Professor Mike Laffan What can go wrong with haemostasis Haemostatic Plug Formation An Overview Vessel constriction Formation of an unstable platelet plug platelet adhesion platelet aggregation ID: 1047494
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1. Why do patients bleed?HaemophiliaProfessor Mike Laffan
2. What can go wrong with haemostasis?
3. Haemostatic Plug Formation: An OverviewVessel constrictionFormation of an unstable platelet plug -platelet adhesion -platelet aggregationStabilisation of the plug with fibrin -blood coagulationDissolution of clot and vessel repair -fibrinolysis
4. Primary haemostasisFormation of the platelet plug
5. plateletplateletPrimary HaemostasisCollagenTissue FactorVWFlumen
6. plateletplateletPrimary Haemostasis: platelet plugCollagenTissue Factorlumenplateletplateletplateletplatelet
7. lumenHaemostasis: fibrin clot stabilising platelet plug
8. plateletplateletVWD: failure of primary haemostasisCollagenTissue Factorlumenplatelet
9. Primary haemostasis failureNumber or function of plateletsQuantity or function of VWFDefects in Collagen or vessel wall
10. CoagulationGeneration of thrombin and hence the fibrin clot
11. Thrombin cleaves fibrinogen:formation of the fibrin clot.
12. TTTTTTTTTissue factorVIIaThrombinFibrin - the ‘clot’FibrinogenCoagulation: fibrin formationFPA & FPB
13. Fibrin mesh
14. lumenHaemophilia: failure to generate thrombin and hence fibrin, to stabilise platelet plug
15. Termination of coagulation
16. TTTTTplateletplateletVIIaTTTT TTMEPCRPCAPCATATATATTermination of Coagulation
17. Bleeding and excess anticoagulant functionNaturally occurring is rareAT Pittsburgh (actually an anti-trypsin)TherapeuticHeparin HirudinActivated protein C
18. TTplateletplateletVIIaFibrinolysisTPgntPAPFDPsT TTMTAFIa-TAFI AP
19. Bleeding and disorders of FibrinolysisDisorders of fibrinolysis can cause abnormal bleeding but are rareHereditary Antiplasmin deficiencyPAI-1 deficiencyAcquired Drugs such as tPADisseminated intravascular coagulationSome tumours precipitate primary fibrinogenolysis
20. Normal haemostasis: a state of equilibriumCoagulation factors,plateletsFibrinolytic factors,anticoagulant proteinsHaemostasis and Thrombosis: a Balance
21. BleedingFibrinolytic factors,anticoagulant proteinsCoagulation factors,platelets
22. Why patients bleed: HaemophiliaUnderstand the coagulation defect in haemophiliaUnderstand the pattern of bleeding in haemophilia (and other coagulation disorders)Understand diagnosis and treatment and complications of treatment Understand the inheritance and molecular genetics of haemophilia
23. lumenHaemophilia: failure to generate fibrin to stabilise platelet plug
24. Why is there a failure of fibrin formation in haemophilia?
25. FVIITFExtrinsic PathwayFVIIaTF-+
26. FXFIXFVIIFIIFXaTHROMBINFibrinFibrinogenTFPLExtrinsic PathwayFVIIaTF-+Ca++Intrinsic PathwayFIXa
27. FXFIXFVIIFIIFXaTHROMBINFibrinFibrinogenTFFIXaExtrinsic PathwayFVIIaTF-+FVIIaTFFXaTFPITFPIIntrinsic Pathway
28. FXFIXFVIIFIIFXaTHROMBINFibrinFibrinogenTFFIXaExtrinsic PathwayFVIIaTF-+FVIIaTFFXaTFPITFPIIntrinsic Pathway
29. FXFIXFVIIFIIFXaTHROMBINFibrinFibrinogenTFPLFIXaFVIIIaPLFVaExtrinsic PathwayFVIIaTF-+Ca++Ca++FVIIaTFFXaTFPITFPIIntrinsic PathwayFVIIIFV
30. FXIFXFIXFVIIIFVIIFIIFXIaFXaTHROMBINFibrinFibrinogenTFPLFIXaFVIIIaPLFVaFVExtrinsic PathwayFVIIaTF-+Ca++Ca++FVIIaTFFXaTFPITFPIIntrinsic Pathway
31. FXIFXFIXFVIIFIIFXIaFXaTHROMBINFibrinFibrinogenTFPLFVaFVExtrinsic PathwayFVIIaTF-+Ca++FVIIIFIXaFVIIIaPLCa++FVIIaTFFXaTFPITFPIIntrinsic PathwayHaemophilia
32. Thrombin generation in haemophilia
33. Thrombin generation and FVIII
34. Why a ‘burst’ of thrombin? Clot formation occurs at end of initiation phase, but, Rapid thrombin generation produces a stronger, denser clot A large amount of thrombin is required to inhibit fibrinolysis (activation of TAFI) Coagulation defects are characterised by a failure of thrombin burst
35. Secondary events securing haemostasis (1)Factor XIII is activated by thrombinFXIII Cross links fibrin monomersCross links a2AP to fibrinPrevents dissolution of clotInhibits fibrinolysis
36. Secondary events securing haemostasis (2)Further thrombin is generated via FXI.This thrombin appears to be important in activating TAFI (thrombin activatable fibrinolysis inhibitor).Inhibits fibrinolysis by removing lysine binding sites for plasminogen on fibrin.Depends on FVIII, IX as well as XI
37. Coagulation in haemophilia Absence of thrombin burst results in:Unstable platelet aggregateLoose fibrin clot Susceptible to fibrinolysisInsecureFailure to activate TAFIFibrinolysis not inhibitedDelayed bleeding
38. Disorders of coagulationFailure of thrombin generation may result from deficiency of any coagulation factorMay be quantitative or qualitativeSeverity depends on degree of deficiency and position in network 40% of most factors is sufficient for haemostasis One functional allele is sufficient Haemophilias are common because X linked
39. Disorders of coagulation: bleeding patternBleeding often delayed (primary haemostasis normal)Typically from deep structuresEg muscles and jointsPattern varies with different deficiencies.
