Force Regulation Mechanisms On The Binding And Cleavage Of VWF-A1A2A3 Tridomain By ADAMTS-13

Adhesion of platelest to the surface of vascular injury is critical for hemostasis and thrombosis. Platelet adhesion is a multi-step process including tether adhesion, rolling adhesion and firm adhesion, which are mediated by distinct receptors and ligands interaction. The interaction of von Willebrand factor (vWF) and it platelet receptor GPIb mediates platelets tethering to and rolling on the vascular surface, playing an important role on the initial adhesion of platelets. The activity of vWF depends on its size. vWF proteolytic enzyme ADAMTS-13(A disintegrin and metalloprotease with a thrombospondin type 1 motifs 13) regulates the size of vWF by cleaving the cryptic peptide Tyr1605-Met1606 inside A2 domain, thereof the activity of vWF. Deficiency on concentration, structure or function of vWF due to mutations will induce bleeding disorder, vWD. On the other hand, deficiency of ADAMTS-13 due to mutations or autoantibodies will result in life-threatening disease, Thrombotic Thrombocytopeinic Purpura, TTP.Platelet adhesion, ADAMTS-13 binding to and cleaving vWF occur in the hemodynamic environment, therefore mediated by hemodynamic factors. We employed AFM (Atomic Force Microscopy), flow chamber, and mathematical model in this study, focusing on the force-chemistry coupling hot topic,“force mediates the binding and cleavage of vWF by ADAMTS-13”. First, we investigated the mechanism(s) of force mediation on the binding of ADAMTS-13 to the important polypeptide A1A2A3 of vWF by using AFM to exert forces upon the molecule pair. Then, we investigated the mechanism of force mediation on the cleavage of vWF by ADAMTS-13 by employing AFM pulling on A1A2A3 to induce structure destablilization. Finally, we simulated the motion of a cell (platelet) flowing in the viscous flow near the surface to a solid study the hydrodynamic effects on platelet adhesion.We found that, ADAMTS-13 contains at least two binding sites for A1A2A3, one of them is in the DelCUB portion, which is Ca2+ dependent; the other is in the ADAMTS-13 C-terminal CUB portion, which is not divalent metal ion dependent. Interestingly, the binding of DelCUB to A1A2A3 inhibits the binding of CUB to A1A2A3. In addition, the binding between DelCUB and A1A2A3 is a“catch”(bond life time increases as force increasing)-“slip”(bond life time decreases as force increasing) transition bond. To the best of our knowledge, it is the first time to demonstrate“catch bond”mediating the interaction between enzyme and its substrate. This mechanism increases the duration of binding and thus facility the cleavage of vWF by ADAMTS-13. The increase in molecular length of A1A2A3 distributed around 20 and 50 nm, when it was pulled by AFM. The short increment probably resulted from the domain-domain uncoupling of A1A2A3 or partial unfolding of A2 domain; while the long increment probably resulted from the fully unfolding of A2. For the first time, we demonstrated that“catch bond”mediated the interaction within A1A2A3 to stabilize its structure. ADAMTS-13 cleaved A1A2A3 only after structure destablilizatin of A1A2A3, but not before. Furthermore, the cleavage became evident when A2 was fully unfolded exposing the cleavage site. Analysis with a model for single-substrate trimolecualr enzymatic kinetics estimated a cleavage rate kcat=2.9 per seconds and dissociation constant Kd=5.6 nM. We also found that the cleavage rate deceased with force increasing.By simulating the motion of a cell near a surface, we proposed that the sliding velocity and contact area between a cell and surface are the main factors in the flow-enhanced cell tethering, while the contact frequency plays a less role. The mean contact area between a cell and a surface is 0.004-0.01μm2, which means only few even one molecule on that area. In this thesis, we studied the binding and cleavage of vWF by ADAMTS-13,investigated the force regulation mechanisms. We also investigated the effects of factors on flow-enhanced cell tethering. This study will enhance our knowledge about platelet adhesion and relative physiological and pathological processes.