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.

Prevalence and severity of thrombocytopenia in patients with hyperferritinemia.

BACKGROUND: In patients with tumors, inflammation, and blood disorders, hyperferritinemia has been associated with the severity of the underlying disease and is frequently accompanied by a co-occurring low platelet count or thrombocytopenia. Despite this, no established correlation has been identified between hyperferritinemia and platelet count. In this retrospective, double-center study, we sought to describe the prevalence and severity of thrombocytopenia in patients with hyperferritinemia. STUDY AND DESIGN: A total of 901 samples were enrolled in this study, all of which had significantly high ferritin levels (>2000 µg/L) between January 2019 and June 2021. We analyzed the general distribution, incidence of thrombocytopenia in patients with hyperferritinemia, and the relationship between ferritin level and platelet count. p-values < 0.05 were considered statistically significant. RESULTS: The total incidence of thrombocytopenia in patients with hyperferritinemia was 64.7%. Hematological diseases were the most frequent cause of hyperferritinemia (43.1%), followed by solid tumors (29.5%) and infectious diseases (11.7%). Patients with thrombocytopenia (<150 × 109/L) had significantly higher ferritin levels than those with platelet counts exceeding 150 × 109/L, with median ferritin levels of 4011 and 3221 µg/L, respectively (P < 0.001). Additionally, the results showed that the incidence of thrombocytopenia was higher in hematological patients with chronic transfusion than in those without chronic blood transfusions (93% vs 69%). CONCLUSIONS: In conclusion, our results suggest that hematological diseases are the most common cause of hyperferritinemia and that patients with chronic blood transfusions are more susceptible to thrombocytopenia. Elevated ferritin levels may act as a trigger for thrombocytopenia.

A case of McLeod syndrome caused by a nonsense variation c.942G>A in the XK gene: A case report.

McLeod syndrome is a rare XK gene-related progressive, debilitating disease involving multiple systems. The blood group phenotypes in McLeod syndrome patients usually display the Kx antigen loss and a decrease in the Kell blood group system antigen expression. This paper describes a 41-year-old male Chinese patient with McLeod syndrome. He first attended a hospital in 2015 and developed progressively worsening symptoms 4 years ago. As the disease progressed, the patient exhibited memory loss, unresponsiveness, and chorea and displayed elevated creatine kinase levels. However, McLeod syndrome could not be diagnosed by these signs and laboratory results. The patient was readmitted to the hospital in 2020 and was suspected of having McLeod syndrome. Serological analysis of the Kell blood group system and genotyping for the XK blood group system were performed, revealing the weak expression of the K antigen and the negative K antigen. Sequencing of the coding region of the XK gene showed a hemizygous c.942G>A variation in the XK gene, which resulted in a premature stop codon at position 314 (p.Trp314Ter). Therefore, the patient was diagnosed with McLeod syndrome. In conclusion, this paper presents a case of McLeod syndrome caused by a nonsense variation c.942G>A in the XK gene. The analysis of the XK gene and blood group antigen is helpful for the diagnosis of McLeod syndrome and for distinguishing it from many other diseases.

[Identification of a novel FUT1 allele in a Chinese individual featuring para-Bombay phenotype].

OBJECTIVE: To explore the genetic basis for an individual with a para-Bombay phenotype. METHODS: A proband with mismatched forward and reverse serotypes for the ABO blood group was identified. Weakly expressed ABH blood type antigen on the surface of red blood cells was verified by absorption and release test, and the blood group substances in saliva was detected by sialic acid test. Exons 6 and 7 of the ABO gene and exons of the FUT1 and FUT2 genes were subjected to direct sequencing. RESULTS: The proband was found to be of O type by forward ABO serotyping and AB type by reverse ABO serotyping, though H and substance A and B were detected in her saliva. DNA sequencing revealed that she has harbored c.35C/T, c.328G/A, and c.504delC compound heterozygous variants of the FUT1 gene. Haploid analysis showed that her FUT1 genotype was h328A/h35T+504delC, which has been uploaded to the NCBI website (No. MW323551). CONCLUSION: The para-Bombay phenotype of the proband may be attributed to the novel compound heterozygous variants including c.504delC of the FUT1 gene, which may affect its function by altering the activity of FUT1 glycotransferase.

