vWF Feels the Force

Robert Flaumenhaft, MD, PhD

2009-11-01

Dr. Flaumenhaft indicated no relevant conflicts of interest.

Zhang X, Halvorsen K, Zhang CZ, et al. Mechanoenzymatic cleavage of the ultralarge vascular protein von Willebrand factor. Science. 2009;324:1330-34.

Mechanism of ULVWF cleavage. The blood protein VWF is secreted as an ultralong chain (ULVWF) containing several hundreds of VWF monomers. Because of its large size, ULVWF experiences higher shear forces than shorter VWF polymers. These higher shear forces not only stretch ULVWF, but — as shown by Zhang et al. — they also unfold domain A2 in the VWF monomers. Unfolding exposes a cleavage site for ADAMTS13, which in turn cuts ULVWF to smaller pieces. The smaller VWF pieces can be stretched (activated) under shear flow conditions found at injury sites, but do not cause thrombosis through premature stretching under normal blood flow conditions. Mechanical shear forces hence regulate both activation and degradation of VWF chains. From Christof J, Gebhardt M, Rief M. Force Signaling in Biology. Science. 2009;324:1278-80. Reprinted with permission from AAAS.
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Von Willebrand factor (vWF) is the central shear-sensing coagulation protein in blood. It is secreted from endothelial cells as ultra-large vWF (ULVWF), which is cleaved to yield smaller multimers. The metalloproteinase ADAMTS13 cleaves ULVWF by proteolysis at a site within the A2 domain of vWF monomers. Since the hemostatic potential of vWF is directly related to its length, cleavage of ULVWF by ADAMTS13 regulates vWF function by modifying the size of vWF multimers. Deficiency of ADAMTS13 results in excess ULVWF and can cause thrombotic thrombocytopenic purpura. Thus, regulation of vWF multimer length by ADAMTS13 is critical to normal coagulation under flow conditions. Yet the mechanisms that control cleavage of vWF by ADAMTS13 are not well understood. Studies using single molecule measurements to monitor the response of the vWF A2 domain to mechanical force now provide new insight into how shear forces in the circulation regulate the cleavage of vWF by ADAMTS13. Zhang et al., from Tim Springer’s lab at Harvard, used a sophisticated biophysical technique called laser optical tweezers to apply force to single A2 domains suspended between two beads. They found that at a force of approximately 11 pNewton an abrupt increase in the length of the A2 domain occurred, the extent of which indicated complete unfolding of the domain. When clamped at a low force, the A2 domain remained unfolded for about two seconds, but then refolded into its original conformation. Thus, the A2 domain is capable of undergoing a reproducible, reversible, and force-mediated conformational change. The authors next determined whether unfolding of the A2 domain influences its susceptibility to cleavage by ADAMTS13. A2 domains were mechanically unfolded in the presence or absence of ADAMTS13. In the presence of ADAMTS13, cleavage was detected as an abrupt decrease in force following exposure to enzyme. In contrast, no cleavage of the folded A2 domain was observed. The calculated efficiency of cleavage of the unfolded domain by ADAMTS13 was significantly better than that of previous estimates of ADAMTS13 cleavage efficiency, suggesting improved recognition by the enzyme of the unfolded A2 domain. These observations demonstrate that force acts as a co-factor to facilitate cleavage of the A2 domain by vWF. That the single molecule measurements are relevant to cleavage of circulating vWF in vivo is evidenced by calculations indicating that an 11 pN unfolding force would lead to an upper length limit of 200 monomers for circulating vWF. This calculated length corresponds to the observed maximum length of vWF multimers found in the circulation.

Regulation of vWF length by ADAMTS13-mediated cleavage is critical for normal hemostasis. Investigators have speculated that force-induced unfolding of the A2 domain within vWF renders it susceptible to cleavage by ADAMTS13. However, this premise has been difficult to prove directly. By stretching single molecules of the A2 domain, Zhang et al. demonstrate that force acts as a co-factor in the activity of ADAMTS13, serving to expose a buried cleavage site within the domain. These studies establish the mechanistic basis for how fluid dynamics within the circulation create forces that regulate the ability of ADAMTS13 to control vWF length and function.

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