scholarly journals A Novel Allogeneic Cell Therapy That Targets Pathogenic Autoantibodies for the Treatment of Immune Thrombocytopenia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1001-1001
Author(s):  
Sunita Patel-Hett ◽  
Silvia Giannini ◽  
Christian Peters ◽  
Brad Dykstra ◽  
Marcus Lehmann ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Immune Thrombocytopenia ◽  
Enzyme Linked Immunosorbent Assay ◽  
Particle Sizes ◽  
Vitro Assay ◽  
Platelet Surface ◽  
Platelet Production ◽  
Platelet Counts ◽  
Nsg Mice ◽  
Increased Risk

Abstract Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by low platelet counts (<100x10 9/L) and increased risk of bleeding. ITP is predominantly driven by immunoglobulin G (IgG)-autoantibodies directed against platelet surface antigens resulting in platelet destruction by the spleen and/or impaired platelet production by bone marrow megakaryocytes. Approximately 60% of ITP patients have measurable levels of anti-platelet autoantibodies in plasma and about 70% of them have autoantibodies directed against the fibrinogen receptor glycoprotein (GP) IIbIIIa. Current treatment strategies for ITP include non-specific immunosuppression (steroids, rituximab), inhibition of platelet clearance (immunoglobulins, splenectomy, anti-D immunoglobulin, and the Syk inhibitor fostamatinib) and stimulation of platelet production (thrombopoietin receptor agonists). In addition, therapeutics targeting the neonatal Fc receptor, responsible for IgG recycling, are under clinical investigation for ITP. Despite these treatment options, some patients with ITP are refractory or have inadequate responses to existing therapy. Here we describe a novel allogeneic cellular therapy, entitled platelet-like cells (PLC), that specifically targets pathogenic IgG for the treatment of thrombocytopenia in ITP. PLC are produced by differentiating a human induced pluripotent stem cell (hiPSC) line into megakaryocyte-like cells (MLC) which are further processed in a proprietary bioreactor that mimics the bone marrow environment and induces the release of anucleate PLC which are then cryopreserved. Analyses of PLC demonstrate sizes ranging between 65 nm and 10 μm. Expression of GPIIbIIIa protein as measured by enzyme-linked immunosorbent assay (ELISA) is evident in all particle sizes. PLC were administered intravenously (IV) into NOD-scid IL2Rgamma null (NSG) mice to evaluate their pharmacokinetics and biodistribution. PLC were rapidly cleared from the mouse circulation and predominantly distributed to the mouse liver where they colocalized primarily with Kupffer cells. PLC clearance through the liver was significantly inhibited by pre-treating mice with liposomes expressing phosphatidylserine (PS), indicating that one mechanism of PLC clearance is dependent on PS exposure on the PLC outer plasma membrane. The ability of PLC to bind anti-GPIIbIIIa autoantibodies was evaluated in an in vitro assay where PLC were incubated with ITP patient plasmas and subsequently removed by centrifugation and filtration. The concentration of anti-GPIIbIIIa autoantibodies remaining in the plasmas decreased following PLC treatment (96.6% ± 2.4%). The ability of PLC to clear anti-human GPIIbIIIa antibodies in circulation was evaluated by using a passive mouse model of ITP. Here, a fluorescently labeled mouse anti-human GPIIbIIIa monoclonal antibody (PAB-1) was dosed intravenously (IV) into NSG mice to achieve a circulating level of antibody comparable to those observed in ITP patients. The GPIIbIIIa antibody persisted in circulation for over 24 hours following a single dose in untreated mice and did not induce clearance of mouse platelets. Following administration of PLC in the passive ITP model, a decrease in antibody fluorescence in blood was observed within 2 minutes and full antibody clearance was observed at 3 hours post dosing. Furthermore, clearance of the antibody was observed to be both dose responsive and specific since PLC treatment did not affect the antibody concentration of an isotype matched control antibody. PLC activity in clearing the anti-GPIIbIIIa antibody was observed in all particle sizes. Fluorescence quantification of liver and spleen tissue demonstrated that PLC driven anti-GPIIbIIIa antibody clearance occurred preferentially in the liver. Taken together, these data indicate that by specifically binding and rapidly removing anti-platelet antibodies from circulation, PLC may disrupt the platelet degradation mechanisms underlying ITP pathogenesis and restore platelet counts. Disclosures Patel-Hett: PlateletBio: Current Employment. Giannini: Platelet Biogenesis: Current Employment. Peters: Platelet Biogenesis: Current Employment. Dykstra: Platelet Biogenesis: Current Employment. Lehmann: Platelet Biogenesis: Current Employment. Russo: Platelet Biogenesis: Current Employment. Kohnke: Platelet Biogenesis: Current Employment. Carpenter: Platelet Biogenesis: Current Employment. Tomczak: Platelet Biogenesis: Current Employment. Lazarus: Platelet Biogenesis: Consultancy. Masuko: Platelet Biogenesis: Current Employment. Leung: Platelet Biogenesis: Current Employment. Meredith: Platelet Biogenesis: Current Employment. Pete: Platelet Biogenesis: Current Employment. Lee: Platelet Biogenesis: Current Employment. Falb: Platelet Biogenesis: Current Employment.

scholarly journals Megakaryocyte Size in Thrombocytopenic and Normal Rats

Blood ◽  
1968 ◽  
Vol 32 (3) ◽  
pp. 383-392 ◽  
Author(s):  
SHIRLEY EBBE ◽  
FREDERICK STOHLMAN ◽  
JOAN OVERCASH ◽  
JANET DONOVAN ◽  
DONALD HOWARD
Keyword(s):  
Bone Marrow ◽  
Exchange Transfusion ◽  
Recovery Period ◽  
Precursor Cell ◽  
Rapid Rise ◽  
Cell Level ◽  
Homologous Blood ◽  
Platelet Production ◽  
Platelet Counts ◽  
Heterologous Antiserum

Abstract Thrombocytopenia was produced in rats by exchange transfusion with platelet-poor homologous blood or heterologous antiserum. The subsequent rapid rise in platelet counts to thrombocytotic levels suggested accelerated platelet production. During this recovery period macromegakaryocytes were present in the bone marrow. The findings suggested that thrombocytopenia affected megakaryocytopoiesis at the precursor cell level with resultant macrocytosis.

