Platelet-associated Antibodies Research Articles

Platelet-Associated Antibodies By Flow Cytometry in Adult Patients with Immune Thrombocytopenia before and after Splenectomy

Introduction Immune thrombocytopenic purpura (ITP), or primary immune thrombocytopenia is an autoimmune disease characterized by isolated thrombocytopenia (number of platelets in peripheral blood is less than 100×109/L) and splenic production of antibodies against platelet glycoprotein complexes and megakaryocytes, resulting in hemorrhagic syndrome. Circulating platelets attached by autoantibodies that leads to accelerated removal of these cells by spleen macrophages. The therapy for patients with newly diagnosed ITP with hemorrhagic syndrome and/or severe thrombocytopenia (number of platelets in peripheral blood <10-20×109/L) includes corticosteroids and characterized by a low rate of remission (just around 20-30%). For cases of ITP resistant to corticosteroid therapy recommends one of the second-line method of treatment - splenectomy (SE). Approximately 60-80% of the patients achieve complete remission after splenectomy. There is a technique for assessment of platelet-associated antibodies (PAA) classes IgG, IgM and IgA on platelets by flow cytometry. This method is a commodious, easy, quick, and relatively cheap and applied to estimate autoimmunity status of patients with thrombocytopenia. However, this method characterized by low specificity. Aim The aim of the study was to determine the correlation between level of PAA of IgG, IgM, and IgA classes in the peripheral blood of adults with ITP before SE, 5-7 days and 3 months after SE. Patients and methods The study included 21 patients with ITP (4 cases of persistent ITP and 17 cases with chronic form). Median age was 36.9 years, M:F ratio was 1: 4.25 (men was older than women - 46.0 years old versus 34.7). All patients underwent from 1 to 3 lines of therapy and were recommended for SE due to resistance to treatment. The PAA level was measured at three time points (before SE, 5-7 days, and three months after SE) by flow cytometry (Becton Dickinson FACS Canto II). Goat polyclonal antibodies against human IgG, IgM, IgA labeled with fluorescein isothiocyanate (FITC) (Cedarlane) were used to determine antibodies of various classes. Anti-CD41a labeled with phycoerethrin (Becton Dickinson) was used to determined platelets. PAA level was assessed based on the mean of fluorescence intensity (MFI) of the FITC-channel. Statistic analysis was carried out using GraphPad Prism 6.01. Wilcoxon signed-rank test had been used for pair comparison. The value of 0.05 had been taken as reliable. Results MFI levels of PAA IgA (391 vs 198, p = 0.005) and IgM (275 vs 142, p<0.0001) significantly decreased in patients after SE compared with the initial level (level before SE). Level of MFI PAA IgM also remained reduced (275 vs 138, p=0.0084) three months after SE (Fig. 1). MFI levels of PAA IgG did not change. Conclusions Using of flow cytometry to determinate platelet-associated immunoglobulins for diagnostic of ITP remains controversial. Despite the fact, this test can be recommended for monitoring of PAA from patients with ITP after SE. In addition, the results confirm the fact that most cells producing antiplatelet antibodies seems to be residing in a spleen. Disclosures No relevant conflicts of interest to declare.

Reevaluation of platelet function in chronic immune thrombocytopenia: impacts of platelet size, platelet-associated anti-αIIbβ3 antibodies and thrombopoietin receptor agonists.

Platelet function of immune thrombocytopenia (ITP) has been controversial because of methodological problems associated with low platelet counts. In this study, we evaluated platelet function in 21 patients with chronic ITP (cITP) using the recently developed flow cytometry (FCM)-based platelet aggregation assay (FCA) along with a PAC1/CD62P assay. Since ITP platelets are larger than controls, whole platelets (whole gating method) and size-adjusted platelets (size-adjusted method) were analysed in the PAC1/CD62P via FCM. We found that: (i) aggregation was equivalent [phorbol myristate acetate (PMA) or adenosine diphosphate (ADP)-induced] or enhanced [protease-activated receptor 1-activating peptide (PAR1AP)-induced] in cITP compared with control by FCA; (ii) PAC1 or CD62P was also equivalent or enhanced in cITP in the whole gating method; and (iii) in sharp contrast, the size-adjusted method revealed that ADP-, PAR1AP-, and collagen synthetic liquid reactive peptide (SRP)-induced PAC1 and ADP-induced CD62P were impaired in cITP. These data suggested that an increase in the number of larger-sized platelets may compensate for the impaired platelet function of cITP, leading to non-inferiority of overall platelet function in cITP. Furthermore, we revealed that ADP-induced aggregation was impaired in the patients with thrombopoietin receptor agonists (TPO-RAs) or platelet-associated anti-αIIbβ3 antibodies compared with the control, suggesting that the presence of anti-αIIbβ3 autoantibodies and/or administration of TPO-RAs may have a negative impact on platelet function.

