Creation of a recombinant peptide substrate for fluorescence resonance energy transfer-based protease assays

Zhang, Lin; Lawson, Heather L.; Harish, Vallathucherry; Huff, Jason D.; Knovich, Mary Ann; Owen, John

doi:10.1016/j.ab.2006.06.022

Cited by 29 publications

(35 citation statements)

References 7 publications

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“…1C). All scFvs were screened for their ability to inhibit the cleavage of the fluorogenic substrate rF-VWF73, a 73-aa fluorescein-labeled peptide that contains the A2 cleavage site of VWF and mimics its ADAMTS13 binding sites (24)(25)(26).…”

Section: Resultsmentioning

confidence: 99%

“…Proteolytic activity was determined by the cleavage of MDTCS and a fluorogenic substrate derived from the VWF A2 domain, as previously described (24). Polyclonal antibody preparations were from 23 patients with acquired TTP, mostly collected before their first plasma exchange and occasionally during treatment or in remission, as detailed in Table S2.…”

Section: Methodsmentioning

confidence: 99%

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High-resolution epitope mapping by HX MS reveals the pathogenic mechanism and a possible therapy for autoimmune TTP syndrome

Casina

Mao

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Acquired thrombotic thrombocytopenic purpura (TTP), a thrombotic disorder that is fatal in almost all cases if not treated promptly, is primarily caused by IgG-type autoantibodies that inhibit the ability of the ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) metalloprotease to cleave von Willebrand factor (VWF). Because the mechanism of autoantibody-mediated inhibition of ADAMTS13 activity is not known, the only effective therapy so far is repeated whole-body plasma exchange. We used hydrogen-deuterium exchange mass spectrometry (HX MS) to determine the ADAMTS13 binding epitope for three representative human monoclonal autoantibodies, isolated from TTP patients by phage display as tethered single-chain fragments of the variable regions (scFvs). All three scFvs bind the same conformationally discontinuous epitopic region on five small solvent-exposed loops in the spacer domain of ADAMTS13. The same epitopic region is also bound by most polyclonal IgG autoantibodies in 23 TTP patients that we tested. The ability of ADAMTS13 to proteolyze VWF is impaired by the binding of autoantibodies at the epitopic loops in the spacer domain, by the deletion of individual epitopic loops, and by some local mutations. Structural considerations and HX MS results rule out any disruptive structure change effect in the distant ADAMTS13 metalloprotease domain. Instead, it appears that the same ADAMTS13 loop segments that bind the autoantibodies are also responsible for correct binding to the VWF substrate. If so, the autoantibodies must prevent VWF proteolysis simply by physically blocking normal ADAMTS13 to VWF interaction. These results point to the mechanism for autoantibody action and an avenue for therapeutic intervention.ADAMTS13 | von Willebrand factor | thrombotic thrombocytopenic purpura | hydrogen exchange | autoimmunity A cquired thrombotic thrombocytopenic purpura (TTP) is characterized by severe thrombocytopenia and microangiopathic hemolytic anemia, resulting from disseminated microvascular thrombosis. Patients with TTP may suffer from widespread organ damage, resulting in death if not aggressively treated (1, 2). In most patients, thrombotic microangiopathy is caused by IgG-type autoantibodies against the plasma metalloprotease ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) (3-5). ADAMTS13 regulates the function of the multidomain von Willebrand factor (VWF) (∼600 to ∼20,000 kDa) by cleaving it at the central A2 domain to regulate platelet-induced blood clot formation (3, 4).Immunological studies show that many acquired TTP patients with low plasma ADAMTS13 activity (less than 10%) harbor IgG autoantibodies that bind ADAMTS13 especially at the Cysrich and/or the spacer domain (4, 6). Deletion of these domains or grouped mutations therein can eliminate the binding of polyclonal anti-ADAMTS13 IgGs derived from many patients (4, 7-11). However, the exact ADAMTS13 binding sites of these autoantibodies are not known. Current treatment...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

