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Catherine P M Hayward - One of the best experts on this subject based on the ideXlab platform.
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Thrombin generation abnormalities in Quebec Platelet Disorder.
International journal of laboratory hematology, 2020Co-Authors: Justin Brunet, Georges-etienne Rivard, Tanmya Sharma, Subia Tasneem, Minggao Liang, Michael D. Wilson, Catherine P M HaywardAbstract:INTRODUCTION Calibrated automated thrombograms (CAT) with Platelet-poor (PPP) and Platelet-rich plasma (PRP) have provided useful insights on bleeding Disorders. We used CAT to assess thrombin generation (TG) in Quebec Platelet Disorder (QPD)-a bleeding Disorder caused by a PLAU duplication mutation that increases Platelet (but not plasma) urokinase plasminogen activator (uPA), leading to intraPlatelet (but not systemic) plasmin generation that degrades α-granule proteins and causes Platelet (but not plasma) factor V (FV) deficiency. METHODS Calibrated automated thrombograms was used to test QPD (n = 7) and control (n = 22) PPP and PRP, with or without added tranexamic acid (TXA). TG endpoints were evaluated for relationships to Platelet FV and uPA, plasma FV and tissue factor pathway inhibitor (TFPI) levels, and bleeding scores. RESULTS Quebec Platelet Disorder PPP TG was normal whereas QPD PRP had reduced endogenous thrombin potential and peak thrombin concentrations (P values
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thrombin generation abnormalities in quebec Platelet Disorder
International Journal of Laboratory Hematology, 2020Co-Authors: Justin Brunet, Tanmya Sharma, Subia Tasneem, Minggao Liang, Michael D. Wilson, Georges E Rivard, Catherine P M HaywardAbstract:INTRODUCTION Calibrated automated thrombograms (CAT) with Platelet-poor (PPP) and Platelet-rich plasma (PRP) have provided useful insights on bleeding Disorders. We used CAT to assess thrombin generation (TG) in Quebec Platelet Disorder (QPD)-a bleeding Disorder caused by a PLAU duplication mutation that increases Platelet (but not plasma) urokinase plasminogen activator (uPA), leading to intraPlatelet (but not systemic) plasmin generation that degrades α-granule proteins and causes Platelet (but not plasma) factor V (FV) deficiency. METHODS Calibrated automated thrombograms was used to test QPD (n = 7) and control (n = 22) PPP and PRP, with or without added tranexamic acid (TXA). TG endpoints were evaluated for relationships to Platelet FV and uPA, plasma FV and tissue factor pathway inhibitor (TFPI) levels, and bleeding scores. RESULTS Quebec Platelet Disorder PPP TG was normal whereas QPD PRP had reduced endogenous thrombin potential and peak thrombin concentrations (P values < .01), proportionate to the Platelet FV deficiency (R2 ≥ 0.81), but unrelated to Platelet uPA, plasma FV, or bleeding scores. QPD TG abnormalities were not associated with TFPI abnormalities and were not reproduced by adding uPA to control PRP. TXA increased QPD and control PRP TG more than PPP TG, but it did not fully correct QPD PRP TG abnormalities or improve TG by plasminogen-deficient plasma. CONCLUSION Quebec Platelet Disorder results in a Platelet-specific TG defect, proportionate to the loss of Platelet FV, that is improved but not fully corrected by TXA. Our study provides an interesting example of why it is important to assess both PRP and PPP TG in bleeding Disorders.
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molecular phenotype and bleeding risks of an inherited Platelet Disorder in a family with a runx1 frameshift mutation
Haemophilia, 2017Co-Authors: M S Badin, Andrew D Paterson, John S Waye, J K Iyer, M Chong, L Graf, G E Rivard, Guillaume Pare, Catherine P M HaywardAbstract:Introduction Inherited defects in RUNX1 are important causes of Platelet function Disorders. Aim Our goals were to evaluate RUNX1-related Platelet Disorders among individuals evaluated for uncharacterized, inherited Platelet function Disorders and test a proof of concept that bleeding risks could be quantitatively estimated for typical families with an inherited Platelet function Disorder. Methods Index cases with an uncharacterized inherited Platelet function Disorder were subjected to exome sequencing with confirmation of RUNX1 mutations by Sanger sequencing. Laboratory findings were obtained from medical records and persistence of Platelet non-muscle myosin heavy chain IIB (MYH10), a biomarker of RUNX1 defects, was assessed by Western blotting. Bleeding histories were assessed using standardized assessment tools. Bleeding risks were estimated as odds ratios (OR) using questionnaire data for affected individuals compared to controls. Results Among 12 index cases who had their exomes sequenced, one individual from a family with eight study participants had a c.583dup in RUNX1 that segregated with the disease and was predicted to cause a frameshift and RUNX1 haploinsufficiency. Unlike unaffected family members (n = 2), affected family members (n = 6) had increased bleeding scores and abnormal Platelet aggregation and dense granule release responses to agonists but only some had thrombocytopenia and/or dense granule deficiency. This family's mutation was associated with persistence of MYH10 in Platelets and increased risks (OR 11–440) for wound healing problems and mild bleeding symptoms, including bleeding interfering with lifestyle in women. Conclusion Inherited Platelet dysfunction due to a RUNX1 haploinsufficiency mutation significantly increases bleeding risks.