PF693 PRIMARY IMMUNE THROMBOCYTOPENIA (ITP) IN THE ELRDELRY AND YOUNGER ADULTS: REAL‐WORLD COMPARATIVE RETROSPECTIVE STUDY FROM THE ITP REGISTRY OF THE HELLENIC SOCIETY OF HEMATOLOGY

Background:As life expectancy increases, elderly patients (pts) with immune thrombocytopenia (ITP) are increasingly being encountered in everyday practice. However, the features of ITP in older adults have not been fully elucidated thus far.Aims:To describe real world characteristics at diagnosis and evaluate disease outcome in a group of elderly pts (> 65 years) with primary ITP as compared to younger pts (>16 years‐ 65 years), using data from the national database (ITP registry) operated under the auspices of the Hellenic Society of Hematology.Methods:The Greek ITP registry recruits pts (n = 1317, to date) nationally through a network of 21 sites. In the present study we retrospectively analyzed data from pts with primary ITP aged over 16 years, who were diagnosed from 1979 to 2018.Results:The total number of evaluable pts was 843. Pts were divided in 2 groups based on age: Group‐1 consisted of 540 pts aged 16–65 years and Group‐2 of 303 pts aged > 65 years. The mean age at diagnosis was 36 years (16.1–64.7) in Group‐1 and 76 years (65.1–99) in Group‐2. The female to male ratio in Group‐1 was 1.9 and in Group‐2 1.1 (P = 0.0012). The median platelet count at diagnosis was significantly higher in Group‐1 (16x109/L, interquartile range: 8–35.7x109/L) than in Group‐2 (12x109/L, interquartile range: 5–29x109/L; P = 0.0014). As expected, significant fewer pts in Group‐1 had co‐morbidities as compared to Group‐2 (54.3% and 87.1%, respectively; P < 0.0001). Concurrently used medications, including vitamin K antagonists, platelet antagonists, other anticoagulants or non‐steroidal anti‐inflammatory drugs were more frequently reported in Group‐1 than in Group‐2 pts (P < 0.0001). Bleeding manifestations at diagnosis were observed at a similar frequency between the two groups, with the exception of menometrorrhagia which was significantly more frequent in Group‐1 that in Group‐2 pts, as expected (P < 0.0001). Bone marrow examination was performed more frequently in Group‐1 pts, whereas antiphospholipid antibody, platelet associated antibody, antinuclear antibody, HIV and hepatitis C testing were more frequently performed in Group‐2 pts. Treatment was given in 88% of Group‐1 and in 91% of Group‐2 patients at diagnosis. Overall response rate did not differ between the two Groups. A similar proportion of Group‐1 and Group‐2 patients were treated with corticosteroids, intravenous IgG or both, rituximab, anti‐D immunoglobulin or thrombopoietin receptor agonists, whereas significant more Group‐1 pts underwent splenectomy (P < 0.0001). Follow‐up of at least 1 year from diagnosis revealed that a similar proportion of Group‐1 and Group‐2 patients had developed chronic ITP. During follow‐up, 2 Group‐1 pts and 11 Group‐2 pts died (1.4% and 12%, respectively; P = 0.0003). Three deaths in Group‐2 were considered to be related to ITP.Summary/Conclusion:Elderly pts (Group‐1) presented with lower platelet counts at diagnosis had a higher frequency of comorbidities and used more often anti‐platetet and anticoagulant agents. However, neither the frequency nor the location of bleeding differed between Group‐1 (younger pts) and Group‐2 pts. Age influenced the choice of diagnostic procedures but not that of treatment, with the exception of splenectomy which was performed at significantly lower rate in Goup‐2 pts. Neither the treatment response nor the frequency of chronic ITP differed between Group‐1 and Group‐2 pts. 27% of deaths in Group‐2 pts were ITP‐related, whereas none in Group‐1 pts, thereby underscoring the need for age‐adapted management in elderly pts with ITP.

The spleen dictates platelet destruction, anti-platelet antibody production, and lymphocyte distribution patterns in a murine model of immune thrombocytopenia.

