OBJECTIVE: To construct a mouse model of Glanzmann's thrombasthenia (GT) with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation by CRISPR/Cas9 technology, and then further explore the expression and function of glycoprotein αIIbβ3 on the surface of platelet membrane. METHODS: The donor oligonucleotide and gRNA vector were designed and synthesized according to the ITGA2B gene sequence. The gRNA and Cas9 mRNA were injected into fertilized eggs with donor oligonucleotide and then sent back to the oviduct of surrogate mouse. Positive F0 mice were confirmed by PCR genotyping and sequence analysis after birth. The F1 generation of heterozygous GT mice were obtained by PCR and sequencing from F0 bred with WT mice, and then homozygous GT mice and WT mice were obtained by mating with each other. The phenotype of the model was then further verified by detecting tail hemorrhage time, saphenous vein bleeding time, platelet aggregation, expression and function of αIIbβ3 on the surface of platelet. RESULTS: The bleeding time of GT mice was significantly longer than that of WT mice (P<0.01). Induced by collagen, thrombin, and adenosine diphosphate (ADP), platelet aggregation in GT mice was significantly inhibited (P<0.01, P<0.01, P<0.05). Flow cytometry analysis showed that the expression of αIIbβ3 on the platelet surface of GT mice decreased significantly compared with WT mice (P<0.01), and binding amounts of activated platelets to fibrinogen were significantly reduced after thrombin stimulation (P<0.01). The spreading area of platelet on fibrinogen in GT mice was significantly smaller than that in WT mice (P<0.05). CONCLUSION A GT mouse model with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation has been established successfully by CRISPR/Cas9 technology. The aggregation function of platelet in this model is defective, which is consistent with GT performance.
Animals ; CRISPR-Cas Systems ; Codon, Nonsense ; Disease Models, Animal ; Fibrinogen/genetics* ; Humans ; Integrin alpha2/genetics* ; Mice ; Oligonucleotides ; Platelet Glycoprotein GPIIb-IIIa Complex/genetics* ; RNA, Guide ; Thrombasthenia/genetics* ; Thrombin/genetics*

OBJECTIVE: To examine the antiplatelet and antithrombotic activity of Rumex acetosella extract. METHODS: Standard light aggregometry was used for platelet aggregation, intracellular calcium mobilization assessed using Fura-2/AM, granule secretion (ATP release) by luminometer, and fibrinogen binding to integrin α_IIbβ₃ detected using flow cytometry. Western blotting is carried out to determine the phosphorylation of mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/Akt signaling. RESULTS: Rumex acetosella displayed the ability to inhibit platelet aggregation, calcium mobilization, granule secretion, and fibrinogen binding to integrin α_IIbβ₃. Rumex acetosella has also down-regulated MAPK and PI3K/Akt phosphorylation (all P<0.01). CONCLUSION Rumex acetosella extract exhibits antiplatelet activity via modulating GPVI signaling, and it may protect against the development of platelet-related cardiovascular diseases.
Blood Platelets/metabolism* ; Calcium/metabolism* ; Fibrinogen/metabolism* ; Mitogen-Activated Protein Kinases/metabolism* ; Phosphatidylinositol 3-Kinase/pharmacology* ; Phosphatidylinositol 3-Kinases/metabolism* ; Phosphorylation ; Plant Extracts/pharmacology* ; Platelet Aggregation ; Platelet Aggregation Inhibitors/pharmacology* ; Platelet Glycoprotein GPIIb-IIIa Complex/pharmacology* ; Proto-Oncogene Proteins c-akt/metabolism* ; Rumex/metabolism*

OBJECTIVE: To establish 293T cell lines stably expressing Calpain-cleavage related α3 cytoplasmic tail mutants, and to explore the effect of amino acid motifs in integrin β3 cytoplasmic tail on αⅡbβ3-mediated cell function. METHODS: 293T cell lines stably co-expressing human wild type integrin αⅡb and full length β3 or mutant β3, including β3-ΔNITY (β3 cytoplasmic tail NITY motif deleted), β3-Δ754 (β3 cytoplasmic tail TNITYRGT motif deleted) and β3-Δ759 (β3 cytoplasmic tail RGT motif deleted) were established. Spreading and adhesion of these stable cell lines on immobilized fibrinogen were tested. RESULTS: 293T-αⅡbβ3ΔNITY, 293T-αⅡbβ3Δ754, 293T-αⅡbβ3Δ759 and 293T-αⅡbβ3 cell lines were successfully established. Compared with the 293T cells, 293T-αⅡbβ3 cells which expressed full β3, possessed well adhesion and spread ability on immobilized fibrinogen, suggesting it can be as a surrogate for platelet. Compared with 293T-αⅡbβ3 cells, the 293T-αⅡbβ3ΔNITY cells showed a partial impairment of adhesion and spreadability on immobilized fibrinogen. while the 293T-αⅡbβ3Δ754 cells and 293T-αⅡbβ3Δ759 cells failed to adhere or spread on immobilized fibrinogen. CONCLUSION To the cell spreading function mediated by integrin β3, RGT motif is vital, while NITY can be dispensable. These established 293T cell lines stably expressing different β3 mutants provide a solid basis for a further analysis of mass spectrometry.
Amino Acid Motifs ; Animals ; CHO Cells ; Cell Adhesion ; Cricetinae ; Cricetulus ; HEK293 Cells ; Humans ; Integrin beta3 ; genetics ; metabolism ; Platelet Glycoprotein GPIIb-IIIa Complex ; genetics ; metabolism ; Signal Transduction

