COAGULATION

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COAGULATION

• Masatoshi Kida, M.D.
• Dept. of Pathology
• University of Vermont

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HEMOSTASIS

• The bodys intrinsic ability to slow down or stop
  hemorrhage
• normal hemostasis involves a delicate balance
  between factors that promote blood coagulation
  and thrombus stabilization and factors that
  inhibit blood coagulation and promote thrombus
  dissolution

RESOLUTION
FORMATION
clot
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COAGULATION

• vascular wall
• coagulation factors
• platelet

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COAGULATION
FIRST STEP

• Smooth muscle
• muscle constriction
• luminal diameter reduction
• retarding blood loss
• enhancing platelet adherence
• Endothelial cells
• VIII-vWF synthesis
• thromboplastin (III) release
• ? activation of ext pathway
• activation of Factor IX X
• Subendothelium
• collagen ? platelet adherence, activation

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PRIMARY HEMOSTASIS

• Platelet
• Adherence
• Activation
• Aggregation
• (Contraction)
• (Stabilization)

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Platelet Adhesion
mediated by GP-Ib on platelet surface and vWF
fibrinogen
C O L L A G E N
C O L L A G E N
GP-Ib
von Willebrand Factor
endothelial cells
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von Willebrand Factor

• Synthesized stored in endothelial cells and
  megakaryocytes (also stored in platelets)
• Forming bridge between subendothelial collagen
  and Plt
• Carrier molecule for Factor VIII Coagulant
  Protein (vWF is also known as Factor VIII
  Related Antigen)
• Circulates as series of multimers (in various
  sizes)
• Large, high molecular weight multimers required
  for normal hemostasis

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Platelet Activation
release of a and electron dense granules

• Release of platelets cytoplasmic products
• activation of platelet factor III (PF-3)
• PF-3 serves as a binding site for cofactor V
  VIII
• production of thrombin

C O L L A G E N
GP-IIb, IIIa
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Platelet Aggregation
GP IIb-IIIa - fibrinogen interaction

• formation of primary plug
• activated platelets synthesize and secrete TXA2
• TXA2
• 1. promotes plt aggregation
• 2. vasoconstriction
• 3. release of plt factor (ADP)

C O L L A G E N
Aspirin inhibits TXA2 and ADP
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Coagulation Factors
XII
XI
IX
extrinsic pathway
intrinsic pathway
VIII
VII
X
V
PT
aPTT
I
XIII
Stable clot
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Coagulation Factors
negative charged surface
XII

• Intrinsic Pathway
• Extrinsic Pathway
• Common Pathway
• majorities are serine proteases
• circulate as inactive forms (require activation
  to function)
• majority produced in liver

XI
tissue factor
IX
intrinsic pathway
extrinsic pathway
VIII
VII
X
V
PT
aPTT
II
I
XIII
Stable clot
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Coagulation Factors
negative charged surface

• Intrinsic Pathway
• Extrinsic Pathway
• Common Pathway
• majorities are serine proteases
• circulate as inactive forms (require activation
  to function)
• majority produced in liver
• many require vit K for synthesis
• II, VII, IX, X
• protein C, S

XII
XI
tissue factor
IX
intrinsic pathway
extrinsic pathway
VIII
VII
X
V
PT
aPTT
II
I
XIII
Stable clot
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Secondary Hemostasis

• tissue factor
• phospholipid complex
• thrombin activation
• fibrin polymerization
• thrombin, ADP, TXA2
• Contraction
• contraction of intraplatelet actomyosin
• formation of secondary plug
• uncover plt membrane receptors (GPIb, IIIa)
• Stabilization
• activation of XII thrombin
• cross linking of fibrin monomers

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A
A
fibrinopeptide A B
fibrinogen
B
B
D domain
E domain
fibrin monomer
Spontaneous aggregation
soluble fibrin polymer
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Stabilization of Fibrin Clot
soluble fibrin clot
Thrombin
Factor XII I Factor XIIIa
stabilized fibrin clot
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Other Proteins in Blood Coagulation

• Prekallikrein
• activates XII and prekallikrein
• High molecular wt. Kininogen (HMWK) (binding
  protein)
• supports reciprocal activation of XII, XI and
  prekallikrein

