血小板特异性抗体对特发性血小板减少性紫癜诊断价值的研究

本研究的目的是比较特发性血小板减少性紫癜 (ITP)、慢性再生障碍性贫血 (CAA)、恶性血液病患者及健康志愿者特异性抗体水平 ,以评价血小板特异性抗体在ITP诊断中的价值。用改良单克隆抗体特异性俘获血小板抗原 (MAIPA)技术同时检测血小板GPIb/Ⅸ、GPIIb/Ⅲa、GPⅣ、GPⅤ的特异性抗体。结果表明 :ITP组、CAA组、恶性血液病患者及健康志愿者血小板特异性抗体总阳性率分别为 6 9.99% ,10 % ,2 0 %和 0 %。ITP组与CAA组存在显著性差异 ( χ2 =2 0 .71,P <0 .0 0 5 ) ,ITP组与恶性血液肿瘤化疗组存在显著性差异 ( χ2 =12 .2 2 ,P <0 .0 0 5 )。健康志愿组无 1例阳性。结论 :多种抗体同时检测可提高敏感性 ,血小板特异性抗体对ITP是一种特异性高、敏感性强的实验室诊断指标。     

HLA-DRB1等位基因与儿童特发性血小板减少性紫癜

目的：研究人类白细胞抗原（LA）DRB1等位基因与儿童特发性血小板减少性紫癜（ITP）的关系。方法：用PCR－SSO法对42例ITP患儿进行HLA－DRB1等位基因分型，同时用改良的血小板抗原单抗特异性固相化法（MAIPA）检测其中36例ITP患儿血清中的抗GPⅡb/Ⅲa和GPⅠb/Ⅸ自身抗体。结果：（1）与健康对照相比，ITP患儿HLA－DRB1＊17基因型显著升高（P＜0．05，RR＝2．76，EF＝0．1064），而HLA－DRB1＊1202基因型显著降低（P＜0．025，RR＝0．20，PF＝0．7616）。（2）慢性难治性ITP患儿与非难治性患儿相比，HLA－DRB1＊11基因型显著升高（P＜0．025），且具有DRB1＊11的患儿主要（5／6）为女性年长患儿；（3）抗GPⅡb/Ⅲa及抗GPⅠb/Ⅸ自身抗体的阳性率都与HLA－DRB1＊02（15／16）基因型显著相关（P分别为0．02和0．01），但难治性和非难治性ITP患儿间抗体阳性差异无显著性（P＞0．1）。结论：（1）DRB1＊17可能与儿童ITP的易感性有关，而DRB1＊1202则可能对儿童ITP的发病具有保护作用；（2）具有DRB1＊11基因型的患儿易发展为慢性难治性ITP；（3）血小板自身抗体与抗原表位的反应可能受DRB1＊02限制，但自身抗体阳性与否并不能预示ITP患儿的预后。     

血小板特异性抗体与ITP发病、临床特点、治疗及预后效果的关系

免疫性血小板减少症（immune thrombocytopenia,ITP）是一种自身免疫性出血性疾病。目前认为血小板自身抗体的形成是本病主要的发病机制之一,其一线治疗中激素和丙种球蛋白均主要通过抑制自身抗体产生、封闭网状内皮系统FC受体等作用以减少血小板的破坏,但有部分患者对一线治疗无效,病程迁延,预后不良,最终进展为慢性/难治性ITP（chronic/refractory ITP,C/RITP）。研究发现,血小板膜糖蛋白GPIIb/IIIa和GPIbα是本病最常见的抗原靶位,而一线治疗对具有抗GPIbα的ITP患者疗效欠佳。进一步研究发现抗GPIIb/IIIa抗体与抗GPIbα抗体导致血小板破坏的途径不尽相同：前者主要为依赖FC途径,而后者主要通过FC非依赖性途径清除血小板。以上研究结果提示早期发现ITP血小板抗体类型对于治疗和预后该疾病起关键作用。本文主要对ITP特异性血小板抗体与疾病的发生、临床特点、治疗及预后的关系进行综述。     

Preliminary laboratory based diagnostic criteria for immune thrombocytopenic purpura: evaluation by multi-center prospective study