For many years, splenectomy has been used to treat immune thrombocytopenia (ITP), and this procedure benefits approximately two-thirds of the treated patients. Although splenectomy may raise platelet counts, antibody-coated platelets and cytotoxic T lymphocytes appear to persist or can change over time. To better understand how the spleen may affect anti-platelet immune responses, we used a murine model of ITP demonstrating both antibody-mediated and T lymphocyte-mediated thrombocytopenia. Mice with severe combined immunodeficiency (SCID) were either splenectomized or not and transfused with splenocytes from CD61 (GPIIIa) knockout mice immunized against CD61(+) platelets. Platelet counts and anti-platelet antibody levels were performed weekly. After 4weeks, the mice were sacrificed, and lymphoid organs were harvested and examined by flow cytometry to quantify CD4(+)CD25(+)FoxP3(+) Tregs and conventional cross-presenting XCR1(+) and tolerizing SIRPα+ dendritic cells. The results indicate that compared with control non-splenectomized mice, thrombocytopenia was improved and anti-platelet antibody production was significantly diminished in all splenectomized mice that received immune splenocytes. Splenectomized SCID mice also had a marked reduction in Tregs in the thymus together with an increased proportion of both thymic dendritic cell subsets that correlated with increased platelet counts. Of interest, although splenectomy diminished anti-platelet antibody production and raised platelet counts, marrow megakaryocyte densities were still significantly reduced in mice that received immune splenocytes. These results suggest that the spleen in murine ITP not only is the primary site responsible for platelet destruction, but it also controls, to a significant extent, the antibody response against platelets and the migration patterns of lymphocyte subsets.

Absence of response to rituximab in patients with refractory primary immune thrombocytopenia

To explore the possible causes for an absence of response to rituximab (RTX) in patients with refractory immune thrombocytopenia (ITP). A total of 20 refractory ITP patients on RTX therapy and 10 health volunteers were recruited from 2008 to 2013. Memory T lymphocytes (Tm) (CD4(+)CD45RO(+)) and B lymphocytes (CD19(+)CD20(+)) in peripheral blood (PB) were examined by flow cytometry (FCM). And enzyme-linked immunosorbent assay (ELISA) was employed for detecting the platelet-associated antibodies IgG/IgM (PAIgG/IgM). (1) 12 patients with refractory ITP achieved a good response after therapy with RTX (effective RTX group) and 8 cases failed to respond (ineffective RTX group). The PB levels of CD4(+)CD45RO(+)Tm were 41.33% ± 9.62% and 45.36% ± 8.57% respectively at pre-therapy and post-therapy in ineffective RTX group. And significant differences existed between ineffective and effective RTX groups (22.36% ± 11.57%, 26.65% ± 6.32%) versus health volunteers (19.72% ± 7.35%, 20.64% ± 7.35%) (all P < 0.05) . No statistical differences existed in the PB level of CD4(+)CD45RO(+)Tm between pre-therapy and post-therapy in ineffective or effective RTX group (both P > 0.05); (2) The PB level of CD19(+)CD20(+)B lymphocytes in RTX ineffective group was lower than that at pre-therapy in RTX effective group (16.36% (6.17%-27.53%) vs 45.72% (35.80%-57.19%), P < 0.05) . No significant difference existed in the level of CD19(+)CD20(+)B at post-therapy between ineffective and effective RTX groups (6.67% (4.09%-19.17%) vs 5.19% (3.78%-17.39%), P > 0.05). And significant differences existed in the PB level of CD19(+)CD20(+)B between pre-therapy and post-therapy in ineffective or effective RTX group (both P > 0.05); (3) PAIgG/IgM was not detected in RTX ineffective group. And PAIgG/IgM was found in 11 cases at pre-therapy versus 1 case at post-therapy in RTX effective group. CD4(+)CD45RO(+)Tm is probably associated with an absence of response to RTX in patients with refractory ITP.

Effects of low concentrations of benzene exposure on levels of platelet-associated antibodies and platelet parameters.

To evaluate the impact of exposure to low concentrations of benzene on the platelet-associated antibodies and platelet parameters. We carried out an analysis on 121 benzene-exposed workers and 110 healthy workers whose blood samples were collected and the levels of platelet-associated antibodies and platelet parameters were assessed. Benzene emissions were monitored over 5 years. Large-platelet cell ratios (P-LCR), platelet distribution width (PDW), and mean platelet volume (MPV) were significantly higher in benzene-exposed participants than in control participants. In participants who smoke cigarettes or drank alcohol, P-LCR, PDW, and MPV were more significantly elevated in the benzene-exposed group than in nonsmokers and nondrinkers. Platelet-associated immunoglobulin (PAIg) levels in benzene-exposed participants were higher than those in the control group, and PAIgA and PAIgM levels correlated with cumulative benzene exposure. Exposure to low concentrations of benzene can induce changes in PAIg levels and platelet parameters.