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ANTICOAGULATION FIBRINOLYSIS

• Vascular Wall
• Anti-Coagulation Factors
• Fibrinolytic Factors

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ANTICOAGULATION FIBRINOLYSISVascular Wall

• Endothelial Cell
• prostacyclin (PGl2)
• heparan sulfate
• thrombomodulin
• tissue plasminogen activator (tPA)
• Muscle
• muscular dilation

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ANTICOAGULATION FIBRINOLYSISAnti-Coagulation
Factors

• Antithrombin III (with thrombin heparin)
• negative feedback on thrombin
• inactivates Xa (XIIa, XIa, IXa)
• Prostacyclin (PGl2) from endothelial cells
• vasodilation
• conversion of ADP into products that inhibit plt
  aggregation
• thrombin endothelial cells
• thrombomodulin binds activates Protein C
• inactivates Va VIIIa
• inhibits thrombin

XII
XI
IX
VIII
VII
X
V
II
I
XIII
Stable clot
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• Protein C
• vit K dependent zymogen
• produced in liver
• inactivates Va and VIIIa
• Protein S
• vit K dependent binding protein
• co-factor for protein C
• binds C4b-binding protein

XII
XI
IX
VIII
VII
X
V
II
I
XIII
Stable clot
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AnticoagulationHeparin

• Heparin activates Antithrombin III (AT III)
• AT III inactivates Thrombin and Factor Xa
• rapid onset of action
• Laboratory monitoring
• aPTT 1.5X 2.5X normal mean
• heparin level
• 0.2 0.4 U/mL by protamine titration
• 0.35 0.70 by Factor Xa inactivation assay

XII
XI
IX
VIII
VII
X
aPTT
V
II
I
XIII
Stable clot
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AnticoagulationHeparin
AT
AT
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II
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AT
II
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II
AT
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AT
II
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AT
II
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AT
II
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Coumadin (Warfarin) Anticoagulants
XII

• inhibits hepatic synthesis of vit K-dependent
  clotting factors (II, VII, IX, X)
• competitive inhibition of g-carboxylation
• inactivate acarboxy forms synthesized
• onset delayed 3 to 5 days
• also inhibits synthesis of protein C S

XI
IX
VIII
VII
X
V
PT
II
I
XIII
Stable clot
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Thrombolytic (Fibrinolytic) Factors

• Urokinase released from endothelial cells and
  monocytes
• Tissue plasminogen activator (tPA)
• conversion of plasminogen to plasmin
• cleavage of fibrinogen fibrin into
  fibrin split products
• inhibit plt aggregation
• thrombin activity
• fibrin strands cross linking

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Plasmin Degradation of Fibrin Clot
plasmin
plasmin
plasmin
Fibrin Degradation Products
D-dimer
DED complex
E-fragment
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Bleeding Disorders
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Procoagulant Platelets Factors Fibrinogen von
Willebrand Factor
Profibrinolytic Plasminogen tPA Fibrin Fragment
D-dimer
RESOLUTION
FORMATION
clot
Anticoagulant Antithrombin III Protein C Protein S
Antifibrinolytic PAI-1 Alpha-2 Antiplasmin
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Vessel Abnormalitiesincreased vascular fragility

• manifested by petechial hemorrhages of
  skin/mucous membranes
• bleeding time, plt count, PT, aPTT --- normal
• not life threatening bleeding
• 1. congenital
• a. Ehlers-Danlos syndrome (AD)
• b. hereditary hemorrhagic telangiectasia (AD)
• 2. acquired
• a. hypersensitivity vasculitis
• (1) drug reaction immune complex deposit in
  vessel walls
• (2) Henoch-Schonlein purpura
• b. scurvy (vit C deficiency)

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Henoch-Schonlein purpura

• generalized hypersensitivity vasculitis
• uncertain cause
• clinical Sx
• purpura
• colicky abdominal pain
• polyarthralgia
• acute glomerulonephritis

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Coagulation Factor Abnormality

