OBJECTIVE: To investigate the effect of zedoarondiol on neovascularization of atherosclerotic (AS) plaque by exosomes experiment. METHODS: ApoE^-/- mice were fed with high-fat diet to establish AS model and treated with high- and low-dose (10, 5 mg/kg daily) of zedoarondiol, respectively. After 14 weeks, the expressions of anti-angiogenic protein thrombospondin 1 (THBS-1) and its receptor CD36 in plaques, as well as platelet activation rate and exosome-derived miR-let-7a were detected. Then, zedoarondiol was used to intervene in platelets in vitro, and miR-let-7a was detected in platelet-derived exosomes (Pexo). Finally, human umbilical vein endothelial cells (HUVECs) were transfected with miR-let-7a mimics and treated with Pexo to observe the effect of miR-let-7a in Pexo on tube formation. RESULTS: Animal experiments showed that after treating with zedoarondiol, the neovascularization density in plaques of AS mice was significantly reduced, THBS-1 and CD36 increased, the platelet activation rate was markedly reduced, and the miR-let-7a level in Pexo was reduced (P<0.01). In vitro experiments, the platelet activation rate and miR-let-7a levels in Pexo were significantly reduced after zedoarondiol's intervention. Cell experiments showed that after Pexo's intervention, the tube length increased, and the transfection of miR-let-7a minics further increased the tube length of cells, while reducing the expressions of THBS-1 and CD36. CONCLUSION Zedoarondiol has the effect of inhibiting neovascularization within plaque in AS mice, and its mechanism may be potentially related to inhibiting platelet activation and reducing the Pexo-derived miRNA-let-7a level.
Animals ; MicroRNAs/genetics* ; Exosomes/drug effects* ; Plaque, Atherosclerotic/genetics* ; Neovascularization, Pathologic/genetics* ; Human Umbilical Vein Endothelial Cells/metabolism* ; Humans ; Blood Platelets/drug effects* ; Apolipoproteins E/deficiency* ; Thrombospondin 1/metabolism* ; CD36 Antigens/metabolism* ; Platelet Activation/drug effects* ; Male ; Mice ; Mice, Inbred C57BL

OBJECTIVE: To study the polymorphism of human platelet antigen (HPA) system 10 among ethnic Han Chinese from Shandong, China so as to supplement the data of platelet donor bank in the region. METHODS: Peripheral blood samples of platelet donors from the region were genotyped for HPA-10 alleles by PCR-sequence specific primer (PCR-SSP) and direct sequencing. RESULTS: Among 1401 donors, a rare heterozygote carrier of HPA-10w (a+b+) was identified, which gave an allelic frequency of approximately 0.035%. CONCLUSION The detection of rare HPA-10bw antigen allele among ethnic Han Chinese from Shandong is useful for the diagnosis and prevention of neonatal alloimmune thrombocytopenia and post-transfusion purpura in the region.
Alleles ; Antigens, Human Platelet/genetics* ; Asians/genetics* ; Gene Frequency ; Genotype ; Humans ; Infant, Newborn ; Polymorphism, Genetic

