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Maureane Hoffman, 2009
Mechanisms ofMechanisms ofHemostasisHemostasis
Maureane HoffmanMaureane HoffmanProfessor of Pathology
Duke University Medical CenterPath & Lab Medicine Service
Durham [email protected]
286-0411 x6494
Maureane Hoffman, 2009
How can weHow can wemake sense ofmake sense ofhemostasis?hemostasis?
Maureane Hoffman, 2009
ObjectivesObjectives
• Understand how hemostasis relatesto the body’s response to tissue injury
• Differentiate the newer cell-mediatedmodel from the classic cascade model
• Describe the basic coagulation testsand how they relate to the clottingcascade
Maureane Hoffman, 2009
Coagulation:Coagulation: Host Response to InjuryHost Response to Injury
Maureane Hoffman, 2009
Primary HemostasisPrimary Hemostasis
Platelets Adhere & Activate at Sites of Injury
Maureane Hoffman, 2009
Secondary HemostasisSecondary HemostasisCoagulation proteins act on platelet surfaces to
form fibrin, which stabilizes the platelet plug
Maureane Hoffman, 2009Factor VI was at one time used to designate activated Factor V.
cofactorAntihemophilic factoror globulin
VIII
proteaseProconvertin, stablefactor
VII
cofactorAccelerator globulin.proaccelerin, labilefactor
V
CalciumIV
cofactorTissuethromboplastin,tissue factor
III
proteaseProthrombinII
polymer unitFibrinogenI
FunctionSynonymsFactor
Coagulation ProteinsCoagulation Proteins
proteasePrekallikrein (Fletcherfactor)
----
proteasePlasma thromboplastinantecedent
XI
proteaseStuart factor, Stuart-Prower factor
X
proteaseChristmas factor, plasmathromboplastincomponent
IX
cofactorHigh-molecular-weight kininogen(Fitzgerald factor)
----
Fibrincrosslinker
Fibrin stabilizingfactor, fibrinoligase
XIII
proteaseHageman factorXII
FunctionSynonymsFactor
Maureane Hoffman, 2009
Eventually the manyEventually the manycoagulation factors werecoagulation factors wereorganized into a cascadeorganized into a cascade
modelmodel……
Maureane Hoffman, 2009
Earl W. DavieOscar D. Ratnoff
Science 1964;145:1310-1312
LandmarkLandmarkdescriptiondescription
ofofcoagulationcoagulation
as aas abiologicalbiologicalamplifieramplifier
Maureane Hoffman, 2009
The The ““cascadecascade”” model evolved model evolvedinto what my generation ofinto what my generation of
medical students was taught medical students was taught ……..
Maureane Hoffman, 2009
Extrinsic PathwayPT
Factor IXaFactor VIIIa
Factor XIaFactor XI
Fibrinogen
Factor XII/HMK/PK
Factor IX
Factor XaFactor Va
Factor X
ThrombinProthrombin
Fibrin
Factor X
Factor VIIaTissue Factor
Intrinsic PathwayaPTT
Maureane Hoffman, 2009
Homologous Coagulation FactorsHomologous Coagulation Factors• Vitamin K-Dependent Serine Proteases:
– Factors II, VII, IX & X– Structurally similar– Circulate as inactive zymogens– Activated by proteolysis– Work best in complex with a protein cofactor on
lipid surface containing phosphatidyl serine– Activity is calcium dependent
Maureane Hoffman, 2009Roberts, Monroe & Hoffman: Molecular Biology and Biochemistry of the Coagulation Factors andPathways of Blood Coagulation. In William’s Hematology 7th ed, 2005
Maureane Hoffman, 2009
Why should I care about theWhy should I care about thebiochemistry of biochemistry of coag coag factors?factors?
Maureane Hoffman, 2009
Why should I care about theWhy should I care about thebiochemistry of biochemistry of coag coag factors?factors?
• It helps explain some things that are very useful– How does Coumadin (Warfarin) work?– How do calcium chelators act as
anticoagulants?
