Dynamic Changes in Platelet Responsiveness to Thrombin and Coated Platelet Potential May Inform Risk of Hemorrhage and/or Thrombosis in a Novel Canine Mode of Immune Thrombocytopenia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2193-2193
Author(s):  
Marshall A. Mazepa ◽  
Dana N LeVine ◽  
Adam J Birkenheuer ◽  
Marjory B Brooks ◽  
Shila K Nordone ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Count ◽  
Exploratory Study ◽  
Immune Thrombocytopenia ◽  
Treated Group ◽  
Canine Model ◽  
Severe Thrombocytopenia ◽  
Dynamic Changes ◽  
Platelet Recovery ◽  
Time Zero

Abstract Abstract 2193 In both canine and human patients with Immune Thrombocytopenia (ITP), bleeding risk is challenging to predict, and potentially leads to over-treatment of patients at low risk. Conversely, recent studies have highlighted the risk of thrombosis in ITP during platelet recovery. Given these clinical observations, we hypothesized that in ITP, changes in platelet response to agonists may occur in addition to changes in platelet numbers. In response to dual agonist activation (thrombin and convulxin), a subpopulation of platelets in both humans and dogs develops enhanced procoagulant activity. This subpopulation is termed coated platelets, and differences in individuals' potential to form coated platelets have been correlated with both hemorrhagic and thrombotic outcomes. In this exploratory study, we serially evaluated ex vivo platelet responsiveness to both thrombin and dual agonists (termed coated platelet potential) in a novel canine model of ITP. Dogs (n=4) were infused with a murine monoclonal anti-GPIIb antibody (2F9) in order to model ITP and generate predictable severe thrombocytopenia. Control dogs (n=3) were infused with a control antibody. Platelet count, thrombin responsiveness, and coated platelet potential were measured at baseline, time zero, 6 hours, 24 hours, and every 24hrs thereafter until the platelet count was ≥ baseline for at least two consecutive measures (recovery). Time zero was defined as the time when platelet count first fell to ≤ 30,000/μl following 2F9 infusion, or 1 hour following control antibody infusion. For platelet thrombin responsiveness, a monoclonal antibody to P-selectin was used to determine platelet P-selectin surface expression by flow cytometry after stimulation with graded doses of thrombin. The ED50 Thrombin was defined as the concentration of thrombin required for half-maximal P-selectin expression. Coated platelet potential was defined as the percent of platelets activated to the highly procoagulant state after dual stimulation with thrombin and convulxin, as determined by binding of biotinylated fibrinogen by platelets by flow cytometry. All dogs in the treated group developed severe thrombocytopenia (median=6×103, range=4–11×103 platelets/uL); no dogs in the control group developed thrombocytopenia. All treated dogs had platelet recovery by 240 hours (median=132 hours, range 120–240hours). Of interest, at 6 hours, ED50 Thrombin in the treated group increased nearly twofold (fig 1A) (ratio of median ED50 Thrombin treated/baseline=1.6, range 1.3–2.3), which correlated with a decline in coated platelet potential by nearly half of baseline (fig 1B) (median 52.4% of baseline, range 19.6–61.5%); minimal change from baseline was observed in controls. In both groups, ED50 Thrombin was lower at recovery than baseline (fig 1A) (treated median ED50 Thrombin=71.5% of baseline; control median ED50 Thrombin=67% of baseline). A trend of rising coated platelet potential was also noted as platelets recovered in the treated group. In conclusion, in this exploratory study of a canine model of ITP, we observed dynamic changes in platelet responsiveness. During severe thrombocytopenia, we observed a rise in ED50, indicating a decline in response to thrombin, which correlated with a fall in coated platelet potential. We speculate that this early fall in platelet thrombin response and coated platelet potential could contribute to hemorrhage risk in ITP. As a complement to this finding, in the treated group, there was a rise in coated platelet potential as platelets rebounded and coated platelet potential was slightly greater than baseline at recovery. This is consistent with others' observation that younger platelets are more likely to have coated platelet potential. We also observed a decline in ED50 Thrombin at recovery, not only in the treated dogs, but also control dogs. Thus, at recovery, the decline in ED50 Thrombin was independent of treatment group. However, this may be an artifact of our small sample size. Our observed increase in coated platelet potential during platelet recovery could potentially contribute to the thrombotic tendency of some ITP patients. Future studies are planned to explore the relationship of hemorrhagic and thrombotic risk with platelet thrombin responsiveness and coated platelet potential in this model of ITP and clinical studies of canine and human ITP. Disclosures: No relevant conflicts of interest to declare.

P8: CHEMOTHERAPY INDUCED IMMUNE THROMBOCYTOPENIA-AN ENTITY TO KEEP IN MIND!