OBJECTIVE: To investigate the pathogenesis and clinical diagnosis of fetal/neonatal alloimmune thrombocytopenia (FNAIT) and analyze the laboratory test results and clinical data related to the disease, so as to provide reference for clinical treatment and improvement of prognosis. METHODS: The clinical data of six neonatal patients with FNAIT in the Neonatology Department of our hospital from March 2017 to September 2020 were retrospectively analyzed, which included laboratory diagnosis, clinical symptoms, treatment, and prognosis. RESULTS: Among six patients, two cases occurred in the first pregnancy and four cases in the second pregnancy. The platelet count of six cases were decreased at admission or during hospitalization and maternal and neonatal serum autoimmune platelet antibody tests were positive. Five cases were accompanied by different degrees of skin and facial bleeding spots or petechiae and ecchymosis, intracranial hemorrhage. Four cases were treated with immunoglobulin and/or steroid hormone therapy (one of them received cross-matched platelets transfusion), while the symptoms of the other two cases improved spontaneously. Five cases recovered and were discharged from the hospital, while one case had not recovered but the family members requested to be discharged forwardly. Four cases were hospitalized within two weeks, but two cases were hospitalized for more than two weeks due to other diseases or factors (e.g., neonatal sepsis, neonatal enteritis, congenital heart disease, neonatal asphyxia, etc.). CONCLUSION FNAIT is characterized by decreased platelet count, with or without bleeding symptoms, and may occur in the first and following pregnancy. FNAIT can recover spontaneously or have a good prognosis after treatment. However, the complication with other diseases or factors may affect the prognosis.
Adult ; Aged ; Antigens, Human Platelet ; Data Analysis ; Female ; Hemorrhage ; Humans ; Infant, Newborn ; Middle Aged ; Platelet Count ; Platelet Transfusion/adverse effects* ; Pregnancy ; Retrospective Studies ; Thrombocytopenia, Neonatal Alloimmune/therapy*

OBJECTIVE: To analyze the polymorphism of the HPA1-5,15 system of the donors in Zhangjiakou area. METHODS: DNA was extracted from the blood samples of the donors, PCR- SSP method was used to divide HPA1-6, 15 genotype. The gene frequency and genotype frequency were calculated, compared with the difference and regiahal specificity of the populations in our country and foregiens was compared other populations. RESULTS: The gene expression in the HPA-1, HPA-2 and HPA-4 systems were all homozygous aa, and the donors who expressed homozygous bb was not exessed. Among them, one heterozygous ab expression was found in both HPA-1 and HPA-4 systems (1%), and 14 cases of heterozygous ab expression were found in HPA-2 system (14%). The gene expression in the HPA-5 system was mainly homozygous aa (98%), and a very few expressed homozygous bb (2%) was found. The degree of heterozygosity of gene expression in the HPA-3 and HPA-15 systems was relatively high. The proprotion of the expression of aa, ab and bb in the HPA-3 system was respectively 46%, 40% and 14%, the proprotion of the expression of aa, ab and bb in the HPA-15 system was respectively 21%, 64% and 15%. CONCLUSION The gene frequency of platelet-specific antigen HPA1-5,15 system in zhangjiakou region shows local characteristics. The heterozygosity degree of gene expression in the HPA-3 and HPA-15 systems are both high, suggesting that they are more likely to result in alloimmunization and ineffective platelet transfusion, which should be pays attention to.
Antigens, Human Platelet/genetics* ; Blood Donors ; Gene Frequency ; Genotype ; Humans ; Polymorphism, Genetic

OBJECTIVE: To establish quantitative surface plasmon resonance (SPR) assay for antibodies against human platelet antigen-1a (HPA-1a). METHODS: Recombinant protein was fixed on the chip surface by amino coupling method. SPR assay was used to detect the standard antibodies against HPA-1a at different conceatration. The optimal experimental parameters were determined, and standard curves were constructed with linear regression. Moreover, the sensitivity, specificity, accuracy and precision of the assay were evaluated. RESULTS: The quantitative SPR assay for HPA-1a antibodies was established. The determination ranges were 0-20 IU, with accuracy (recovery rate) was 97.75%-103.08%. The intra-assay precision [coefficients of variation (CV)] was 3.53%-4.29%, and the inter-assay precision (CV) was 2.08%-4.40%. For specificity test, several kinds of monoclonal and human antibodies against platelet membrane protein were tested and no positive result was observed. CONCLUSION The established quantitative SPR assay for HPA-1a antibodies shows good sensitivity, specificity, accuracy and precision, and this rapid and simple method provides a new reference method for scientific research and clinical antibody detection.
Antigens, Human Platelet ; Blood Platelets ; Humans ; Isoantibodies ; Surface Plasmon Resonance

