2. Topics for Discussion
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4. Virchow’s Triad
Changes in blood coagulability
Platelets, Coagulation Factors & Inhibitors, Fibrinolysis
Changes in vessel wall Changes in blood flow
Endothelial changes due to Rheology in vessels
inflammation or atherogenesis
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5. Vessel Wall
Tunica intima
Endothelium - inner most layer of cells
that separate the remainder of the
vessel from the lumen
Basement membrane – thin layer of
spongy connective tissue that secretes
elastic collagen
Tunica adventitia
Surrounds the tunica media
Connective tissues – produce elastic
http://www.alegent.com/122711.cfm and non-elastic collagen fibers
Tunica intermedia Prevents ballooning of vessel with high
systolic blood pressure
Surrounds the tunica intima
Aneurysm – weaknesses in the tunica
Smooth muscle – layer of smooth muscle cells adventitia
that are under involuntary control and can
dilate or constrict
Connective tissue – produces collagen fibers
whose elasticity is reduced by hypertension
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6. Rheology
Region 1 Region 2
In this range of shear rates: As shear rate increases:
Cells are in large Cells are disaggregating
aggregates (rouleaux Applied forces (yield
formation) stress) are forcing cells to
As shear rate increases, orient and deform
size of aggregates diminish Blood viscosity decreases
Viscoelasticity is strongly
influenced by aggregation
tendency of RBCs
Region 3
With increasing stress:
Cells deform
With normal deformability,
cells will form layers that
Adapted from : slide on layers of plasma
http://www.vilastic.com and align in the direction
/tech10.html of flow
Science of the deformation and flow of matter
Blood is a fluidized suspension of elastic cells that demonstrates both a viscous
and elastic effect
– Elastic effect makes blood a non-Newtonian fluid (plasma is a Newtonian fluid)
– Blood has a yield stress that depends on hematocrit and fibrinogen concentration
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7. Shear & Vessel Wall
Endothelial Cells
At Wall Surface: Greatest Platelet Flowing Blood
Shear Rate & Least Velocity
Shear
At Vessel Center: Greatest Platelet
Flow
Velocity & Least Shear Rate
Shear Stress
Pressure- induced Force
Between 2 Laminae
Laminae (Arrow Length = Velocity)
Endothelial Cells
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8. Hemostasis
Process by which blood is maintained in a fluid state and
confined to the circulatory system
Goal is to stop bleeding and to do so only at the site of
injury
Components
– Platelets
• Involved in Primary Hemostasis
– Coagulation system
• Involved in Secondary Hemostasis
– Fibrinolytic system Platelet / fibrin mesh
– Inflammatory processes
– Wound healing processes
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10. Primary Hemostasis
First physiological response to vascular injury, which is
mediated by platelets, in order to arrest bleeding
Mechanism
– Activation of platelets via stimulators such as thrombin
– Adhesion of platelets to subendothelium via interaction
between GPIb and von Willebrand Factor (VWF)
– Release of platelet granule products in order to recruit
more platelets to the injured site
– Aggregation of platelets via interaction between
GPIIb/IIIa (αIIbβ3) and fibrinogen to form the initial plug
Triggers secondary hemostasis (coagulation proteins)
Affected by medications, platelet function status, and
vessel wall status
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11. Platelets
Disk-shaped cell
fragments produced in GPIIb/IIIa
the bone marrow by
megakaryocytes Alpha Dense
Life span ~ 10 days granules granules
Glycoprotein Ib (GPIb)
is involved in adhesion
Glycoprotein IIb/IIIa
(GPIIb/IIIa) is the
primary receptor for
fibrinogen (aggregation
phase)
Provide procoagulant
surface on which
coagulation proteins Coagulation
can interact proteins GPIb
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12. Platelet Adhesion
Platelet
GPIb
Endothelium VWF Injury site
collagen exposed
COL
COL
COL CO
L COL
VWF VWF
VWF
VWF
Subendothelial extracellular matrix