40. Factor VIII Deficiency100 90 80 70 60 50 40 30 20 10 0 Nose Uterus Haem- GI Joint Muscle CNS Cord Postop/ Oral aturia part cavity 15%16%25%75%81%N.A13%0%75%90%
41. Coagulation factor deficiencies are not all the sameFactor VIII and IX (Haemophilia)Severe but compatible with lifeSpontaneous joint and muscle bleedingProthrombin (Factor II)LethalFactor XIBleed after trauma but not spontaneouslyFactor XIINo excess bleeding at all
42. XXaXFibrinogenFibrinCrosslinked fibrinXIIIaXIIIProthrombinthrombin (IIa)IXaIXVIIIaPlCa 2+XIaXIXIIaXIICOMMON PATHWAYVaPlCa 2+thrombinBlood coagulationTissue factor(vessel damage)VIIaCa 2+EXTRINSIC PATHWAYVIIaCa 2+INTRINSIC PATHWAY
43. Haemophilia A and BFactor VIII and Factor IX deficiency are clinically indistinguishable pattern of bleeding severity coagulation screen results mode of inheritance
44. IXIIFibrinogenFibrinPKHMWKXIXIIVIIaTFVIIIXVCONTACTPT 10.6 (9.6-11.6)APTT 85 (26-32)TT 16 (15-19)Diagnosis of haemophilia: prolonged APTTPTAPTTTT
45. Classification of HaemophiliaType DeficiencySeverity Factor levelHaemophilia A Factor VIIIHaemophilia B Factor IX(Christmas disease)Severe ≤1%Moderate 2-5%Mild 6-40%Normal 40-150%
46. Bleeding in haemophiliaSevere (≤1%) Moderate (2-5%)Mild (6-40%)‘spontaneous’ bleedsJoints and muscleEasy bruisingBleeding often delayed, but prolongedBleeding time normalProlonged or excessive bleeding after minor traumaRarely bleeds into jointsExcessive bleeding only after major trauma or surgery
47. Thrombin generation and FVIIIFEDP 1 pMTF012.551050020040060080010001200140016000102030405060FVIII %ETP
48. Deep; muscle and jointDelayedNot from superficial cuts or small vessels (eg nosebleeds)Bleeding in haemophilia
49. Taking blood samples is usually safe
50. Do not mistake haemophilia for NAI
51. Cause of death recorded for 113 cases of HaemophiliaCarol Birch 1937
52. Bleeding in haemophilia1. Haemarthrosis begin at age approx 1 year apparently spontaneous may be preceded by ‘tingling’ blood fills joint cavity rise in pressure is excruciatingly painful pressure eventually stop bleeding blood damages cartilage joint becomes prone to recurrent bleeds
53. Haemarthrosis: the hallmark of haemophilia
54.
55. Target joints: a vicious cycleBleedingSynovial hypertrophyFriable and expandedsynoviumArthritis
56. Joint deformationcaused by haemophilia AScar on right lower thigh is site ofpreviously excisedpseudotumour.
57.
58. Haemophilicarthropathy
59.
60. Bleeding in haemophilia2. Muscle bleeds often apparently spontaneous may result from exertion blood fills muscle capsule or compartment ‘compartment sydrome’ may result pressure eventually stops bleeding Psoas bleed is typical example
61. Intramuscular injections should be avoided
62. Bleeding into muscle: a psoas bleed
63. Psoas haematomaJOL 2011
64. Treatment for haemophilia
65. History of haemophilia treatmentpre 1940 snake venom1950’s whole blood, plasma1960’s cryoprecipitate1970’s factor concentrates1984 factor VIII cloned1990’s recombinant FVIII and FIX2000 protein free production
66. Factor replacement – half livesFactor VIII 8-12 hoursFactor IX 18-24 hours
67. Using factor replacement therapytime (hours)factorlevel (%)0257510050036241224489660VIIIIX
68. Using factor replacement therapy 21: Treat bleeds as they occur “on demand” therapy2: Treat before bleeds occur “prophylactic” therapy
69. Advantages of prophylactic therapy no painful bleeds treat when well and not in pain no haemophilic arthropathy less concern re-activity less need to carry concentrate around life approaches 'normal'Disadvantages of prophylactic therapy regular injections need to start at early age difficult in young children may need portacath relatively expensive (?)