• hematomas/ecchymoses after minor trauma
• often severe bleeding
• 1. congenital usually single factor deficiency
• a. sex-linked
• (1) hemophilia A (Factor VIII def.)
• (2) hemophilia B (Christmas disease, Factor IX
  def.)
• b. autosomal dominant
• (1) von Willebrands disease
• c. autosomal recessive
• 2. acquired usually multi-factor deficiency and
  clotting abnormalities
• a. vitamin K deficiency
• b. severe liver disease

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Hemophilia A (Factor VIII deficiency)

• bleeding into joints ? crippling arthropathy
• sex-linked inheritance
• high rate of spontaneous mutation
• 25 of pts do not have family history of
  hemophilia
• decreased VIII-C, near normal VIII-vWF
• gt50 severe deficiency
• increased aPTT
• normal bleeding time, plt, PT

XII
XI
IX
VIII
VII
X
aPTT
V
II
I
XIII
Stable clot
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hemophilia A
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Hemophilia B(Christmas disease, Factor IX def.)

• less common than hemophilia A
• similar clinical Sx and inheritance pattern as
  hemophilia A (sex-linked)

XII
XI
IX
VIII
VII
X
V
II
I
XIII
Stable clot
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von Willebrands disease

• easy bruisability (no bleeding into joints)
• unable to release VIII-vWF
• intact VIII-vWF synthesis
• VIII-C level is also decreased (unknown reason)
• autosomal dominant
• 1 in 30,000 population
• usually diagnosed in childhood or young adults
• increased bleeding time
• normal plt, PT
• normal or increased aPTT

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Vitamin K Deficiencyvitamin K dependent factors
II, VII, IX, X

• acquired disorder
• may occur in malnutrition, malabsorption, biliary
  obstruction, drug
• increased PT
• normal bleeding time, plt
• normal or increased aPTT

XII
XI
IX
VIII
VII
X
V
PT
II
I
XIII
Stable clot
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Severe Liver Diseasefactors synthesized in
liver II, V, VII, IX, X, fibrinogen
XII

• increased PT, aPTT
• normal bleeding time, plt

XI
IX
VIII
VII
X
V
PT
aPTT
II
I
XIII
Stable clot
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PLATELET ABNORMALITIES

• 1. Thrombocytopenia
• a. decreased production
• b. increased utilization
• c. increased destruction
• (1) isoimmune thrombocytopenia
• (2) idiopathic thrombocytopenic purpura (ITP)
• (3) thrombotic thrombocytopenic purpura (TTP)
• (4) drug reaction
• (5) mechanical destruction
• (6) hypersplenism
• 2. Functional abnormalities
• a. congenital
• (1) defective adhesion (Bernard-Soulier)
• (2) defective aggregation (thrombasthenia)
• b. acquired
• (1) aspirin
• (2) thrombocythemia

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Thrombocytopenia

• decreased in number of platelets
• bleeding from small vessels (skin, GI, mucous
  membrane, GU, brain)
• normal or increased bleeding time
• decreased platelet
• normal PT, aPTT

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Thrombocytopenia

• decreased production
• diffuse bone marrow disease (aplastic anemia,
  tumor)
• megakaryocyte disorder
• increased utilization
• DIC

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Thrombocytopenia
increased destruction

• isoimmune thrombocytopenia
• neonatal
• PLA 1 neg. mother PLA 1 pos. baby
• production of anti-PLA 1 Ab (IgG)
• post-transfusion
• PLA 1 neg. recipient PLA 1 pos. platelet
• destruction of PLA 1 platelets and recipient's
  own platelets

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Thrombocytopenia
increased destruction

• idiopathic thrombocytopenic purpura (ITP)
• acute ITP
• children following a viral infection
• self-limiting disease
• ? platelet as an innocent bystander
• chronic ITP
• adults (often premenopausal females)
• may be associated with other autoimmune
  diseases
• production of autoantibody against Pts own
  platelets
• removal of opsonized platelets by
  reticuloendothelial system
• decreased circulating platelet, but increased BM
  megakaryocytes

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• idiopathic thrombocytopenic purpura (ITP)
• clinical
• easy bruising and bleeding after minor trauma
• treatment
• steroid
• splenectomy

