Coagulation Cascade Amplification Initiation Introduction Congenital bleeding disorder

Coagulation Cascade Amplification Initiation

Introduction • Congenital bleeding disorder caused by low levels of specific coagulation factors • Hemophilia A: 85%, factor VIII deficiency Third most common X-linked disorder • Hemophilia B: 10%-15%, factor IX deficiency

Genetics • X-linked recessive disorder • FVIII gene is on Xq 28; most common (45%) defect is inversion and translocation of exons 122 away from 23 -26, others: point mutation • 1/10, 000 live male births • 女性也可能為symptomatic carriers (ex. extreme “Lyonisation” of the normal X chromosome) • 約1/3為mutation

Classifications 依血液中凝血因子的剩餘含量而定 • Severe: < 1 unit/d. L (1% activity) • Moderate: 1%-5% • Mild: > 5% Definition • 1 unit = the amount found in 1 ml of normal pool plasma • 100% activity = the actvity found in 1 ml of normal pool plasma

Bleeding Manifestations in Hemophilia Sites of bleeding • Serious Joints (hemarthrosis), muscle/soft tissue, mouth/gum/nose, hematria • Life-threatening CNS, GI, neck/throat, severe trauma • Incidence of different sites of bleeding Hemarthrosis: 70 -80% Muscle/soft tissue: 5 -10% CNS: <5%

Clinical Manifestations • 嚴重者，出生時就可能出現subgaleal hematoma and/or ICH〈應特別留意嬰兒室 頭圍迅速增大的新生兒〉 • 症狀可能多在開始爬及走路時才出現 • The hallmark of hemophilic bleeding: 自發 性的關節出血 (hemarthrosis) and intramuscular hematoma • 1 -2 % ICH

Diagnosis and Laboratory Tests • • • Family history Normal platelet count, bleeding time, PT Prolonged PTT Specific factor assays Genetic testing Prenatal diagnosis

Management • History of hemophilia treatment Decade Milestone (s) 1840 s First transfusion administered 1940 s Transfusion therapy estabilished 1950 s FFP; early factor concentrates 1960 s Cryoprecipitate 1970 s Intermediate-purify factor concentrates; DDAVP 1980 s Monoclonal antibody-purified and high-prify factor VIII concentrates; effective viral inactivation 1990 s High-purify factor IX concentrates; recombinant factor VIII and IX threapy 2000 s Improved recombinant products; gene therapy ?

治療 • A型： FFP，cryoprecipitate 或 factor VIII concentrate (dose: desired rise level % × BW × 0. 5) • B型：FFP或factor IX concentrate (dose: desired rise level % × BW × 1. 2~1. 5) • Factor concentrate: plasma-derived or recombinant product • DDAVP for mild or moderate form • Antifibrinolytic therapy

Recommeded Plasma Factor Level and Duration of Administration Type of hemorrhage Hemophilia A Hemophilia B Desired level Duration (days) Joint 40 -60% 1 -2 or longer Muscle 40 -60% 2 -3 or longer Initial 80 -100% 1 -2 60 -80% 1 -2 Maintenance 30 -60% 3 -5 or longer 80 -100% 1 -7 60 -80% 1 -7 50% 8 -14 30% 8 -14 Iliopsoas CNS/head Initial Maintenance

Type of hemorrhage Hemophilia A Hemophilia B Desired level Duration (d) 80 -100% 1 -7 60 -80% 1 -7 50% 8 -14 30% 8 -14 80 -100% 1 -6 60 -80% 1 -6 Maintenance 50% 7 -14 30% 7 -14 Kidney 50% 3 -5 40% 3 -5 Throat and neck Initial Maintenance GI Initial Surgery (major) Pre-op 80 -100% Post-op 60 -80% 40 -60% 30 -50% 60 -80% 1 -3 4 -6 7 -14 40 -60% 30 -50% 20 -40% 1 -3 4 -6 7 -14

Prophylaxis • Initial observation: persons with moderate hemophilia (1 -5% FVIII) have decreased joint disease • Hypothesis: converting a person from severe hemophilia to moderate with prophylaxis would decrease incidence of joint disease • Goat: to raise FVIII above 1% was commenced in Malmo, Sweden in 1958 • Lovqvist, et al: J Intern Med 1997

When to Start: The Swedish Experience Conclusion: prophylaxis should be started in the first years of life, before age 3 Astermark et al: Br J Hematol 1999

