NATIONAL STUDY OF INHERITED PLATELET FUNCTION DISORDERS IN THE NETHERLANDS

NATIONAL STUDY OF INHERITED PLATELET FUNCTION DISORDERS IN THE NETHERLANDS TROMBOCYTOPATHY IN THE NETHERLANDS TiN STUDY Version 1.1, 26 November 2015 Corresponding author: M.W. Blaauwgeers, MD University Medical Center Utrecht (UMCU), Van Creveldkliniek Heidelberglaan 100 3584 CX Utrecht T: +31-88 75 584 50 F: +31-88 75 55438 Email: m.w.blaauwgeers@umcutrecht.nl

PROTOCOL TITLE National study of inherited platelet function disorders in the Netherlands Protocol ID ABR number 53207.041.15 / METC nr 15-597 Short title Thrombocytopathy in the Netherlands (TiN) Version 1.1 Date 26-11-2015 Coordinating investigator/ project leader Principal investigator TiN studygroup Sponsor M.W. Blaauwgeers, MD University Medical Center Utrecht (UMCU), Van Creveldkliniek Heidelberglaan 100 3584 CX Utrecht T: +31-88 75 51709 F: +31-88 75 55438 Email: m.w.blaauwgeers@umcutrecht.nl R.E.G. Schutgens, MD PhD MSc Van Creveldkliniek UMCU T: +31-88 75 584 50 Email: r.schutgens@umcutrecht.nl Dr. R.T. Urbanus, assistant professor, department of clinical chemistry and haematology Drs. I. van Asten, PhD student Dr. A. Huisman, clinical chemist Dr. J.K. Ploos van Amstel, professor genome diagnostics, clinical laboratory geneticist Dr. A.J. Willemze, MD PhD Each HTC will have one representative in the TiN studygroup Van Creveldkliniek UMCU Subsidising party Independent expert (s) Partly funded by Sanquin Drs. D. Dekker, MD, department of internal medicine and dermatology, acute medicine D.dekker@umcutrecht.nl Laboratory sites Van Creveld laboratory (Laboratory of clinical chemistry and haematology. UMCU and Sanquin Research)

PROTOCOL SIGNATURE SHEET Name Signature Date Director of the Van Creveldkliniek UMCU: REG Schutgens, MD PhD MSc Principal Investigator REG Schutgens, MD PhD MSc Van Creveldkliniek, UMCU Coordinating Investigator/project leader MW Blaauwgeers, MD Van Creveldkliniek UMCU

TABLE OF CONTENTS 1. INTRODUCTION AND RATIONALE... 8 1.1 Platelet function... 8 1.2 Classification of platelet function disorders... 8 1.2.1 Disorders of platelet adhesion... 9 1.2.2 Disorders of platelet activation and signal transduction... 9 1.2.3 Disorders of platelet secretion...10 1.2.4 Disorders of platelet aggregation...10 1.2.5 Disorders of cytoskeletal organization...11 1.3 Symptoms...11 1.4 Diagnosis...12 1.4.1 Initial screening...12 1.4.2 Bleeding time and platelet function analyzer...12 1.4.3 Light transmission platelet aggregation...12 1.4.4 Platelet nucleotides content and release...13 1.4.5 Flow cytometry...13 1.4.6 Next-generation sequencing (NGS)...13 1.4.7 Recommendations by the ISTH...13 1.5 Treatment...14 1.6 Situation in the Netherlands and rationale...15 2. OBJECTIVES...16 3. STUDY DESIGN...16 4. STUDY POPULATION...16 4.1 Population...16 4.2 Inclusion criteria...16 4.3 Exclusion criteria...16 4.4 Sample size calculation...17 5. METHODS...17 5.1 Study parameters...17 5.2 Study procedures...17 5.2.1 Patient selection...17 5.2.2 Informed consent procedure...17 5.2.3 Data collection...18 5.2.4 Diagnostic tests...18 5.2.5 Instruments...20 5.2.6 Total Burden...20 5.2.7 Follow up...21 5.3 Withdrawal of individual subjects...21 5.4 Replacement of individual subjects after withdrawal...21 6. SAFETY REPORTING...21 6.1 Section 10 WMO event...21 6.2 Adverse events and serious adverse events...21

6.3 Follow-up of adverse events...22 7. STATISTICAL ANALYSIS...22 8. ETHICAL CONSIDERATIONS...22 8.1 Regulation statement...22 8.2 Recruitment and consent...23 8.3 Benefits and risks assessment, group relatedness...23 8.4 Compensation for injury...23 9. ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION...23 9.1 Handling and storage of data and documents...23 9.2 Monitoring and Quality Assurance...24 9.3 Amendments...24 9.4 Annual progress report...24 9.5 End of study report...24 9.6 Public disclosure and publication policy...24 10. REFERENCES...25 APPENDIX 1 Flow chart blood samples

LIST OF ABBREVIATIONS AND RELEVANT DEFINITIONS AE BAT BS BSS CHS EM FACS GP HPS HTC ISTH LTA METC NGS NVHB NVHP NVHV PFA PFD PT-VWD SAE SPD UMCU VCK VWD VWF WAS WBP WFH WMO Adverse event Bleeding Assessment Tool Bleeding score Bernard Soulier syndrome Chediak-Higashi syndrome Electron microscopy Fluorescence-activated cell sorting Glycoprotein Hermansky-Pudlak syndrome Hemophilia Treatment Center International Society of Thrombosis and Haemostasis Light Transmission platelet Aggregation Medical research ethics committee (MREC); in Dutch: Medisch Ethische Toetsing Commissie (METC) Next-Generation Sequencing Nederlandse Vereniging voor Hemofilie Behandelaren Nederlandse Vereniging voor Hemofilie Patiënten Nederlandse Vereniging voor Hemofilie Verpleegkundigen Platelet Function Analyzer Platelet function disorder Platelet type von Willebrand disease Serious adverse event Storage pool disease University Medical Center Utrecht Van Creveldkliniek Von Willebrand Disease Von Willebrand Factor Wiskott-Aldrich syndrome Personal Data Protection Act (in Dutch: Wet Bescherming Persoonsgevens) World Federation of Hemophilia Medical Research Involving Human Subjects Act (in Dutch: Wet Medischwetenschappelijk Onderzoek met Mensen) Version 1.1: November 2015 6 of 28

