PB0813 - Using flow assays to probe Von Willebrand Factor function

Background: Flow based assays are commonly used to investigate the function of VWF, however results can vary due to the type of surface coating used and the source and concentration of VWF used.

Aims: To investigate different parameters affecting flow assays to probe VWF function

Methods: Ibidi VI0.1 chambers were coated collagen and 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 VWD causing mutations were generated and expressed in HEK293T cells.

Results: Larger, more discrete thrombi formed when whole blood was perfused over collagen type I and PSC ranged from 5 – 40% but did not associate with plasma VWF levels. Interestingly while more PSC and larger thrombi were seen at pathological shear rates and did correlate with VWF levels, some individuals demonstrated reduced PSC at high shear rates. When using PFB, although PSC was affected by VWF concentration, even low concentrations of recombinant VWF (0.5µg/ml) gave good PSC and purified VWF enriched for the highest weight multimers demonstrated less PSC than VWF containing a full range of multimers. With PFB higher background binding in the absence of VWF was seen with collagen type I due to contaminating VWF, while collagen type III gave little or no background binding. As expected type 2M mutations demonstrated reduced PSC, while type 2B mutations caused large rolling aggregates at 1500s-1 that were readily digested by ADAMTS13.

Conclusion(s): Together this data confirms the variability in in-vitro thrombus formation and indicates that some individuals do not readily form thrombi at pathological shear rates in-vitro. This work also indicates that when investigating VWF function in flow assays collagen type III should be used and high protein concentrations are not necessary.