Platelet Antibodies, Activated Platelets and Serum Leptin in Childhood Immune Thrombocytopenic Purpura

Background/Aim: The aim of this study was to evaluate the levels of platelet-associated antibodies (PAIgG and PAIgM), activated platelets and serum leptin in children with acute immune thrombocytopenic purpura (ITP). Methods: The study included 40 patients with ITP and 40 healthy age- and sex-matched controls. PAIgG, PAIgM and activated platelet levels were estimated by flow cytometry, and serum leptin levels were estimated by ELISA. Results: Activated platelets and serum leptin were significantly higher in the ITP patients than in the controls. The percentage and mean florescence intensity of PAIgG and PAIgM staining were significantly higher in the patients than in the controls. Serum leptin and activated platelet levels in patients with thrombocytopenia of brief duration were significantly lower than those in patients with thrombocytopenia of prolonged duration. The levels of activated platelets, serum leptin and PAIgG were positively correlated, and the levels of serum leptin, activated platelets and platelet counts were negatively correlated. Conclusion: The increased levels of activated platelets, serum leptin and platelet-associated antibodies in children with acute ITP suggest that these factors could play a role in ITP pathogenesis. Additionally, activated platelets and serum leptin could have prognostic significance in paediatric acute ITP.

Diagnosis of von Willebrand disease in Argentina: a single institution experience

Background: von Willebrand disease (VWD) is the most common autosomal bleeding disorder, mostly inherited as dominant trait. VWD is due to deficiency/abnormality of von Willebrand factor (VWF). The true prevalence of VWD is unknown, but estimated as 0.1% to 1% of the general population. Methods: The bleeding score (BS) was used to evaluate the bleeding status of patients presenting at our institution. Laboratory analyses include: VWF:Ag, VWF:RCo, VWF:CB, multimeric pattern, VWF propeptide, Desmopressin challenge test. Genotypic analysis comprises the study of specific exons of VWF, depending on the suspected variant. Results: We describe the main characteristics of our VWD population, including phenotypic and genotypic methods used to achieve the diagnosis. From our 2,482 patients registered, quantitative variants account for 83.2% of cases, and qualitative, 16.8% of cases. Platelet-type VWD (PT-VWD) was diagnosed in two patients and acquired von Willebrand syndrome (AVWS) in six. From quantitative variants, relative frequencies are: possible type 1, 78.7%; type 1, 14.9%; type 1S, 1.5%; type 1C, 0.4%; type 3, 1.6%. From qualitative variants: 2M, 21.9%; 2B, 10.3%; 2A, 8.4%; 2N, 3.4%; patients currently without complete diagnosis: 56%. Family history of bleeding: 64.7% of cases. Type 3, 2A and 2B VWD showed the highest major bleeding (MB) frequency. Genotypic diagnosis was reached in eight type 1C, 72 type 2M, 38 type 2B, 30 type 2A, 14 type 2N, and two PT-VWD patients. Five de novo mutations were identified among 52 families (9.61%): two mutations were associated with type 2B VWD (p.V1316M and p.S1310F), one mutation associated with type 1C (Vicenza) (p.R1205H) described in combination with p.R924Q, one deletion associated to type 1 VWD according to the ISTH VWF database (p.Pro1648fs*45) in combination with p.R1426C resulting in a 2M phenotype and one mutation related to type 2A VWD (p.Y1542D). Six patients are under prophylactic treatment due to their severe bleeding: four are type 3 VWD, one type 2M VWD, and one, the patient with p.Pro1648fs*45 and p.R1426C. Conclusions: The wide heterogeneity of clinical symptoms and laboratory data from possible type 1 VWD implies a risk for bleeding that becomes crucial in challenging situations. In our population, type 2M was found to be more frequent than the type 2A variant. DDAVP (1-deamino-8-D-arginine vasopressin) challenge test, VWF propeptide/antigen ratio and genotypic studies are useful tools for discriminating cases representing difficult diagnoses such as type 2M versus type 1C VWD. Elevated propeptide ratio, the detection of antiphospholipid antibodies, platelet-associated antibodies or anti-VWF immunoglobulins and negative genotypic screening is useful to discriminate inherited VWD from AVWS. Genotypic testing assisted discrimination between types 2B versus platelet-type-VWD and type 2N VWD versus mild hemophilia A.

