Domain-specific mechanical modulation of VWF–ADAMTS13 interaction

Abstract

Hemodynamic forces activate the Von Willebrand factor (VWF) and facilitate its cleavage by a disintegrin and metalloprotease with thrombospondin motifs-13 (ADAMTS13), reducing the adhesive activity of VWF. Biochemical assays have mapped the binding sites on both molecules. However, these assays require incubation of two molecules for a period beyond the time allowed in flowing blood. We used a single-molecule technique to examine these rapid, transient, and mechanically modulated molecular interactions in short times under forces to mimic what happens in circulation. Wild-type ADAMTS13 and two truncation variants that either lacked the C-terminal thrombospondin motif-7 to the CUB domain (MP-TSP6) or contained only the two CUB domains (CUB) were characterized for interactions with coiled VWF, flow-elongated VWF, and a VWF A1A2A3 tridomain. These interactions exhibited distinctive patterns of calcium dependency, binding affinity, and force-regulated lifetime. The results suggest that 1) ADAMTS13 binds coiled VWF primarily through CUB in a calcium-dependent manner via a site(s) outside A1A2A3, 2) ADAMTS13 binds flow-extended VWF predominantly through MP-TSP6 via a site(s) different from the one(s) at A1A2A3; and 3) ADAMTS13 binds A1A2A3 through MP-TSP6 in a Ca2+-dependent manner to autoinhibit another Ca2+-independent binding site on CUB. These data reveal that multiple sites on both molecules are involved in mechanically modulated VWF–ADAMTS13 interaction.

Mechanical Forces Impacting Cleavage of Von Willebrand Factor in Laminar and Turbulent Blood Flow

“…To unfold the A2 domain, 10-22 pN [44][45][46][47] of tension must exist locally across the domain, which is expected to occur near the multimer center. In addition, the average lifetime of VWF and ADAMTS13 bonds is maximized at 22 pN [48]. For high 5500-10,000 kDa multimers, cleavage is expected when shear rates exceed 10,000 s −1 , but could theoretically commence at shear rates greater than physiological values of 2800 s −1 .…”

Insights Into Immunothrombosis: The Interplay Among Neutrophil Extracellular Trap, von Willebrand Factor, and ADAMTS13

“…ADAMTS13 is a multi-domain protein including a metalloprotease domain (M), a disintegrin-like domain (D), a thrombospondin Type-1 domain (T), a cysteine-rich domain (C), a spacer domain (S), followed by seven additional thrombospondin Type-1 repeats (T2-8) and two CUB (complement c1r/c1s, sea urchin epidermal growth factor, and bone morphogenetic protein) domains (71,72). Evidence shows that various domains of ADAMTS13 bind to VWF (73,74). Notably, the lack of spacer domain results in a dramatic reduction in its proteolytic activity, suggesting spacer domain is vital (75,76).…”

“…It has been well established that blood flow mediates the recognition and cleavage processes of VWF by ADAMTS13 ( 73 ). As mentioned above, high shear stress unfolds VWF A2 domain to expose the scissile bond Tyr1605-Met1606 for ADAMTS13 cleavage, down-regulating the activity of VWF multimers and preventing excessive platelet aggregation.…”

“…By the use of single-molecule techniques, we and other groups have demonstrated that A2 unfolding is the prerequisite for ADAMTS13 cleavage ( 92 – 94 ). Recently, we reported, for the first time, that the interactions between VWF and ADAMTS13 were modulated by mechanical forces, exhibiting multiple bond characteristics: slip bonds, catch bonds, biphasic bonds, and triphasic bonds ( 73 ). These dynamic bonds might be critical, as such interaction can be mechanically strengthened or weakened by the formation of various dynamic bonds between different binding sites in circulation.…”

From DNA damage to epithelial integrity: new roles for cell forces

“…The net effect leads to a nontrivial feedback between binding and microvillus motion, which selectively stabilizes scanning microvilli for antigen discrimination. Zhenhai Li and coworkers devote their attention to the molecular physics of the large blood polymer Von Willebrand factor, which has long been known to not just extend under flow but then be cleaved and activated by protease. Single-molecule force measurements are applied to various constructs to uncover the stretch-dependent interactions and their force-dependent lifetime, revealing site interactions that depend on forced conformation and sometimes calcium too.…”