In vitro effects of Gla-domainless factor Xa analog on procoagulant and fibrinolytic pathways in apixaban-treated plasma and whole blood.

Andexanet alfa is a Gla-domainless FXa (GDXa) analog used as an antidote to FXa inhibitors. Despite its clinical use, laboratory monitoring for anti-Xa reversal and the effect of andexanet on fibrinolysis has not been explored. We used a GDXa with a serine-to-alanine mutation at position 195 (chymotrypsin numbering) to model the interaction between andexanet and apixaban. Six batches of pooled plasma, and whole blood from healthy volunteers were treated with increasing concentrations of apixaban with/without GDXa. Thrombin generation and plasmin generation (TG and PG) were tested in plasma, and whole blood thrombus formation was tested using thromboelastometry or a flow-chamber system. FXa was also tested in isolation for its ability to support plasmin activation with/without apixaban and GDXa. Apixaban (250–800 nM) concentration-dependently decreased the velocity and peak of TG in plasma. Apixaban prolonged the onset of thrombus formation in thromboelastometry and flow-chamber tests. GDXa normalized apixaban-induced delays in TG and whole blood thrombus formation. However, GDXa minimally affected the low PG velocity and peak caused by apixaban at higher concentrations (500–800 nM). FXa promoted plasmin generation independent of fibrin that was inhibited by apixaban at supratherapeutic concentrations. This study demonstrated the feasibility of assessing coagulation lag time recovery in plasma and whole blood following in vitro apixaban reversal using GDXa, a biosimilar to andexanet. In contrast, GDXa-induced changes in plasmin generation and fibrinolysis were limited in PG and tPA-added ROTEM assays, supporting the endogenous profibrinolytic activity of FXa and its inhibition at elevated apixaban concentrations. Schemata of putative mechanisms of factor Xa modulation of plasmin generation. A trace amount of fibrin initiates the formation of plasmin (Pln), which proteolyzes phospholipid-bound factor Xaα into Xaβ. The latter is further proteolyzed by plasmin into a heterodimer of 33 kDa and 13 kDa fragments (Xa33/13). Both Xaβ and Xa33/13 express carboxy-terminal lysine residues (⊕ in yellow) to which plasminogen (Plg) can be bound and subsequently activated by tissue plasminogen activator (tPA), facilitating the initiation of fibrinolysis. After binding of apixaban to factor Xaα, thrombin (IIa) generation is slowed down (|| in red), and Xa33/13 formation is blocked (× in red), resulting in reduced velocities of plasmin generation. [Display omitted] • Gla-domainless factor Xa S195A (GDXa) is useful in modeling a factor Xa inhibitor reversal with andexanet alpha in vitro • Profibrinolytic activity of factor Xa is inhibited by apixaban. • Despite the recovery of thrombin generation, GDXa had minimal effect on the changes in plasmin generation and fibrinolysis at elevated apixaban concentrations. [ABSTRACT FROM AUTHOR]