Coagulation Cascade

Coagulation Cascade

Initiation

Am

plification

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 Xq28; most common (45%) defe

ct is inversion and translocation of exons 1-22 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/dL (1% activity)• Moderate: 1%-5%• Mild: > 5%

Definition• 1 unit = the amount found in 1ml of normal pool plasma• 100% activity = the actvity found in 1ml of normal pool pl

asma

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

其他出血的警訊• 腦出血： infants with “meningitis”

• 後腹腔出血：嚴重腹痛，可能誤為盲腸炎• 頸部、咽喉出血：喉痛、吞嚥困難，可能阻塞呼吸道

• Toddler: ankle most, than knee

• Child: knee most, than ankle

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)

1840s First transfusion administered

1940s Transfusion therapy estabilished

1950s FFP; early factor concentrates

1960s Cryoprecipitate

1970s Intermediate-purify factor concentrates; DDAVP

1980s Monoclonal antibody-purified and high-prify factor VIII concentrates; effective viral inactivation

1990s High-purify factor IX concentrates; recombinant factor VIII and IX threapy

2000s Improved recombinant products; gene therapy ?

治療• A 型： FFP ， cryoprecipitate 或 factor VIII co

ncentrate (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 recombin

ant product• DDAVP for mild or moderate form• Antifibrinolytic therapy

On-demand Therapy

• 一有症狀出現就及早注射（即補充）凝血因子

• 劑量及次數視體重、病情輕重、出血程度及出血部位而定

Recommeded Plasma Factor Level and Duration of Administration

Type of hemorrhage

Hemophilia A Hemophilia B

Desired level

Duration (days)

Desired level

Duration (days)

Joint 40-60% 1-2 or longer 40-60% 1-2 or longer

Muscle 40-60% 2-3 or longer 40-60% 2-3 or longer

Iliopsoas

Initial 80-100% 1-2 60-80% 1-2

Maintenance 30-60% 3-5 or longer 30-60% 3-5 or longer

CNS/head

Initial 80-100% 1-7 60-80% 1-7

Maintenance 50% 8-14 30% 8-14

Type of hemorrhage

Hemophilia A Hemophilia B

Desired level Duration (d) Desired level Duration (d)

Throat and neck

Initial 80-100% 1-7 60-80% 1-7

Maintenance 50% 8-14 30% 8-14

GI

Initial 80-100% 1-6 60-80% 1-6

Maintenance 50% 7-14 30% 7-14

Kidney 50% 3-5 40% 3-5

Surgery (major)

Pre-op 80-100% 60-80%

Post-op 60-80%

40-60%

30-50%

1-3

4-6

7-14

40-60%

30-50%

20-40%

1-3

4-6

7-14

Prophylactic Therapy

• 原理：將重度缺乏者的凝血因子提升至 >1%

• 有效減少自發性出血• 避免關節出血及進一步關節病變• 瑞典最早開始， A 型血友病自 1958 年起，

B 型血友病自 1972 年起

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

一般照護• 預防注射 : 最小的針頭接種• 不可接受其他肌肉注射• 勿吃含有阿斯匹靈的止痛藥，但 Panadol,

Ponstan, Codeine 可以服用• 牙齒的保護

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 morbi

dity

• 預防是最佳的處理方法• Arthroscopic synovectomy

• Joint replacement

輸血引發的感染• 經加熱等特殊程序處理血液製品後， 1985年以後出生的血友病患者，至今並無因注射凝血因子而感染愛滋病的報告

• HBV, HCV infection

(vaccination using is recommended)

• Recombinant product 可完全免除這種潛在的危險

凝血因子抗體 • 14-25% of severe hemophilia A, very rare i

n hemophilia B

• 可能與基因有關• 輕者可能需要大量而頻繁的注射才可能止血

• 嚴重的就可能對注射凝血因子無效，必須使用其他凝血因子 (by-pass)

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 functio

n

Factor VIII inhibitors

• The most common inhibitor

• Polyclonal IgG antibodies, esp IgG4

• Bleeding is more severe in autoantibody patients than in hemophilia A inhibitor patients

EtiologyDefinite Factors Involved

• FVIII gene mutation No FVIII protein means high risk• Adjuvants in FVIII products• Race Higher in African-Americans• HLA status• Immune modifiers IL10 polymorphism

Classification of Inhibitors

Definitions• “High” responders IgG 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 High-responder 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 High-responder Inhibitors

• Immune tolerance induction• Rituximab Anti-CD20 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 (chrom

osome 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 2A Decreased platelet-dependent function with absence of largest multimers