BACKGROUND: We proposed diagnostic criteria for immune thrombocytopenic purpura (ITP) by modifying the existing guidelines for diagnosis of ITP and by incorporating laboratory tests found useful for predicting its diagnosis, for example erythrocyte count, leukocyte count, anti-GPIIb/IIIa antibody-producing B cells, platelet-associated anti-GPIIb/IIIa antibodies, percentage of reticulated platelets, and plasma thrombopoietin. OBJECTIVE AND METHODS: To validate our criteria, we conducted a multi-center prospective study involving 112 patients with thrombocytopenia and a morphologically normal peripheral blood film at the first visit. Each patient underwent a physical examination, routine laboratory tests, and specialized tests for the anti-GPIIb/IIIa antibody response and platelet turnover. RESULTS: Ninety-one patients (81%) satisfied the proposed criteria at first visit. Clinical diagnosis was made by skilled hematologists > 6 months after the first visit; ITP was diagnosed in 88 patients and non-ITP disorders in 24. The proposed criteria had 98% sensitivity, 79% specificity, a 95% positive predictive value, and a 90% negative predictive value. A relatively low specificity appears to be attributed to a few patients who had both ITP and aplastic anemia or myelodysplastic syndrome. CONCLUSIONS: Our preliminary diagnostic criteria based on ITP-associated laboratory findings were useful for the differential diagnosis of ITP, but additional evaluations and modifications will be necessary to develop criteria that can be used routinely.     

Advances in immunological aspects of idiopathic thrombocytopenic purpura

Idiopathic thrombocytopenic purpura(ITP) is an autoimmune disease characterized by increased platelet destruction caused by anti-platelet autoantibodies, which mainly target a platelet surface antigen, GP IIb-IIIa. CD4+ T cells reactive with GP IIb-IIIa play a primary role in the disease process, since these autoreactive T cells are involved in the production of pathogenic anti-platelet autoantibodies. GP IIb-IIIa-reactive T cells mainly recognize 'cryptic' determinants on the amino-terminal extracellular domains of both GP IIb alpha and GP IIIa, and are activated primarily in the spleen. These findings are particularly useful for clarifying the pathogenic process in ITP patients and for developing a therapeutic approach that blocks pathogenic anti-platelet antibody production.     

Significance of detection of anti-GPIIb/IIIa antibody secreting B cells and platelet-specific antibody in patients with idiopathic thrombocytopenic purpura

OBJECTIVE: To 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. METHODS: The 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. RESULTS: The 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. CONCLUSION: The 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.     

Modulation of immune response with cytotoxic T-lymphocyte-associated antigen 4 immunoglobulin-induced anergic T cells in chronic idiopathic thrombocytopenic purpura.

BACKGROUND: Platelet glycoprotein (GP)-reactive CD4+ T cells are essential for the stimulation and maintenance of antiplatelet autoantibody production in chronic idiopathic thrombocytopenic purpura (ITP). Blocking costimulatory signals could result in platelet-specific T-cell anergy. METHODS: GP-specific CD4+ T cells from patients with ITP were made anergic using cytotoxic T-lymphocyte-associated antigen 4 immunoglobulin (CTLA4-Ig). The CTLA4-Ig-induced GP-specific anergic T cells were investigated for their inhibitory function on GP-reactive T-cell proliferation and antibody production with in vitro culture systems. To further analyze their tolerizing mechanisms, we cocultured GP-anergic T cells with dendritic cells (DCs) from patients with ITP. RESULTS: Our studies demonstrated that the anergized GP-specific T cells have profound effects on both GP-specific T-cell proliferation and antibody production. These anergic T cells exerted their suppressive effects mainly in a cell contact-dependent manner, and they were not constitutively suppressive but required specific antigen stimulation to make DCs tolerogenic. The anergic T-cell-modulated DCs could induce the autoreactive T cells to be tolerant, and this effect was not restricted to T cells of the same specificity. CONCLUSION: Our studies demonstrate the efficacy of CTLA4-Ig in suppressing the pathologic autoimmune responses in ITP. These findings provide new insights into the underlying mechanisms of anergy induction in chronic ITP.     

