INHERITED COAGULATION DISORDERS

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INHERITED COAGULATION DISORDERS

• Kevin A. Rickard
• Royal Prince Alfred Hospital

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INHERITED COAGULATION DISORDERS

• HAEMOPHILIA A, HAEMOPHILIA B von WILLEBRANDS
  disease
• Haemophilia is the most severe and most important
  inherited bleeding disorder
• Inherited as sex linked recessive condition
• Males affected
• Females carriers
• Bleeding manifestations of both are identical
• Cannot differentiate clinically Haemophilia A
  from Haemophilia B

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HAEMOPHILIA A

• HAEMOPHILIA A
• Commonest severe inherited bleeding disorder
• Bleeding due to deficiency of FVIII coagulant
  activity
• Severity of bleeding is related to FVIII conc in
  blood
• Low levels of FVIII (lt1iu/dl)
• Frequent bleeding
• spontaneous bleeding joints or muscles
• PREVALENCE
• Rely on diagnostic figures from UK, Europe, USA
• Haemophilia A - 90 per million of population
• Or 20 per 100,000 males
• 1 per 5,000 male births
• 1 per 10,000 population
• FVIII deficiency 80-85 of cases of haemophilia
• FVIII deficiency 40 severe
• ie. FVIII lt 2 of normal

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HAEMOPHILIA A

• Made of Inheritance
• Daughters of a haemophiliac are carriers
• Sons of a haemophiliac are normal
• Female carriers
• Sons 5050 normal or affected
• Daughters 5050 normal or carriers
• Severity of bleeding symptoms runs true in a
  family
• Severe - Severe
• Mild - Mild
• Important for
• Transmissions
• Prenatal diagnosis
• ? Termination

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HEAMOPHILIA A

• Inheritance
• Transmitted as a sex linked disorder
• Gene for FVIII production
• Long arm of X chromosomes (Xq 2.8)
• Gene large and complex
• 186kb, 26 exons
• Encodes for large protein
• 2332AA
• 6 Domains
• A1 A2 B A3 C1 C2
• B little part in clotting
• Lost during thrombin activation

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HAEMOPHILIA A

• Gene Defects
• Large number of gene defects identified
• Most mutations single nucleotides substitutions
• 5 mutations are insertions or deletions

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TYPES OF GENETIC MUTATIONS

• Partial gene deletion or rearrangement
• FVIII/FIX genes. No breakpoint hot spots,
  fragile sites
• Large insertions, inversions, complex
  rearrangements
• Subtle defect
• Micro deletions or insertions
• single base mutation (point mutations)
• Frame shift mutations
• Single base transitions
• Purine - Purine (A-G)
• Pyrimidine - Pyrimidine (T-C)
• Transitions
• Purinte ? Pyrimidine
• ?
• Missense mutation

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• MUTATIONS OF THE FVIII GENE
• Methods of detection
• Gene sequencing
• Screening procedures
• Sequencing
• Direct examination of products of PCR
  amplification
• or
• PCR amplification - transcription - transcript
  sequencing
• Screening
• Amplification mismatched detection
• Cleavage of DNA - presence, size, position
• Direct sequencing of amplified mismatched
  region
• Denaturing gradient gel electrophoresis
• Migration of DNA retarded
• Entering gel region with denaturant
• Meets stable domain

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GENETIC MUTATIONS

• Mutation type generally true in families
• Phenotype true in families
• Intron 22 Inversion
• Gene within a gene
• F8 supergene
• Transcribed in opposite direction
• Responsible for 1/3 of severe haemophilia

