B-cell activating factor (BAFF) expression in ITP

Thesis submitted for Partial Fulfillments For master degree (M. Sc.) in clinical and chemical pathology.

Presented by: Marianne Edward Gad El-Kareem (M. B., B. Ch. Cairo University)

Supervised by: Prof Dr. Tayssir Kamel Eyada Professor of clinical and chemical pathology Faculty of medicine, Cairo University.

Dr. Dalia Gamil Amin Dr. Ihab Sameeh Amin Lecturer of clinical and Lecturer of internal Medicine chemical pathology Faculty of medicine Faculty of medicine Cairo university Cairo university

Faculty of medicine Cairo university 2012 Acknowledgement

First and foremost, thanks are to """GOD"GODGOD" for his many indescribable gifts. I can do everything through Him who gives me strength and always leads to triumphal procession.

I would like to express my deepest gratitude and respect to Prof.

Dr. Tayssir Kamel EyadaEyada,,,, Professor of Clinical Pathology, Faculty of

Medicine, Cairo University, , for her moral and scientific support and for giving me the honor of working under her supervision and valuable guidance.

My sincere thanks and deep appreciation goes to Dr. Dalia Gamil

AminAmin, Ass. Professor of Clinical Pathology, Faculty of Medicine, Cairo

University, for her faithful guidance and careful supervision. Her generous contributions and meticulous revisions helped to clarify this study.

Many Special thanks and deepest gratitude to Dr. IhabIhab Sameh

AminAmin,, Lecturer of internal medicine, Faculty of medicine, Cairo

University, for his moral and scientific support

Last but not least, I would like to thank my family for their continuous help, patience, care, support and love.

TABLE OF CONTENTS

Page ‹‹‹LIST OF Abbreviations------I

‹‹‹LIST OF Tables ------V

‹‹‹LIST OF Figures ------VІІ

‹ ABSTRACT------ІX

‹‹‹INTRODUCTION AND AIM OF THE WORK------1

‹‹‹REVIEW OF LITERATURE------

óóó Chapter 1: Immune Thrombocytopenic Purpura------4

óóó Chapter 2: BAFF------57

‹‹‹SUBJECTS AND METHODS------93

‹‹‹RESULTS------106

‹‹‹DISCUSSION------131

‹‹‹SUMMARY------137

‹‹‹REFRENCES------140

‹‹‹ARABIC SUMMARY------

List of abbreviations

‹ A/WySnJ strain :BAFF-R-mutant strain of mice. ‹ A-623 :Peptide fusion . ‹ AchR :Acetylcholine receptor. ‹ Act1 :Adaptor protein actin 1. ‹ ADCC :Induce antibody dependent cell mediated cytotoxicity. ‹ Akt :Serine/threonine kinase. ‹ ANA :Anti- Nuclear Antibodies. ‹ APLA :Antiphospholipid antibodies. ‹ APRIL :A proliferation-inducing ligand. ‹ APS :Antiphospholipid Syndrome. ‹ ASH :American Society of Hematology. ‹ Bad :Bcl-2-associated death promoter protein. ‹ BAFF-R/BR3 :BAFF receptor. ‹ Bak :Bcl-2 homologous antagonist/killer. ‹ Bcl-2 :B-cell lymphoma 2 ‹ B-CLL :B-cell chronic lymphocytic leukemia. ‹ Bcl-xl :B-cell lymphoma-extra-large. ‹ BCMA :B-cell maturation antigen. ‹ BCR :B-cell receptor. ‹ BlyS :B-lymphocyte stimulator. ‹ BM :Bone marrow. ‹ BR3-Fc :BAFF receptor fusion protein. ‹ CAMT :Congenital amegakaryocytic thrombocytopenia. ‹ CCP antibodies :Anti-cyclic citrullinated peptide (CCP) antibodies. ‹ CIC :Circulating immune complexes. ‹ CR :Complete response. ‹ CRD :Cysteine-rich domain. ‹ CRP :C-reactive protein. ‹ CSR :Class switch recombination. ‹ CVID :Common variable immunodeficiency. ‹ DAT :Direct Antiglobulin test. ‹ DCs :Dendritic cells. ‹ DIC :Disseminated Intravascular Coagulation. ‹ DITP :Drug-induced thrombocytopenia. ‹ DTH :Delayed type hypersensitivity. ‹ EBV :Epstein- Barr virus. ‹ EDTA :Ethylenediaminetetraacetic acid. ‹ ES :Evans syndrome .