OBJECTIVE: To study the Polymorphism of the human platelet antigen(HPA) gene 1-17 and human leukocyte antigen(HLA) gene-A and B locus in Shandong Han population. METHODS: A total of 962 samples from routine voluntary platelet donors were genotyped for HPA1-17 system and HLA-A site, B by PCR-SSP and PCR-SSOP respectively．Gene frequencies were calculated by counting. HPA1-17 and HLA genotype combinations were analyzed by Arelequin 3.5. RESULTS: The gene frequencies of HPA-la, -1b, HPA-2a, -2b, HPA-3a, -3b, HPA-4a, -4b, HPA-5a, -5b, HPA-6a, -6b, HPA-15a, -15b were 0.9918, 0.0082, 0.9419, 0.0592, 0.5841, 0.4174, 0.9969, 0.0031, 0.9892, 0.0108, 0.9835, 0.0175,0.5488 and 0.4512, respectively． The most common HPA genotype combination was HPA-(1, 2, 4, 5, 6, 7-14, 16, 17) aa-3ab-15ab (0.2048). Moreover, HLA-A*2(0.3094) and HLA-B*13(0.1513) showed the highest frequency in their respective locus. The most common HLA genotype combination was HLA-A*2-B*13(0.1397) . CONCLUSION Distributions of HPA and HLA show high polymorphism in Shandong Han population. The ethnic and territorial difference of HPA distribution is also confirmed. It is imperative to establish local genetic database of volunteer platelet donors.
Alleles ; Antigens, Human Platelet/genetics* ; Gene Frequency ; Genotype ; Humans ; Polymorphism, Genetic

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

OBJECTIVETo generate recombinant GPⅢa as an alternative source for HPA-1a antigen and combine it with Luminex xMAP beads for the detection of HPA-1a-specific alloantibody.

METHODSThe full coding region of ITGB3 gene was amplified and ligated with pcDNA3.1. The recombinant plasmid was transfected into CHO cells, and those with stable expression were screened with G418. Expressed protein was identified and coupled with Luminex xMAP beads, which were then reacted with sera samples. Subsequently, phycoerythrin-labeled anti-species IgG antibody was added to the reaction wells and the median fluorescence was determined on a Luminex-100 analyzer.

RESULTSDNA sequencing confirmed that the cloned ITGB3 gene was HPA-1aa. The recombinant GPⅢa was coupled with Luminex xMAP beads. The sensitivity of Luminex beads assay to detect HPA-1a antibody was dilution 1/32 (3.125 U/mL). The Luminex beads assay could specifically identify the HPA-1a antibody from the test sera, and the results were consistent with that of monoclonal antibody-specific immobilization of platelet antigens (MAIPA) technology. Cross-reactivity was not observed with the samples containing HLA, ABO and other HPA antibodies (HPA-3a and HPA-5b). The results illustrated that to detect HPA antibody with Luminex xMAP beads technology is feasible.

CONCLUSIONRecombinant GPⅢa was successfully obtained and used to establish a Luminex technology-based method for the detection of HPA antibodies.

Animals ; Antigens, Human Platelet ; immunology ; Autoantibodies ; immunology ; Base Sequence ; Blotting, Western ; CHO Cells ; Cricetinae ; Cricetulus ; Humans ; Immunoassay ; methods ; Integrin beta3 ; genetics ; immunology ; metabolism ; Microspheres ; Recombinant Proteins ; immunology ; metabolism ; Reproducibility of Results

OBJECTIVETo study the gene polymorphism distribution characteristics of human platelet HPA-1-5 and 15 blood group antigens and construct a certain scale of platelet HPA database in the north area of Henan Province so as to provide platelet apheresis for clinical departments.