Specificity of antibody responses affected by extracorporeal immunoadsorption of plasma over columns of protein A silica

A relationship is described between the interaction of circulating immune complexes (CIC) from plasma with staphylococcal protein A immunoadsorption treatment columns and modulation of antibody responses related to the specific CIC. Eluates from the initial immunoadsorption columns used to treat a series of patients with breast adenocarcinoma, cancer chemotherapy-associated thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (C-TTP/HUS), or immune thrombocytopenic purpura (ITP) were evaluated for disease-specific CIC containing Le^x glycosphingolipid (Le^x gl) adenocarcinoma-associated antigens or platelet autoantibody (anti-GPIIb/IIIa), together with the corresponding neutralizing antibody [anti-F(ab′)₂], and for nonspecific CIC containing cytomegalovirus (CMV) or herpes simplex virus type 1 (HSV-1) antigens. In addition, the levels of antibodies directed against CMV, HSV-1. Le^x gl, and GPIIb/IIIa antigens, as well as anti-F(ab′)² antibodies, were compared in pretreatment and posttreatment serum samples. Columns used to treat breast adenocarcinoma patients contained only Le^x gl CIC, and the only immunologic change observed after treatment was significant increases in anti-Le^x gl antibodies in some patients. Columns used to treat C-TTP/HUS patients contained anti-GPIIb/IIIa-anti-F(ab′)₂ CIC, in addition to Le^x gl CIC. After treatment, significant increases in anti-Le^x gl and anti-F(ab′)₂ antibodies and significant decreases in anti-GPIIb/IIIa antibodies were observed in some patients. Columns used to treat ITP patients only exhibited anti-GPIIb/IIIa-anti-F(ab′)₂ CIC, and after treatment only decreases in anti-GPIIb/IIIa and increases in anti-F(ab′)₂ antibodies were observed in some patients. None of the treatment columns exhibited CIC with CMV or HSV-1 antigens, and no effects on pretreatment levels of anti-CMV or anti-HSV-1 antibodies were observed in any patients posttreatment. Immunologic responses were correlated closely with clinical responses in each group of patients. The study showed that neither stimulation nor depression of antibody responses occurred in the absence of an interaction of corresponding specific CIC with the matrix. The results suggest that nonspecific stimulation of a variety of antibody responses is not the usual mechanism by which clinical responses to protein A immunoadsorption treatment are obtained. © 1992 Wiley-Liss, Inc.     

特发性血小板减少性紫癜患者脾脏CD5+和CD5-B细胞共同参与血小板自身抗体的产生

目的：研究慢性特发性血小板减少性紫癜（ITP）患者脾脏CD5^ B细胞水平的变化及CD5^ 和CD5^-B细胞与血小板膜糖蛋白（GP）特异性自身抗体产生的关系，以识别致病B细胞亚群。方法：应用双色流式细胞仪检测8例慢性ITP患者脾脏CD5^ B细胞水平。选择4例血浆抗GPⅡb/Ⅲa和抗GP Ⅰb/Ⅸ抗体双阳性ITP切脾患者，应用Ficoll密度梯度离心及花环形成分离法分离脾脏B淋巴细胞，继而采用镝产珠分选法分选、纯化CD5^ B细胞和CD5^-B细胞，并分别进行体外培养，应用改良MAIPA法检测血浆和细胞培养上清液的血小板特异性抗体。结果：ITP患者脾脏CD5^ B细胞水平圈晨自身免疫性疾病患者略有增高，二者之间差异无统计学意义。CD5^ B细胞水平与患者血小板计数无相关性。4例血浆抗GPⅡb/Ⅲa抗体和抗GPⅠb/Ⅸ抗体双阳性。另外1例CD5^ B细胞培养液抗GPⅡb/Ⅲa抗体阴性，抗GPⅠb/Ⅸ抗体阳性;CD5^-B细胞培养液抗GPⅡb/Ⅲa抗体和抗GPⅠb/Ⅸ抗体双阳性。结论：脾脏CD5^ 和CD5^-B细胞 均可产生血小板GP特异性自身抗体，抗体产生种类和滴度无明显差异。提示二者共同参与了ITP的发病过程。     

Autoreactive T cells to platelet GPIIb-IIIa in immune thrombocytopenic purpura. Role in production of anti-platelet autoantibody.