2016 ◽  
Vol 64 (3) ◽  
pp. 820.2-820
Author(s):  
P Draksharam ◽  
J Park ◽  
G Sidhu
Keyword(s):  
Flow Cytometry ◽  
Platelet Count ◽  
Recovery Time ◽  
Immune Thrombocytopenia ◽  
Chemotherapeutic Agents ◽  
Metastatic Colon Cancer ◽  
Platelet Recovery ◽  
Platelet Antibodies ◽  
Drug Dependent ◽  
Platelet Transfusions

Purpose of StudyThrombocytopenia during chemotherapy is not always due to myelosuppression. We report an unusual case of isolated acute thrombocytopenia after oxaliplatin and irinotecan administration. We reviewed 11 reported cases to better understand the nature of the presentation and variability in response to treatment.Case ReportPatient is a 63 year old female with metastatic colon cancer treated with palliative chemotherapy with FOLFOX. Follwing her 14th cycle she had an episode of acute drop in platelet count to 8,000/microliter. Peripheral smear revealed no evidence of thrombotic microangiopathy. She was managed with supportive platelet transfusions with slow recovery of platelet count. Subsequently she was treated with second line chemotherapy with FOLFIRI. Following the first cycle of Irinotecan, she again had a catastrophic drop in platelets from 136,000/microliter to 6,000/microliter within 10 hours. Due to this recurrent episode, a drug mediated thrombocytopenia was suspected and work up was initiated. She was initially treated with dexamethasone without a significant response. Platelet count normalized after 7 days with supportive platelet transfusions.Methods UsedBlood was tested for drug dependent platelet antibodies by Flow Cytometry at the Platelet and Neutrophil Immunology Laboratory at the Blood Center of Wisconsin.Summary of ResultsThe patient's serum showed evidence of drug dependent platelet antibodies to both oxaliplatin and irinotecan.ConclusionsDrug mediated immune thrombocytopenia is not uncommon. Time to severe acute thrombocytopenia and platelet recovery time varied post exposure of the drug. It is unclear whether steroid or IVIG administration had any effect on the platelet recovery time. Recovery from thrombocytopenia was observed in all 11 cases after the discontinuation of the insulting agent. Confirmation of the presence of drug dependent platelet antibodies against the chemotherapeutic agent by flow cytometry essential for diagnosis. This would be the first reported case of acute thrombocytopenia to two different chemotherapeutic agents in the same patient. Whether the reaction is two different mechanisms or if there is a cross reactivity between Oxaliplatin and Irinotecan has yet to be investigated.

scholarly journals Regulation of thrombopoiesis: effects of the degree of thrombocytopenia on megakaryocyte ploidy and platelet volume

Blood ◽  
1987 ◽  
Vol 70 (1) ◽  
pp. 177-185 ◽  
Author(s):  
L Corash ◽  
HY Chen ◽  
J Levin ◽  
G Baker ◽  
H Lu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Platelet Count ◽  
Immune Thrombocytopenia ◽  
Temporal Relationship ◽  
Recovery Phase ◽  
Severe Thrombocytopenia ◽  
Detectable Change ◽  
Platelet Volume ◽  
Murine Bone Marrow ◽  
Ploidy Class

Abstract We have established a murine model and techniques with which to serially study thrombocytopoiesis after induction of experimental immune thrombocytopenia of variable severity and duration. Bone marrow megakaryocyte ploidy distribution was determined by using unfractionated bone marrow, a polyclonal megakaryocyte-specific probe, and two-color, fluorescence-activated flow cytometry. With these techniques, the modal megakaryocyte ploidy class in normal murine bone marrow was 16N. Serial studies of bone marrow megakaryocyte ploidy after the induction of acute, severe thrombocytopenia (platelet count, less than 0.05 X 10(6) microL) demonstrated no detectable change in the ploidy distribution at 12, 24, and 36 hours after the onset of thrombocytopenia. At 48 hours, the modal ploidy class shifted from 16N to 32N, and the 64N class increased significantly (P less than .001). The ploidy distribution returned to normal 120 hours after the onset of thrombocytopenia. A lesser degree of thrombocytopenia (platelet count reduction to 0.100 to 0.200 X 10(6)/microL) delayed the modal ploidy class shift from 16N to 32N until 72 hours after the onset of thrombocytopenia. Chronic, severe thrombocytopenia (platelet count, less than 0.05 X 10(6)/microL for seven days) resulted in a modal ploidy class shift from 16N to 32N during the thrombocytopenic phase and an enhanced increase in the 64N megakaryocyte class during the recovery phase. Mean platelet volume (MPV) was simultaneously measured on isolated total platelet populations after induction of thrombocytopenia. MPV was significantly increased (P less than .001) as early as eight hours after the onset of acute, severe thrombocytopenia, 40 hours before a shift in the ploidy distribution. Mild thrombocytopenia (platelet count reduction to 0.400 X 10(6)/microL) was not associated with a ploidy shift but did result in a significantly increased MPV (P less than .001). These studies demonstrate that the temporal relationship and magnitude of the effects of thrombocytopenia upon megakaryocyte ploidy distribution are dependent upon the degree and the duration of the thrombocytopenic stimulus and that the effects of experimental thrombocytopenia on platelet volume and megakaryocyte ploidy are dissociated.

