Thrombin-mediated in vitro processing of pro-von Willebrand factor

Area of Science:

• Biochemistry
• Hematology
• Molecular Biology

Background:

• Von Willebrand factor (vWF) is synthesized as pre-von Willebrand factor (pre-provWF) and processed into propeptide (vWFpp) and mature vWF.
• Previous research suggests recombinant provWF can be processed extracellularly in vivo.

Purpose of the Study:

• To investigate the in vitro processing of recombinant provWF.
• To elucidate the mechanisms and factors involved in extracellular vWF processing.

Main Methods:

• Incubation of recombinant provWF (rpvWF) with vWF-deficient plasma.
• Multimer analysis and SDS-polyacrylamide gel electrophoresis using 125-labeled provWF.
• Dose-dependent experiments with purified thrombin and meizothrombin.
• In vivo studies using hirudin preconditioned vWF-deficient mice.

Main Results:

• rpvWF processing in plasma resulted in decreased provWF antigen and increased vWFpp antigen.
• Multimer analysis demonstrated conversion of provWF to mature vWF multimers.
• Processing was calcium-dependent, inhibited by thrombin inhibitors, and did not occur in prothrombin-depleted plasma.
• Thrombin and meizothrombin induced dose-dependent propeptide removal.
• Collagen binding reduced the thrombin concentration required for propeptide removal.
• Hirudin attenuated in vivo rpvWF processing.

Conclusions:

• Extracellular processing of von Willebrand factor (vWF) is mediated by thrombin.
• Thrombin cleaves the vWF propeptide (vWFpp) from precursor vWF (provWF).
• This processing pathway is relevant both in vitro and in vivo, with implications for hemostasis.