T cell proliferative responses to platelet membrane GPIIb-IIIa were examined in 14 patients with chronic immune thrombocytopenic purpura (ITP), 7 systemic lupus erythematosus (SLE) patients with or without thrombocytopenia, and 10 healthy donors. Although peripheral blood T cells from all subjects failed to respond to the protein complex in its native state, reduced GPIIb-IIIa stimulated T cells from three ITP patients and one SLE patient with thrombocytopenia, and tryptic peptides of GPIIb-IIIa stimulated T cells from nearly all subjects. The specificity of the responses for GPIIb-IIIa was confirmed by activation of GPIIb-IIIa-primed T cells by a recombinant GPIIbalpha fragment in secondary cultures. Characterization of T cell response induced by modified GPIIb-IIIa showed that the response was restricted by HLA-DR, the responding T cells had a CD4(+) phenotype, and the proliferation was accelerated only in ITP patients, suggesting in vivo activation of these T cells. In vitro IgG anti-GPIIb-IIIa synthesis in PBMC cultures was induced by modified GPIIb-IIIa specifically in ITP patients with platelet-associated anti-GPIIb-IIIa antibody. Anti-GPIIb-IIIa antibody produced in supernatants was absorbed by incubation with normal platelets. In summary, CD4(+) and HLA-DR-restricted T cells to GPIIb-IIIa are involved in production of anti-platelet autoantibody in ITP patients and are related to the pathogenic process in chronic ITP.     

Glycoprotein IIb/IIIa inhibition attenuates platelet-activating factor-induced platelet activation by reducing protein kinase C activity

Summary.  Glycoprotein (GP)IIb/IIIa inhibition may abolish activated leukocyte-induced platelet activation, in which leukocyte-released platelet-activating factor (PAF) is a major mediator. The present study thus investigated if and how GPIIb/IIIa inhibitors interfere with PAF-induced platelet activation. Platelet and leukocyte activation were monitored by flow cytometry and immunoblotting. GPIIb/IIIa inhibitors (c7E3, non-peptide SR121566, and MAb RFGP56) attenuated PAF-induced, but not adenosine diphosphate (ADP)- or thrombin receptor activating peptide ( TRAP)-induced platelet P-selectin expression in whole blood. GPIIb/IIIa blockade enhanced ADP- or TRAP-induced leukocyte CD11b expression, but not the response to PAF. GPIIb/IIIa blockade attenuated PAF-induced, but enhanced ADP- or TRAP-induced platelet–leukocyte aggregation. Under the present experimental conditions, thromboxane A₂ receptor antagonism did not significantly influence PAF-induced platelet activation, and GPIIb/IIIa inhibition did not interfere with calcium mobilization/influx in platelets. Protein kinase C (PKC) blockade inhibited PAF-induced platelet P-selectin expression, and PAF-induced PKC activity was reduced by GPIIb/IIIa inhibition. PAF (=1 µ m ) did not induce MEK 1/2 or ERK 1/2 phosphorylation, whilst thrombin induced marked responses, which were enhanced by GPIIb/IIIa blockade. Thus, GPIIb/IIIa inhibition attenuates PAF-induced platelet activation via inhibiting PKC activity. GPIIb/IIIa blockade enhances thrombin-induced platelet MEK 1/2 and ERK 1/2 activation, and augments ADP- and TRAP-induced leukocyte activation by enhancing platelet–leukocyte aggregation.     

Protein C inhibitor and serum amyloid A in immune thrombocytopaenic purpura

In immune thrombocytopaenic purpura (ITP), phagocytic cells prematurely destroy platelets opsonized by anti-platelet auto-antibodies, while residual platelets rescued from these autoimmune attacks are hyperfunctioning. The exact pathobiological basis of this phenomenon is unknown. Protein C inhibitor (PCI), a platelet alpha-granule pro-coagulant molecule, is released on activation of platelets. Serum amyloid A (SAA; an acute phase protein), however, inhibits platelet aggregation and modulates platelet adhesion. We aimed to assess circulating soluble plasma PCI and SAA concentrations in 17 patients with newly diagnosed ITP and ten healthy volunteers. Plasma PCI concentrations tended to be higher in ITP patients, despite absolute thrombocytopaenia, than in normal controls. SAA levels were significantly higher in ITP patients compared with the control group. We conclude that secretion of the alpha-granule PCI content of platelets could result from platelet activation, and that PCI may be the link between platelet microparticles and haemostatically active ITP platelets. Increased concentrations of SAA and PCI may interfere with the disordered and compensatory pro-coagulant mechanisms of ITP.     

