Humans ; Platelet Aggregation Inhibitors ; therapeutic use ; Platelet Glycoprotein GPIIb-IIIa Complex ; antagonists & inhibitors

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

This study was aimed to investigate the influence of different platelet membrane glycoprotein monoclonal antibodies (McAb) which are common used in laboratories on the monoclonal antibody-specific immobilization of platelet antigens (MAIPA) technique according to the request of 14th International Society of Blood Transfusion Platelet Immunology Workshop. 30 participant laboratories were provided with 10 known human platelet antigen (HPA) antibodies, 1 normal serum, 9 different McAbs (against GPIIb/IIIa, GPIa/IIa, GPIb/IX and GPIV respectively), and the same protocol. Each participant laboratory carried out the test as the protocol to compare the results of different McAbs against the same glycoprotein and submitted the data to organizer. The results indicated that in McAbs against GPIIb/IIIa, AP2, Gi-5 and PL2-73 showed higher mean S/CO than that of others; in GPIa/IIa, MBC202.2 and 143.1 showed higher mean S/CO than that of others; in GPIb/IX, 142.11 and CLB-MB45 (CD42b) showed higher mean S/CO than that of others; as to GPIV, 131.4 showed higher mean S/CO. In conclusion, capture effects of various McAbs are different, so that different products of McAbs exert influences on the sensitivity of MAIPA. To use a panel of McAbs against the same glycoprotein may avoid the false negative results.
Antibodies, Monoclonal ; classification ; Antigens, Human Platelet ; immunology ; Humans ; Indicators and Reagents ; Platelet Glycoprotein GPIIb-IIIa Complex ; immunology ; Platelet Glycoprotein GPIb-IX Complex ; immunology ; Platelet Membrane Glycoproteins ; classification ; immunology

OBJECTIVETo investigate a specific and sensitive assay for the diagnosis of autoimmune thrombocytopenic purpura (AITP).

METHODSGlycoprotein specific autoantibodies in platelet eluate and plasma were detected by a modified monoclonal antibody immobilization of platelet antigens assay (MAIPA).

RESULTSThe overall positive rate of specific autoantibodies against platelet GPIIb/IIIa and GPIb/IX in plasma was 38.89% and in eluated platelet membrane was 68.52%. The difference between them was significant (corrected chi(2) = 19.39, P < 0.005). The proportion of positive MAIPA results between primary AITP and secondary AITP was not significantly different. There was a significant inverse correlation between antibody level and platelet count.

CONCLUSIONDetection of eluated GP-specific autoantibodies by MAIPA is highly specific and much more sensitive as compared with the measurement of their plasma counterparts in the diagnosing and therapeutic monitoring of AITP.

Adolescent ; Adult ; Autoantibodies ; blood ; Blood Platelets ; immunology ; Female ; Humans ; Male ; Middle Aged ; Platelet Glycoprotein GPIIb-IIIa Complex ; immunology ; Platelet Glycoprotein GPIb-IX Complex ; immunology ; Purpura, Thrombocytopenic, Idiopathic ; immunology

Platelet plays an important role in bleeding and thrombotic diseases. Humanized anti-platelet antibodies have great clinical effects in treatment of ITP and preventing thrombosis. The important role of platelet in bleeding and thrombotic diseases, the present status of development on study of humanized anti-platelet antibody and its application in treatment of bleeding and thrombotic diseases were summarized in this review.
Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; therapeutic use ; Autoantibodies ; immunology ; Blood Platelets ; immunology ; Humans ; Platelet Glycoprotein GPIIb-IIIa Complex ; biosynthesis ; immunology ; Platelet Glycoprotein GPIb-IX Complex ; immunology

OBJECTIVETo detect the frequencies of anti-GPIIb/IIIa antibody secreting B cells and platelet-specific antibody in patients with idiopathic thrombocytopenic purpura (ITP) and non-immune thrombocytopenia, and to evaluate their roles in the diagnosis of ITP and their clinical significance.