OBJECTIVE: To explore the clinical significance of platelet membrane glycoprotein (GPIIb/IIIa) detection by flow cytometry (FCM) combined with enzyme-linked immunosorbent assay (ELISA) in diagnosis and treatment of immune thrombocytopenia(ITP). METHODS: Fifty-two patients with immunological thrombocytopenia admitted to the Department of Hematology in our hospital during October 2014-October 2018 were enrolled in ITP group. Thirty healthy people from the physical examination center were enrolled in control group. The positive expression rate of platelet membrane glycoprotein was measured by FCM, and the peripheral platelet count was detected by automatic analyzer, and the plasma membrane glycoprotein level was measured by ELISA. The difference between the two groups was compared. The platelet membrane glycoprotein levels in ITP patients before and after treatment were compared. RESULTS: The positive expression rate of GPIIb/IIIa (i,e CD41/CD61) and plasma GPIIb/IIIa levels in ITP patients were significantly lower than those in the healthy controls. The increased expression of platelet GPIIb/IIIa was associated with the response to therapy. The positive expression rate and level of GPIIb/IIIa postively correlated with its platelet count. The sensitivity of platelet GPIIb/IIIa combined with platelet count for diagnosis of ITP was 90.38%, the specificity was 93.33%, the positive likelihood ratio was 13.57, and the positive predictive value was 95.92%. CONCLUSION The platelet membrane glycoprotein detection as a preliminary screening method used for diagnosis of immune thrombocytopenia is simple, convenient, sensitive and rapid, thus may be considered as a new method for clinical diagnosis.
Autoantibodies ; Blood Platelets ; Humans ; Platelet Glycoprotein GPIIb-IIIa Complex ; Purpura, Thrombocytopenic, Idiopathic

Objective: To review the clinical characteristics of a pedigree with inherited hemorrhagic disease to explore its molecular pathogenesis. Methods: The clinical data of the pedigree with inherited hemorrhagic disease were collected. After extracting DNA, next generation sequencing was utilized to detect the potential gene mutation. The changes of RASGRP2 transcript of this proband and his parents were detected using RT-PCR to compare with normal control. Results: The phenotype of the proband in this pedigree with inherited platelet dysfunction and bleeding disorder was similar to variant Glanzmann's thrombasthenia, the maximum aggregations of platelet in response to the physiological agonists including ADP, epinephrine and arachidonic acid were significantly lower, leading to severe spontaneous mucosal bleeding. Integrin αIIbβ3 gene mutation was not detected, but another gene mutation RASGRP2 IVS3-1 stood out. The mutation was homozygous in the proband and heterozygosis in both of his parents. Two transcript types were detected in the proband, without transcripts coding functional RASGRP2 protein, however, his parents had functional transcripts and abnormal transcripts, with the normal transcripts in the majority. Conclusions: The RASGRP2 IVS3-1 gene mutation was responsible for the inherited hemorrhagic disease. The RASGRP2 IVS3-1 gene mutation led to abnormal alternative splicing, without formation of functional RASGRP2 protein. The RASGRP2 protein is at the nexus of calcium-dependent platelet activation and hemostasis after damage of blood vessels. Spontaneous mucosal bleeding was a result of the lack of the functional RASGRP2 protein. This was the first report of RASGRP2 gene mutation resulting in bleeding disorder in China, and also the first report of the mutation type of RASGRP2 IVS3-1.
Blood Platelets ; Guanine Nucleotide Exchange Factors ; Humans ; Pedigree ; Platelet Glycoprotein GPIIb-IIIa Complex ; Thrombasthenia

UNLABELLEDOBJLECTIVE: To investigate the effect of integrin β3 cytoplasmic NITY motif on αIIbβ3-mediated cell functions.