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Figure Mechanism of crossover between a copy of
F8A within the factor VIII gene and one of the
two telomeric copies of F8A.After the event two
separate mRNA species representing the two 5 and
3 halves can still be detected but are
unconnected, hence giving rise to severe
haemophilia A
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CLINICAL FEATRUESSeverity of Haemophilia
relates to Factor VIII/IX level
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• CLINICAL FEATURES
• Bleeding as a baby rare
• No circumcision if family history
• First bleeding in childhood
• Dental eruption
• Trauma with walking
• Bleeding in Haemophilia
• Acute Haemarthrosis
• Chronic haemophilic arthropathy
• Bleeding into muscles
• Haemophilic pseudo tumour - cysts
• Haematuria
• Gastrointestinal bleeding
• Intracranial bleeding

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BLEEDING INTO JOINTS

• Most characteristic feature of severe haemophilia
• Seen in majority of patients
• Factor VIII (or IX) lt 2
• STAGES
• Initial bleed into joint - haemarthrosis
• Inflammatory stage affecting
• Synovium (synovial hypertrophy)
• Cartilage
• Bone
• Final Stage
• Permanent joint changes
• Erosion destruction
• Cartilage
• Bone
• Knees Wrists
• Ankles Shoulders
• Elbows Hips

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CLINICAL FEATURES OF ACUTE HAEMARTHROSIS

• Premonitary abnormal sensation
• Pain and swelling of joint
• Limitation of movement - especially flexion
• Tenderness and heat in the joint
• Before HIV - Symptoms - Acute haemarthrosis
• Now - always ? Septic arthritis
• NB. Generally do NOT aspirate joint
• Rx with factor replacement
• Bleeding from synovial tissue
• Frequent bleeding - synovial hypertrophy
• - boggy synovium
• - swollen joint
• Haemosiderin deposition

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CHRONIC HAEMPHILIC ARTHROPATHY

• Repeated bleeds - many years
• Chronic degenerative changes
• Chronic haemophilic arthritis
• Loss of joint movement
• Fixed flexion contractures
• Severe muscle wasting
• Muscle action imbalance
• Valgus deformities
• Subluxation tibia on femur
• Crippling deformities
• Wheel chair ridden
• Above changes before adequate treatment an
  prophylaxis available

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CHRONIC HAEMOPHILIC ARTHROPATHYRADIOLOGICAL
CHANGES

• Epiphyseal overgrowth
• Enlargement of bone ends
• Loss of cartilage (joint space)
• Gross irregularity articular surface
• Subchondral collapse
• Subchondral cysts
• Osteophyte formation
• Osteoporosis
• Changes in joint alignment

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• HAEMOPHILIC PSEUDO TUMOURS (BLOOD CYSTS)
• Cysts within the fascial muscle envelope
• Cysts arising in muscles
• Cysts arising from sub-periosteal haemorrhage
• Pseudo tumours in bone
• Gross destruction of normal architecture of
  bone
• Large bone cysts
• Pathological fracture
• HAEMATURIA
• Common 3rd most frequent bleed - pain
• Frightening - needs management
• Careful with treatment
• Careful to avoid ureteric obstruction
• Reflux renal shutdown
• Dont treat till good urine flow

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• THERAPY FOR HAEMOPHILIA
• DEMAND THERAPY ? Hospital
• ? Home
• Long standing approach to haemophilia
• Patient treats when bleeds
• Bleeds produce tissue damage
• Arrest acute bleed
• No arrest, long term sequelae
• PROPHYLACTIC THERAPY
• Home - self or patient infused
• Small dose FVIII (FIX) ? 20-30 x 2-3 week
• Prevents bleeds
• Prevents long term sequelae
• 20 year follow up Sweden
• Cohort of men severe haemophilia. Normal joints
• Problems
• Small children
• Venous access

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BENEFITS OF TREATMENT HAEMOPHILIA 1 B, vWD

• Treatment necessary to stop bleeding
• Repeated bleeding causes tissued
  damage/destruction
• often irreversible
• Haemophilic arthropathy
• Bleeding
• Synovial hypertrophy
• Thin wall vessels proliferate
• New bleed
• Cartilage damage and destruction
• Loss of joint space
• Subchondral cyst formation
• New bone growth
• Collapse of weight bearing surfaces
• Chronic haemophilic arthropathy
• Treatment
• Demand
• Prophylaxis
• Prevent the above joint changes

