1.Anti-CD36 Mediated Platelet Transfusion Refractoriness and Related Cases After Stem Cell Transplantation.
Yan ZHOU ; Li-Lan LI ; Zhou-Lin ZHONG ; Xue-Jun LIU ; Jin-Lian LIU ; Wei-Dong SHEN ; Guo-Guang WU
Journal of Experimental Hematology 2018;26(2):541-546
OBJECTIVETo analyse the cases of platelet transfusion refractoriness after received HLA-matched unrelated donor hematopoietic stem cell transplantation, to analyze and identify the phenotype and genotype of CD36 in both the patient and stem cell donor, as well as the characteristic of antibody induced platelet transfusion refractoriness, and to analyse the efficacy of matched CD36-deficiency platelets transfusions.
METHODSThe CD36 expression on platelet and monocyte was analyzed by flow cytometry (FCM) in both patient and donor. Polymerase chain reaction sequence-based typing (PCR-SBT) was used to analyze the exons sequence of CD36 and HPA. Fast monoclonal antibody-specific immobilization of platelet antigen (F-MAIPA) and FCM were used to identify platelet antibodies in the patient. Short tandem repeat polymerase chain reaction (STR-PCR) was applied to monitor engraftment evidence. The platelet level was monitored. CD36- deficiency donor's platelets were selected from CD36- deficiency donor blood bank.
RESULTSThe donor was CD36 positive and the patient was typed I CD36 deficiency. The anti-CD36 antibody was identified in patient's serum (after transplantation), while the HLA and HPA-related antibodies were excluded. Sequence analysis of CD36 exon in the patient showed Exon 6 -1G>C(Change in splicing site) homozygote, which was a novel CD36 mutation. STR, HPA and CD36 of the patient (complete chimerism) were conversed to that of donor gene types on day 18 after allo-HSCT. The positive CD36 expression on platelet and monocyte in the patient was observed on day 96 after allo-HSCT. The patient showed the platelet transfusion refractoriness which was significantly improved after platelets transfusions from CD36 deficiency donors.
CONCLUSIONStem cell transplants resulted in anti-CD36 and caused platelet transfusion refractoriness, that was first reported in China. To ensure the efficacy of platelet transfusion, the CD36-deficiency patient should receive CD36 deficiency platelets for transfusion.
Antigens, Human Platelet ; Blood Platelet Disorders ; Blood Platelets ; CD36 Antigens ; China ; Humans ; Platelet Transfusion ; Thrombocytopenia
2.Construction of eukaryotic expression vector for human platelet CD36 gene 220C>T and 429+4insg variants and analysis of their expressions in HEK293T cells.
Xiuzhang XU ; Haoqiang DING ; Jing LIU ; Wenjie XIA ; Jing DENG ; Yangkai CHEN ; Jiali WANG ; Yuan SHAO ; Dawei CHEN ; Xin YE
Chinese Journal of Medical Genetics 2019;36(2):124-127
OBJECTIVE:
To construct eukaryotic expression vectors for human platelet CD36 gene 220 C>T and 429+4insg variants and analyze their expressions in HEK293T cells.
METHODS:
RNA was isolated from human platelets and reversely transcribed into cDNA. Sequences of 220C>T and 429+4insg variants were derived by PCR amplification. The target sequence was ligated into a pcDNA3.1/V5-His-TOPO vector by TA cloning, which was transformed into TOP10 E. coli. Positive plasmids were screened by blue-white selection. After sequencing, plasmid DNA carrying 220C>T or 429+4insg variant was used to transfect HEK293T cells with the help of effectene. Expression of CD36 protein was then analyzed by flow cytometry and Western blotting.
RESULTS:
An eukaryotic expression vector pcDNA3.1/V5-His-CD36 (220C>T/429+4insg) was constructed by TA cloning. After transfected into HEK293T cells, the 220C>T and 429+4insg variants resulted in CD36 deficiency in HEK cells, which was confirmed by flow cytometry and Western blotting.
CONCLUSION
The 220C>T and 429+4insg variants can cause CD36 deficiency in human platelets. This system may be used for assessing the effect of 220C>T, 429+4insg, and other variants on the expression of CD36.