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the duplication mutation of quebec Platelet Disorder dysregulates plau but not c10orf55 selectively increasing production of normal plau transcripts by megakaryocytes but not granulocytes
PLOS ONE, 2017Co-Authors: Catherine P M Hayward, Subia Tasneem, Minggao Liang, Georges E Rivard, Asim Soomro, Andrew D Paterson, John S Waye, Michael D. WilsonAbstract:Quebec Platelet Disorder (QPD) is a unique bleeding Disorder that markedly increases urokinase plasminogen activator (uPA) in megakaryocytes and Platelets but not in plasma or urine. The cause is tandem duplication of a 78 kb region of chromosome 10 containing PLAU (the uPA gene) and C10orf55, a gene of unknown function. QPD increases uPA in Platelets and megakaryocytes >100 fold, far more than expected for a gene duplication. To investigate the tissue-specific effect that PLAU duplication has on gene expression and transcript structure in QPD, we tested if QPD leads to: 1) overexpression of normal or unique PLAU transcripts; 2) increased uPA in leukocytes; 3) altered levels of C10orf55 mRNA and/or protein in megakaryocytes and leukocytes; and 4) global changes in megakaryocyte gene expression. Primary cells and cultured megakaryocytes from donors were prepared for quantitative reverse polymerase chain reaction analyses, RNA-seq and protein expression analyses. Rapidly isolated blood leukocytes from QPD subjects showed only a 3.9 fold increase in PLAU transcript levels, in keeping with the normal to minimally increased uPA in affinity purified, QPD leukocytes. All subjects had more uPA in granulocytes than monocytes and minimal uPA in lymphocytes. QPD leukocytes expressed PLAU alleles in proportions consistent with an extra copy of PLAU on the disease chromosome, unlike QPD megakaryocytes. QPD PLAU transcripts were consistent with reference gene models, with a much higher proportion of reads originating from the disease chromosome in megakaryocytes than granulocytes. QPD and control megakaryocytes contained minimal reads for C10orf55, and C10orf55 protein was not increased in QPD megakaryocytes or Platelets. Finally, our QPD megakaryocyte transcriptome analysis revealed a global down regulation of the interferon type 1 pathway. We suggest that the low endogenous levels of uPA in blood are actively regulated, and that the regulatory mechanisms are disrupted in QPD in a megakaryocyte-specific manner.
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quebec Platelet Disorder is associated with greater than expected increases in urokinase plasminogen activator in granulocytes and monocytes
Blood, 2013Co-Authors: Subia Tasneem, Georges E Rivard, Andrew D Paterson, Bhupinder Bharaj, John S Waye, Catherine P M HaywardAbstract:Myeloid leukocytes produce urokinase plasminogen activator (uPA), an important activator of fibrinolysis. Stimuli such as lipopolysaccharide and formyl-methionyl-leucyl-phenylalanine (LPS/fMLP), increase the expression of PLAU by myeloid leukocytes. We postulated that the basal, and/or stimulus-induced, uPA production by myeloid leukocytes would be increased in Quebec Platelet Disorder (QPD), a congenital bleeding Disorder caused by duplication of PLAU , the uPA gene. In QPD, plasma and urine uPA levels are within the expected range. However, overexpression of PLAU in QPD was found to emerge during megakaryopoiesis and QPD Platelets to contain >100-fold increased uPA. We investigated PLAU expression by Platelet-free, granulocytes and monocytes that were differentiated in culture, from peripheral blood CD34+ cells, without added thrombopoietin and erythropoietin. QPD and control (C) myeloid cells were harvested on day 7 or 14 of culture (n= 6 samples per subject for each endpoint). Granulocytes and monocytes were isolated by affinity purification. For some experiments, granulocytes from day 14 cultures were treated with: LPS/fMLP; complement and heat activated immunoglobulin (C5a/IgG); thrombin, thrombin plus fibrinogen, or thrombin plus plasma (IIa, IIa/Fg or IIa/plasma). PLAU expression was characterized by real time quantitative polymerase chain reaction and uPA was quantified by enzyme-linked immunosorbent assays. The lysis of plasma clots (optical density endpoint) that had been spiked with day 14 granulocytes or uPA, was assessed with or without added tranexamic acid. Data were expressed as means ± standard error (SEM) and analyzed by Mann-Whitney tests. Higher PLAU expression by QPD myeloid leukocytes was evident in day 7 (23-fold, p = 0.002) and day 14 (13-fold; p = 0.002) myeloid leukocyte cultures, accompanied by increased cellular uPA (day 7, 16-fold increase [pg/106 cells, QPD: 162 ± 11; C: 10.2 ± 0.3, p = 0.002]; day 14, 9-fold increase [pg/106 cells, QPD: 458 ± 8, C: 50.9 ± 2.5, p = 0.002]). In day 14 cultured cells, PLAU expression was higher in QPD granulocytes (57-fold) and monocytes (9-fold)(p values ≤ 0.005), and allele specific analysis confirmed overexpression of PLAU by the disease chromosome. The higher PLAU expression was associated with higher levels of uPA (pg/106 day 14 cultured cells) in QPD granulocytes (170-fold [QPD: 477 ± 33, C: 2.8 ± 0.4, p = 0.005]) and monocytes (5-fold increase [QPD: 208 ± 11; C: 41.5 ± 4.3; p = 0.002]). Like control granulocytes, QPD granulocytes expressed and secreted much more uPA after exposure to immune, infectious and procoagulant stimuli ([Table 1][1]). Interestingly, some of these stimuli reduced the differences between QPD and control granulocytes in PLAU expression and secreted uPA ([Table 1][1]). In fibrin clots, QPD granulocytes triggered more fibrinolysis than control granulocytes, and release higher levels of uPA. Tranexamic acid inhibited the fibrinolysis-induced by QPD and control granulocytes. View this table: Table1 Effect of stimuli on PLAU expression and uPA secreted by granulocytes We conclude that the greater than expected effect of the QPD PLAU duplication on PLAU expression during myeloid differentiation extends to granulocytes and monocytes. In QPD, PLAU dysregulation was associated with much higher uPA levels in granulocytes than in monocytes. Based on changes to PLAU expression and uPA in response to procoagulant, immune or septic stimuli, we conclude that QPD has greater effects on the basal expression of PLAU by myeloid cells. The upregulation of PLAU expression by granulocytes exposed to thrombin, fibrin clots, immune and septic stimuli, may contribute to the increased fibrinolysis in some pathological conditions. Disclosures: No relevant conflicts of interest to declare. [1]: #T1
Georges E Rivard - One of the best experts on this subject based on the ideXlab platform.
