Zhenni Ma1, Jian Su1, Jingyu Zhang2, Jing Ling3, Jie Yin1, Xia Bai1, Changgeng Ruan4. 1. MOH Key Lab of Thrombosis and Hemostasis, Jiangsu Institute of Hematology, the First Affiliated Hospital of Soochow University, 188 Shizi Street, Suzhou 215006, China; Collaborative Innovation Center of Hematology, Soochow University, 1 Shizi Street, Suzhou 215006, China. 2. MOH Key Lab of Thrombosis and Hemostasis, Jiangsu Institute of Hematology, the First Affiliated Hospital of Soochow University, 188 Shizi Street, Suzhou 215006, China; Collaborative Innovation Center of Hematology, Soochow University, 1 Shizi Street, Suzhou 215006, China; Department of Hematology, Hebei Institute of Hematology, The Second Hospital of Hebei Medical University, 215 Hepingxi Road, Shijiazhuang 050000, China. 3. MOH Key Lab of Thrombosis and Hemostasis, Jiangsu Institute of Hematology, the First Affiliated Hospital of Soochow University, 188 Shizi Street, Suzhou 215006, China; Collaborative Innovation Center of Hematology, Soochow University, 1 Shizi Street, Suzhou 215006, China; Department of Hematology and Oncology, Children's Hospital of Soochow University, 303 Jingde Road, Suzhou 215003, China. 4. MOH Key Lab of Thrombosis and Hemostasis, Jiangsu Institute of Hematology, the First Affiliated Hospital of Soochow University, 188 Shizi Street, Suzhou 215006, China; Collaborative Innovation Center of Hematology, Soochow University, 1 Shizi Street, Suzhou 215006, China. Electronic address: changgengruan@hotmail.com.
Abstract
INTRODUCTION: In plasma, the size of the von Willebrand factor (VWF) multimer is down-regulated by ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13). The binding of platelets or glycoprotein (GP) Ibα recombinant fragment to VWF domain A1 may increase the cleavage by ADAMTS13 to VWF. Both type 2B and type 2M von Willebrand disease (VWD) result in bleeding disorders with the diathesis of increased and decreased binding affinity between GPIbα and VWF, respectively. However, the influence of 2B/2M VWD mutations in the A1 domain and GPIbα on cleavage by ADAMTS13 to VWF needs further study. MATERIALS AND METHODS: Different types of full-length human recombinant VWF (rVWF) were expressed, including three type 2B mutations (P1337L, H1268D, and R1308C), one type 2M mutation (D1302G), and wild type (WT). The three characterized types of rVWF were digested by ADAMTS13 under static conditions or high-shear stress. The interaction of rVWF and ADAMTS13 was also tested by plate-binding assays. RESULTS: Under static (natured) conditions or high-shear stress, type 2B mutants exhibited a higher susceptibility to ADAMTS13 than rVWF-WT, whereas type 2M mutant was normal. While under static (denatured) conditions or high-shear stress (with GPIbα fragment) rVWF-WT showed an even higher susceptibility to ADAMTS13 than the two type 2B mutants studied. CONCLUSION: Type 2B mutations localized in the A1 domain could enhance the sensitivity to ADAMTS13-mediated proteolysis. When GPIbα participated, there was a dramatically increased proteolytic cleavage of VWF by ADAMTS13 to rVWF-WT, excluding some type 2B mutants.
INTRODUCTION: In plasma, the size of the von Willebrand factor (VWF) multimer is down-regulated by ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13). The binding of platelets or glycoprotein (GP) Ibα recombinant fragment to VWF domain A1 may increase the cleavage by ADAMTS13 to VWF. Both type 2B and type 2M von Willebrand disease (VWD) result in bleeding disorders with the diathesis of increased and decreased binding affinity between GPIbα and VWF, respectively. However, the influence of 2B/2M VWD mutations in the A1 domain and GPIbα on cleavage by ADAMTS13 to VWF needs further study. MATERIALS AND METHODS: Different types of full-length human recombinant VWF (rVWF) were expressed, including three type 2B mutations (P1337L, H1268D, and R1308C), one type 2M mutation (D1302G), and wild type (WT). The three characterized types of rVWF were digested by ADAMTS13 under static conditions or high-shear stress. The interaction of rVWF and ADAMTS13 was also tested by plate-binding assays. RESULTS: Under static (natured) conditions or high-shear stress, type 2B mutants exhibited a higher susceptibility to ADAMTS13 than rVWF-WT, whereas type 2M mutant was normal. While under static (denatured) conditions or high-shear stress (with GPIbα fragment) rVWF-WT showed an even higher susceptibility to ADAMTS13 than the two type 2B mutants studied. CONCLUSION: Type 2B mutations localized in the A1 domain could enhance the sensitivity to ADAMTS13-mediated proteolysis. When GPIbα participated, there was a dramatically increased proteolytic cleavage of VWF by ADAMTS13 to rVWF-WT, excluding some type 2B mutants.