Investigating inter-domain regulation of von Willebrand factor interactions with platelets Ryan M Harrison 1, An-Yue Tu 2, Wendy E Thomas 2 1 Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 2 Department of Bioengineering, University of Washington, Seattle, WA

How Stuff Works: Blood Clotting von Willebrand factor (vWF)

How Stuff Works: Blood Clotting D’ D3 von Willebrand factor Not to Scale A1 Platelet Not to Scale

How Stuff Works: Blood Clotting Glycoprotein Ib α vWF-A 1 Platelet Not to Scale D’ D3 von Willebrand factor Not to Scale

Understanding blood clotting Relevance! 1. Basic Science = Inherently Cool 2. Molecular understanding of blood clotting    Molecular pathology

Flow chamber setup Schematic Real McCoy

Platelet Not to Scale Platelet Not to Scale Flow chamber setup Flow Plate Chamber

Gin Rummy on science There are known knowns. These are things we know that we know. There are known unknowns. That is to say, there are things that we know we don't know. But there are also unknown unknowns. There are things we don't know we don't know. - Donald Rumsfeld - “Known-Known” Platelet protein (GP Ib a ) binds von Willebrand factor (vWF-A1) shear dependently “Known-Unknown” -Does isolated protein function? - Does isolated protein adhere to plate? - What other domains affect vWF binding? “Unknown-Unknown” ¿¿¿42 ???

Does isolated protein function? % Aggregation Platelets + vWF + Ristocetin + A1 Platelets + Ristocetin Time (seconds) Platelets + vWF + Ristocetin + A1A2A3 Platelets + vWF + Ristocetin

Does isolated protein adhere to plate? Uncoated polystyrene (blue) Ab anti-vWF coated polystyrene (gold). vWF A1A1 A1A2A3A1A2A3 D’D 3 A 1 A 2 A 3 Fraction of vWF A650

This is Wrong  Shear Stress (Pa) Platelets bound in 120 seconds

Mistakes 

Mistake #1: Flow Rate (ml/min)  Shear Stress (Pa) Conversion Gasket A (0.5mm thickness?) versus Gasket B (1mm thickness?) Mistake #2: Platelet Re-suspension Re-suspended platelets in H/T + BSA + PGE-1 + Apyrase instead of H/T + BSA.

Yesterday’s Results Mistake #1 Fixed!!!

Today’s Results! Mistake #1 & 2 Fixed!!!

Miscellaneous Domains surrounding vWF-A1 are likely to play a role in mediating its interactions with platelets Special Thanks Kaoru Kusuma Dr. Shivani Sharma Dr. Olga Yakovenko Dr. An-Yue Tu Dr. Wendy Thomas Greg Mize Ethan Allen (NNIN-UW)