Procoagulant Actions of Circulating Microparticles in Sickle Cell Anemia and Sepsis Associated Coagulopathy and Their Modulation By a Triblock Polymer MST 188

Abstract

Introduction:Circulating microparticles (MPs) represent vesicles and cellular fragments of less than 1um which are generated in microvascular angiopathic conditions in both sickle cell anemia (SCA) and sepsis associated coagulopathy (SAC). MPs mediate pro-coagulant actions via the expression of their surface phospholipids and tissue factor. In SCA, MPS are mainly of erythrocytic origin generated during hemolysis and contribute to thrombin generation. In SAC, MPs are generated through the fragmentation of both blood and endothelial cells and contribute to thrombogenic and inflammatory responses. MST-188 is a triblock polymer with affinity to hydrophobic surfaces which are generated in microangiopathic conditions and may also interact with the phospholipid component of MPs. To test the hypothesis that MST-188 may decrease the pro-coagulant effects of MPs, a functional method to access MPs levels and a thrombin generation assay was used.Materials & Methods:Citrated and EDTA anticoagulated whole blood samples (N =10 -15) were collected from healthy normal individuals, SCA patients and confirmed SAC patients. Individual aliquots of these samples were supplemented with saline and MST 188 at a concentration range of 0.1-10mg/ml. After 30mins of incubation these mixtures were centrifuged at 2,500g and plasma was retrieved and frozen. These samples were analyzed for functional microparticle levels using an annexin binding/chromogenic substrate based method for microparticle mediated thrombin generation (Hyphen Biomedical Paris France). The results are expressed in terms of nM of microparticles present. The same samples were also evaluated for thrombin generation using a commercially available flurometric method (Technoclone Vienna Austria). The results are compiled in the total amount thrombin generated and thrombokinetic profile.Results:In comparison to normal (16.3 ±4.2 nm), the MPs levels were significantly higher in the SCA (34.2±8.4 nm) and SAC patients (22.6±9.1 nm). The functional level of MPs was markedly reduced in the MST 1-88 supplemented normal (9.3 ± 1.6 nm), SCA (18.1± 4.6 nm) and SAC (14.6 ± 3.1 nm). This decrease ranged from 36-48%. In the thrombin generation assays in contrast to normal (250±38 nm) SCA patients (348±58 nM) and is SAC patient’s plasma (368±71 nm) generated higher levels of thrombin. Supplementation of MST 1-88 resulted in significant decrease in thrombin generation in normal (110±14 nm), SCA patient’s plasma (160±21 nm) and SAC patient’s (168±26 nm) groups. The decrease in thrombin generation ranged from 24-55%. These results show that both the microparticle facilitated and tissue factor mediated generation of thrombin is decreased by supplementing MST 1-88.Discussion:Both SCA and SAC are associated with vaso-occlusive complications due to the deposition of fibrin in the microvasculture resulting in the generation of microparticles from cellular deformation and damage. The microparticles further augment thrombogenesis and contribute to vascular complications in SCA and SAC. In this study, both SCA and SAC groups exhibited higher levels of microparticles compared to normals. Supplementation of MST 188 to both groups of blood samples resulted in a marked decrease of functioning microparticles and thrombin generation. This may be due to the interaction of MST 188 with hydrophobic moieties of the microparticles. . DisclosuresEmanuele:Mast Therapeutics: Employment.