Dominant

Type 2B Increased VWF affinirty for platelet GPIb

Dominant. May be associated with thrombocytopenia, especially after DDAVP

Type 2M Decreased platelet-dependent function with presence of largest multimers

Dominant

Type 2N 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.

vWF Genetics

• http://www.vwf.group.shef.ac.uk/pictures.html

Location of Gene: chromosome 12 (p13.3)

vWF Protein

• A1: binds to Gp IB alpha• A3: Collagen binding Domain• D’/D3: Interacts with Factor VIII• C2: Interacts with GpIIb/IIIa


vWF Protein

• A1: type 2B and 2M• A2: type 2A, cleavage site for ADAMTS 13• D’/D3: type 2N


Diagnosis

vWD Tests: Initial Work-up

• Quantitative

• Factor VIII level

• vWF Antigen level

• vWF Multimers

• Qualitative

• Ristocetin Cofactor Assay: studies function of Vwf/Platelet interaction

Lab Values in vWD Subtypes

Variants of vWD

Official Abbreviated Terms as Designated by ISTH

Factor VIII Von Willebrand factor

Type of Test Official Old or informal

Official Old or informal

Immunological

(total amount, functional or not)

FVIII:Ag FVIII:CAg VWF:Ag FVIIIR:Ag

AHF:Ag

Functional

(functional assay)

FVIII FVIII:C, AHF, AHG

VWF:RCo

(ristocetin cofactor)

VWF:CB(collagen binding)

VWF:FVIIIB

(factor VIII binding)

FVIIIR:RCo and others

VWF:CBA

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 vWF) for surgery, traum

a• Platelet for pseudo-vWD• Recombinant factor 7a, correct underlying disord

er (hypothyroidism) for acquired vWD

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, vWF, 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, vWF Lysosomes, peroxisomes

Hemostasis: Adhesion

• Initial event in hemostasis• Platelets contact subendothelial components ex

posed after vessel injury• vWF secreted into extracellular matrix from endo

thelial cells binds to GPIb-V-IX on platelet surface

• vWF 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, vWF, thrombospondin, fa

ctor 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• vWD 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 tend

ency• Abnormality of the GP Ib-IX-V complex• Normally binds to vWF• 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, aracha

donic acid, collagen• Fails in response to ristocetin• Cannot be corrected by the addition of normal pl

asma containing vWF• Abnormal flow cytometry

Glanzmann Thrombasthenia

• First described in 1918• AR• Present with mucocutaneous bleeding as neonat

e or infant• Bleeding tendency, normal platelet count• Deficiency of GPIIb/IIIa• Normally binds to fibrinogen and vWF• Cross links adjacent platelets to form platelet plu

g

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 risto

cetin• Flow cytometry abnormal

Storage Pool DisordersGray 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 DisordersDense 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


Chediak-Higash• Partial oculocutaneous al

binism• Frequent pyogenic infecti

on• 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


• 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 ActivityScott syndrome

• AR• Severe bleeding• Decrease transport of phospholipids to surface o

f activated platelet• Decreased expression of factor Xa binding sites• Failure of factor Xa to bind• Incapacity of the activated cell surface to transfo

rm prothrombin to thrombin• Prothrombin consumption test is the only abnor

mal 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 DestructionNonimmune

• Hemolytic-uremic syndrome

Microangiopathic anemia

Bloody diarrhea (E coli O157:H7)

• Disseminated intravascular coagulation

Microangiopathic anemia; low fibrinogen

Sepsis

• Cyanotic heart disease

Increased Platelet DestructionImmune

• 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 w

ith 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 transfusi

on for bleeding• Severity increases with s

ubsequent 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 > 30k• Asymptomatic or only minor purpura: no treatment• Platelet counts < 20k• Significant mucosal bleeding• Treatment with IVIG or steroids• Platelet counts < 10k• 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 a

pproximately 2.5 times than steroid gr• Average platelet count rose over 30k by 24 hrs i

n IVIG, and by 48 in steroid gr. At one week the levels were equal

• IVIG will not affect subsequent BM biopsy results

• Dosing: 1g/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: 2mg/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 secretio

n in response to many agonists• May be caused by both dialyzable and nondialyz

able substances• Cardiopulmonary bypass surgery• SLE• Chronic myeloproliferative disorders and acute l

eukemia

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, larg

e vascular aneurysms)• Severe hepatic faliure• Severe toxic or immunologic reactions (snake bites, tran

sfusion 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: 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)• 4G/5G 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)

Score 0 1 2

Platelet () > 100 < 100 < 50

FDP No increase Moderate Strong

PT (sec.) < 3 3-6 >6

Fibrinogen (g/L) >1 <1

• 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

Coagulation Cascade

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