Administration of Anti-Platelet Antibodies Prevents the Anti-Platelet Immune Response and Bleeding Complications of Neonatal Immune Thrombocytopenia in a Murine Model.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 223-223
Author(s):  
Heidi Tiller ◽  
Pingguo Chen ◽  
Bjorn Skogen ◽  
Mette Kjaer Killie ◽  
Anne Husebekk ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Immune Response ◽  
Platelet Count ◽  
Murine Model ◽  
Immune Thrombocytopenia ◽  
Human Platelet ◽  
Bleeding Complications ◽  
Platelet Antibodies ◽  
Β3 Integrin ◽  
Equity Ownership

Abstract Abstract 223 Background: The human platelet antigen (HPA) 1a is a potent immunogen located on the β3 integrin. Ten % of pregnant HPA1a negative women produce antibodies against the HPA1a antigen if the foetus is HPA1a positive. Fetal/neonatal immune thrombocytopenia (FNIT) can occur if the mother develops alloantibodies against fetal platelets, with intracranial haemorrhage as the most severe complication. The current opinion has been that immunization against the HPA1a antigen takes place during the first HPA1 non-compatible pregnancy. However, results from a large and recent screening study in Norway found that the majority (75%) of women were immunized around time of delivery, and not so often during pregnancy. This indicates that FNIT could be more similar to haemolytic disease of the newborn (HDN) than previously thought. To prevent HDN, antibody mediated immune suppression (AMIS) is induced by administration of anti-D antibodies in connection with RhD-negative pregnancies. The same principle could be used to prevent FNIT by administration of anti-HPA1a antibodies in HPA1a-negative pregnancies. We have previously established a murine model of FNIT using β3 integrin-deficient (β3−/−) mice. The first aim of the current project was to test whether administration of human anti-HPA1a IgG could suppress the anti-human platelet immune response in β3−/− mice after transfusion of human HPA1a positive platelets. For the second part of the project, we used a pure murine model to test whether administration of murine anti-β3 antibodies transfused after delivery could induce AMIS and prevent bleeding complications of FNIT in the subsequent pregnancies. Methods: Human/murine model: Human IgG from 5 donors with high levels of anti-HPA1a antibodies was purified by Protein G affinity chromatography. Purified IgG from one male donor without detectable anti-platelet specific antibodies was used as control IgG. Human platelets were isolated from an HPA1a positive donor. β3−/− mice were immunized by one tail vein transfusion with 2 × 106 human HPA1a positive platelets, with or without subsequent transfusion of 900ug human IgG (100% saturation). After 7 days, the mice were bled and sera collected. The anti-human platelet immune response was analyzed via flow cytometry, using FITC-conjugated goat anti-mouse IgG as detection antibody. Six mice were injected with anti-HPA1a containing IgG. Control IgG (n=6) or no IgG (n=4) were used as negative controls. Pure murine model: High-titer anti-β3 sera were produced by 4 weekly transfusions of 108 wild type (WT) platelets to β3−/− mice. Naïve β3−/− female mice were bred with naïve β3−/− male mice. Within 24 hours of delivery, the mother was transfused with 108 WT platelets with or without immediate transfusion of anti-β3 sera. The transfusions were repeated one week after delivery and the same females were bred again with WT male BALB/c mice. The anti-β3 immune response was analyzed via flow cytometry, using FITC-conjugated goat anti-mouse IgG. The FNIT phenotype was monitored and all live pups were bled from the carotid vein to determine platelet count. Results: Administration of purified anti-HPA1a IgG significantly suppressed the anti-human platelet immune response in β3−/− mice after transfusion of HPA1a positive platelets as compared with control IgG (p < 0.05). In the pure murine model of FNIT, the anti-β3 immune response was markedly suppressed during the subsequent pregnancy in the mice treated with anti-β3 sera. Two out of three mice receiving anti-β3 sera treatment delivered live pups with moderate thrombocytopenia without signs of haemorrhage (mean platelet count 217 ×106/mL). The third mouse receiving anti-β3 sera delivered dead pups. In contrast, all female mice (n = 3) without anti-β3 sera treatment miscarried. Conclusions: We have demonstrated in vivo that AMIS can be induced by administration of anti-platelet antibodies using a murine model of FNIT. Preliminary data indicates that bleeding complications of FNIT can be prevented with this prophylactic approach. Disclosures: Skogen: Prophylix Pharma a/s: Employment, Equity Ownership. Killie:Prophylix Pharma a/s: Equity Ownership. Husebekk:Prophylix Pharma a/s: Equity Ownership. Kjeldsen-Kragh:Prophylix Pharma a/s: Equity Ownership.

Association of Interleukin-18 Gene Polymorphism with Severity of Chronic Immune Thrombocytopenia (ITP).

