Altered plasma factors in congenital thrombophilia suppressing platelet glycoprotein VI activation

Introduction Congenital deficiencies in protein C or protein S are established risk factors for thrombophilia and venous thromboembolism (VTE), but not for arterial thrombosis as a platelet-driven process. Prior investigations indicated that patients with these defects have a partial impairment in collagen- and glycoprotein VI (GPVI)-induced thrombus formation and platelet phosphatidylserine exposure [ 1 ]. This impairment was found to be plasma-based rather than platelet-based. Here, we identified several plasma proteins with altered expression in thrombophilia patients able to modulate GPVI-induced platelet activation. Method Plasmas were collected from 64 patients with genetic defects in protein C, protein S, antithrombin or compound heterozygous factor V Leiden together with plasmas from 29 matched healthy control subjects. Coagulation profiles and thrombin generation parameters were measured. Fibrinogen was assessed using the Clauss method. Plasma proteomic profiles were analyzed by mass spectrometry on Data-Independent Acquisition mode. To measure GPVI-induced platelet activation, patient plasmas were reconstituted with platelets from a healthy subject and measurement by flow cytometry and luminescent ATP release. Results In control plasmas, purified fibrinogen dose-dependently enhanced platelet activation and thrombin generation upon low-dose GPVI triggering or GPVI inhibition with a single-chain antibody. Control platelets reconstituted with plasmas from protein C/S deficient patients were impaired in GPVI-induced platelet activation and ATP release, but not with fibrinogen as a co-variable. Hence, fibrin(ogen) was excluded as a modulator of platelet activation in these thrombophilia patients. Proteomic analysis of 93 plasma samples produced levels of 527 (550) unique plasma-based proteins, of which 35 (38) were significantly altered in protein C deficiency (all thrombophilias), which were in majority liver-secreted. In vitro studies of two of the altered plasma proteins (acronymed Ppf1 and Ppf2) – with structural evidence for platelet receptor interactions – demonstrated dose-dependent suppression of platelet ATP release in response to CRP-XL, collagen and the pentameric single-chain antibody Nb2c5. Proteins Ppf1 and Ppf2 also interfered with other GPVI-induced platelet activation parameters, independently of the fibrinogen concentration. Conclusion These findings identify two novel liver-produced plasma proteins as modulators of GPVI-induced platelet activation independently of fibrinogen. The increased levels of Ppf1-2 in patients with congenital thrombophilia, by suppressing GPVI activity, will act as compensation mechanism for in the thrombotic propensity. These findings can also help to refine the thrombosis risk stratification in affected patients. Publication History Article published online: 17 February 2026 © 2026. Thieme. All rights reserved. Georg Thieme Verlag KG Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany