Conformational Stability and Domain Unfolding of the Von Willebrand Factor A Domains

Matthew T Auton, Miguel A. Cruz, Joel Moake

Research output: Contribution to journalArticle

63 Citations (Scopus)

Abstract

Von Willebrand factor (VWF), a multimeric multidomain glycoprotein secreted into the blood from vascular endothelial cells, initiates platelet adhesion at sites of vascular injury. This process requires the binding of platelet glycoprotein Ib-IX-V to the A1 domain of VWF monomeric subunits under fluid shear stress. The A2 domain of VWF monomers contains a proteolytic site specific for a circulating plasma VWF metalloprotease, A Disintegrin and Metalloprotease with Thrombospondin motifs, member #13 of the ADAMTS enzyme family (ADAMTS-13), that functions to reduce adhesiveness of newly released, unusually large (UL), hyperactive forms of VWF. Shear stress assists ADAMTS-13 proteolysis of ULVWF multimers allowing ADAMTS-13 cleavage of an exposed peptide bond in the A2 domain. Shear stress may induce conformational changes in VWF, and even unfold regions of VWF monomeric subunits. We used urea as a surrogate for shear to study denaturation of the individual VWF recombinant A domains, A1, A2, and A3, and the domain triplet, A1-A2-A3. Denaturation was evaluated as a function of the urea concentration, and the intrinsic thermodynamic stability of the domains against unfolding was determined. The A1 domain unfolded in a 3-state manner through a stable intermediate. Domains A2 and A3 unfolded in a 2-state manner from native to denatured. The A1-A2-A3 triple domain unfolded in a 6-state manner through four partially folded intermediates between the native and denatured states. Urea denaturation of A1-A2-A3 was characterized by two major unfolding transitions: the first corresponding to the simultaneous complete unfolding of A2 and partial unfolding of A1 to the intermediate state; and the second corresponding to the complete unfolding of A3 followed by gradual unfolding of the intermediate state of A1 at high urea concentration. The A2 domain containing the cleavage site for ADAMTS-13 was the least stable of the three domains and was the most susceptible to unfolding. The low stability of the A2 domain is likely to be important in regulating the exposure of the A2 domain cleavage site in response to shear stress, ULVWF platelet adherence, and the attachment of ADAMTS-13 to ULVWF.

Original languageEnglish (US)
Pages (from-to)986-1000
Number of pages15
JournalJournal of Molecular Biology
Volume366
Issue number3
DOIs
StatePublished - Feb 23 2007
Externally publishedYes

Fingerprint

von Willebrand Factor
varespladib methyl
Urea
Platelet Glycoprotein GPIb-IX Complex
Metalloproteases
Blood Platelets
Thrombospondins
Disintegrins
Adhesiveness
Vascular System Injuries
Thermodynamics
Proteolysis
Glycoproteins
Endothelial Cells
Peptides

Keywords

  • ADAMTS-13
  • linear extrapolation method
  • protein unfolding
  • shear stress
  • thrombotic thrombocytopenia purpura
  • urea
  • Von Willebrand factor
  • Von Willebrand's disease

ASJC Scopus subject areas

  • Virology

Cite this

Conformational Stability and Domain Unfolding of the Von Willebrand Factor A Domains. / Auton, Matthew T; Cruz, Miguel A.; Moake, Joel.

In: Journal of Molecular Biology, Vol. 366, No. 3, 23.02.2007, p. 986-1000.

Research output: Contribution to journalArticle

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N2 - Von Willebrand factor (VWF), a multimeric multidomain glycoprotein secreted into the blood from vascular endothelial cells, initiates platelet adhesion at sites of vascular injury. This process requires the binding of platelet glycoprotein Ib-IX-V to the A1 domain of VWF monomeric subunits under fluid shear stress. The A2 domain of VWF monomers contains a proteolytic site specific for a circulating plasma VWF metalloprotease, A Disintegrin and Metalloprotease with Thrombospondin motifs, member #13 of the ADAMTS enzyme family (ADAMTS-13), that functions to reduce adhesiveness of newly released, unusually large (UL), hyperactive forms of VWF. Shear stress assists ADAMTS-13 proteolysis of ULVWF multimers allowing ADAMTS-13 cleavage of an exposed peptide bond in the A2 domain. Shear stress may induce conformational changes in VWF, and even unfold regions of VWF monomeric subunits. We used urea as a surrogate for shear to study denaturation of the individual VWF recombinant A domains, A1, A2, and A3, and the domain triplet, A1-A2-A3. Denaturation was evaluated as a function of the urea concentration, and the intrinsic thermodynamic stability of the domains against unfolding was determined. The A1 domain unfolded in a 3-state manner through a stable intermediate. Domains A2 and A3 unfolded in a 2-state manner from native to denatured. The A1-A2-A3 triple domain unfolded in a 6-state manner through four partially folded intermediates between the native and denatured states. Urea denaturation of A1-A2-A3 was characterized by two major unfolding transitions: the first corresponding to the simultaneous complete unfolding of A2 and partial unfolding of A1 to the intermediate state; and the second corresponding to the complete unfolding of A3 followed by gradual unfolding of the intermediate state of A1 at high urea concentration. The A2 domain containing the cleavage site for ADAMTS-13 was the least stable of the three domains and was the most susceptible to unfolding. The low stability of the A2 domain is likely to be important in regulating the exposure of the A2 domain cleavage site in response to shear stress, ULVWF platelet adherence, and the attachment of ADAMTS-13 to ULVWF.

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KW - thrombotic thrombocytopenia purpura

KW - urea

KW - Von Willebrand factor

KW - Von Willebrand's disease

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