Abstract
Introduction: PMP, EMP and LMP are known to be sensitive markers of thrombotic and inflammatory disorders, but their respective functional activities are obscure. Recent data indicate that these microparticles (MP) possess procoagulant, proaggregatory, and proinflammatory activities. In this study, we have compared these activities in MP derived from different cells.
Methods: PMP were prepared by stimulating platelet-rich plasma with 10 μM ADP plus 5 μg/mL collagen. EMP were prepared by incubating renal endothelial cells (EC) with 10 ng/mL of TNF-α for 24 hrs. LMP were prepared by incubating U937 monocytic cells or neutrophils with 10 ng/mL LPS for 1 hr. Cells were removed by centrifugation (1000xg for 10 min), MP were sedimented (15,000xg for 30 min), pellets were washed twice, then resuspended to equal MP concentration (1 x 108 counts/μL, final concentration) based on counts by flow cytometry. The MP were then tested for (ia) tissue factor (TF) antigen expression (TF:Ag) by flow cytometry, (ib) TF activity by recalcified clotting time in presence of corn trypsin inhibitor, (ii) platelet factor 3 (PF3) procoagulant activity by RVVT [Thromb Res 80:471, 1995], (iii) von willebrand factor (vWF)-dependent platelet aggregating activity by a ristocetin flow cytometric method [J Thromb Haemost 3:1301, 2005], and (iv) binding of MP to leukocytes, induced expression of CD11b and enhanced transendothelial migration (TEM) [Front Biosci 9:3137, 2004].
Results: In vivo, we found that the relative abundance of PMP, EMP, and LMP numbers in normal plasma is 50–70%, 5–10%, and 5–15% of total MP, respectively. In vitro results were as follows: (i) LMP exhibited the highest TF:Ag per MP followed by EMP, >PMP. However, PMP produced the shortest recalcified clotting time, indicating PMP had the highest apparent TF activity. (ii) PF3 activity was highest in PMP, followed by EMP, > LMP. In view of the abundance of circulating PMP (50–70% of total MP), it appears that PMP are mainly responsible for hemostatic activity in cell-free coagulation. (iii) On the other hand, EMP had the highest specific activity in promoting vWF-dependent platelet aggregation, > PMP, > LMP. (iv) Both PMP and EMP showed high affinity in binding monocytes and neutrophils, and inducing expression of CD11b, as well as promoting TEM. LMP had little effect on leukocyte activation.
Discussion: Our results show that MP of different cell origins have distinctive activities in promoting coagulation, platelet aggregation, leukocyte activation, and TEM. These differences may reflect their distinctive membrane compositions. Overall, PMP and EMP seem to play more active roles in hemostasis and inflammation, as judged by these measures. Since LMP expressed the highest TF:Ag, they may serve to initiate coagulation during leukocyte activation. But their net procoagulant and proinflammatory activities are relatively small. A better understanding of the functional activities of the growing number of recognized MP species is expected to provide new insights on their roles in hemostasis and inflammation.
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