METHODSThe frequencies of circulating B cells secreting anti-GPIIb/IIIa antibody and platelet-specific antibody in 58 ITP patients, 33 non-ITP patients and 31 healthy controls were tested by Enzyme-linked Immunospot Assay (ELISPOT) and modified monoclonal antibody immobilization of platelet antigens assay (MAIPA) respectively.

RESULTSThe frequencies of circulating B cells secreting anti-GPIIb/IIIa antibody in ITP patients \[(6.6 ± 4.2)/10(5) PBMNC\] were significantly increased (P < 0.05) than that of the controls \[(1.3 ± 0.5)/10(5) PBMNC\] and non-immune thrombocytopenic purpura patients \[(2.2 ± 2.0)/10(5) PBMNC\]. However there was no apparent difference between the latter two groups (P > 0.05). ELISPOT had a sensitivity of 70.69%, a specificity of 90.91% for the diagnosis of ITP, the sensitivity being higher than that of modified MAIPA's (43.10%) (χ(2) = 7.03, P < 0.05). The ROC curve showed the discriminative validity of cytometric bead array was 0.886.

CONCLUSIONThe frequencies of circulating B cells secreting anti-GPIIb/IIIa antibody may reflect the pathogenesis of ITP. ELISPOT assay have high sensitivity and specificity than modified MAIPA for the diagnosis of ITP and the guidance for clinical therapy.

Autoantibodies ; immunology ; B-Lymphocytes ; Blood Platelets ; Humans ; Platelet Glycoprotein GPIIb-IIIa Complex ; immunology ; Purpura, Thrombocytopenic, Idiopathic ; immunology

Platelet glycoprotein VI (GPVI) is a major receptor for collagen on the platelet surface. It mediates the initial platelet contact with collagen, generates intracellular signals, increases the affinity of integrin receptor, and causes platelet aggregation and thrombosis. Suppression of GPVI function can significantly inhibit collagen-induced platelet adhesion, aggregation and thrombosis, so GPVI has become a novel target for antiplatelet therapy. Within the last few years, major advances have been made in understanding platelet-collagen interactions. In this paper, the advances of study on GPVI, including composition of GPVI, functions of GPVI, factors related with functions of GPVI, GPVI and clinic were summarized.
Humans ; Platelet Adhesiveness ; physiology ; Platelet Aggregation ; physiology ; Platelet Glycoprotein GPIIb-IIIa Complex ; metabolism ; physiology ; Platelet Glycoprotein GPIb-IX Complex ; metabolism ; physiology ; Platelet Membrane Glycoproteins ; chemistry ; metabolism ; physiology ; Protein Binding ; physiology

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

Integrin alphaIIbbeta3 of the platelet surfaces regulates the thrombosis formation. alphaIIbbeta3 binds to the RGD sequence (Arg-Gly-Asp) of fibrinogen, promotes the platelet aggregation and finally leads to the thrombus. We obtained the three-dimensional molecular structure of alphaIIbbeta3 using homology-modeling (modeller8v2 software), with integrin alphavbeta3 (pdb code 1JV2) as the template. Accordingly, a cyclic RGD(RGD-c) peptide was designed to bind alphaIIbbeta3 as an antagonist and to block the formation of thrombus. We added two amino acids X, Y to both sides of RGD-c. X and Y could bind to each other by disulfide bond that finally made RGD-c a cyclic peptide. The optimum structure of RGD-c was obtained from the energetic optimization processes. All amino acids were placed at the X and Y to conduct Molecular Docking to the integrin alphaIIbbeta3 We got the optimum structure of RGD-c by energetic optimization and the antagonistic combination analysis. The results might provide an insight into designing and screening integrin alphaIIbbeta3 antagonists.
Amino Acid Sequence ; Drug Design ; Humans ; Models, Molecular ; Oligopeptides ; chemistry ; Platelet Aggregation Inhibitors ; chemical synthesis ; chemistry ; Platelet Glycoprotein GPIIb-IIIa Complex ; antagonists & inhibitors ; chemistry