Immune Thrombocytopenia in Type I Gaucher Disease.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3200-3200 ◽  
Author(s):  
Hanna Rosenbaum ◽  
Tina Napso ◽  
Lilach Bonstein
Keyword(s):  
Bone Marrow ◽  
Gaucher Disease ◽  
Immune Modulation ◽  
Hematological Parameters ◽  
Autoimmune Disorder ◽  
Type I ◽  
Bleeding Tendency ◽  
Platelet Surface ◽  
Platelet Counts ◽  
Enzyme Replacement

Abstract Backgound: Type I Gaucher disease (GD) the non-neuronopathic form is characterized by hepatosplenomegaly, pancytopenia and skeletal complications due to the accumulation of glucocerebroside in macrophages. Thrombocytopenia is usually related to hypersplenism and/or infiltration of bone marrow by the lipid-laden macrophages namely Gaucher cells. Enzyme replacement therapy (ERT) restores the hemoglobin and platelet count in treated GD patients within 12–24 months of treatment. In GD patients, including ERT treated, with persistent low platelet counts other ethiological factors should be considered. Goals: To determine the etiology of persistent thrombocytopenia in Type I GD patients and to evaluate their clinical course and hematological parameters. Methods: Flow cytometric technique was used to detect platelet-surface associated IgG/M (PSIgG/M) in a cohort of 24 Type I GD patients followed at the Gaucher clinic in Haifa, Israel. The evaluated hematological parameters of the thrombocytopenic GD patients include: bleeding phenomena, concurrence of autoimmune phenomena, hematological malignancies and bone marrow findings. Results: Twenty four Type I GD patients, 15 females and 9 males with an age range of 35 to 80 years (median 53 years) were included in the study. Seventeen of the evaluated 24 patients were thrombocytopenic with platelet counts less than <100×109/l and 7/24 were in the normal range. Bone marrow aspirate was performed in 16 of the 17 thrombocytopenic patients and showed normal or hyperplasic megakariopoiesis together with Gaucher cells infiltrates. Six of the 17 thrombocytopenic patients received ERT for at least 24 months with no effect on the low platelet counts. Elevated platelet surface IgG was detected in 16/17(94%) of GD patients with thrombocytopenia and in only 1/7 (14%) of non thrombocytopenic patients (p<0.0001). In 6/17 of the thrombocytopenic patients, surface IgM (PSIgM) was found, in addition to the PSIgG. Those six patients are known with monoclonal IgM (concomitant Waldenstrom macroglobulinemia), markedly elevated polyclonal IgM levels, or lupus like autoimmune disorder which may have been responsible for the positive PSIgM. Only three thrombocytopenic patients with platelet counts less than 40×109/l had bleeding tendency (mainly purpura) with no response to steroid treatment (two of them were also resistant to ERT concerning their thrombocytopenia). Conclusions: Thrombocytopenia in Type I GD is related to either infiltration of bone marrow compromising megakariopoiesis or hypersplenism, but immune factors should also be considered. Despite the lack of response to steroids, the observed megakaryocytic hyperplasia in Gaucher infiltrated marrows, the failure to respond to ERT, and the presence of platelet surface antibodies in the thrombocytopenic patients, strongly implicate autoimmune etiology. The present study demonstrates that surface platelet antibodies may play a role in refractory thrombocytopenic GD patients. Since the role of splenectomy is controversial in GD, immune modulation approach should be considered.

Thrombopoietin Stimulation Leads to Enhanced Polyploidization in p53-/- Bone Marrow Megakaryocytes: In Vivo and in Vitro Studies.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2531-2531
Author(s):  
Pani A. Apostolidis ◽  
Stephan Lindsey ◽  
William M. Miller ◽  
Eleftherios T. Papoutsakis
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Platelet Surface ◽  
Platelet Counts ◽  
Reticulated Platelets ◽  
High Ploidy ◽  
And Control ◽  
Platelet Formation

