Abstract
Abstract
INTRODUCTION. Microparticles (MP) derived from the plasma membranes of platelets (PMP), leukocytes (LMP), red cells (RMP) and endothelial cells (EMP) are known to be associated with a variety of thrombotic and inflammatory disorders. Most studies have measured their numbers in circulation but few have addressed their function and contribution to clinical thrombosis (TBS). We developed an assay to measure microparticle-mediated thrombin generation (MMTG) and evaluated it in patients (pts) with TBS. We report here that MMTG can distinguish pts at high risk of recurrent TBS from those with stable diseases.
METHODS. (a) MMTG assay. Platelet-poor plasma (PPP) was obtained from citrated blood by centrifuging platelet-rich plasma (PRP) at 3000xg for 10min, and total MP were sedimented from 1.0 mL of PPP at 20,000xg for 15min. The supernatant was considered particle-free plasma (PFP). The MP were resuspended in normal pooled PFP containing 25 ug/mL corn trypsin inhibitor, then MP sample was tested for MMTG initiated by calcium employing the calibrated automated thrombogram (CAT) method of Hemker et al in a Fluoroskan Ascent plate reader. Measures recorded were the lag times, peaks, and rates (slope) of thrombin generation based on CAT data. In addition, populations and cell origins of the MP were determined by flow cytometry using cell-specific fluorescent antibodies. (b) Clinical study. Sixty-six pts with TBS were studied: 29 with antiphospholipid syndrome, 11 high homocysteine, 17 hypercoagulable state due to defective clotting factors (FVLeiden etc) or natural anticoagulants (ATIII etc), and 9 idiopathic. Of the total, 25 suffered multiple (2–8) thrombotic events within the 5-yr study period (recurrent TBS); the other 41 had only a single episode (stable TBS). All measures were done at least 4 wk after acute events. There were 34 normal controls (NC).
RESULTS. (1) MMTG data: Recurrent TBS group showed the highest MP-dependent thrombin generation (lag = 9.1 min, peak = 299.9 nM, and rate = 120.2 nM/min) followed by stable TBS (lag = 12.4, peak = 244.3, and rate = 84.6), and NC (lag = 13.4, peak = 198.6, and rate = 43.1). The difference between groups (recurrent vs. stable; stable vs. NC; recurrent vs. NC) on all three measures were statistically significant, with exception of lag time between stable and NC. (2) MP profiles: Our data showed that recurrent TBS also had highest counts of EMP, PMP, LMP, and RMP, followed by stable TBS, and NC. This trend parallels MMTG data. (3) Sensitivity of MMTG assay: We found that MMTG rate in recurrent TBS was about triple the NC (120.2 vs. 43.1, p<0.0001), with little overlap of standard deviation (SD) indicating good sensitivity to distinguish between these two groups: 76% of recurrent TBS pts had MMTG rate >2 SD above normal mean. (4) We analyzed correlations between MMTG measures and MP counts in all TBS patients: the best correlation was LMP with lag time (p=0.00014), or with rate (p=0.008), suggesting that LMP may carry tissue factor to initiate coagulation. PMP also correlated with rate and peak (p=0.01).
CONCLUSIONS. To our knowledge, this is the first laboratory assay to differentiate stable from recurrent thrombotic pts. MMTG assay can be utilized as a global screening test for risk of recurrent TBS. Microparticle mediated thrombin generation (MMTG) may play an important role in the pathogenesis of recurrent TBS.
