Circulating Microparticles and Microparticle Procoagulant Activity in Patients with Antiphospholipid Antibodies

Abstract

Abstract 5244 Background:Microparticles are membrane fragments from cells and microparticles derived from platelets, monocytes and endothelial cells have been implicated in prothrombotic states. Their procoagulant activity is thought to be through exposure of procoagulant phospholipids. Elevated microparticle levels have been found in artherosclerosis, inflammatory states and pregnancy loss. More recently, elevated levels of both endothelial and platelet microparticles have been implicated in APS and thrombosis and in APS and recurrent miscarriage. Microparticles may play a role in the pathogenesis of APS. Aims:The aim of this study was to assess levels of circulating endothelial and platelet microparticles and to measure microparticle procoagulant activity in patients with aPL. Materials & Methods:We obtained samples from 69 patients (69 females, median age 45 (range 19–73) years) who had PAPS according to International Consensus statement criteria, or had persistent aPL without associated complications. 18 healthy controls (18 females, median age 37.5 (range 20–58) years) were recruited and were not known to have antiphospholipid antibodies. Patients with PAPS included 38 with thrombotic complications, 12 with obstetric complications and 19 with isolated aPL. 37/69 patients with thrombotic complications of APS were receiving vitamin K antagonists and 9/19 of those with isolated aPL were receiving aspirin.Blood was drawn by flawless venepuncture into tubes containing citrate. Samples were centrifuged at 3000 rpm for 15 min at 4°C and stored at −80°C until use. A functional ELISA assay of microparticle procoagulant activity (based on prothrombinase activity stimulated by microparticle phospholipid exposure on binding to annexin V immobilised on the ELISA plates) was performed on platelet depleted plasma samples according to manufacturer’s protocol (Hyphen Biomed, UK). Intra-assay CV was 3–8%.Endothelial and platelet derived microparticles were detected by flow cytometry, specific surface markers were used to identify platelet microparticles (CD41&CD61, Beckman Coulter, UK) and endothelial microparticles (CD51&CD105, Beckman Coulter, UK). Samples were thawed at room temperature and incubated with CD41-PC, CD61-PC, CD51-FITC and CD105-PE. Enumeration beads were added prior to analysis by flow cytometry for quantification of microparticle levels. The unpaired t-test was used to compare platelet microparticle levels between groups and a p-value of <0.05 was considered statistically significant. Results:Results are shown below in table 1 and are represented as medians and 95% confidence intervals.Patient groupaPL/PAPS n=69Thrombotic APS n=38Obstetric APS n=12Isolated aPL n=19Controls n=18Procoagulant activity (nM PS)11.1 11.6–15.911.6 10.8–15.320.3 9.3–28.09.9 8.7–15.914 14.1–23.01PMP CD41 (MP/ul)1452 1918–36781622 1697–43831837 630–5567868 895–3338742 623–2170PMP CD61631 795–1454754 770–1797510 190–1866351 417–1296305 329–905EMP CD511522 1940–37011673 1725–44451639 500–49401107 1026–3653561 612–1725EMP CD105702 961–1752705 911–2168594 344–1550630 585–1877365 379–1039Microparticle procoagulant activity was lower in patients with aPL/PAPS but did not reach statistical difference(p=0.05). No significant difference was observed in platelet microparticle levels(CD41, CD61) between patients with aPL/PAPS. Endothelial microparticle(CD51) levels were significantly higher in patients with aPL/PAPS compared to healthy controls(p=0.02) but endothelial microparticles(CD105) were not significantly different(p=0.08). No statistical differences were seen in procoagulant activity, platelet or endothelial microparticle levels between patients with aPL according to complication(thrombotic APS, obstetric APS or isolated aPL). Conclusions:Endothelial microparticles (CD51) were elevated but no differences were observed in endothelial microparticles (CD105), platelet microparticles or microparticle procoagulant activity in patients with aPL/PAPS. Most of these patients were receiving anticoagulants and antiplatelet agents but this has not been demonstrated to have an effect on microparticle activity. There are currently a variety of methods to quantify microparticles and as standardisation of methods improve, so too will understanding of relevance of results from this clinical study. Disclosures:No relevant conflicts of interest to declare.