[Analysis of protein kinase C activity of peripheral blood T lymphocytes in children with acute idiopathic thrombocytopenic purpura].
OBJECTIVE
Acute idiopathic thrombocytopenic purpura (AITP) is a common autoimmune disease in children. Thrombocytes decrease extremely in serious patients, its pathogenesis involves abnormal activation of T lymphocytes and T cell-dependent production of autoantibody. The aim of the present study was to investigate changes of protein kinase C (PKC) activity in peripheral blood T lymphocytes in children with AITP and the relationships between PKC activity and T lymphocytes activation and thrombocytopenia.


METHODS
Peripheral blood specimens were collected from children with acute ITP (n = 35) and healthy children (n = 30), and T lymphocytes were isolated and purified by using T cells Segregation Enrichment Column. PKC activity was detected by using PepTag Assay, a non-radioactive detection method. The reaction mixture, in a final volume of 25 microl, consisted of 5 microl reaction buffer, PepTag C1 5 microl (0.4 microg/microl), PKC activator solution (DG) 5 microl, peptide protection solution 1 microl and sample 9 microl. Phosphorylation reaction was allowed to continue for 30 minutes, then 25 microl reaction mixture was subjected to electrophoresis on a 0.8% agarose gel at 100 V for about 20 minutes. After electrophoresis, the PepTag C1 peptides which were phosphorylated and non-phosphorylated were separated, phosphorylated PepTag C1 peptide with negative electricity migrated toward the anode (+), but nonphosphorylated PepTag C1 peptide with positive electricity migrated toward cathode (-), the gel was photographed. Electrophoresis bands on anode represented PKC activity and were analyzed quantitatively. FasL, which is T cell activation marker, was determined by flow cytometer and platelet was counted by cell counting meter.


RESULTS
Compared with healthy children, children with AITP had significantly higher PKC total activity [(0.97 +/- 0.21) nmol/(min.ml) vs. (0.55 +/- 0.13) nmol/(min.ml), (P < 0.05)]. Expression of FasL on T cell subpopulation in children with AITP was significantly higher [Th FasL: (32.7 +/- 3.4) vs. (14.7 +/- 4.2); Tc FasL: (17.3 +/- 9.7) vs. (11.6 +/- 8.5)%, (P < 0.05)]. Besides, relationships between the changes of PKC activity, Th FasL and Tc FasL had positive correlation (r(1) = 0.68, r(2) = 0.53, P < 0.05). However, PKC activity and platelet count had a significantly negative correlation (r = -0.75, P < 0.05).


CONCLUSIONS
Increased PKC activity was seen in children with AITP, which can cause damage to thrombocytes and reduction of thrombocytes. PKC signal transduction pathway might play an important role in the immunopathogenesis of AITP.
Liu
Fang
F
Department of Microbiology and Immunology, Guangdong Medical College, Zhanjiang 524023, China.
Wu
Chang-lin
CL
Xiao
Hong
H
Chen
Qun
Q
D000208:Acute Disease; D016022:Case-Control Studies; D002469:Cell Separation; D002648:Child; D004586:Electrophoresis; D005434:Flow Cytometry; D006801:Humans; D008213:Lymphocyte Activation; D010976:Platelet Count; D011493:Protein Kinase C / Q000378:metabolism*; D016553:Purpura, Thrombocytopenic, Idiopathic / Q000201:enzymology* / Q000276:immunology; D013601:T-Lymphocytes / Q000201:enzymology*
D011493:Protein Kinase C