Dataset Information

Single particle tracking of ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type-1 repeats) molecules on endothelial von Willebrand factor strings.

ABSTRACT: von Willebrand factor (VWF) strings are removed from the endothelial surface by ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type-1 repeats)-mediated proteolysis. To visualize how single ADAMTS13 molecules bind to these long strings, we built a customized single molecule fluorescence microscope and developed single particle tracking software. Extensive analysis of over 6,000 single inactive ADAMTS13(E225Q) enzymes demonstrated that 20% of these molecules could be detected in at least two consecutive 60-ms frames and followed two types of trajectories. ADAMTS13(E225Q) molecules either decelerated in the vicinity of VWF strings, whereas sometimes making brief contact with the VWF string before disappearing again, or readily bound to the VWF strings and this for 120 ms or longer. These interactions were observed at several sites along the strings. Control experiments using an IgG protein revealed that only the second type of trajectory reflected a specific interaction of ADAMTS13 with the VWF string. In conclusion, we developed a dedicated single molecule fluorescence microscope for detecting single ADAMTS13 molecules (nm scale) on their long, flow-stretched VWF substrates (μm scale) anchored on living cells. Comprehensive analysis of all detected enzymes showed a random interaction mechanism for ADAMTS13 with many available binding sites on the VWF strings.

SUBMITTER: De Ceunynck K

PROVIDER: S-EPMC3979392 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Single particle tracking of ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type-1 repeats) molecules on endothelial von Willebrand factor strings.

De Ceunynck Karen K Rocha Susana S De Meyer Simon F SF Sadler J Evan JE Uji-i Hiroshi H Deckmyn Hans H Hofkens Johan J Vanhoorelbeke Karen K

The Journal of biological chemistry 20140218 13

von Willebrand factor (VWF) strings are removed from the endothelial surface by ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type-1 repeats)-mediated proteolysis. To visualize how single ADAMTS13 molecules bind to these long strings, we built a customized single molecule fluorescence microscope and developed single particle tracking software. Extensive analysis of over 6,000 single inactive ADAMTS13(E225Q) enzymes demonstrated that 20% of these molecules could be detected in a ...[more]

PMID: 24550384

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Project description:Objective- ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats-13) cleaves VWF (von Willebrand factor). This process is essential for hemostasis. Severe deficiency of plasma ADAMTS13 activity, most commonly resulting from autoantibodies against ADAMTS13, causes thrombotic thrombocytopenic purpura. Therapeutic plasma exchange is the standard of care to date, which removes autoantibodies and replenishes ADAMTS13. However, such a therapy is often ineffective to raise plasma ADAMTS13 activity, and in-hospital mortality rate remains as high as 20%. Approach and Results- To overcome the inhibition by autoantibodies, we developed a novel approach by delivering rADAMTS13 (recombinant ADAMTS13 ) using platelets as vehicles. We show that both human and murine platelets can uptake rADAMTS13 ex vivo. The endocytosed rADAMTS13 within platelets remains intact, active, and is stored in α-granules. Under arterial shear (100 dyne/cm2), the rADAMTS13 in platelets is released and effectively inhibits platelet adhesion and aggregation on a collagen-coated surface in a concentration-dependent manner. Transfusion of rADAMTS13-loaded platelets into Adamts13-/- mice dramatically reduces the rate of thrombus formation in the mesenteric arterioles after FeCl3 injury. An ex vivo transfusion of rADAMTS13-loaded platelets to a reconstituted whole blood containing plasma from a patient with immune-mediated thrombotic thrombocytopenic purpura and the cellular components (eg, erythrocytes and leukocytes) from a healthy individual, as well as a fresh whole blood obtained from a patient with congenital or immune-mediated thrombotic thrombocytopenic purpura also dramatically reduces the rate of thrombus formation under arterial flow. Conclusions- Our results demonstrate that transfusion of rADAMTS13-loaded platelets may be a novel and potentially effective therapeutic approach for arterial thrombosis, associated with congenital and immune-mediated thrombotic thrombocytopenic purpura.

Dataset Information

Single particle tracking of ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type-1 repeats) molecules on endothelial von Willebrand factor strings.

Publications

Single particle tracking of ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type-1 repeats) molecules on endothelial von Willebrand factor strings.

Similar Datasets

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