長期的關節病痛問題 • Target joints: 膝關節、踝關節及肘關節 • 惡性循環下，發生慢性關節炎 • Arthropathy: most significant chronic morbidity • 預防是最佳的處理方法 • Arthroscopic synovectomy • Joint replacement

What are inhibitors ? • Antibodies directed against coagulation factors Alloantibody in patients with hemophilia A or B Autoantibody in people without hemophilia • Incidence Antifactor VIII inhibitors in hemophilia A: 25% Antifactor IX inhibitors in hemophilia B: 1 -3% Antifactor VIII autoantibody inhibitors: 1/106/year • Usually result in loss of coagulation factor function

Factor VIII inhibitors • The most common inhibitor • Polyclonal Ig. G antibodies, esp Ig. G 4 • Bleeding is more severe in autoantibody patients than in hemophilia A inhibitor patients

Etiology Definite Factors Involved • FVIII gene mutation No FVIII protein means high risk • Adjuvants in FVIII products • Race Higher in African-Americans • HLA status • Immune modifiers IL 10 polymorphism

Classification of Inhibitors Definitions • “High” responders Ig. G inhibitors of titer > 5 Bethesda Units (BU) Inability to overwhelm with native factor • “Low” responders < 5 BU Transient Less likely to have anamnestic responses Amenable to treatment by overwhelming inhibitor with native factor VIII or XI

Treatment Options for Highresponder Inhibitors Bypassing agents • Low-purity, plasma-derived concentrates Prothrombin complex concentrates Activated prothrombin complex concentrates Recombinant VIIa • Emergency treatments Recombinant VIIa Plasmapheresis Porcine VIII and new recombinant procine VIII

Treatment Options for Highresponder Inhibitors • Immune tolerance induction • Rituximab Anti-CD 20 chimeric antibody reliably depletes peripheral B cells Several reports of success in acquired hemophilia (an autoimmune disorder) NHLBI-sponsored clinical trial through Transfusion Medicine/Hemostasis research network to begin May 2006 • Fox et al, Hemophilia 2006

von Willebrand Disease • Disorder first described by Erik von Willebrand in 1925 in persons living off the coast of Finland • Marked heterogeneity in phenotype, autosomal Dominant or Recessive Inheritance • Deletion in chromosome 12 is most common • Overall prevalence 1: 100 to 1: 500 • Incidence equal among Man and Women (chromosome 12)

1994 Classifications of VWD by TSTH 1994 Term 1994 Definition Genetics, Comment Type 1 Partial quantitative deficiency Dominant with variable expression; phenotype influenced by multiple genes Type 2 Qualitative defect Type 2 A Decreased platelet-dependent function with absence of largest multimers Dominant Type 2 B Increased VWF affinirty for platelet GPIb Dominant. May be associated with thrombocytopenia, especially after DDAVP Type 2 M Decreased platelet-dependent function with presence of largest multimers Dominant Type 2 N Decreased VWF affinity for FVIII Recessive, often mistaken for mild-moderate hemophilia A Type 3 Virtually complete deficiency Recessive: homozygous or doubly heterozygous Platelet-type (pseudo-VWD) Not a defect of VWF, not to be considered a form of VWD Dominant. A platelet disorders: increased affinity of platelet GPIb for VWF. Thrombocytopeinia may be present.

v. WF Genetics Location of Gene: chromosome 12 (p 13. 3) • http: //www. vwf. group. shef. ac. uk/pictures. html

v. WF Protein • • A 1: binds to Gp IB alpha A 3: Collagen binding Domain D’/D 3: Interacts with Factor VIII C 2: Interacts with Gp. IIb/IIIa • http: //www. vwf. group. shef. ac. uk/pictures. html

v. WF Protein • A 1: type 2 B and 2 M • A 2: type 2 A, cleavage site for ADAMTS 13 • D’/D 3: type 2 N • http: //www. vwf. group. shef. ac. uk/pictures. html

Diagnosis

v. WD Tests: Initial Work-up • • • Quantitative Factor VIII level v. WF Antigen level v. WF Multimers Qualitative Ristocetin Cofactor Assay: studies function of Vwf/Platelet interaction

Lab Values in v. WD Subtypes

Variants of v. WD

Official Abbreviated Terms as Designated by ISTH Factor VIII Von Willebrand factor Type of Test Official Old or informal Immunological (total amount, functional or not) FVIII: Ag FVIII: CAg VWF: Ag FVIIIR: Ag AHF: Ag VWF: RCo (ristocetin cofactor) VWF: CB (collagen binding) VWF: FVIIIB (factor VIII binding) FVIIIR: RCo and others VWF: CBA FVIII Functional (functional assay) FVIII: C, AHF, AHG ISTH: International Society on Thrombosis and Hemostasis

