Microparticle-mediated thrombin generation assay: increased activity in patients with recurrent thrombosis

Background: Circulating cell-derived microparticles (MP) are important players in thrombogenesis, attributed in part to tissue factor (TF) carried on them. We developed MP-mediated thrombin generation assay (TGA) and measured a series of patients with thrombosis (TBS) and normal controls (NC). Methods: MP were isolated from plasma of 66 patients with TBS and 34 NC. The MP were resuspended in normal pooled particle-free plasma (PFP) containing corn trypsin inhibitor (to inhibit contact pathway). MP mediated TGA yields three parameters: lag time, peak and rate. This method is not influenced by anticoagulant therapy. Of the TBS patients, 41 had only a single thrombosis (S-TBS) and 25 had recurrences (R-TBS) within a 5-year period. In parallel, MP were quantitated by flow cytometry, and cell origin was determined: endothelial cells (EMP), leukocytes (LMP), red cells (RMP) and platelets (PMP). Results: MP from all TBS patients exhibited higher thrombin generation than NC by all three TGA parameters. R-TBS had significantly greater TGA values than S-TBS, reflected in higher peak and rate, and shorter lag time. MP numbers were also higher in TBS vs. NC, for all MP subtypes, and were significantly higher in R-TBS than S-TBS (except LMP). All MP levels correlated with thrombin generation (P < 0.0001), most closely between PMP and peak (R = 0.47) and rate (R = 0.43). Conclusions: MP-mediated TGA is a novel way to assess functional procoagulant activity of MP. Enhanced MP-mediated TGA was demonstrated in TBS patients, and significantly higher activity in R-TBS. These findings support a major role of MP in thrombogenesis.