Expression and activity of membrane surface tissue factor in peripheral blood cells of patients with cerebral infarction / 中国实验血液学杂志

This study was aimed to investigate the expression and activity of membrane surface tissue factor (TF) of monocytes and platelets in peripheral blood cells from patients with cerebral infarction and their clinical significance. The TF expressions in monocytes and platelets from 25 patients with cerebral infarction were detected by flow cytometry, the TF activity was detected by chromogenic reaction method, and compared with 24 normal people used as control. The results showed that the TF expressions of monocytes and platelets in peripheral blood cells from patients with cerebral infarction were significantly higher than that in normal controls (p<0.01), and TF activity was also higher in patients than that in controls (p<0.01). In conclusion, the expression and activity of membrane surface in patients with cerebral infarction were enhanced, the hematocyte-derived tissue factor as a trigger in coagulation pathway is involved in pathological thrombosis in patients with cerebral infarction.

Establishment and evaluation of a rabbit model of arterial thrombosis by collagen encapsulated thread-drawing / 中南大学学报(医学版)

OBJECTIVE@#To establish and evaluate a rabbit model of arterial thrombosis by modified thread-drawing.@*METHODS@#Fifty-three rabbits were randomly divided into 6 groups: a normal group, a ligating group,a collagen encapsulated thread-drawing group,a aspirin group,a clopidogrel group, and an aspirin clopidogral group. The endovascular pathological changes in the rabbits were observed, and D-fructose-1,6-diphosphate trisodium salt octahydrate (FDP), D-Dimer and tissue factor (TF) were detected with enzyme linked immunosorbent assay.@*RESULTS@#In the thread-drawing group, thrombus was obvious, and the endovascular elastic membrane was injured seriously compared with the ligating group. After being treated with aspirin and clopidogrel, most arterial thrombus was softened, dissolved and absorbed. Compared with that in the modified thread-drawing group,wet and dry weight of thrombus increased,and the level of D-Dimer, FDP and TF also increased in the modified thread-drawing group (P<0.01). After being treated by aspirin and/or clopidogrel, the wet and dry weight of thrombus and the level of D-Dimer, FDP and TF decreased compared with the control (P<0.01). Aspirin plus clopidogral could obviously reduce the wet and dry weight of thrombus, and reduce the level of D-Dimer and FDP (P<0.01). Aspirin plus clopidogral could obviously inhibit the formation of TF compared with aspirin (P<0.05).@*CONCLUSION@#Arterial thrombosis model by collagen encapsulated thread-drawing which is visible, repeatable and effective is better than thread-drawing. It is suitable for screening anti-thrombosis drugs and evaluating their effect.

Effect of glycoprotein alpha II bA2334C mutation on the biosynthesis and transportation of alpha II bbeta3 complex / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the effect of glycoprotein (GP) alpha II bA2334C mutation on the biosynthesis and expression of alpha II bbeta3 complex.</p><p><b>METHODS</b>The GP alpha II bA2334C eukaryotic expression plasmid pc3.1-2334M2b was constructed. Chinese hamster ovary (CHO) cells were transfected with the plasmid with or without integrin beta3 expression plasmid pc3.1-3a. The whole expression of alpha II bA2334C was confirmed by Western blot and the membrane expression was analyzed by flow cytometry. A newly constructed alpha II bA2334C GFP fusion protein expressing plasmid was used to determine its subcellular localization by laser confocal scanning microscopy.</p><p><b>RESULTS</b>Expression of the mutant protein, alpha II bA2334C, in the transfected CHO cells was confirmed by Western blot with a lower rate of the mature type than the wild type control. The expression on membrane was only 25% of the normal. Subcellular localization analysis showed that alpha II bA2334C GFP was able to be expressed in CHO cells and could be transported from endoplasmic reticulum to Golgi apparatus.</p><p><b>CONCLUSIONS</b>The mutant alpha II bA2334C can be synthesized in CHO cells and form alpha II bbeta3 complex. However, only a small fraction of the premature alpha II bA2334C can be transported to Golgi apparatus and transformed to mature alpha II b. The possible pathogenesis of this type II thrombasthenia may be that the misfolded alpha II bA2334C is partially degraded in the endoplasmic reticulum causing lower expression of alpha II bbeta3 complex on the membrane and resulting in impared function of platelets than normal alpha II b.</p>

GFP fused to the cytoplasmic tail of integrin alphaIIb allows the normal expression of alphaIIb beta3 compound in CHO cells / 中国实验血液学杂志