Blood Platelets
;
CD36 Antigens
;
Cloning, Molecular
;
Escherichia coli
;
Eukaryota
;
Genetic Vectors
;
HEK293 Cells
;
Humans
;
Plasmids
;
Transfection
3.CD36, a scavenger receptor implicated in atherosclerosis.
Experimental & Molecular Medicine 2014;46(6):e99-
CD36 is a membrane glycoprotein that is present on various types of cells, including monocytes, macrophages, microvascular endothelial cells, adipocytes and platelets. Macrophage CD36 participates in atherosclerotic arterial lesion formation through its interaction with oxidized low-density lipoprotein (oxLDL), which triggers signaling cascades for inflammatory responses. CD36 functions in oxLDL uptake and foam cell formation, which is the initial critical stage of atherosclerosis. In addition, oxLDL via CD36 inhibits macrophage migration, which may be a macrophage-trapping mechanism in atherosclerotic lesions. The role of CD36 was examined in in vitro studies and in vivo experiments, which investigated various functions of CD36 in atherosclerosis and revealed that CD36 deficiency reduces atherosclerotic lesion formation. Platelet CD36 also promotes atherosclerotic inflammatory processes and is involved in thrombus formation after atherosclerotic plaque rupture. Because CD36 is an essential component of atherosclerosis, defining the function of CD36 and its corresponding signaling pathway may lead to a new treatment strategy for atherosclerosis.
Animals
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Antigens, CD36/chemistry/genetics/*metabolism
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Atherosclerosis/*metabolism/pathology
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Humans
;
Macrophages/metabolism/pathology
;
Plaque, Atherosclerotic/*metabolism/pathology
4.Establishment of method detecting CD36 expression on human platelet and its application.
Ying LIU ; Xian-Guo XU ; Xiao-Fei LAN ; Kai-Rong MA ; Shu CHEN ; Xiao-Zhen HONG ; Ji HE ; Fa-Ming ZHU ; Hang-Jun LYU
Journal of Experimental Hematology 2013;21(4):1042-1045
The individual with the deficiency of CD36 antigen on platelet displayed the risk of anti-CD36 immune reaction induced by transfusion, which is one of the reasons for platelet transfusion refractoriness (PTR). This study was purposed to detect the expression level of CD36 antigen on platelet by flow cytometry among apheresis platelet donors of Hangzhou area, and the frequency of CD36 deficiency was analyzed. Platelet-rich plasma (PRP) was separated from fresh anticoagulant whole blood by centrifugation, then the platelets were washed and adjusted to 1×10(6). The platelets were incubated with FITC-labeled CD36 and PE-labeled CD41 monoclonal antibodies, then the expression level of CD36 was detected by flow cytometry. The CD36 expression on monocytes for the samples of CD36-deficiency on the platelets was further analyzed. The results showed that 7 samples with CD36 antigen deficiency were found in 192 apheresis platelet donors. The frequency of CD36 deficiency was 3.6% and all of them were typeII deficiency. The significant difference of CD36 antigen expression was observed in the platelet donors of Hangzhou population, among them 59 individuals with low expressed CD36 antigen and 126 individuals with highly expressed CD36 antigen were found according to the geometric mean fluorescence intensity. It is concluded that the CD36 antigen deficient phenotype existed in the population, these data will provide the information for research of the CD36 antigen distribution and help to solve the platelet transfusion refractoriness.
Blood Platelet Disorders
;
diagnosis
;
Blood Platelets
;
metabolism
;
CD36 Antigens
;
metabolism
;
Flow Cytometry
;
methods
;
Genetic Diseases, Inborn
;
diagnosis
;
Humans
5.Inhibitory effect of BF523 from Ilex hainanensis on ox LDL-induced foam cells formation.