Platelet‐associated antibodies, cellular immunity and FCGR3a genotype influence the response to rituximab in immune thrombocytopenia

Rituximab is widely used in autoimmune diseases including immune thrombocytopenia (ITP), although the mechanism of effect remains unclear. This study describes the effects of rituximab on platelet-associated antibodies (PA-APAs), B and T cell counts and clonality ( IGHV and TRG@ gene rearrangements), FCGR3A (FcγRIIIa) and FCGR2A (FcγRIIa) polymorphisms and correlation to anti-CD40 ligand (CD40L) response. PA-APA levels fell more frequently in responders (6/8) than in non-responders (2/10: P = 0·08-0·15). Two responders had no PA-APAs. Two non-responders with a fall in PA-APAs had very high CD8 levels. One non-responder had a B cell clone, one responder and one non-responder had a T cell clone. 15/16 patients had the same responses to rituximab and antiCD40L. Patients with FCGR3A V/V polymorphisms were more likely to respond to rituximab (P = 0·03). In summary, the fall in PA-APAs in responders confirms the humoural effect of rituximab. Failure to respond in patients with very high CD8 levels, despite PA-APA fall indicates a role for T cell-mediated platelet/megakaryocyte destruction. Concordance of response to anti-CD40L suggests autoantibody-producing cells are under T cell control. Finally, the effect of FCGR polymorphisms on response confirms the importance of FCGR-mediated depletion of B cells in autoimmunity. This has implications on the pathology of ITP as well as the immunological effect of B cell depletion.

Discussion on the relationship between platelet-associated antibody and effect of platelet transfusion

Objective To investigate effect of platelet-associated antibody to the platelet transfusion.Methods Platelet-associated antibody in 120 patients who needed repeated platelet transfusion for long-time was determined by enzyme-linked immunosorbent assay (ELISA),and platelet count was assayed before and after blood transfusion,and the effect of platelet transfusion was investigated by corrected platelet count.Crossmatch approcah was used to screen compatible platelet for patients with platelet relative antibody positive.Results The ratio of positive platelet-aasociated antibody in patients who needed repeated platelet transfusion was 65.0％,and the ratio of platelet transfusion resistance was 61.7％.The ratios of platelet transfusion resistance were 84.6％ and 19.0％ respectively in patients with positive platelet-associated antibody and in patients with negative platelet-associated antibody,which were different in statistic in both groups( x2 =,46.914,P ＜0.0l ).The effective ratio of platelet transfusion in patients with positive platelet-associated antibody after crossmatch approach was 85.9％.Conclusion Positive platelet-associated antibody was related to platelet transfusion resistance with high risk,and crossmatch approach could increase the effect of platelet transfusion. Key words: Platelet-associated antibody; Platelet transfusion; Enzyme-linked immunosorbent assay

Critical role of CD4+CD25+ regulatory T cells in preventing murine autoantibody-mediated thrombocytopenia

Autoimmune response suppression by regulatory T cells (Tregs) helps to maintain peripheral immune tolerance, and defects in this mechanism are thought to play a role in the pathogenesis of various autoimmune diseases. In patients with immune thrombocytopenia, naturally occurring CD4(+)CD25(+) Tregs are both functionally impaired and reduced in number. Thisstudy was undertaken to investigate Tregs' role in preventing immune thrombocytopenia in mice. Treg-deficient mice were prepared by inoculation of Treg-depleted CD4(+)CD25(-) Tcells isolated from BALB/c mice into syngeneic nude mice intravenously. Platelet count, proportion of reticulated platelets, platelet-associated IgG, platelet-associated anti-platelet antibodies, and IgG anti-platelet antibody production in splenocyte cultures were examined by flow cytometry. Of 69 Treg-deficient mice, 25 (36%) spontaneously developed thrombocytopenia that lasted at least 5 weeks. The platelet-associated IgG level and proportion of reticulated platelets were elevated in the thrombocytopenic mice. Platelet eluates and splenocyte culture supernatants prepared from thrombocytopenic mice, but not from nonthrombocytopenic mice, contained IgG antibodies capable of binding to intact platelets. Simultaneous transfer of Tregs completely prevented the onset of thrombocytopenia, but Treg transfer after the onset of thrombocytopenia had no apparent effect. Treatment with IgG anti-cytotoxic Tlymphocyte-associated antigen 4 antibody canceled this Treg-governed suppressive effect.In summary, these results indicate that Tregs play a critical role in preventing murine autoantibody-mediated thrombocytopenia by engaging cytotoxic T lymphocyte-associated antigen4.