Anti-platelet antibody and platelet function]

The effect of anti-platelet antibodies, including murine monoclonal antibodies, autoantibodies and alloantibodies, on platelet function was analyzed. The target antigen of these antiplatelet antibodies, investigated in the present study, was a glycoprotein IIb/IIIa, which is a receptor of fibrinogen and plays an important role in platelet aggregation. Some of these antibodies inhibited agonist-induced platelet aggregation. The target antigen of one murine monoclonal antibodies, designated OP-G2, was a glycoprotein IIb/IIIa and interestingly, this antibody induced platelet aggregation, which required divalent cation and fibrinogen. We compared the epitope of these antibodies by inhibition assay and found the epitope of these antibodies to be very close. The binding of OP-G2 to the platelets required Ca2+. These data suggest that OP-G2 recognizes an epitope at or in very close proximity to the fibrinogen binding site of GPIIb/IIIa, as compared with other antibodies.     

Comparison of platelet immunity in patients with SLE and with ITP

Idiopathic thrombocytopenic purpura (ITP) is characterized by the development of a specific anti-platelet autoantibody immune response mediating the development of thrombocytopenia. Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of a wide variety of autoantibodies. In 15-20% of SLE cases, patients develop thrombocytopenia which appears to be autoimmune in nature (SLE-TP). To better understand the pathogenesis of the thrombocytopenia associated with SLE, we investigated the overlapping platelet and cellular immune features between SLE and ITP. Thirty-one patients with SLE, eight with SLE-TP, and 17 with ITP, were studied and compared to 60 healthy controls. We evaluated platelet-associated IgG, platelet microparticles, reticulated platelets, platelet HLA-DR expression, in vivo cytokine levels, lymphocyte proliferation, and the T lymphocyte anti-platelet immune response in these patients. Patients with SLE-TP and those with ITP had increased platelet-associated IgG, an increased percentage of platelet microparticles, a higher percentage of reticulated platelets and larger platelets, suggesting antibody-mediated platelet destruction and increased platelet production. More than 50% of patients with ITP had increased HLA-DR on their platelet surface whereas subjects with SLE-TP did not. Analysis of serum cytokines demonstrated increased levels of IL-10, IL-15 and TNF-alpha in patients with SLE, but in those with ITP, only increased levels of IL-15 were seen, no increases in any of these cytokines were observed in patients with in SLE-TP. The ability of lymphocytes to proliferate in response to phorbol myristate acetate (PMA) stimulation was increased in SLE-TP, but was normal in both SLE and ITP. Lymphocytes from subjects with ITP displayed an increased ability to proliferate on exposure to platelets, in contrast, those with SLE-TP did not. While the number of subjects evaluated with SLE-TP was small, these data reveal a number of differences in the immunopathogenesis between SLE-TP and ITP.     

Studies of activated GPIIb/IIIa receptors on the luminal surface of adherent platelets. Paradoxical loss of luminal receptors when platelets adhere to high density fibrinogen.

The accessibility of activated GPIIb/IIIa receptors on the luminal surface of platelets adherent to damaged blood vessels or atherosclerotic plaques is likely to play a crucial role in subsequent platelet recruitment. To define better the factors involved in this process, we developed a functional assay to assess the presence of activated, luminal GPIIb/IIIa receptors, based on their ability to bind erythrocytes containing a high density of covalently coupled RGD-containing peptides (thromboerythrocytes). Platelets readily adhered to wells coated with purified type I rat skin collagen and the adherent platelets bound a dense lawn of thromboerythrocytes. With fibrinogen-coated wells, platelet adhesion increased as the fibrinogen-coating concentration increased, reaching a plateau at about 11 micrograms/ml. Thromboerythrocyte binding to the platelets adherent to fibrinogen showed a paradoxical response, increasing at fibrinogen coating concentrations up to approximately 4-6 micrograms/ml and then dramatically decreasing at higher fibrinogen-coating concentrations. Scanning electron microscopy demonstrated that the morphology of platelets adherent to collagen was similar to that of platelets adherent to low density fibrinogen, with extensive filopodia formation and ruffling. In contrast, platelets adherent to high density fibrinogen showed a bland, flattened appearance. Immunogold staining of GPIIb/IIIa receptors demonstrated concentration of the receptors on the filopodia, and depletion of receptors on the flattened portion of the platelets. Thus, there is a paradoxical loss of accessible, activated GPIIb/IIIa receptors on the luminal surface of platelets adherent to high density fibrinogen. Two factors may contribute to this result: engagement of GPIIb/IIIa receptors with fibrinogen on the abluminal surface leading to the loss of luminal receptors, and loss of luminal filopodia that interact with thromboerythrocytes. These data provide insight into the differences in thrombogenicity between surfaces, and may provide a mechanism for purposefully passivating platelet-reactive artificial surfaces.     

