PB0327 - Modulating ADAMTS13 Activity Using Organic Solvents

Background: ADAMTS13 is a metalloprotease that regulates Von Willebrand Factor (VWF) by cleaving the shear-unfolded A2 domain, however, cannot be inactivated by broad-spectrum protease inhibitors. The cleavage site within VWF is enriched with aliphatic amino acids, a feature that reflects its location within the hydrophobic core of the A2 domain. This unusual substrate property implies that the active site of ADAMTS13 evolved to accommodate the hydrophobicity of its substrate. How the hydrophobicity of its active site contributes to ADAMTS13 substrate recognition or protection from inhibitors is not known.

Aims: Assess the potential of various chaotropic agents, organic solvents, and alcohols to modulate ADAMTS13 activity.

Methods: Full-length ADAMTS13 (10nM) was incubated with increasing concentrations of various solvents for 5 minutes. The activity was measured using 1uM FRETS-VWF73 at pH 7.4. Additional experiments with 10% ethanol were conducted using MDTCS (10 nM) or MD (100 nM) at pH 7.4 and FL-ADAMTS13 at pH 6.0.

Results: ADAMTS13 activity was undetectable at all tested concentrations of acetonitrile, guanidine hydrochloride, SDS, and methanol. By contrast, ADAMTS13 activity increased to 112% in 10% isopropanol (v/v) and 140% with 0.2M DMSO but attenuated at higher concentrations of these solvents. Interestingly, ADAMTS13 activity increased to 300% (p < 0.05) with 10% ethanol (v/v) and was attenuated with ethanol above 30% (v/v) (Figure 1). The presence of 10% ethanol decreased the activity of MDTCS to 50% but increased the activity of MD to 146% (Figure 2). In addition, 10% ethanol (v/v) decreased the activity of full-length ADAMTS13 to 20% when its activity was measured at pH 6.0.

Conclusion(s): Some alcohols and organic solvents can increase ADAMTS13 activity at pH 7.4. The increase in ADAMTS13 activity observed with 10% ethanol (3-fold) is comparable to changes observed when ADAMTS13 adopts an open conformation. Future studies will examine the mechanism of this observation.