTS1, a closely related member of ADAMTS family, have provided hints to investigate the role of the amino acid residues between Arg(659) and Glu(664) of the ADAMTS13 spacer domain in substrate recognition. A deletion of all these 6 amino acid residues (ie, Arg(659)-Glu(664)) from the ADAMTS13 spacer domain resulted in dramatically reduced proteolytic activity toward VWF73 peptides, guanidine-HCl denatured VWF, and native VWF under fluid shear stress, as well as ultralarge VWF on endothelial cells. Site-directed mutagenesis, kinetic analyses, and peptide inhibition assays have further identified a role for amino acid residues Arg(659), Arg(660), and Tyr(661) in proteolytic cleavage of various substrates under static and fluid shear stress conditions. These findings may provide novel insight into the structural-function relationship of ADAMTS13 and help us to understand pathogenesis of thrombotic thrombocytopenic purpura and other arterial thromboses associated with compromised VWF proteolysis.

Factor VIII and Platelets Synergistically Accelerate Cleavage of von Willebrand Factor by ADAMTS13 under Fluid Shear Stress

Previous studies have demonstrated that factor VIII (FVIII) or platelets alone increase cleavage of von Willebrand factor (VWF) by ADAMTS13 under mechanically induced shear stresses. We show in this study that the combination of FVIII and platelets at the physiological concentrations is more effective than either one alone. In the absence of FVIII, lyophilized platelets increase the formation of cleavage product by 2–3-fold. However, in the presence of physiological concentration of FVIII (1 nm), the formation of VWF cleavage product increases dramatically as a function of increasing platelets with the maximal rate enhancement of ∼8-fold. Conversely, in the presence of a physiological concentration of lyophilized platelets (150 × 103/μl), the half-maximal concentration of FVIII required to accelerate VWF proteolysis by ADAMTS13 reduces by ∼10-fold (to ∼0.3 nm) compared with that in the absence of platelets (∼3.0 nm). Further studies using the FVIII derivative that lacks an acidic region (a3), an antiplatelet glycoprotein 1bα IgG, and a purified recombinant VWF-A1 domain or glycoprotein 1bα-stripped platelets demonstrate that the synergistic rate-enhancing effect of FVIII and platelets depends on their specific binding interactions with VWF. Our findings suggest that FVIII and platelets are cofactors that regulate proteolysis of multimeric VWF by ADAMTS13 under physiological conditions.

Correction of ADAMTS13 Deficiency by In Utero Gene Transfer of Lentiviral Vector encoding ADAMTS13 Genes

Deficiency of A Disintegrin And Metalloprotease with ThromboSpondin (ADAMTS13) results in thrombotic thrombocytopenic purpura (TTP). Plasma infusion or exchange is the only effective treatment to date. We show in this study that an administration of a self-inactivating lentiviral vector encoding human full-length ADAMTS13 and a variant truncated after the spacer domain (MDTCS) in mice by in utero injection at embryonic days 8 and 14 resulted in detectable plasma proteolytic activity (approximately 5-70%), which persisted for the length of the study (up to 24 weeks). Intravascular injection via a vitelline vein at E14 was associated with significantly lower rate of fetal loss than intra-amniotic injection, suggesting that the administration of vector at E14 may be a preferred gestational age for vector delivery. The mice expressing ADAMTS13 and MDTCS exhibited reduced sizes of von Willebrand factor (vWF) compared to the Adamts13(-/-) mice expressing enhanced green fluorescent protein (eGFP). Moreover, the mice expressing both ADAMTS13 and MDTCS showed a significant prolongation of ferric chloride-induced carotid arterial occlusion time as compared to the Adamts13(-/-) expressing eGFP. The data demonstrate the successful correction of the prothrombotic phenotypes in Adamts13(-/-) mice by a single in utero injection of lentiviral vectors encoding human ADAMTS13 genes, providing the basis for developing a gene therapy for hereditary TTP in humans.

von Willebrand factor cleaved from endothelial cells by ADAMTS13 remains ultralarge in size