Abstract Abstract 2531 Poster Board II-508 BACKGROUND AND HYPOTHESIS. We have previously shown that tumor suppressor p53 is activated in differentiating megakaryocytic (Mk) cells and its knock-down (KD) leads to increased polyploidization and delayed apoptosis in CHRF, a human Mk cell line. Furthermore, bone marrow (BM)-derived Mks from p53−/− mice reach higher ploidy classes in culture. Accordingly, we hypothesized that the role of p53 during megakaryopoiesis is to delimit polyploidization and control the transition from endomitosis by inhibiting DNA synthesis and promoting apoptosis. Here, we test this hypothesis by examining the differential effect of mouse thrombopoietin (rmTpo) on the ploidy of p53−/− and p53+/+ mouse Mk cells. METHODS. 8–10 week-old, male p53−/− mice and p53+/+ littermates were injected once with 1.2 μg rmTpo or saline. On days 2 and 5 after Tpo/saline treatment, tail-bleeding assays were performed to measure bleeding times/volumes, mice were bled for platelet counts and sacrificed to harvest BM. We employed flow cytometry to examine baseline ploidy in BM-resident Mks in p53−/− and p53+/+ mice as well as Mk cells generated from BM progenitors after 4 and 6 days of culture with rmTpo. RESULTS. At steady state, ploidy in BM-resident CD41+ Mk cells was similar in p53−/− and p53+/+ mice: 11.8±2.3% and 10.7±1.3% of p53−/− and p53+/+ Mks, respectively, reaching a ploidy of ≥32N (n=3-4). Platelet counts were 1.3×106±1×105/μl (12.5±1.0% reticulated) and 1.1×106±5×104/μl (12.4±1.3% reticulated) in p53−/− and p53+/+ mice, respectively (n=8). Two days following Tpo treatment of the mice, we did not observe significantly increased platelet levels, while ploidy was marginally affected. However, 5 days following Tpo treatment, we found greater ploidy in the BM in the absence of p53: 22±1.6% 16N and 10.1±0.8% ≥32N Mks in the p53−/− versus 18.6±3.3% 16N and 7.1±1.4% ≥32N Mks in the p53+/+ (n=2). This was accompanied by increased platelet formation: 23.6±8.3% reticulated platelets in the p53−/− versus 17.8±2.6% in the p53+/+ (n=2). Culture of BM cells from non-Tpo treated mice with 50ng/ml rmTpo resulted in a 50% increase in total Mks and increased polyploidy by day 6 of culture: 38.6±4.6% of p53−/− versus 19.2±2.3% of p53+/+ Mks reached ploidy classes of ≥32N (n=3-4, p < 0.01). Lack of p53 led to hyperploid Mk cells; by day 6 of culture 10.3±2.2% of p53−/− Mks were in ploidy classes of 128N and higher, while only 0.6±0.1% p53+/+ Mks achieved such high ploidy (n=3-4). In addition, a 6 day culture with Tpo of BM cells derived from p53−/− and p53+/+ mice pre-treated with Tpo 5 days prior to sacrifice led to more profound polyploidization compared to Mks generated from the non-Tpo treated mice but only in the p53−/− Mks: 48.8±1.1% of p53−/− versus only 17.6±0.2% of p53+/+ Mks reached ploidy ≥32N (n=2). Microarray analysis comparing p53KD to control CHRF cells undergoing Mk differentiation revealed down-regulation of genes coding for platelet surface complex CD41/CD61 and CD62P in the p53KD cells. To examine the possibility of altered functionality of platelets in p53−/− mice, we performed tail-bleeding assays on the mice that did not receive Tpo. Bleeding times and volumes were generally prolonged in the absence of p53 (all p53−/− mice exceeded the 10 min duration of the assay; mean p53−/− and p53+/+ blood loss was 17μl and 10μl, respectively, n=3-4). CONCLUSIONS. Our data indicate that in vivo polyploidization and platelet formation from Mks is increased in the p53−/− relative to p53+/+ mice after Tpo administration. These data are in line with our hypothesis that p53 activation decreases the ability of Mks to respond to Tpo and undergo polyploidization. Additionally, our preliminary data on platelet functionality suggest that p53 may have a role in hemostasis. Disclosures: No relevant conflicts of interest to declare.

Final Results from an International, Multi-Center, Single-Arm Study Evaluating the Safety and Efficacy of Romiplostim in Adults with Primary Immune Thrombocytopenia (ITP),

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3279-3279 ◽  
Author(s):  
Ann Janssens ◽  
Michael D. Tarantino ◽  
Robert Bird ◽  
Maria Gabriella Mazzucconi ◽  
Ralph Vincent V. Boccia ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Platelet Count ◽  
Board Of Directors ◽  
Research Funding ◽  
Platelet Response ◽  
Employment Equity ◽  
Advisory Committees ◽  
Platelet Production ◽  
Platelet Counts ◽  
Equity Ownership

Abstract Abstract 3279 Background: ITP is an autoimmune disorder characterized by increased platelet destruction and suboptimal platelet production. Romiplostim stimulates platelet production via the TPO-receptor, and is recommended for second- and third-line treatment of chronic ITP in adults. We report final data from a large prospective study of romiplostim in adults with ITP of varying duration and severity. Methods: Eligibility criteria were broad: patients ≥18 years of age, who had received prior ITP therapies (final protocol amendment: ≥1, previous amendments: ≥3), with low platelet counts (final amendment: ≤ 30 × 109/L, previous amendments: ≤ 10, ≤ 20 × 109/L) or experiencing uncontrolled bleeding. The only excluded comorbidities were: hematological malignancy, myeloproliferative neoplasms, MDS and bone marrow stem cell disorder. Romiplostim was initiated at 1 (final amendment) or 3 (previous amendments) μg/kg/week, with dose adjustments allowed to maintain platelet counts ≥50 × 109/L. Patients could continue on study until they had access to commercially available romiplostim. Rescue medications were allowed at any time; concurrent ITP therapies could be reduced when platelet counts were > 50 × 109/L. Primary endpoint was incidence of adverse events (AEs) and antibody formation. Secondary endpoint was platelet response, defined as either (1) doubling of baseline count and ≥ 50 × 109/L or (2) ≥20 × 109/L increase from baseline. Results: A total of 407 patients received romiplostim, 60% of whom were female. Median (Q1, Q3) time since ITP diagnosis was 4.25 (1.20, 11.40) years (maximum 57.1 years), with 51% of patients splenectomised and 39% receiving baseline concurrent ITP therapies. Seventy-one percent of patients completed the study, with requirement for alternative therapy and withdrawn consent the most common reasons for discontinuation (5% each). Median (Q1, Q3) on-study treatment duration was 44.29 (20.43, 65.86) weeks (maximum 201 weeks), with a total of 20,201 subject-weeks on study. Incidence and type of AEs were consistent with previous studies. The most common serious treatment-related AEs were cerebrovascular accident, headache, bone marrow reticulin fibrosis (with no evidence of positive trichrome staining for collagen and no evidence suggesting primary idiopathic myelofibrosis), nausea, deep vein thrombosis, hemorrhage and pulmonary embolism, with each reported in 2 of 407 (0.5%) patients. All other serious treatment-related AEs were each reported in one patient. Eighteen patients died; 3 deaths (hemolysis, intestinal ischaema, aplastic anemia) were considered treatment-related. No neutralizing antibodies to romiplostim or TPO were reported. Approximately 90% of patients achieved each of the platelet response definitions, regardless of splenectomy status. Overall, median (Q1, Q3) time to response was 2 (1, 4) weeks for response definition 1, and 1 (1, 3) week for response definition 2. Median (Q1, Q3) baseline platelet count was 14 (8, 21) × 109/L. After 1 week of treatment median (Q1, Q3) platelet count had increased to 42 (18, 101) × 109/L. From week 8 onwards, and excluding counts within 8 weeks of rescue medication use, median platelet counts were consistently above 100 × 109/L (range 101.0–269.5 × 109/L). Median (Q1, Q3) average weekly romiplostim dose was 3.62 (1.99, 6.08) μg/kg. Summary/conclusions: This is the largest prospective study in adult ITP reported to date. The data reported here are similar to those reported for previous romiplostim studies, with romiplostim able to safely induce a rapid platelet response in adult ITP patients with low platelet counts or bleeding symptoms. Romiplostim is an important, well-tolerated, treatment option for adult ITP patients, which significantly increases and maintains platelet counts. Adverse Event Subject Incidence Platelet Response Disclosures: Janssens: Amgen: Consultancy; Roche: Speakers Bureau; GSK: Membership on an entity's Board of Directors or advisory committees. Tarantino:Cangene corporation: Research Funding; Baxter: Research Funding; Talecris: Honoraria, Speakers Bureau; Up-to-date: Patents & Royalties; The Bleeding and Clotting Disorders Institute: Board Member. Bird:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; GSK: Membership on an entity's Board of Directors or advisory committees. Boccia:Amgen: Equity Ownership, Honoraria, Speakers Bureau. Lopez-Fernandez:Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Kozak:Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Steurer:Amgen: Honoraria. Dillingham:Amgen Limited: Employment, Equity Ownership. Lizambri:Amgen: Employment, Equity Ownership.

