Mechanical Activation of a Multimeric Adhesive Protein Through Domain Conformational Change

Abstract

The mechanical force-induced activation of the adhesive protein von Willebrand Factor (VWF), which experiences high hydrodynamic forces, is essential in initiating platelet adhesion. The importance of the mechanical force-induced functional change is manifested in the multimeric VWF’s crucial role in blood coagulation, when high fluid shear stress activates plasma VWF (pVWF) multimers to bind platelets. Here we showed that a pathological level of high shear stress exposure of pVWF multimers results in domain conformational changes, and the subsequent shifts in the unfolding force allow us to use force as a marker to track the dynamic states of multimeric VWF. We found that shear-activated pVWF multimers (spVWF) are more resistant to mechanical unfolding than non-sheared pVWF multimers, as indicated in the higher peak unfolding force. These results provide insight into the mechanism of shear-induced activation of pVWF multimers.

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

“…3(b)]. This is consistent with one to two unfolding peaks per VWF monomer in a DVWF [13]. RVWF multimers had more unfolding force peaks in the force-extension curves [ Fig.…”

“…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.…”

“…RVWF multimers had more unfolding force peaks in the force-extension curves [ Fig. 3(c)] than RDVWF, compatible with the stretching of single molecules containing a larger number of repeating VWF monomeric subunits (and unfolding domains) [13].…”

“…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.…”

“…VWF multimers can then mediate the adhesion of platelets. It has been shown that VWF multimers change from a globular to an elongated conformation under shear stress [10][11][12] and remain * chkiang@rice.edu in their activated, more adhesive state for hours [13]. Prolonged activation may coincide with the lateral association of VWF multimers into a fibrillar form of VWF [3,8,14].…”