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13. Platelet Aggregation
Activated
Platelet GPIb Fibrinogen
(Granule contents released)
VWF GPIIb/IIIa
COL
COL
COL CO
L COL
VWF VWF
VWF
VWF
Subendothelial extracellular matrix
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14. Tests for Primary Hemostasis
Bleeding Time
– Assesses all components of Virchow’s triad
– in vivo test – performed directly on patient
– Has fallen into disrepute and replaced by instruments that perform “in
vitro” bleeding times
Platelet Aggregation studies
– Measure ability of platelets to aggregate, in vitro, when subjected to
various stimulators (agonists)
– Predominantly assesses function of platelet glycoprotein IIb/IIIa
receptor
Von Willebrand Factor (VWF) assays
– Measure amount and function of VWF, a protein that works with
platelets so that they adhere to site of injury
– Assesses function of VWF ligand in its interaction with platelet
glycoprotein Ib receptor
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15. Platelet Appearance / Function
Electron Micrographs Aggregation Tracings
Resting platelets
Normal platelet Aspirin-like defect
function
Activated platelets
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17. Secondary Hemostasis
Process of blood coagulation
Mechanism
– Coagulation proteins work in
concert to generate thrombin
– Thrombin converts fibrinogen to
fibrin
– Fibrin consolidates the platelet plug
made in primary hemostasis such Credit: Weisel JW. University of Pennsylvannia
that a thrombus (secondary
hemostatic plug) is formed
Prevents further blood loss from
the injury site
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18. Coagulation Factors
Factor XII (FXII) activated FXII (FXIIa)
Factor XI (FXI) activated FXI (FXIa)
Factor X (FX) activated FX (FXa)
Factor IX (FIX) activated FIX (FIXa)
Factor VIII (FVIII) activated FVIII (FVIIIa)
Factor VII (FVII) activated FVII (FVIIa)
Factor V (FV) activated FV (FVa)
Factor II (prothrombin) is converted to thrombin (FIIa)
Factor I (fibrinogen) is converted to fibrin
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19. “in vivo” v “in vitro” Coagulation
Physiologic (“in vivo”) coagulation is dependent upon
the tissue factor pathway
– Goal is to form a thrombus
Laboratory (“in vitro”) coagulation is dependent upon
the contact system
– Classical “waterfall” or cascade concept
• Step-by-step biochemical reactions in which an inactive proenzyme
is converted to a reactive enzyme which, in turn, converts another
proenzyme to its active form
– Amplification process (very minute amounts of Factor XII
yield large amounts of thrombin)
– Goal is to form a clot
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20. Waterfall Scheme of Coagulation
FXII FXIIa
FXI FXIa
FVIIIa
FIX Ca++ PL FIXa FVIIa FVII
TF
Ca++ PL
FX FXa FX
FVa
FII Ca++ PL Thrombin
TF = Tissue Factor
Ca+ += Calcium ion
After Macfarlane RG. Nature 1964;202:498-9
Fibrinogen Fibrin PL = Phospholipid
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24. Propagation Phase
Monroe DM, et al.
ATVB 2006;26:41-8
“Intrinsic Pathway”
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25. Recapping Secondary Hemostasis
Tissue Factor complexed with FVIIa initiates coagulation at site
of injury
Small amounts of thrombin are generated that activate platelets
Coagulation factors form complexes on platelet surfaces
Very large amounts of thrombin are formed to convert
fibrinogen to fibrin
Fibrin reinforces platelet plug (primary hemostasis) and
hemostasis is achieved
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27. First the Specimen
Blood is collected into Plasma is used for testing
a tube that contains – PLASMA contains
sodium citrate, an FIBRINOGEN
anticoagulant
– Serum does not contain
Blood fluidity is Fibrinogen
maintained because
sodium citrate binds Plasma is “platelet poor”
calcium ions, which are since platelets remain in
critical to the buffy coat
coagulation process
Tube is centrifuged in
order to separate
plasma from buffy coat Plasma
(white blood cells & Buffy Coat
platelets) and red
blood cells Red Cells
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28. Coagulation in the Laboratory