Differential Diagnosis Hemophilia A & von Willebrand Disease

Management • Education • Cryoprocipitate (dose: desired rise level % × BW × 0. 75) • DDAVP for type 1 • Amicar (antifibrinolytic agent) for mucosal bleeds • Humate-P (factor 8 and v. WF) for surgery, trauma • Platelet for pseudo-v. WD • Recombinant factor 7 a, correct underlying disorder (hypothyroidism) for acquired v. WD

Platelet

Platelet Anatomy • • Disc-shaped, anuclear fragment Size: 1. 5 μm Normal maturation time 4 -5 days Circulating life span 9 -10 days

Platelet Anatomy • Peripheral zone Plasma membrane Open canalicular system Extension of the plasma membrane Forms interconnecting network, greatly increases the surface area Membrane proteins: receptors for agonists and adhesive glycoproteins, signal transduction molecules IIb-IIIa: fibrinogen, v. WF, fibronectin Ib-IIa: collagen Ib-IX-V: insoluble Vwf VI: collagen

Platelet Anatomy • Submembranous zone Contractile protein system: regulates shape and carry out events such as secretion of granules and retraction of clots • Organelle zone Platelet specific storage granules Dense bodies: serotonin, ADP, ATP, Ca α granules: platelet factor 4, thromboglobulin, PDGF, v. WF Lysosomes, peroxisomes

Hemostasis: Adhesion • Initial event in hemostasis • Platelets contact subendothelial components exposed after vessel injury • v. WF secreted into extracellular matrix from endothelial cells binds to GPIb-V-IX on platelet surface • v. WF on endothelial cells forms a bridge between the subendothelium and platelet

Hemostasis: Activation • Interaction between GP Ia-IIa and GP VI with collagen results in platelet arrest and activation • Forms a firm adherence • Leads to intracellular signaling processes that initiate secretion • Activated platelets express a procoagulant surface

Hemostasis: Secretion • • • Undergo shape change Spherical Pseudopods Spread over the exposed subendothlium Contents of platelet granules are released α granules: fibrinogen, v. WF, thrombospondin, factor V, vitronectin • Dense granules: ADP, ATP, serotonin, calcium

Hemostasis: Aggregation • Once activated, platelets become adhesive to each other • Interact via fibrinogen bound to their GPIIb -IIIa receptors • Microthrombus of aggregated platelets is formed

Presentation of Disorders of Platelet Function • • Mucocutaneous bleeding Gingiva Epistaxis Menorrhagia Petechiae Ecchymoses Bleeding after trauma and surgery Rare: ICH, joint, muscle

Clinical Presentation of Bleeding Disorder Clinical signs Disorders of coagulation Disorder of platelets or vessles Petechiae Rare Characteristics Ecchymoses Common, large Characteristics, small Bleeding from superficial cuts Minimal Persistent Delayed bleeding Common Rare Deep hematomas Characteristics Rare Hemothrosis Characteristics Rare

Diagnostic work up • • • Initial Platelet count and morphology PT, PTT (mixing studies) Platelet function analyzer (PFA)-100 Bleeding time v. WD panel Platelet aggregation Flow cytometry, electron microscopy Detailed drug history

Inherited Platelet Function Disorders • Adhesion Bernard Soulier Syndrome Collagen receptor deficiency • Aggregation Glamzman’s Thrombasthenia • Secretion Storage pool disorders • Coagulant activity Scott syndrome

Bernard-Soulier Syndrome • • First described in 1948 AR Present in infancy or early childhood Thrombocytopenia, giant platelets, bleeding tendency Abnormality of the GP Ib-IX-V complex Normally binds to v. WF Initial platelet adhesion to the subendothelium Mutations in Ibα, Ibβ, or IX

Bernard-Soulier Syndrome • • • Prolonged bleeding time Thrombocytopenia, variable Abnormal smear, enlarged platelets Aggregation Normal in response to ADP, epinephrine, arachadonic acid, collagen • Fails in response to ristocetin • Cannot be corrected by the addition of normal plasma containing v. WF • Abnormal flow cytometry

