PB0810 - Investigating ADAMTS13 cleavage of Von Willebrand Factor under flow

Background: ADAMTS13 regulates the multimeric size of VWF by cleaving VWF in its A2 domain. While much is understood about the interaction between ADAMTS13 and VWF, less is known about the factors affecting ADAMTS13 function under shear stress.

Aims: To investigate the impact of ADAMTS13 on VWF function under flow

Methods: Ibidi VI0.1 flow chambers coated with collagen were perfused with whole blood from healthy donors, or plasma free blood (PFB) supplemented with plasma derived or recombinant VWF. VWF mediated platelet capture was measured as platelet surface coverage (PSC). A panel of type 2A VWD causing mutations were generated and expressed in HEK293T cells. Blood group specific VWF was purified using gel filtration and recombinant ADAMTS13 expressed in HEK293T cells.

Results: ADAMTS13 failed to digest pre-formed thrombi, but slowed down the initial rate of platelet capture during the first 60sec of perfusion over collagen, but once a thrombi was forming it only appeared to slow its growth resulting in smaller less dense thrombi. Interestingly, most of the type 2A variants tested demonstrated normal platelet capture over collagen in the presence of ADAMTS13, however when circulated around a closed system with ADAMTS13 they were readily cleaved. Significantly, purified blood group O-VWF demonstrated less PSC in the presence of ADAMTS13 compared to blood group AB-VWF indicating more proteolysis and moreover VWF lacking its D4 domain and therefore the ADAMTS13 docking site was proteolysed to a similar extent as wild type VWF under flow conditions.

Conclusion(s): Together this data highlight the role of ADAMTS13 in modulating the initial rate of thrombus formation and confirms that under flow conditions ABO group can alter ADAMTS13 proteolysis. Our data also questions the significance of the ADAMTS13 docking site in the D4 domain.