Novel Protein Agonist of Thrombopoiesis Acts Via a Physiocrine Pathway Distinct From That of Thrombopoietin

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2376-2376
Author(s):  
Minh-Ha T Do ◽  
Wei Zhang ◽  
Kyle Chiang ◽  
Chi-Fang Wu ◽  
Chulho Park ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Board Of Directors ◽  
Employment Equity ◽  
Advisory Committees ◽  
Platelet Production ◽  
Platelet Counts ◽  
Normal Platelet ◽  
Naturally Occurring ◽  
Equity Ownership

Abstract Abstract 2376 Thrombopoietin (TPO) is recognized as the main regulator of platelet production, yet its genetic ablation in mice does not completely obliterate thrombopoiesis, suggesting that alternate pathways could lead to platelet formation. We recently identified a naturally-occurring protein that acts as a potent agonist of platelet production by a mechanism distinct from that of TPO. This protein belongs to a novel class of human extracellular signaling proteins called physiocrines that are generated from tRNA synthetases by alternative splicing or proteolysis. Physiocrines interact with several classes of receptors through unique mechanisms to modulate cellular differentiation and tissue homeostasis in normal and pathological processes. The newly identified thrombopoietic physiocrine, termed ATYR0030, is an engineered version of a naturally-occurring physiocrine derived from the tyrosyl tRNA synthetase (YRS). In vivo, systemic administration of ATYR0030 or YRS physiocrine to rats led to an increase in platelets counts comparable to that seen with TPO treatment, but with a greater effect in animals with low baseline platelet levels. When injected into normal animals preselected for low platelet counts, ATYR0030 treatment resulted in an increase in platelets up to, but not beyond, normal levels (Figure 1), suggesting a role in platelet homeostasis and differentiating its effects from the known activity of TPO. Intravenous administration of ATYR0030 also accelerated recovery of platelet counts in carboplatin-treated rats, indicating a possible role in bone marrow reconstitution after chemical insult. Consistent with homeostatic properties, no toxicity was seen in a repeat-dose 28-day non-GLP safety study in rats dosed up to 100-fold above the efficacious range. Histopathology assessment revealed no tissue abnormalities, no increase in bone marrow reticulin and no hyperplasia of myeloid precursors. Clinical chemistry and hematology parameters were in the normal range with a modest increase in platelet counts, as anticipated in animals with normal platelet levels. Our in vitro data suggest that ATYR0030 may play a role in megakaryopoiesis by facilitating cell migration and adhesion to the vasculature. In contrast to TPO, ATYR0030 does not directly signal through the TPO receptor and does not activate the JAK/STAT pathway but rather appears to engage specific G-protein coupled receptors. In vitro, ATYR0030 does not stimulate proliferation of cultured M07e human megakaryoblasts or primary bone marrow cells isolated from AML patients (Figure 2). The parent synthetase is present in human platelets and is secreted in response to platelet activation, perhaps providing a feedback mechanism to stimulate the release of new platelets. In an effort to link the biological activity of ATYR0030 and the role that the parent synthetase plays in human physiology, we have begun to analyze samples from patients with abnormal platelets counts to determine circulating levels of the parent synthetase. The unique thrombopoietic activity of ATYR0030 may lead to an orthogonal approach to restoring normal platelet levels in thrombocytopenic patients who currently have limited treatment options. For example, in the myelodysplastic syndrome population, TPO-receptor agonists carry a risk of stimulating blast proliferation and accelerating disease progression to acute myeloid leukemia (AML). The distinct proliferation profile of ATYR0030 may translate into important safety benefits by reducing the risk of progression to AML. In addition, the potential role of ATYR0030 in regulating platelet homeostasis may provide a greater safety margin in the normalization of platelet levels, thereby also limiting the risk of thrombosis. Leveraging the therapeutic potential of this thrombopoietic physiocrine may lead to the development of a novel treatment option with a favorable safety profile. Disclosures: Do: aTyr Pharma: Employment, Equity Ownership, Patents & Royalties. Zhang:aTyr Pharma: Employment, Equity Ownership. Chiang:aTyr Pharma: Employment, Equity Ownership. Wu:aTyr Pharma: Employment, Equity Ownership, Patents & Royalties. Park:aTyr Pharma: Equity Ownership. Yang:aTyr Pharma: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. Kunkel:aTyr Pharma: Consultancy, Stock Ownership. Ashlock:aTyr Pharma: Employment, Equity Ownership. Mendlein:aTyr Pharma: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Belani:Atyr Pahrma: Consultancy, Equity Ownership, Patents & Royalties. Vasserot:aTyr Pharma: Employment, Equity Ownership, Patents & Royalties. Watkins:aTyr Pharma: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Association Of Platelet Function Markers, Independent Of Platelet Count, With Bleeding Score In Patients With Immune Thrombocytopenia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3534-3534
Author(s):  
Andrew L. Frelinger ◽  
Anja J Gerrits ◽  
Michelle A. Berny-Lang ◽  
Travis Brown ◽  
Sabrina L. Carmichael ◽  
...  
Keyword(s):  
Platelet Count ◽  
Platelet Activation ◽  
Platelet Function ◽  
Research Funding ◽  
Immune Thrombocytopenia ◽  
Univariate Analysis ◽  
Blood Draw ◽  
Platelet Surface ◽  
Platelet Counts ◽  
Bleeding Score

