High Autophagy Patterns in Swelling Platelets During Apheresis Platelet Storage.

Platelets undergo remarkable morphological changes during storage. Platelets change into different sizes and densities and differ in their biochemistry and functions. However, the correlation between structural heterogeneity and platelet autophagy is largely unknown. The aim of this study was to investigate the autophagy process in vitro, such as routine storage of platelets, and explore the role of reactive oxygen species (ROS) involved in the regulation of platelet autophagy. The ROS and autophagy levels of platelet concentrates from apheresis platelets were evaluated through flow cytometry. The expression levels of autophagy-associated proteins (LC3I, LC3II, Beclin1, Parkin, and PINK1) were measured via Western blot. All biomarkers were dynamically monitored for seven days. Moreover, the morphological characteristics of platelet morphology during storage were analyzed through transmission electron microscopy (TEM). Flow cytometry showed that the levels of total cell ROS and mitochondria ROS increased in the stored platelets. Together with the increase in mitochondrial ROS, the autophagy signal LC3 in the platelets was strongly amplified. The number of swollen platelets (large platelets) considerably increased, and that of autophagy signal LC3 was remarkably higher than that of the normal platelets. Western blot revealed that the expression levels of Beclin1 and LC3 II/LC3 I ratio were enhanced, whereas those of Parkin and PINK1 almost did not change during the seven days of storage. The existence of autophagosomes or autophagolysosomes in the platelets at the middle stage of platelet storage was observed via TEM. Our data demonstrated that the subpopulation of large (swollen) platelets exhibited different autophagy patterns. Furthermore, increased platelet autophagy was associated with mitochondrial ROS. These preliminary results suggest that swelling platelets have a higher autophagy pattern than normal platelets during storage.

Methodological Study on the Establishment of HLA/HPA Gene Bank of Platelet Donors and Its Clinical Application.

The current study aimed at establishing a large-scale platelet donor database with known HPA and HLA genotypes in Ningbo, to provide matched platelets for the clinic and prevent the occurrence of ineffective immune platelet transfusion refractoriness (PTR). The platelet banks of clinical donor wtih HLA-A, HLA-B, HPA1, HPA2, HPA3, HPA4, HPA5, HPA6, HPA10, HPA15 and HPA21 genotyps were established. Meanwile, the platelet gene matching was performed on the donor. It was found that there were phenotype polymorphisms in 1000 donnors with HPA1, HPA2, HPA3, HPA4, HPA5, HPA6, HPA10, HPA15 and HPA21 genotyps, and allel polymorphism distribution in donnors with HPA1, HPA2, HPA3, HPA4, HPA5, HPA6, HPA15, HPA21 genotyps. The frequency of HPA10 was a gene, and not b gene, showing a single linear distribution. The HPA 2, HPA3, HPA15 system were the most polymorphic with three phenotypes: aa, ab, bb. In the HLA-A allele, the highest frequency is A*11:01 (24.25%). There were 13 alleles that were greater than 1%, such as A*24:02, A*02:01, A*33:03, and the accumulated frequency reached 96.20%. In the HLA-B allele, the highest gene frequency was B*40:01 (13.40%). There were 24 alleles that were greater than 1%, such as B*46: 01, B*58: 01, B*15: 01, and the accumulated frequency reached 91.60%. Platelet antibody cross matching was performed on 100 blood samples from patients with thrombocytopenia after multiple platelet transfusions. The number of consistent samples was 46 (46%). Twenty patients were transfused with platelet cross matching. Among them, 18 patients had obvious improvement in clinical symptoms and good hemostatic effect after transfusion, which was judged to be effective. Platelet donor HPA and HLA-A, B antigen genotyping database provided patients with individual appropriate platelets, and provided the effectiveness of immune platelet infusion ensuring effective platelet transfusion.

[Molecular genetic analysis of two individuals with weak D variant of the Rh blood type].

OBJECTIVE: To explore the molecular basis of two individuals with weak D variant of the Rh blood type. METHODS: Routine serological testing was carried out to detect the D, C, c, E and e antigens of the Rh blood group. The D antigen was further detected with an indirect antiglobulin test. The presence of Rhesus box was detected by PCR to determine the homozygosity of the RHD gene. RESULTS: Both samples were determined as weak D phenotype by the indirect antiglobulin test. DNA sequencing revealed that case 1 harbored a heterozygous 208C>T variant in exon 2 and a heterozygous 1227G>A variant in exon 9; while case 2 harbored homozygous 779A>G variants of exon 5 of the RHD gene. Case 1 was determined as RHD+/RHD+, while case 2 was determined as RHD+/RHD-. The two samples were respectively named as weak D type 122 and weak D type 149 based on the rules of Rhesus Base Nomenclature. CONCLUSION: D negative blood donors should subject to indirect antiglobulin testing and molecular analysis for safer transfusion.

[A case with a novel weak D type].

