Intrinsically impaired platelet production in some patients with persistent or chronic immune thrombocytopenia

Persistent or chronic immune thrombocytopenias (P/C‐ITP) are acquired blood disorders lasting more than 3 months or 1 year, respectively. The pathogenesis of these disorders is thought to be immunological. We hypothesized that some patients with P/C‐ITP might have an intrinsic megakaryopoiesis defect. We identified a group of P/C‐ITP patients with acquired isolated mild thrombocytopenia (30–100 × 10⁹/l), undetectable anti‐platelet antibodies, negative autoimmune investigations and no need for treatment. We examined in vitro megakaryocyte differentiation and compared these patients' results with those of acute‐ITP patients and healthy controls. No difference in proliferation, ploidy or expression of surface markers was found. In contrast, P/C‐ITP patients had significantly fewer proplatelet‐forming megakaryocytes. This novel observation demonstrated that some patients diagnosed with P/C‐ITP have an intrinsic megakaryopoiesis defect independent of the bone‐marrow environment. Further investigations are needed to dissect mechanisms underlying this impaired proplatelet formation in these patients.     

Pathophysiology and management of chronic immune thrombocytopenia: focusing on what matters

Immune thrombocytopenia (ITP) is a common autoimmune disease characterized by low platelet counts and an increased risk of bleeding. Antibody-mediated platelet destruction has been the prevailing hypothesis to explain ITP pathogenesis, supported by the efficacy of B-cell depletion therapy; however, the recent success of thrombopoietin receptor agonists lends support to the notion that platelet production is also insufficient. Best practice for the management of chronic ITP has not yet been established because data from comparative trials are lacking. Despite renewed interest in novel drugs capable of increasing platelet counts, ultimate treatment goals for ITP patients must be kept in mind: to improve patients' health and well-being. In this article, the pathophysiology of ITP is reviewed and key remaining questions about mechanism are explored. A rational approach to the management of ITP in adults is outlined, acknowledging evidence and evidence gaps, and highlighting the need for clinically important endpoints in future clinical trials.     

Remission and platelet responses with romiplostim in primary immune thrombocytopenia: final results from a phase 2 study

In anecdotal reports, some patients with immune thrombocytopenia (ITP) maintained platelet counts after discontinuing romiplostim. Here, we examined rates of platelet response (≥50 × 10⁹/l), remission , splenectomy and adverse events in patients with primary ITP duration ≤6 months who were treated with romiplostim for ≤12 months. The starting dose of romiplostim was 1 μg/kg; concomitant and rescue treatments were permitted to maintain platelet counts. Patients with platelet counts ≥50 × 10⁹/l at the end of 12 months entered a dose taper in which the romiplostim dose was decreased as long as platelet counts were maintained. Remission (platelet count ≥50 × 10⁹/l for 24 consecutive weeks with no ITP treatments) was evaluated in patients once romiplostim was discontinued. Over the 12 months, a high response rate (>90%) was observed. Platelet response occurred quickly (median, ~2 weeks) and was observed for a cumulative median of 11 months. Remission was observed in 24 patients (32%); there were no significantly predictors of remission. Most (20/24) patients had remission start before the forced taper. No new safety signals were identified. Thus, in patients with early‐stage ITP, romiplostim was well tolerated and induced rapid responses, with remission occurring in approximately one‐third of patients (NCT01143038, Amgen 20080435).     

Investigation of platelet apoptosis in adult patients with chronic immune thrombocytopenia

Objectives: Immune thrombocytopenia (ITP) is an acquired and heterogeneous autoimmune-mediated hematological disease typically characterized by a low platelet count. Emerging evidence over the past several years suggests that platelet biogenesis and ageing are regulated, at least in part, by apoptotic mechanisms. However, the association between decreased platelets and apoptosis in ITP patients is poorly understood. To better understand the role of platelet apoptosis in ITP pathophysiology, we investigated apoptotic markers in platelets acquired from 40 chronic ITP patients. Furthermore, the results of ITP patients were compared to those from 40 healthy individuals.