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‹ ESR :Erythrocyte sedimentation rate. ‹ FDCs :Follicular dendritic cells. ‹ FPD/AML :Familial platelet disorder with predisposition to acute myelogenous Leukemia. ‹ G6PD deficiency :Glucose-6-Phosphate Dehydrogenase deficiency. ‹ GC :Germinal centers. ‹ GP :Glycoprotein. ‹ GPS :Gray Platelet Syndrome. ‹ H. pylori :Helicobacter Pylori. ‹ HCV :Hepatitis C virus. ‹ HIT :Heparin-induced thrombocytopenia. ‹ HIV :Human immunodeficiency virus. ‹ HSPGs :Heparin sulphate proteoglycans. ‹ HUS :Hemolytic-Uremic Syndrome. ‹ IFNs :Interferons. ‹ IgAD :IgA deficiency. ‹ Igs :Immunoglobulins. ‹ INR :International Normalized Ratio. ‹ ISC :Immunoglobulin (Ig)-secreting cells. ‹ ITP :Immune Thrombocytopenic Purpura. ‹ IVIG :Intravenous Immunoglobulin. ‹ LGL :Large granular lymphocytic leukemia. ‹ LPS :Lipopolysaccharides. ‹ Mcl-1 :Induced myeloid leukemia cell differentiation protein. ‹ MDS :Myelodysplastic syndromes. ‹ MKs :Megakaryocytes. ‹ MPV :Mean Platelet Volume. ‹ MS :Multiple sclerosis. ‹ MZ :Marginal zone. ‹ NEMO :NF-kB essential modulator. ‹ NF-kB :Nuclear factor kappa B. ‹ NHL :Non-Hodgkin’s lymphoma. ‹ NK :Natural killer. ‹ NMMHC :Nonmuscle myosin II-A heavy chain. ‹ NR :No response. ‹ PAIgG :Platelet associated IgG. ‹ Pax5 :Paired box protein. ‹ PBC :Primary biliary cirrhosis. ‹ PBMNCs :Peripheral blood mononuclear cells. ‹ PC :Plasma cells.

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‹ PDW :Platelet Distribution Width. ‹ PF4 :platelet factor 4. ‹ Pim2 :Serine/threonine-protein kinase. ‹ PMA :Phorbol myristol acetate. ‹ PTP :Post transfusion purpura. ‹ PTS :Paris-Trousseau Syndrome. ‹ R :Response. ‹ RA :Rheumatoid arthritis. ‹ RAEB :Refractory anemia with excess blasts. ‹ RelB :Transcription factor RelB is a protein that in humans is encoded by the RELB . ‹ SF :Synovial fluid. ‹ SG :Salivary gland. ‹ SjS :Sjogren’s syndrome. ‹ SLE :Systemic Erythematosus. ‹ SN :Supernatant. ‹ SOCS-1 :Suppressor of cytokine signaling-1. ‹ SSc :Systemic sclerosis. ‹ T1 :Type 1. ‹ T2 :Type 2. ‹ T3 :Type 3. ‹ TACI :Transmembrane activator and calcium modulator and cyclophilin Ligand (CAML) interactor. ‹ TALL-1 :TNF and ligand–related leukocyte-expressed ligand-1. ‹ TAR :Thrombocytopenia with absent radius syndrome. ‹ TD Ag :T-cell dependent antigen. ‹ Tg :Transgenic. ‹ Th :T-helper cells. ‹ Th1 :T-helper-1. ‹ THANK :TNF homologue that activates apoptosis, nuclear factor kB and c-Jun and N-terminal kinase. ‹ TI Ag :T-cell independent Ag. ‹ TLR :Toll like receptor. ‹ TNF :Tumour necrotic factor. ‹ TNF-R :Tumour necrosis factor receptor. ‹ TNFSF 13b :TNF superfamily member 13B. ‹ TPO :. ‹ TRAF3 :TNFR-associated factor 3. ‹ TRAFs :TNFR-associated factors. ‹ TTP :Thrombotic Thrombocytopenic Purpura.