To investigate the effect of GFP fused to C terminal of integrin alpha(IIb) on the biosynthesis and expression of alpha(IIb) beta(3) compound, the alpha(IIb) GFP expression plamid, named palpha(IIb) GFP, the cDNA of alpha(IIb) was constructed from p3.1-2b and fused to pEGFP-N1 in frame. When the sequence of palpha(IIb) GFP was confirmed by sequencing it was transferred to Chinese Hamster Ovary (CHO) cells with or without p3.1-3a expressing integrin beta(3). Then the expression of alpha(IIb) GFP fusion protein was confirmed by Western blot and then its subcellular localization was determined with laser confocal scanning microscopy. The results showed that the target gene was cloned into recombinant vector by restriction analysis and sequencing. Overexpression of the fusion protein in the transfected CHO cells was identified with Western blot. Subcellular localization analysis confirmed that alpha(IIb) GFP was expressed in CHO cells and could be transferred from endoplasmic reticulum to Golgi apparatus. It is concluded that the eukaryotic expression plasmid containing alpha(IIb) GFP fusion gene is successfully constructed. GFP fused to the cytoplasmic tail of integrin alpha(IIb) allows the normal expression of alpha(IIb) beta(3) in CHO cells.

Study on platelet-associated tissue factor and its significance / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore whether normal platelet contains tissue factor (TF), and the significance of platelet-associated TF (PATF).</p><p><b>METHODS</b>Platelets were isolated by Sepharose 2B gel column. ELISA was used to detect the TF content in the lysates of washed platelets. Procoagulant activity of PATF was measured by one stage clotting time assay. The mRNA of TF was detected by reverse transcription polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>A certain amount of TF antigen (16.37 +/- 6.39) ng/L was detected in the washed-platelet lysates. Upon activation by collagen, platelets released TF and caused a marked increase in TF level in plasma (P <0.05). Resting platelets had no TF procoagulant activity, while procoagulant activity of platelets activated by collagen increased significantly, which could be blocked by TF McAb and poor VII plasma. TF mRNA could not be detected in washed platelets. TF content in platelets from patients with coronary heart disease was significantly higher than that from normal controls (P < 0.05). Resting platelets from the patients showed a higher procoagulant activity, which could be inhibited by TF McAb.</p><p><b>CONCLUSION</b>Platelets contain TF and the latter released by activated platelet was functionally active. Platelet itself might not synthesize TF. Protein content and procoagulant activity of PATF in patients with coronary heart disease were higher than that in controls. All these indicate that platelet may be involved in coagulation and thrombosis by releasing TF.</p>

Expression levels of CXCR4 on megakaryocytes and its ligand in bone marrow in children with acute idiopathic thrombocytopenic purpura / 中华儿科杂志

<p><b>OBJECTIVE</b>Chemokine receptor CXCR4 and its ligand stromal-derived factor 1 alpha (SDF-1alpha) have been paid increasing attention for their involvement in megakaryocytic hematopoiesis. It has been revealed in recent years that they can induce mature and immature megakaryocytes (MKs) to migrate through bone marrow endothelial cells (BMEC) by increasing the affinity of MKs for BMEC. Thus MKs maturity and eventual release of platelet from MKs ensues. While maturity disturbance of MKs and impaired production of platelets have been regarded as the main pathogenesis of ITP, the mechanism of which still remains unclear. Therefore, a clear understanding of the levels of CXCR4 and SDF-1alpha within bone marrow in children with ITP will help us to elucidate further the mechanism of ITP as well as to provide direct theoretical evidence for predicting treatment effect and evaluating prognosis.</p><p><b>METHODS</b>Bone marrow were aspirated from 28 children with AITP and 12 normal children. Percoll density gradient and immunomagnetic beads method were used to purify megakaryocytes from the bone marrow. The immune cytochemistry was used to detect CXCR4 on megakaryocytes. The levels of SDF-1alpha were detected by ELISA. SPSS10.0 statistical software was used to deal with the experimental data.</p><p><b>RESULTS</b>Before the treatment in children with AITP, both the CXCR4 expression on megakaryocytes and the SDF-1alpha level in bone marrow plasma were markedly decreased compared with the normal controls (P < 0.05). As to the cases who were sensitive to the high-dose intravenous immunoglobulin (HDIVIgG), the CXCR4 and SDF-1alpha levels were much higher in children after the treatment than those before the treatment (P < 0.05). In 6 cases insensitive to HDIVIgG, before the treatment the CXCR4 level was much lower than the children sensitive to HDIVIgG (P < 0.05).</p><p><b>CONCLUSIONS</b>The low levels of CXCR4/SDF-1alpha system in bone marrow may be one of the factors which contribute to the maturity disturbance of megakaryocytes and disturbance of platelets production in AITP, while decreased CXCR4/SDF-1alpha system may be caused by the effect of autoantibody against platelet. The mechanism of HDIVIgG in the treatment of AITP may involve in the increasing expression of CXCR4/SDF-1alpha system. The level of CXCR4 on megakaryocytes may play a certain role in predicting the treatment effect of immunoglobulin.</p>