METHODSStable Chinese hamster ovary (CHO) cell lines that co-express human wild type integrin αIIb and wild type β3 or mutant β3ΔNITY (β3 deleting cytoplasmic NITY motif) were established. Expression of αIIb and β3 were tested by Western blot and flow cytometry in CHO cell lines. Spreading and adhesion of stable cell lines on immobilized fibrinogen were examined. The co-immunoprecipitation was used to detect protein interactions.

RESULTSCHO-αIIbβ3, CHO-αIIbβ3ΔNITY cells were successfully established. The CHO cells transfected with wild type αIIbβ3 had the ability of adhesion and spreading. Compared with CHO-αIIbβ3 cells, CHO-αIIbβ3ΔNITY cells showed an impaired capacity of adhesion but no significant difference was observed in spreading of adhered cells. The co-immunoprecipitation showed that kindlin-2 associated with wild type integrin αIIbβ3. The β3ΔNITY mutation substantially reduced kindlin-2 association.

CONCLUSIONDeletion of NITY motif causes an impaired ability of adhesion. The deletion mutation can suppress kindlin-2 binding to integrin β3, thereby partially inhibit the integrin β3 signaling.

Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Fibrinogen ; Humans ; Integrin alpha2 ; Integrin beta3 ; Platelet Glycoprotein GPIIb-IIIa Complex ; Protein Structure, Tertiary ; Signal Transduction

OBJECTIVETo investigate the influence of divalent cation chelator EDTA and heparin sodium on the detection of ITP platelet-specific autoantibodies by the modified monoclonal antibody immobilization of platelet antigen assay (MAIPA) and to explore the relationship between types of platelet specific autoantibodies and glucocorticoid efficacy.

METHODSThe samples were obtained from EDTA- and heparin- anticoagulant ITP patients, respectively, so as to detect the platelet-specific autoantibodies (GPIIb/IIIa and GPIbα) in 140 ITP samples by modified MAIPA, then the differences between these two methods were compared.

RESULTSOut of 140 cases in EDTA group, 55 cases were positive for GPIIb/IIIa, while 76 cases in heparin group were positive for GPIIb/IIIa, 42 cases in both group were repeatable; among them 13 cases were positive in EDTA group and negative in heparin group, while 34 cases were positive in heparin group and negative in EDTA group, there was significant difference between them (x (2) = 9.38, P < 0.05), 62 cases in 140 cases of EDTA group were positive for GPIba, while 51 cases in heparin group were positive for GPIba, 42 cases in both group were repeatabe; among them 20 cases were positive in EDTA group and negative in heparin group, while 9 cases were positive in heparin group and negative in EDTA group, there was no significant difference between them (x (2) = 3.44, P > 0.05). A total of 320 cases received a standard glucocorticoid treatment, out of them 143 cases were positive for GPIbα with effective rate 39.9%, 177 cases were negative for GPIbα with effective rate 79.7%, there was statisticalty significant difference between them (x (2) = 53.115, P < 0.05).

CONCLUSIONEDTA anticoagulant (a divalent cation chelator) has a significant influence on detection of ITP platelet-specific autoantibodies (GPIIb/IIIa) by a modified MAIPA method and the GPIbα antibody positive may be one of the important factors that results in un-sensitivity of ITP patients to glucocorticoid treatment.

Antibodies, Monoclonal ; Anticoagulants ; therapeutic use ; Antigens, Human Platelet ; Autoantibodies ; blood ; Blood Platelets ; immunology ; Fibrinolytic Agents ; Glucocorticoids ; therapeutic use ; Heparin ; Humans ; Platelet Glycoprotein GPIIb-IIIa Complex ; Purpura, Thrombocytopenic, Idiopathic ; blood ; immunology

Although the effects of the rice bran have recently been investigated, there is no information regarding platelet physiology available. However, it is well known that fermented natural plants have a beneficial effect on cardiovascular diseases. Therefore, this study was conducted to investigate whether fermented rice bran extract (FRBE) with several plants (Artemisia princeps, Angelica Gigantis Radix, Cnidium officinale, and Camellia sinensis) affected agonist-induced platelet aggregation, and if so, what the underlying mechanism of its activity was. We performed several experiments, including in vitro platelet aggregation, intracellular calcium concentration and adenosine triphosphate release. In addition, the activation of integrin alphaIIbbeta3 was determined using fibrinogen binding. Thrombus formation was also evaluated in vivo using an arterio-venous shunt model. The FRBE inhibited collagen-induced platelet aggregation in a concentration-dependent manner. FRBE significantly and dose dependently attenuated thrombus formation using rat arterio-venous shunt. FRBE suppressed the intracellular calcium mobilization in collagen-stimulated platelets. We also found that FRBE inhibited extracellular stimuli-responsive kinase 1/2, p38-mitogen-activated protein kinases and c-Jun N-terminal kinase phosphorylation. These results suggested that FRBE inhibited collagen-induced platelet aggregation, which was mediated by modulation of downstream signaling molecules. In conclusion, FRBE could be developed as a functional food against aberrant platelet activation-related cardiovascular diseases.
Adenosine Triphosphate ; Angelica ; Animals ; Blood Platelets ; Calcium ; Camellia ; Cardiovascular Diseases ; Cnidium ; Collagen ; Fibrinogen ; Functional Food ; JNK Mitogen-Activated Protein Kinases ; Phosphorylation ; Phosphotransferases ; Physiology ; Platelet Aggregation ; Platelet Glycoprotein GPIIb-IIIa Complex ; Protein Kinases ; Rats ; Thrombosis