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‹ UFH :Un-fractionated heparin. ‹ WAS :Wiskott-Aldrich syndrome. ‹ WM :Waldenstrom’s macroglobulinaemia.

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List of Tables

Page Table (1): Pathophysiologic Classification of Thrombocytopenia………………………..……...5 Table (2): syndromes caused by MYH9 Gene defect…………………………………………….8 Table (3): Classification and causes of immune thrombocytopenia………………………….....12 Table (4): Features of Acute and Chronic Idiopathic Thrombocytopenic Purpura (ITP)……….13 Table (5 ): Clinical features of Idiopathic Thrombocytopenic Purpura in Children and Adults…………………………………………………………………………………………….14 Table (6): Characteristics of Platelet Autoantibodies in Idiopathic Thrombocytopenic Purpura ……………………………………………………………………………………………………20 Table (7): Recommendations for Treatment of Idiopathic Thrombocytopenic Purpura ……….33 Table (8): Therapeutic Agents and Their Dosing Schedules…………………………………....37 Table (9): Specific functions of BAFF-R, TACI and BCMA on human and murine B cells...... 76 Table (10): Pathological role of BAFF in various diseases……………………………….…81-82 Table (11): Molecules in development to target BAFF/APRIL………………………………...87 Table (12): Gender distribution in ITP patients and controls………………………………….106

Table (13): Age distribution in ITP patients and controls……………………………………..107

Table (14): Different ages at onset of the disease and the time of sampling together with the disease duration in the ITP patients…………………………………………………………….107

Table (15 ): The platelet counts of ITP patients at onset of the disease, at sampling as well as at follow up………………………………………………………………………………………..107

Table (16): Hematological data of the ITP patients……………………………………………108 Table (17): BAFF and BAFF-R mRNA expressions in the studied ITP patients and controls………………………………………………………………………………………….110 Table (18): Descriptive classifications of the studied ITP patients group…………………….111 Table (19): Comparison between children ( ≤12y) and adults (>12y) ITP patients as regards different laboratory data as well as BAFF and BAFF-R expression levels…………………….112 Table (20): Comparison between males & females ITP patients as regards different laboratory data as well as BAFF and BAFF-R expression levels………………………………………….113

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Table (21): Comparison between patients with acute and chronic phase disease as regards different laboratory data as well as BAFF and BAFF-R expression levels…………………….114

Table (22): Comparing between patients with active disease and those in remission as regards different laboratory data as well as BAFF and BAFF-R expression levels…………………….116

Table (23): Comparing ITP patients during activity and those during remission with controls as regards BAFF and BAFF-R expression levels…………………………………………………117

Table (24): Comparing between bleeders and non-bleeders as regards different laboratory data as well as BAFF and BAFF-R expression levels……………………………………………….118

Table (25): comparison between patients who responded clinically (cessation of bleeding) and those who didn’t respond as regards different laboratory data as well as BAFF and BAFF-R expression levels………………………………………………………………………………..119

Table (26): comparison between patients with different responses as regards different laboratory data as well as BAFF and BAFF-R expression levels………………………………………….120

Table (27): Lines of management of the studied ITP patients…………………………………122

Table (28): comparison between treated patients and untreated patients (observed) as regards BAFF and BAFF-R expression levels………………………………………………………….123

Table (29): comparison between patients who received Imuran and patients who didn’t receive Imuran as regards BAFF and BAFF-R expression levels…………………………………….125

Table (30): comparison between patients under observation, patients treated with steroids and those treated with both steroids and Imuran as regards BAFF and BAFF-R expression levels……………………………………………………………………………………………126

Table (31): Correlation between BAFF mRNA expression with different clinical and laboratory data among ITP patients………………………………………………………………………...127

Table (32): Correlation between BAFF-R mRNA expression with different clinical and laboratory data among ITP patients…………………………………………………………….129