Methods: Markers of apoptosis, including phosphatidylserine (PS) exposure and mitochondrial inner membrane potentials (ΔΨm), were examined using flow cytometry. The expression of pro-apoptotic molecules such as Bak and Bax and anti-apoptotic molecules such as Bcl-xL were determined using quantitative real-time PCR (qRT-PCR) and Western blotting.

Results: Our study demonstrated that the platelet mitochondrial membrane depolarization in chronic ITP patients tended to be higher than in healthy controls. Additionally, the proportion of platelets with surface-exposed PS in chronic ITP was significantly higher than that of controls. The results showed that the expression levels of Bak and Bax were significantly higher in chronic ITP patients than in healthy controls; Bcl-xL expression levels were significantly decreased in the platelets of chronic ITP patients compared to healthy controls.

Discussion and conclusion: study indicates that the enhancement of platelet apoptosis observed in patients with chronic ITP may be one of the pathogenic mechanisms of chronic ITP.     

Stromal cell-derived factor-1 rs2297630 polymorphism associated with platelet production and treatment response in Chinese patients with chronic immune thrombocytopenia

Stromal cell-derived factor-1 (SDF-1), signaling through CXCR4, is implicated in megakaryopoiesis and platelet production. SDF-1 rs2297630 is a functional polymorphism in linkage disequilibrium with other functional variants in SDF-1. This study aimed to investigate the role of SDF-1 rs2297630 in chronic ITP. The genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism and confirmed by direct sequencing. Immature platelet fraction (IPF) was performed using Sysmex XE-2100. Anti-platelet autoantibodies were assayed by enzyme-linked immunosorbent assay. The main characteristics at diagnosis and the outcome of chronic ITP in 201 Chinese patients were retrospectively reviewed. There was no significant difference in either genotype or allelic distribution between ITP patients and the controls (p = 0.114; p = 0.787). However, both heterozygote (GA) and homozygote minor allele (AA) patients had significantly increased megakaryocyte quantity compared to homozygote genotype (GG) patients at diagnosis (p = 0.011). The mean IPF values of GA and AA genotype patients were higher than those observed in the GG genotype patients when platelet counts ≤50 × 10⁹/L at diagnosis (p = 0.007). Patients with GA and AA genotype showed a higher response rate to standard treatments than patients with GG genotype (p < 0.001). In particular, GA and AA genotype patients had a significantly increased chance of responding to steroids, intravenous immunoglobulin (IVIG), and thrombopoietin analogs (p = 0.007; p = 0.029; p = 0.034, respectively). No significant difference was found between anti-platelet antibodies and genotypes (p = 0.296). In summary, the SDF-1 rs2297630 was associated with platelet production and treatment response in Chinese patients with chronic ITP.     

Development of romiplostim for the treatment of patients with chronic immune thrombocytopenia: from bench to bedside

Immune thrombocytopenia (ITP) is an autoimmune disorder characterised by abnormally low platelet counts (<100 × 10⁹/l), purpura, and bleeding episodes, and can be categorised in three phases: newly‐diagnosed, persistent, and chronic. As many patients become refractory to standard treatments (corticosteroids, danazol, azathioprine, splenectomy), there is an urgent need for alternative treatments. The successful isolation and cloning of thrombopoietin (TPO) in the mid‐1990s and identification of its key role in platelet production was a major breakthrough, rapidly followed by the development of the recombinant thrombopoietins, recombinant human TPO and a pegylated truncated product, PEG‐rHuMGDF. Both agents increased platelet counts but development was halted because of the development of antibodies that cross‐reacted with native TPO, resulting in prolonged treatment‐refractory thrombocytopenia. Experimentation with novel platforms for extending the circulating half‐life of therapeutic peptides by combining them with antibody fragment crystallisable (Fc) constructs led to the development of a new family of molecules termed ‘peptibodies’. The 60Da recombinant peptibody romiplostim was finally produced by linking several copies of an active TPO‐binding peptide sequence to a carrier Fc fragment. In clinical trials, romiplostim was effective in ameliorating thrombocytopenia in patients with chronic ITP, was well tolerated and did not elicit cross‐reacting antibodies. Romiplostim has recently been approved for the treatment of adults with chronic ITP.     

