Proteolytic signatures of coagulation identified and monitored by plasma peptidomics

Background: Coagulation comprises a network of proteolytic processes initiated by tissue factor (TF) that regulate the function of plasma proteins and result in the formation of a fibrin clot. Altered proteolysis of coagulation proteins is implicated in hemostatic and fibrinolytic disorders. Here, we employed plasma peptidomics to identify the proteolytic products of coagulation and subsequently monitor the impact of tissue factor concentrations and thrombin inhibition in proteolytic processing.

Methods: Pooled citrated plasma from healthy donors was clotted in vitro by recalcification and addition of increasing concentrations of tissue factor (TF), in the absence or presence of a thrombin inhibitor hirudin. Endogenous peptides were enriched by solid phase extraction (SPE), analysed by LC-MS/MS in DDA mode (Orbitrap Fusion Lumos) and identified using PEAKS X. Thrombin generation in clotting plasma was measured using the Calibrated Automated Thrombogram (CAT) assay with a microtiter plate reading fluorometer (Fluoroskan Ascent, ThermoLab systems) and Thrombinoscope® software (Thrombinoscope BV).

Results: Plasma coagulation revealed a distinct and richer endogenous peptidome in which fibrinopeptides A and B were most abundant. Unexpected proteolytic hotspots were identified in the C-terminal region of fibrinogen α chain. The activation peptides of coagulation factors prothrombin (316TAT-DGR363) and FXIIIA (6TAF-VPR38) were recognized as well as their proteolytic degradation products. Peptides derived from protease inhibitors mapping within their internal bait (e.g. 705VGF-MGR715 of alpha 2 macroglobulin) or C-terminal regions (e.g. 426SLN-CVK464 of antithrombin) were observed exclusively upon coagulation and were sensitive to increasing TF concentrations. Thrombin inhibition blocked the formation of coagulation activation peptides and the proteolytic processing of hemostatic regulators, but not inflammatory proteins such as acute phase SAA4. Finally, quantitative monitoring of coagulation with calibrated automated thrombograms showed rapid cleavage of fibrinopeptides correlated with initial thrombin generation followed by cleavages in prothrombin, FXIIIA, and protease inhibitors.

Conclusion: Plasma peptidomics exposed a distinct endogenous peptidome upon clotting, enriched with activation peptides from zymogen-to-enzyme transitions and proteolytic products of protease inhibitors. Monitoring changes in the peptidome under different coagulating conditions provided insights into thrombin and tissue factor sensitive proteolytic processing in plasma. This peptidomics strategy can be leveraged to study proteolytic processing of protease networks in bleeding or thrombosis.