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Lists of figures

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Figure (1): Pathogenesis of epitope spread in ITP………………………………………………22 Figure (2): Peripheral blood and Bone marrow smears in ITP………………………………….28 Figure (3): Estimated fraction of the various forms of secondary ITP………………………….46 Figure (4): APRIL and BAFF bind to multiple receptors……………………………………….62 Figure (5): Effects of BAFF on various immune cells...……………………………………….66 Figure (6): Distinct effects of BAFF on activation of human B cells…………………………...70 Figure (7): Model of the signaling pathways implicated in BAFF-R-mediated survival signals in primary B cells………………………………………….………………………………………..75 Figure (8): Differential expression of BAFF receptors during human development and differentiation……………………………………………………………………………………76 Figure (9): the gender distribution in ITP patients and controls……………………………….106 Figure (10): The platelet counts of ITP patients at diagnosis, at sampling & at follow up...….108 Figure (11): Hematological data of the ITP patients at sampling and at follow up……………109 Figure (12): BAFF and BAFF-R mRNA median expressions in ITP patients and controls…..110 Figure (13): Representation of different classifications of the studied ITP patients group according to the parameters mentioned in table (18)…………………………………………...111 Figure (14): Relation of age at sampling in ITP patients with BAFF and BAFF-R…………..113 Figure (15): Comparing ITP male & female patients as regards BAFF&BAFF-R expressions……………………………………………………………………………………...114 Figure (16): Relation of phase of the disease in ITP patients with BAFF and BAFF-R………115 Figure (17): Relation of disease state at sampling in ITP patients with BAFF and BAFF-R …………………………………………………………………………………………………..117 Figure (18): Relation of disease state at sampling in ITP patients and controls with BAFF & BAFF-R.………………………………………………………………………………………..117 Figure (19): Relation of severity of the disease in ITP patients with BAFF and BAFF-R...…119 Figure (20) Relation of clinical response at follow up with BAFF and BAFF-R.…………….120 Figure (21 ): Relation of the quality of response at follow up in ITP patients with BAFF and BAFF-R……………………………………………………………………………………...…121

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Figure (22): different lines of management of the studied ITP patients……………………….123 Figure (23): relation of treatment in ITP patients with BAFF and BAFF-R…………..…….124 Figure (24): Relation of BAFF and BAFF-R with the response to Imuran in the studied ITP patients………………………………………………………………………………………….125

Figure (25): Relation of BAFF and BAFF-R with the response to observation, steroids and steroids + Imuran in the studied ITP patients……………….………………………………..126

Figure (26): Correlation between BAFF mRNA expression and BAFF in ITP patients………128

Figure (27): Correlation between BAFF mRNA expression and age at sampling in ITP patients………………………………………………………………………………………….129

Figure (28): Correlation between BAFF mRNA expression and age at onset in ITP patients …………………………………………………………………………………………………..129 Figure (29): Amplification plot of; (A) BAFF, (B) BAFF-R and (C) the house keeping gene B- actin. …………………………………………………………………………………….....…...130

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Abstract:

B-cell activating factor (BAFF) plays a crucial role in B-cell development, survival and immunoglobulin production. Excess BAFF results in the rescue of self-reactive B cells from anergy, and the rescue of autoreactive T cells from the suppressing effect of dendritic cells, thus implicating a role in the development of autoimmunity. The aim of this study was to evaluate BAFF and its receptor (BAFF–R) mRNAs expression in patients with idiopathic thrombocytopenic purpura, and also to study the potential association of their expression with variation in disease severity, chronicity and response to treatment. Evaluation of BAFF and BAFF-R expression was done using quantitative real-time polymerase chain reaction (qRT- PCR), in 79 ITP patients as well as 20 age- and sex-matched control volunteers. The median expression level of BAFF and of BAFF-R in ITP patients was significantly higher compared to the control group. Children had a significantly lower mean BAFF expression level compared to adults with ITP. Female patients had a significantly higher mean BAFF-R expression level compared to male patients. Patients with active ITP had a significantly higher BAFF expression compared to those in remission and those of control group. Mean expression level of BAFF-R was significantly higher in Patients with active ITP and in those in remission when each was compared to the control group. BAFF-R expression was significantly higher in steroid treated patients compared to untreated patients. A significant positive correlation was found between BAFF and BAFF-R mRNA expression levels. BAFF expression was positively correlated with the median age of patients at sampling time and at diagnosis. E levated BAFF expression in patients with active ITP indicates its possible role in the pathogenesis of ITP. Hence, selective antagonistic targeting of BAFF or BAFF-R in ITP patients with high levels of BAFF expression might be considered as a novel therapeutic strategy. Key words: BAFF, BAFF-R, qRT-PCR, ITP.

