Microparticles Are a Major Determinant of Thrombin Generation Measured by Technothrombin®TGA.

We have recently shown that a thrombin generation assay using low concentrations of phospholipids (3,2 mM; 20% PS) can discriminate patients with an increased risk for recurrent venous thromboembolism after cessation of anticoagulant therapy from those with a lower risk (Hron et al JAMA 2006). Patients with peak thrombin of ≥400nM had a ~3-fold higher relative risk of recurrence than those with peak thrombin <400nM. This difference was not explained by a larger proportion of patients with clotting factor abnormalities among those with high peak thrombin. We therefore investigated which other factors might influence peak thrombin generation. For this purpose 35 healthy male plasma donors were analyzed. Thrombin generation was measured in standard platelet poor plasma and in plasma made microparticle (MP) free by centrifugation for 120 minutes at 35,250 x g using low (3,2μM) and high (32μM ) phospholipids concentrations at the same tissue factor concentration (71,6 pM). For control purposes, peak thrombin was measured under similar conditions in 12 individuals each having either heterozygous FV_Leiden mutation, being on vitamin K-antagonist therapy (VKA; mean INR=3.2) or having a lupus anticoagulant. In healthy plasma donors peak thrombin as generated by tissue factor and low phospholipids (RCL) in platelet poor plasma was 110±40 nM and increased to 306±99 nM in the presence of high phospholipids (RCH). When MP free plasma was used, however, peak thrombin dropped to 23±13 nM (RCL) and 109±43 nM thrombin (RCH), respectively. When MPs were re-added to MP-free plasma samples, a dose dependent increase in peak thrombin was found both for RCL and RCH reagents. At a MP concentration of 25,000 added per 1 ml MP free plasma, peak thrombin was 381±24 nM for RCL and 399±63 nM for RCH. The effects of MPs on peak thrombin using RCL were much more pronounced than the differences in peak thrombin seen between plasmas from individuals with FV_Leiden, during anticoagulant therapy or with lupus anticoagulant as compared to controls (FV_Leiden : 161±50 nM; VKA: 50±12 nM; lupus: 37±23 nM; controls: 110±40 nM). These results indicate that MPs contained in platelet poor plasma are a major determinant of peak thrombin in thrombin generation assays when low phospholipids are used and might cause increased peak thrombin levels found in patients with recurrent venous thromboembolism.