[Identification of A Case of DEL Blood Group with DAT Positive Phenotype and Pedigree Analysis].

OBJECTIVE: To identify the blood group of a patient with DEL phenotype combined with positive direct anti-human globulin test and to analyze the pedigree. METHODS: Routine serological reagents were used for serological analysis of RhD blood group in the pedigree members. Exons and flanking sequences of RHD gene were amplified, sequenced and analyzed for heterozygosity. The familial genetic state of DEL phenotype was further analyze in the family members. RESULTS: The DAT was strongly positive in the proband. The 1227G>A allele (RHD*DEL1) was present in the exon 9 of RHD gene, and the mother was the carrier of RHD*DEL1. The proband was identified as RHD+/RHD-, suggesting the CD1227Ae/Cde haplotype. CONCLUSION: The proband is DEL phenotype (RHD*DEL1).

[Identification of Phenotype and Genotype in One Case with Weak D blood group].

OBJECTIVE: To investigate the phenotype and genotype of the weak D blood group in one case of Chinese Han people. METHODS: Phenotype of blood sample was identified with serologic tests; PCR-SBT was applied for the analysis of genotype and RhD zygosity. RESULTS: Both saline and gel card tests demonstrated this case to be dCcee, which was contrary to glass bead card result. Some of the RBC D epitopes were negative.c.1022T>A allele was identified with PCR-SBT and the zygosity analysis showed this case to be D/d. CONCLUSION: RHD*1022 A is more suitable to be categorized as weak partial D other than weak D in a Chinese Han people.

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.

[Experimental Research of Circular RNA hsa_circ_0000254 in Acute Myeloid Leukemia].

OBJECTIVE: To clone the circular RNA hsa_circ_0000254 and construct its lentiviral over-expression vector. METHODS: The sequence of hsa_circ_0000254 (a total of 524 bp long) was synthesized and cloned by using pGH vector. The vector was cut by EcoR I and BamH I, and artificial hsa_circ_0000254 was obtained, then inserted in pLCDH-ciR to construct the recombinant expression vector pLCDH-circ254(C254), which was confirmed by DNA sequencing. The lentiviral expression vectors pLCDH-circ254(C254) and NC(pLCDH-ciR) were cotransfected into 293T cells by lipofectamineTM 2000(lipo2000). After transfection for 40 hours, the cells were collected and verified by PCR and sequencing. RESULTS: Restriction analysis and DNA sequencing demonstrated that the lentiviral vector pLCDH-circ254(C254) was constructed successfully, the expression efficiency increased 10000 times after transfection of cells. CONCLUSION: The successful construction of the lentiviral expression vector pLCDH-circ254(C254) results in the production of high-titer lentivirus, so as to facilitate further study of the molecular functions of hsa_circ_0000254.

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.

miR-326 targets antiapoptotic Bcl-xL and mediates apoptosis in human platelets.

Platelets play crucial roles in hemostasis, thrombosis, wound healing, inflammation, angiogenesis, and tumor metastases. Because they are anucleated blood cells, platelets lack nuclear DNA, but they do contain mitochondrial DNA, which plays a key role in regulating apoptosis. Recent evidence has suggested that miRNAs are also involved in regulating gene expression and apoptosis in platelets. Our previous study showed that the expression of miR-326 increased visibly when apheresis platelets were stored in vitro. The antiapoptotic Bcl-2 family regulator Bcl-xL has been identified as a putative target of miR-326. In the present study, dual reporter luciferase assays were used to characterize the function of miR-326 in the regulation of the apoptosis of platelet cells. These assays demonstrated that miR-326 bound to the 3'-translated region of Bcl-xL. To directly assess the functional effects of miR-326 expression, levels of Bcl-xL and the apoptotic status of stored apheresis platelets were measured after transfection of miR-326 mimic or inhibitor. Results indicated that miR-326 inhibited Bcl-xL expression and induced apoptosis in stored platelets. Additionally, miR-326 inhibited Bcl-2 protein expression and enhanced Bak expression, possibly through an indirect mechanism, though there was no effect on the expression of Bax. The effect of miR-326 appeared to be limited to apoptosis, with no significant effect on platelet activation. These results provide new insight into the molecular mechanisms affecting differential platelet gene regulation, which may increase understanding of the role of platelet apoptosis in multiple diseases.