OBJECTIVE: To report on a novel weak D type identified in a Chinese individual. METHODS: Peripheral blood sample was collected for a voluntary blood donor with weakened expression of D antigen. Routine serological testing was carried out to determine the D, C, c, E and e antigens of the Rh blood group. A D-screening kit was used to analyze the RhD epitopes. The 10 exons and flanking intronic regions of the RHD gene were sequenced. The zygosity of RHD was determined with a sequence-specific primer PCR method. RESULTS: A novel RHD allele, RHD (1022T>A), was found in the subject with a weak D phenotype. Serological testing of the RhD epitopes has coined with the weak D phenotype. CONCLUSION: A novel weak D allele has been identified in Chinese population.

[Identification of a novel FUT1 allele of para-Bombay phenotype].

OBJECTIVE: To explore the molecular basis for an individual with para-Bombay phenotype of the H blood group. METHODS: Intron 5 to 3'-UTR of the ABO gene and exon 4 of the FUT1 gene were amplified with PCR and subjected to direct sequencing. Mutations of the FUT1 gene were identified by TOPO cloning sequencing. RESULTS: Direct sequencing showed that her ABO genotype was B101/O01. TOPO cloning sequencing found that this individual had three mutations of the FUT1 gene, including an heterozygous AG deletion (CAGAGAG→CAGAG) at position 547 to 552, and two C→T mutations at positions 35 (C35T) and 293 (C293T) on the other homologous chromosome. The two alleles comprised a new recombination of mutations c.35T>C and c.293C>T, and the sequence has been submitted to NCBI (No. MG597611). CONCLUSION: A novel combination of FUT1 alleles with c.35 C>T and c.293C>T has been identified in an individual with para-Bombay phenotype.

[Identification of a novel Ax allele of the ABO blood group].

OBJECTIVE: To explore the molecular basis for an individual with Ax28 phenotype of the ABO subtype. METHODS: The ABO group antigens on red blood cells of the proband were identified by monoclonal antibodies. The ABO antibody in serum was detected by standard A, B, O cells. Exons 1 to 7 of the ABO gene were respectively amplified by PCR and directly sequenced. Amplicons for exons 5 to 7 were also sequenced after cloning. RESULTS: Weakened A antigen was detected on red blood cells from the proband. Both anti-A and anti-B antibodies were detected in the serum. Heterozygous 261G/del was detected in exon 6, while heterozygous 467C/T and 830T/C were detected in exon 7 by direct DNA sequencing. After cloning and sequencing, two alleles (O01 and Ax28) were obtained. Compared with A102, the sequence of Ax28 contained one nucleotide changes (T to C) at position 830, which resulted in amino acid change (Val to Ala) at position 277. CONCLUSION: The novel mutation c.830T>C of the galactosaminyltransferase gene may give rise to the Ax28 phenotype.

Effect of Nitric Oxide Donor on Metabolism of Apheresis Platelets.

The aim of this study is to investigate the effects of a nitric oxide (NO) donor S-nitrosoglutathione (GSNO) on the metabolism and improvement of preservation quality in apheresis platelets. A GSNO solution with a certain concentration was added into fresh apheresis platelets, and the parameters associated with platelet morphology, metabolism and function were temporally monitored for 7 days. The results showed that the NO level in GSNO group were remarkably higher than those in the control group during the whole storage stage. No significant morphology or function difference was observed between the control and GSNO groups such as Platelet count, platelet distribution width, mean platelet volume and mitochondrial metabolic activity. But in metabolism there are something differences from morphology data: pCO2, pO2, cHCO3- were also found to have no clear difference between the control and GSNO groups; the lactic acid content, sugar consumption and Lactate dehydrogenase activity in the GSNO group were lower than that in the control group at some time point; and pH values in the GSNO group were higher than the control group. Our study discovered that the NO donor GSNO can reduce the metabolism and maintain the cellular characteristics of platelets in vitro during the platelet storage period.

Effects of nitric oxide donor S-nitrosoglutathione on apoptosis of apheresis platelets.

OBJECTIVES: The aim of this study is to investigate the effects of a Nitric oxide (NO) donor, S-nitrosoglutathione (GSNO), on apoptosis and the improvement of preservation quality in apheresis platelets. METHODS: A GSNO solution - to make the final GSNO concentration of 100 uM was added into fresh apheresis platelets, and the parameters associated with platelet morphology, metabolism, and apoptosis were dynamically monitored for seven days. RESULTS: The results showed that the NO level was remarkably higher during the whole storage stageafter GSNO injection. The number of depolarized platelets and platelets with phosphatidylserine valgus were significantly reduced in the GSNO group compared to that of the control group at some time point. The expression of Bcl-xL mRNA on day 5 of storage in the experimental group was significantly higher than that of the control group, but the expression of Bcl-xL protein was not significantly higher than that in the control group. In addition, Bak and Bax mRNA expression levels in the experimental group were significantly lower than those in the control group, but Bak and Bax protein expression levels were not statistically different. Meanwhile, caspase-3 activity was significantly inhibited. DISCUSSION AND CONCLUSION: These data suggest that the addition of a certain dose of GSNO as a NO donor during platelet storage could inhibit platelet apoptosis and reduce platelet storage lesion (PSL) to a certain extent.

[Identification of a novel Bx allele of the ABO blood group].