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thrombin generation abnormalities in quebec Platelet Disorder
International Journal of Laboratory Hematology, 2020Co-Authors: Justin Brunet, Tanmya Sharma, Subia Tasneem, Minggao Liang, Michael D. Wilson, Georges E Rivard, Catherine P M HaywardAbstract:INTRODUCTION Calibrated automated thrombograms (CAT) with Platelet-poor (PPP) and Platelet-rich plasma (PRP) have provided useful insights on bleeding Disorders. We used CAT to assess thrombin generation (TG) in Quebec Platelet Disorder (QPD)-a bleeding Disorder caused by a PLAU duplication mutation that increases Platelet (but not plasma) urokinase plasminogen activator (uPA), leading to intraPlatelet (but not systemic) plasmin generation that degrades α-granule proteins and causes Platelet (but not plasma) factor V (FV) deficiency. METHODS Calibrated automated thrombograms was used to test QPD (n = 7) and control (n = 22) PPP and PRP, with or without added tranexamic acid (TXA). TG endpoints were evaluated for relationships to Platelet FV and uPA, plasma FV and tissue factor pathway inhibitor (TFPI) levels, and bleeding scores. RESULTS Quebec Platelet Disorder PPP TG was normal whereas QPD PRP had reduced endogenous thrombin potential and peak thrombin concentrations (P values < .01), proportionate to the Platelet FV deficiency (R2 ≥ 0.81), but unrelated to Platelet uPA, plasma FV, or bleeding scores. QPD TG abnormalities were not associated with TFPI abnormalities and were not reproduced by adding uPA to control PRP. TXA increased QPD and control PRP TG more than PPP TG, but it did not fully correct QPD PRP TG abnormalities or improve TG by plasminogen-deficient plasma. CONCLUSION Quebec Platelet Disorder results in a Platelet-specific TG defect, proportionate to the loss of Platelet FV, that is improved but not fully corrected by TXA. Our study provides an interesting example of why it is important to assess both PRP and PPP TG in bleeding Disorders.
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the duplication mutation of quebec Platelet Disorder dysregulates plau but not c10orf55 selectively increasing production of normal plau transcripts by megakaryocytes but not granulocytes
PLOS ONE, 2017Co-Authors: Catherine P M Hayward, Subia Tasneem, Minggao Liang, Georges E Rivard, Asim Soomro, Andrew D Paterson, John S Waye, Michael D. WilsonAbstract:Quebec Platelet Disorder (QPD) is a unique bleeding Disorder that markedly increases urokinase plasminogen activator (uPA) in megakaryocytes and Platelets but not in plasma or urine. The cause is tandem duplication of a 78 kb region of chromosome 10 containing PLAU (the uPA gene) and C10orf55, a gene of unknown function. QPD increases uPA in Platelets and megakaryocytes >100 fold, far more than expected for a gene duplication. To investigate the tissue-specific effect that PLAU duplication has on gene expression and transcript structure in QPD, we tested if QPD leads to: 1) overexpression of normal or unique PLAU transcripts; 2) increased uPA in leukocytes; 3) altered levels of C10orf55 mRNA and/or protein in megakaryocytes and leukocytes; and 4) global changes in megakaryocyte gene expression. Primary cells and cultured megakaryocytes from donors were prepared for quantitative reverse polymerase chain reaction analyses, RNA-seq and protein expression analyses. Rapidly isolated blood leukocytes from QPD subjects showed only a 3.9 fold increase in PLAU transcript levels, in keeping with the normal to minimally increased uPA in affinity purified, QPD leukocytes. All subjects had more uPA in granulocytes than monocytes and minimal uPA in lymphocytes. QPD leukocytes expressed PLAU alleles in proportions consistent with an extra copy of PLAU on the disease chromosome, unlike QPD megakaryocytes. QPD PLAU transcripts were consistent with reference gene models, with a much higher proportion of reads originating from the disease chromosome in megakaryocytes than granulocytes. QPD and control megakaryocytes contained minimal reads for C10orf55, and C10orf55 protein was not increased in QPD megakaryocytes or Platelets. Finally, our QPD megakaryocyte transcriptome analysis revealed a global down regulation of the interferon type 1 pathway. We suggest that the low endogenous levels of uPA in blood are actively regulated, and that the regulatory mechanisms are disrupted in QPD in a megakaryocyte-specific manner.