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• THERAPY FOR HAEMOPHILIA
• Factor replacement
• Demand - If bleeding occurs
• Prophylaxis - Prevent bleeding
• Regular minimal administration
• Factor VIII standard activity
• 1ml fresh normal plasma
• 1 unit of Factor VIII coagulant activity
• 100 iu/dl (100)
• FACTOR RECOVERY AFTER I.V. INFUSION
• Factor VIII 100
• Factor IX 30-50
• DOSAGE CALCULATION
• FVIII 1u/kg Increase plasma factor VIIII by 2 -
  2iu/dl
• FIX 1u/kg Increase plasma factor IX by 1 -
  1iu/dl
• PLASMA VOLUME
• Weight in kg x 41 plasma volume - eg. 70 x 41
  3000ml

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LEVELS OF FVIII OR FIX FOR HAEMOSTASIS
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THERAPEUTIC MATERIALS

• Fresh frozen plasma and cryoprecipitate virtually
  abandoned
• Spread of infection
• HIV Hepatitis
• Non inactivated products
• CONCENTRATES
• Produced from pooled donor plasma
• Donors are completely screened
• Final products virally inactivated
• Products vary in purity
• SPECIFIC ACTIVITY (S.A.)
• Units of FVIII/mg protein
• INTERMEDIATE PURITY CONCENTRATES (Australia)
• S.A. 1-5u.mg of protein
• Chromatographic precipitation techniques

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• HIGH PURITY CONCENTRATES
• S.A. 25 - 50u/mg of protein
• (1) conventional chromatographic techniques
• (2) Immuno-affinity chromatography
• Monoclonal antibody immobilised
• eg. To FVIII or vWF
• RECOMBINANT PRODUCTS
• rFVIII r FIX
• vWF
• Monoclonally purified
• Recombinant FVIII products
• Contain virtually no vWF

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• VIRUCIDAL PROCESSES
• Blood and blood products can transmit infection
  or disease
• Hepatitis, HIV
• Virucidal processes developed
• Sterilize factor concentrates
• Coag factor concentrates are heat labile
• HIV
• Most virucidal processes seem to be effective
• Dry heat at only 60 for 30 hours
• Associated with no seroconversion
• RELATIVE RISK OF DONOR INFECTION
• eg. 1991
• USA 50 of lots of FVIII considered HIV infected
• UK 1 of lots of FVIII considered HIV
  infected

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VIRAL INACTIVATION PROCESS CONC STATUS
Probably effective but patients immunised
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PREVENTION OF POST-TRANSFUSION HEPATITIS

• Screening of Donor Units
• Introduction of HBsAg screening reduced PTH -
  1970s
• Surrogate marker screening - enzymes ALT
• ? Reduced incidence of PTH
• Anti HCV testing
• ? Reduced incidence of PTH
• Donor HCV Ab ve - 0.02
• General population - 0.5

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FACTOR IX CONCENTRATES

• Prothrombin complex concentrates (PCC)
• Contain FIX Vitamin K dependent factors
• I.e. II, VII, IX, X
• Australia Prothrombinex' II, IX, X
• Contains activated factor FXA II
• Thrombogenic in high doses
• Pure factor IX concentrates available
• Currently high cost

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RECOMBINANT CLOTTING FACTORS

• RFVIII Available in US 10 Years
• Available in Australia in part, 1-2 years
• Inhibitor development
• In PUPS - 20
• Continued use, tolerance
• RFIX Clinical trials now complete
• Available in USA
• Not available in Australia
• Recombinant products favoured by patients and
  families
• Free of concern with plasma products
• Problems with expense 1.14/unit

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PHARMACOLOGICAL AGENTS FOR THE MANAGEMENT OF
HAEMOPHILIA ANTIFIBRINOLYTIC AGENTS

