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Clinical Lab: Coagulation


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Coagulation results in..
What is needed for clot formation to proceed?
What breaks down the clot?
Thrombolysis (Fibrinolysis)
Primary Hemostasis
-platelet adhesion
-granule adhesion
-platelet aggregation
What forms inital platelet plug?
-aggregated platelets
Secondary hemostasis
-coagulation cascade
What starts the coagulation cascade and leads to clot formation?
-injury to the epithelial cell
Endothelial cell help control clotting cascade by making...
-tissue factor
-Vonwillebrands factor
-Anti-thrombin III
-tissue plasminogen activator
What is released when endothelial lining is disrupted?
-tissue factor and vW (a cofactor of VIII)
Aspects of Coagulation
-extrinisic pathway
-instrinsic pathway
-common pathway
-fibrinolytic pathway
How were clotting factors numbered?
-in the order of their discovery
How many factors are there?
-there are 12 factors (even though 13 numbers, because VI was dropped)
Which factor is NOT made in the liver?
-factor VIII
How many of the factors are enzymes?
-9 of the 12 factors are enzymes
Which factors are vitamin K dependent?
-factors II, VII, IX, X
Factor I
Factor II
Factor III
Factor IV
Factor V
Proaccelerin, labile factor
Factor VII
Proconvertin, stable factor
Factor VIII
-antihemophilic factor (AHF)
-antihemophilic factor A
-antihemophilic globulin (AHG)
Factor IX
-Plasma thromboplastic component (PTC)
-Christmas Factor
-antihemophilic factor B
Factor X
-Stewart Prower Factor
Factor XI
-Plasma Thromboplastin Antecedent (PTA)
-Antihemophilic Factor C
Factor XII
-Hageman Factor
-glass factor
Factor XIII
-Fibrin Stabilizing Factor
-High molecular weight kininogen
-fitzgerald factor
-Fletcher Factor
-platelet phospholipid
Clotting cascade
1. platelets are recruited and release tissue thromboplastin
2. thromboplastin (factor III) is formed by cascade involving calcium and factors V, VII,X, XI, XII
3. Prothrombin (factor II) is activated by thromboplastin formation
4. Thrombin is formed from prothrombin with the help of calcium
5. thrombin interacts with fibinogen to form clot
6. fribrinolytic system is activated
Clotting Cascade:
Platelets are recruited and release...
tissue thromboplastin
Clotting Cascade:
Thromboplastin (factor III) is formed by cascade involving...
-factors V, VIII, X, XI, XII
Clotting Cascade:
Prothrombin (factor II) is activated by ...
thromboplastin formation
Clotting Cascade:
Thrombin is formed from .... with the help of ....
formed from Prothrombin with the help of calcium
Clotting Cascade:
Thrombin interacts with .... to form a clot
Extrinsic Pathway
1. thromboplastin (TPL) is released from damaged skin
2. TPL converts Factor VII to VIIa (initial step in extrinsic system)
3. TPL and VIIa convert X to Xa (inital step in common pathway)
Extrinsic pathway:
...... is released from damaged skin
Thromboplastin (TPL)
Extrinsic pathway:
TPL convert ..... to .....
converts factor VII to VIIa
(initial step in extrinsic system)
Extrinsic pathway:
TPL and VIIA convert .... to ....
convert X to Xa
(inital step in common pathway)
INtrinsic Pathway trigger
collagen exposure is the trigger
Intrinsic Pathway
1. kallikrein converts XII to XIIa
2. XIIa convert pre-kallikrein to kallikrein via HMWK
3. VonWillebrands factor helps platelets adhere to exposed collagen
4. Platelets release PF-3
5. PF-3, IXa, VIII, Calcium convert X to Xa (inital step in common pathway)
6. intact intimal lining has anti-platelet aggregating factor
Intrinsic Pathway:
kallikrein converts ....