SUMMARY Rationale: Inherited platelet function disorders (PFDs) are rare bleeding disorders caused by genetic defects, resulting in dysfunction of adhesion, activation or aggregation of platelets. This group of disorders is heterogeneous in severity, mechanisms and frequency. In the Netherlands little is known about prevalence, clinical aspects, burden of disease and quality of life of patients with these disorders. Objective: Primary objective: to register and investigate Dutch patients suspect for an inherited platelet function disorder, to assess clinical presentation, bleeding score, previous treatment, burden of disease and quality of life. Secondary objectives: to investigate if diagnostic approaches can be improved and optimized using tests additional to the standard diagnostic tests. To search for a possible relationship between type of PFD and bleeding phenotype and to study genotype-phenotype relationships. Study design: Cross-sectional study coordinated at the Van Creveldkliniek of the University Medical Center Utrecht. Study population: Patients over 18 years with a bleeding diathesis suspected for an inherited PFD. Study procedures: Patients will fill out a questionnaire on treatment history, social activities and quality of life, including items of the RAND-36 health survey. Blood will be drawn to perform routine laboratory testing and additional tests (including Platelet Activition Test, electron microscopy, mass spectrometry, RNA diagnostics and MYH9 immunofluorescence analysis). Plasma will be stored in the biobank for additional testing in the future. Study parameters/endpoints: Frequency and severity of bleeding symptoms: the bleeding score using the ISTH-BAT. Treatment of bleeding diathesis: type and frequency of treatment received in the past (local treatment, antifibrinolytics, DDAVP, platelet transfusion). Impact of PFD on quality of life: RAND-36 health survey score. Diagnostic utility of additional platelet tests as compared to standard Light Transmission platelet Aggregation. Relation between type of PFD and bleeding score. Genotype-phenotype relationships. Nature and extent of the burden and risks associated with participation, benefit and group relatedness: This study will be the first to asses clinical presentation, quality of life and diagnostic approaches of patients with inherited PFDs in the Netherlands. The participating patients may benefit directly from participation as the use of new tests in this study may lead to diagnosing patients who were previously undiagnosed, resulting in better understanding and treatment options. The study consists of a questionnaire and drawing extra blood for additional tests. Risks imposed by participation are considered negligible. Version 1.1: November 2015 7 of 28

1. INTRODUCTION AND RATIONALE 1.1 Platelet function Platelets are small anucleated cells with a discoid shape and measure approximately 2.0-4.0 x 0,5 µm. They are derived from bone marrow megakaryocytes. The normal platelet count is 150-450 x 10 9 /l and their lifespan is 7-10 days 1. Approximately one-third of the total platelet mass is normally sequestered in an exchangeable splenic pool 2. Old platelets are destroyed by phagocytosis in the liver and spleen 3. Platelets play an important role in primary hemostasis. Under normal circumstances circulating platelets do not encounter the connective tissue matrix that lies beneath vascular endothelial cells. Upon tissue damage, platelets are recruited to the site of injury. Initially they tether and roll over the exposed extracellular matrix, a process eventually resulting in firm adhesion 4,5. This process is primarily mediated by two platelet adhesion receptors: glycoprotein (GP) Ib-IX-V that binds to von Willebrand factor (VWF) and GPVI that binds to collagen 6. Adhesion triggers rapid signal transduction, mediated by tyrosine kinases and G-protein coupled receptors, leading to platelet activation 4-6. Activation results in shape change, degranulation of granules, intracellular calcium fluxes, thromboxane formation, exposure of procoagulant surface and inside-out activation of integrins, supporting aggregation 6,7. The cytoplasm of platelets contains three types of granules: dense granules, α-granules and lysosomal granules. Upon activation the granules release their contents 8,9. Release of ADP leads to secondary activation of platelets via binding on the platelet surface receptors P2Y 2 and P2Y 6,10 12. A second positive feedback loop is established by the conversion of arachidonic acid into thromboxane-a 2 by cyclooxygenase-1. Once formed, thromboxane-a 2 can diffuse across the plasma membrane and activate other platelets by binding to its surface thromboxane receptor 10,11. The ultimate step in the activation cascade is the transformation of the αiibβ3 receptor into a competent receptor for fibrinogen. Activation of platelets converts the αiibβ3 receptor to a high-affinity state, exposing the binding side and thereby allowing fibrinogen to bind. Fibrinogen binds to the αiibβ3 receptor on two or more adjacent platelets resulting in platelet aggregation 4,5,12. 1.2 Classification of platelet function disorders Disorders of primary hemostasis are characterized by an increased mucocutaneous bleeding tendency and prolonged bleeding after surgery or trauma. The most common inherited disorder of primary hemostasis is von Willebrand disease (VWD) with a prevalence of 1% 13 Version 1.1: November 2015 8 of 28