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quebec Platelet Disorder is associated with greater than expected increases in urokinase plasminogen activator in granulocytes and monocytes
Blood, 2013Co-Authors: Subia Tasneem, Georges E Rivard, Andrew D Paterson, Bhupinder Bharaj, John S Waye, Catherine P M HaywardAbstract:Myeloid leukocytes produce urokinase plasminogen activator (uPA), an important activator of fibrinolysis. Stimuli such as lipopolysaccharide and formyl-methionyl-leucyl-phenylalanine (LPS/fMLP), increase the expression of PLAU by myeloid leukocytes. We postulated that the basal, and/or stimulus-induced, uPA production by myeloid leukocytes would be increased in Quebec Platelet Disorder (QPD), a congenital bleeding Disorder caused by duplication of PLAU , the uPA gene. In QPD, plasma and urine uPA levels are within the expected range. However, overexpression of PLAU in QPD was found to emerge during megakaryopoiesis and QPD Platelets to contain >100-fold increased uPA. We investigated PLAU expression by Platelet-free, granulocytes and monocytes that were differentiated in culture, from peripheral blood CD34+ cells, without added thrombopoietin and erythropoietin. QPD and control (C) myeloid cells were harvested on day 7 or 14 of culture (n= 6 samples per subject for each endpoint). Granulocytes and monocytes were isolated by affinity purification. For some experiments, granulocytes from day 14 cultures were treated with: LPS/fMLP; complement and heat activated immunoglobulin (C5a/IgG); thrombin, thrombin plus fibrinogen, or thrombin plus plasma (IIa, IIa/Fg or IIa/plasma). PLAU expression was characterized by real time quantitative polymerase chain reaction and uPA was quantified by enzyme-linked immunosorbent assays. The lysis of plasma clots (optical density endpoint) that had been spiked with day 14 granulocytes or uPA, was assessed with or without added tranexamic acid. Data were expressed as means ± standard error (SEM) and analyzed by Mann-Whitney tests. Higher PLAU expression by QPD myeloid leukocytes was evident in day 7 (23-fold, p = 0.002) and day 14 (13-fold; p = 0.002) myeloid leukocyte cultures, accompanied by increased cellular uPA (day 7, 16-fold increase [pg/106 cells, QPD: 162 ± 11; C: 10.2 ± 0.3, p = 0.002]; day 14, 9-fold increase [pg/106 cells, QPD: 458 ± 8, C: 50.9 ± 2.5, p = 0.002]). In day 14 cultured cells, PLAU expression was higher in QPD granulocytes (57-fold) and monocytes (9-fold)(p values ≤ 0.005), and allele specific analysis confirmed overexpression of PLAU by the disease chromosome. The higher PLAU expression was associated with higher levels of uPA (pg/106 day 14 cultured cells) in QPD granulocytes (170-fold [QPD: 477 ± 33, C: 2.8 ± 0.4, p = 0.005]) and monocytes (5-fold increase [QPD: 208 ± 11; C: 41.5 ± 4.3; p = 0.002]). Like control granulocytes, QPD granulocytes expressed and secreted much more uPA after exposure to immune, infectious and procoagulant stimuli ([Table 1][1]). Interestingly, some of these stimuli reduced the differences between QPD and control granulocytes in PLAU expression and secreted uPA ([Table 1][1]). In fibrin clots, QPD granulocytes triggered more fibrinolysis than control granulocytes, and release higher levels of uPA. Tranexamic acid inhibited the fibrinolysis-induced by QPD and control granulocytes. View this table: Table1 Effect of stimuli on PLAU expression and uPA secreted by granulocytes We conclude that the greater than expected effect of the QPD PLAU duplication on PLAU expression during myeloid differentiation extends to granulocytes and monocytes. In QPD, PLAU dysregulation was associated with much higher uPA levels in granulocytes than in monocytes. Based on changes to PLAU expression and uPA in response to procoagulant, immune or septic stimuli, we conclude that QPD has greater effects on the basal expression of PLAU by myeloid cells. The upregulation of PLAU expression by granulocytes exposed to thrombin, fibrin clots, immune and septic stimuli, may contribute to the increased fibrinolysis in some pathological conditions. Disclosures: No relevant conflicts of interest to declare. [1]: #T1
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Simultaneous measurement of adenosine triphosphate release and aggregation potentiates human Platelet aggregation responses for some subjects, including persons with Quebec Platelet Disorder.