Beyond the platelet count: immature platelet fraction and thromboelastometry correlate with bleeding in patients with immune thrombocytopenia

Platelet counts (PC) estimate bleeding risk in Immune Thrombocytopenia (ITP). We investigated whether measures of thromboelastometry and absolute immature platelet fraction (A‐IPF) would correlate better with acute bleeding score (ABS) than PC or mean platelet volume (MPV). Simultaneous determination of ABS, complete blood count and thromboelastometry was performed in 141 ITP patients; 112 underwent A‐IPF testing. Subgroup analyses were performed for paediatric subjects, PC <60 × 10⁹/l and <30 × 10⁹/l. PC significantly inversely correlated with ABS in all subjects, PC <30 × 10⁹/l and total paediatric cohort. MPV did not correlate with ABS in any subgroup. Thromboelastometry measures of clot firmness, but not PC, significantly correlated with ABS in all subjects with PC <60 × 10⁹/l, and children with PC <60 × 10⁹/l and <30 × 10⁹/l. A‐IPF demonstrated stronger correlation with ABS than did PC among all subjects, those with PC <60 × 10⁹/l, all children and children with PC <30 × 10⁹/l (r = ?0·37; r = ?0·34; r = ?0·44; r = ?0·60) versus ABS with PC (r = ?0·36; ns; r = ?0·32; ns). Stronger correlations of both thromboelastometry measures of clot firmness and A‐IPF than PC with ABS suggest factors beyond PC, i.e. related to platelet function, contribute to ITP bleeding pathophysiology. Thromboelastometry, A‐IPF and ABS can be incorporated into routine or acute visits.     

Markers of refractory primary immune thrombocytopenia

Refractory immune thrombocytopenia (ITP) is a challenging disease that can be defined by refractoriness to second-line treatments. In this review, we list and comment available evidence about clinical and biological factors associated with refractoriness to splenectomy, thrombopoietin receptor agonists (TPO-RAs), rituximab and fostamatinib, as well as those associated with multirefractory ITP (active disease with failure of rituximab, TPO-RAs and splenectomy).     

Detection of platelet associated IgG in immune thrombocytopenia: A new assay employing protein a and peroxidase anti-peroxidase (PROA-PAP)

Immune thrombocytopenia is frequently encountered in medical practice and is generally accepted as being caused by an IgG antibody. The capability of detecting platelet-bound IgG as a diagnostic and therapeutic modality is critical for appropriate care and management of patients with idiopathic thrombocytopenic purpura (ITP), as well as other immune thrombocytopenias We have modified our previous assay (Br J Haematol 37:265, 1977) by employing protein A and PAP as a labeled antibody. Surface bound platelet IgG was quantitated by phase contrast microscopy after incubation with PAP, graded per 100 platelets and expressed as a reactive index (RI). Controls (n=13) had RIs ranging from 0.49 to 0.72 (mean 0.63 ± 0.02 SE). The nonimmune thrombocytopenic group (n=7) had an RI ranging from 0.58 to 0.72 (mean 0.64 ± 0.01 SE). In contrast, the immune thrombocytopenic group (n=28) had RIs ranging from 1.04 to 1.75 (mean 1.43 ± 0.03 SE). Platelet-associated IgG was evaluated further by absorbing representative sera samples from each group against washed granulocytes, red cells and platelets Only when sera from the immune thrombocytopenic group were absorbed against platelets did the reactive indices of pre- and postabsorption samples change significantly. These findings suggest that our assay is clinically applicable in detecting platelet-associated IgG in immune thrombocytopenia and has the advantage of being rapid, reproducible and easy to perform in a clinical laboratory.     

