Basic regions of factor V and tissue factor pathway inhibitor mediate heavy chain and acidic region interactions on factor V revealed by tethered chemical cleavage.

BACKGROUND

Factor (F)V plays a pivotal role in coagulation, having both pro- and anticoagulant functions. Conserved acidic and basic regions (AR and BR) within FVs central B-domain play a key role in regulating procoagulant function. Removing the BR (residues 963-1008) through proteolysis or splicing, as in FV-short, produces an active cofactor, which is partly regulated by tissue factor pathway inhibitor-α (TFPIα) through its C-terminal BR. Although AR2 (residues 1493-1537) is known to be involved, other FV regions that may interact with the FV-BR or TFPIα-BR have not been defined.

OBJECTIVES

To uncover binding interactions between the BRs and other regions on FV-short.

METHODS

We used a unique chemical cleavage agent, FeBABE (Fe-p-bromoacetamidobenzyl-ethylenediaminetetraacetic acid) tethered to the FV-BR or TFPIα-BR (BR-FeBABE) to footprint areas of engagement with FV-short using biochemical methods and western blotting.

RESULTS

BR-FeBABE bound FV-short and FV-810, another B-domainless form of FV, with high affinity and blocked procoagulant activity, similar to untethered BR. When BR-FeBABE was stimulated, it cut FV-short and FV-810 at 3 sites including the beginning of AR2, near residues 678 to 692 comprising part of AR1 (residues 659-697), and at the beginning of the A2 domain. Cleavage by BR-FeBABE was specific as untethered FV-BR, TFPIα-BR, or TFPIα blocked proteolysis, and forms of FV that do not bind BR-FeBABE, such as FV, FVa, and any form of FV lacking AR2, were not cut.

CONCLUSION

These results define areas of interaction between the BR and FV-short and advance our knowledge about how FV is regulated by the B-domain and TFPIα.