Background: Unfractionated heparin (UFH) is the primary anticoagulant for highly procoagulant indications including cardiopulmonary bypass (CPB) and severe COVID-19. UFH is unable to fully inhibit thrombin generation increasing the risk for thrombo-inflammatory adverse events (AEs).
We attempt to address this limitation by evaluating the FX/Xa aptamer, 11F7t. We hypothesized that combinations of UFH and 11F7t would achieve enhanced anticoagulation while minimizing thrombin generation in models of CPB and COVID-19 associated coagulopathy (CoAC).

Aims: We determined optimal combinations of UFH and 11F7t that produced CPB-sufficient anticoagulation and minimized thrombin generation in whole blood in vitro and ex vivo models of CPB. Furthermore, we aimed to determine the utility of this anticoagulation strategy in addressing CoAC.

Methods: We employed whole blood coagulation assays to determine the optimal doses of UFH and 11F7t. Fresh human blood was treated with UFH+11F7t combinations and continuously circulated in an ex vivo oxygenator circuit for 2 hours to model CPB. Thereafter, thrombin generation was assessed using ELISAs. For CoAC studies, thrombin generation from severely ill COVID-19 patient plasma was determined using the Calibrated Automated Thrombogram (CAT) assay.

Results: In in vitro and ex vivo models of CPB, a combined dose of 2U/mL UFH with 2μM 11F7t was able to achieve anticoagulation similar to the therapeutic dose of UFH (5U/mL). Further investigation into thrombin generation with ex vivo experiments showed that the combinations of UFH and 11F7t are more effective than UFH alone at limiting thrombin generation (Figure 1). Similar anticoagulation and reduced thrombin generation was observed when blood from severely ill COVID-19 patients on heparin therapy was treated with 2μM 11F7t (Figure 2).
Thrombin generation during extracorporeal circulation of human blood. a) Levels of prothrombin fragment F1+2 in plasma prepared from blood that went through circulation for 120 minutes and that which did not were measured. b) Levels of thrombin-antithrombin (TAT) in plasma prepared from blood that went through circulation for 120 minutes and that which did not were measured. In both A and B, (n=3) for 5U/mL UFH, 2μM 11F7t, 2U/mL UFH +2μM 11F7t, and 5U/mL UFH +2μM 11F7t. (n=4) for 2U/mL UFH. Statistical analysis was performed by one way ANOVA followed by Sidak’s multiple comparisons test. For panel A, (prothrombin F1+2) comparison between 2U/mL UFH versus 2U/mL UFH + 2μM 11F7t, P <0.0001 and, comparison 5U/mL UFH vs. 5U/mL UFH + 2μM 11F7t, P=0.0404. For panel B, (TAT) comparison between 2U/mL UFH versus 2U/mL UFH + 2μM 11F7t, P= 0.0006 and, comparison 5U/mL UFH vs. 5U/mL UFH + 2μM 11F7t, P= 0.0109.

Effect of 11F7t on thrombin generation in heparin-treated COVID-19 patient blood. Calibrated Automated Thrombogram assays were performed to measure thrombin generation in platelet poor plasma from severely ill COVID-19 patients. All COVID-19 patients were treated with heparin the day before a) Thrombograms of plasma from a severely ill COVID-19 negative patient (n=1), COVID-19 positive patients (n=5) treated with either Vehicle or 2µM 11F7t. b) Plotted is the area under the curve or ETP for each treatment group. Statistical analysis was performed by paired t-test with P=0.0025. 

Conclusions: This study demonstrates that combining UFH with a FX/Xa aptamer produces potent anticoagulation with reduced thrombin generation and has the potential to address CPB-associated AEs and has potential utility in other procoagulant settings such as CoAC.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/combining-heparin-with-a-fx-fxa-aptamer-to-reduce-thrombin-generation-in-models-of-cardiopulmonary-bypass-and-covid-19-associated-coagulopathy/