converts XII to XIIa
Intrinsic Pathway:
XIIa converts...
converts pre-kallikrein to kellikrein via HMWK
Intrinsic Pathway:
VonWillebrand factor is derived from..... and attached to ....
derived from endothelium and attached to platelets
Intrinsic Pathway:
VonWillebrand's factor helps .... adhere to .....
helps platelets adhere to exposed collaged
Intrinsic Pathway:
platelets release...
Intrinsic Pathway:
PF-3, IXa, VIII and Calcium convert...
convert X to Xa
(intial step in common pathway)
If adventita layer is exposed....
platelets will aggregate
Common Pathway: Goal
-formation of stable clot
Common pathway: Initial Step
-activation of X to Xa
Common pathway:
1. activation of X to Xa
2. Xa converts II (prothrombin) into IIa (thrombin)
3. Thrombin converts fibrinogen to fibrin and XIII to XIIIa
4. XIIIa stabilizes the fibrin clot
Common pathway:
Xa converts ... to ...
converts prothrombin (II) to thrombin (IIa)
Common pathway:
thrombin convert ... to .... and .... to ....
convert fibrinogen to fibrin
Common pathway:
XIIIa ....
stablizes the fibrin clot
Disorders of hemostasis
-vascular abnormailities (congenital and acquired)
-connective tissue disorders (congenital and aquired)
-qualitative platelet abnormalities
-quantitative platelet abnormalities
-coagulation abnormalities (congenital and acquired)
Congenital coagulation abnormailites
-van vonwillebrands
Acquired coagulation abnormalities
-circulating anticoagulants
-vitamin D deficiency
-liver disease
Important historical questions for the bleeding patient?
-circumcicion (look for factor deficieny)
-prior surgery
-dental procedures
-easy bruising
-irregular menses
Reasons for bleeding
-liver disease
-anticaogulant therapy/drugs
-inherited factor deficeincy
Screening tests for Hemostasis
-platelet count, size, morphology
-bleeding time
-fibringen level
Other tests for Hemostasis
-factor assays
-fibinolysis studies (fibrin split products)
Tests for hypercoagulable states
-antiplatelet factors (antithrombin II, protein C, protein S, lupus anticoagulant)
-fibrinolysis tests (fibrin split products, D-dimer)
PT relationship to Coagulation Cascade
PT assesses Extrinsic sys and common pathway
APTT relationship to Coagulation Cascade
APTT assesses Intrinsic sys and common pathway
Ivy Bleeding relationship to Coagulation Cascade
Ivy Bleeding time assesses platlet function
Prothrombin test mechanism
-mixture of tissue thromboplastin and calcium are added to pt plasma to induce extrinsic pathway
-formation of fibrin clot is the endpoint
PT assesses the extrinsic pathways by..
-assessing defects in Factors II, V, VII, X
PT is most sensitive to which factor?
Factor VII
PT is used to monitor which type of therapy?
-coumadin therapy
Uses for PT?
-screenging for coagulation disorder
-assesses extrinsic system
-monitors coumadin therapy
-assesses for perenchymal liver disease (factos VII)
-assesses for vitamin K deficiency (since prothombin is vit K dependent)
Causes of increased PT
-deficient of factor II, V, VII, X
-decreased vitamin K
-liver disease
-coumadin therapy
-biliary obstruction
-decreased fibrinogen
-circulating anticogulants
Caused of decreased PT
-ovarian hyperfunction
-regional enteritis
Prothrombin time INR: purpose
-designed to measure PT consistently from lab to lab for coumadin management
Prothrombin time INR: How calculated
-pts PT is divided by the midpoint of the Laboratories normal range then multiplied by an exponent representing the specific TPL reagent utilized
Prothrombin time INR: Normal range
Prothrombin time INR: therapeutic range
2.0-3.0 x control for most clinical situations