Intrinsic Pathway Extrinsic Pathway
XII
XI VII Tissue Factor
IX
APTT VIII
PT
Intrinsic X Extrinsic
+
Common V Pathway +
Common
II Common
Fibrinogen Fibrin Clot
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29. Routine Coagulation Assays
Prothrombin Time (PT)
Activated Partial Thromboplastin Time (APTT)
Quantitative Fibrinogen (FIB)
Thrombin Time (TT)
Assays for specific coagulation factors
– Factors assessed by a PT-based test system: FVII, FV, FX,
and FII
– Factors assessed by an APTT-based test system: FXII,
FXI, FIX, and FVIII
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30. Prothrombin Time (PT)
Time for clot formation
~ 12 seconds
Incubate at 37 oC for ~3 minutes
0.1 ml Thromboplastin + Ca++
0.1 ml Plasma
PT Reagent Composition
Thromboplastin
Credit: PNAS; Collet JP and
− Tissue Factor (recombinant/human or animal brain) Weisel JW. Un Pennsylvannia
− Lipid (source of phospholipid since platelets were removed from plasma)
− CaCl2 – used to reintroduce calcium ions that were chelated by sodium citrate
Historically referred to as “complete” since both phospholipid and apoprotein
make up the reagent
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31. Causes for Prolonged PT
Deficiencies or abnormalities in:
– FVII (Extrinsic Pathway)
– FV, FX, FII (prothrombin), and FI (fibrinogen)
• Both PT and APTT will be prolonged
Vitamin K antagonists
– PT sensitive to reductions in three of four vitamin K-dependent
procoagulant proteins: FVII, FX, and FII
• FIX measured by APTT
– Pharmacologic anticoagulants that modify vitamin K-dependent
proteins such that they do not bind calcium thereby reducing blood
coagulability
Liver disease
– Site for synthesis of vitamin K-dependent proteins
– Site for clearance of coumarins (warfarin) and coagulation proteins
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32. Biochemistry: Vit K-Dep Proteins
Addition of an extra carboxyl group to glutamate (Glu)
residues at their amine termini gives rise to a novel
amino acid called gamma-carboxyglutamate (Gla)
– Presence of Gla enables proteins to undergo a calcium-
dependent conformational change that allows for their
binding to
phospholipid
surfaces and
generation of
membrane
bound
macromolecular
complexes
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33. Vitamin K Antagonists (AVK)
DICUMAROL: 3,3’-methylenebis (4-hydroxycoumarin)
– Isolated by Karl Link (University of Wisconsin-1939) as the anti-
vitamin K agent responsible for hemorrhagic disorder in cattle
– Coumarin derivatives
• Bishydroxycoumarin (Dicumarol)
• Warfarin (Coumadin®)
– Analog #42 of many coumarins synthesized by Dr Link and named by him as
WARFarin for the Wisconsin Alumni Research Foundation and coumARIN
Require monitoring because:
– Vitamin K-dependent proteins have different half-lives
– Differences in drug absorption and clearance
– Levels affected by concomitant medications, comorbid conditions,
changes in diet, patient compliance
– PT (thromboplastin) reagents vary in their reaction to clotting defects
produced by AVK (warfarin)
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34. Warfarin Sodium
Oral anticoagulant of choice in North America
Pharmacologic properties more favorable than Dicumarol
– Warfarin is 5-10x more potent
Racemic mixture (~1:1) of R & S isomers
– S isomer is 5x more potent than R isomer
• Hepatic microsomal enzyme cytochrome P450 2C9 is responsible for the
oxidative metabolism of S isomer
Bioavailability
– Absorbed from gastrointestinal tract
– Maximal blood concentrations reached in 90 minutes
– Half-life (T½) is 36-42 hours
– At therapeutic concentrations, 99% of warfarin is bound to albumin
and 1% is free and can bind to its receptor on hepatic cells
Anticoagulant effects are reversed by administration of vitamin K
or biologic products that contain vitamin K-dependent proteins
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35. Warfarin Mode of Action
CO2 H2O
O2
Des-Carboxy Prothrombin
Prothrombin (“Glu”) γ-Carboxylase (“Gla”)
Vitamin K Vitamin K
hydroquinone Vitamin K 2, 3 epoxide
(active form K1H2) (inactive form K1O)
epoxide reductase