Von Willebrand factor (vWF) is synthesized in all vascular endothelial cells and megakaryocytes 1. The mature vWF is stored in the Weibel-Palade bodies of endothelial cells and the α-granules of platelets. A small fraction of vWF is constitutively secreted from the cells. Upon stimulation by thrombin, hormones, and inflammatory cytokines, vWF is released from the Weibel-Palade bodies of the endothelial cells as ultra large (UL) vWF, which forms polymers and remains attached to the endothelial cell surface via poorly characterized mechanisms, although recent reports have suggested that UL-vWF might be anchored on endothelial cells via interactions with P-selectin 2 and αvβ3 integrin 3. Proteolytic cleavage of the newly synthesized UL-vWF on endothelial cells by ADAMTS13 may be critical to maintain normal hemostasis and inflammatory response. For instance, certain mutations in the vwf gene may result in increased proteolysis of vWF by ADAMTS13 and significant reduction of vWF multimer sizes, leading to compromised hemostatic function as seen in patients with von Willebrand disease 4. Conversely, an inability to cleave the newly released UL-vWF strings 5;6 due to hereditary or acquired deficiency of ADAMTS13 may result in an accumulation of UL-vWF that results in formation of disseminated thrombi in the microvasculature as in patients with thrombotic thrombocytopenic purpura (TTP) 7. Moreover, ADAMTS13 deficiency appears to result in increased leukocyte rolling on unstimulated veins, leukocyte adhesion and extravasation of neutrophils in the inflamed veins 8. All these processes seem to depend on the presence of UL-vWF multimers 8. Therefore, the removal of cell bound UL-vWF by ADAMTS13 may help attenuate systemic inflammatory responses in addition to arterial thrombosis. In contrast to proteolytic cleavage of vWF in solution, which requires denaturant (urea 9 or guanidine 10) in a low ionic and alkaline buffer or high shear stress 11;12, the removal of UL-vWF polymers on cultured endothelial cells by ADAMTS13 occurs very efficiently under various shear stresses 5;6. The rapid removal of the UL-vWF strings by infused recombinant ADAMTS13 also occurs in both arteries (high shear) and venules (low shear) in Adamts13-/- mice 13. A recent study has shown that UL-vWF strings on histamine stimulated endothelial cells can be removed by ADAMTS13 in the absence of flow shear stress 14. It remains poorly understood what ADAMTS13 domains are required for cleavage of cell bound UL-vWF, and whether proteolytic cleavage of UL-vWF by ADAMTS13 on endothelial cell membranes is sufficient to reduce the multimer sizes of UL-vWF in the presence or absence of flow shear stress. In this study, we demonstrated by immunofluorescent microscopy that UL-vWF polymers were readily formed on the membrane of human umbilical vein endothelial cells (HUVECs) after being stimulated for 2 min with histamine (100 μM) in phosphate buffered saline (Fig. 1A-i). Recombinant ADAMTS13 (10 nM) (Fig. 1A-ii) or normal human plasma (NHP) (1:2 dilution) (not shown) in an assay buffer (20 mM HEPES, pH 7.4, 150 mM NaCl and 5 mM CaCl2) within 5 min could efficiently remove all surface bound UL-vWF polymers in the absence of flow shear stress. Plasma (1:2 dilution) from a patient with acquired idiopathic TTP with known high titer of anti-ADAMTS13 IgG autoantibodies, however, did not result in the removal of the cell bound UL-vWF polymers under the same conditions (not shown). These results are consistent with what has been reported by Turner et al 14, and are the premises for our further study of the structural and functional relationship of ADAMTS13 in cleavage of UL-vWF at the cellular levels. Figure 1 Cleavage of cell bound UL-vWF by recombinant ADAMTS13 and variants To better quantify the amount of UL-vWF released from endothelial cells, we employed an enzyme linked immunoassay (ELISA) using two polyclonal anti-vWF antibodies as described previously 15. In these experiments, HUVECs cultured on 6-well plate were stimulated with histamine (100 μM) for 2 min, washed with PBS and then incubated for 0~60 minutes without or with a fixed concentration (10 nM) of purified recombinant ADAMTS13 in 20 mM HEPES, 150 mM NaCl and 5 mM CaCl2, pH 7.4 (Fig. 1B-i) or for 5 min with various concentrations of recombinant ADAMTS13 (0~ 40 nM) (Fig. 1B-ii). The amount of vWF antigen in the conditioned media was increased in an incubation time and ADAMTS13 concentration dependent manner (Fig. 1B). After 60 min of incubation with purified recombinant ADAMTS13 (10 nM), the vWF antigen in the conditioned media (~24 ng/ml) was approximately 4-fold higher than in the buffer-treated control (~6 ng/ml) (Fig. 