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Introduction and aim of work

Introduction:

Idiopathic thrombocytopenic purpura (ITP) is an autoimmune-specific disorder in which platelets are opsonized by autoantibodies directed against platelet glycoproteins followed by their prematurely destruction by phagocytic cells in the reticuloendothelial system (George, 2006).

The autoantibodies, produced by autoreactive B cells against self-antigens, specifically immunoglobulin G (IgG) antibodies against glycoprotein IIb (GPIIb)/IIIa and/or GPIb/IX, are considered to play a crucial role in the pathogenesis of ITP (Cines & Blanchette, 2002).

In addition, several abnormalities involving the cellular mechanisms of immune modulation, such as the T helper 1 (Th1) bias, (Semple et al., 1996), the decreased number or defective suppressive function of regulatory T cells , (Sakakura et al., 2007, Stasi et al., 2008, Yu et al., 2008) and the platelet destruction by cytotoxic T cells, (Olsson et al., 2003, Zhang et al., 2006, Zhao et al., 2008) have been described. The cause for these abnormalities remains unknown.

The reason for the production of autoantibodies remains unknown and the clinical course is extremely variable. Some patients remain asymptomatic, whereas others develop life-threatening bleeding episodes. Moreover, therapy including prednisolone, intravenous immuno-globulin G, anti-D and splenectomy is not always effective, and only one-third of adult patients achieve long-term remission (Bellucci et al., 1988; Berchtold & McMillan, 1989).

It is increasingly recognized that B cells have multiple functions that contribute to the pathogenesis of autoimmunity. B-cell activating factor (BAFF) ), is a 285 amino acid type II membrane-bound protein, it belongs to the family of tumour necrosis factor (TNF). BAFF is also known as TALL-1, Blys, and zTNF4, and is expressed by several cell types, including monocytes, macrophages, neutrophils, dentritic cells and T lymphocytes (Mackay & Browning,

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2002; Schneider & Tschopp, 2003) . BAFF binds to three receptors: BAFF receptor, (BAFF-R), B-cell maturation antigen (BCMA) and transmembrane activator and calcium modulating cyclophilin ligand interactor (CAML), these receptors is mainly expressed on B lymphocytes (Thompson et al., 2001; Mackay & Mackay, 2002) . BAFF plays a crucial role in B-cell development, survival and immunoglobulin production ( Harless et al., 2001; Hsu et al ., 2002; O’Connor et al, 2004 ).

Moreover, excess amounts of BAFF results in the rescue of self-reactive B cells from anergy, thus implicating a role in the development of autoimmunity ( Thien et al., 2004 ). Mice overexpressing BAFF display increased B-cell numbers and immunoglobulin levels as well as clinical features similar to that observed in patients with systemic lupus erythematosus (SLE) (Mackay et al., 1999; Gross et al ., 2000; Khare et al ., 2000 ). In accordance with such data, some studies demonstrated elevated BAFF serum levels in patients with systemic autoimmune diseases such as SLE, rheumatoid arthritis (RA) and Sjo¨gren syndrome ( Cheema et al., 2001; Zhang et al., 2001; Groom et al ., 2002; Mari ette et al., 2003; Tan et al ., 2003 ). However, patients with primary biliary cirrhosis and autoimmune diabetes displayed normal BAFF levels, discounting a general association between autoimmune diseases with elevated BAFF ( Mackay et al, 2002 ).

