Lisa A. Westfield

Allosteric activation of ADAMTS13 by von Willebrand factor

Significance The blood protein von Willebrand factor (VWF) is required for platelets to stop bleeding at sites of injury, and the metalloprotease ADAMTS13 limits platelet adhesion by cleaving VWF only when flowing blood stretches it, especially within a growing thrombus. This feedback inhibition is essential because ADAMTS13 deficiency causes fatal microvascular thrombosis. How ADAMTS13 recognizes VWF so specifically is not understood. We now find that ADAMTS13 is folded roughly in half so that its distal domains inhibit the metalloprotease domain. VWF relieves this autoinhibition and promotes its own destruction by allosterically activating ADAMTS13. Thus, VWF is both a substrate and a cofactor in this critical regulatory process. The metalloprotease ADAMTS13 cleaves von Willebrand factor (VWF) within endovascular platelet aggregates, and ADAMTS13 deficiency causes fatal microvascular thrombosis. The proximal metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C), and spacer (S) domains of ADAMTS13 recognize a cryptic site in VWF that is exposed by tensile force. Another seven T and two complement C1r/C1s, sea urchin epidermal growth factor, and bone morphogenetic protein (CUB) domains of uncertain function are C-terminal to the MDTCS domains. We find that the distal T8-CUB2 domains markedly inhibit substrate cleavage, and binding of VWF or monoclonal antibodies to distal ADAMTS13 domains relieves this autoinhibition. Small angle X-ray scattering data indicate that distal T-CUB domains interact with proximal MDTCS domains. Thus, ADAMTS13 is regulated by substrate-induced allosteric activation, which may optimize VWF cleavage under fluid shear stress in vivo. Distal domains of other ADAMTS proteases may have similar allosteric properties.

Unfolding the A2 Domain of Von Willebrand Factor with the Optical Trap

Von Willebrand factor (VWF) is a multimeric plasma glycoprotein involved in both hemostasis and thrombosis. VWF conformational changes, especially unfolding of the A2 domain, may be required for efficient enzymatic cleavage in vivo. It has been shown that a single A2 domain unfolds at most probable unfolding forces of 7-14 pN at force loading rates of 0.35-350 pN/s and A2 unfolding facilitates A2 cleavage in vitro. However, it remains unknown how much force is required to unfold the A2 domain in the context of a VWF multimer where A2 may be stabilized by other domains like A1 and A3. With the optical trap, we stretched VWF multimers and a poly-protein (A1A2A3)3 that contains three repeats of the triplet A1A2A3 domains at constant speeds of 2000 nm/s and 400 nm/s, respectively, which yielded corresponding average force loading rates of 90 and 22 pN/s. We found that VWF multimers became stiffer when they were stretched and extended by force. After force increased to a certain level, sudden extensional jumps that signify domain unfolding were often observed. Histograms of the unfolding force and the unfolded contour length showed two or three peaks that were integral multiples of approximately 21 pN and approximately 63 nm, respectively. Stretching of (A1A2A3)3 yielded comparable distributions of unfolding force and unfolded contour length, showing that unfolding of the A2 domain accounts for the behavior of VWF multimers under tension. These results show that the A2 domain can be indeed unfolded in the presence of A1, A3, and other domains. Compared with the value in the literature, the larger most probable unfolding force measured in this study suggests that the A2 domain is mechanically stabilized by A1 or A3 although variations in experimental setups and conditions may complicate this interpretation.

Phylogenetic and Functional Analysis of Histidine Residues Essential for pH-dependent Multimerization of von Willebrand Factor

von Willebrand factor (VWF) is a multimeric plasma protein that mediates platelet adhesion to sites of vascular injury. The hemostatic function of VWF depends upon the formation of disulfide-linked multimers, which requires the VWF propeptide (D1D2 domains) and adjacent D′D3 domains. VWF multimer assembly occurs in the trans-Golgi at pH ∼6.2 but not at pH 7.4, which suggests that protonation of one or more His residues (pKa ∼6.0) mediates the pH dependence of multimerization. Alignment of 30 vertebrate VWF sequences identified 13 highly conserved His residues in the D1D2D′D3 domains, and His-to-Ala mutagenesis identified His395 and His460 in the D2 domain as critical for VWF multimerization. Replacement of His395 with Lys or Arg prevented multimer assembly, suggesting that reversible protonation of this His residue is essential. In contrast, replacement of His460 with Lys or Arg preserved normal multimer assembly, whereas Leu, Met, and Gln did not, indicating that the function of His460 depends primarily upon the presence of a positive charge. These results suggest that pH sensing by evolutionarily conserved His residues facilitates the assembly and packaging of VWF multimers upon arrival in the trans-Golgi.

