09 hemostasis

Maureane Hoffman, 2009

Mechanisms ofMechanisms ofHemostasisHemostasis

Maureane HoffmanMaureane HoffmanProfessor of Pathology

Duke University Medical CenterPath & Lab Medicine Service

Durham [email protected]

286-0411 x6494


How can weHow can wemake sense ofmake sense ofhemostasis?hemostasis?


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


Coagulation:Coagulation: Host Response to InjuryHost Response to Injury


Primary HemostasisPrimary Hemostasis

Platelets Adhere & Activate at Sites of Injury


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


Eventually the manyEventually the manycoagulation factors werecoagulation factors wereorganized into a cascadeorganized into a cascade

modelmodel……


Earl W. DavieOscar D. Ratnoff

Science 1964;145:1310-1312

LandmarkLandmarkdescriptiondescription

ofofcoagulationcoagulation

as aas abiologicalbiologicalamplifieramplifier


The The ““cascadecascade”” model evolved model evolvedinto what my generation ofinto what my generation of

medical students was taught medical students was taught ……..


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


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


Why should I care about theWhy should I care about thebiochemistry of biochemistry of coag coag factors?factors?


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?


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


Vitamin K-dependent factorsVitamin K-dependent factorscontain contain Gla-residuesGla-residues


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


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


Coag Coag proteins work asproteins work asprotease/cofactor complexesprotease/cofactor complexes


The Coagulation CascadeThe Coagulation Cascade

• Helps us interpret clinical laboratory tests– Prothrombin time (PT)– Activated Partial Thromboplastin Time (aPTT)


Extrinsic PathwayPT


Factor XIaFactor XI

Fibrinogen

Factor XII/HMK/PK

Factor IX

Factor XaFactor Va

Factor X

ThrombinProthrombin

Fibrin

Factor X




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



Factor XIaFactor XI

Fibrinogen

Factor XII/HMK/PK

Factor IX

Factor XaFactor Va

ThrombinProthrombin

Fibrin

Factor X


Prolonged aPTTVariable bleeding

Prolonged aPTTSevere bleeding

Prolonged aPTT only


How does it really workHow does it really workin the body?in the body?


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.


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


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


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


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


Fibrin Clot FormationFibrin Clot Formation

FibrinogenFibrinogen

IIaIIa


POLYMERIZING FIBRINPOLYMERIZING FIBRIN

POLYMERIZATIONPOLYMERIZATION

FIBRINOGENFIBRINOGEN

D DE

FIBRIN MONOMERFIBRIN MONOMER

THROMBINTHROMBIN


Factor XIIIFactor XIII

• Activated by thrombin during coagulation• Has transglutaminase activity• Covalently crosslinks fibrin strands to

stabilize the clot


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


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


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


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


P L A S M I N

TISSUE PLASMINOGEN ACTIVATOR (tPA)STREPTOKINASEUROKINASEFACTOR XIIaKALLIKREIN

FIBRINFRAGMENTS

FibrinolysisFibrinolysis

FIBRIN CLOT

PLASMINOGENPLASMINOGEN


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


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


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


BleedingBleeding DisordersDisorders

• We are only going to talk aboutinherited (not acquired) disorders

• Platelet problems• Coagulation factor problems


Clinical BleedingClinical Bleeding

• Platelet Problem– Petechiae & purpura– Mucocutaneous bleeding

• Coagulation Factor Problem– Bruises (ecchymoses)– Soft tissue hemorrhage


Descriptors of BleedingDescriptors of Bleeding

http://www.uptodate.com

• Petichiae are < 3 mm,Purpura are 0.3-1 cm,Ecchymoses are > 1 cm


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


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


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


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


Congenital Bleeding DisordersCongenital Bleeding Disorders

• vonWillebrand Disease• Hemophilia A & B• FXI deficiency• Rare coagulation deficiencies• Rare platelet defects


Hemophilia A & BHemophilia A & B

Deficiency of FVIII or FIX


Extrinsic PathwayPT


Factor XIaFactor XI

Fibrinogen

Factor XII/HMK/PK

Factor IX

Factor XaFactor Va

Factor X

ThrombinProthrombin

Fibrin

Factor X




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.


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


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-)


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)


Inheritance of Inheritance of HemophiliasHemophilias


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


vonWillebrand DiseasevonWillebrand Disease


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


FXI deficiencyFXI deficiency



Factor XIaFactor XI

Fibrinogen

Factor XII/HMK/PK

Factor IX

Factor XaFactor Va

ThrombinProthrombin

Fibrin

Factor X


Prolonged aPTTVariable bleeding

Prolonged aPTTSevere bleeding

Prolonged aPTT only


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

09 hemostasis

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