Glanzmann Thrombasthenia • First described in 1918 • AR • Present with mucocutaneous bleeding as neonate or infant • Bleeding tendency, normal platelet count • Deficiency of GPIIb/IIIa • Normally binds to fibrinogen and v. WF • Cross links adjacent platelets to form platelet plug

Glanzmann Thrombasthenia • • Normal platelet count and morphology Prolonged bleeding time PFA-100 COL/EPI abnormal COL/ADP abnormal Aggregation Abnormal in response to all agonists except ristocetin • Flow cytometry abnormal

Storage Pool Disorders Gray Platelet Syndrome • • • Absence of αgranules (normal ~ 50) AR Molecular defect unknown Mild mucocutaneous bleeding Variably prolonged bleeding time Moderate thrombocytopenia Reticulin fibrosis of BM Large gray platelet EM: small, empty or absent αgranules

Storage Pool Disorders Dense Granule Disorders • • • Normal dense granules 3 -6/platelet Serotonin, ADP, ATP, Ca Heterogeneous group of disorders Molecular defect unknown Mild to moderate bleeding

Storage Pool Disorders • Two autosomal recessive syndromes associated with albinism Chediak-Higash Hermansky-Pudlack • Non-albino syndromes Wiskott-Aldrich Thrombocytopenia absent radii Osteogenesis imperfecta

Storage Pool Disorders Chediak-Higash • Partial oculocutaneous albinism • Frequent pyogenic infection • Giant lysosomal granules in cells • Thrombocytopenia • Dense granule deficiency Hermansky-Pudlack • Oculocutaneous albinism • Inclusions in the cells of RES • Thrombocytopenia • Dense granule deficiency • Common in Puerto Rico

Storage Pool Disorders • • • Clinical presentation Platelet morphology normal Bleeding time usually, not always prolonged Aggregation Marked impairment with weak agonists ADP, epinephrine and low concentrations of collagen • Response to higher concentration may be normal • Absent second wave of aggregation when stimulated by ADP and epinephrine

Disorders of Procoagulant Activity Scott syndrome • AR • Severe bleeding • Decrease transport of phospholipids to surface of activated platelet • Decreased expression of factor Xa binding sites • Failure of factor Xa to bind • Incapacity of the activated cell surface to transform prothrombin to thrombin • Prothrombin consumption test is the only abnormal test

Thrombocytopenia • Increase platelet destruction Immune Non-immune • Decreased platelet production Congenital Acquired • Sequestration • Qualitative platelet disorders

Qualitative Platelet Disorders • Wiskott-Aldrich syndrome X-linked; small platelet • Bernard-Soulier syndrome AD, large platelets • May-Hegglin anomaly AD, giant platelet, Dohle bodies • Gray platelet syndrome Pale/oval platelet • Glandzmann’s thrombesthenia

Sequestration • Kasabach-Merritt syndrome • Hypersplenism May be associated with infiltrative disease (leukemia) May arise from liver disease, portal hypertension • In Vitro platelet clumping • Easily diagnosed by peripheral smear evaluation

Decreased Platelet Production Congenital • Thrombocytopenia-Absent Radius syndrome Absence of radii at birth Association with congenital heart disorder • Amegakaryocytic Thrombocytopenia Presents in neonatal period No skeletal anomalies • Fanconi Anemia Due to aplastic anemia Short stature, thumb and radii hypoplasia, microcephaly

Thrombocytopenia-Absent Radius

Decreased Platelet Production Acquired • • Leukemia Aplastic anemia Neuroblastoma Due to bone marrow metastasis Drugs Nutritional deficiency Megaloblastic anemia

Increased Platelet Destruction Nonimmune • Hemolytic-uremic syndrome Microangiopathic anemia Bloody diarrhea (E coli O 157: H 7) • Disseminated intravascular coagulation Microangiopathic anemia; low fibrinogen Sepsis • Cyanotic heart disease

Increased Platelet Destruction Immune • • • HIV Post transfusion Drugs Collagen-vascular disease Neonatal alloimmune thrombocytopenia Idiopathic thrombocytopenia

Heparin and Thrombocytopenia • Immune Heparin-induced thrombocytopenia • Initially presents as decrease in platelet with or without thrombosis • Two distinct syndromes: • 1 Uaually mild and transient thrombocytopenia with rapid recovery upon discontinuation of heparin • 2 Severe thrombocytopenia often complicated by thrombosis or DIC