Rearranging Exosites in Noncatalytic Domains Can Redirect the Substrate Specificity of ADAMTS Proteases

Background: ADAMTS metalloproteases are multidomain proteins with remarkable substrate specificity. Results: Swapping noncatalytic domains between ADAMTS13 and ADAMTS5 causes reciprocal changes in the cleavage of their natural substrates. Conclusion: ADAMTS exosites in noncatalytic domains are portable modifiers of proteolytic activity. Significance: Shuffling and recombination of ADAMTS ancillary structural domains may be exploited to evolve or engineer new protease functions. ADAMTS proteases typically employ some combination of ancillary C-terminal disintegrin-like, thrombospondin-1, cysteine-rich, and spacer domains to bind substrates and facilitate proteolysis by an N-terminal metalloprotease domain. We constructed chimeric proteases and substrates to examine the role of C-terminal domains of ADAMTS13 and ADAMTS5 in the recognition of their physiological cleavage sites in von Willebrand factor (VWF) and aggrecan, respectively. ADAMTS5 cleaves Glu373–Ala374 and Glu1480–Gly1481 bonds in bovine aggrecan but does not cleave VWF. Conversely, ADAMTS13 cleaves the Tyr1605–Met1606 bond of VWF, which is exposed by fluid shear stress but cannot cleave aggrecan. Replacing the thrombospondin-1/cysteine-rich/spacer domains of ADAMTS5 with those of ADAMTS13 conferred the ability to cleave the Glu1615–Ile1616 bond of VWF domain A2 in peptide substrates or VWF multimers that had been sheared; native (unsheared) VWF multimers were resistant. Thus, by recombining exosites, we engineered ADAMTS5 to cleave a new bond in VWF, preserving physiological regulation by fluid shear stress. The results demonstrate that noncatalytic thrombospondin-1/cysteine-rich/spacer domains are principal modifiers of substrate recognition and cleavage by both ADAMTS5 and ADAMTS13. Noncatalytic domains may perform similar functions in other ADAMTS family members.

An optimized fluorogenic ADAMTS13 assay with increased sensitivity for the investigation of patients with thrombotic thrombocytopenic purpura.

Most ADAMTS13 assays use non‐physiological conditions (low ionic strength, low pH, barium chloride), are subject to interference from plasma proteins, hemoglobin and bilirubin, and have limited sensitivity, especially for inhibitors.

Molecular biology of von Willebrand factor.

von Willebrand factor is a complex, multimeric glycoprotein that performs at least two essential functions for hemostasis: it is required for the normal adhesion of platelets to areas of vascular damage, and it binds to and stabilizes blood clotting factor VIII in the circulation. I von Willebrand factor is synthesized by vascular endothelial and by megakary~cytes.~ Mature von Willebrand factor isolated from plasma consists of apparently identical subunits with molecular weight -250,000. These occur in a series of multimers that range in size from dimers to species of over 20 subunits. The biosynthesis of von Willebrand factor is a complicated process that includes proteolytic processing, the formation of many disulfide bonds within and between subunits, both N-linked and 0-linked glycosylation, and sulfation.5*6 The inherited deficiency of von Willebrand factor, or von Willebrand disease, is the most common genetic bleeding disorder of humans. Abnormalities of von Willebrand factor function can be demonstrated in approximately 8,000 persons per million population. Clinically significant von Willebrand disease is much less common, with a prevalence of approximately 125 per million persons. von Willebrand disease is a very heterogeneous disorder. Three major types of von Willebrand disease are currently recognized, and each of them is genetically and phenotypically heterogeneous. Simple quantitative deficiency of von Willebrand factor is referred to as von Willebrand disease type I. Qualitative abnormalities of von Willebrand factor are classified separately as von Willebrand disease type 11. von Willebrand disease types I and I1 usually show dominant inheritance. von Willebrand disease type I11 refers to a particularly severe phenotype characterized by essentially absent von Willebrand factor antigen and function, with recessive inheritance.‘” In the early 1980s, studies were initiated in several laboratories to clone cDNA for human von Willebrand factor and thereby to determine the primary structure of the protein. The ensuing years have seen remarkable progress in understanding the molecular biology of von Willebrand factor. Besides providing information concerning the structure of the von Willebrand factor gene and its protein product, these studies have shown that structural motifs first identified in von Willebrand factor are shared by many proteins in otherwiseunrelated gene families. In addition, recent studies have determined the cause of von Willebrand disease at the level of gene sequence in a small but rapidly growing number of patients.