Thrombosis and haemostasis, 2012Co-Authors: Catherine P M Hayward, Subia Tasneem, Karen A. Moffat, Jean-francois Castilloux, Yang Liu, Jodi Seecharan, Stephen A. Carlino, Anik Cormier, Georges E RivardAbstract:Platelet aggregometry and dense granule adenosine triphosphate (ATP) release assays are helpful to diagnose Platelet Disorders. Some laboratories simultaneously measure aggregation and ATP release using Chronolume® a commercial reagent containing D-luciferin, firefly luciferase and magnesium. Chronolume® can potentiate sub-maximal aggregation responses, normalising canine Platelet Disorder findings. We investigated if Chronolume® potentiates human Platelet aggregation responses after observing discrepancies suspicious of potentiation. Among patients simultaneously tested by light transmission aggregometry (LTA) on two instruments, 18/43 (42%), including 14/24 (58%) with Platelet Disorders, showed full secondary aggregation with one or more agonists only in tests with Chronolume®. As subjects with Quebec Platelet Disorder (QPD) did not show the expected absent secondary aggregation responses to epinephrine in tests with Chronolume®, the reason for the discrepancy was investigated using samples from 10 QPD subjects. Like sub-threshold ADP (0.75 μM), Chronolume® significantly increased QPD LTA responses to epinephrine (p
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simultaneous measurement of adenosine triphosphate release and aggregation potentiates human Platelet aggregation responses for some subjects including persons with quebec Platelet Disorder
Thrombosis and Haemostasis, 2012Co-Authors: Catherine P M Hayward, Subia Tasneem, Karen A. Moffat, Jean-francois Castilloux, Yang Liu, Jodi Seecharan, Stephen A. Carlino, Anik Cormier, Georges E RivardAbstract:Platelet aggregometry and dense granule adenosine triphosphate (ATP) release assays are helpful to diagnose Platelet Disorders. Some laboratories simultaneously measure aggregation and ATP release using Chronolume® a commercial reagent containing D-luciferin, firefly luciferase and magnesium. Chronolume® can potentiate sub-maximal aggregation responses, normalising canine Platelet Disorder findings. We investigated if Chronolume® potentiates human Platelet aggregation responses after observing discrepancies suspicious of potentiation. Among patients simultaneously tested by light transmission aggregometry (LTA) on two instruments, 18/43 (42%), including 14/24 (58%) with Platelet Disorders, showed full secondary aggregation with one or more agonists only in tests with Chronolume®. As subjects with Quebec Platelet Disorder (QPD) did not show the expected absent secondary aggregation responses to epinephrine in tests with Chronolume®, the reason for the discrepancy was investigated using samples from 10 QPD subjects. Like sub-threshold ADP (0.75 μM), Chronolume® significantly increased QPD LTA responses to epinephrine (p<0.0001) and it increased both initial and secondary aggregation responses, leading to dense granule release. This potentiation was not restricted to QPD and it was mimicked adding 1-2 mM magnesium, but not D-luciferin or firefly luciferase, to LTA assays. Chronolume® potentiated the ADP aggregation responses of QPD subjects with a reduced response. Furthermore, it increased whole blood aggregation responses of healthy control samples to multiple agonists, tested at concentrations used for the diagnosis of Platelet Disorders (p values <0.05). Laboratories should be aware that measuring ATP release with Chronolume® can potentiate LTA and whole blood aggregation responses, which alters findings for some human Platelet Disorders, including QPD.
Akihide Yoshimi - One of the best experts on this subject based on the ideXlab platform.
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genetic basis of myeloid transformation in familial Platelet Disorder acute myeloid leukemia patients with haploinsufficient runx1 allele
Blood Cancer Journal, 2016Co-Authors: Masatoshi Sakurai, Hidenori Kasahara, Kenichi Yoshida, Akihide Yoshimi, Hiroyoshi Kunimoto, Naohide Watanabe, Yuichi Shiraishi, Kenichi Chiba, Hirotoshi Tanaka, Yuka HaradaAbstract:Genetic basis of myeloid transformation in familial Platelet Disorder/acute myeloid leukemia patients with haploinsufficient RUNX1 allele
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Genetic basis of myeloid transformation in familial Platelet Disorder/acute myeloid leukemia patients with haploinsufficient RUNX1 allele.
Blood cancer journal, 2016Co-Authors: Masatoshi Sakurai, Hidenori Kasahara, Kenichi Yoshida, Akihide Yoshimi, Hiroyoshi Kunimoto, Naohide Watanabe, Yuichi Shiraishi, Kenichi Chiba, Hirotoshi Tanaka, Yuka HaradaAbstract:Genetic basis of myeloid transformation in familial Platelet Disorder/acute myeloid leukemia patients with haploinsufficient RUNX1 allele
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targeted gene correction of runx1 in induced pluripotent stem cells derived from familial Platelet Disorder with propensity to myeloid malignancy restores normal megakaryopoiesis
Experimental Hematology, 2015Co-Authors: Hiromitsu Iizuka, Akihide Yoshimi, Yuki Kagoya, Keisuke Kataoka, Masashi Miyauchi, Kazuki Taoka, Keiki Kumano, Takashi Yamamoto, Akitsu HottaAbstract:Familial Platelet Disorder with propensity to acute myeloid leukemia (FPD/AML) is an autosomal dominant disease associated with a germline mutation in the RUNX1 gene and is characterized by thrombocytopenia and an increased risk of developing myeloid malignancies. We generated induced pluripotent stem cells (iPSCs) from dermal fibroblasts of a patient with FPD/AML possessing a nonsense mutation R174X in the RUNX1 gene. Consistent with the clinical characteristics of the disease, FPD iPSC-derived hematopoietic progenitor cells were significantly impaired in undergoing megakaryocytic differentiation and subsequent maturation, as determined by colony-forming cell assay and surface marker analysis. Notably, when we corrected the RUNX1 mutation using transcription activator-like effector nucleases in conjunction with a donor plasmid containing normal RUNX1 cDNA sequences, megakaryopoiesis and subsequent maturation were restored in FPD iPSC-derived hematopoietic cells. These findings clearly indicate that the RUNX1 mutation is robustly associated with thrombocytopenia in patients with FPD/AML, and transcription activator-like effector nuclease-mediated gene correction in iPSCs generated from patient-derived cells could provide a promising clinical application for treatment of the disease.
