ALBERT

Description: Abstract 195 ADAMTS13 contains multiple free thiols on its surface, which may form disulfide bonds with surface-exposed free thiols on plasma-derived von Willebrand factor (VWF). This interaction may prevent lateral association of apposed VWF under arterial shear stress. However, the functional consequence of ADAMTS13-VWF interaction without proteolysis is not known. We hypothesize that the interaction between the C-terminus of ADAMTS13 and the C-terminus of VWF inhibits thrombus formation under shear stress. Using a BioFlux microfluidic system, we showed that under arterial shear stress, 10 dyn/cm2, fluorescein-labeled platelets from PPACK (thrombin inhibitor) anti-coagulated human whole blood adhered to collagen (type I)-coated surface in a time-dependent manner. Addition of human recombinant full-length ADAMTS13 (10 nM) into whole blood dramatically reduced the surface coverage of fluorescein-labeled platelets. Conversely, addition of an inhibitory polyclonal anti-ADAMTS13 IgGs (150 ug/ml) to whole blood dramatically accelerated the accumulation of fluorescein-labeled platelets. These results suggest that this microfluidic system is highly sensitive for the assessment of anti-thrombotic function of ADAMTS13. Under the same conditions, we were able to further show that addition of recombinant C-terminal fragment of ADAMTS13 comprising of the 5th to 8th thrombospondin type 1 (TSP1) repeats and two CUB domains (T5C) or the 2nd to 8th TSP1 repeats and two CUB domains (T2C) into whole blood also inhibited the surface coverage of fluorescein-labeled platelets on collagen-coated surface in a concentration-dependent manner. In the presence of 0.1 μM and 0.5 μM of recombinant T2C or T5C, the surface coverage of fluorescein-labeled platelets was reduced by ∼40% and ∼60%, respectively. The inhibitory activity of these recombinant C-terminal fragments was nearly abolished if pre-treated with 40 mM of N-ethylmaleimide which blocked surface-exposed free thiols. Moreover, recombinant CUB domains at the highest concentration tested (1.0 μM) did not appear to alter the surface coverage of fluorecein-labeled platelets under the same conditions. These results suggest that the C-terminal TSP1 repeats of ADAMTS13 inhibit platelet adhesion and aggretion or thrombus formation through thiol-thiol interactions between ADAMTS13 and VWF (or other proteins). We conclude that the C-terminal TSP1 repeats may modulate thrombus formation independent of proteolytic activity. Disclosures: No relevant conflicts of interest to declare.