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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 measurement opens a new door for investigating detailed biomolecular interactions and their thermodynamic properties by pulling molecules apart while monitoring the force exerted on them. Recent advances in the nonequilibrium work theorem allows one to determine the free-energy landscapes of these events. Such information is valuable for understanding processes such as protein and RNA folding and receptor-ligand binding. Here, we used force as a physical parameter under the traditional chemical and temperature denaturing environment to alter the protein folding energy landscape and compared the change in the unfolding free-energy barrier of the I27 domain of human cardiac titin. We found that the trends in protein unfolding free-energy barriers are consistent for single-molecule force measurements and bulk chemical and temperature studies. The results suggest that the information from single-molecule pulling experiments are meaningful and useful for understanding the mechanism of folding of titin I27.

Hemostasis is initiated by tethering platelets to the site of vessel injury, a process mediated by the interaction of the GP Ib-IX-V complex on platelets and von Willebrand Factor (VWF) in the subendothelial matrix. The adhesion activity of VWF depends on the multimer size: those freshly secreted from activated endothelial cells are ultra-large (UL) and overly adhesive, capable of forming spontaneous high strength bonds with the platelet receptor. In contrast, VWF multimers circulating in blood (pVWF) is required to be activated by high fluid shear stress or modulators in order to bind and aggregate platelets. Shear stress has previously been demonstrated to convert globular shape VWF multimers to elongated rope-like structures, but whether this structural change correlates with VWF adhesion activity remains largely unknown. We have showed that, upon secretion, ULVWF multimers form long string-like structures on activated endothelial cells that can be viewed under a regular light microscope, suggesting laterally association between ULVWF multimers. Furthermore, pVWF multimers can be induced to laterally associate with each other by shear stress, potentially resulting in the formation of VWF fibrils. We hypothesize that by forming laterally associated strings, ULVWF multimers require a greater physical force to unfold as compared to pVWF, which can be induced to form fibrillar structures that become more resistant to force-induced unfolding. Here, we present experimental data to support this hypothesis. First, we found that shear stress covalently aggregated pVWF multimers and the process was prevented rADAMTS-13 or disulfide reducing agents. VWF multimers were not cleaved by ADAMTS-13 under this experimental condition, suggesting a non-proteolytic activity. Second, when pVWF multimers are captured and subjected to physical pulling force on an atomic force microscope, the length of VWF multimers extended sequentially in response to increasing force. Plasma VWF multimers were unfolded at the peak forces of 115 pN, which increased to 153 pN after they were exposed to a pathological level of shear stress (100 dyn/cm2) for 3 min at 37°C. The peak force required to unfold pVWF after shear exposure (153 pN) was very similar to that of ULVWF multimers (152 pN). Third, recombinant (r) ADAMTS-13 did not reduce the peak force for unfolding pVWF multimers (115 pN) before but it lessened the increase in magnitude in the peak force required to unfold pVWF exposed to shear stress. This ADAMTS-13 activity was not inhibited by 5 mM of EDTA. Similarly, the peak force for unfolding ULVWF multimers was reduced from 152 pN to 127 pN when ULVWF was pulled in the presence of an equal molar concentration of rADAMTS-13. Taken together, these data demonstrate that shear stress significantly increases the peak force required to unfold pVWF multimers to the levels similar to ULVWF multimers, likely by promoting the formation of laterally associated VWF fibrils. ADAMTS-13 prevents the shear-induced increase in the peak force for unfolding sheared pVWF multimers, independent of the VWF proteolytic activity of the metalloprotease. The results reveal a mechanism for shear-induced VWF activation. They also suggest that ADAMTS-13 contains a non-proteolytic activity that plays a role in cleaving ULVWF strings and preventing pVWF multimers to be activated by lateral association induced by high fluid shear stresses.