Differences in platelet function in patients with acute myeloid leukemia and myelodysplasia compared to equally thrombocytopenic patients with immune thrombocytopenia

Summary. Background: Severe thrombocytopenia is a major risk factor for hemorrhage, but platelet function and bleeding risk at low platelet counts are poorly understood, because of the limitations of platelet function testing at very low platelet counts. Objectives: To examine and compare platelet function in severely thrombocytopenic patients with acute myeloid leukemia (AML) or myelodysplasia (MDS) with that in patients with immune thrombocytopenia (ITP). Methods: Whole blood flow cytometric measurement of platelet activation and platelet reactivity to agonists was correlated with the immature platelet fraction (IPF) and bleeding symptoms. Results: Patients with AML/MDS had smaller platelets, lower IPF and substantially lower platelet surface expression of activated glycoprotein (GP)IIb–IIIa and GPIb, both with and without addition of ex vivo ADP or thrombin receptor‐activating peptide, than patients with ITP. In both ITP and AML/MDS patients, increased platelet surface GPIb on circulating platelets and expression of activated GPIIb–IIIa and GPIb on ex vivo activated platelets correlated with a higher IPF. Whereas platelet reactivity was higher for AML/MDS patients with bleeding than for those with no bleeding, platelet reactivity was lower for ITP patients with bleeding than for those with no bleeding. Conclusions: AML/MDS patients have lower in vivo platelet activation and ex vivo platelet reactivity than patients with ITP. The proportion of newly produced platelets correlates with the expression of platelet surface markers of activation. These differences might contribute to differences in bleeding tendency between AML/MDS and ITP patients. This study is the first to define differences in platelet function between AML/MDS patients and ITP patients with equivalent degrees of thrombocytopenia.     

Response of neonatal platelets to nitric oxide in vitro

OBJECTIVES: Several studies have demonstrated altered platelet function during nitric oxide inhalation (iNO) in adults and neonates. In vitro NO inhibits activation of fibrinogen receptor glycoprotein (GP) IIb/IIIa in a dose-dependent manner. In neonates GPIIb/IIIa response to stimulation is physiologically attenuated during the first days after birth in comparison to adults; the effects of NO on GPIIb/IIIa in neonates, however, are less established. We investigated the response of platelets from neonates, their mothers, and nonpregnant controls to the NO donor SIN-1 in vitro. DESIGN: Umbilical cord and venous (mother, controls) platelet-rich plasma was stimulated in vitro with 10 microM ADP or 0.05 U/ml thrombin in the presence or absence of 10 microM SIN-1. GPIIb/IIIa activation was determined by two-color flow cytometry. SETTING: Delivery department of an university hospital. PATIENTS AND PARTICIPANTS: Ten healthy term neonates, their mothers and nonpregnant controls. MEASUREMENTS AND RESULTS: NO significantly reduced GPIIb/IIIa activation in thrombin- and ADP-stimulated platelets in all groups (p < 0.001). Neonatal platelets were significantly hyporeactive to stimulation (p < 0.05), but the relative response to SIN-1 was similar in all three groups (70 +/- 5 %). CONCLUSIONS: The relative amount of NO-induced inhibition of GPIIb/ IIIa activation in neonates is thus similar to that of adults. However, due to the intrinsic hyporesponsiveness of neonatal platelets and NO-synergistic pharmacodynamic profiles of other drugs (e.g., prostacyclin), possible adverse effects of iNO must be considered.     