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3693-3693
Author(s):  
Takayuki Saitoh ◽  
Norihiko Moriyama ◽  
Tomonori Takani ◽  
Takeki Mitsui ◽  
Takumi Hoshino ◽  
...  
Keyword(s):  
Platelet Count ◽  
Immune Thrombocytopenia ◽  
Initial Diagnosis ◽  
Severe Thrombocytopenia ◽  
Interleukin 18 ◽  
Single Nucleotide ◽  
Chronic Itp ◽  
Significant Difference ◽  
Haplotype Frequencies ◽  
And Control

Abstract Abstract 3693 Introduction: Immune thrombocytopenia (ITP) is a chronic acquired organ-specific autoimmune disorder characterized by the production of antibodies against antigens on the membranes of platelets. Several cytokine studies have shown Th1 polarization in ITP patients. Interleukin-18 (IL-18) plays an important role in Th1 and Th2 immune response. Recent studies showed that single-nucleotide promoter polymorphisms influence the transcriptions of IL-18 mRNA. IL-18 polymorphism has been implicated in autoimmunity, including Crohn's disease, rheumatoid arthritis, and asthma. We examined the single nucleotide polymorphisms (SNPs) in the promoter regions of the IL-18 genes in patients with ITP, and analyzed the relationship between IL-18 SNPs and clinical features. Patients and Methods: One hundred patients (male/female; 22/78, median age; 54.5) diagnosed as chronic ITP and 151 healthy controls were included. Chronic ITP was defined as thrombocytopenia (platelet count < 100×109/L) persisting greater than 12 months, normal or increased marrow megakaryocytes, and no secondary immune or non-immune abnormality that could account for the thrombocytopenic state. ITP with severe thrombocytopenia was defined as thrombocytopenia (platelet count < 10×109/L) at presentation of ITP. The response criteria of the ITP International Working Group was used. A complete response (CR) is defined as any platelet count of at least 100×109/L, and a response (R) was defined as any platelet count between 30 and 100×109/L and at least doubling of the baseline count. Allparticipants gave written informed consent about the study. Genomic DNA was isolated from peripheral blood using the DNA Kit (QIAGEN, Hilden, Germany). An allele-specific polymerase chain reaction was used to analyze polymorphism in IL-18 –607A/C and -137G/C. Genotype and allele frequencies were compared between the study groups using Χ2-test. The characteristics and laboratory features of the ITP patients with each IL-10 promoter polymorphism were compared using X2-tests and student t-tests. Probability values <0.05 were considered statistically significant. Results: The platelet count was at an initial diagnosis ranged from 1×109/L to 98 ×109/L, with a median of platelet count of 15×109/L. Thirty-five patients (35%) had severe thrombocytopenia. Steroid treatment was given to 68 patients (68%), while splenectomy was used in 11 patients (11%).The frequencies of the genotypes were as follows: AA (34%), AC (57%), and CC (9%) for -607; GG (77%), GC (21%), and CC (2%) for -137 loci. The frequencies of each haplotype were as follows: C-G/C-G haplotype (9%), A-G/C-G haplotype (47%), A-C/C-G haplotype (10%), A-G/A-G haplotype (21%), A-G/A-C haplotype (11%) and A-C/A-C haplotype (2%). No significant differences in the genotype or haplotype frequencies demonstrated between chronic ITP patients and control group. However, patients with -137CC genotypes showed severe thrombocytopenia at initial diagnosis compared to those with -137GG/GC genotypes (5×109/L vs. 22×109/L, p=0.002). Furthermore, patients with A-C/A-C haplotype showed severe thrombocytopenic state (5×109/L vs. 22×109/L, p=0.002) compared to those without A-C/A-C haplotype. No significant difference of treatment response was observed according to IL-18 polymorphism. Conclusion: No significant differences in the genotype or haplotype frequencies demonstrated between chronic ITP patients and control. However, -137CC genotypes or AA/CC haplotype was associated with severity of chronic ITP. Our data suggest that the group with low IL-18 inducibility (i.e. -137CC genotype, A-C/A-C haplotype) may have more severe thrombocytopenia. Disclosures: No relevant conflicts of interest to declare.

The Cytokine Polymorphisms Affect the Severity of Thrombocytopenia at Diagnosis in Chronic Immune Thrombocytopenia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2194-2194
Author(s):  
Takayuki Saitoh ◽  
Chiaki Ushie ◽  
Atsushi Iwasaki ◽  
Norihiko Moriyama ◽  
Tomonori Takani ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Platelet Count ◽  
Clinical Features ◽  
Immune Thrombocytopenia ◽  
Steroid Treatment ◽  
Severe Thrombocytopenia ◽  
Bleeding Tendency ◽  
Chain Reaction ◽  
Chronic Itp ◽  
Polymerase Chain