A diagnostic approach that may help to discriminate inherited thrombocytopenia from chronic immune thrombocytopenia in adult patients

Inherited thrombocytopenia (IT) is a heterogeneous group of rare diseases that are often confused with immune thrombocytopenia (ITP). The objective of this study was to supply clinicobiological elements that allow a distinction to be drawn between IT and chronic ITP. We then compared 23 adult patients with IT and 9 patients with chronic ITP. Our study revealed six discriminating criteria: (i) an age of discovery <34 years: positive predictive value (PPV) = 88.2% [63.6; 98.5], (ii) a family history of thrombocytopenia: PPV = 100.0% [82.4; 100.0], (iii) a personal history of bleeding: PPV = 100% [76.8; 100.0], (iv) a mean platelet volume >11 fL: PPV = 93.3% [68.1; 99.8], (v) an excess of giant platelets on blood smear: 100.0% [76.8; 100.0], and (vi) a percentage >44% of platelets with a surface area >4 µm² in electron microscopy: PPV = 83.3% [58.6; 96.4]. If at least three of these criteria were combined, it was possible to distinguish IT from chronic ITP with 91.3% [72.0; 98.9] sensitivity and PPV = 100.0% [66.4; 100.0] specificity. The secondary objective of this study was to assess the prevalence of potential IT diagnosis in patients with chronic thrombocytopenia of uncertain origin. Applying our diagnostic approach to a series of 20 cases allowed us to estimate that 40% of them could be suffering from IT. Finally, our diagnostic approach may help to correctly distinguish IT from chronic ITP, particularly in the context of macrothrombocytopenia.     

Evaluation of the immature platelet fraction in the diagnosis and prognosis of childhood immune thrombocytopenia

Rapid assessment of platelet production would distinguish between thrombocytopenia due to decreased platelet production or increased peripheral platelet destruction. We evaluated the value of immature platelet fraction (IPF) in differentiating immune thrombocytopenia (ITP) from thrombocytopenia secondary to bone marrow failure and its potential use as a prognostic marker. Forty-one young patients with ITP were compared with 14 patients with hematological malignancies under chemotherapy, representing a control group with thrombocytopenia due to bone marrow suppression and 30 age- and sex-matched healthy controls. Patients were studied stressing on bleeding manifestations, organomegaly/lymphadenopathy and therapy. Complete blood count including IPF was performed using Sysmex XE-2100. ITP patients were classified into two subgroups: acute ITP with spontaneous resolution within 3 months from diagnosis and chronic ITP that lasted ≥1 year from diagnosis. Median IPF was 11.8% in patients with ITP, 7% in those with hematological malignancy and 3% in the control group (p?<?0.001). ITP patients had significantly higher mean platelet volume (MPV), platelet distribution width (PDW), platelet large cell ratio (P-LCR) and IPF compared with patients with malignancy or healthy controls, while plateletcrit (PCT) was significantly lower in ITP patients than other groups (p?<?0.001). IPF was increased in patients with chronic ITP compared with acute ITP group (p?<?0.001). Patients with active ITP had the highest IPF followed by those in partial remission, while ITP patients in remission had the lowest IPF. IPF was positively correlated to the number of lines of treatment used, MPV, PDW and P-LCR, while negatively correlated to platelet count and PCT among ITP patients (p?<?0.001). Multiple regression analysis showed that platelet count and P-LCR were independently related to IPF. ROC curve analysis revealed that the cut-off value of IPF at 9.4% could be diagnostic for ITP patients with a sensitivity of 88% and a specificity of 85.7%. We suggest that IPF may be a rapid and inexpensive automated marker for etiology of thrombocytopenia and can be integrated as a standard parameter to evaluate the thrombopoietic state of the bone marrow. It may be considered as a potential prognostic marker for the development of chronic ITP.     