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Recurrent CDC25C mutations drive malignant transformation in FPD/AML
Nature communications, 2014Co-Authors: Akihide Yoshimi, Masahiro Nakagawa, Yasuhito Nannya, Takashi Toya, Shunya Arai, Hiromitsu Iizuka, Masahito Kawazu, Toshihide Ueno, Ayato Tsukamoto, Hironori HaradaAbstract:Familial Platelet Disorder (FPD) with predisposition to acute myelogenous leukaemia (AML) is characterized by Platelet defects with a propensity for the development of haematological malignancies. Its molecular pathogenesis is poorly understood, except for the role of germline RUNX1 mutations. Here we show that CDC25C mutations are frequently found in FPD/AML patients (53%). Mutated CDC25C disrupts the G2/M checkpoint and promotes cell cycle progression even in the presence of DNA damage, suggesting a critical role for CDC25C in malignant transformation in FPD/AML. The predicted hierarchical architecture shows that CDC25C mutations define a founding pre-leukaemic clone, followed by stepwise acquisition of subclonal mutations that contribute to leukaemia progression. In three of seven individuals with CDC25C mutations, GATA2 is the target of subsequent mutation. Thus, CDC25C is a novel gene target identified in haematological malignancies. CDC25C is also useful as a clinical biomarker that predicts progression of FPD/AML in the early stage.
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recurrent cdc25c mutations drive malignant transformation in fpd aml
Nature Communications, 2014Co-Authors: Akihide Yoshimi, Masahiro Nakagawa, Yasuhito Nannya, Takashi Toya, Shunya Arai, Hiromitsu Iizuka, Masahito Kawazu, Toshihide Ueno, Ayato Tsukamoto, Hironori HaradaAbstract:Familial Platelet Disorder (FPD) with predisposition to acute myelogenous leukaemia (AML) is characterized by Platelet defects with a propensity for the development of haematological malignancies. Its molecular pathogenesis is poorly understood, except for the role of germline RUNX1 mutations. Here we show that CDC25C mutations are frequently found in FPD/AML patients (53%). Mutated CDC25C disrupts the G2/M checkpoint and promotes cell cycle progression even in the presence of DNA damage, suggesting a critical role for CDC25C in malignant transformation in FPD/AML. The predicted hierarchical architecture shows that CDC25C mutations define a founding pre-leukaemic clone, followed by stepwise acquisition of subclonal mutations that contribute to leukaemia progression. In three of seven individuals with CDC25C mutations, GATA2 is the target of subsequent mutation. Thus, CDC25C is a novel gene target identified in haematological malignancies. CDC25C is also useful as a clinical biomarker that predicts progression of FPD/AML in the early stage.
Subia Tasneem - One of the best experts on this subject based on the ideXlab platform.
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enhancer gene rewiring in the pathogenesis of quebec Platelet Disorder
Blood, 2020Co-Authors: Minggao Liang, Subia Tasneem, Asim Soomro, Luis E Abatti, Azad Alizada, Xuefei Yuan, Liis Uuskulareimand, Lina Antounians, Sana Akhtar Alvi, Andrew D PatersonAbstract:Abstract Quebec Platelet Disorder (QPD) is an autosomal dominant bleeding Disorder with a unique, Platelet-dependent, gain-of-function defect in fibrinolysis, without systemic fibrinolysis. The hallmark feature of QPD is a >100-fold overexpression of PLAU, specifically in megakaryocytes. This overexpression leads to a >100-fold increase in Platelet stores of urokinase plasminogen activator (PLAU/uPA); subsequent plasmin-mediated degradation of diverse α-granule proteins; and Platelet-dependent, accelerated fibrinolysis. The causative mutation is a 78-kb tandem duplication of PLAU. How this duplication causes megakaryocyte-specific PLAU overexpression is unknown. To investigate the mechanism that causes QPD, we used epigenomic profiling, comparative genomics, and chromatin conformation capture approaches to study PLAU regulation in cultured megakaryocytes from participants with QPD and unaffected controls. QPD duplication led to ectopic interactions between PLAU and a conserved megakaryocyte enhancer found within the same topologically associating domain (TAD). Our results support a unique disease mechanism whereby the reorganization of sub-TAD genome architecture results in a dramatic, cell-type–specific blood Disorder phenotype.
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Thrombin generation abnormalities in Quebec Platelet Disorder.