Abstract Abstract 2194 Introduction: The severity of immune thrombocytopenia (ITP) depends on the degree of the thrombocytopenia and the extent of bleeding. Some investigators have reported the association between the thrombocytopenia and cytokine dysregulation in ITP. We investigated the association between the severity of thrombocytopenia at diagnosis in ITP patients and several cytokine polymorphisms, including IL-10-1082A/G, -819T/C, -592A/C, IL-17F-7488T/C and IL-18-607A/C, −137G/C. Patients and methods: We examined 102 patients (male/female, 24/78; median age, 42) diagnosed with chronic ITP. The definition, response criteria, including complete response (CR)and response (R), loss of CR,and “corticosteroid-dependence” were assessed according to the criteria of the ITP International Working Group. ITP with severe thrombocytopenia (ST group)was defined as thrombocytopenia (platelet count < 10×109/L) at the initial diagnosis of ITP. Genotyping of IL-10 (rs1800870 − 1082 A/G, rs1800871 − 819 T/C, and rs1800872 − 592 A/C) and IL-17F (rs763780, 7488 T/C) polymorphisms were determined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and the genotyping of the IL-18 polymorphism (rs187238 −137G/C and rs1946518−607 A/C) was determined by the allelic specific polymerase chain reaction technique. To confirm the accuracy of the assay, amplification products of several individuals were sequenced using an ABI Prism Genetic Analyzer. Genotype and allele frequencies were compared between the study groups using χ2-test. The characteristics and laboratory features of ITP patients with each polymorphisms were compared using χ2-tests and student t-tests. Odds ratios (OR) and 95% confidence intervals (CIs) were estimated for each study. All patients were provided written information about the study. This study was approved by the Institutional Research Board of Gunma University Hospital. Results: Clinical features of chronic ITP: The platelet count ranged from 1×109/L to 98×109/L with a mean of platelet count of 32×109/L at the initial diagnosis. Fifty seven patients (49%) had bleeding tendency. Steroid treatment was given to 68 patients (66.7%) and eradication of Helicobacter pylori (H. pylori) was performed in 32 patients (31.4%), while splenectomy was performed in only 11 patients (10.8%). Clinical features of ST group vs. non-ST group in chronic ITP: Of these 102 patients, 17 (16.7%) had severe thrombocytopenia (platelet count < 10×109/L) (ST group). ST group were significantly older (ST group: median 59 years vs. non-ST group: 41 years, p<0.01) and had more severe bleeding tendency (ST group: 100% vs. non-ST group: 54%, p<0.0001). Steroid treatment was frequently given to ST group than to non-ST group (ST group: 100% vs. non-ST group: 59.5%, p<0.001). Though the response to corticosteroids treatment was not significantly different between ST group and non-ST group (CR rate, ST group: 50% vs. non-ST group: 51.0%, p=0.94), corticosteroid-dependent patients in ST group was significantly higher than in non-ST group (76.9% vs. 25.3%, p<0.005). Polymorphism study of ST group vs. non-ST group in chronic ITP: The frequencies of genotypes of cytokines in patients with chronic ITP according to the definition of criteria of ST were as follows: AA (93.3% vs. 97.1%) and AG (6.7% vs. 2.9%, p=0.48) for IL-10–1082; TT (46.7% vs. 33.3%), TC (33.3% vs.55 %) and CC (20% vs. 11.7%) for IL-10–819; AA (46.7% vs. 33.3%), AC (33.3% vs.55 %) and CC (12.2% vs. 11.5%) for IL-10–592; TT (100% vs. 81%) and TC (0% vs. 19%) for IL-17F; GG (82.4% vs. 74.4%), GC (17.6% vs. 23.2%) and CC (0% vs. 2.4%) for IL-18–137; AA (35.3% vs. 34.1%), AC (58.8% vs. 53.7%) and CC (5.9% vs 12.2%) for IL-18–607 loci (ST group vs. non-ST group, respectively). No significant difference was observed between ST group and non-ST group according to IL-10–1082A/G, −819T/C, −592A/C, and IL-18–607A/C, −137G/C polymorphism. However, the numbers of IL-17F 7488TT genotype (higher function type) in ST group were significantly higher than in non-ST group (ST group: 100% vs. non-ST group: 81% p<0.05). Conclusion: These findings suggest that severe thrombocytopenia at diagnosis have an impact of bleeding tendency and corticosteroid-dependency of chronic ITP. Furthermore, IL-17F polymorphism may affect the severity of thrombocytopenia of chronic ITP. Disclosures: No relevant conflicts of interest to declare.

A Novel Canine Model of Immune Thrombocytopenia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3326-3326
Author(s):  
Dana N LeVine ◽  
Adam J Birkenheuer ◽  
Marjory B Brooks ◽  
Shila K Nordone ◽  
Dwight A Bellinger ◽  
...  
Keyword(s):  
Platelet Count ◽  
Immune Thrombocytopenia ◽  
Canine Model ◽  
Target Range ◽  
Dose Titration ◽  
Platelet Glycoprotein ◽  
Control Group ◽  
Large Animal ◽  
Isotype Control ◽  
Platelet Counts