(VKOR)
Regeneration
of active form NAD+ NADH
is sensitive to
warfarin WARFARIN
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36. International Normalized Ratio
ISI
Patient PT
INR =
Mean Normal PT
“Normalizes” the PT by mathematically considering
differences in PT reagents (thromboplastins)
Only to be used to monitor long term anticoagulant
effects for patients stabilized on oral anticoagulant
therapy
– Prevent recurrence of thrombosis caused by under
anticoagulation
– Prevent hemorrhagic complications caused by over
anticoagulation
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37. Activated Partial Thromboplastin Time
Time for clot formation
~ 30 seconds
0.1 ml CaCl2
Incubate at 37 oC for ~5 minutes
0.1 ml Activator
0.1 ml Plasma
APTT Reagent Composition
Activator to convert FXII to FXIIa
Phospholipid (replaces “in vivo” platelet surface on which coagulation reactions
occur)
CaCl2 – used to reintroduce calcium ions that were chelated by sodium citrate
Referred to as “partial thromboplastin” since no Tissue Factor is used
− Two-stage assay (activation and re-calcification)
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38. Intended Use for APTT
Screening test
– Intrinsic (and severe common pathway) factor deficiencies
Laboratory monitoring
– Unfractionated heparin
– Other antithrombotic agents (Direct Thrombin Inhibitors)
Laboratory detection of the Lupus Anticoagulant
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39. Causes for Prolonged APTT
Most common causes
– Heparin (contamination from lines or therapeutic)
– Lupus Anticoagulant
Other causes
– Deficiencies of coagulation factors
• FVIII (Hemophilia A or Von Willebrand Disease), FIX, FXI, FXII
– Liver disease (site of production for most coagulation
factors)
– Consumption of coagulation factors as seen in
Disseminated Intravascular Coagulation (DIC)
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40. Heparin
Heparin is a heterogeneous group of straight-chain anionic
mucopolysaccharides (glycosaminoglycans)
– Molecular weights range from 5 – 40 kiloDaltons
– Composed of alternating D-glucosamine residues linked 1 → 4
to either L-iduronic acid or D-glucuronic acid
Heparin is highly acidic therefore binds to positively charged
amino acids such as arginine & lysine
– Pentasaccharide sequence
• Comprisies ~30% of heparin
• Binds to antithrombin (AT)
• Accelerates AT inhibition of
activated factors XII, XI, IX, X, and II (thrombin)
thus serving as an anticoagulant
Pharmaceutical heparins are extracted from pig intestinal
mucosa (source of mast cells)
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42. APTT Monitoring of Heparin
Assumes antithrombotic (anti-IIa) effect parallels
anticoagulant effect
Limitations
– Pre-treatment APTT of patient
• Baseline APTT of patient prolonged due to Lupus Anticoagulant
• Baseline APTT of patient sample below or at low end of reference
interval due to high levels of FVIII (apparent “heparin resistance”)
– APTT reagents vary in sensitivity to heparin
• Laboratories must determine responsiveness of their APTT reagent
to unfractionated heparin
• Determine APTT therapeutic interval (seconds) for reagent used to
monitor heparin therapy
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43. Direct Thrombin Inhibitors
Hirudin [Lepirudin (rDNA)–trade name: Refludan®]
– Approved in USA, Canada, and EU for Heparin Induced
Thrombocytopenia (HIT) complicated by thrombosis
– Target APTT is 1.5-2.5 x patient baseline APTT
– In absence of severe thrombosis, some experts recommend a target
APTT of 1.5-2.0 x patient baseline APTT and monitor every 4 hours
Argatroban [non-US trade name: Novastan]
– Approved for HIT with or without thrombosis and also for
anticoagulation during percutaneous coronary intervention (PCI) in
patients with, or at risk for, HIT
– Target APTT 1.5-3.0 x patient baseline APTT (maximum 100 seconds)
Hirulog [Bivalirudin-trade name: Angiomax™]
– Undergoing evaluation for use as an anticoagulant for “on-pump” and
“off-pump” cardiac surgery in patients with HIT
– Target APTT is 1.5-2.5 x patient baseline APTT
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44. “Recap”ping the PT and APTT