Maureane Hoffman, 2009
Things that are necessary forThings that are necessary forcoagulation proteases to workcoagulation proteases to work
• Post-translational modification to producegamma-carboxy glutamic acid (Gla)residues, which is vitamin K – dependent
• Calcium to bind to Gla’s and hold theprotein in the right conformation
• Phospholipid surface for the proteases tobind to along with their cofactors
Maureane Hoffman, 2009
Vitamin K-dependent factorsVitamin K-dependent factorscontain contain Gla-residuesGla-residues
Maureane Hoffman, 2009
WarfarinWarfarin: Commonly Used: Commonly UsedOral Anti-CoagulantOral Anti-Coagulant
• Warfarin alters synthesis of vitamin K-dependent factorsby preventing vitamin K-dependent carboxylation of– Factors II, VII, IX, X– Protein C & Protein S
• Result: no longer bind calcium• New proteins must be synthesized to overcome the
warfarin effect
Maureane Hoffman, 2009
Vitamin K-dependent proteasesVitamin K-dependent proteases(FII, VII, IX and X)(FII, VII, IX and X)
Roberts, Monroe & Hoffman: Molecular Biology and Biochemistry of the Coagulation Factorsand Pathways of Blood Coagulation. In William’s Hematology 7th ed, 2005
Maureane Hoffman, 2009
Coag Coag proteins work asproteins work asprotease/cofactor complexesprotease/cofactor complexes
Maureane Hoffman, 2009
The Coagulation CascadeThe Coagulation Cascade
• Helps us interpret clinical laboratory tests– Prothrombin time (PT)– Activated Partial Thromboplastin Time (aPTT)
Maureane Hoffman, 2009
Extrinsic PathwayPT
Factor IXaFactor VIIIa
Factor XIaFactor XI
Fibrinogen
Factor XII/HMK/PK
Factor IX
Factor XaFactor Va
Factor X
ThrombinProthrombin
Fibrin
Factor X
Factor VIIaTissue Factor
Intrinsic PathwayaPTT
Maureane Hoffman, 2009
The Coagulation The Coagulation ““CascadeCascade”” Doesn Doesn’’t Explaint ExplainHow Blood Clots How Blood Clots in vivoin vivo
• Patients lacking FXII, HMK, or PK have along aPTT but no bleeding
• Patients lacking FXI have a long aPTT andmay or may not have bleeding
• Patients lacking FVIII or FIX have anequally long aPTT and serious bleeding
Maureane Hoffman, 2009
Factor IXaFactor VIIIa
Factor XIaFactor XI
Fibrinogen
Factor XII/HMK/PK
Factor IX
Factor XaFactor Va
ThrombinProthrombin
Fibrin
Factor X
Intrinsic PathwayaPTT
Prolonged aPTTVariable bleeding
Prolonged aPTTSevere bleeding
Prolonged aPTT only
Maureane Hoffman, 2009
How does it really workHow does it really workin the body?in the body?
Maureane Hoffman, 2009
Cells areCells are important in theimportant in thebody, but arenbody, but aren’’t includedt included
in the coagulationin the coagulationcascade or the clinicalcascade or the clinical
lab tests.lab tests.
Maureane Hoffman, 2009
IIa
Hemostasis Occurs on Two Surfaces:Hemostasis Occurs on Two Surfaces:TF-bearing Cells and PlateletsTF-bearing Cells and Platelets
1. Initiation
2. Amplification 3. PropagationIIa
Maureane Hoffman, 2009
VIIa
VIIa
VIIa
X
Xa
IX
TF-Bearing Cell
IXa
TF
TF TF
Hoffman M, Monroe DM: A Cell-Based Model of Hemostasis. Thromb Haemostas, 85:958-65, 2001
Initiation
Maureane Hoffman, 2009
IIIIa
VIII/vWF
XI
XIaPlatelet
Va
V
VIIIa+Free vWF
V
VIIa
VIIa
VIIa
XXa
TF-Bearing Cell
TF TF
TF
Va
Activated Platelet
VaXIa
VIIIa
Priming Amountof Thrombin
Amplification
Maureane Hoffman, 2009
VIIa
XIa
XaIXa
X IIIIa
IX
IX
Activated Platelet
VaXIa VIIIa
IIa
VIII/vWF
XI
XIaPlatelet
Va
V
VIIIa + Free vWF
V
TFPI
TF-Bearing Cell
TF
VIIaVIIaXaXa
TF TFVa
Propagation
Large amountof thrombin
Maureane Hoffman, 2009
Fibrin Clot FormationFibrin Clot Formation
FibrinogenFibrinogen
IIaIIa
Maureane Hoffman, 2009
POLYMERIZING FIBRINPOLYMERIZING FIBRIN
POLYMERIZATIONPOLYMERIZATION
FIBRINOGENFIBRINOGEN
D DE
FIBRIN MONOMERFIBRIN MONOMER
THROMBINTHROMBIN
Maureane Hoffman, 2009
Factor XIIIFactor XIII
• Activated by thrombin during coagulation• Has transglutaminase activity• Covalently crosslinks fibrin strands to
stabilize the clot
Maureane Hoffman, 2009
TF-Bearing CellTF-Bearing Cell
Activated PlateletActivated Platelet
PlateletPlateletTFTF
VIIIaVIIIa VaVa
VaVa
VIIaVIIa
TFTF VIIaVIIa
XX
Xa
IIIIIIa
IXIX VV VaVa
IIII
VIII/vWFVIII/vWF
VIIIaVIIIa
IXaIXa XX
IXa IIaIIaXa
A Cell-Based Model ofA Cell-Based Model ofHemostasisHemostasis
Hoffman M, et al. Hoffman M, et al. Blood Blood Coagul Coagul Fibrinolysis.Fibrinolysis. 1998;9(suppl 1):S61-S65. 1998;9(suppl 1):S61-S65.