Abstract Abstract 3326 Immune thrombocytopenia (ITP) is a relatively prevalent disease in dogs with significant morbidity and mortality. Canine ITP is clinically analogous to human ITP, with heterogeneity in bleeding manifestations in individuals with similar platelet counts. With a view to ultimately investigate this bleeding heterogeneity, we set out to develop a canine model of ITP. There are currently no existing large animal models of ITP. An induced canine ITP model would be representative of ITP without the confounding co-morbidities seen in clinical cases. Since spontaneous ITP occurs in both dogs and humans, the dog is an ideal translational model. We hypothesized that 2F9, a murine IgG2a monoclonal antibody to the canine platelet glycoprotein GPIIb (a common target of autoantibodies in ITP), would induce predictable dose-dependent thrombocytopenia (TCP) in healthy dogs. 2F9 had not been previously administered in vivo. We produced highly purified 2F9 and αYFA antibodies from the 2F9 hybridoma (gift of David Wilcox, Blood Research Institute, Wisconsin) and an isotype control murine anti-yellow fever antibody (αYFA) hybridoma. A dose titration (2 dogs) and a dose repeatability study (3 dogs) were performed in healthy adult research dogs by repeated intravenous infusion (≤ 6 doses) of 2F9 antibody until a target nadir of 5–30 × 103 platelets/μl was reached. Platelet counts were performed hourly until the platelet count reached the desired nadir range (t=0 hrs), after which complete blood counts were performed at 2, 4, 6, 8, 12, 24 hours, then q 24 hours for 10 days. The following were evaluated throughout the study: physical examination, buccal mucosal bleeding time (BMBT, baseline and t=0 only), serum cytokines and chemokines (INFγ, Interleukin (IL) 2, 6, 7, 8, 10, 15, 18, KC, IP-10, MCP-1, GM-CSF, TNFα; Milliplex CCYTOMAG-90K), fibrinogen, and D-dimers. Specificity of the 2F9 effect was confirmed by IV infusion of the isotype control (αYFA) to 3 dogs at the highest cumulative effective dose of 2F9 (167 μg/kg); all parameters were measured as above (t=0 hrs was one hour after αYFA dosing). Within 2 hours of a median cumulative 2F9 administration of 63 μg/kg (range 50.0–166.6 μg/kg), all dogs developed profound TCP (range 11–28 × 103/μl). Compared to the control group, platelet nadir was significantly lower (median (range): 6 (4–11) × 103/μl vs. 200 (179–209) × 103/μl; p= 0.036) and change in platelet count from baseline to nadir was significantly greater in the 2F9-treated group (median (range): 238 (179–325) × 103/μl vs. 4 (0–10) × 103/μl; p=0.036) (Fig 1); p-values were calculated using the exact Wilcoxon rank-sum test. Platelet nadir was in our target range and platelet count remained < 40 × 103/μl in all 2F9-treated dogs for 24 hours. Dosing was predictable: in each dog, after an initial dose of 50 μg/kg 2F9, the second dose needed to reach the target nadir could be accurately calculated from the initial platelet decrease. 2F9-treated dogs developed a range of clinical bleeding from none to petechiae, ecchymoses, melena, and hematuria. At t=0 hrs, BMBT increased 3–8 fold in treated dogs, compared to < 2 fold in control dogs. Dogs had no changes in vital signs or demeanor and did not require any transfusion support. The model does not appear pro-thrombotic as fibrinogen and D-dimers were similar over time in 2F9-treated vs. control dogs. 2F9 infusion also generated negligible systemic inflammation, as assessed by white blood cell count and serum cytokine measurement. Unexpectedly, however, serum IL8 tracked faithfully with platelet count, demonstrating that platelets are a major source of serum IL8 in dogs (Fig 2). Although α granules are known to contain IL8, platelets have not been previously described as a significant serum IL8 source. Since IL8 is an important neutrophil chemokine, our finding may illuminate a novel mechanism of platelet-neutrophil cross-talk. In summary, we have developed a novel large animal ITP model that is highly representative of the spontaneous disease. Like naturally-occurring ITP, dogs demonstrate bleeding heterogeneity despite similar platelet counts (data not shown). We expect our model to lead to further insights into bleeding mechanisms in ITP. Ultimately, understanding what factors predispose certain patients to bleed will allow us to exploit these factors therapeutically as novel ITP treatments. Disclosures: No relevant conflicts of interest to declare.

Severe Thrombocytopenia Delays but Does Not Prevent the Occlusion of an Arterial Prosthesis in Rats

1981 ◽  
Vol 46 (02) ◽  
pp. 558-560 ◽  
Author(s):  
I Reyers ◽  
L Mussoni ◽  
M B Donati ◽  
G de Gaetano
Keyword(s):  
Platelet Count ◽  
Abdominal Aorta ◽  
Immune Thrombocytopenia ◽  
Severe Thrombocytopenia ◽  
Thrombotic Occlusion ◽  
Arterial Prosthesis ◽  
Low Platelet Count ◽  
Experimentally Induced

