Collagen,VWF-A1A2A3-mediated Expression Of Platelet P-selectin Under Flow Shear Stresses

In the initial hemostatic processes,platelets adhere to the injured vascular site through interactions with collagen,VWF under the flow shear stress environment.Platelets are further activated by mechanical-chemical coupling processes,and platelets express P-selectin on its membrane surface.P-selectin serves as a marker for activated platelets and plays a important role in hemostasis.Mature VWF molecules consist of A,B,C,and D domains.The A domains are key domains in VWF,including A1A2A3-tridomain,of which the A1 domain and A3 domain contain the binding sites for platelet membrane receptor GPIbα and collagen,respectively.VWF-A1A2A3 is like a bridge connecting platelets and collagen.Given that the interactions between collagen,VWF-A1A2A3 and platelets play a key role in the early hemostatic processes,interactions between collagen,VWF-A1A2A3 and platelets are investigated by the flow chamber system in this thesis.Our research focused on platelets adhesions and the expression of platelet P-selectin induced by collagen,VWF-A1A2A3 under various shear stresses,which would help us to understand the mechanism of how collagen,VWF-A1A2A3 mediates platelet activation under shear stresses,and how it leads to hemostasis under physiological conditions.Firstly,VWF-A1A2A3 was efficiently expressed by exploring the expression conditions(Collagen is obtained by purchase,but VWF-A1A2A3 is obtained by expression).Then,the proteins were purified by Ni-affinity chromatography.The purity and specificity of purified products was identified by SDS-PAGE and Western blotting,respectively.BCA Protein Quantification results showed that we obtained about 53.85 mg per 100 milliliter VWF-A1A2A3 in culture supernatants.Secondly,the biological activity of purified proteins was assessed by testing their abilities to mediate platelet adhesion using parallel-plate flow chamber system.Purified platelets(extracted from healthy donors)were perfused at various shear stresses(1~20dyn/cm2)on different substrates(incubated with 1% BSA,PBS,200μg/mL collagen,VWF-A1A2A3 and VWF-A1A2A3/ collagen)in the flow chamber.Control experiments were designed to test whether the interaction between platelets and proteins were specific.The results showed that 1% BSA can effectively block the non-specific adhesion between platelet and flow chamber substrates,without affecting the specific interaction between platelet and proteins.Thus,we used this method to functionalize flow chamber floor in subsequent experiments.Lastly,platelets were perfused at low shear stresses on the functionalized surfaces coated with the 200μg/mL collagen,VWF-A1A2A3 and VWF-A1A2A3/ collagen,respectively(all containing 1% BSA)in flow chamber,and platelet were incubated for 10 minuts to interact with substrates fully.Different chemical agonists induced expression level of platelet surface P-selectin under different wall shear stresses(0~20dyn/cm2)for various time(0min,2.5min,5min,7.5min,10 min,12.5min,15min)were observed and analyzed.To verify whether the expression of platelet P-selectin depended on the force and understanding the differences of the expression level of platelet P-selectin induced by different agonists under various shear stresses,activation rate,time-to-peak and relative intensity of fluorescence were analyzed.The results showed that platelet activation not only depended on force,but also the flow shear stress regulated platelet P-selectin expression rates.Different agonists regulated time-to-peak of P-selectin,and VWF-A1A2A3/collagen activated platelets more quickly.