Abstract
Abstract 2256
Blood coagulation is marked by sequential proteolytic activation of the coagulation factors and complex feedback modulation which is central to haemostasis. The fine balance between the coagulation cascade and the fibrinolytic system is also an integral component in haemostasis. Multiple coagulation factors, including FIIa (thrombin), FXa, FXIa, and FXIIa, are attractive targets for treating thrombosis, and novel inhibitors for several targets are already in late stage development or on the market. However, the responsiveness of these inhibitors to intrinsic vs. extrinsic cues warrants further study because in different disease settings the relative signaling intensities from the two pathways could be different in vivo. It is also poorly understood how these inhibitors might differentially modulate fibrinolytic activity, and a dysregulated fibrinolytic cascade is a potential contributing factor to both thrombosis and bleeding. We therefore sought to gain insight by comparing effects of four agents (dabigatran, apixaban, FXIa inhibitory antibody (FXIa Ab), and FXIIa inhibitory antibody (FXIIa Ab)) in whole blood coagulation and fibrinolysis in vitro using thromboelastography (TEG).
We first established optimal clot-triggering conditions for TEG by using either a low concentration of kaolin (approximately 100-fold less kaolin than in standard aPTT assay), or a low concentration of tissue factor (TF) (1:8000 dilution of Recombiplastin in blood), along with recalcification. We then performed titration of tissue plasminogen-activator (tPA) with these clotting conditions and identified a tPA concentration of 125 ng/ml as optimal in inducing a fibrinolytic phase without significantly altering the clotting phase. Compound profiling was then pursued by dose titration with the individual agent in normal human donor's blood and tested TEG responses to kaolin+/− tPA and TF+/−tPA. Key TEG parameters that were assessed include R (clotting time), reflecting anticoagulant effect, MA (maximal amplitude), representing clot strength, and Ly30 (percent lysis at 30 minutes after MA), reflecting fibrinolytic activity.
For clotting time, dabigatran and apixaban both delayed R in response to both kaolin and TF. FXIa Ab and FXIIa Ab delayed R only in response to kaolin and not to TF. This is consistent with the understanding that thrombin and FXa are in the common pathway, and FXIa and FXIIa are in the intrinsic pathway. For agents that affected the response to kaolin, stack-ranking of the magnitude of effect was FXIa Ab > dabigatran > apixaban > FXIIa Ab. For agents that affected the response to TF, dabigatran was slightly more effective than apixaban. Regarding clot strength, none of the agents appreciably altered MA in the absence of tPA; in the presence of tPA, dabigatran, apixaban, and FXIa Ab reduced MA to a greater extent than FXIIa Ab. Regarding fibrinolytic activity, none of the agents modulated Ly30 in the absence of tPA, consistent with the notion that these inhibitors alone are not fibrinolytic agents. However, in the presence of tPA, dabigatran, apixaban, FXIa Ab, but not FXIIa Ab, enhanced Ly30, suggesting that inhibition of thrombin, FXa, or FXIa may potentiate tPA-induced fibrinolysis.
In summary, each inhibitory agent examined with this whole blood spike-in paradigm exhibited a distinct profile of effects on clotting time, clot strength and fibrinolysis with intrinsic and extrinsic cues in the presence and absence of tPA. Because the thrombotic cues in different patient segments are highly complex, and the endogenous fibrinolytic activities in different tissues may vary considerably, our results provide a benchmarking dataset for these inhibitory agents and their respective targets and represent a step forward in differentiating specific agents and targets in our effort to optimize safety and efficacy with novel anticoagulant therapies.
Disclosures:
Chen: Merck Research Laboratories: Employment. Wu:Merck Research Laboratories: Employment. Xu:Merck Research Laboratories: Employment. Ogletree:Merck Research Laboratories: Employment. Plump:Merck Research Laboratories: Employment. Graziano:Merck Research Laboratories: Employment. Wang:Merck Research Laboratories: Employment.
Blood Coagulation, Fibrinolytic Activity, Platelet Function and Immunoglobulins in Frostbite