1B-i). The concentration of ADAMTS13 that achieved the half maximal levels of vWF released from endothelial cells was approximately 3.0 nM (Fig. 1B-ii), which is within the physiological range of ADAMTS13 in plasma. To determine whether or not the C-terminal domains of ADAMTS13 were required for cleavage of cell bound UL-vWF, histamine-stimulated and washed HUVECs were incubated with 10 nM of purified ADAMTS13 and various C-terminal truncated variants that were well characterized previously 12. The remaining UL-vWF on endothelial cells was determined by immunofluorescent microscopy using polyclonal anti-vWF IgG (Dako). We showed that the variants lacking the CUB domains (delCUB) (Fig. 1D-i) and truncated after the spacer domain (MDTCS) (Fig. 1D-ii) also efficiently removed UL-vWF polymers from histamine-stimulated endothelial cells, similar to full-length ADAMTS13 (Fig. 1A-ii). However, the variant truncated after the first thrombospondin type 1 (TSP1) repeat (MDT) (Fig. 1D-iii) or the metalloprotease domain (M) (Fig. 1D-iv) exhibited markedly impaired activity in removing the cell bound UL-vWF under the same conditions. The removal of cell bound UL-vWF polymers was consistent with the about 3~4 fold of increase in vWF antigen in the conditioned medium after incubation of the histamine-stimulated endothelial cells with full-length ADAMTS13, delCUB and MDTCS, but not with MDT and M or buffer alone (Fig. 1C-i). The proteolytic cleavage products (176kDa and 140 kDa) were also detectable in the conditioned media of HUVECs treated with FL-A13, delCUB and MDTCS, but not with MDT and M or buffer alone (Fig. 1C-ii). The amount of vWF antigen detected in the MDTCS-treated medium appeared to be slightly low compared with the FL-A13 and delCUB-treated media (Fig. 1C-ii), but such a difference was not statistically significant (p>0.05). The reduced cleavage products in the MDTCS-treated medium compared with full-length ADAMTS13 and delCUB may be a result of imperfect protein loading, rather than the lower proteolytic activity. Thus, our results suggest that the Cys-rich and spacer domains are required for recognition of the cell bound UL-vWF, but the TSP1 2-8 repeats and the CUB domains are dispensable. These results, however, do not fully agree with those reported 14. The reason for the discrepancy is not completely understood, but may be related to the assay methodologies being used. The domain requirement for proteolytic cleavage of cell bound UL-vWF is reminiscent of that seen in cleavage of soluble vWF by ADAMTS13 under denaturing conditions 16;17, suggesting that membrane association of UL-vWF results in a conformational change that permits ADAMTS13 binding in the absence of fluid shear stress. The mechanism underlying such a conformational change remains to be determined. To determine whether proteolytic cleavage of cell bound UL-vWF by ADAMTS13 was sufficient to reduce UL-vWF multimer size, the conditioned media were assessed by SDS-agarose (1%) gel electrophoresis and Western blot as previously described 15. Unexpectedly, whether in the absence of flow (Fig. 1E-i) or in the presence of flow (2.5 dynes/cm2) (Fig. 1E-ii), vWF multimers released into the conditioned media after being incubated with ADAMTS13 (10 nM) were indistinguishable from those in the conditioned media after being incubated with buffer alone or immediately after histamine stimulation (Fig. 1E-i & ii). When compared with normal human plasma, the conditioned media were highly enriched with UL-vWF as indicated by UL (Fig. 1E-i & ii). Incubation of the histamine-stimulated endothelial cells with recombinant ADAMTS13 (10 nM) for up to 60 min in the absence of flow (Fig. 1E-i, lanes 2-8) or for 5 min under various flow shear stresses up to ~25 dynes/cm2 (Fig. 1E-ii, lanes 3-7) did not appear to alter the vWF multimer distribution in the conditioned media, as compared with the UL-vWF released from the Weibel-Palade bodies after histamine stimulation (Fig. 1E-i, lane 9 & Fig. 1E-ii, lane 8), although some of the low molecular weight vWF (L) may be resulted from proteolytic degradation of the UL-vWF in the conditioned medium during storage. Addition of 0.1% protease inhibitor cocktail (Sigma) reduced the intensity of the smaller vWF bands. Nevertheless, the vWF cleaved off from endothelial cells by ADAMTS13 remains ultra large in size. This suggests further proteolytic processing downstream by ADAMTS13 or other leukocyte proteases 18, likely to occur in the small arteries and capillaries, where high fluid shear stress and perhaps physiological cofactors such as factor VIII 19 and platelets 20 are present, may be necessary to eliminate the ultra large vWF polymers.

