The Purification Of VWF-A1 Mutations R1308L And G1324S And Their Function Study

von Willebrand factor(VWF) is a multimeric glycoprotein,mature VWF consists of a 2050 residue subunit that having repeated of A, B, C and D type domains in the order D1-D2-D’-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK.The A1-domain play a central role during the platelet adhesion, activation and aggregation as it contains the VWF-binding site for platelet surface receptor GPIbα.When blood vessel are disrupted, circulating platelets adhere to subendothelial surfaces through the interaction of the GPIbα with VWF-A1. GPIbα/VWF-A1 interaction also induces platelet activation signaling events, leading to stable platelet adhesion and aggregation. This is the early stage of hemostatic processes. The most common inherited bleeding disorder, von Willebrand disease(VWD) is arises from quantitative or qualitative defects in plasma VWF. VWD is classified into 1, 2, 3 three different types according to the deficiency degree of VWF, and type 2 VWD characterized by qualitative defects, including type 2B and type 2M VWD. In particular, 2B VWD shows a gain-of- function phenotype and 2M VWD shows a loss-of- function phenotype. Mutation in VWF can disrupt the interaction of the VWF and platelet, resulting in the bleeding and coagulopathy. Type 2B mutant R1308 L in VWF A1 domian shows an increased ability to inhibit ristocetin- induced platelet agglutination, while the type 2M mutant G1324 S shows a decreased ability. As for the importance of the VWF-A1 in hemostasis, we use parallel-plate flow chamber system to investigate the point mutations R1308 L and G1324 S bonding behavior with platelet. Then contrast the difference between mutations and wide type A1(WT-A1) in mediating the platelet adhesion and activation. This study discussed the molecular structure of VWD, and established a better understanding of hemostasis. The results will be helpful for the development of new generations of VWD therapies.Firstly, VWF-A1 mutations R1308 L and G1324 S recombinant plasmids were transformed into competent cells DH5α, clonal and extract the plasmids. Then the extracted plasmids were transformed into E.coli M15. In the optimized expression condition, soluble protein was expressed. Then, the proteins were purified by Ni-affinity chromatography, and we detect the purity and immunological activity of the purified products by SDS-PAGE and Western Blot. BCA Protein Quantitation result show that R1308 L protein concentration is 316.50μg/ml, and G1324 S is 204.83μg/ml.Next, the platelet adhesion to R1308 L and G1324 S was observed under 100s-1、1000 s-1、2000 s-1 flow shear rate condition. Our research shows that the VWF-A1 mutations could interaction with platelet specificity and have good biological activity. Compared with WT-A1, as the shear rate increased, the decline(58.2%) of the number of adhesive platelets in G1324 S mutation group was higher than that of wild type group(36.5%). While the R1308 L group showed a stronger interaction with platelet under shear stress, and easy to induced platelet aggregation.Finally, we observed the progress of VWF-A1 mutations R1308 L and G1324 S induced platelet activation under shear rate in real-time. A calcium indicator Fluo4-AM was used to visualize intracelluar calcium variations during platelet adhesion. After that, the activity ratio of the platelet in different shear rate was got, we also analyzed the delay time, peak time, half time and fluorescence increase ratio of the platelet. Compared with WT-A1, mutant in VWF-A1 had influence on the delay time, peak time, half time. The results indicated that activity ratio of the platelet was regulated b y shear rate. As the shear rate increased, the activity ratio shows a increased tendency, and the delay time shows a decreased tendency.