International journal of laboratory hematology, 2020Co-Authors: Justin Brunet, Georges-etienne Rivard, Tanmya Sharma, Subia Tasneem, Minggao Liang, Michael D. Wilson, Catherine P M HaywardAbstract:INTRODUCTION Calibrated automated thrombograms (CAT) with Platelet-poor (PPP) and Platelet-rich plasma (PRP) have provided useful insights on bleeding Disorders. We used CAT to assess thrombin generation (TG) in Quebec Platelet Disorder (QPD)-a bleeding Disorder caused by a PLAU duplication mutation that increases Platelet (but not plasma) urokinase plasminogen activator (uPA), leading to intraPlatelet (but not systemic) plasmin generation that degrades α-granule proteins and causes Platelet (but not plasma) factor V (FV) deficiency. METHODS Calibrated automated thrombograms was used to test QPD (n = 7) and control (n = 22) PPP and PRP, with or without added tranexamic acid (TXA). TG endpoints were evaluated for relationships to Platelet FV and uPA, plasma FV and tissue factor pathway inhibitor (TFPI) levels, and bleeding scores. RESULTS Quebec Platelet Disorder PPP TG was normal whereas QPD PRP had reduced endogenous thrombin potential and peak thrombin concentrations (P values
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thrombin generation abnormalities in quebec Platelet Disorder
International Journal of Laboratory Hematology, 2020Co-Authors: Justin Brunet, Tanmya Sharma, Subia Tasneem, Minggao Liang, Michael D. Wilson, Georges E Rivard, Catherine P M HaywardAbstract:INTRODUCTION Calibrated automated thrombograms (CAT) with Platelet-poor (PPP) and Platelet-rich plasma (PRP) have provided useful insights on bleeding Disorders. We used CAT to assess thrombin generation (TG) in Quebec Platelet Disorder (QPD)-a bleeding Disorder caused by a PLAU duplication mutation that increases Platelet (but not plasma) urokinase plasminogen activator (uPA), leading to intraPlatelet (but not systemic) plasmin generation that degrades α-granule proteins and causes Platelet (but not plasma) factor V (FV) deficiency. METHODS Calibrated automated thrombograms was used to test QPD (n = 7) and control (n = 22) PPP and PRP, with or without added tranexamic acid (TXA). TG endpoints were evaluated for relationships to Platelet FV and uPA, plasma FV and tissue factor pathway inhibitor (TFPI) levels, and bleeding scores. RESULTS Quebec Platelet Disorder PPP TG was normal whereas QPD PRP had reduced endogenous thrombin potential and peak thrombin concentrations (P values < .01), proportionate to the Platelet FV deficiency (R2 ≥ 0.81), but unrelated to Platelet uPA, plasma FV, or bleeding scores. QPD TG abnormalities were not associated with TFPI abnormalities and were not reproduced by adding uPA to control PRP. TXA increased QPD and control PRP TG more than PPP TG, but it did not fully correct QPD PRP TG abnormalities or improve TG by plasminogen-deficient plasma. CONCLUSION Quebec Platelet Disorder results in a Platelet-specific TG defect, proportionate to the loss of Platelet FV, that is improved but not fully corrected by TXA. Our study provides an interesting example of why it is important to assess both PRP and PPP TG in bleeding Disorders.
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the duplication mutation of quebec Platelet Disorder dysregulates plau but not c10orf55 selectively increasing production of normal plau transcripts by megakaryocytes but not granulocytes
PLOS ONE, 2017Co-Authors: Catherine P M Hayward, Subia Tasneem, Minggao Liang, Georges E Rivard, Asim Soomro, Andrew D Paterson, John S Waye, Michael D. WilsonAbstract:Quebec Platelet Disorder (QPD) is a unique bleeding Disorder that markedly increases urokinase plasminogen activator (uPA) in megakaryocytes and Platelets but not in plasma or urine. The cause is tandem duplication of a 78 kb region of chromosome 10 containing PLAU (the uPA gene) and C10orf55, a gene of unknown function. QPD increases uPA in Platelets and megakaryocytes >100 fold, far more than expected for a gene duplication. To investigate the tissue-specific effect that PLAU duplication has on gene expression and transcript structure in QPD, we tested if QPD leads to: 1) overexpression of normal or unique PLAU transcripts; 2) increased uPA in leukocytes; 3) altered levels of C10orf55 mRNA and/or protein in megakaryocytes and leukocytes; and 4) global changes in megakaryocyte gene expression. Primary cells and cultured megakaryocytes from donors were prepared for quantitative reverse polymerase chain reaction analyses, RNA-seq and protein expression analyses. Rapidly isolated blood leukocytes from QPD subjects showed only a 3.9 fold increase in PLAU transcript levels, in keeping with the normal to minimally increased uPA in affinity purified, QPD leukocytes. All subjects had more uPA in granulocytes than monocytes and minimal uPA in lymphocytes. QPD leukocytes expressed PLAU alleles in proportions consistent with an extra copy of PLAU on the disease chromosome, unlike QPD megakaryocytes. QPD PLAU transcripts were consistent with reference gene models, with a much higher proportion of reads originating from the disease chromosome in megakaryocytes than granulocytes. QPD and control megakaryocytes contained minimal reads for C10orf55, and C10orf55 protein was not increased in QPD megakaryocytes or Platelets. Finally, our QPD megakaryocyte transcriptome analysis revealed a global down regulation of the interferon type 1 pathway. We suggest that the low endogenous levels of uPA in blood are actively regulated, and that the regulatory mechanisms are disrupted in QPD in a megakaryocyte-specific manner.