Abstract Background Immune thrombocytopenia (ITP) patients with similarly low platelet counts differ in their tendency to bleed. Aim To determine if differences in platelet function in ITP patients with similarly low platelet counts partly account for the variation in bleeding tendency. Methods The relationship between bleeding scores and platelet function markers was investigated in a single center cross-sectional study of pediatric patients with ITP. Following informed consent, blood was collected from ITP patients and bleeding was graded using the Buchanan and Adix Score (J Pediatr 2002) at routine clinic visits or while admitted to the hospital. Bleeding scores were obtained by one of three hematologists blinded to platelet function results, and investigators performing platelet function tests were blinded to clinical results. Platelet function was assessed by whole blood flow cytometric measurement of unstimulated, ADP- or TRAP-stimulated platelet surface activated GPIIb-IIIa (as measured by PAC1 binding), P-selectin, and GPIb and by unstimulated, convulxin-, or ADP plus TRAP-stimulated platelet surface phosphatidylserine expression (as determined by annexin V binding). Platelet count, immature platelet fraction (IPF) and mean platelet volume (MPV) were determined by a Sysmex XE-2100, and platelet forward angle light scatter (FSC) was measured by flow cytometry. Results Platelet function and bleeding scores were evaluated in 34 consecutive consenting pediatric ITP patients (16 female, 18 male, age 9.7 ± 5.7 years [mean ± SD]). ITP was newly diagnosed (< 3 months) in 10 patients, persistent (3 -- 12 months) in 7 patients, and chronic (>12 months) in 17 patients. Platelet count at the time of the blood draw was 47 ± 55 x 109/L. The median bleeding score on day of blood draw was 1 (range 0 to 4). By univariate analysis, higher IPF, and lower platelet count were significantly associated with a higher bleeding score (odds ratio [OR] >1, p<0.05) but MPV was not. Multiple measures of platelet function were associated with bleeding scores by univariate analysis: higher levels of platelet FSC (a measure affected by multiple variables including size) surface GPIb on unstimulated, ADP- or TRAP-stimulated platelets, surface P-selectin on unstimulated platelets, and platelet FSC were associated with increased odds for higher bleeding scores (ORs each >1, p<0.05), while higher ADP- and TRAP-stimulated platelet surface activated GPIIb-IIIa and P-selectin were associated with reduced odds of higher bleeding scores (ORs each <1, p<0.05). After adjustment for platelet count, higher levels of platelet surface P-selectin on unstimulated platelets, GPIb on TRAP-stimulated platelets, and FSC remained significantly associated with increased odds for higher bleeding scores (Figure), but IPF did not. Similarly, after adjustment for platelet count, higher TRAP-stimulated percentage of P-selectin and activated GPIIb-IIIa positive platelets remained significantly associated with reduced odds of higher bleeding scores (Figure). These findings were independent of recent ITP-related treatment. Conclusions In this study of pediatric ITP patients, we identified selected platelet function markers which, independent of platelet count, are associated with increased (platelet FSC, platelet surface P-selectin on unstimulated platelets, and GPIb on TRAP-stimulated platelets) or decreased (TRAP-stimulated percent P-selectin and GPIIb-IIIa positive platelets) odds of high bleeding scores. Possible hypotheses to explain these associations are as follows: 1) Increased P-selectin on unstimulated platelets demonstrates in vivo platelet activation, possibly as a consequence of the recent bleeding. 2) Because platelet activation results in a reduction in platelet surface GPIb and increases in platelet surface activated GPIIb-IIIa and P-selectin, the ORs associated with all of these markers could be explained by reduced ability of platelets in patients with higher bleeding scores to respond to agonists. 3) While platelet FSC is partly related to size, the finding that MPV and IPF, adjusted for platelet count, were not associated with bleeding score suggests that factors other than size account for the association of platelet FSC with higher bleeding scores. Further study is required to validate these findings and determine if differences in platelet function are associated with future risk for bleeding. Disclosures: Off Label Use: Eltrombopag was given to WAS/XLT patients for treatment of thrombocytopenia. Neufeld:Shire: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Apopharma: Consultancy. Michelson:Sysmex: Honoraria.

