INTRODUCTION. Cell-derived microparticles (MP) such as from platelets (PMP), endothelia (EMP) and leukocytes (LMP) are increasingly recognized as useful biomarker and important mediators of thrombosis and inflammation. However, little attention has been paid to the possible role of MP from RBC (RMP) in vascular disorders. RMP were identified by glycophorin (GPH) in flow cytometry in most studies. We reported heterogeneity of RMP in size and phenotypes and that GPH is expressed predominately in larger RMP, not in smaller RMP and that GPH+ RMP are more active than GPH- RMP in thrombin generation. Since acetylcholinesterase (AChE) activity has been measured on RMP, and was recently proposed as a marker of some inflammatory states, we investigated AChE activity of RMP compared to platelet-derived MP (PMP). AChE of PMP has not previously been reported. METHODS. RMP were prepared from intact washed RBC at 18% Ht exposed to calcium ionophore (4uM) in presence of calcium (2mM) for 30 min. PMP were prepared from 20 mL citrated blood, and exposing the platelet-rich plasma to 1 uM calcium ionophore (without added Ca2+) and collagen, 4ug/mL, for 20 min. AChE assay was based on Ellman’s method and reagent (DTNB), run in 96-well plates, 300uL. Substrate was acetylthiocholine iodide (1 mM f.c.). DTNB was used at 0.67 mM f.c. Tests were run +/− quinidine (Q) (1.2 uM) and some tests were in presence of saponin 0.01%. Q is known to inhibit AChE of plasma but RBC activity is insensitive. Activity is expressed in umols substrate cleaved /min per 108 MP, with provisos below. Flow cytometry using FITC labeled lectin, Ulex europaeus (Ulex) was used to quantitate RMP and PMP. RESULTS. As expected, Q inhibited AChE in plasma by >90% but not AChE of RMP. On contrary, RMP were consistently stimulated by Q, up to 150% activity +Q; some preparations of PMP were also stimulated. Saponin, which has been used in assay of RBC AChE, had little effect on PMP or RMP activity. In 12 experiments, AChE of PMP exhibited marked concentration-dependence. The apparent activity per mL of suspension was greater with lesser volumes, by as much as 3-fold between 2.5uL and 20uL added. This could not be explained by substrate inhibition since the effect varied in different preparations, was absent in particle-free plasma, and did not diminish in low substrate. This suggests the presence of a natural inhibitor. Calculation of specific activity of the MP was complicated by the dependence of apparent activity on volume assayed. However, when equal dilutions were compared, a representative experiment showed RMP had about 6-fold greater activity than PMP per 108 MP: 36.0 vs. 5.88 for 2.5uL suspension; and 29.0 vs. 3.9 for 20 uL assayed, in units above. CONCLUSIONS / DISCUSSION. The AChE activity of RMP is about 6-fold greater than PMP. Weaker activity on PMP is possibly attributed to a previously unreported natural inhibitor. Blood AChE activity has been shown to reflect inflammatory states. Since AChE is a GPI-anchored protein, it is preferentially depleted from cells on the MP shed off. Assay of this activity in patient cell-free plasma, +/− Q, may be a useful biomarker. It is well known that hemolytic anemia, where RMP are elevated, is often associated with thrombotic complications, whereas ITP, where PMP are frequently elevated, rarely is. Further study to characterize AChE in RMP and other MP, and to clarify the physiological role of MP- and cell-associated AChE in thrombosis, inflammation, and cardiovascular disease is in progress.
2991 Poster Board II-967 Background. The natural function of ACTH in blood is not well defined. However elevated plasma level was reported in patients with chronic inflammation, suggesting as one of inflammatory markers. Erythrocytes possess membrane-bound acetylcholinesterase (AChE) while plasma contains a less specific cholinesterase (ChE). We reported at a previous meeting [Blood 2008, 112(11) Abst #3849] that AChE activity of RBC-derived microparticles (RMP) is 6 times higher than those in PMP. In this study we measured plasma ACTH levels in several groups and encountered unexpectedly high plasma ChE activities in patients with TIA and other thrombosis. Methods. (i) Patient population. A series of n=108 consenting patients were recruited sequentially having various disorders For purpose of analysis, they were divided in 2 main groups: n=53 with thrombosis (TBS), F/M 27/26; and n=55 non-TBS, F/M 24/31. Thrombosis group consists of venous and arterial thrombosis including those with TIA. Non TBS group consists of anemias, thrombocytopenias including ITP, MDS. (ii) Assay was essentially by Ellman's method. In our system, milli-absorbance units/min (mA/min) x 0.065 = umols substrate cleaved/min. Values reported here are in unitsof mA/min per mL plasma. Normal controls (NC, n=14) had a cutoff of 3000 mA/min per mL plasma, = mean +2SD. (iii) Sample handling. Platelet-poor plasma (PPP) was prepared by centrifuging 10 min at 1800 xg, then frozen in aliquots. For assay, it was diluted 1:20 with saline, then 5 uL and 10 uL were used in 96-well microtiter plate. Results: (i) Significantly higher ChE activity was observed in thrombosis (TBS) patients compared to non-TBS. Mean value ±SD in TBS was 2928 ±773, and in Non-TBS was 1897 ±713 (units as above). This difference was significant, p<0.001. (ii) When analyzed elevated levels of ACTH above 2SD of normal controls (n=26) between 2 groups, the TBS group had elevated ChE (>3000) in 30% of patients, while the Non-TBS had elevated ChE in 7.8%. (iii) Further analysis among thrombosis group revealed that the subgroup with neurological TBS (TIA, minor strokes) had the most consistent elevations of ACTH; 10 of15 with TIA group (66%) had ChE activities >3000. Summary/ Discussion. Although ChE and AChE in blood has long been studied, to our knowledge, this is the first report to show a relation between plasma ChE and thrombosis. Its elevation is very pronounce in thrombosis of the CNS, manifesting as TIA. Further study is warranted to elucidate how ChE is related to AChE of the CNS and on red cells. Disclosures: No relevant conflicts of interest to declare.
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