Inhibition of Red Blood Cell-Prothrombinase by Heparin and a Covalent Antithrombin-Heparin Complex
Authors: Stevic Ivan , Chan Howard H.W. , Berry Leslie R. , Chan Anthony K.C. 
Published: October 2019

Abstract
Abstract 1179

Introduction:
Various hematological conditions, including sickle cell anemia, beta-thalassemia and preeclampsia, have been linked with the presence of procoagulant red blood cells (RBCs). Overt exposure of phosphatidylserine (PS) on surfaces of RBCs has been a suspect for increased thrombotic complications in these patients. Asymmetrical distribution of PS at the membrane surface may provide docking sites for the formation of the procoagulant prothrombinase complex, which further aids in amplification of the coagulation cascade leading to subsequent thrombosis. In acute thrombotic complications, heparin is used as the first line of treatment in these patients. However, little is known regarding heparin's efficiency to pacify the procoagulant RBC-prothrombinase system. Therefore, we sought to determine the rate of RBC-prothrombinase inhibition by antithrombin and unfractionated heparin (AT+UFH) versus a novel covalent complex of AT and heparin (ATH).

Methods:
Thrombin generation was used to assess functionality of the phosphatidic acid (PA)-induced RBC-prothrombinase system, using a thrombin-specific substrate S-2238™. Discontinuous second order rate constant assays were carried out to obtain k2 values for inhibition of free or prothrombinase-bound Xa by AT+UFH or ATH. Freshly isolated RBCs were activated using PA and calcium for 15 min at room temperature. Activated RBCs were incubated with factors Xa and Va, Ca2+ and pefabloc®-TH (thrombin inhibitor) in different wells of a 96-well plate for 3 min at 37 °C. Prothrombin was then simultaneously added to all wells and allowed to react for 3 min at 37 °C, followed by addition of AT+UFH or ATH inhibitors to each well at specific time intervals. Reactions were neutralized by simultaneous addition of polybrene, Na2EDTA and Xa-specific substrate S-2222™ in buffer. The remaining Xa enzyme activity was obtained and final k2 values calculated. To investigate the roles of individual components of the prothrombinase complex on the anticoagulant effects of heparin and ATH, additional experiments were performed where components of the complex (prothrombin, activated RBCs or Va) were removed prior to reaction with the inhibitors.

Results:
PA-induced RBC-prothrombinase yielded a 4-fold greater thrombin generation compared to non-activated RBCs (p<0.05). The k2 values (x 108 M−1min−1) for inhibition of Xa alone by AT+UFH was 3.14 ± 0.11, whereas inhibition of RBC-prothrombinase significantly decreased this value to 2.12 ± 0.22, p<0.02. In comparison, ATH inhibited both free Xa and prothrombinase-complexed Xa at significantly faster rates compared to AT+UFH reactions (5.12 ± 0.27 and 4.85 ± 0.24 (p<0.0001), respectively). No significant differences were observed for free Xa or RBC-prothrombinase inhibition by ATH (p<0.46). When components of the RBC-prothrombinase were removed from reactions, the greatest impact on Xa inhibition was observed from factor Va omission. A significant decrease in inhibition rate was shown for both AT+UFH and ATH (4-fold and 2-fold, respectively) due to factor Va absence.

Conclusion:
In this study, we report inhibition of the prothrombinase complex on the surface of procoagulant RBCs. Consistent with vesicle and activated platelet systems, RBC-prothrombinase seems to protect Xa from inhibition by AT+UFH, which suggests that heparin efficiency is also compromised when prothrombinase forms on the surface of procoagulant RBCs. Relative to AT+UFH reactions, RBC-prothrombinase was significantly enhanced and no protection of Xa was observed with ATH. From our findings, factor Va may be the major component moderating the protective effect. Overall, these data indicate a clinical advantage for ATH over heparin in treatment of blood-based coagulation in vivo. This study also provides some understanding of how heparin or ATH treatment may interact with procoagulant complexes on the surface of RBCs in patients with red cell disorders that have thrombotic complications.

Disclosures:
No relevant conflicts of interest to declare.