SummaryThis study shows that experimentally-induced immune thrombocytopenia significantly delayed occlusion of an arterial prosthesis inserted in rat abdominal aorta. Thrombocytopenia was effective when induced several hours or shortly, or even several hours after the insertion of the prosthesis. Maintenance of severe thrombocytopenia by daily administrations of antiplatelet antiserum appeared to further delay thrombotic occlusion.However, though delayed, occlusion eventually occurred in all rats, even in those with very low platelet count. This would imply that any attempt to prevent arterial prosthesis thrombosis solely by interfering with platelets is ultimately bound to fail.

scholarly journals Helicobacter pylori eradication affects platelet count recovery in immune thrombocytopenia

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Ayoung Lee ◽  
Junshik Hong ◽  
Hyunsoo Chung ◽  
Youngil Koh ◽  
Soo-Jeong Cho ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Platelet Count ◽  
Immune Thrombocytopenia ◽  
Complete Response ◽  
Long Term Effects ◽  
Infection Group ◽  
Platelet Recovery ◽  
First Line Therapy ◽  
H Pylori ◽  
Count Recovery

Abstract Helicobacter pylori (H. pylori) infection is on the rise as a cause of immune thrombocytopenia (ITP). It has been suggested that platelet recovery can be achieved following successful microbial eradication, although, the exact pathophysiology has yet to be fully elucidated. This study evaluated the long-term effects of H. pylori eradication monotherapy on platelet count recovery in patients with ITP. H. pylori eradication was analysed in 61 ITP patients. Patients who maintained a complete response (CR) for more than six months were classified as sustained responders (SR). The prevalence of H. pylori infection was 54.3% (75/138), and the success rate of eradication with first-line therapy was 71.4% (35/49). Patients who had achieved a CR at 2 months maintained a higher platelet count thereafter. At 1 year following eradication, platelet counts had increased 2.78 times in the eradicated group, 1.36 times in the sustained infection group, and 1.33 times in the no infection group compared with the baseline (P = 0.016).

Transgenic Expression of Human Platelet FcγRIIA (CD32A) in Mice Does Not Affect the Thrombocytopenia Associated with WASp Deficiency.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1106-1106
Author(s):  
Michael P. Marchetti ◽  
Francois Maignen ◽  
Herve Falet
Keyword(s):  
Flow Cytometry ◽  
Body Weight ◽  
Platelet Count ◽  
Weight Ratio ◽  
Analysis Of Covariance ◽  
Spleen Weight ◽  
Severe Thrombocytopenia ◽  
Body Weight Ratio ◽  
Igg Antibody ◽  
Normal Platelet

Abstract Wiskott-Aldrich Syndrome (WAS) is an X-linked hematopoietic disorder that is characterized by immune deficiency, eczema and severe thrombocytopenia with small platelets. Platelets isolated from WAS patients are cleared rapidly from the circulation when transfused autologously. However, the role of WASp, the protein mutated or absent in WAS, is unclear since platelets isolated from WAS patients function normally. WASp knockout (KO) mice only have a mild thrombocytopenia. Because 1) human, but not mouse platelets express the Fc receptor for IgGs, FcγRIIA (CD32A), and 2) platelet-associated IgGs (PAIgGs) are often observed in WAS patients, we sought to determine whether FcγRIIA expression in humans was involved in the severe thrombocytopenia associated with WAS. WASp KO mice expressing human FcγRIIA on the surface of their platelets were generated by breeding female mice carriers for WASp deficiency with heterozygous FcγRIIA transgenic (TG) males. A total of 221 offsprings were obtained, of which only 97 were males. WASp KO / FcγRIIA TG males were a minority, with a total of 19 animals, compared to 24 WASp WT / FcγRIIA WT, 30 WASp WT / FcγRIIA TG and 24 WASp KO / FcγRIIA WT males. WASp KO / FcγRIIA WT and WASp KO / FcγRIIA TG males had about 70% of normal platelet count compared to WASp WT / FcγRIIA WT and WASp WT / FcγRIIA TG mice (Table 1). Thus, FcγRIIA expression did not affect the thrombocytopenia associated with WASp deficiency. PAIgGs were not detected on the surface of WASp KO / FcγRIIA TG platelets, as evaluated by flow cytometry using an anti-mouse IgG antibody. Spleen weight was increased in WASp KO / FcγRIIA TG compared to WASp WT / FcγRIIA WT and WASp WT / FcγRIIA TG males, but similar to that of WASp KO / FcγRIIA WT males (Table 1). Age of the mice was not involved since similar results were obtained with 6-, 12- or 24-weeks old mice. However, an increased population of macrophages appeared in the spleen of mice lacking WASp as they aged, as evidenced by flow cytometry using anti-mouse Mac-1 (CD11b) and Gr-1 (Ly-6G) antibodies. Our data indicate that expression of platelet FcγRIIA alone does not explain the difference observed between the severe thrombocytopenia of WAS patients and the mild thrombocytopenia of WASp KO mice. WASp deficiency may affect the surface organization of platelets such that clearance is accelerated by spleen macrophages. Table 1. Platelet count and spleen/body weight ratio in WASp KO / FcγRIIA TG mice relative to controls. WASp WT / FcγRIIA WT WASp WT / FcγRIIA TG WASp KO / FcγRIIA WT WASp KO / FcγRIIA TG Results represent mean ± SD. The statistical analysis was performed by analysis of covariance (ANCOVA) with an adjustment on the spleen/body weight ratio. The family error rate for the multiple comparisons was maintained below 0.05 (Sidak method). Platelet count (× 103/μl) 998 ± 145 963 ± 172 677 ± 147 682 ± 120 p &lt; 10−7 Spleen/body weight ratio (mg/g) 2.9 ± 0.6 2.9 ± 0.5 4.1 ± 1.6 3.8 ± 1.8 p &lt; 0.05 Number of males (adjusted) 31 35 30 30