Detection of apoptosis-associated microRNA in human apheresis platelets during storage by quantitative real-time polymerase chain reaction analysis.

BACKGROUND: Platelet transfusion is an essential part of the treatment of a variety of conditions such as thrombocytopenia and qualitative platelet disorders. As indicated in previous reports, during in vitro storage, platelets undergo morphological and physiological changes collectively known as the platelet storage lesion. Apoptosis is a programmed process of cell death, which has been considered as an important cause of platelet storage lesion under the common storage conditions in standard blood banks. Platelets are anucleate blood cells, but contain significant amounts of microRNA (miRNA, miR), which may play an important role in the regulation of gene expression. Drawing on previously published reports on cell apoptosis, we selected 49 miRNA for analysis to explore whether miRNA are of importance during the storage of platelets. MATERIALS AND METHODS: We used quantitative real-time polymerase chain reaction analysis to determine the levels of expression of miRNA in apheresis platelets at different times of storage. Bioinformatics analysis was applied to explore target genes and the main functions of the selected miRNA. RESULTS: Our observations suggest that apheresis platelets contain large amounts of apoptosis-associated miRNA. The levels of expression of 25 miRNA remained high and ten of these miRNA showed different expression from that at day 0. Of these ten miRNA, hsa-miR-326, hsa-miR-96, hsa-miR-16, hsa-miR-155 and hsa-miR-150 were up-regulated, while hsa-miR-7, hsa-miR-145, hsa-miR-24, hsa-miR-25 and hsa-miR-15a were down-regulated. The markedly increased expression of hsa-miR-326 in all platelets is noteworthy (p<0.001). DISCUSSION: Since Bcl-xl and Bak1, members of the Bcl-2 family, are the targets of hsa-miR-326, our findings suggest that hsa-miR-326 may be involved in platelet apoptosis during storage.

[Preparation of monoclonal antibodies against the adhesion protein 33 of Trichomonas vaginalis].

OBJECTIVE: To prepare and characterize the monoclonal antibodies (McAbs) against recombinant adhesion protein 33 (AP33) of Trichomonas vaginalis. METHODS: The purified recombinant fusion protein AP33 was used as antigen to immunize BALB/c mice. Sp2/0 myeloma cells were fused with the splenocytes from immunized BALB/c mice. After ELISA screening and 4 times of limited dilution, 5 positive hybridoma cell lines were obtained, and the biological properties of the McAbs were identified by Western blotting. Indirect immunofluorescent antibody test (IFAT) was performed and the inhibition effect of McAbs on the cytoadherence of Trichomonas vaginalis to HeLa cell was assayed. RESULTS: Western blotting demonstrated that 5 McAbs, designated as 4A2, 4F11, 4F8, 4E7 and 4H11, specifically combined with the recombinant AP33 of T. vaginalis. The McAbs were IgG1 isotypes. Four of them (4F11, 4F8, 4E7 and 4H11) showed parasite recognition by IFAT. Parasite cytoadherence to a monolayer of HeLa cells was inhibited in vitro with a inhibition rate of 50.08%, 65.03%, 50.70% and 49.08% by the 4 McAbs under a concentration of 200, 200, 400 and 200 microg/ml, respectively. CONCLUSIONS: The prepared McAbs against the recombinant AP33 show a protective inhibition on cytoadherence of Trichomonas vaginalis in vitro.