Novel clearance mechanisms of platelets

Blood platelets are involved in primary and secondary hemostasis and thus maintain the integrity of the vasculature. They circulate with an average lifespan of 5-9 days in humans. Thus, the body must generate and clear platelets daily to maintain normal physiological blood platelet counts. Known platelet clearance mechanisms include antibody-mediated clearance by spleen macrophages, as in immune thrombocytopenia, and platelet consumption due to massive blood loss. New concepts in the clearance mechanisms of platelets have recently emerged. New evidence shows that platelets desialyted due to chilling or sepsis are cleared in the liver by macrophages, that is Kupffer cells, as well as hepatocytes, through lectin-mediated recognition of platelet glycans. On the contrary, platelet-associated antibodies normalize the clearance of platelets in a mouse model for Wiskott-Aldrich syndrome. The goal of this review is to summarize the latest findings in platelet clearance mechanisms with a focus on lectin-mediated recognition of platelet glycans. Transfusion medicine and treatments of hematopoietic disorders associated with severe thrombocytopenia may benefit from a better understanding of these mechanisms.

Platelet production and destruction in liver cirrhosis

Thrombocytopenia is common in liver cirrhosis (LC) but the mechanisms are not fully understood. The purpose of our work was to evaluate platelet kinetics in LC with different etiologies by examining platelet production and destruction. Ninety-one consecutive LC patients (36 HCV, 49 alcoholics, 15 HBV) were enrolled. As controls, 25 subjects with idiopathic thrombocytopenic purpura, 10 subjects with aplastic anemia, and 40 healthy blood donors were studied. Plasma thrombopoietin (TPO) was measured by ELISA. Reticulated platelets (RP) were determined using the Thiazole Orange method. Plasma glycocalicin (GC) was measured using monoclonal antibodies. Platelet associated and serum antiplatelet antibodies were detected by flow cytometry. B-cell monoclonality in PBMC was assessed by immunoglobulin fingerprinting. Serum TPO was significantly lower in LC (29.9±18.1 pg/ml) compared to controls (82.3±47.6 pg/ml). The GC levels were higher in LC (any etiology) than in healthy cases. Conversely, the absolute levels of RP were lower in LC (any etiology) than in healthy controls. The platelet-associated and serum anti-platelet antibodies were higher in HCV+ LC compared to healthy subjects (p<0.0064), alcoholic LC (p<0.018), and HBV+ LC (p<0.0001). B-cell monoclonality was found in 27% of the HCV+LC, while it was not found in HBV+ or alcoholic LC. Patients with LC present decreased plasma TPO, accelerated platelet turnover, and reduced platelet production. This indicates that LC thrombocytopenia is a multifactorial condition involving both increased platelet clearance and impaired thrombopoiesis.

Specific cross-reaction of anti-dsDNA antibody with platelet integrin GPIIIa49-66

Anti-platelet autoantibodies are frequently found in systemic lupus erythematosus (SLE) patients and contribute to the development of SLE-associated immunologic thrombocytopenia (SLE-ITP). Although the correlation of anti-dsDNA autoantibody with platelet-associated antibody has been reported, the potential mechanism underlying such a correlation is incompletely understood. We have reported that anti-platelet integrin GPIIIa49-66 (CAPESIEFPVSEARVLED) autoantibodies play a major role in the development of HIV-1-related thrombocytopenia (HIV-1-ITP). The strong negative charge of GPIIIa49-66 prompts us to investigate whether GPIIIa49-66 can be an epitope mimicking dsDNA. We report here that anti-GPIIIa49-66 antibodies are found in three out of nine SLE-ITP patients. Double-stranded (ds) DNA competitively inhibited the binding of purified patient anti-dsDNA antibodies to GPIIIa49-66 peptide. Both polyclonal and monoclonal anti-GPIIIa49-66 antibodies are able to cross-react with dsDNA. Consistent with previous reports, the DNA binding activities of anti-GPIIIa49-66 antibodies are mainly dependent on the positively charged amino acid in the heavy-chain complementarity-determining region 3 (HCDR3). The HCDR3 of human SLE anti-dsDNA monoclonal antibody (mAb) 412.67 demonstrates a similar positively charged amino acid chain orientation compared with that of anti-GPIIIa49-66 mAb A11, and it cross-reacts with GPIIIa49-66 peptide. Purified anti-GPIIIa49-66 antibodies from SLE-ITP patients are able to induce platelet fragmentation in vitro and to induce thrombocytopenia in vivo. Thus, our data suggest that specific epitope cross-reaction between GPIIIa49-66 and dsDNA could be a mechanism involved in the development of SLE-associated thrombocytopenia.