Novel Management of Immune Thrombocytopenia in Gaucher Disease Patients

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5006-5006
Author(s):  
Hanna Rosenbaum
Keyword(s):  
Bone Marrow ◽  
Platelet Count ◽  
Avascular Necrosis ◽  
Gaucher Disease ◽  
Immune Thrombocytopenia ◽  
High Dose ◽  
Type I ◽  
Platelet Counts ◽  
Skeletal Complications

Abstract Type I Gaucher disease (GD) the is characterized by hepatosplenomegaly, pancytopenia and skeletal complications due to the accumulation of glucocerebroside in macrophages. Thrombocytopenia is usually related to hypersplenism and infiltration of bone marrow by lipid-laden macrophages namely Gaucher cells. Enzyme replacement therapy (ERT) restores the hemoglobin and platelet count in GD patients. In GD ERT treated patients, manifesting persistent low platelet counts, immune thrombocytopenia (ITP) should be considered.Treatment of GD with concomitant ITP is a challenge. Splenectomy may worsen bone manifestations in GD patients and is controversial. Steroids should be used with caution because of possible induction of osteopenia and joints avascular necrosis. Thrombopoietin receptor analogues (TPO-RA) are therapeutic option in GD patients with ITP. Beneficial use of TPO-RA is reported in 2 cases. Patient 1: 39 YO male with new onset of purpura and low platelet count failed treatment with 1 mg/kg of Prednisone. Bone marrow biopsy (BM) showed Gaucher cells infiltration, numerous atypical megakaryocytes, normal erythropoiesis and myelopoiesis with no fibrosis. Low level of ß-glucocerebrosidase activity with compound heterozygosity for 84GG /R495H mutations, established the diagnosis of Type I GD. Low C4 and detection of IgG platelet antibodies added to the diagnosis of concomitant immune thrombocytopenia. ERT with taliglucerase alfa (ElelysoTM) 60 Units/kg/month was given with Prednisone for six weeks. Occurrence of retinal bleeding and purpura, with decrease of platelet count necessitated addition of high-dose IVIG with no response regarding platelet counts. Splenectomy was not considered due to known bony complication risk in splenectomised GD patients. Rituximab was given to prevent wet purpura recurrence with short response regarding platelet count. Romiplostim was initiated raising platelet count from 29,000/µL to 60,000/µL after 3 wks. and to 90,000/µL after 8 wks. enabling corticosteroids withdrawal. Same dose Romiplostim is maintained for the last 30 months with platelet counts of 90,000 - 110,000/µL with no bleeding events. Repeated BMB showed no increase in collagen fibrosis. Patient 2: 63 YO female patient diagnosed with Gaucher at age 33 with a history of purpura, ecchymosis, and occasional vaginal bleeding episodes. At age 53 the platelet count dropped to < 20,000/µL with presence of Anti Platelets Ab (IgG). BMB revealed megakaryocytic hyperplasia with atypical forms, focal infiltration by Gaucher cells and no fibrosis. Combined therapy by ERT (Imiglucerase® followed by Velaglucerase Alfa®), Prednisone (1mg/kg/d for 2 months) and one course of IVIG yielded no increase in platelet count. The patient refused Rituximab®. Romiplostim was initiated increasing platelet count to100,000/µL maintained throughout a year of follow up. Repeated BMB showed slight increase of fibrosis and marked hyperplasia of atypical megakaryocytes. Discussion: Thrombocytopenia is often present in GD and may be severe in approximately 15% of the patients. Persistent cytopenias may be caused by other underlying pathologies such as autoimmune disorders and are important to be recognized and addressed. Before ERT era GD patients with hypersplenism and severe cytopenia were splenectomised. Risks of splenectomy include serious bacterial infection and vascular complications limiting its use in chronic refractory ITP. Splenectomy is avoided in Gaucher patients, due to risk of exacerbating skeletal complications (bone infarcts, avascular necrosis). Stable bone marrow results regarding fibrosis in our patients are consistent with data from a recent 2-year follow-up of 100 ITP patients receiving Romiplostim treatment with no evidence of BM fibrosis. Conclusion: In patients with type I Gaucher disease and concomitant ITP, adjunctive treatment with Romiplostim was successful in maintaining haemostatic platelet counts with no adverse effects. Traditional treatment regimens of corticosteroids and splenectomy should be used with caution or avoided in GD patients due to possible aggravation of Gaucher skeletal disease and the risk of osteopenia and avascular necrosis resulting in increased morbidity in this cohort of patients. Use of TPO-RA should be considered in GD patients with ITP. Disclosures Off Label Use: Romiplostim in gaucher patients.

Abnormalities of Bone Marrow Immune Micro-Environment in Patients with Immune Thrombocytopenia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3464-3464
Author(s):  
Yang Song ◽  
Yu-tong Wang ◽  
Xiao-jun Huang ◽  
Yuan Kong
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Endothelial Cells ◽  
Th17 Cells ◽  
Immune Thrombocytopenia ◽  
Perivascular Cells ◽  
Platelet Production ◽  
Cd34 Cells ◽  
Significant Difference

