The duplication mutation of Quebec platelet disorder dysregulates PLAU, but not C10orf55, selectively increasing production of normal PLAU transcripts by megakaryocytes but not granulocytes.

Catherine P M Hayward,Andrew D Paterson,Michael D Wilson,John S Waye,Georges E Rivard,Minggao Liang,Subia Tasneem,Asim Soomro,Christian Schulz

doi:10.1371/journal.pone.0173991

Abstract

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.

Highlights

Urokinase-type plasminogen activator is an important activator of fibrinolysis
To further determine if Quebec platelet disorder (QPD) selectively dysregulates PLAU in megakaryocytes, we investigated if QPD leads to: 1) markedly increased levels of PLAU transcripts and Urokinase-type plasminogen activator (uPA) in leukocytes, and in cells of the granulocyte and monocyte lineages as like megakaryocytes, these cells are derived from myeloid differentiation of a common hematopoietic progenitor cell; 2) overproduction of specific PLAU transcripts by the disease chromosome; 3) altered levels of C10orf55 mRNA and protein in megakaryocytes/platelets and/or leukocytes and 4) global gene expression differences in megakaryocytes
Western blots indicated that the uPA in QPD and control leukocytes had the same mobility and confirmed that the amount of uPA was only modestly increased in QPD granulocyte and monocytes (Fig 1B and 1C)

Summary

Introduction

Urokinase-type plasminogen activator (uPA) is an important activator of fibrinolysis. The uPA from blood cells is important for fibrinolysis as bone marrow transplantation corrects the fibrinolytic defect of uPA deficient mice [1]. Granulocytes contain most of the cellular uPA and fibrinolytic activity in blood [2, 3]. Platelets inhibit fibrinolysis by releasing large amounts of active plasminogen activator inhibitor 1 (PAI-1) but little uPA [4]. Inherited bleeding disorders that increase uPA in blood cells are rare: the only known example is Quebec platelet disorder (QPD), an autosomal dominant bleeding disorder with a unique, gain-of-function defect in fibrinolysis due to increased platelet uPA [5]. Persons with QPD suffer delayed-onset bleeding following hemostatic challenges unless treated with fibrinolytic inhibitors [6]

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Discussion

Conclusion