Role of sequence and position of the cleavage sites in prothrombin activation

Bosko M Stojanovski,Enrico Di Cera

doi:10.1016/j.jbc.2021.100955

Abstract

In the penultimate step of the coagulation cascade, the multidomain vitamin-K-dependent zymogen prothrombin is converted to thrombin by the prothrombinase complex composed of factor Xa, cofactor Va, and phospholipids. Activation of prothrombin requires cleavage at two residues, R271 and R320, along two possible pathways generating either the intermediate prethrombin-2 (following initial cleavage at R271) or meizothrombin (following initial cleavage at R320). The former pathway is preferred in the absence of and the latter in the presence of cofactor Va. Several mechanisms have been proposed to explain this preference, but the role of the sequence and position of the sites of cleavage has not been thoroughly investigated. In this study, we engineered constructs where the sequences 261DEDSDRAIEGRTATSEYQT279 and 310RELLESYIDGRIVEGSDAE328 were swapped between the R271 and R320 sites. We found that in the absence of cofactor Va, the wild-type sequence at the R271 site is cleaved preferentially regardless of its position at the R271 or R320 site, whereas in the presence of cofactor Va, the R320 site is cleaved preferentially regardless of its sequence. Additional single-molecule FRET measurements revealed that the environment of R271 changes significantly upon cleavage at R320 due to the conformational transition from the closed form of prothrombin to the open form of meizothrombin. Detailed kinetics of cleavage at the R271 site were monitored by a newly developed assay based on loss of FRET. These findings show how sequence and position of the cleavage sites at R271 and R320 dictate the preferred pathway of prothrombin activation.

Highlights

In the penultimate step of the coagulation cascade, the multidomain vitamin-K-dependent zymogen prothrombin is converted to thrombin by the prothrombinase complex composed of factor Xa, cofactor Va, and phospholipids
Characterization of the activation pathway of proT320/320 provides further evidence that the sequence makes an important contribution in directing the sequential cleavage by factor Xa (fXa). proT320/320 is activated by fXa along the prethrombin-2 pathway, but the initial cleavage at R271 is significantly slower and the appearance of the prethrombin-2 band is delayed compared with wild-type (Figs. 2 and 3)
Because the P1, P2, and P4 residues are conserved between the R271 and R320 sites and the P3 residue is acidic in both cases, our data show that the distal residues that occupy the P5–P11 positions play an important role for the preferential recognition of the R271 site by fXa

Summary

Introduction

In the penultimate step of the coagulation cascade, the multidomain vitamin-K-dependent zymogen prothrombin is converted to thrombin by the prothrombinase complex composed of factor Xa, cofactor Va, and phospholipids. If the sequential cleavage is caused by differences in the amino acid sequence at the two sites of prothrombin, replacing the sequence at the R320 site with that of the R271 site should accelerate cleavage by fXa at the swapped region in proT271/271 and give evidence of activation along the meizothrombin pathway.

Results

Conclusion