XI XI XIa
XIa
IXIX
Maureane Hoffman, 2009
PT: measures extrinsic/initiationPT: measures extrinsic/initiationpathway*pathway*
*short name (PT) = short pathway
Monroe, DM and Hoffman, M: What does it take to make the perfect clot? Arterio Thromb Vasc Biol 26:41-48, 2006
Maureane Hoffman, 2009
aPTT: measures intrinsic/plateletaPTT: measures intrinsic/plateletpathway*pathway*
*long name (aPTT) = long pathway
Monroe DM and Hoffman, M: What does it take to make the perfect clot? Arterio Thromb Vasc Biol 26:41-48, 2006
Maureane Hoffman, 2009
The intrinsic andThe intrinsic andextrinsic pathways areextrinsic pathways are
not redundant,not redundant, but have distinct roles inbut have distinct roles in
hemostasis hemostasis in vivoin vivo
Maureane Hoffman, 2009
P L A S M I N
TISSUE PLASMINOGEN ACTIVATOR (tPA)STREPTOKINASEUROKINASEFACTOR XIIaKALLIKREIN
FIBRINFRAGMENTS
FibrinolysisFibrinolysis
FIBRIN CLOT
PLASMINOGENPLASMINOGEN
Maureane Hoffman, 2009
FIBRINOGEN
+ 2 FRAGMENTSA,B,C
PLASMIN
D DE
D DE
D DE
D E
FRAGMENT X
FRAGMENT Y
PLASMIN
FRAGMENT D
PLASMIN
FRAGMENT DFRAGMENT E
Maureane Hoffman, 2009
Control of Clot FormationControl of Clot FormationSeparation of Initiation & Propagation
Presence of plasma coagulation inhibitors– Antithrombin (AT or ATIII)
• Activity increased by heparin & LMWH
– Tissue Factor Pathway Inhibitor (TFPI)
Anti-thrombotic mechanisms on healthy vascularendothelial cells
– Thrombomodulin (TM)/Protein C/S system– Heparin sulfates that bind AT-3
Maureane Hoffman, 2009
Hemostasis Sets the Stage Hemostasis Sets the Stage forfor
Thrombinhas manybiologicalactivities
Fibrin isthe matrix
forhealing
Plateletsrelease
cytokines& growthfactors
Hemostaticdefects can
lead todefectivewoundhealing
Inflammatory Cell Influx & Effective Wound Healing
Maureane Hoffman, 2009
BleedingBleeding DisordersDisorders
• We are only going to talk aboutinherited (not acquired) disorders
• Platelet problems• Coagulation factor problems
Maureane Hoffman, 2009
Clinical BleedingClinical Bleeding
• Platelet Problem– Petechiae & purpura– Mucocutaneous bleeding
• Coagulation Factor Problem– Bruises (ecchymoses)– Soft tissue hemorrhage
Maureane Hoffman, 2009
Descriptors of BleedingDescriptors of Bleeding
http://www.uptodate.com
• Petichiae are < 3 mm,Purpura are 0.3-1 cm,Ecchymoses are > 1 cm
Maureane Hoffman, 2009
ThrombocytopeniaThrombocytopenia leads to leads toendothelial changesendothelial changes
• The top shows an EM of acapillary from a thyroidperfused with PRP for 5h
• The bottom shows a capillaryfrom a thyroid perfused withPPP for 5h. Note the disruptionin the endothelium.– Gimbrone et al: Preservation of vascular
integrity in organs perfused in vitro with aplatelet-rich medium. Nature, 222:13-4,1969
Maureane Hoffman, 2009
ThrombocytopeniaThrombocytopenia leads to leads toendothelial changesendothelial changes
• This picture shows a RBCextravasating from a capillaryof a thrombocytopenicmouse
• RBC appear to traversesmall channels in theendothelial cells.– Aursnes & Pedersen: Petechial
hemorrhage in the ciliary process ofthrombocytopenic rabbits. An EMstudy. Microvascular Res, 17:12-21,1979
Maureane Hoffman, 2009
Maureane Hoffman, 2009
Factor Deficiencies:Factor Deficiencies:General ConsiderationsGeneral Considerations
Deficiencies of each of the following exist:• Factor VIII Factor IX• Factor XI Factor VII• Prothrombin Factor X• Factor V Factor XII• Fibrinogen Factor XIII
Maureane Hoffman, 2009
Factor Deficiencies:Factor Deficiencies:General ConsiderationsGeneral Considerations
• Inheritance: Most are inherited asautosomal recessive disorders
