Leiden University Medical Center, Leiden University Medical Center LEIDEN, Netherlands
Shuzhen Cheng (Leiden University Medical Center, Leiden University Medical Center)| Geraldine Poenou (Leiden University Medical Center, Leiden University Medical Center)| Ka Cheung (Leiden University Medical Center)| Mark Schreuder (Leiden University Medical Center)| Mettine Bos (Leiden University Medical Center)
The serine protease factor Xa plays an important role in blood coagulation as it, in complex with its cofactor Va, proteolytically activates prothrombin to thrombin, the latter being a key enzyme in coagulation. To perform this vital function, factor Xa undergoes numerous posttranslational modifications including vitamin K-dependent γ-carboxylation of glutamic acid (Glu) residues into so-called Gla residues. A correctly γ-carboxylated GLA domain is essential for interaction with the negatively charged membrane surface of activated platelets or endothelial cells at the site of injury where clot formation is required. Moreover, assembly of factor Xa with the cofactor Va into the prothrombinase enzyme complex occurs on a negatively charged membrane surface only. Interestingly, the venom of some Australian Elapid snakes comprises powerful prothrombin-activating enzyme complexes consisting of factor Xa- and Va-like proteins that are specifically expressed in the venom gland. We have previously demonstrated that this venom enzyme complex is capable of functioning independently of a negatively charged phospholipid membrane.
We aim to uncover the putative differential role of the GLA domain between human and snake venom (Pseudonaja textilis, pt) factor Xa. To do so, we exchanged the human factor Xa (hFX) GLA domain for that of the snake venom ortholog (ptFX), thereby generating hFX-wt, hFX-ptGLA, ptFX-wt, ptFX-hGLA. The FX variants were recombinantly expressed and purified employing chromatography techniques.
FX activation by either the human extrinsic or intrinsic coagulation tenase complex was analyzed using a peptidyl substrate specific for activated FX. Introduction of the ptGLA domain into hFX modestly improved the catalytic rate of the extrinsic reaction, while for the intrinsic reaction the substrate binding affinity was enhanced. Conversely, using similar conditions no FXa formation could be detected following incubation of ptFX or ptFX-hGLA with the human intrinsic or extrinsic tenase complex. Assessment of the FX-specific plasma clotting activity in which thrombin-dependent fibrin clot formation is monitored following either an extrinsic or intrinsic coagulation trigger revealed that while the extrinsic clotting activity of hFX was drastically reduced when hGLA was exchanged for ptGLA (3% residual activity), its intrinsic activity was unperturbed. Use of human versus rabbit reagents for the extrinsic pathway may explain the different observations. Conversely, exchange of ptGLA for hGLA resulted in an overall substantial increase in the FX-specific clotting activity of ptFX-hGLA. Finally, exchanging the ptGLA domain for the hGLA domain did not abolish the requirement for a negatively charged membrane surface in Va-dependent prothrombin activation.
These data indicate that the snake venom GLA domain interacts with the intrinsic tenase complex and contributes to substrate conversion and subsequent thrombin formation, which may point to evolutionary adaptation of this snake venom GLA domain.