Ling-Xiao WANG ; Zhen WU ; Zi-Yu LIU ; Feng-Yu JIN ; Yun-Fang ZHAO ; Peng-Fei TU ; Jiao ZHENG
China Journal of Chinese Materia Medica 2019;44(13):2680-2685
Cardio-cerebral vascular disease induced by atherosclerosis is a serious cause of human health. The pathogenesis of AS is very complex,and the oxidized low-density lipoprotein( ox LDL) induced foam cells formation is considered to be the most important cytological change in AS. Based on the definition of " TCM chemical biology",we clarified the chemical composition of Ilex hainanensis,the effective substances of I. hainanensis on the activity of anti-AS were screened. Then we found that saponin BF523 had the good inhibitory effect on foam cell formation. In this research,we studied the BF523 as the research object to clarify the molecular target of the active compound of I. hainanensis by foam cell formation model. The results showed that BF523 significantly inhibited the oxidation of ox LDL-induced macrophage foaming and decreased the lipid content in macrophages. BF523 had inhibited the phagocytosis of ox LDL in macrophages by reducing the mRNA and protein levels of scavenger receptor CD36,thereby inhibiting the occurrence and development of AS. These findings not only clarified the mechanism of the inhibition of foam cell formation by saponin BF523,but also provided a useful exploration for the enrichment of the theory of " TCM chemical biology".
Atherosclerosis
;
CD36 Antigens
;
metabolism
;
Cells, Cultured
;
Foam Cells
;
cytology
;
drug effects
;
Humans
;
Ilex
;
chemistry
;
Lipoproteins, LDL
;
adverse effects
6.Molecular polymorphism Analysis on CD36 Deficiency among Platelet Blood Donors in Shenzhen.
Yun-Ping XU ; Ze-Tao SUN ; Long PENG ; Shuang LIANG ; Fan WU ; Zhen LI ; Da-Cheng LI
Journal of Experimental Hematology 2022;30(3):884-889
OBJECTIVE:
To analyze the molecular polymorphisms of CD36 among 58 blood donors with CD36 deficiency and compare with CD36 positive controls.
METHODS:
A total of 58 donors with CD36 deficiency during a screening conducted in the laboratory from September 2019 to December 2020 were enrolled as the test group, including 39 males and 19 females, while 120 platelet donors with CD36 positive were randomly selected as the controls, including 76 males and 44 females. All of the subjects were Han nationality. The PCR-SBT method was used to detect coding region of CD36 gene, and molecular mutations were compared with those CD36 positive controls.
RESULTS:
Among the 58 donors with CD36 deficiency, mutations appears in 32 individuals. The detection rate for type I was 71.43% (5/7), and type II was 51.92% (27/52), while among the 120 controls, mutations appears in 12 donors (10%). In the CD36 antigen-deficient donors, 16 variations were found, in which 329-330 del AC with the highest frequency accounted for 20.69%, followed by 1228-1239 del ATTGTGCCTATT(15.52%) and 1156 C>T(10.34%). Two variations, 198-205 del GATCTTTG and 220 C>T, led to premature termination of translation; four mutations, 329-330 del AC, 560 ins T, 1011-1049 39bp dupl and 1343-1344 ins TCTT, caused translation frame shift; 1228-1239 del ATTGTGCCTATT led to deletion of four amino acids (Ile-Val-Pro-Ile) at sites 410-413 of the peptide chain. The 1140 T>A and 1275 G>A were synonymous mutations, and the other 7 mutations resulted in the substitution of single nucleotide. The platelet expression in the donors of CD36 positive with 329-330 del AC or 1228-1239 del ATTGTGCCTATT mutation (heterozygote) was lower than those CD36 positive individuals without mutations (homozygote).
CONCLUSION
Multiple gene mutations in the CD36 coding region may cause CD36 deficiency, and the heterozygous individuals with mutations may lead to CD36 antigen reduction or deletion. Mutation is not detected in 44.83% of CD36 deficient individuals, there may be some other reasons for the CD36 antigen deficiency.
Blood Donors
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Blood Platelet Disorders/metabolism*
;
Blood Platelets/metabolism*
;
CD36 Antigens/metabolism*
;
Female
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Genetic Diseases, Inborn
;
Humans
;
Male
7.Apoptosis of Megakaryocytic Leukemia Cell Line Meg-01 Induced by TSP-1 Via CD36/Caspase-3.