Therefore, BAFF overexpression alters immune tolerance in the periphery and predisposes to the development of autoimmune disease ( Mackay et al., 2003, Thien et al., 2004, Lesley et al., 2004 ). BAFF overexpression impairs self-tolerance by promoting enhanced survival and proliferation of activated autoreactive B cells and suppressing the protective effects of dendritic cells against the emergence of autoreactive T cells ( Mackay et al., 1999).

Recent study has shown that circulating level of BAFF significantly increased in ITP, which offers important new insights into the pathogenesis of ITP (Emmerich et al., 2007) , therefore, BAFF might serve as a new rational therapeutic target for ITP.

In vivo, administration of soluble decoy receptors for BAFF effectively decreases disease progression in some autoimmune diseases mouse models, such as lymphoid cancers (Reyes et

2 2 al., 2006 ), systemic lupus erythematosus (SLE) (Sabahi & Anolik, 2006, Matsushita et al., 2007) , rheumatoid arthritis (Bosello et al., 2007 ) and sjogren’s syndrome (Szodoray & Jonsson, 2005). These evidences render BAFF as a potentially new therapeutic target for autoimmune diseases.

Taken together, these evidences have led us to hypothesize that BAFF plays an important role in the pathology of ITP. Most probably, blocking the expression of BAFF may induce B cells depletion; therefore, it may be an effective therapeutic target molecule in patients with ITP.

Aim of the work:

The objective of the study is to evaluation BAFF and BAFF–R mRNAs expression in patients with ITP & study of the potential association of their expression with variation in disease severity, chronicity and response to treatment.

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The Review of Literature

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Thrombocytopenia Backgrounds: Thrombocytopenia is one of the most frequent causes for hematologic consultation in the practice of medicine, and potentially one of the most life-threatening. Although the normal platelet count in humans (150,000–400,000/ µl) far exceeds the minimal level required to avoid pathologic hemorrhage (<50,000/ µl), a number of medical conditions cause either increased destruction or reduced production of platelets, increasing the risk of pathologic bleeding (Chen et al., 2010).

Immune (autoimmune, idiopathic) thrombocytopenic purpura is a common acquired autoimmune disorder defined by a low platelet count secondary to accelerated platelet destruction or impaired thrombopoiesis by antiplatelet antibodies (George et al., 1998). The site of destruction is usually the reticuloendothelial system of the spleen and, less commonly, the liver (Thienelt & Calverley, 2009).

The term idiopathic thrombocytopenic purpura refers to thrombocytopenia in which apparent exogenous etiologic factors are lacking and in which diseases known to be associated with secondary thrombocytopenia have been excluded (George et al., 2000; Cines & Blanchette, 2002), which might include infections, collagen vascular diseases, lymphoproliferative disorders (chronic lymphocytic leukemia or lymphoma), or drugs (Thienelt & Calverley, 2009).

The diagnosis of autoimmune thrombocytopenic purpura is primarily a diagnosis of exclusion, because currently available clinical assays for platelet-associated antibodies or serum antiplatelet antibodies/immune complexes are neither specific nor sensitive enough for routine clinical use (Thienelt & Calverley, 2009).

The diagnosis of ITP requires decreased platelets on the blood film, normal or increased numbers of marrow megakaryocytes and the exclusion of other causes of thrombocytopenia (George et al., 1998).

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In some instances, ITP may be the presenting manifestation of an underlying disease, and additional manifestations appear weeks to months later (Thienelt & Calverley, 2009). (Table 1): Pathophysiologic Classification of Thrombocytopenia: Artifactual thrombocytopenia: Platelet clumping caused by anticoagulant-dependent immunoglobulin (pseudothrombocytopenia) Platelet satellitism Giant platelets Decreased platelet production: hypoplasia of megakaryocytes Ineffective thrombopoiesis Disorders of thrombopoietic control Hereditary thrombocytopenias Abnormal platelet distribution or pooling: Disorders of the spleen (neoplastic, congestive, infiltrative, infectious, of unknown cause) Hypothermia Dilution of platelets with massive transfusions Increased platelet destruction: Caused by immunologic processes: Autoimmune: Idiopathic Secondary: Infections, pregnancy, collagen vascular disorders, lymphoproliferative disorders, drugs, miscellaneous Alloimmune: Neonatal thrombocytopenia Posttransfusion purpura Caused by nonimmunologic processes: Thrombotic microangiopathies Disseminated intravascular coagulation (Neeraj & George, 2009).