Platelet function disorders (PFDs) can be classified into inherited and acquired disorders, the latter caused by a variety of factors including certain drugs, uremia and liver disease 14. Inherited platelet function disorders are a heterogeneous group of diseases. Although rare, the prevalence is probably underestimated due to under-diagnosis. While the diagnosis of severe defects is rather straightforward, mild defects are often difficult to diagnose because of the mild bleeding pattern and normal platelet count 15,16. Inherited platelet function disorders can be classified according to platelet function into adhesion, activation, secretion and aggregation defects 17. 1.2.1 Disorders of platelet adhesion Bernard-Soulier syndrome (BSS) is a rare congenital bleeding disorder caused by the absence or decreased expression of the GPIb/IX/V complex, resulting in deficient binding of the platelet to VWF, leading to deficient adhesion 18. The condition is inherited in an autosomal recessive manner. A number of causative mutations have been identified 1,16,19. BSS is characterized by thrombocytopenia and large platelets. Diagnosis can be confirmed by aggregation studies, platelets fail to aggregate with ristocetin, and flowcytometry analysis 1,17. The estimated prevalence is less than one in a million 20. Platelet type von Willebrand disease (PT-VWD) is characterized by platelet hyperresponsiveness rather than decreased function. It is caused by gain-offunction mutations in the platelet GPIBα gene, leading to an enhanced binding between von Willebrand factor and its platelet ligand GP1Bα. Features are a low to normal platelet count, decreased high-molecular-weight VWF multimers and increased ristocetin-induced platelet agglutination at low ristocetin concentrations. Distinction between PT-VWD and type 2B VWD can be challenging due to similar bleeding phenotype and laboratory findings. Definitive diagnosis can be made by identifying the defective gene 21-23. Platelets possess two different types of collagen receptors that mediate adhesion to collagen: integrin α2β1 en GPVI. Integrin α2β1 is important for platelet adhesion to collagen, GPVI is primary responsible for collagen-induced platelet activation. Deficiency or dysfunction of either one of these receptors leads to impaired adhesion and activation 24. 1.2.2 Disorders of platelet activation and signal transduction Several PFDs have been described caused by dysfunctional activating receptors. Patients with ADP receptor dysfunction have impaired or absent aggregation with ADP and variable impairment in aggregation to other agonists 25. The ADP receptors P2Y 2 and P2Y 12 are G-protein coupled receptors and mediate calcium mobilization, reversible and transient aggregation and eventually sustained platelet aggregation. Only a few patients with defects of the ADP receptor have been described 17,26,27. Version 1.1: November 2015 9 of 28

Defects in the thromboxane-a2 formation or receptor function are associated with impaired signal transduction and impaired platelet aggregation to arachidonic acid and thromboxane-a2 26. The signaling pathways after platelet activating is highly complex and involves Src family kinases (SFKs), phosphoinositide 3-kinases (PI3Ks) and the ITAM signaling pathway 24. 1.2.3 Disorders of platelet secretion Defects of platelet secretion are referred to as storage pool disease (SPD) and can be a result of deficiencies in the number or the content of granules or failure of normal secretory mechanisms. Disorders of the dense granules (δ-spd) include Hermansky-Pudlak syndrome (HPS), Chediak-Higashi syndrome (CHS) and isolated dense granule deficiency. Dense granules store serotonin and nucleotides like ATP and ADP 26. HPS is characterized by oculocutaneous albinism, bleeding diathesis due to δ-spd and lysosomal storage disease 28. CHS is, like HPS, characterized by oculocutaneous albinism and bleeding diathesis due to δ-spd. In addition, it is associated with severe life-threatening infections associated with neutropenia, impaired chemotaxis and bactericidal activity, and lack of natural killer cell function 17,29. Isolated dense granule deficiency is featured by a defect in the dense granules without the presence of albinism 30. Disorders of the α-granules (α-spd) include Gray platelet syndrome and Quebec syndrome. α-granules store platelet factor 4, platelet-derived growth factor, β- thromboglobulin and VWF among others. Gray platelet syndrome is an extremely rare condition and is associated with an absence of α-granules and their contents, resulting in diminished secretion-dependent aggregation 31,32. In Quebec syndrome, platelets have a protease-related degradation of many α-granule proteins 33. 1.2.4 Disorders of platelet aggregation Glanzmann thrombastenia is an autosomal recessive disorder caused by a functional defect or deficiency of the membrane αiibβ3 complex, resulting in impaired platelet aggregation. Genes that encode αiib and β3 are located on chromosome 17. Over 100 mutations have been reported. Platelet count and morphology are normal. Aggregation studies show failure to aggregate in response to agonists but platelets do agglutinate in the presence of ristocetin. Definitive diagnoses is made by flowcytometry analysis 1,34. Version 1.1: November 2015 10 of 28

