The Co-Influence of VWD Type 2B and Type 2M Mutations and Platelet GPIbα On the Susceptibility of VWF to ADAMTS13 Under Shear Stress

Von Willebrand Factor (VWF) is one kind of vital proteins involved in thrombosis, which exists in the form of multimeric glycoprotein in plasma of human. The size of VWF multimer is down regulated by metalloproteinase ADAMTS13. This physiological process is modulated by multiple factors. It was reported that the binding of platelets or GPIbα recombinant fragment to VWF domain A1 may increase the cleavage by ADAMTS13 to recombinant A1A2A3 segment in the condition of static state. Due to the mutations, VWF synthesized by patients of von Willebrand disease (VWD) type 2B and type 2M may enhance and decrease the binding of platelet membrane glycoprotein Ib (GPIb), respectively. This study aims to observe the influence of 2B/2M VWD mutations and recombinant GPIbα active fragment on the susceptibility of VWF to ADAMTS13, in the condition of high shear stress.

We constructed and expressed different forms of recombinant full-length human VWF (rhVWF) including three type 2B mutations (P1337L, H1268D, R1308C), three type 2M mutations (M740I, R1205H, D1302G) and wide-type (WT) in Hela cells. Recombinant human ADAMTS13 (rhADAMTS13) and GPIbα (H1-V289) were both from our own lab. The different types of rhVWF were characterized by multimeric analysis and ristocetin-induced platelet aggregation (RIPA). We use rhADAMTS13 to digest seven full-length rhVWF in the reaction Buffer (20 mM Tris-HCl, 150mM NaCl, pH7.4, 1% bovine serum albumin) in the absence and presence of GPIbα (H1-V289). To develop a more physiological assay under fluid shear stress, we assessed the proteolytic cleavage of rhVWF by rhADAMTS13 under constant vortexing (2,500 rpm) in a PCR tube mini-mixer that mimics arterial fluid shear stress (30 dynes/cm²). The 176kDa fragment resulting from the cleavage of rhVWF at the Y1605-M1606 bond by rhADAMTS13 was visualized by 5% SDS-PAGE under reducing conditions and Western blot analysis.

SDS-agarose gel electrophoresis showed that all rhVWF mutants were in the similar multimeric distribution to the rhVWF-WT. In the RIPA assay, type 2B rhVWF exhibited clearly enhanced RIPA responsiveness with ristocetin at 0.5 mg/ml (R1308C > P1337L > H1268D > WT), while type2M rhVWF showed decreased RIPA responsiveness with ristocetin at 1.0 mg/ml (WT > M740I > R1205H > D1302G). At physiological salt concentration (150nM NaCl), type 2B mutants exhibited more susceptibility to rhADAMTS13 than WT and type 2M mutants in the absence of GPIbα (H1-V289). In the presence of GPIbα (H1-V289), the higher susceptibility of mutants to rhADAMTS13 was detected, which decreased in the following order: type 2B > WT > type 2M, whereas no obvious enhancement happened to the two type 2M mutants (R1205H and D1302G).

Our study further confirmed that platelet GPIbα binding to VWF A1 domain could increase the susceptibility to ADAMTS13 at physiological conditions under high stress. The three type 2B mutations are all localized in the A1 domain and these mutations alone may also increase the susceptibility to ADAMTS13. While type 2M mutations M740I, R1205H and D1302G are localized in the D2, D3 and A1 domain, respectively. It is concluded that type 2M mutations localized close to or in the A1 domain may exhibit significantly lower susceptibility to ADAMTS13 in the presence of GPIbα (H1-V289). It may partially give the explanation that some VWD type 2M may display larger than normal VWF multimers in plasma.

No relevant conflicts of interest to declare.