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Thrombocytopenia
increased destruction

• thrombotic thrombocytopenic purpura (TTP)
• abnormal platelet aggregation in microcirculation
• microangiopathic hemolytic anemia
• fever
• transient neurologic deficits
• renal failure
• hemolytic uremic syndrome (HUS)
• platelets start to aggregate in small vessels
  without particular reason

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Disseminated Intravascular Coagulation (DIC)

• - an acute, subacute, or chronic
  thrombohemorrhagic disorder occurring as a
  secondary complication in a variety of diseases
• - activation of clotting system resulting in wide
  spread formation of microthrombi throughout the
  microcirculation
• - as a consequence, causing consumption of
  platelets, fibrin and coagulation factors, and
  activation of thrombolytic mechanism
• Two major triggering mechanisms
• 1. release of tissue factor or thromboplastic
  substance
• 2. widespread endothelial injury

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DIC

• Triggering Mechanisms
• 1. release of tissue factor or thromboplastic
  substance
• - placental tissue
• - granules from leukemic cells
• - bacterial endotoxin
• - mucus from adeno CA
• 2. widespread endothelial injury
• - Ag-Ab immune complex deposit
• - extreme temperature
• - microorganisms

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DIC

• Pathology - wide spread thrombi
• (brain, heart, lungs, kidneys, adrenals,
  spleen , liver)
• - microinfarcts
• Clinical - 50 associated with obstetric
  complications
• - 30 with carcinomatosis
• - microangiopathic anemia
• - dyspnea, cyanosis
• - convulsions, coma
• - oliguria, acute renal failure
• - shock, circulatory failure

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DIC

• Clinical acute DIC with a predominance of
  thrombin generation and consumption of
  coagulation factors
• bleeding tendency
• (oozing from venopuncutres or operating site)
• subacute and chronic DIC
• thrombotic tendency

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DIC

• Lab - fibrinogen
• - platelet
• - PT
• - aPTT
• - fibrin degradation product
• acute DIC - prolongation of aPTT, PT and TT
• - reduction of platelets, AT III and protein
  C
• - decreased fibrinogen
• - elevated fibrin degradation products
• chronic DIC - aPTT and PT may be within normal
  ranges
• - slightly decreased platelets
• - elevated fibrin degradation products and
  D-dimer

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Platelet Functional Abnormalities congenital
1

• Bernard-Soulier syndrome
• defect in platelet adhesion
• autosomal recessive
• defect in platelet membrane glycoprotein (GP Ib)
• thrombasthenia
• defect in platelet aggregation
• autosomal recessive
• defect in platelet membrane glycoprotein (GP IIb
  IIIa)
• no fibrinogen linking of platelets
• easy bleeding and no clot retraction

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Platelet Functional Abnormalities acquired

• aspirin
• inhibits cyclooxygenase
• suppression of TXA2 synthesis
• effect lasts for 72 hours
• thrombocythemia
• platelet gt3,000,000/ml
• functionally abnormal platelets
• occasionally seen in myeloproliferative disorders

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Coagulation Tests

• 1. Bleeding Time in vivo test
• measures adequacy of plt function
• normal lt6 min.
• 2. Platelet Count normal gt200,000/mL
• 3. aPTT intrinsic pathway (XII, XI, IX, VIII,
  X, V)
• used to guide heparin therapy
• 4. 50/50 mixing study
• pts plasma nl. plasma
• if mixing correct aPTT Pt is deficient in
  intrinsic factor(s)
• no correction circulating anticoagulants or
  inhibitors
• 5. Prothrombin Time (PT) extrinsic pathway (II,
  VII, V, X)
• monitoring warfarin/coumadin effects

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Coagulation Tests

• 6. Fibrinogen Level normal 200 500
  mg/dL
• 7. ADP platelet aggregation
• 8. Ristocetin aggregation test
• test for presence or activity of vWF
• 9. Thrombin Time (TT) normal 20 30 sec
• measures 3rd stage of coagulation
• prolonged if
• def or abnormality of fibrinogen
• presence of fibrin split products
• presence of heparin

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History Physical Examaremost importantmost
sensitivemost specificTests of Hemostasis
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