1.2.5 Disorders of cytoskeletal organization The platelet cytoskeleton consists of microtubules, actin filaments, and other cytoskeletal proteins such as myosin heavy chain (MYH) and filamin (FLN). These structures are important for both maintaining the discoid shape of the circulating platelet and for the rapid morphological changes after activation 35. MYH9-related disorders include May-Hegglin anomaly, Fechtner syndrome, Sebastian syndrome and Epstein syndrome. The MYH9 gene encodes non-muscle myosin heavy chain IIA (NMMHC-IIA) and is located on chromosome 22. Symptoms may consist of a mild to moderate bleeding tendency, hearing loss, cataracts and progressive nephritis. These disorders are characterized by macrothrombocytopenia and Döhle-like granulocyte inclusions due to aggregates of the abnormal NMMHC- IIA peptide. Diagnosis can be made using immunofluorescence analysis and nextgeneration sequencing 17,36. Wiskott-Aldrich syndrome (WAS) is a recessive disorder characterized by a bleeding tendency due to microthrombocytopenia and impaired platelet aggregation, recurrent infections due to immune deficiency, eczema, autoimmune manifestations and malignancies. It is caused by mutations in the WAS gene located on the short arm of the X-chromosome 26. Other disorders of cytoskeletal organization include mutations in the FLNA, TUBB1 and ACTN1 gene 35. 1.3 Symptoms Typical bleeding manifestations of platelet function defects are unexplained or extensive bruising, bleeding from mucous membranes like epistaxis, gastro-intestinal bleeding, oral cavity bleeding and menorrhagia, and bleeding following an appropriate challenge like dental extraction, invasive procedures or childbirth. Severe platelet disorders are usually identified in childhood due to bleeding problems at any age, including the neonatal period. Particular attention should be paid to intracranial hemorrhage, umbilical stomp bleeding and bleeding after circumcision. Mild platelet disorders can be a challenge to diagnose and may only present after an appropriate challenge like dental extraction or surgery 1,37. Documentation of bleeding symptoms is not standardized. Recently, the International Society of Thrombosis and Haemostasis (ISTH) developed a bleeding assessment tool (BAT) to accurately record bleeding symptoms in all hemorrhagic disorders and to help diagnose patients referred with a possible bleeding disorder 38. The utility of the ISTH-BAT for PFD has been evaluated in 76 patients with a suspected platelet disorder, where it appeared to be a powerful tool for documenting bleeding history, but not to be predictive for the presence of a platelet defect detected by lumiaggregometry 39. This indicates that either the ISTH-BAT is not able to discriminate between platelet defects and other Version 1.1: November 2015 11 of 28

hemostatic defects, or that current platelet function assays are insufficient to detect the underlying platelet defects. 1.4 Diagnosis Diagnosing platelet function disorders is challenging due to a lack of agreement about classification and poor standardization of laboratory tests 40. A variety of methods and tests are available to assess platelet function. 1.4.1 Initial screening Evaluation of patients with abnormal bleeding requires clinical evaluation and careful history with attention to personal and family bleeding history. Associated medical complications consistent with some of the more common platelet function disorders should be evaluated. The use of a BAT is encouraged, as BATs have proven to be helpful in assessing severity of symptoms. Preliminary laboratory tests include full blood count, blood smear (platelet morphology), prothrombin time, activated partial thromboplastin time and von Willebrand factor screening tests. If normal, physicians should proceed with specific platelet function tests 37,41. 1.4.2 Bleeding time and platelet function analyzer Bleeding time was the first test available to asses platelet function 42. It assesses in vivo hemostasis. Bleeding time is prolonged in severe PFD but can be normal or only minimally prolonged in mild forms 1. Since the test is invasive, highly influenced by different variables and poorly standardized, it has become obsolete over the years. Platelet function analyzer (PFA) is the in vitro standardized bleeding time. It is easy and sensitive to severe platelets disorders. However, platelet count and hematocrit can influence the results and PFA can be falsely negative in storage pool diseases 43. Currently, both the bleeding time and PFA have low sensitivity and specificity to detect PFD 44. 1.4.3 Light transmission platelet aggregation Light transmission platelet aggregation (LTA) is regarded as the gold standard for platelet function testing 45. It measures the increase in light transmission through the optical dense sample of platelet-rich plasma after the addition of exogenous platelet agonists. Typical agonist for platelet aggregation are ADP, adrenaline, collagen, arachidonic acid, ristocetin and thrombin receptor activating peptide. After addition of the agonist, the plasma becomes clearer due to precipitation of platelet aggregates and light transmission through the plasma sample increases. The device records the rate and maximal percentage of this increase from 0% to 100% by a photometer and this signal is converted in a graphic curve. LTA is considered as the most complete assay, because it is easy to use and investigates different platelet pathways 43. However, despite its widespread use, experience and expertise of laboratory staff varies and the test is still poorly standardized 44. Version 1.1: November 2015 12 of 28

1.4.4 Platelet nucleotides content and release Normal aggregometry does not exclude deficiencies in granule number or content (storage pool disease) or specific defects in degranulation (release defects). Studies show that these conditions can be missed or misdiagnosed if relying on platelet aggregometry alone 46,47. Lumiaggregometry measures ATP release from activated platelets and normally demonstrate release of ATP during the secondary aggregation phase in LTA. Using this approach, it is impossible to distinguish between storage and release defects. Therefore, it is recommended to determine the total platelet content and release of both ADP and ATP and calculate the ATP/ADP ratio. Storage defects are associated with an increased ratio, release defects show a normal ratio but decreased ADP release 43,44. 1.4.5 Flow cytometry In flow cytometry, platelets are identified via analysis of samples incubated with a fluorescent monoclonal antibody directed against an antigen on the surface of the platelet. It can be a useful tool in the quantification of membrane glycoprotein receptors like GPIB/IX/V and αiibβ3 for diagnosing Bernard-Soulier syndrome and Glanzmann thrombasthenia. It can also be used to assess other glycoprotein receptors as well as platelet activation in response to agonists, dense granule content and release and phospholipid expression. The main benefit of the test is the use of small quantities of blood, making it useful in thrombocytopenic individuals. On the other hand, flow cytometry is an expensive test and requires specialized training 43,44,48. 1.4.6 Next-generation sequencing (NGS) PFDs are most likely caused by a mutation in one or more of the genes involved in megakaryocyte development, platelet formation or platelet function. Identification of these genetic defects is complicated by the redundancy in platelet receptor and signaling pathways, the overwhelming number of candidate genes and the heterogeneity and complexity of these disorders. Next-generation sequencing technologies permit the simultaneous analysis of large numbers of genes. Recent studies have found a functional defect in ~60% of patients using platelet function tests in combination with next-generation sequencing. 63 candidate defects, affecting 40 genes, have been identified among patients with Gi signaling abnormalities and 53 candidate defects, affecting 49 genes, were identified among patients with secretion abnormalities 16,49. 1.4.7 Recommendations by the ISTH There is a high variability in the diagnostic flow chart used to evaluate patients for a suspected PFD 15. There is no widely accepted international guideline and many laboratory tests used for assessment are insufficiently standardized, technically challenging and poorly reproducible. Therefore, the ISTH recently developed a guideline, trying to standardize the approach to the diagnosis of inherited PFDs. A Version 1.1: November 2015 13 of 28

