Membrane binding by prothrombin mediates its constrained presentation to prothrombinase for cleavage

Area of Science:

• Biochemistry
• Molecular Biology
• Hemostasis

Background:

• Prothrombin (factor II) activation to thrombin is crucial for blood coagulation.
• Membrane binding of prothrombin is traditionally thought to be essential for efficient thrombin generation by prothrombinase.
• The role of Gla-domain-mediated membrane interactions in prothrombin activation kinetics and pathway remains incompletely understood.

Purpose of the Study:

• To investigate the impact of impaired membrane binding on prothrombin activation kinetics and cleavage pathway.
• To elucidate the mechanistic basis for altered substrate cleavage in the absence of Gla-domain-dependent functions.
• To explore the implications of these findings for prothrombinase activity on physiological cell surfaces.

Main Methods:

• Utilized full-length prothrombin variants lacking gamma-carboxyglutamate modifications (desGla prothrombin) with impaired membrane binding.
• Employed desGla prothrombin variants with individually rendered uncleavable cleavage sites (Arg320 and Arg271).
• Analyzed prothrombinase action on both modified and unmodified prothrombin on cell surfaces (platelets, endothelial cells).

Main Results:

• DesGla prothrombin exhibited a modest decrease in thrombin formation rate but a significant alteration in the cleavage pathway.
• Loss of membrane binding led to decreased cleavage at Arg320 and increased cleavage at Arg271.
• These effects were attributed to reduced substrate tethering and relaxed presentation of cleavage sites.
• Prothrombin cleavage pathway and intermediate formation switched without a profound rate decrease.
• Cell-dependent prothrombinase function varied based on prothrombin binding support and intermediate accumulation.

Conclusions:

• Membrane binding's contribution to prothrombin activation rate is less profound than dogma suggests, but it critically influences the activation pathway.
• Gla-domain-dependent functions modulate substrate presentation and tethering, impacting cleavage site accessibility and catalysis.
• Cellular context significantly influences prothrombinase activity through differential prothrombin binding and pathway selection.