Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 103, Issue 50, Pages 19099-19104Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0607264104
Keywords
enzyme kinetics; thrombotic thrombocytopenic purpura; fluid shear stress
Categories
Funding
- NCI NIH HHS [P30 CA 91842, P30 CA091842] Funding Source: Medline
- NHLBI NIH HHS [R01 HL072917, HL 72917] Funding Source: Medline
Ask authors/readers for more resources
Von Willebrand factor (VWF) is a multimeric protein that mediates platelet adhesion at sites of vascular injury, and ADAMTS13 (a disintegrin and metalloprotease with thrombospondin)is a multidomain metalloprotease that limits platelet adhesion by a feedback mechanism in which fluid shear stress induces proteolysis of VWF and prevents disseminated microvascular thrombosis. Cleavage of the Tyr(1605)-Met(1606) scissile bond in the VWF A2 domain depends on a Glu(1660)-Arg(1668) segment in the same domain and on the noncatalytic spacer domain of ADAMTS13, suggesting that extensive enzyme-substrate interactions facilitate substrate recognition. Based on mutagenesis and kinetic analysis, we find that the ADAMTS13 spacer domain binds to an exosite near the C terminus of the VWF A2 domain. Deleting the spacer domain from ADAMTS13 or deleting the exosite from the VWF substrate reduced the rate of cleavage approximate to 20-fold. A cleavage product containing the exosite was a hyperbolic mixed-type inhibitor of ADAMTS13 proteolysis of either VWF multimers or model peptide substrates but only if the ADAMTS13 enzyme contained the spacer domain. The specificity of this unique mechanism depends on tension-induced unfolding of the VWF A2 domain, which exposes the scissile bond and exosite for interaction with complementary sites on ADAMTS13.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available