• Tranexamic acid
• Used for external bleeding
• eg. Mouth teeth extraction
• Not for internal bleedings
• Inhibits fibrinolysis
• Dental extractions TA mandatory
• DDAVP D-amino D-arginine Vasopressin
• Found to raise FVIII levels
• Normals
• Mild haemophilia
• Mild von Willebrands disease
• PRIMARY ACTION
• Release of vWF from stores
• Weibel Palade bodies endothelial cells
• Stabilisation of FVIII
• Patient response variable
• Side effects, headache, increased BP
• Tachyphylaxis

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PATIENTS WITH INHIBITORS

• Antibodies in transfused patients
• Transfused factor recognised as foreign
• Patient sever gene defect
• Lack of mRNA for appropriate factor
• Mutated factor present - differs from transfused
  factor
• Patients fail to respond to factor VIII
• Inhibitor inactivates factor VIII
• Time dependent fashion
• Simple or complex reaction kinetics
• Expressed as units - Bethesda Units
• The amount of inhibitor that inactivates half
  the FVIII at 2 hrs in a standard incubation
  mixture (100u/dl)

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MANAGEMENT OF PATIENTS WITH INHIBITORS

• After HIV and hepatitis most sever complications
  of haemophilia treatment.
• METHODS
• Attempt to reduce inhibitor permanently
• Immunosuppression immune tolerance
• Treatment of acute bleeds in presence of
  inhibitors
• REDUCING FVIII INHIBITOR PREMANENTLY
• Immunosuppression with drugs
• Immuno depletion plus immunosuppression
• Immunosuppression plus FVIII I.V. IgG
• Cyclophosphamide
• Immune modulation
• Anti-idiotypic antibodies by I.V. IgG

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MANAGEMENT OF ACUTE BLEEDS IN INHIBITOR PATIENTS

• Local measures
• Pharmacological methods
• Russells viper venom. Antifibrinolytic agents
• Factor VIII concentrates
• Human and porcine
• Plasmapheresis
• Extracorporeal immunodepletion
• Intravenous IgG
• Methods of bypassing FVIII and its inhibitor
• PCC Activated and non-inactivated
• FEIBA Autoplex
• rFVIIa
• Experimental methods

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von WILLERBRANDS DISEASE

• 1926 von Willebrand
• 5 year old girl sever spontaneous bleeding
• Aland Isles, Gulf of Bothnia Sweden Finland
• Strong family history, both sexes
• Bleeding from
• Skin
• Mucous membranes
• Post trauma
• Soft tissue Joint Bleeds uncommon compared
  with haemophilia
• INHERITANCE
• Mild vWD, autosomal dominance
• Severe, life threatening vWD, autosomal recessive

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von WILLEBRANDS DISEASE LABORATORY EVALUATION

• Bleeding time prolonged. Template
• Ristocetin cofactor activity
• vWF function
• vWF support platelet aggregation, Gl 1b
• Aggregation quantitative cf with standard dose
• vWF Antigen
• vWF Protein
• Laurell immunoelectrophoresis
• Factor VIIIc
• VWF Multimers
• Multimer distribution in plasma
• Gel electrophoresis
• Low resolution differentiates types I from II
• High resolution differentiates types II

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CLASSIFICATION OF MAJOR TYPES OF vWD
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Blood Products Containing vWFTreatment of
Bleeding in v WD

• Cryoprecipitate Traditional Rx of choice
• Untreated plasma derivatives discontinued
• Increasing purity of FVIII concentrate
• Decreasing levels of vWF
• Intermediate purity factor VIII reasonable
• Monoclonal plasma derived FVIII concentrate
• Lack sufficient vWF to support platelet
  adhesions
• Not enough vWF to bind stabilise administered
  FVIII
• FVIII concs to type III or IIN
• Reduced initial recovery
• Rapid clearance
• vWF concentrates being prepared, France
• rvWF is available in Germany
• Must avoid DDAVP in type IIB
• Increase platelet binding ? thrombotic
  complications