Evaluation of platelet function in thrombocytopenia

Whole blood aggregometry is a functional assay for determination of platelet function. Until now, whole blood aggregometry has not been considered feasible at low platelet counts. Hence, the objectives of the present study were to explore platelet function in thrombocytopenia using a novel index of impedance aggregometry adjusted for platelet count and evaluate the association to platelet function assessed by flow cytometry. Hirudin anticoagulated blood was collected from 20 healthy volunteers, 20 patients with primary immune thrombocytopenia (ITP), and 17 hematological cancer patients. Platelet function was analyzed by impedance aggregometry and by flow cytometry. Collagen, adenosine diphosphate, thrombin receptor agonist peptide-6, and ristocetin were used as agonists for both analyses. Thrombocytopenia in healthy whole blood was induced in vitro employing a recently published method. Platelet aggregation of thrombocytopenic patients was evaluated relative to the aggregation of healthy volunteers at the same platelet count. In flow cytometry, platelet function was described as expression of the platelet surface glycoproteins: bound fibrinogen, CD63, and P-selectin. Similar platelet counts were obtained in the patient groups (p = 0.69) (range: 13–129 × 10⁹/l). Aggregation adjusted for platelet count was significantly increased in ITP patients compared to healthy platelets across all agonists. The platelet aggregation was high in the 95% prediction interval, with 18 ITP patients above the prediction interval in at least two agonists. In contrast, the platelet aggregation was low in the prediction interval in cancer patients, and three cancer patients with platelet aggregation below the prediction interval in at least one agonist. ITP patients displayed increased expression of bound fibrinogen and CD63 following activation, compared with particularly cancer patients, but also compared with healthy platelets. This study demonstrated the feasibility of a novel approach to perform platelet function analyses in thrombocytopenia using impedance aggregometry adjusted for platelet count.     

Impaired proplatelet formation in immune thrombocytopenia: a novel mechanism contributing to decreased platelet count

The pathophysiological mechanisms contributing to the decreased platelet count in immune thrombocytopenia (ITP) are not entirely understood. Here, we investigated the key step of proplatelet formation (PPF) by studying the effect of ITP plasma in thrombopoiesis. Normal cord blood‐derived mature megakaryocytes were cultured in the presence of recalcified plasma from ITP patients, and PPF was evaluated by microscopic analysis. Patient samples induced a dose‐dependent inhibition in PPF, as well as decreased complexity of proplatelet architecture. Although slightly increased, plasma‐induced megakaryocyte apoptosis was not related to PPF impairment. Purified IgG reproduced the inhibitory effect, while platelet‐adsorbed plasma induced its reversion, suggesting the involvement of auto‐antibodies in the inhibition of thrombopoiesis. Impaired PPF, induced by ITP plasmas bearing anti‐GPIIb‐IIIa antibodies, was related to their ability to interfere with the normal function of this integrin, as assessed by megakaryocyte PAC‐1 binding and β3 integrin phosphorylation while the presence of anti‐glycoprotein Ia‐IIa auto‐antibodies was associated with loss of normal inhibition of PPF induced by type I collagen. In conclusion, abnormal thrombopoiesis comprising decreased PPF and morphological changes in proplatelet structure are induced by patient samples, unveiling new mechanisms contributing to decreased platelet count in ITP.     

Mean platelet volume at baseline and immune thrombocytopenia relapse in Chinese newly-diagnosed patients: a retrospective cohort study

ABSTRACT

Objective: In China, the ability of the current immune thrombocytopenia (ITP) guideline to stratify recurrent risk at diagnosis is limited. This study aimed to investigate whether mean platelet volume at diagnosis (MPV) is a risk factor for ITP relapse in Chinese.

Methods: The present study was a retrospective cohort study. Two hundred thirty-three adult patients with newly diagnosed ITP were consecutively and nonselectively collected from March 2013 to June 2017. The exposure and outcome variable were MPV at baseline and relapse-free survival at 6 months. Other covariants included demographic data, general information, variables that can affect MPV reported by previous literature and risk factors of ITP relapse.

Results: After adjusting potential confounders, the non-linear relationship was detected between MPV and ITP relapse, and inflection point was 21. The effect sizes and the confidence intervals on the left and right sides of inflection point were 1.30 (1.22–1.39) and 0.89 (0.76–1.04), respectively. Subgroup analysis showed, in subjects with hyperuricemia (1.54 (1.24, 1.90)), MPV showed significant differences from non-hyperuricemia (1.19 (1.13, 1.25)), and the p for interaction was less than 0.05.

Conclusion: The relationship between MPV and ITP relapse is non-linear. MPV is an independent risk factor of ITP relapse when MPV is less than 21?fl.