METHODSUsing polymerase chain reaction with sequence-specific primers (PCR-SSP), the genotyping of HPA-1-5 and 15 system was carried out; the periperal blood of 500 healthy Han donors in north area of Henan Province was collected randomly, the gene and genotype frequencies were detected by direct counting method, and the population distribution frequncy of HPA genes was analyzed by Hardy-Weinberg balance test, and compared with other regions and ethnics by using χ(2) test.

RESULTSThere was statistically significant (P < 0.05) of increase HPA-3b and HPA-5a in North area of Henan Province, compared with Chinese Han population; the HPA-3b and 5a increase and HPA-2a decrease were statistically significant (P < 0.05), compared with Ethnic minority of China. There was partly increase of HPA-1a, 2a, 3a and 5a, compared with different regions and ethnic in abroad. HPA allele genes of 500 Han donors in the North area of Henan Province were as follows: 0.985 and 0.015 for 1a and 1b; 0.924 and 0.076 for 2a and 2b; 0.469 and 0.531 for 3a and 3b; 1.000 and 1.000 for 4a and 5a; 0.532 and 0.468 for 15a and 15b, respectively. HPA allele gene frequencies were 1aa0.970, 1ab0.030; 2aa0.848, 2ab0.152; 3aa0.222, 3ab0.494, 3bb0.284; 4aa1.000; 5aa1.000; 15aa0.282, 15ab0.500, 15bb0.218. Compared with other regions and ethnic, HPA gene frequencies partly had statistical significance.

CONCLUSIONDistribution of HPA allele frequencies in the North area of Henan province is in accordence with the Hardy-Weinberg law. There are race and regional differences in HPA allele gene frequencies, compared with other regions and countries. And the HPA systems HPA-3 and 15 display the genetic polymorphisms, which provides a theoretical basis for the relevant research of the same type platelet infusion and alloimmune thrombocytopenia.

Alleles ; Antigens, CD ; genetics ; Antigens, Human Platelet ; genetics ; Blood Platelets ; China ; DNA Primers ; Ethnic Groups ; GPI-Linked Proteins ; genetics ; Gene Frequency ; Genotype ; Humans ; Neoplasm Proteins ; genetics ; Plateletpheresis ; Polymerase Chain Reaction ; Polymorphism, Genetic

OBJECTIVETo study the polymorphisms of human platelet antigen (HPA) 1-16 and human leukocyte antigen (HLA)-A and -B loci among ethnic Han population from Shandong.

METHODSA total of 588 samples from platelet donors were genotyped for the above loci with sequence-specific primer PCR and sequence-specific oligonucleotide probe PCR.

RESULTSThe frequencies of HPA-la, -1b, HPA-2a, -2b, HPA-3a, -3b, HPA-4a, -4b, HPA-5a, -5b, HPA-6a, -6b, HPA-15a, -15b were 0.9974, 0.0026, 0.9456, 0.0544, 0.5417, 0.4583, 0.9983, 0.0017, 0.9889, 0.0111, 0.9903, 0.0097, 0.5434 and 0.4583, respectively. The HPA-7-14 and HPA-16 showed no heterozygosity as the b allele was not detected in such loci. The most common genotypic combination for HPA was HPA-(1,4,7-14,16,17) aa-2aa-3ab-5aa -6aa-15ab (0.1820). HLA-A2 (0.3070) and HLA-B13 (0.1361) demonstrated the highest frequencies at their respective loci.

CONCLUSIONThe HPA and HLA loci are highly polymorphic among ethnic Hans from Shandong. The distribution of HPA polymorphisms also shows a great ethnic and territorial difference. It is important to construct regional database for the genotypes of HPA and HLA loci for platelet donors.

Alleles ; Antigens, Human Platelet ; genetics ; Asian Continental Ancestry Group ; genetics ; statistics & numerical data ; Blood Donors ; China ; Female ; Gene Frequency ; Genetics, Population ; Genotype ; HLA-A Antigens ; genetics ; HLA-B Antigens ; genetics ; Humans ; Linkage Disequilibrium ; Male ; Polymorphism, Genetic