Abstract Background: Immune thrombocytopenia (ITP) is an immune-mediated disease that is characterized by excessive platelet destruction and decreased platelet production. Although antiplatelet antibodies are considered as the primary immunologic defect in ITP, dysfunctional cellular immunity is also important in the pathophysiology of ITP. The current publications have observed excessive activation and proliferation of platelet auto-antigen-reactive CTLs, production abnormal Th cells, abnormal numbers and function of Tregs in peripheral blood of ITP, but no one focus on the bone marrow (BM) micro-environment in ITP patients. Many cell types including osteoblastic, perivascular, endothelial cells, and various mature immune cells contribute to the BM micro-environment. We have recently reported that the impaired BM vascular micro-environment may affect the thrombopoiesis of CD34+ cells by disrupting the interaction between megakaryocytes and BM endothelial cells (BMECs), resulting in the delayed platelet engraftment in allotransplant patients with prolonged isolated thrombocytopenia (Kong Y, et al. Biol Blood Marrow Transplant. 2014; 20:1190-1197). In mice model, the cross-talk between megakaryocytes and BMECs in BM vascular micro-environment regulates the megakaryocyte maturation and thrombopoiesis. Therefore, we hypothesized that the abnormal BM vascular micro-environment and immune micro-environment may operate in the occurrence of ITP. Aims: To investigate whether abnormal BM vascular and immune micro-environment are involved in ITP patients. Methods: The compartments of BM immune micro-environment were analyzed by flow cytometry in 26 untreated ITP patients and 26 healthy donors (HD). The fractions of T cells, including Th1, Tc1,Th2, Tc2 ,Th17 and Treg were identified as CD3+ CD8- IFN-gama+, CD3+ CD8- IFN-gama+, CD3+ CD8+ IL4+, CD3+ CD8+ IL-4+, CD3+ CD8- IL17A+ and CD3+ CD4+ CD25+ Foxp3+, respectively. The BMECs and perivascular cells, acting as key elements of vascular micro-environment, were identified as CD45- CD34+ VEGFR2+ and CD45- CD34- CD146+, respectively. Hematoxylin-eosin (H&E) staining and immunohistochemistry (IHC) using rabbit anti-human CD34 and CD146 primary antibodies were performed on each BM trephine biopsies (BMB) derived from the patients and controls. Results: The proportion of Th1 cells and Tc1 cells among the bone marrow mononuclear cells (BMMNCs) was significantly increased in ITP patients compared to HD (27.7% ± 11.6% vs. 16.3% ± 7.7%, P<0.001; 39.8%±17.7% vs. 24.1%±11.8%, P<0.005), whereas there was no significant difference in the percentages of Th2 and Tc2 cells. In addition, the proportion of Th17 cells in ITP patients was remarkable higher than HD (3.2%±0.51%1.5%vs 1.7%±1.0%, P<0.0001). We also found the significantly decreased percentage of Treg in ITP patients compared to HD (2.5%±2.0% vs 3.7%±2.6%, P<0.001). However, the frequency of CD34+ cells as well as BMECs and perivascular cells were similar in BM between the ITP patients and HD. Consistent with our flow cytometry data, histological analysis of the recipient BMBs in situ showed no significant differences in CD34-positive BMECs and CD146-positive perivascular cells between ITP patients and HD. Summary/Conclusion: The BM CD34+ cells and vascular micro-environment were normal in ITP patients. However, the abnormal BM immune micro-environment, including the excessive polarization of Th1, Tc1 and Th17 cells and a remarkable decrease of Treg cells were observed in ITP patients. Our data indicated that the desregulated T cells responses in BM may abrogate the thrombopoiesis through the impaired megakaryocytes maturation and decreased platelet production, and eventually contributing to the occurrence of ITP. Acknowledgment: Supported by the National Natural Science Foundation of China (grant nos. 81370638&81230013), and the Beijing Municipal Science and Technology Program (grant nos. Z141100000214011& Z151100004015164& Z151100001615020). Disclosures No relevant conflicts of interest to declare.

Recognition of Megakaryocyte-Specific T-Antigen By Macrophages Negatively Regulates Platelet Production in Bone Marrow

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 420-420
Author(s):  
Melissa M. Lee-Sundlov ◽  
Renata Grozovsky ◽  
Silvia Giannini ◽  
Martina McGrath ◽  
Haley Elizabeth Ramsey ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Flow Cytometric Analysis ◽  
Antigen Expression ◽  
Blood Platelet ◽  
T Antigen ◽  
Severe Thrombocytopenia ◽  
Extrinsic Factors ◽  
Platelet Production ◽  
Platelet Counts

Abstract Glycosylation defects have been associated with low platelet counts. Six genes encoding sialyltransferases (ST), ST3gal1 to 6, that synthesize an α2,3 sialic acid (SA) linkage have been identified in the mammalian genome, and deletion of St3gal1 and St3gal4 genes has been associated with macrothrombocytopenia in mice. Despite the similarity in transferring SA in a α2,3-linkage to terminal galactose residues, St3gal1 and St3gal4 sialylate distinct glycans: St3gal1 is associated with core 1 O-glycan Galβ1,3GalNAcα1-Ser/Thr expression, also known as tumor-associated or Thomsen-Friedenreich antigen (T-antigen), whereas St3gal4 sialylates lactosaminyl Galβ1,4GlcNAc N-glycans. It has been previously shown that St3gal4-null platelets are cleared by the hepatic Ashwell-Morell receptor, causing severe thrombocytopenia in these mice. Herein, we generated St3gal1loxP/PF4+ mice specifically lacking ST3Gal1 in the megakaryocyte (MK) lineage to investigate the detailed mechanisms of macrothrombocytopenia associated with St3gal1 deficiency. Both St3gal1loxP/PF4+ circulating platelets and bone marrow (BM) MKs had increased T-antigen expression, compared to control, as evidenced by peanut agglutinin (PNA) binding. As expected, other blood cell lineages had no increase in T-antigen expression. Blood platelet counts were reduced by ~50% and platelets were enlarged in St3gal1loxP/PF4+ mice, compared to control, despite a virtually indistinguishable platelet clearance. BM MK numbers were normal despite the observed thrombocytopenia, BM MK colony forming units (CFUs) were reduced and in vitro proplatelet production was normal in St3gal1loxP/PF4+ mice, suggesting that extrinsic factors in the St3gal1loxP/PF4+ BM environment affected platelet production. We hypothesize that recognition of the T-antigen epitope on MKs mediate phagocytosis by macrophages. Macrophages in St3gal1loxP/PF4+ mice had increased expression of CD68 (macrosialin), indicative of an activated macrophage state. Flow cytometric analysis of BM derived macrophages of St3gal1loxP/PF4+ mice showed an increased population of resolving M2-type macrophages, which are normally involved in apoptotic cell clearance. Additionally, St3gal1loxP/PF4+ BM smears revealed increased hemophagocytosis, as evidenced by May-Grunwald/Giemsa, indicative of an unspecific increase in phagocytic macrophages. Macrophage ablation by in vivo injection of clodronate-encapsulated liposomes significantly reduced the numbers of activated macrophages in St3gal1loxP/PF4+ mice, thereby normalizing blood platelet counts and size. Taken together data show the contrasting effects of different SA loss on platelet homeostasis: Platelets lacking α2,3-linked SA on N-glycans have increased platelet clearance, whereas a lack of α2,3-linked on O-glycans do not affect platelet half-life, but cause defective thrombopoiesis in MKs. Disclosures No relevant conflicts of interest to declare.