Neonatal Thrombocytopenia Child born to Mother with ITP • Mother with thrombocytopenia • Resolves within about 6 weeks • Risk of ICH 1% • Avoid maternal platelet transfusion Neonatal alloimmune thrombocytopenia • Mother with normal platelet • Resolves with about 6 weeks • Risk of ICH 10 -30% • Maternal platelet transfusion for bleeding • Severity increases with subsequent siblings

Idiopathic Thrombocytopenia Purpura • Diagnosis is based primarily on the history, PE, CBC, and peripheral smear examination • CBC must show isolated and usually severe thrombocytopenia • Bone marrow aspiration should be performed in patients with thrombocytopenia lasting more than 6 to 12 months, and in those unresponsive to IVIG therapy

Idiopathic Thrombocytopenia Purpura • • • Clinical features M=F Child is well with rapid onset of thrombocytopenia Generally seen in children 1 to 9 years old Peak incidence is between 2 to 5 y/o 1 in 1500 persons with get ITP in childhood Seasonal presentation More common in winter and fall Platelet-specific autoantibodies seen 4 -8 weeks following a viral illness or exposure

ITP Treatment • • • Severe life-threatenin bleeding IVIG, steroids, platelet transfusion Platelet counts > 30 k Asymptomatic or only minor purpura: no treatment Platelet counts < 20 k Significant mucosal bleeding Treatment with IVIG or steroids Platelet counts < 10 k Only minor purpura Treatment with IVIG or steroids

ITP Treatment • IVIG • More rapid increase in platelet counts • The mean platelet count in the IVIG group was approximately 2. 5 times than steroid gr • Average platelet count rose over 30 k by 24 hrs in IVIG, and by 48 in steroid gr. At one week the levels were equal • IVIG will not affect subsequent BM biopsy results • Dosing: 1 g/k on 1 day

ITP Treatment • Steroids • IVIG costs about 150 times as much as treatment with steroids • IVIG can cause aseptic meningitis • Causing an emergent diagnostic evaluation to rule out ICH • Dosing: 2 mg/k/day

Chronic ITP • Defined as thrombocytopenia lasting longer than 6 months • Child may present with recurrent upper an lower respiratory tract infections, GERD, and FTT • Family history is often positive for ITP or other autoimmune disease (SLE)

Acquired Platelet Defects • Medications • Chronic renal failure • Abnormal platelet aggregation, reduced secretion in response to many agonists • May be caused by both dialyzable and nondialyzable substances • Cardiopulmonary bypass surgery • SLE • Chronic myeloproliferative disorders and acute leukemia

DIC

Clinical Conditions Associated with DIC • • • Sepsis/severe infection (any microorganism) Trauma (polytrauma, fat embolism) Organ destruction (severe pancreatitis) Malignancy (solid tumors, hematological malignancy) Obstetrical calamities (amniotic fluid embolism, abruptio placentae) • Vascular abnormalities (Kasabach-Merritt syndrome, large vascular aneurysms) • Severe hepatic faliure • Severe toxic or immunologic reactions (snake bites, transfusion reactions, transplant rejection)

Pathogenesis: Initiation of Fibrin Deposition Hambleton, J. et al. Hematology 2002

Pathogenesis: Amplification of Fibrin Deposition Hambleton, J. et al. Hematology 2002

Pathogenesis: Amplification of Fibrin Deposition Hambleton, J. et al. Hematology 2002

Pathogenesis: Propagation of Fibrin Deposition • Fibrinolytic system is downregulated at the time of max. activation of coagulation • In baceremia, endothelial cells release plasminogen activators • Increase plasminogen activator inhibitor, type 1 (PAI-1) immediately to suppress of fibrinolytic activity • High PAI-1 level strongest predictors of mortality (ref 3) • 4 G/5 G polymorphism, functional mutation of PAI-1 gene indicated higher PAI-1, increased risk of death in bacteremia (ref 38)

Diagnosis • 1 Underlying disorder • 2 Global coagulation tests (platelet; PT; fibrinogen; FDP) Platelet () FDP PT (sec. ) Fibrinogen (g/L) Score 0 > 100 No increase <3 1 < 100 Moderate 3 -6 >1 <1 2 < 50 Strong >6 • If score ≧ 5: DIC, repeat daily • If score < 5: non-overt DIC, repeat next 1 -2 days

Management • 1. Plasma and platelet substitution therapy • Only in active bleeding, requiring an invasive procedure, at risk for bleeding complications • 2. Anticoagulants • 3. Restoration of anticoagulant pathways