Ion-Switchable Quantum Dot Förster Resonance Energy Transfer Rates in Ratiometric Potassium Sensors.

The tools for optically imaging cellular potassium concentrations in real-time are currently limited to a small set of molecular indicator dyes. Quantum dot-based nanosensors are more photostable and tunable than organic indicators, but previous designs have fallen short in size, sensitivity, and selectivity. Here, we introduce a small, sensitive, and selective nanosensor for potassium measurements. A dynamic quencher modulates the fluorescence emitted by two different quantum dot species to produce a ratiometric signal. We characterized the potassium-modulated sensor properties and investigated the photonic interactions within the sensors. The quenchers protonation changes in response to potassium, which modulates its Förster radiative energy transfer rate and the corresponding interaction radii with each quantum dot species. The nanosensors respond to changes in potassium concentrations typical of the cellular environment and thus provide a promising tool for imaging potassium fluxes during biological events.

Enzyme-linked DNA dendrimer nanosensors for acetylcholine.

It is currently difficult to measure small dynamics of molecules in the brain with high spatial and temporal resolution while connecting them to the bigger picture of brain function. A step towards understanding the underlying neural networks of the brain is the ability to sense discrete changes of acetylcholine within a synapse. Here we show an efficient method for generating acetylcholine-detecting nanosensors based on DNA dendrimer scaffolds that incorporate butyrylcholinesterase and fluorescein in a nanoscale arrangement. These nanosensors are selective for acetylcholine and reversibly respond to levels of acetylcholine in the neurophysiological range. This DNA dendrimer architecture has the potential to overcome current obstacles to sensing in the synaptic environment, including the nanoscale size constraints of the synapse and the ability to quantify the spatio-temporal fluctuations of neurotransmitter release. By combining the control of nanosensor architecture with the strategic placement of fluorescent reporters and enzymes, this novel nanosensor platform can facilitate the development of new selective imaging tools for neuroscience.

Quadruplex Integrated DNA (QuID) Nanosensors for Monitoring Dopamine

Dopamine is widely innervated throughout the brain and critical for many cognitive and motor functions. Imbalances or loss in dopamine transmission underlie various psychiatric disorders and degenerative diseases. Research involving cellular studies and disease states would benefit from a tool for measuring dopamine transmission. Here we show a Quadruplex Integrated DNA (QuID) nanosensor platform for selective and dynamic detection of dopamine. This nanosensor exploits DNA technology and enzyme recognition systems to optically image dopamine levels. The DNA quadruplex architecture is designed to be compatible in physically constrained environments (110 nm) with high flexibility, homogeneity, and a lower detection limit of 110 µM.

Compromised shear-dependent cleavage of type 2N von Willebrand factor variants by ADAMTS13 in the presence of factor VIII

Compromised shear-dependent cleavage of type 2N von Willebrand factor variants by ADAMTS13 in the presence of factor VIII -