2.5-3.5 x control for pts with mechanical heart valves

>3x control for pts with antiphospholipid antibody syndrome
Absolute contraindications to Coumadin
-bleeding diathesis
-previous purple toe syndrome or skin necrosis d/t coumadin
Relative containdications to Coumadin
-HTN systolic > 180
-HTN diastolic > 100
-gait disturbances
-severe liver disease
-aortic aneurysm
-recent surgery on nervous system or eye
Overall general use of Coumadin is to....
prevent blod clots (indirect anticoagulant)
Coumadin acts by...
-interfering with factors II, VII, IX, X (vitamin K dependent factors) in the liver
How long until Coumadin provides a measurable change?
48-72 hours
Acute Coumadin therapy
-perioperative anticoagulation (DVT prophylaxis)
-treatment of acute DVT
-acute MI
Chronic Coumadin Therapy
-atrial fibrillation
-mechanical heart valve
Dose for chronic coumadin therapy
-start coumadin at maintenance dose since there is no acute urgency
-dose is based on serial PT and associated INRs
-fixed (ie 5 mg/day) vs alternating (ie 5 mg odd days and 3 mg even days)
Coumadin Complication
-life threatening bleeds
-non life threatengin bleeds
Treatment of non-life threatening bleeds d/t coumadin (hematurea or epistaxis)
1.2 mg vitamin K (SQ)

should decrease INR within 6-8 hours
Treatment of life threatening bleeds d/t coumadin
10 mg vitamin K (IV)

give factor VII or FFP
ACtivated Partial Thromboplastin Time
APTT Uses:
-to assess coagulation abnormalities in the intrinsic and common pathways
-to monitor heparin therapy
APTT: how the test works
-Add cephaloplastin and calcium to plasma to initiate the coagulation cascade
APPT: normal ranges
21-35 seconds
APTT: therapeutic range
1.5 -2.5 x control

calculated as the ratio of the pts PTT to mean of the normal laboratory control
Causes of elevated APTT
-lupus anticoagulant
-factor deficiency (XII, XI, IX, VIII)
-VonWillbrands Disease (VIII vw)
-Liver disease
-heparin therapy/coumadin therapy
-vitamin K deficiency
-decreased fibrinogen
-fibrin breakdown products
Causes of decreased APTT
-Cancer (not liver cancer)
-following acute hemorrhage
-early DIC
Heparin therapy: how does it work?
-prevents further clotting but does not lyse existing clots
-direct acting anticoagulant
-combines with alpha globulin to form a potent anti-thrombin
Heparin therapy: Low dose refers to..
<5000 units
Heparin therapy: high dose refers to..
>5000 units (IV)
Heparin therapy: bolus dose
7500 - 10,000 units/hr IV
Heparin therapy: maintenance dose
1000-1,500 units/hour
Heparin therapy: Typically high dose therapy
-loading dose: 7500-10,000 Units IV bolus
-continuous maintenance infusion

25,000 Units in 250 ml, ie 100 units/ml

then run at 1000- 1500 units/hr infusion rate, ie 10-15 ml/hr)
Laboratory eval with HMW Heparin
-obtain APPT as base before heparin therapy
-obtain APPT 6 hrs p initial bolus
-after steady state is reached (~4-6 hrs), obtain APTT qd
-monitor platelet count qd

remember coumadin can raise APTT
Coagulation in newborns
-increase APTT compared to adults
-factors XI, XII, HMWK decreased by 30%
-reach adult levels by 6 months
-vitamin K factors decreased at birth 20-60%
-decreased AT III, protein C
vonWillebrand Factor (VIII vw)
-originally secreted by endothelium
-resides on platelets
-adhesive component helps platelets stick to damaged epthelium
Pro-coagulant factor (VIII-pc) works via..
intrinsic pathway
Von Willebrand disease is a defect in..
VIII-vw (vonWillebrands factor) and VIII-pc (pro-coagulant factor)
Defect in VIII vw causes
increased bleeding time
Defect in VIII pc causes
elevated APTT
Hemophilia d/t VIII deficiency
-defect is only in VIII pc
-bleeding time is normal
-PTT is increased
-PT is normal
Coagulation Factor Inhibitors
-antiphospholipid antibodies
-factor VIII-pc antibodies
Anti-phospholipid antibodies
-lupus anticoagulant (LAC)
(can inc APTT)