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Artifactual Thrombocytopenia: Artifactual thrombocytopenia, or falsely low platelet counts, occurs ex vivo when platelets are not counted accurately. This mechanism should be considered in patients who have thrombocytopenia but no petechiae or ecchymoses. Although inaccurate counting may occur in the presence of giant platelets (Muraoka et al., 1997), or with platelet satellitism (Ihara et al., 1999), the most common cause of artifactual thrombocytopenia is platelet clumping (pseudothrombocytopenia) (Ballmaier et al., 2001) . Platelet clumping in pseudothrombocytopenia appears to be caused by anticoagulant-dependent platelet agglutinins that are immunoglobulins (Igs) of IgG, IgA, or IgM subtypes. Although clumping is most commonly seen when blood is collected into ethylenediaminetetraacetic acid (EDTA) anticoagulant, other anticoagulants may cause clumping. Platelet clumping is also time dependent and varies with the type of instrumentation used for automatic counting (Gandhi et al., 2005) . There is evidence that the autoantibodies bind to glycoprotein IIb/IIIa (Drachman, 2000) , and in one study, there was over 80% concordance between the presence of anticardiolipin antibody and platelet agglutinins in individual patient plasmas (Germeshausen et al., 2006).

Deficient Platelet Production: Deficient platelet production may result from any of a number of processes. Those that depopulate the stem cell or megakaryocyte compartments are the most common, such as marrow injury by myelosuppressive drugs or irradiation, and aplastic anemia. Deficient platelet production also may be the consequence of disordered proliferation within a precursor compartment of normal or even increased size. For example, in disorders characterized by megaloblastic hematopoiesis, hypertrophy of the precursor compartment occurs in response to thrombopoietic stimuli, but thrombopoiesis is ineffective and platelet production is insufficient. Rarely, abnormalities of the processes that normally regulate thrombopoiesis appear to underlie deficient platelet production, such as deficiency of thrombopoietin and cyclic thrombocytopenia (Neeraj & George, 2009).

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Hereditary thrombocytopenia includes: 1) Autosomal recessive thrombocytopenias: A) Congenital amegakaryocytic thrombocytopenia (CAMT): It results from mutations in the thrombopoietin (TPO) receptor c-Mpl, rendering it deficient (typeI CAMT) or of reduced function (type II CAMT) (Tonelli et al., 2000) . TPO affects megakaryocytes but also multipotent hematopoietic stem and progenitor cells (van den Oudenrijn et al., 2000). Mutations of the TPO receptor are associated with greatly reduced megakaryopoiesis, the disorder progresses to aplastic anemia before age 3 to 5 years in most patients. Marrow transplantation is the only curative therapy for CAMT (Chen et al., 2010).

B) Thrombocytopenia with absent radius (TAR) syndrome: It is characterized by bilateral absence of the radii but with thumbs present, hypomegakaryocytic thrombocytopenia, and a number of additional features including skeletal and cardiac anomalies (Hall et al., 1969).

C)Bernard-Soulier syndrome: It is an autosomal recessive bleeding disorder associated with deficiency of platelet membrane GPIb, GPIX, and GPV. This results in abnormal platelet interaction with ligands of these receptor proteins, which include thrombin, von Willebrand factor, P-, and leukocyte integrin αMβ2, in addition to causing thrombocytopenia (Neeraj & George, 2009).

2) Autosomal dominant thrombocytopenia: A) Mutations in MYH9 Gene for Nonmuscle Myosin Heavy Chain: These include May-Hegglin anomaly, Sebastian syndrome and its variants, Epstein syndrome, and Fechtner syndrome. These disorders are defined by the presence of mutations involving the MYH9 gene located at 22q12.3–13.1. The MYH9 gene encodes nonmuscle myosin II-A heavy chain (NMMHC-IIA) (Seri et al., 2003) which is a component of the contractile cytoskeleton in megakaryocytes, platelets, and other tissues. All of these disorders have various

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