Inhibition of Platelet-Prothrombianse by a Covalent Antithrombin-Heparin Complex.
Authors: Stevic Ivan , Chan Howard H.W. , Chander Ankush , Berry Leslie R. , Chan Anthony K.C. 
Published: October 2019

Abstract
Abstract 2212

Introduction:
Factor Xa is protected within the prothrombinase complex from inhibition by heparin. We have developed a covalent antithrombin-heparin complex (ATH) with enhanced anticoagulant activity. Previously, we have shown that ATH is able to inhibit coagulation enzymes much more efficiently than regular antithrombin+heparin (AT+UFH). For example, ATH inhibited TF/VIIa ∼30-fold faster compared to AT+UFH. Furthermore, we have also demonstrated that ATH is capable of inhibiting Xa within a prothrombinase complex assembled on synthetic phospholipid vesicles better than AT+UFH. However, ATH's effect on prothrombinase when the complex is formed on a more native system such as platelets has never been explored. Thus, the objective of the present study is to determine the ability of ATH vs AT+UFH to inhibit Xa within the prothrombinase complex when the enzyme complex is assembled on the platelet system.

Methods:
Discontinuous second order rate constant assays were performed to obtain k2-values for inhibition of free or prothrombinase-bound Xa by AT+UFH or ATH. Freshly prepared resting platelets were subjected to inhibition analysis by first incubating them with Xa, 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 to initiate thrombin generation, 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. For experiments requiring activated platelets, freshly isolated platelets were activated with 5 μM calcium ionophore A23187 + 4 mM CaCl2 for 15 min at room temperature. The activated platelets were then tested in inhibition assays as described above. To investigate the roles of individual components of the prothrombinase complex on the anticoagulant effects of AT+UFH and ATH, additional experiments were performed where components of the complex (prothrombin, activated platelets or Va) were omitted prior to reaction with inhibitors. Thrombin generation was used to assess functionality of the activated platelet-prothrombinase system in the presence of inhibitors using a thrombin-specific substrate S-2238™.

Results:
The k2-values (×108M−1min−1) for inhibition of free Xa or resting platelet-prothrombinase were similar for both inhibitors, although the overall inhibition rates achieved by the ATH were 2-fold faster than AT+UFH (p<0.001). Since activated platelets are required for enhanced prothrombinase function, we then compared inhibition of free vs activated platelet-prothrombinase by the two inhibitors (platelet activation was confirmed with flow cytometry using an anti CD-41 antibody). No differences were observed in the k2-values between free Xa (3.96±0.23) and activated platelet prothrombinase (3.83±0.39) for ATH reactions. However, the k2-values for inhibition of free Xa by AT+UFH was 2.37±0.32, and assembly of Xa within the activated platelet-prothrombinase resulted in a reduction in the k2-values to 0.99±0.22 (p<0.001), thus confirming a moderate 60% protection of Xa by the prothrombinase components. However, omitting the components (prothrombin, activated platelets or Va) from the complex resulted in higher k2-values (1.76±0.37, 2.29±0.26 and 2.52±0.32, respectively p<0.01) for AT+UFH, and as expected, no net effect was observed for ATH. Thrombin generation was inhibited significantly by both AT+UFH and ATH compared to the control (p<0.001), but further analysis of thrombin potential yielded greater inhibition by ATH compared to AT+UFH (p<0.05).

Conclusion:
In this study, we report inhibition of the prothrombinase complex on the surface of resting and activated platelets. Consistent with previous investigations, a moderate protection of Xa was observed when the activated platelet-prothrombinase was inhibited by AT+UFH. ATH on the other hand, targets and inhibits prothrombinase complexed-Xa as fast as free Xa, and at inhibition rates that were significantly faster than AT+UFH. Thus, overall the covalent conjugate enhances anticoagulation of surface-bound enzymes and offers advantages over conventional heparin for the treatment of cell-based coagulation in vivo.

Disclosures:
No relevant conflicts of interest to declare.