diagnostic flowchart was developed, distinguishing first-step, second-step and thirdstep tests. Systematic application of this staged approachis likely to increase the diagnosis of PFDs and the identification of cases worthy of further investigation 41. 1.5 Treatment Inherited PFDs are rare and require specialized management. According to the Dutch national guideline for hemophilia and allied disorders, all patients with PFD should be registered with a hemophilia center with 24 hour access to appropriate facilities for investigation and treatment. There is a lack of adequate evidence (i.e. randomized controlled trials) upon which to base recommendation for management. Most advice is based on expert opinions. General measures mainly include proper lifestyle education. Contact sports should be avoided by patients with severe disorders. Patients must be informed about medication which interferes with platelet function. These medications should be used with caution and use must be balanced against the risks 1,19. Menorrhagia may decrease by the use of hormonal contraceptives. Proper dental hygiene is important in preventing invasive dental procedures later in life 37. The number of therapeutic options is limited and little is specific to a single disorder. Treatment must be personalized since type and severity of bleeding differ between patients 17. The following options may be considered 1,19. - Local measures, e.g. proper compression in the case of epistaxis, will usually be sufficient in the case of mild bleeding - Antifibrinolytic agents, e.g. tranexamic acid, can be given for mild bleeding manifestations from mucous membranes, such as epistaxis or menorrhagia - Desmopressin (DDAVP) may be the treatment of choice for mild bleeding problems where tranexamic acid alone is ineffective. It has been reported to be effective in patients with storage pool disease and in preventing bleeding after dental extraction and minor surgery 50 - Platelet transfusion is indicated for the treatment of major bleeding and when other agents have failed. Transfusion should be considered carefully, since there is a risk of transfusion-transmitted infections and allergic reactions. Repeated transfusion puts patients at risk of alloimmunization, where antibodies are formed against either HLA antigens or missing GPs (in Bernard-Soulier syndrome and Glanzmann thrombasthenia) - Recombinant factor VIIa (rfviia) is licensed for use in Glanzmann thrombasthenia where refractoriness to platelet transfusions has been demonstrated 51. Experience in platelet disorders is limited, although use in Bernard-Soulier syndrome and storage pool disease has been described 52 Version 1.1: November 2015 14 of 28

1.6 Situation in the Netherlands and rationale In the Netherlands care for all patients with hemophilia and allied bleeding disorders, such as von Willebrand disease and platelet function disorders, is concentrated in specialized Hemophilia Treatment Centers (HTCs). Currently there are seven HTCs. One or two representatives of each HTC are organized in the Dutch Hemophilia Treaters Society (Nederlandse Vereniging voor Hemofilie Behandelaren, NVHB). Other organizations involved in the optimization of management of patients with bleeding disorders are the Dutch Hemophilia Nurses Society (Nederlandse Vereniging voor Hemofilie Verpleegkundigen, NVHV) and The Netherlands Hemophilia Patients Society (Nederlandse Vereniging voor Hemofilie Patiënten, NVHP). In 2000 the Ministry of Health has written a hemophilia management policy which states that all patients must have an individual treatment protocol and are regularly seen in a HTC, at least once a year. Treatment with coagulation factor concentrate takes places in a HTC or under responsibility of a HTC. All HTCs have 24 hours facilities for investigation and treatment 53. In contrast to hemophilia and von Willebrand disease, prevalence and characteristics of patients with inherited platelet function disorders are poorly described in the Netherlands 54,55. The World Federation of Hemophilia reports 46 patients in the Netherlands with bleeding disorders other than hemophilia and von Willebrand disease, of which 16 patients with Glanzmann thrombasthenia. No numbers have been reported on Bernard-Soulier syndrome or other platelet defects 56. Currently a national registry of patients with bleeding disorders does not exist. Hardly any information on clinical aspects, bleeding phenotype, burden of disease and quality of life of patients with PFD in the Netherlands is available. There is no standardized diagnostic or therapeutic flowchart available 19. Therefore, a more systematic en nationwide approach is required in the form of this study. This study will obtain more insight and understanding in clinical presentation and impact of the disease in patients with an inherited PFD in the Netherlands. It will explore if diagnostic approaches can be improved and standardized. It will study phenotypegenotype relationships. Previous similar studies to hemophilia (Hemophilia in the Netherlands (HIN-study)) and von Willebrand disease (von Willebrand in the Netherlands (WIN-study)) have provided us with a wealth of information on medical and social aspects of these diseases. Expertise gained with these previous studies can be used in the current study. Version 1.1: November 2015 15 of 28