Platelet-Associated IgG May Be Associated with Thrombocytopenia In Patients with Myelodysplastic Syndromes

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2943-2943
Author(s):  
Simone Gilli ◽  
Samuel de Souza Medina ◽  
Vagner Castro ◽  
Sara T. Olalla-Saad
Keyword(s):  
Bone Marrow ◽  
Platelet Count ◽  
Myelodysplastic Syndromes ◽  
Screening Test ◽  
Immune Thrombocytopenia ◽  
Severe Thrombocytopenia ◽  
Platelet Indices ◽  
Wilcoxon Rank Sum Test ◽  
Highly Sensitive ◽  
Spearman Test

Abstract Abstract 2943 Background. Myelodysplastic Syndromes (MDS) are a heterogeneous group of bone marrow disorders characterized by ineffective and dysplastic hematopoiesis, progressive bone marrow failure, cytopenias and a high risk of transformation into acute leukemia. Thrombocytopenia is detected in up to two thirds of patients with MDS and severe thrombocytopenia is present in approximately 10%. Besides ineffective thrombopoiesis, immune destruction of platelets could be an additional factor in the genesis of thrombocytopenia, since immunological abnormalities are also frequent in patients with MDS. The detection of platelet associated IgG (PAIgG) by immunofluorescence (platelet immunofluorescence test or PIFT) is a highly sensitive assay. In addition, some morphological platelet indices (PDW and MPV) are correlated with the occurrence of immune thrombocytopenia. We prospectively analysed platelet-bound IgG and platelet indices (PDW and MPV) in 35 patients with MDS. Methods: Thirty-five patients with MDS (mean age ± SD: 63 ± 19 yo; range 21–89 yo; 15female/20male) were evaluated. According to FAB, 27 patients were classified as RA, 5 as RARS and 3 as RAEB. Clinical manifestations of immunological disorder were not present in this population. Blood samples were analyzed by PIFT, in order to detect platelet associated IgG and results were expressed as a ratio of patient fluorescence/negative control fluorescence (R). Cell-dyn Sapphire blood cell analyzer (Abbott, Illinois, USA) was used to measure platelet count, mean platelet volume (MPV) and platelet size deviation width (PDW). Thrombocytopenia was defined as a platelet count <100 × 109/L. All samples were analyzed on the day of collection. Results. Platelet counts of the entire population ranged from 6.7 to 708 ×109/L, with median of 95.4 × 109/L. Eighteen patients (51.43%) had platelet count <100 × 109/L. A strong association between thrombocytopenia and PAIgG measured by PIFT (R) was demonstrated taking into account three analysis: an inverse correlation between the number of platelets and the fluorescence ratio (p=0.01, r=-0.39, Spearman test), the higher positivity of PIFT in patients with platelet count <100 × 109/L (p=0.007, Wilcoxon rank sum test) and a lower platelet count in patients with positive PIFT (p=0.059, Wilcoxon rank sum test). MPV was significantly higher in patients with platelet count <100 × 109/L (median, min-max: 9.38, 6.9–23.1 vs 8.46, 4.88–12.83; p<0.001, Wilcoxon rank sum test). PDW showed no statistical difference between these groups. Discussion: Immune thrombocytopenia is a relatively frequent hematological disorder of unknown origin and until today depends mainly upon clinical diagnosis. Assays for the detection of glycoprotein-specific antibodies such as MAIPA (monoclonal antibody-specific immobilization of platelet antigens) are highly specific but less sensitive than PIFT. MAIPA is laborious and require a certain amount of platelets, which is not always available in thrombocytopenia Thus, PIFT could be recommended as a screening test to discriminate patients with MDS in whom the hyperdestructive component of thrombocytopenia is important. Our results also showed a higher MPV in patients with platelet count <100 × 109/L, suggesting that this index may be of interest for detection of immune-mediated hyperdestructive thrombocytopenia in MDS. Conclusion: Thus, we propose that a combination of a simple index as MPV and a highly sensitive and easy to perform screening test for PAIgG as PIFT could be applied to select a subset of MDS patients in which we would be able to prevent the overuse of unnecessary platelet transfusions and who could be candidates for an immunosuppressive therapeutic approach. Supported by INCTS, FAPESP, CNPq. Disclosures: No relevant conflicts of interest to declare.

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