OBJECTIVE: To identify a novel Bx13 allele. METHODS: Serological characteristics was determined with standard serological methods. All of the seven exons and flanking regions of the ABO gene were analyzed with PCR and direct sequencing. The amplicon of exon 7 was also cloned and sequenced. RESULTS: The individual was determined as with a rare Bx phenotype by serological tests. Direct DNA sequencing showed that the individual was heterozygous for the B/O01 allele, while there was a novel 893C>T mutation in the B101 allele, which has led to an amino acid substitution Ala298Val in the α,3-D-galactosyl-transferase. The mutation was not found among 100 randomly selected blood donors. CONCLUSION: A novel Bx13 allele has been identified. Substitution of amino acid in the conserved region of the enzyme may reduce the activity of α,3-D-galactosyl-transferase.

MicroRNA profiling of platelets from immune thrombocytopenia and target gene prediction.

Immune thrombocytopenia (ITP) is an autoimmune disease characterized by a low platelet count and insufficient platelet production. Previous studies identified that microRNAs (miRNAs/miRs) are important for platelet function. However, the regulatory role of miRNAs in the pathogenesis of thrombocytopenia in ITP remains unclear. The aim of the present study is to isolate differentially expressed miRNAs, and identify their roles in platelets from ITP. A total of 5 ml blood from 22 patients with ITP and 8 healthy controls was isolated for platelet collection. A microarray assay was performed to analyze the differentially expressed miRNAs in the patients with ITP and healthy patients. Furthermore, the expression of differentially expressed miRNAs was verified by reverse transcription­quantitative polymerase chain reaction. In addition, the target mRNAs of the differentially expressed miRNAs were predicted via miRWalk databases, and the target genes and miRNAs were classified by Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes analyses. In the present study, 115 miRNAs were identified to be differentially expressed in platelets from patients with ITP compared with the healthy controls (>3­fold alteration; P<0.05). Among them, 57 miRNAs were upregulated in ITP, while 58 miRNAs were downregulated. Bioinformatic prediction demonstrated that hsa­miR­548a­5p, hsa­miR­1185­2­3p, hsa­miR­30a­3p, hsa­miR­6867­5p, hsa­miR­765 and hsa­miR­3125 were associated with platelet apoptosis and adhesion in ITP. The present study performed miRNA profiling of platelets from patients with ITP and the results may aid in the understanding of the regulatory mechanism of ITP.

Investigation of platelet apoptosis in adult patients with chronic immune thrombocytopenia.

OBJECTIVES: Immune thrombocytopenia (ITP) is an acquired and heterogeneous autoimmune-mediated hematological disease typically characterized by a low platelet count. Emerging evidence over the past several years suggests that platelet biogenesis and ageing are regulated, at least in part, by apoptotic mechanisms. However, the association between decreased platelets and apoptosis in ITP patients is poorly understood. To better understand the role of platelet apoptosis in ITP pathophysiology, we investigated apoptotic markers in platelets acquired from 40 chronic ITP patients. Furthermore, the results of ITP patients were compared to those from 40 healthy individuals. METHODS: Markers of apoptosis, including phosphatidylserine (PS) exposure and mitochondrial inner membrane potentials (ΔΨm), were examined using flow cytometry. The expression of pro-apoptotic molecules such as Bak and Bax and anti-apoptotic molecules such as Bcl-xL were determined using quantitative real-time PCR (qRT-PCR) and Western blotting. RESULTS: Our study demonstrated that the platelet mitochondrial membrane depolarization in chronic ITP patients tended to be higher than in healthy controls. Additionally, the proportion of platelets with surface-exposed PS in chronic ITP was significantly higher than that of controls. The results showed that the expression levels of Bak and Bax were significantly higher in chronic ITP patients than in healthy controls; Bcl-xL expression levels were significantly decreased in the platelets of chronic ITP patients compared to healthy controls. DISCUSSION AND CONCLUSION: study indicates that the enhancement of platelet apoptosis observed in patients with chronic ITP may be one of the pathogenic mechanisms of chronic ITP.

[Molecular genetic analysis of four cases with weak D variant of Rh blood type].

OBJECTIVE: To explore the molecular basis of 4 cases with weak D variant of Rh blood type. METHODS: Routine serological testing was applied to determine the D, C, c, E and e antigens of the Rh blood group. The D antigen was further detected with an indirect antiglobulin test. RHD zygosity was detected by sequence-specific primer PCR method. All exons and flanking intron regions of the RHD gene were sequenced. RESULTS: The samples were determined as weak D phenotype by serological testing. DNA sequencing showed that the 4 cases were heterozygous for 17C>T mutation in exon 1, 29G>C mutation in exon 1, 1212C>A mutation in exon 9, and IVS4+5G>A mutation in intron 4 of the RHD gene, respectively. According to the rule of Rhesus Base Nomenclature, the 4 samples were respectively named as weak D type 31, weak D type 71, weak D type 72, and weak D type 82. CONCLUSION: Serological and molecular testing for the weak D can facilitate in-depth understanding of its immunology and genetics, and provide guidance for clinical blood transfusion and prevention of hemolytic disease in newborns.