2. OBJECTIVES Our primary objective is to register and investigate Dutch patients suspect for an inherited platelet function disorder, to assess clinical presentation, bleeding score, treatment, burden of disease and quality of life. Our secondary objectives are: To investigate if diagnostic approaches can be improved and optimized using tests additional to the standard diagnostic tests available. To search for a possible relationship between type of PFD and bleeding phenotype. To study phenotype-genotype relationships 3. STUDY DESIGN This is a cross-sectional study coordinated at the Van Creveldkliniek (VCK) of the University Medical Center Utrecht (UMCU) 4. STUDY POPULATION 4.1 Population Dutch patients with bleeding diathesis suspected for an inherited platelet function disorder. 4.2 Inclusion criteria In order to be eligible to participate in this study, a subject must meet all of the following criteria: - Age 18 years - History of bleeding diathesis suspected for a PFD 4.3 Exclusion criteria A potential subject who meets any of the following criteria will be excluded from participation in this study: - Inability to give informed consent - Bleeding diathesis due to an acquired platelet function disorder - Bleeding diathesis due to von Willebrand disease - Bleeding diathesis due to hemophilia or other disorders of secondary hemostasis or fibrinolysis - Current use of antiplatelet therapy Version 1.1: November 2015 16 of 28

4.4 Sample size calculation As the primary objective of this study is mainly descriptive, a power calculation is not applicable. Since there are no population-based data, the prevalence remains unknown. We hope to include at least 150 patients over a 5-year period 5. METHODS 5.1 Study parameters Main study parameters/endpoints Frequency and severity of bleeding symptoms: bleeding score using the ISTH-BAT Treatment of bleeding diathesis: type and frequency of treatment received in the past (local treatment, antifibrinolytics, DDAVP, platelet transfusion) Impact of PFD on quality of life: RAND-36 health survey score Secondary parameters/endpoints Diagnostic utility of additional platelet tests as compared to standard LTA Relation between type of PFD and bleeding phenotype Genotype-phenotype relationship 5.2 Study procedures 5.2.1 Patient selection Patients over 18 years old with a bleeding diathesis suspected for an inherited platelet function disorder in the Netherlands can be selected. Currently, general practitioners and physicians are referring their patients with unexplained or undiagnosed bleeding symptoms for additional diagnostic workup to the UMC Utrecht. We will ask all eligible patients with suspected PFD to participate in the study. We will also include patients with a previously diagnosed platelet function disorder. These patients will be asked to participate by their treating physician. 5.2.2 Informed consent procedure All patients referred to the VCK for additional diagnostic workup will receive written information on the study, given by their referring specialist or send to them by postal mail. This provides patients with at least one week to consider their decision. During their visit at the VCK, they will be seen by a physician for taking medical history and physical examination. After that, they will be verbally informed about the study and informed consent will be asked and signed by one of the investigators or GCP certified research nurse. Consideration of participation will be allowed as long as the patient need time to think about participating. Version 1.1: November 2015 17 of 28

The patients with a previously diagnosed platelet function disorder will receive written information on the study by postal mail. As indicated in the information letter, patients will be contacted by one of the investigators within one to three weeks after having received the written information. This provides patients with one to three weeks to consider their decision. The investigator will inquire about the patient s interest to participate and, if applicable, schedule an appointment at the VCK. During their visit at the VCK, they will be verbally informed about the study and informed consent will be asked and signed by one of the investigators or GCP certified research nurse. 5.2.3 Data collection Data will be collected using a questionnaire. Data of the BAT will be used to evaluate bleeding symptoms. Data that will be collected includes: Patient characteristics: initials, year of birth to calculate age, body weight and length (to calculate BMI), medical history, use of medication Bleeding history: bleeding score (using the ISTH-BAT), age at time of first bleeding symptoms Treatment history: previous treatment for bleeding diathesis, date/age of first treatment, number of bleedings needing treatments, side-effects of treatment Sports activities: which sport and frequency (mean self-reported times per month) Occupation: mean number of working hours a week, number of sick days, highest level of education completed Risk of cardiovascular disease: risk factors for cardiovascular disease, date/age of cardiovascular event, family history of cardiovascular disease Items of the RAND-36 health survey Items of the Brief Illness Perception Questionnaire (B-IPQ) 5.2.4 Diagnostic tests Next to the questionnaire, blood samples will be drawn during the visit at the VCK to obtain blood and DNA for further diagnostic tests. The routine diagnostic tests include complete blood count, MPV, morphology, PT, APTT, von Willebrand screening test (vwf ristocetin), aggregation tests (ADP/collagen/AA/ristocetin), ADP/ATP content and ratio and NGS. The additional tests for this study include Platelet Activition Test (using FACS), electron microscopy, mass spectrometry for proteomics, MYH9 immunofluorescence analysis, RNA diagnostics and perfusion studies. We will perform batch analysis on the isolated platelets. Remaining material will be stored in the biobank afterwards. Plasma will be stored in the biobank for additional platelet function tests in the future (see Biobankprotocol). DNA obtained for NGS will be stored for additional DNA tests in the future. To perform the additional tests 25 ml extra blood is needed (see Appendix 1). Version 1.1: November 2015 18 of 28

