6 research outputs found
VWF processing by ADAMTS13: unraveling the mode of action.
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mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}Von Willebrand factor (VWF) plays a crucial role during haemostasis andits activity is regulated by the length of this multimeric protein. The size ofVWF is after secretion reduced by ADAMTS13. ADAMTS13 consists of ametalloprotease domain, a disintegrine-like domain, a thrombospondine type 1repeat (TSP1), a cysteine rich and a spacer domain, seven additional TSP1repeats and two CUB domains. The function of the different domains of ADAMTS13on its activity in vitro has beenstudied, but their contribution to the proteolysis of VWF remains largelyuncovered.Seven C-terminally truncated murine ADAMTS13(mADAMTS13) mutants were constructed and expressed in HEK293T cells. Allmutants were characterised in vitro:both proteolysis of the truncated substrate GST-VWF73 and VWF multimers wasinvestigated. The above tests are only qualitative and for kinetic analysis,FRETS-VWF73 was used. As could be expected, murine MDTCS (devoid of the T2-8and CUB domains) retained full enzyme activity and like seen for human ADAMTS13,mADAMTS13 without spacer domain (MDT and M) had reduced catalytic efficiency(fifty fold reduction in kcat/KM). Similar domains inmurine and human ADAMTS13 are important for the activity in vitro which motivates the use of murine models to study ADAMTS13in vivo. Using intravital fluorescencemicroscopy, platelet-decorated VWF multimers ( strings ) which form on theactivated/damaged endothelium of the mesenteric venules in mice, were visualised.These VWF strings are long-lived in Adamts13-/-mice, while they are rapidly digested in wild type mice and thus can beused as a measure for ADAMTS13 activity invivo. Surprisingly the absence of the CUB domains completely abolished theADAMTS13 activity, while MDTCS retained full functionality and the partial(del(T6-CUB)) or complete (delCUB) addition of the tandem T2-8 repeats resultedin a gradual decrease of the activity. ADAMTS13 CUB and T2-8 domains influencethe proteolysis of platelet-decorated VWF strings in vivo and emphasise the importance of these domains. Possibly theTSP1 repeats shield the spacer or the catalytic site and is this interactiononly broken after binding of the CUB domains to VWF. An interaction between theN- and C-terminal domains of ADAMTS13 is currently under investigation andbinding of the TSP1 repeats and CUB domains will be examined in the currentmodel.status: publishe
Paratope and epitope mapping of the antithrombotic antibody 6B4 in complex with platelet glycoprotein Ibalpha
No full textThe monoclonal antibody 6B4 has a potent antithrombotic effect in nonhuman primates by binding to the flexible loop, also known as the beta-switch region (amino acids 230-242), of glycoprotein Ibalpha (GPIbalpha). This interaction blocks, in high shear stress conditions, the specific interaction between GPIbalpha and von Willebrand factor suppressing platelet deposition to the damaged vessel wall, a key event in the pathogenesis of arterial thrombosis. To understand the interactions between this antibody and its antigen at the amino acid level, we here report the identification of the paratope and epitope in 6B4 and GPIbalpha, respectively, by using computer modeling and site-directed mutagenesis. The docking programs ZDOCK (rigid body docking) and HADDOCK (flexible docking) were used to model the interaction of 6B4 with GPIbalpha and to delineate the respective paratope and epitope. 6B4 and GPIbalpha mutants were constructed and assayed for their capacity to bind GPIbalpha and 6B4, respectively. From these data, it is found that the paratope of 6B4 is mainly formed by five residues: Tyr(27D), Lys(27E), Asp(28), and Glu(93) located in light chain CDR1 and -3, respectively, and Tyr(100C) of the heavy chain CDR3. These residues form a valley, where the GPIbalpha flexible loop can bind via residues Asp(235) and Lys(237). The experimental results were finally used to build a more accurate docking model. Taken together, this information provides guidelines for the design of new derivatized lead compounds with antithrombotic properties.status: publishe
The distal carboxyterminal domains of murine ADAMTS13 influence proteolysis of platelet-decorated VWF strings in vivo