Hui-Min KONG ; Wei-Qing SU ; Yi LUO ; Hui GE ; Liang LI ; Mo YANG ; Qian-Li JIANG
Journal of Experimental Hematology 2022;30(4):998-1004
OBJECTIVE:
To investigate the effect of thrombospondin-1 (TSP-1) on apoptosis of human megakaryocytic leukemia cell line Meg-01 and its possible mechanism.
METHODS:
The expression of CD36 antigen in Meg-01 cells was detected by flow cytometry and immunocytochemistry. Meg-01 cells were cultured for 48 hours with TSP-1 and CD36 antibody FA6-152 at different concentrations. The early apoptosis and activity of caspase-3 were detected by flow cytometry. The effect of TSP-1 on the growth and differentiation of megakaryocytes was investigated by cell counting and CFU-MK culture.
RESULTS:
The flow cytometry and immunocytochemistry showed that CD36 antigen was expressed on the surface of Meg-01 cells. TSP-1 (5 μg/ml) inhibited the growth of Meg-01 cells, but had unobvious effect on M-07e cells. After addition of CD36 antibody FA6-152 (5, 10, and 25 μg/ml), the inhibition effect of TSP-1 was significantly reduced. TSP-1 (2.5, 5, and 7.5 μg/ml) increased the positive expression of Annexin V (P<0.01) and caspase-3 activity (P<0.01), which indicated that TSP-1 had a significant effect on inducing apoptosis. After addition of CD36 antibody FA6-152 (25 μg/ml), the apoptosis induced by TSP-1 in Meg-01 cells was significantly reduced. TSP-1 (5, 10, and 25 μg/ml) could significantly inhibit the formation of CFU-MK in mouse bone marrow cells, while β-TG could not. CD36 antibody FA6-152 (25 μg/ml) could significantly reduce the inhibition of TSP-1 on CFU-MK.
CONCLUSION
TSP-1 may induce apoptosis of megakaryocytic leukemia cell line Meg-01 cells via CD36/caspase-3, which provides a potential new drug development and treatment target for clinical treatment of megakaryocytic leukemia.
Animals
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Apoptosis
;
CD36 Antigens/metabolism*
;
Caspase 3/metabolism*
;
Cell Line
;
Humans
;
Leukemia, Megakaryoblastic, Acute
;
Mice
;
Thrombospondin 1/pharmacology*
8.Preliminary study on human mature placenta tissue-derived hematopoietic stem/progenitor cells.
Yu-Feng LIU ; Yong-Zhuo ZHANG ; Chuang-Xin ZHANG ; Diao WANG
Journal of Experimental Hematology 2006;14(1):98-101
Clinical transplantation has indicated that cord blood (CB) can be used in the hematopoietic reconstitution in the children, but not well used in the adult patients because of the low cell amount. The present study aimed to explore the capability of proliferation and differentiation of the hematopoietic stem/progenitor cells derived from human mature placenta tissue (PT) in vitro, and to find a new source of hematopoietic/progenitor cells for clinical transplantation. CD34(+) cells in human mature placenta tissue were isolated and characterized by using enzyme-digestion method and flow cytometry. A long culture system without cytokines was established with human mature placenta tissue-derived mononucleated cells and cord blood mononuclear cells. The number of nucleated cells was weekly counted in culture for 14 weeks. The number of CFC was counted in culture for 2 weeks. The results showed that the CFC yields (CFU-GM, 186.90 +/- 24.52; BFU-E, 101.40 +/- 13.35) and the percentage of CD34(+) cells (2.74 +/- 0.61%) and CD34(+)/CD38(-) cells (2.46 +/- 0.42%) in placenta tissue (PT) were higher than CFC (CFU-GM, 136.90 +/- 25.15; BFU-E, 49.20 +/- 8.13), CD34(+) cells (1.73 +/- 0.32%) and CD34(+)/CD38(-) cells (0.80 +/- 0.25%) in cord blood (CB). The MNCs from PT have shown more survival ability than the cells from CB in the long-term cell culture condition; and the cells from PT increased by 2 times. It is concluded that the placenta may be another hematopoietic organ in ontogeny. The cells from placenta were more juvenile, and may be favorable source for clinical stem cell transplantation.