• Factors VIII and IX are encoded on theX chromosome and their deficienciesare sex-linked recessive
• While bleeding is the hallmark of thesedisorders, its severity and pattern varydepending on the involved factor
Maureane Hoffman, 2009
Congenital Bleeding DisordersCongenital Bleeding Disorders
• vonWillebrand Disease• Hemophilia A & B• FXI deficiency• Rare coagulation deficiencies• Rare platelet defects
Maureane Hoffman, 2009
Hemophilia A & BHemophilia A & B
Deficiency of FVIII or FIX
Maureane Hoffman, 2009
Extrinsic PathwayPT
Factor IXaFactor VIIIa
Factor XIaFactor XI
Fibrinogen
Factor XII/HMK/PK
Factor IX
Factor XaFactor Va
Factor X
ThrombinProthrombin
Fibrin
Factor X
Factor VIIaTissue Factor
Intrinsic PathwayaPTT
Maureane Hoffman, 2009
TF-Bearing CellTF-Bearing Cell
Activated PlateletActivated Platelet
PlateletPlateletTFTF
VaVa
VaVa
VIIaVIIa
TFTF VIIaVIIa
X
Xa
IIIIIIaIIa
IXIXVV VaVa
IIIIIXa XX
IXa
Hemophilia Is a Failure of Hemophilia Is a Failure of PlateletPlateletSurfaceSurface Thrombin Generation Thrombin Generation
Hoffman M, et al. Blood Coag Fibrinolys. 1998;9(suppl 1):S61-S65.
Maureane Hoffman, 2009
Much early coagulationMuch early coagulationresearch was driven by theresearch was driven by the
presence of hemophilia in thepresence of hemophilia in theroyal families of Europeroyal families of Europe
Maureane Hoffman, 2009
Maureane Hoffman, 2009
Hemophilia A and BHemophilia A and B
• X-linked• Up to 30% from de novo mutation i.e. no
family history• Mild, moderate and severe forms• Dysfunctional molecules – Cross-reacting
material positive (CRM+)• Reduced level of a normal molecule -
Cross-reacting material negative (CRM-)
Maureane Hoffman, 2009
Inheritance of HemophiliaInheritance of Hemophilia
•• Hemophilia A (FVIII deficiency) 1 inHemophilia A (FVIII deficiency) 1 in10,000 live male births10,000 live male births
•• Hemophilia B (FIX deficiency) 1 inHemophilia B (FIX deficiency) 1 in30,000 live male births30,000 live male births
•• Inherited as sex linked recessive traitsInherited as sex linked recessive traitsAn affected male will produce only normal males and
carrier females (with a normal female)
A carrier female will produce offspring of which half the femalesare carriers and half the males are affected (with a normal male)
Maureane Hoffman, 2009
Inheritance of Inheritance of HemophiliasHemophilias
Maureane Hoffman, 2009
Hemophilia A and BHemophilia A and B
• Clinical picture is identical in A & B• Prolonged aPTT in both, need factor
assays to distinguish• Severely affected have spontaneous soft
tissue and joint hemorrhage• Severely deficient may develop antibody
inhibitors
Maureane Hoffman, 2009
vonWillebrand DiseasevonWillebrand Disease
Maureane Hoffman, 2009
vonWillebrand DiseasevonWillebrand Disease
• Autosomal• Most common inherited bleeding disorder• vWF mediates platelet adhesion under high
shear - bleeding is typical of platelet defects• vWF is the carrier for FVIII - FVIII level may
be reduced and aPTT may be prolonged• Subdivided into several types based on
multimer pattern and antigen level
Maureane Hoffman, 2009
FXI deficiencyFXI deficiency
Maureane Hoffman, 2009
Factor IXaFactor VIIIa
Factor XIaFactor XI
Fibrinogen
Factor XII/HMK/PK
Factor IX
Factor XaFactor Va
ThrombinProthrombin
Fibrin
Factor X
Intrinsic PathwayaPTT
Prolonged aPTTVariable bleeding
Prolonged aPTTSevere bleeding
Prolonged aPTT only
Maureane Hoffman, 2009
FXI DeficiencyFXI Deficiency
• Autosomal• Common in certain populations -
Ashkenazi Jews, some Arabpopulations
• Bleeding with trauma or surgery,especially if on aspirin
• Bleeding risk not predictable fromaPTT or FXI level