INTRODUCTION: Cell derived microparticles (MP) are submicroscopic vesicles released druing stimulation and carries different adhesion molecules and membrane phospholipids. Although the procoagulant activity (PCA) of PMP and EMP was well recognized, little is known about PCA of RMP. We investigated PCA properties of RMP and compare them with PMP and EMP in relation to their TF expression and Annexin V binding.

METHODS: Thrombin generation was assayed by calibrated automated thrombogram (CAT) system (

Hemker et al, Pathophysiol Haemost Thromb. 2002;32:249
). RMP were generated by exposure of washed RBC to calcium ionophore (10 μM). PMP were generated by exposure of PRP to ADP (10 μM) or calcium ionophore (2 μM). EMP were obtained from the supernatant of cultured renal microvascular endothelial cells activated by TNF-α (10 nM). MP were sedimented down at 20,000xg for 15 min. The MP pellets were resuspended in corn trypsin inhibitor (25 μg/mL)-treated particle free plasma (PFP), then were initiated for CAT test by addition of calcium. MP concentrations and phenotypes were evaluated by flow cytometry. Two measured parameters are of interest, the lag time and maximum peak height of thrombin generation.

RESULTS:

  1. Lag time of thrombin generation. Of 3 species of MP were compared at similar concentrations, EMP gave the shortest lag time (3–5 min), followed by PMP (11–17 min), and RMP (23–27 min).

  2. Peak of thrombin: High thrombin peaks were generated by RMP (278–403 nM) as well as PMP (286–385 nM) even though they have much longer lag time. On the other hand, EMP gave a much lower thrombin peak (10–20 nM), despite having the shortest lag time.

  3. Effect of anti-TF: Anti-TF had little effect on lag time of RMP, but weakly delayed lag time of PMP by 2–3 min. EMP were largely inhibited by anti-TF (↑ lag time by 5–9 min).

  4. Effect of factor deficient plasma: FVIII- or FIX-deficient plasma completely abolished thrombin generation by RMP, and inhibited PMP generated peak by 20%, but have no effect on EMP.

  5. Mixing experiments: When PMP were mixed with RMP (50:50), the mixture behaved like PMP in lag time, but produced higher peak (↑ 25–20%).

Mixing RMP with EMP (50:50) resulted in similar lag time with EMP but greatly increased thrombin peak (from 10–20 nM to 315– 360 nM). (vi) Expression of TF and annexin V: Flow cytometric analysis showed that EMP possessed highest TF (15–21% of total EMP), followed by PMP and RMP (2–5%, and <0.1% of their total MP, respectively). For Annexin V binding, RMP carried highest binding sites (85–94%of total RMP), followed by PMP (70–86%), and EMP (15–28%).

SUMMARY:

  1. Each species of C-MP (PMP, RMP, EMP) have distinct profiles of thrombin generation: EMP is rapid in thrombin generation with shortest lag time but produce very small peak. In contrast, PMP and RMP take longer time to initiate thrombin generation but produce much higher thrombin peak. Mixing study of MP indicates that MPs can work synergistically by combining high TF with high anionic PL for optimum thrombin generation.

  2. TF plays a dominant role in initiating rapid thrombin generation as shown by the lag time, i.e. EMP was most efficient followed by PMP, RMP.

  3. Anionic PS (annexin V binding) appears to play an important role in propagation of thrombin peak, as demonstrated by RMP or PMP.

Topics:
blood platelets, cell-derived microparticles, earth microbiome project, endothelium, erythrocytes, thrombin, mechlorethamine, annexin a5, calcium, ionophores

Disclosure: No relevant conflicts of interest to declare.

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2006, The American Society of Hematology
2006
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