-anticardiolipin antibody
Anti-phospholipid antibodies (lupus anticoagulants and anti-cardiolipin antibodies) work by..
-interfere with phospholipid interaction with coagulation factors

-results in thrombosis and sponantenous abortion

-no increased tendancy for bleeding
Factor VIII-pc antibodies
-occurs in 5-10% of hemophelia pts
-may be seen post-partum and in autoimmune disorders
-blocks or inactivates VIII-pc (intrinsic pathway) which results in bleeding
Test results:
normal PT
inc PTT
normal or inc bleeding time
-vW disease
-lupus anticoagulant
-heparin effect
Test results:
normal PT
normal or inc PTT
inc bleeding time
-drug effect
-platelet function defect
-vW disease
Test results:
inc PT
normal PTT
normal bleeding time
-factor 7 deficiency
-liver disease
-vitamin K deficiency
Test results:
inc PT
inc PTT
normal or inc bleeding time
-Liver Disease
-Factor 2,5,10 deficeincy
-Vitamin K deficiency
Which 2 systems act to keep clotting in check?
-plasma inhibitors of activated factors
Plasma inhibitors of activated factors include:
-antithrombin III and other plasma inhibitors
-protein c and protein s
Plasma Factor Inactivation
1. thrombin binds to thrombomodulin
2.thrombin/thrombomodulin complex activates protein C
3. activated protein C destoryes activated factors V and VIII to stop to formation of thrombin
4. Protein S binds to protein C to enhance its activity
5. Activated protein C also enhances the fibrinolytic pathway
Plasma Factor Inactivation:
Thrombin binds to...
Thrombomodulin (thrombomodulin is a receptor on the endothelial surface)
Plasma Factor Inactivation:
Thrombin/thrombomodulin complex activates....
Protein C (protein C is a vitamin K dependent protein)
Plasma Factor Inactivation:
Activated protein C destroys...
Factors V and VIII (to stop the formation of thrombin)
Plasma Factor Inactivation:
Destruction of factors V and VII stop the fomration of ...
Plasma Factor Inactivation:
Protein S (surface component of platelets) bind to ....
bind to protein c to enhance its activity
Plasma Factor Inactivation:
Activated protein C also enhances....
the fibrinolytic pathway
1. Plasminogen is converted to plasmin by factor XIIa and kallikrein (intrinsic system) and by tissue plasminogen activator (tPA)(extrinsic system)
2. Plasmin causes the breakdown of fibrin into fibrin split products
3. tPA is inactivated by circulating plasminogen activator inhibitor 1 (PAI-1)
Plasminogen is converted to ...
Plasminogen is converted to plasmin by..
-factor XIIa and kalliekrein (intrinsic system)

-tissue plasminogen activator (extrinsic system); released from endothelial cells during injury; activated protein C destroys inhibitors of TPA to increase its activity
Plasmin causes the breakdown of... into ...
breakdown of fibrin into fibrin split products
TPA is inactivated by ...
circulating plasminogen actovator inhibitor (PAI-1) (released by platelets)
TPA has to be shut down or...
system goes on and on and on
Fibrinolytic System Simplified
1. Plasmin lyses fibrin