Factor H binds to washed human platelets.

BACKGROUND: Factor H regulates the alternative pathway of complement. The protein has three heparin-binding sites, is synthesized primarily in the liver and copurifies from platelets with thrombospondin-1. Factor H mutations at the C-terminus are associated with atypical hemolytic uremic syndrome, a condition in which platelets are consumed. Objectives The aim of this study was to investigate if factor H interacts with platelets. METHODS: Binding of factor H, recombinant C- or N-terminus constructs and a C-terminus mutant to washed (plasma and complement-free) platelets was analyzed by flow cytometry. Binding of factor H and constructs to thrombospondin-1 was measured by surface plasmon resonance. RESULTS: Factor H bound to platelets in a dose-dependent manner. The major binding site was localized to the C-terminus. The interaction was partially blocked by heparin. Inhibition with anti-GPIIb/IIIa, or with fibrinogen, suggested that the platelet GPIIb/IIIa receptor is involved in factor H binding. Factor H binds to thrombospondin-1. Addition of thrombospondin-1 increased factor H binding to platelets. Factor H mutated at the C-terminus also bound to platelets, albeit to a significantly lesser degree. CONCLUSIONS: This study reports a novel property of factor H, i.e. binding to platelets, either directly via the GPIIb/IIIa receptor or indirectly via thrombospondin-1, in the absence of complement. Binding to platelets was mostly mediated by the C-terminal region of factor H and factor H mutated at the C-terminus exhibited reduced binding.     

Autoimmune thrombocytopenia

Idiopathic thrombocytopenic purpura (ITP) is an autoimmune disorder in which platelets coated with mainly antibodies against platelet GPIIb/IIIa and GPIb/IX are destroyed in the spleen. Recent evidence suggests that platelets are also destroyed by cytotoxic T cells. The diagnosis is made by exclusion for other causes of thrombocytopenia. As routine blood counts are becoming more available, many mild cases of ITP (platelets >30 x 10(9) L(-1)) are being diagnosed and they usually do not require treatment. In patients with platelet counts persistently <30 x 10(9) L(-1), treatment with corticosteroids, and/or intravenous immunoglobulin (IVIG) or anti-D may be required. The primary goal of treatment is to maintain the platelet count at a safe level with minimal side effects. After 3-6 months, if spontaneous remission has not occurred and if the side effects are significant, splenectomy is recommended. This is the single most effective treatment of ITP. The refractory patients who fails splenectomy and subsequently first- and second-line therapies, is a management dilemma. Therapeutic options are limited, available treatments potentially toxic and the chances of sustained response low. Observation with no active treatment is a reasonable option. With the increased availability of the thrombopoietic agents in the future, there may be a good prospect of keeping the platelet counts of these refractory patients at a safe long-term level with one of these drugs.     

Detection of various platelet-associated immunoglobulins by flow cytometry in idiopathic thrombocytopenic purpura

BACKGROUND: In 1975, Dixson reported that anti-platelet IgG on platelets from patients with idiopathic thrombocytopenic purpura (ITP) is greater than in normal people, by determining anti-platelet antibodies directly on the platelet surface with a quantitative complement lysis-inhibition-assay. Since then, platelet-associated IgG (PAIgG) has been thought of as evidence of ITP. Although platelets from ITP patients show significantly higher PAIgG values than from normal control individuals, PAIgG is not specific for autoantibody because it increases in other than immune ITP patients. METHODS: We analyzed positive platelet percentage with various platelet-associated immunoglobulins: IgG, IgM, IgA, and total immunoglobulins, in the blood from 17 normal donors and 23 ITP patients. RESULTS: The specificity for ITP disease was better in flow cytometry than in ELISA, because, other than ITP, only aplastic anemia was positive in flow cytometry; however, various disorders (aplastic anemia, chronic lymphocytic leukemia, acute myeloid leukemia, and myelodysplastic syndrome) showed positive in ELISA. Flow cytometry methods had the same sensitivity for ITP disease as ELISA. However, it is supposed that there was no nonimmune ITP in this study because the PAIgG negative patients (n = 1) showed positive results in flow cytometry. CONCLUSION: Flow cytometry method was effective for ITP screening, especially for specificity.