If patients who are referred to the VCK for additional diagnostic work up decide to participate in the study, then 25 ml extra blood will be drawn for research purposes. The 45 ml of blood for the routine diagnostic tests will be drawn regardless of participation in the study. For patients with a previously diagnosed PFD, all the diagnostic tests (routine and research) are qualified as research tests, since they are already diagnosed and don t require routine diagnostic tests. Thus, if these patients participate in the study, 70 ml of blood will be drawn for research purposes. Electron microscopy (EM) Electron microscopic techniques are used for assessing defects of platelet organelle, cytoskeleton or membrane. Most organelles are visualized in thin sections of fixed embedded platelets. Whole-mount preparations are useful for quantifying dense-granule numbers. Platelet disorders that can be diagnosed using electron microscopy include α- and δ-storage pool disease, MYH9-related disorders and Wiskott-Aldrich syndrome 57,58. Proteomics and mass spectrometry The proteome is the entire set of proteins expressed at a given time and circumstance. Proteomics is the large-scale study of visualization, quantitation and identification of these proteins. Proteins can be separated with several techniques such as electrophoresis and chromatographic methods. Mass spectrometry is used to identify the proteins in conjunction with protein sequence databases. Proteomics can be used in platelet science for deciphering signaling cascades and to identify secreted proteins released from activated platelets 59,60. MYH9 immunofluorescence analysis Originally the diagnosis of MYH9-related disorders has been made by staining peripheral blood smear with May-Giemsa. Features are macrothrombocytopenia and granulocyte inclusion bodies. These inclusion bodies can be identified as aggregates of abnormal NMMHC-IIA peptides by immunofluorescence analysis. An immunofluorescence analysis can detect a minute amount of abnormal NMMHC-IIA peptides, even in cases with no inclusion bodies detected with May-Giemsa staining. Negative immunofluorescence analysis findings for NMMHC-IIA can rule out MYH9-related disorders 36. Perfusion studies Platelet adhesion and aggregation under flow can be measured in vitro by flow chamber-based methods. These methods are based on the perfusion of whole blood over a platelet-activating surface at physiological shear rates. Most used coatings are collagen, endothelial cells, fibrinogen and von Willebrand factor. Rate of adhesion and aggregation, surface coverage by platelet thrombi and thrombus volumes can be measured 61. Version 1.1: November 2015 19 of 28

5.2.5 Instruments ISTH-BAT Bleeding symptoms will be evaluated using the ISTH-BAT. The BAT systematically evaluates the number and severity of 14 different bleeding symptoms: epistaxis, cutaneous bleeding, minor cutaneous wound, oral cavity bleeding, hematemesis/melena/hematochezia, hematuria, tooth extraction, surgical bleeding, menorrhagia, post-partum bleeding, muscle hematomas, hemarthrosis, CNS bleeding and other bleeding. Symptoms will be scored on a scale ranging from 0 to 4 points. Higher scores reflect more severe or frequent bleeding. The total of all 14 items results in a bleeding score 38. Although the ISTH-BAT is not validated in a large cohort of patients 39, this tool is used at the VCK to document bleeding symptoms. RAND-36 health survey The RAND 36-items health survey is a generic measure of physical and mental health comprising 8 scales, roughly covering all ICF domains, measuring quality of life 62,63. This instrument is not specific to a particular condition. We chose the RAND- 36 because it is a widely used questionnaire, also in hemophilia patients 64. Brief Illness Perception Questionnaire (B-IPQ) The Brief Illness Perception Questionnaire (Brief IPQ) is a nine-item scale designed to rapidly assess the cognitive and emotional representations of illness 65. This instrument is not specific to a particular condition. 5.2.6 Total Burden For patients referred to the VCK for additional diagnostic workup, the physician at the VCK will fill out the BAT as part of normal care. After being seen by the physician, patients will be verbally informed about the study and informed consent will be asked. Participants will receive the questionnaire. Filling out this questionnaire will take approximately 20 minutes. Afterwards, one of the investigators or GCP certified nurse will go through the questionnaire with the participant and complete missing data where possible. Next, venipuncture will take place at the VCK. The risk associated with this venipuncture is negligible. The total amount of time required for the visit to the VCK is minimum 1 hour and maximum 2,5 hours. For patients with a previously diagnosed PFD, an appointment at the VCK will be scheduled. During their visit, they will be verbally informed about the study and informed consent will be asked. The BAT will be filled out by one of the investigators or GCP certified nurse. After that, participants will fill out the questionnaire and one of the investigators or GCP certified nurse will go through the questionnaire with the participant and complete missing data where possible. Next, venipuncture will take place at the VCK. The total amount of time required for the visit to the VCK is minimum 1 hour and maximum 2 hours. Version 1.1: November 2015 20 of 28

5.2.7 Follow up A few weeks after their visit, the treating physician will give the patients the results of the tests by telephone. It may be required to confirm the diagnosis or to perform additional tests. If so, patients will be asked to revisit the VCK and another venipuncture may be necessary. This is all part of normal care. If possible, patients will be referred back to their treating physician. 5.3 Withdrawal of individual subjects Subjects can leave the study at any time for any reason if they wish to do so without any consequences. The investigator can decide to withdraw a subject from the study for urgent medical reasons. 5.4 Replacement of individual subjects after withdrawal Since this study is observational, subjects will not be replaced after withdrawal. 6. SAFETY REPORTING 6.1 Section 10 WMO event In accordance to section 10, subsection 4, of the WMO, the sponsor will suspend the study if there is sufficient ground that continuation of the study will jeopardise subject health or safety. The sponsor will notify the accredited METC without undue delay of a temporary halt including the reason for such an action. The study will be suspended pending a further positive decision by the accredited METC. The investigator will take care that all subjects are kept informed. 6.2 Adverse events and serious adverse events Adverse events (AE) are defined as any undesirable experience occurring to a subject during the study, whether or not considered related to the investigational tests. All adverse events reported spontaneously by the subject or observed by the investigator or his staff will be recorded. A serious adverse event (SAE) is any untoward medical occurrence or effect that at any dose: - results in death; - is life threatening (at the time of the event); - requires hospitalization or prolongation of existing inpatients hospitalization; - results in persistent or significant disability or incapacity; - is a congenital anomaly or birth defect; - Any other important medical event that may not result in death, be life threatening, or require hospitalization, may be considered a serious adverse experience when, based upon appropriate medical judgment, the event may Version 1.1: November 2015 21 of 28

