Mechanoenzymatic Cleavage of the Ultralarge Vascular Protein von Willebrand Factor

Abstract

Von Willebrand factor (VWF) is secreted as ultralarge multimers that are cleaved in the A2 domain by the metalloprotease ADAMTS13 to give smaller multimers. Cleaved VWF is activated by hydrodynamic forces found in arteriolar bleeding to promote hemostasis, whereas uncleaved VWF is activated at lower, physiologic shear stresses and causes thrombosis. Single-molecule experiments demonstrate that elongational forces in the range experienced by VWF in the vasculature unfold the A2 domain, and only the unfolded A2 domain is cleaved by ADAMTS13. In shear flow, tensile force on a VWF multimer increases with the square of multimer length and is highest at the middle, providing an efficient mechanism for homeostatic regulation of VWF size distribution by force-induced A2 unfolding and cleavage by ADAMTS13, as well as providing a counterbalance for VWF-mediated platelet aggregation.

The effects of shear flow on protein structure and function

“…The results showed that only the unfolded A2 domain is cleaved by ADAMST-13. 62 These observations corroborated previous studies which found that shear-induced stretching of large vWF multimers results in the exposure of the scissile Tyr 1605 -Met 1606 bond within the A2 domain, facilitating cleavage by ADAMST-13 in normal plasma. 56,57 Smaller vWF multimers in microcirculation equally undergo stretching in regions of high shear, such as a bleeding arteriole, which expose bonding sites in the A1 domain of vWF for complexation with platelets during haemostasis.…”

“…62 Specifically, the study examined the folded and unfolded states of a single A2 domain of vWF, which contains the site of cleavage for ADAMST-13, in the presence and absence of the metalloprotease. The A2 domain of vWF was unfolded by directly applying force with laser tweezers.…”

“…63,114 On the other hand, the smaller size of circulating vWF multimers, resulting from proteolysis, enhance their haemostatic potential in high shear regimes as occurs during arteriolar bleeding. 62 The influence of shear stress on the activity of platelets and vWF has been discussed in other review articles. 57,58,63,115 Others have linked the deterioration in cerebral microcirculation (cerebral hypoperfusion) to the onset of protein conformational disorders such as Alzheimer's disease.…”

Single-molecule force measurements of the polymerizing dimeric subunit of von Willebrand factor

“…The unfolding force, L c , and L p distributions are consistent with protein domain unfolding. This is supported by: (i) at 1000 nm/s pulling velocity, the unfolding force varies between 100-130 pN [13], (ii) the increase in the contour length for each added peak L c is 25 nm, consistent with the contour length of an unfolded full or partial VWF domain [13]; and (iii) the persistence length, L p , of 0.3 nm is consistent with an unfolded protein [13,20,[22][23][24][25][26]. Force-extension curves demonstrate that the VWF domains unfold in a sequential manner, producing one to four peaks for RDVWF [ Fig.…”

“…3(d)]. These values correspond to the length of unfolded protein domains of 60 and 190 amino acids (aa), respectively, assuming 0.36 nm per residue [13,20,21]. The persistence length, L p , of the RDVWF was 0.3 nm [ Fig.…”