Antiplatelet antibodies in WASP(−) mice correlate with evidence of increased in vivo platelet consumption

To study the role of antiplatelet antibodies in the thrombocytopenia of murine Wiskott-Aldrich syndrome (WAS). A flow cytometric method was developed for detection of serum antiplatelet antibodies via their binding to intact target platelets lacking surface antibodies. Platelets were labeled with 5-chloromethylfluorescein diacetate (CMFDA) in order to track their clearance from the circulation. WASP(-)muMT(-/-) mice were generated by standard breeding methods. Serum antiplatelet antibodies were detected in approximately 40% of WASP(-) males. The mean level of reticulated platelets is significantly increased in these antibody(+) males. While WASP(-) males show an approximately 50% reduction in platelet counts, 5% to 10% show a more severe thrombocytopenia associated with increased reticulated platelets, suggesting the presence of clearance-inducing antiplatelet antibodies. In support of that inference, 90% of the latter mice show detectable serum antiplatelet antibodies. The antibodies are primarily immunoglobulin G, and are also detected in >30% of CD47(-/-) males. WASP(-)muMT(-/-) males, which demonstrate no serum- or platelet-associated antibodies, show a degree of thrombocytopenia similar to that of WASP(-) males. Their platelet clearance rates remain accelerated--more so in WASP(-)muMT(-/-) than WASP(+)muMT(-/-) recipients. These findings suggest that platelet WASP deficiency results in an increase in platelet clearance rates by two mechanisms: an antibody-independent mechanism that largely requires WASP deficiency in trans, and an antibody-dependent mechanism that does not. Both an increased incidence of antiplatelet antibodies and an increased susceptibility to their effects contribute to antibody-dependent clearance of WASP(-) platelets.

Mice Lacking Wasp-Interacting Protein Evolve Anti-Platelet IgAs That Impair Platelet Responses Mediated by the Collagen Receptor GPVI.

Wiskott-Aldrich Syndrome (WAS) is a hematopoietic disorder characterized by immune deficiency, eczema and severe thrombocytopenia with small platelets. However, the role of the WAS protein (WASp), mutated or absent in WAS, is unclear in platelets because platelets that lack WASp function normally. WASp constitutively associates with WIP in resting and activated platelets. We investigated the role of the platelet WIP-WASp complex by using mice that lack either WIP or WASp. WIP knockout (KO) platelets lack WASp and WIP expression is reduced in WASp KO platelets, indicating a requirement for both proteins for each other's integrity. WIP KO, but not WASp KO mice evolve platelet-associated antibodies of the IgA class, as evidenced by immunoblot analysis and flow cytometry. We present evidence that platelet-associated IgAs specifically target the collagen receptor GPVI in WIP KO mice:protein tyrosine phosphorylation, including that of phospholipase C-γ2, and calcium mobilization are impaired in IgA-presenting WIP KO platelets stimulated through GPVI, resulting in α-granule secretion, integrin αIIbβ3 activation and actin assembly defects;binding of the anti-GPVI antibody JAQ1 to normally expressed GPVI is reduced in IgA-presenting WIP KO platelets;JAQ1 induces the irreversible loss of GPVI from circulating platelets in WT mice, but not in WIP KO mice that bear anti-platelet IgAs.Together, the data indicate that platelet-associated IgAs negatively modulate GPVI signaling in WIP KO mice.

Autoimmune thrombocytopenia: Flow cytometric determination of platelet-associated CD154/CD40L and CD40 on peripheral blood T and B lymphocytes

Background and objectives: The CD40–CD40L system has pleiotropic effects in a variety of cells and biological processes including the immune response. Within the immune system, these molecules represent a critical link between its humoral and cellular arms. Immune or idiopathic thrombocytopenic purpura (ITP) is an autoimmune disorder characterized by antibody-induced platelet destruction and clearance due to anti-platelet autoantibodies, which bind to circulating platelets resulting in their destruction by the reticuloendothelial system. Despite its clinical importance, the diagnosis of ITP is one of exclusion, thus, inevitably associated with potential difficulties. CD40 is a cell surface receptor that belongs to the tumor necrosis factorreceptor (TNF-R) family, and that was first identified and functionally characterized on B lymphocytes. CD40-ligand (CD40L/CD154), a member of the TNF superfamily, is a cell membrane molecule expressed on activated CD4 + T lymphocytes and is essential for the T cell-dependent activation of B lymphocytes. Therefore it is now thought that CD40–CD40L interactions play a more important role in ITP immune regulation.Design and methods: The expressions of CD154 and CD40 on peripheral blood (PB) T and B lymphocytes, respectively, were measured using flow cytometry (FCM). An antigen-specific assay for platelet-associated antibody CD154 (CD40L) on CD4 + T lymphocytes and for CD40 on CD19 + B lymphocytes was tested in 30 pediatric patients with acute ITP, 30 adult patients with chronic ITP, and in 20 age- and sex-matched healthy controls.Results: The expression of CD4 + CD154 + and CD4 + CD154 + /CD4 + on PB T lymphocytes, and CD19 + CD40 + and CD19 + CD40 + /CD19 + on PB B lymphocytes were significantly higher in acute and chronic ITP patients compared to controls, and in acute patients compared to chronics (p < 0.001).Conclusions: CD40–CD40L interaction plays an important role in the pathology of certain autoimmune diseases. ITP is an autoimmune disease characterized by increased platelet destruction caused by anti-platelet autoantibodies, which mainly target a platelet surface antigen. It is speculated that platelet-associated CD154 is competent to induce the CD40-dependent proliferation of B lymphocytes. Therefore, platelet-associated CD154 expression is increased in ITP patients and is able to drive the activation of autoreactive B lymphocytes in this disease. 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. Blockade of the CD40/CD154 signal is a potential immunomodulatory strategy for T cell-mediated diseases, and many findings suggest that CD40/CD154 blockade therapy is potentially effective for ITP through selective suppression of autoreactive Tand B lymphocytes to platelet antigens.

