Erythrocyte Participation in Thrombin Generation
Authors: Whelihan Matthew , Mann Kenneth G. 
Published: October 2019

Abstract
Abstract 374

Introduction:
The potential contributions of erythrocytes (RBCs) to coagulation biochemistry have been controversial. We evaluated the potential for RBCs to participate in thrombin generation using minimally altered whole blood. Methods: Platelet poor plasma (PPP), platelet rich plasma (PRP) adjusted to a physiologic concentration with PPP, washed RBCs (PLTs <0.5%) and washed PLTs were prepared from contact pathway inhibited (corn trypsin inhibitor, CTI) whole blood in the absence of other anticoagulants. RBCs and PLTs were evaluated for phosphatidylserine (PS) exposure by FACS using FITC-labeled bovine lactadherin and their ability to support prothrombinase (1.4μM prothrombin, 20nM factor Va, 200pM factor Xa (FXa), 3.4μM antithrombin (AT) at physiologic concentrations of RBCs and PLTs). CTI whole blood and prepared sub fractions (PRP, PPP, PRP+RBC, PPP+RBC) were subjected to a 5pM tissue factor (TF) stimulus and samples analyzed by Western blotting and α-thrombin (αIIa) antithrombin (αTAT) ELISA (Rand et al. Blood, 1996). Results: CTI whole blood (N=3 donors mean±SD) clotted in 4±1.5 min whereas the matching PRP clotted in 8±2 min. In αTAT ELISA analyses, PRP showed a corresponding increase in lag phase and a 50% decrease in the maximum rate (26±9nM/min vs 58±7nM/min) and extent (263±62nM vs 476±86nM) of αTAT formation compared to whole blood. When PRP was reconstituted with physiologic levels of washed RBCs, the rate and extent of αTAT formation as well as the lag phase were restored to that observed in CTI whole blood. Addition of buffy coat to PRP, to test white blood cell contributions, had no effects on any of these parameters. Western blotting showed a significant decrease in prothrombin consumption in the PRP experiments compared to whole blood and PRP with washed RBCs. When RBCs were added to PPP (N=1), there was no significant prolongation of the lag phase and the rate of αTAT formation (21nM/min) was half of that seen in the corresponding whole blood (41nM/min). However, the maximum αTAT generated in RBCs+PPP (267nM) vs whole blood (343nM) differed by only 22%. Collectively, these data indicate a significant role for RBCs in the propagation of thrombin generation in CTI whole blood. FACS analysis of PS exposure on RBCs showed the following: untreated RBCs showed minimal binding to lactadherin (1.3%) compared to the positive control (99.7%; RBCs treated with 10mM N-ethylmaleimide (NEM) and 4μM ionophore A23187); pretreatment of the RBC population with 10nM αIIa or FXa showed a 5-fold increase in lactadherin binding over the untreated control indicating the presence of surface conditions capable of supporting prothrombinase. This proportion (5%) of PS-expressing RBCs represents a similar proportion of PLTs to RBCs (4%) in whole blood. Prothrombin activation (N=2)(1.4μM II, 20nM FVa, 200pM FXa, 3.4μM AT) on untreated RBCs exhibited a 3.5±0.5 minute lag phase followed by αTAT production which reached a rate of 18nM/min and maximum level of 180nM. In experiments performed in the absence of AT, pretreatment of RBCs with either 10nM αIIa or FXa shortened the lag phase by 1±0.25min. PAR-1 (TFLLRN) activated PLTs or activated PLTs+RBCs showed no lag phase, and no difference in the rate of αTAT production (25nM/min) or maximum level (200nM) of αTAT generated. Conclusions: In contact pathway-inhibited whole blood initiated with TF, RBCs appear essential for normal thrombin generation. Our approach rapidly fractionates CTI blood in the absence of other anticoagulants and yields populations of minimally altered RBCs that when subjected to a Tf stimulus produce thrombin more rapidly than PRP. Physiologic levels of washed RBCs pretreated with αIIa or FXa support a level of prothrombin activation similar to that observed with washed activated PLTs. These findings suggest that RBCs participate in thrombin generation and produce a PS-equivalent membrane when treated with αIIa or FXa.

Disclosures:
Mann: Haematologic Technologies: Chairman of the Board, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; corn trypsin inhibitor: Patents & Royalties; NIH, DOD, Baxter: Research Funding; Merck, Daiichi Sankyo, Baxter, GTI: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.