scholarly journals Pharmacokinetic and Pharmacodynamic Properties of Romiplostim

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1463-1463
Author(s):  
Karen Arkam ◽  
Sameer Doshi ◽  
Bing-Bing Yang
Keyword(s):  
Healthy Volunteers ◽  
Healthy Subjects ◽  
Immune Thrombocytopenia ◽  
Receptor Complex ◽  
Platelet Response ◽  
Employment Equity ◽  
Platelet Production ◽  
Platelet Counts ◽  
Equity Ownership ◽  
Chronic Immune Thrombocytopenia

Abstract Background: Chronic Immune thrombocytopenia (ITP) is characterized by low platelet counts, resulting from increased platelet destruction and inadequate platelet production. Romiplostim is a 59 kDa peptibody which binds to and activates the thrombopoietin (TPO) receptor on platelet precursors in the bone marrow, and increases platelet counts. This analysis integrates the pharmacokinetic (PK) and pharmacodynamic (PD) properties of romiplostim in animals, healthy volunteers and patients with ITP, and describes its intricate PK-PD inter-relationship. Methods and Results: In healthy subjects, over a wide range of doses examined, the PK and PD (platelet response) of romiplostim were dependent on both the dose administered and the baseline platelet counts. Following SC administration, platelet counts increased in a dose-dependent fashion after 4 to 9 days, peaking at 12 to 16 days (Wang Clin Pharmacol Ther. 2004;76:628-38). When romiplostim binds to the TPO receptor on megakaryocytes and platelets, the peptibody-receptor complex is internalized and degraded inside the cells. Therefore, as platelet counts increase, a higher number of free receptors are available to clear romiplostim (Wang AAPS J. 2010;12:729-40). Results from rodent studies suggest that as the dose increases, the TPO receptors become saturated and the contribution of the kidney to clearance increases. Additionally, proteolysis plays a role in the clearance of romiplostim; however, the cytochrome P450 enzymes are not involved in protein catabolism (Wang Pharm Res. 2011;28:1931-8), hence there are no known drug-drug interactions or dietary restrictions (Nplate Prescribing Information 2014). Following SC administration, serum concentrations of romiplostim were markedly lower, however, platelet response was similar after the same dose of intravenous (IV) and SC administration (Wang Clin Pharmacol Ther. 2004;76:628-38). This suggests that the PD response is driven by the length of time that the romiplostim concentrations remained above a threshold rather than by the magnitude of concentrations achieved. This effect was verified in a mechanistic PK-PD modeling study in animals (Krzyzanski Pharm Res. 2013;30:655-69). In patients with ITP receiving SC romiplostim at a dose of 1 mcg/kg, the peak platelet response was achieved at 18 days (range 8 to 43; Bussel N Engl J Med. 2006;355:1672-81). Pharmacodynamic model analysis showed that compared with healthy subjects, patients with ITP had a shorter platelet life span and a decreased rate of production of progenitor cells, but no major difference in the time to maturation of megakaryocytes. The PD response in this modeling analysis was not notably affected by age, body weight, sex, and race (Perez-Ruixo J Clin Pharmacol. 2012;52:1540-51). The frequency of once-weekly dosing was selected because once every 2 weeks dosing was determined to be inadequate to achieve and maintain platelet counts in the therapeutic range (Bussel N Engl J Med. 2006;355:1672-81). A mechanistic PK-PD model based on data from the healthy subjects further suggested that weekly dosing resulted in a sustained platelet response while dosing less frequently resulted in high fluctuation of platelet counts (Wang AAPS J. 2010;12:729-40). Large inter- and intra-individual variability in the PD response was observed at a given dose; therefore, dose adjustments should be made based on a patient's platelet counts, using a titrated dosing scheme to prevent having platelet counts over 400 x 109/L (Perez-Ruixo J Clin Pharmacol. 2012;52:1540-51). Conclusion: Romiplostim is a peptibody that binds and activates the TPO receptor, and consequently increases platelet production in individuals with chronic ITP. The peptibody-receptor complex is internalized and degraded inside the cells, without involvement of the liver. Romiplostim's PD response is driven by the length of time that its concentrations remained above a threshold rather than by the magnitude of concentrations achieved. Moreover, weekly dosing has demonstrated a sustained platelet response while less frequent dosing resulted in fluctuating platelet counts. Disclosures Arkam: Amgen Inc.: Employment, Equity Ownership. Off Label Use: Romiplostim is a thrombopoietin receptor agonist indicated for the treatment of thrombocytopenia in patients with chronic immune thrombocytopenia (ITP) who have had an insufficient response to corticosteroids, immunoglobulins, or splenectomy. This abstract also describes PK data from healthy volunteers.. Doshi:Amgen Inc.: Employment, Equity Ownership. Yang:Amgen Inc.: Employment, Equity Ownership.

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