The association between platelet autoantibody specificity and response to intravenous immunoglobulin G in the treatment of patients with immune thrombocytopenia

We retrospectively investigated the association between platelet autoantibody specificity and response to intravenous immunoglobulin G (IVIG) in 17 patients with immune thrombocytopenia (ITP). Platelet-associated antibodies against glycoprotein (GP) IIb/IIIa, GPIb/IX, and GPIa/IIa were detected in 13, 10, and 8 patients, respectively. A response occurred in 7 of 7 patients without anti-GPIb/IX, but in only 3 of 10 patients with anti-GPIb/IX (p<0.01). There was no difference in the response rates in patients with or without anti-GPIIb/IIIa or anti-GPIa/IIa. We conclude that ITP patients with anti-GPIb/IX may be less responsive to IVIG.

Mycophenolate mofetil as a treatment for refractory idiopathic thrombocytopenic purpura

To determine whether mycophenolate mofetil (MMF) has beneficial effects on refractory idiopathic thrombocytopenic purpura (ITP) and the corresponding cellular mechanism. Twenty refractory ITP patients resistant to corticosteroid and/or splenectomy and chemical therapy were given MMF 1.5-2.0 g/d orally for a 2 to 4-month period. Serum immunoglobulin was detected by rate nephelometry. Platelet-associated antibodies (PAIgG) were assayed by enzyme-linked immunosorbant assay. The immunophenotypic analysis was performed on a flow cytometer and cell apoptosis was detected with transferase mediated dUTP biotin nick end labeling (TUNEL) method. Sixteen of the 20 (80%) patients had responses to MMF treatment; 9 (45%) achieved a complete response, 4 (20%) achieved a partial response, and 3 (15%) achieved a minor response. The therapeutic effects were found to be better in male patients than female patients. The number of CD3+ peripheral blood cells (PBCs) and CD4+ PBCs increased and the number of CD8+ PBCs decreased. The plasma level of IgG, IgM, IgA and platelet associated IgG (PAIgG) decreased in 86% of the patients. TUNEL assay showed that mycophenolate acid (MPA) 0.1 mmol/L induced apoptosis of peripheral blood mononuclear cells isolated from refractory ITP patients. The apoptosis rate was increased in male patients after treatment with MPA, but was unchanged in female patients. Therapy for a period of 8 to 16 weeks with median-dose of MMF was valuable for the treatment of refractory ITP.

Prognostic Factors of Response to Laparoscopic Splenectomy in Patients with Idiopathic Thrombocytopenic Purpura

Laparoscopic splenectomy (LS) has become the treatment of choice for patients with idiopathic thrombocytopenic purpura (ITP) who do not respond to medical treatment. The aim of this study was to identify factors predictive of outcome after LS for ITP. From May 1997 to December 2002, we performed 30 LS on patients with ITP. A positive response was defined as a postoperative platelet count greater than 50,000/µL and no requirement for maintenance therapy. Chi-square testing was performed to determine the predictive effects of the following variables: age, sex, preoperative response to steroids or immunoglobulin, duration of disease, antiplatelet antibody, platelet associated antibody, and antinuclear antibody. LS was successfully performed in all patients. For a mean follow-up interval of 24.3 months, response to LS was 73.3%. Splenectomy for steroid nonresponders resulted in an inferior complete response rate (10 of 18, 55.6%) as compared with those that experienced relapse after steroid treatment (11 of 12, 91.7%) (p=0.042). The other significant predictor of outcome by univariate analysis was the time between diagnosis and surgery (p=0.049). The other variables showed no significant correlation with successful splenectomy. We conclude that LS can be performed safely with a satisfactory remission rate in patients with ITP who do not respond to medical treatment, and that the factors most frequently associated with surgical success are a response to steroid and disease duration.