No full textBackground: The multidomain metalloprotease ADAMTS13 regulates the size of von Willebrand factor (VWF) multimers upon their release from endothelial cells. How the different domains in ADAMTS13 control VWF proteolysis in vivo remains largely unidentified. Methods: Seven C-terminally truncated murine ADAMTS13 (mADAMTS13) mutants were constructed and characterized in vitro. Their ability to cleave VWF strings in vivo was studied in the ADAMTS13-/- mouse. Results: Murine MDTCS (devoid of T2-8 and CUB domains) retained full enzyme activity in vitro towards FRETS-VWF73 and the C-terminal T6-8 (del(T6-CUB)) and CUB domains (delCUB) are dispensable under these assay conditions. In addition, mADAMTS13 fragments without the spacer domain (MDT and M) had reduced catalytic efficiencies. Our results hence indicate that similar domains in murine and human ADAMTS13 are required for activity in vitro, supporting the use of mouse models to study ADAMTS13 function in vivo. Interestingly, using intravital microscopy we show that removal of the CUB domains abolishes proteolysis of platelet-decorated VWF strings in vivo. In addition, whereas MDTCS is fully active in vivo, partial (del(T6-CUB)) or complete (delCUB) addition of the T2-8 domains gradually attenuates its activity. Conclusions: Our data demonstrate that the ADAMTS13 CUB and T2-8 domains influence proteolysis of platelet-decorated VWF strings in vivo.status: publishe
The novel ADAMTS13-p.D187H mutation impairs ADAMTS13 activity and secretion and contributes to thrombotic thrombocytopenic purpura in mice
No full textSummary: Background: Congenital thrombotic thrombocytopenic purpura (TTP) is characterized by mutations in the ADAMTS13 gene, which either impair protein secretion or influence ADAMTS13 (A Disintegrin-like And Metalloprotease domain with ThromboSpondin type-1 motif, member 13) activity. Phenotypic consequences of these mutations have not yet been evaluated in animal models for TTP. Objectives: To identify the in vitro effect of a novel ADAMTS13 mutation and to investigate whether this mutation induces TTP in vivo. Methods: All 29 ADAMTS13 exons with exon-intron boundaries of a patient with pregnancy-onset TTP were sequenced. Wild-type and mutant ADAMTS13 proteins were both transiently and stably expressed in human embryonic kidney cells, and their activity was evaluated in vitro using fluorescence resonance energy transfer and flow assays. Molecular dynamics simulations were performed to study Ca2+ stability. Adamts13-/- mice were hydrodynamically injected with wild-type and mutant expression plasmids and triggered with recombinant human von Willebrand factor. Results: We identified a novel heterozygous c.559G>C mutation in exon 6 of the proposita's ADAMTS13 gene. This mutation resulted in a p.Asp187His substitution (p.D187H), which was located in the high affinity Ca2+-binding site in the metalloprotease domain of ADAMTS13. The homozygous p.D187H mutation down-regulated ADAMTS13 activity in vitro. Impaired proteolytic activity was linked to unstable Ca2+ binding as visualized using a molecular dynamics simulation. In addition, the p.D187H mutation affects protein secretion in vitro. In Adamts13-/- mice, the homozygous p.D187H mutation reduced ADAMTS13 secretion and activity and contributed to TTP when these mice were triggered with recombinant human von Willebrand factor. Conclusions: Our data indicate that the p.D187H mutation impairs ADAMTS13 activity and secretion and is responsible for TTP onset in mice
Development and characterization of new species cross-reactive anti-sialoadhesin monoclonal antibodies
No full textDevelopment and Characterization of New Species Cross-Reactive Anti-Sialoadhesin Monoclonal Antibodies
Get PDFSialoadhesin (Sn) is a surface receptor expressed on a subset of macrophages in steady state conditions. During inflammation and diseases, Sn is highly upregulated on macrophages and blood monocytes. Therefore, therapies using monoclonal antibodies (mAbs) to target Sn-positive (Sn+) cells are a potential strategy for targeted treatment. It has been shown that Sn internalizes after binding with a mAb, though it is not clear whether this is species-specific. In this study, new Sn-specific mAbs were developed and analyzed for cross-reactivity between species. In addition, the newly developed mAbs were compared to mAbs used in previous research for their epitope recognition and other Sn-specific characteristics. Both species-specific and cross-reactive antibodies could be identified. Furthermore, sialic acid-binding of red blood cells (RBC) could be inhibited with mAbs recognizing different epitopes and all mAb showed internalization of Sn. The newly developed mAbs can be used as novel tools for Sn research and further analysis of Sn internalization in different species