jeopardize the subject or may require an intervention to prevent one of the outcomes listed above. The investigator will report all SAEs to the sponsor without undue delay after obtaining knowledge of the events. The sponsor will report the SAEs through the web portal ToetsingOnline to the accredited METC that approved the protocol, within 7 days of first knowledge for SAEs that result in death or are life threatening followed by a period of maximum of 8 days to complete the initial preliminary report. All other SAEs will be reported within a period of maximum 15 days after the sponsor has first knowledge of the serious adverse events. Because of the minor invasive nature of the tests performed, SAEs are considered very unlikely to occur. We will only report SAEs that occur on the day of venipuncture. All adverse events will be reported to the primary investigator. 6.3 Follow-up of adverse events All adverse events will be followed until they have abated, or until a stable situation has been reached. Depending on the event, follow up may require additional tests or medical procedures as indicated, and/or referral to the general physician or a medical specialist. SAEs need to be reported till end of study within the Netherlands, as defined in the protocol 7. STATISTICAL ANALYSIS Data analysis will be performed with SPSS 21.0. Quantitative data will be presented as continuous variables of the bleeding score and RAND-36 score. Data will be assessed for normal distribution. Descriptives and frequencies of several parameters will be calculated as percentage and means. Receiver operators curves will be made for analysis of diagnostic performance of the several platelet function assays, including LTA and PACT. Univariate and multivariate analysis We will use linear regression to model the association of bleeding score with age, sex and type of PFD in a univariate model. Analysis of determinants of bleeding phenotype will be performed by univariate and multivariate linear regression analysis. A significance level of α=0.05 will be used. 8. ETHICAL CONSIDERATIONS 8.1 Regulation statement This study will be conducted in accordance to the principles of the Declaration of Helsinki, ICH guidelines on Good Clinical Practice (GCP) and in accordance with the Medical Research Involving Human Subjects Act (WMO). The local investigator is responsible for Version 1.1: November 2015 22 of 28

ensuring that the study will be conducted in accordance with the protocol, ethical principles, ICH-GCP guidelines and the WMO. 8.2 Recruitment and consent Subjects will be informed about the study by a treating physician, either their referring physician or the physician of the VCK. A minimum of one week will be allowed to consider their decision. Informed consent will be asked and signed with the subject by one of the investigators or GCP certified nurse, not being the treating physician. The patient information letter and informed consent in Dutch are attached as separate documents. 8.3 Benefits and risks assessment, group relatedness This study will contribute to the knowledge on clinical presentation and quality of life of patients with platelet function disorders in the Netherlands. It will contribute to improving the diagnostic approach of patients presenting with bleeding symptoms. The participating patients may benefit directly from participation, as the use of new tests in this study may lead to diagnosing patients who were previously undiagnosed. A correct diagnosis is important for patients to get accurate information and counseling, important for doctors in order to determine the right treatment and important for researchers to determine a possible relationship between clinical phenotype and genotype and to unravel the molecular pathways of PFDs. Risks imposed by participation are considered negligible. 8.4 Compensation for injury Any injury resulting from the study investigations is considered implausible; therefore no special insurance will be necessary to compensate for injury. 9. ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION 9.1 Handling and storage of data and documents The handling of personal data complies with the Dutch Personal Data Protection Act (in Dutch: De Wet Bescherming Persoonsgegevens, Wbp). Patient data will be coded by number of inclusion to protect privacy of the subjects. Subject identification codes will consist of a study code and serial number. Subject identification codes will be ascribed on arrival and these codes will be used throughout the study. The key to the identification code will be safeguarded by the principal investigator. Data will be stored on protected files on personal computers according to Standard Operating Procedures of the Department of Internal Medicine and Dermatology. Research data will be kept until publication of the study results and stored for 15 years at the investigational site where the data was collected. The primary investigator will be responsible for data management. Version 1.1: November 2015 23 of 28

9.2 Monitoring and Quality Assurance This study can be considered a low risk study, and therefore requires minimal monitoring. According to the Monitoring Plan (K6. Monitoringplan versie 1.0), monitoring will take place before the start of the study, after the inclusion of the first 5 patients followed by once a year, and after termination of the study. Details can be found in the Monitoring Plan. Monitoring will be carried out by independent experts of the UMC Utrecht (Monitor Laag Risico Onderzoek, Julius Centrum). 9.3 Amendments Amendments are changes made to the study procedures after a favorable opinion by the accredited METC has been given. All amendments will be notified to the METC that gave a favorable opinion. 9.4 Annual progress report The coordinating investigator will submit a summary of the progress of the trial to the accredited METC once a year. Information will be provided on the date of inclusion of the first subject, numbers of subjects included and numbers of subjects that have completed the trial, serious adverse events/ serious adverse reactions, other problems, and amendments. 9.5 End of study report The investigator will notify the accredited METC of the end of the study within a period of 8 weeks. The end of the study is defined as the last patient s last visit. In case the study is ended prematurely, the investigator will notify the accredited METC, including the reasons for the premature termination. Within one year after the end of the study, the investigator/sponsor will submit a final study report with the results of the study, including any publications/abstracts of the study, to the accredited METC. 9.6 Public disclosure and publication policy The study results will be disclosed unreservedly. After approval of the study protocol by the METC this trail will be registered in a public trail registry. Version 1.1: November 2015 24 of 28