High-resolution epitope mapping by HX MS reveals the pathogenic mechanism and a possible therapy for autoimmune TTP syndrome

Casina

Mao

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…35 Normal human plasma was used for calibration (1 U/mL activity). Murine plasma ADAMTS13 activity was determined by the cleavage of a commercial fluorescent resonance energy transfer VWF73 peptide (Peptide International, Louisville, KY), as described.…”

Section: Assay Of Adamts13 Activitymentioning

confidence: 99%

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

Pickens

Mao

et al. 2015

Blood

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“…[20][21][22] Multimeric human VWF was purified from cryoprecipitate of normal human plasma using ion exchange and gel filtration chromatography as described. 23 Fluorescein-5-malaimide-labeled VWF73 peptide (rF-VWF73) 24 …”

Section: Methodsmentioning

confidence: 99%

“…Proteolytic activity of rADAMTS13 in the absence or in the presence of purified or synthetic HNPs (0-150 mM) was determined by the cleavage of rF-VWF73 24,25 and multimeric VWF 27,28 under denaturing conditions using 1.5 M urea as previously described.…”

Section: Adamts13 Activity Assaysmentioning

confidence: 99%

Human neutrophil peptides inhibit cleavage of von Willebrand factor by ADAMTS13: a potential link of inflammation to TTP

Pillai

Bao

Zander

et al. 2016

Blood

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• HNPs inhibit proteolytic cleavage of VWF by ADAMTS13 by physically blocking VWF-ADAMTS13 interactions.• Plasma levels of HNP1, HNP2, and HNP3 are markedly increased in patients with acquired autoimmune TTP.Infection or inflammation may precede and trigger formation of microvascular thrombosis in patients with acquired thrombotic thrombocytopenic purpura (TTP). However, the mechanism underlying this clinical observation is not fully understood.Here, we show that human neutrophil peptides (HNPs) released from activated and degranulated neutrophils inhibit proteolytic cleavage of von Willebrand factor (VWF) by ADAMTS13 in a concentration-dependent manner. Half-maximal inhibitory concentrations of native HNPs toward ADAMTS13-mediated proteolysis of peptidyl VWF73 and multimeric VWF are 3.5 mM and 45 mM, respectively. Inhibitory activity of HNPs depends on the RRY motif that is shared by the spacer domain of ADAMTS13. Native HNPs bind to VWF73 (K D 5 0.72 mM), soluble VWF (K D 5 0.58 mM), and ultra-large VWF on endothelial cells. Enzyme-linked immunosorbent assay (ELISA) demonstrates markedly increased plasma HNPs1-3 in most patients with acquired autoimmune TTP at presentation (median, ∼170 ng/mL; range, 58-3570; n 5 19) compared with healthy controls (median, ∼23 ng/mL; range, 6-44; n 5 18) (P < .0001). Liquid chromatography plus tandem mass spectrometry (LC-MS/MS) reveals statistically significant increases of HNP1, HNP2, and HNP3 in patient samples (all P values <.001).There is a good correlation between measurement of HNPs1-3 by ELISA and by LC-MS/MS (Spearman r 5 0.7932, P < .0001).Together, these results demonstrate that HNPs1-3 may be potent inhibitors of ADAMTS13 activity, likely by binding to the central A2 domain of VWF and physically blocking ADAMTS13 binding. Our findings may provide a novel link between inflammation/ infection and the onset of microvascular thrombosis in acquired TTP and potentially other immune thrombotic disorders. (Blood. 2016;128(1):110-119)

show abstract

Creation of a recombinant peptide substrate for fluorescence resonance energy transfer-based protease assays

Cited by 29 publications

References 7 publications

High-resolution epitope mapping by HX MS reveals the pathogenic mechanism and a possible therapy for autoimmune TTP syndrome

High-resolution epitope mapping by HX MS reveals the pathogenic mechanism and a possible therapy for autoimmune TTP syndrome

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

Human neutrophil peptides inhibit cleavage of von Willebrand factor by ADAMTS13: a potential link of inflammation to TTP

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