70. Prophylactic regimensFactor VIII 3 times per weekFactor IX 2 times per week25-40u/kg i.v.
71.
72. 0312456Group 11974-9Group 21980-5Group 3 1986-990Score/AgeAge at start of prophylaxisTime from bleed to prophylaxisX-Ray ScoreOrthopaedic score
73. Manco-Johnson 2007Prophylaxis versus on demand treatment for haemophilia
74. MRI score and joint bleedsManco-Johnson 2007
75. Secondary prophylaxis in adultsOn demandProphylaxispAll bleeds20.5 (14-37)0 (0-3)<0.001Joint bleeds15.0 (11-26)0 (0-3) <0.001Spontaneous bleeds13.5 (7-29)0 (0-1)<0.001Traumatic bleeds2.5 (0-9)0 (0)<0.001Collins JTH 2009
76. Non-concentrate treatments local measures tranexamic acid DDAVP
77. Tranexamic acidLysine derivativeBinds to plasminogen Blocks binding to fibrinClot lysis is reducedWidely distributedTeratogenicIntravenous: 0.5g tdsOral: 1.5g tdsMouthwash: 1g (10ml 10%) qds
78. Desmopressin (DDAVP)Vasopressin derivativeActs via V2 receptors2-5 fold rise in VWF-VIII (VIII>VWF)Dose 0.3µg/kg i.v300µg i.n.Peak response30-60 mins60-90 mins
79. Mannucci 1981
80. Mortality in Haemophilia
81. Severe haemophiliaAll patientsHIV +veHIV -ve77 79 81 83 85 87 89 91 93120100806040200Standardized death rate per 1000YearDarby 1995
82. Survival of UK men with haemophilia (no HIV) in 1999. Darby 2007
83. Factor VIII usage by UK centres 1989-2010
84. Complications of treatment1. Infection Hepatitis B Hepatitis C HIV Prions?2. Immune Antibody production (inhibitors)
85. Haemophilia A: geneticsFactor VIII and IX are both on the X chromosome
86. Inheritance of haemophiliaYXXXXXYXXXYXYXXXXXYXYX
87. Inheritance of haemophiliaFor a female heterozygote (carrier)Half of sons are affectedHalf of daughters are carriersFor a male haemophiliac:All daughters are heterozygotesAll sons are unaffected Approx one third are new mutations
88. Women and haemophilia sex linked recessive level in males is consistent levels in heterozygotes is extremely variable
89. Factor VIII level in heterozygotes Allele usage FVIIINormal Deficient 50 50 50%10 90 10%20 80 20%60 40 60%80 20 80%
90. Factor levels in haemophilia carriersPlug 2006
91. Graw 2005Severe 15%45%
92. 1-2226-23XqtercencenXqterHomologousrecombinationXqtercendisruptedF8 geneExons 26-2326-2322-122-1Flipping the tipF8Nearly always occurs in spermatogenesisSimilar mechanism in intron 1
93. Haemophilia A Mutations (excluding inversions)Total Reports1104Unique mutations622Unique Deletions169Unique Insertions28Unique Splice variants42Unique Point mutations (mis-sense + stop)383
94. Factor IX geneticsFactor IX deficiency is one-fifth as common as FVIIINo inversionsOtherwise the same range of mutations is seen
95. Inhibitors antibodies to factor VIII/FIX ‘foreign’ molecule to severe patients 40-50% of severe haemophilia ~10-15% are persistent High titre render FVIII/FIX treatment useless Require by-pass therapy – partially effective
96. Developments in haemophiliaAntenatal diagnosisPreimplantation genetic diagnosis (PIGD)New delivery systems – infusion‘Designer molecules’ rFVIIILonger half lifeLow immunogenicity/reactivityGene therapy
97. New approaches to haemophilia FVIII molecules with increased activity/stabilityEg by linking A1 and A2 domainsProlonging half life of FVIII or IXCouple to liposomesCouple to PEGPolysialylationCouple to albuminCouple to Fc
98. FIX-Fc pharmacokineticsShapiro Blood 2012;119:666-672T1/2 = 60 hours(cf 20)
99. Dosing models for prophylaxis with rFIXFc Shapiro Blood 2012;119:666-672
100. Indirect effect on FVIII by prolonging half life of VWFPegulationPolysialylationPeptidomimetics Non-peptide molecules with FIX cofactor activityAllow different routes of administrationAntibodybinds FX and FIXNew approaches to haemophilia
101. Problems with new approachesMay increase immunogenicity (though could decrease too)May be thrombogenic.Cost