OBJECTIVETo investigate the different outcomes by dexamethasone in adults immune thrombocytopenia purpura (ITP) with different types of platelet specific-autoantibodies.

METHODSA total of 185 ITP were enrolled, 61 males and 124 females, with a median age of 42 (18-83) years, including 117 newly diagnosed, 35 persistent, and 33 chronic cases. All the patients received the dexamethasone at an initial dose of 40 mg per day for 4 days and a low dose of 5-10 mg for 3-4 weeks. The platelet specific-autoantibodies were identified by the modified monoclonal antibody-specific immobilization of platelet antigen (MAIPA) assay.

RESULTSAmong the IgG positive patients, the response rates in anti-GPIIb/IIIa antibody, anti-GPIbα antibody, both antibody positive, and both antibody negative were 87.5%, 50.0%, 68.0%, and 72.3% (χ²=11.489, P<0.05), respectively. Among the IgM positive patients, the response rates in the four groups were 82.1%, 71.4%, 61.9%, and 68.9% (χ²=2.719, P=0.437), respectively. Among the GPIbα antibody positive patients, the response rates in IgG alone, IgM alone, both positive, and both negative were 52.4%, 59.1%, 76.1%, and 77.9% (χ²=10.811, P<0.05), respectively. Among the GPIIb/IIIa antibody positive patients, the response rates in the four groups were 73.3%, 71.0%, 78.6%, and 66.3% (χ²=1.374, P=0.719), respectively.

CONCLUSIONITP patients with GPIbα-IgG antibody have worse response to dexamethasone treatment.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Antibodies, Monoclonal ; Autoantibodies ; Blood Platelets ; Dexamethasone ; Female ; Humans ; Male ; Middle Aged ; Platelet Glycoprotein GPIIb-IIIa Complex ; Purpura, Thrombocytopenic, Idiopathic ; Young Adult

OBJECTIVETo evaluate the efficacy and safety of lower doses rituximab(375 mg/m²×1) in primary children immune thrombocytopenia (ITP).

METHODSFifty children [23 male and 27 female, the median age was 9.5 years (rage 3.5-17.0 years)]with persistent and chronic ITP were treated with lower doses rituximab from January 2009 to January 2013 in our hospital. Efficacy and side effects of lower doses rituximab was studied, and factors related to the outcomes were analyzed.

RESULTSAmong fifty patients, 17/50(34%) achieved a complete response (CR) and 15/50 (30%) patients got response (R). Patients with CR continued to maintain a platelet count above 50×10⁹/L at a median 12.3 (6-40) months. Patents with R continued to maintain a platelet count above 30×10⁹/L at a median 6 (2-12) months. The overall response (OR) in 3 and 6 months were 58% (29/50), 64% (32/50) respectively. Six patients have mild and transient side effects, including urticarial rash and fever, which were promptly resolved with appropriate therapy. Sex, age at diagnosis, interval from diagnosis to initial treatment with rituximab, platelet count at treatment and CD19+B cell count did not influence the overall response and complete response (P>0.05). Patients with anti-GPIIb/IIIa autoantibody had a better OR (P<0.05).

CONCLUSIONChildren with persistent and chronic ITP treated by lower doses rituximab had better therapeutic effects. Patients with anti-GPIIb/IIIa autoantibody had better response. Rituximab was well tolerated and no related serious side effects were recorded in the study.

Adolescent ; Antibodies, Monoclonal, Murine-Derived ; Autoantibodies ; Child ; Child, Preschool ; Female ; Fever ; Hospitals ; Humans ; Male ; Platelet Count ; Platelet Glycoprotein GPIIb-IIIa Complex ; Purpura, Thrombocytopenic, Idiopathic ; Remission Induction ; Rituximab ; Thrombocytopenia