Antigens, CD34
;
analysis
;
CD36 Antigens
;
analysis
;
Cell Differentiation
;
physiology
;
Cells, Cultured
;
Hematopoietic Stem Cell Transplantation
;
Hematopoietic Stem Cells
;
cytology
;
Humans
;
Leukocytes, Mononuclear
;
cytology
;
Placenta
;
cytology
9.Purification of thrombospondin receptor (CD36) from human platelet membrane.
Joong Ho KIM ; Kyoung Ho SUK ; Na Young PARK ; Seung Ho KIM
Experimental & Molecular Medicine 1997;29(1):31-34
Thrombospondin receptor (CD36) has been recently identified in platelets and various cell lines as the receptor for thrombospondin, an adhesive protein required for irreversible aggregation of platelets as well as other adhesive processes. Thrombospondin receptor, one of major glycosylated platelet membrane proteins, is thought to play an important role as a cell adhesion molecule in blood coagulation system as well as intercellular signaling. In this work, thrombospondin receptor was purified to homogeneity from human platelet by wheat germ agglutinin (WGA)-affinity chromatography and size exclusion chromatography on Ultrogel-AcA44. The molecular weight of the purified thrombospondin receptor was about 88 kDa on SDS-PAGE and its identity was confirmed by immunoblot analysis and immunodiffusion assay.
Adhesives
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Antigens, CD36*
;
Blood Coagulation
;
Blood Platelets*
;
Cell Adhesion
;
Cell Line
;
Chromatography
;
Chromatography, Gel
;
Electrophoresis, Polyacrylamide Gel
;
Humans*
;
Immunodiffusion
;
Membrane Proteins
;
Membranes*
;
Molecular Weight
;
Thrombosis
;
Thrombospondins*
;
Triticum
10.Characterization of Binding and Phagocytosis of Oxidatively Damaged Erythrocyte to Macrophage.
Hong Sook KO ; In Seop KIM ; Kwang Je LEE ; Sang Wook KIM ; Chee Jeong KIM ; Wang Seong RYU
The Korean Journal of Internal Medicine 2002;17(4):220-226
BACKGROUND: Scavenger receptors are thought to be involved in the recognition of oxidized low-density lipoprotein (oxLDL) and oxidized erythrocyte (oxRBC). However, there are controversies about the kind of receptors and ligands related to the binding. Macrophages lacking class A scavenger receptor show identical binding of oxRBC with wild-type ones. METHODS: RBCs were oxidized with ascorbic acid and CuSO4. Lipid oxidation was measured indirectly by measuring TBARS semiquantitatively. The binding and phagocytosis were measured by counting the number of oxRBC bound or taken up after incubation at 4 degrees C or 37 degrees C for 60 minutes to 100 macrophages differentiated from human monocytic leukemia cell line. RESULTS: The degree of oxidation and the binding of oxRBCs were dependent on the concentration of CuSO4. The binding and phagocytosis of oxRBC were inhibited by 99% with oxLDL. Fucoidan, competing class A scavenger receptor, inhibited the binding by more than 90%. The binding of oxRBC was higher at 37 degrees C than at 4 degrees C by 3 times. The binding of oxRBCs was maximal at pH 6.5 and higher than at physiologic pH by 2.8 times. At pH 8.5 and 9.5, binding decreased by 67 and 88%, respectively. CONCLUSION: OxRBCs might bind and be taken up to macrophages not mainly through class A nor B scavenger receptors, but through other scavenger receptors and/or pathways. These processes are dynamic and ionic strength might be involved.
Antigens, CD36
;
*Erythrocyte Aging
;
Erythrocytes/*metabolism
;
Human
;
Lipoproteins, LDL/metabolism
;
Macrophages/*metabolism
;
Oxidation-Reduction
;
Phagocytosis/*physiology
;
Receptors, Immunologic/metabolism
;
Support, Non-U.S. Gov't
;
Tumor Cells, Cultured/metabolism