OC 55.5 - Prothrombin Circulates in Human Plasma in Multiple Covalent Forms with Differing Activities

Background: Disulfide bonds are covalent links between two cysteine residues and their formation was thought to be complete in the mature functioning protein. Recent studies demonstrated that circulating fibrinogen and human platelet αIIbβ3 integrin are constitutively produced in multiple disulfide-bonded states where different disulfides are unformed in different molecules. Here, we examine the disulfide-bonded state of the coagulation protein prothrombin which is proteolytically converted into the key protease thrombin.

Aims: To determine the redox state of the 12 disulfide bonds in circulating human prothrombin and to define the functions of the different prothrombin covalent states.

Methods: The redox state of disulfide bonds in prothrombin from healthy donors or cultured cells was assessed using differential cysteine labelling and mass spectrometry. Prothrombin variants comprising a single ablated disulfide bond by replacing both Cys with Ala were produced in HEK293 cells.

Results: Six disulfide bonds are unformed in ~1 in 10 to ~1 in 5 molecules of prothrombin from the plasmas of healthy donors. Recombinant prothrombin from HEK293 cells displayed an identical redox state. To assess the functional consequences of these unformed bonds, the six disulfide bond prothrombin mutants were produced. Ablation of one disulfide bond did not significantly alter the redox state of other disulfide bonds, indicating that disulfide formation in prothrombin is not interdependent. Functional assessment of prothrombin variants by the prothrombin time assay indicated that the clotting potential of four disulfide bond-variants are reduced by ~2-30-fold compared to wild-type. Furthermore, the efficiency of hydrolysis of chromogenic substrate for 2 out of 4 thrombin mutants are decreased by 2-5-fold.

Conclusion(s): These findings indicate that prothrombin circulates in multiple covalent forms in human plasma. Functional assays suggest that the covalent forms are less effectively converted to thrombin and exhibit differing proteolytic activities. Potentially, these covalent forms could have distinct functions towards different thrombin ligands.