2. protein C inactivates extrinsic via V and inactivates intrinsic via VIII

3. protein S is a cofactor in protein C synthesis
1 ml of blood contains enough coagulation product to clot the entire circulation for...
10-15 seconds
DIC - what does it stand for?
disseminated intravascular coagulation
-an acquired hemorrhagic syndrome characterized by uncontrolled formation and deposition of fibrin
-thrombin formation causes depletion of clotting factors
DIC causes:
-metastatic CA
DIC triggers:
-gram negative bacterial endotoxin
-tissue factors
-TNF (tumor necrosis factor)
DIC mechanism
1. Fibrin and fibrin clots are deposited throughout the vasculature
2. fibrinolytic system is activated
3. eventually fibrin becomes depleted and blood cannot clot
4. platelets are consumed
5. result is extensive, uncontrolled bleeding
Laboratory picture of DIC
-PT inc
-PTT inc
-bleeding time inc
-fibrinogen dec
-platelets dec
-FSP positive
-thrombin time inc
-clotting factors dec
-D-dimer inc
-anti-thrombin III positive
DIC treatment
-treat the underlying cause
DIC tx: severe
-fresh frozen plasma
DIC tx: mild or chronic
Labs to support DIC work-up
-thrombin time
-fibrin split products
Thrombin Time detects...
-fibrinogen defects
Thrombin Time measures..
time to clot when thrombin is added to plasma
Thrombin time normal value
7-12 seconds
prolonged TT is seen in...
-severe liver disease
-multiple myeloma
-heparin tx
-decreased fibrinogen
shortened TT is seen in..
-elevated Hct
-hyperfibrinogenemia (elevated fibrinogen)
Fibrin split products: normal
negative at 1:4 dilution
Fibrin split products help dx
Fibrin split products: at high levels..
blood will not clot
D-dimer: normal
no d-dimer present
D-dimer produced during...
fibrinolysis when plasmin attacks cross-linked fibrin
D-dimer help dx

more specific than fibrin split products
Fibrinogen: normal values
200-400 mg/dl
Fibrinogen is used when..
-work up for abnormal PT/PTT/TT values
-suspected DIC
Increased fibrinogen with..
-acute MI
-nephrotic syndrome
-nephrotic CA
decreased fibrinogen with..
-liver disease
Overactivity of platelet system (arterial clots) occurs with..
-inc lipids or cholesterol
-elevated platelet levels
-blood flow changes
Accelerated activity of the clotting system (venous clots) occurs with..
-artificial material
-damaged vessels
Clot/DVT work-up
-protein S antigen and free protein S
-protein C acitivity
-antithrombin III activity
-plasminogen level
-others as indicated (lupus anticoagulant, antiphospholipid antibody)
Protein C deficiency due to..
-genetic disorder

manifests with recurrent DVT and PE
Heterozygotes for protein C deficiency can be treated with..
heparin or coumadin
Homozygotes for protein C deficiency should be treated with...
regular plasma infusions
Normal platelet count
150,000 to 450,000 /mm3
Platelet distribution
2/3 in blood

1/3 in spleen
Platelet function
-clot formation
-maintenance of vascular integrity
-formation of platelet plugs
Platelet lifespan
7.5 days
Platelet panic values
<20,000 risk of spontaneous bleed

>1,000,000 risk of spontaneous clots or emboli
Increased platelet counts in...
-polycythemia vera
-splenectomy (post-op)
-collagen diseases
-acute infection
-recovery from bone marrow suppression
Decreased platelet counts in...
-aplastic and hemolytic anemias
-after massive transfusion
-drugs DIC
-inherited conditions
Thrombocytopenia (low platelets) results from three mechanisms...
1. increased destruction
2. abnormal distribution
3. decreased production
Types of Increased destruction of platelets

-non-immune (platelet consumption, platelet destruction)
Types of Decreased production of platelets
-hypoplasia of megakaryocytes

-bone marrow infiltration
Platelet aggregation test is used to..
test platelet function
what happens in Platelet aggregation test
-light transmission through plasma will increase when platelets aggregate
-platelets are challanged with reagents and transmission is measured
Platelet aggregation test: Decreased aggregation with ...
congenital and acquired conditions
Platelet aggregation test:
Increased aggregation with ...
Raynauds phenomenon
Decreased Platelet Aggregation:
COngenital disorders
Decreased Platelet Aggregation:
Acquired disorders
-antiplatelet antibodies
-cardiopulmonary bypass
-myloproliferative disorders
Test of platelet function
-bleeding time (time it takes for bleeding to cease)

ivy method - incision on pts arm

duke method - incision on pts ear
Normal bleeding time
2-10 minutes

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