© Schattauer 2013 Thrombosis and Haemostasis 110.4/2013 751 Red cell-derived microparticles (RMP) as haemostatic agent Wenche Jy; Max E. Johansen; Carlos Bidot Jr.; Lawrence L. Horstman; Yeon S. Ahn The Wallace H Coulter Platelet Laboratory, Division of Hematology/Oncology, Department of Medicine, University of Miami Miller School of Medicine, Miami, Florida, USA Summary Among circulating cell-derived microparticles, those derived from red cells (RMP) have been least well investigated. To exploit potential hae- mostatic benefit of RMP, we developed a method of producing them in quantity, and here report on their haemostatic properties. High-press- ure extrusion of washed RBC was employed to generate RMP. RMP were identified and enumerated by flow cytometry. Their size distribu- tion was assessed by Doppler electrophoretic light scattering analysis (DELSA). Interaction with platelets was studied by platelet aggrego- metry, and shear-dependent adhesion by Diamed IMPACT-R. Thrombin generation and tissue factor (TF) expression was also measured. The effect of RMP on blood samples of patients with bleeding disorders was investigated ex vivo by thromboelastography (TEG). Haemostatic efficacy in vivo was assessed by measuring reduction of blood loss and bleeding time in rats and rabbits. RMP have mean diameter of 0.45 μm and 50% of them exhibit annexin V binding, a proxy for pro- coagulant phospholipids (PL). No TF could be detected by flow cyto- metry. At saturating concentrations of MPs, RMP generated thrombin robustly but after longer delay compared to PMP and EMP. RMP en- hanced platelet adhesion and aggregation induced by low-dose ADP or AA. In TEG study, RMP corrected or improved haemostatic defects in blood of patients with platelet and coagulation disorders. RMP re- duced bleeding time and blood loss in thrombocytopenic rabbits (bu- sulfan-treated) and in Plavix-treated rats. In conclusion, RMP has broad haemostatic activity, enhancing both primary (platelet) and sec- ondary (coagulation) haemostasis, suggesting potential use as hae- mostatic agent for treatment of bleeding. Keywords Animal bleeding models, haemostatic agent, RMP–platelet interac- tion, red cell microparticles (RMP), thromboelastography Correspondence to: Wenche Jy, PhD University of Miami Miller School of Medicine 1600 NW 10th Ave., RMSB 7109, R36-A Miami, FL 33136, USA Tel.: +1 305 243 6617, Fax: +1 305 243 5957 E-mail: wjy@med.miami.edu Financial support: The research is supported and funded by the Wallace H Coulter Foundation and a grant from NIH NHLBI (1R01HL098031). Received: March 2, 2013 Accepted after major revision: July 3, 2013 Prepublished online: September 12, 2013 doi:10.1160/TH12-12-0941 Thromb Haemost 2013; 110: 751–760 Blood Coagulation, Fibrinolysis and Cellular Haemostasis Introduction Cell-derived microparticles (C-MP) are membranous vesicles re- leased during cell activation and apoptosis (1). Release of C-MP is associated with reversal of membrane asymmetry, exposing proco- agulant phospholipids such as phosphatidyl serine (PS) to the ex- ternal medium. This provides negatively charged phospholipid membrane surface for the binding of clotting factors and assembly of tenase and prothrombinase, thereby accelerating thrombin gen- eration and blood clotting (2). They are found in normal circu- lation at low levels and at higher levels in thrombotic, inflamma- tory, and cardiovascular disorders (3). The role of C-MP in haemostasis and thrombosis has been re- ceiving increased attention in recent years (3-5). Among the many species of circulating C-MP, those from red cells (RMP) have been investigated least in regard to their role in haemostasis and throm- bosis. RBCs have traditionally been viewed as comparatively inert in haemostasis and thrombosis (6). However, a number of clinical and laboratory observations suggest a significant role in coagu- lation and inflammation (7, 8). Platelet-derived MP (PMP) has been proposed as an infusible haemostatic agent for treatment of bleeding disorders (9-11) but development was apparently halted. Our studies in vitro, together with in vivo animal studies (12-15), suggested that RMP would have broad haemostatic activity and appear well suited for use as haemostatic agent. In this paper, we report on indicators of haemostatic activity of RMP by in vitro and ex vivo methods, and also document in vivo efficacy in rats and rabbits. Materials and methods Preparation of RMP Packed red blood cells (40 ml) were diluted 2.5-fold with isotonic saline, centrifuged at 650 x g for 10 minutes (min) and supernatant discarded. The RBCs were re-suspended in 40 ml saline and sub- jected to high-pressure extrusion via the TS2 Bench-top Cell Dis- ruptor (Constant Systems, Kennesaw, GA, USA). The output was centrifuged at 250 x g for 10 min to remove large debris; the pellets and bottom 1 ml of each tube was discarded. The remaining super- For personal or educational use only. No other uses without permission. All rights reserved. Downloaded from www.thrombosis-online.com on 2018-03-23 | ID: 1001066444 | IP: 54.70.40.11