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quebec Platelet Disorder is associated with greater than expected increases in urokinase plasminogen activator in granulocytes and monocytes
Blood, 2013Co-Authors: Subia Tasneem, Georges E Rivard, Andrew D Paterson, Bhupinder Bharaj, John S Waye, Catherine P M HaywardAbstract:Myeloid leukocytes produce urokinase plasminogen activator (uPA), an important activator of fibrinolysis. Stimuli such as lipopolysaccharide and formyl-methionyl-leucyl-phenylalanine (LPS/fMLP), increase the expression of PLAU by myeloid leukocytes. We postulated that the basal, and/or stimulus-induced, uPA production by myeloid leukocytes would be increased in Quebec Platelet Disorder (QPD), a congenital bleeding Disorder caused by duplication of PLAU , the uPA gene. In QPD, plasma and urine uPA levels are within the expected range. However, overexpression of PLAU in QPD was found to emerge during megakaryopoiesis and QPD Platelets to contain >100-fold increased uPA. We investigated PLAU expression by Platelet-free, granulocytes and monocytes that were differentiated in culture, from peripheral blood CD34+ cells, without added thrombopoietin and erythropoietin. QPD and control (C) myeloid cells were harvested on day 7 or 14 of culture (n= 6 samples per subject for each endpoint). Granulocytes and monocytes were isolated by affinity purification. For some experiments, granulocytes from day 14 cultures were treated with: LPS/fMLP; complement and heat activated immunoglobulin (C5a/IgG); thrombin, thrombin plus fibrinogen, or thrombin plus plasma (IIa, IIa/Fg or IIa/plasma). PLAU expression was characterized by real time quantitative polymerase chain reaction and uPA was quantified by enzyme-linked immunosorbent assays. The lysis of plasma clots (optical density endpoint) that had been spiked with day 14 granulocytes or uPA, was assessed with or without added tranexamic acid. Data were expressed as means ± standard error (SEM) and analyzed by Mann-Whitney tests. Higher PLAU expression by QPD myeloid leukocytes was evident in day 7 (23-fold, p = 0.002) and day 14 (13-fold; p = 0.002) myeloid leukocyte cultures, accompanied by increased cellular uPA (day 7, 16-fold increase [pg/106 cells, QPD: 162 ± 11; C: 10.2 ± 0.3, p = 0.002]; day 14, 9-fold increase [pg/106 cells, QPD: 458 ± 8, C: 50.9 ± 2.5, p = 0.002]). In day 14 cultured cells, PLAU expression was higher in QPD granulocytes (57-fold) and monocytes (9-fold)(p values ≤ 0.005), and allele specific analysis confirmed overexpression of PLAU by the disease chromosome. The higher PLAU expression was associated with higher levels of uPA (pg/106 day 14 cultured cells) in QPD granulocytes (170-fold [QPD: 477 ± 33, C: 2.8 ± 0.4, p = 0.005]) and monocytes (5-fold increase [QPD: 208 ± 11; C: 41.5 ± 4.3; p = 0.002]). Like control granulocytes, QPD granulocytes expressed and secreted much more uPA after exposure to immune, infectious and procoagulant stimuli ([Table 1][1]). Interestingly, some of these stimuli reduced the differences between QPD and control granulocytes in PLAU expression and secreted uPA ([Table 1][1]). In fibrin clots, QPD granulocytes triggered more fibrinolysis than control granulocytes, and release higher levels of uPA. Tranexamic acid inhibited the fibrinolysis-induced by QPD and control granulocytes. View this table: Table1 Effect of stimuli on PLAU expression and uPA secreted by granulocytes We conclude that the greater than expected effect of the QPD PLAU duplication on PLAU expression during myeloid differentiation extends to granulocytes and monocytes. In QPD, PLAU dysregulation was associated with much higher uPA levels in granulocytes than in monocytes. Based on changes to PLAU expression and uPA in response to procoagulant, immune or septic stimuli, we conclude that QPD has greater effects on the basal expression of PLAU by myeloid cells. The upregulation of PLAU expression by granulocytes exposed to thrombin, fibrin clots, immune and septic stimuli, may contribute to the increased fibrinolysis in some pathological conditions. Disclosures: No relevant conflicts of interest to declare. [1]: #T1
Akitsu Hotta - One of the best experts on this subject based on the ideXlab platform.
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targeted gene correction of runx1 in induced pluripotent stem cells derived from familial Platelet Disorder with propensity to myeloid malignancy restores normal megakaryopoiesis
Experimental Hematology, 2015Co-Authors: Hiromitsu Iizuka, Akihide Yoshimi, Yuki Kagoya, Keisuke Kataoka, Masashi Miyauchi, Kazuki Taoka, Keiki Kumano, Takashi Yamamoto, Akitsu HottaAbstract:Familial Platelet Disorder with propensity to acute myeloid leukemia (FPD/AML) is an autosomal dominant disease associated with a germline mutation in the RUNX1 gene and is characterized by thrombocytopenia and an increased risk of developing myeloid malignancies. We generated induced pluripotent stem cells (iPSCs) from dermal fibroblasts of a patient with FPD/AML possessing a nonsense mutation R174X in the RUNX1 gene. Consistent with the clinical characteristics of the disease, FPD iPSC-derived hematopoietic progenitor cells were significantly impaired in undergoing megakaryocytic differentiation and subsequent maturation, as determined by colony-forming cell assay and surface marker analysis. Notably, when we corrected the RUNX1 mutation using transcription activator-like effector nucleases in conjunction with a donor plasmid containing normal RUNX1 cDNA sequences, megakaryopoiesis and subsequent maturation were restored in FPD iPSC-derived hematopoietic cells. These findings clearly indicate that the RUNX1 mutation is robustly associated with thrombocytopenia in patients with FPD/AML, and transcription activator-like effector nuclease-mediated gene correction in iPSCs generated from patient-derived cells could provide a promising clinical application for treatment of the disease.