PT and APTT are screening assays to determine if a
patient, when challenged, has a potential to bleed
– If warfarin or heparin are not present in sample, then:
• Prolonged PT and normal APTT = deficiency of FVII
• Normal PT and prolonged APTT = deficiencies in any of the intrinsic
pathway factors (FVIII, FIX, FXI, or FXII)
• Prolongation of both PT and APTT = deficiencies of factors common
to both pathways (FX, FV, FII, or fibrinogen)
PT, via the INR, is used to monitor oral anticoagulant
therapy (warfarin)
APTT is used to monitor heparin anticoagulant therapy
APTT is affected by inhibitors such as Lupus Anticoagulant
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45. Tests for Fibrinogen
Quantitative Fibrinogen Thrombin Time (TT)
Measures the amount of Assesses the functionality of
fibrinogen present in plasma fibrinogen in plasma
Low levels, termed TT clotting time prolonged
hypofibrinogenemia, can be – HEPARIN
inherited but generally are due – Direct thrombin inhibitors
to acquired causes such as – Hypofibrinogenemia
DIC, liver disease, or – Dysfibrinogenemia
fibrinolytic therapy
– Elevated fibrin split products
High levels are seen in
inflammatory states since Time for clot formation
~ 15 seconds
fibrinogen is an acute phase
reactant Incubate at 37 oC for ~2 minutes
0.2 ml Diluted Thrombin
0.2 ml Plasma
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46. Quantitative Fibrinogen
Calibrator plasma is serially diluted
Time for clot formation (1:5, 1:10, 1:20, and 1:40) to establish
~ 5-14 seconds a reference curve (see graph below)
Patient plasma is diluted 10 fold (1:10)
in buffer
Incubate at 37 oC for ~3 minutes
0.1 ml Thrombin
0.2 ml Diluted Plasma
100
Patient clotting time in seconds is
Clotting Time-Seconds
35.0
read against the reference curve
18.3
− Patient clotting time of 8.0 9.5
seconds equates to 281 mg/dl 8.0
5.1 Patient
fibrinogen in example at right
Fibrinogen concentration is inversely
1
proportional to clotting time 1
10
10 58 115
100 230 281 460 1000
mg/dl Fibrinogen
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48. Inhibitors
Naturally occurring inhibitors
– Protein C (activated) and Protein S
• Inhibit coagulation cofactors FVIIIa and FVa
– Antithrombin
• Inhibits FXIa, FIXa, FXa, FVIIa/TF, and thrombin (IIa)
Pathologic inhibitors
– Acquired or autoimmune antibodies to specific
coagulation factors
Pharmacologic inhibitors
– Heparin and Low Molecular Weight Heparin
– Warfarin
– Direct Thrombin Inhibitors
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52. Breakdown of Fibrin(ogen)
FPB
FPB FPA & B
FPA & B
Fibrinogen Fibrinogen
Fibrin
Thrombin Fibrin Monomer Clot
Fibrin Monomer
Fragment X
Fragment X
Fibrin Polymer
Fibrin Polymer
D
D Y
Y
FXIII FXIIIa
PLASMIN D
D E
E D
D D
D E
E D
D
D
D E
E D
D
D-dimer D
D D
D E
E D
D D
D E
E D
D
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53. Fibrinolytic Agents
All currently available thrombolytic agents are
plasminogen activators (PA)
– Convert patient plasminogen to plasmin which then acts
on fibrin within a thrombus
– Additionally can breakdown fibrinogen (fibrinogenolysis)
• Commonly referred to as the lytic state (systemic lysis)
• Therapeutic doses of PA overwhelm PAI-1 and α2-antiplasmin
Beneficial effect is reduction of thrombus size
(thrombolysis)
Negative effect is that hemostatic plugs are also lysed
Most commonly used agents are: Streptokinase (SK),
Alteplase (tPA), Reteplase, and Tenecteplase (TNK-tPA)
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55. Time Frame for Hemostasis
Platelets Coagulation Factors Fibrinolytic Proteins
Primary
Primary Secondary
Secondary Fibrinolysis
Fibrinolysis
Hemostasis
Hemostasis Hemostasis
Hemostasis
•• Vessel constriction
Vessel constriction •• Activation of
Activation of •• Activation of
Activation of
occurs immediately
occurs immediately coagulation
coagulation fibrinolytic
fibrinolytic
factors occurs in
factors occurs in proteins happens
proteins happens
•• Platelet adhesion
Platelet adhesion seconds
seconds immediately
immediately
occurs in seconds
occurs in seconds
•• Fibrin forms in
Fibrin forms in •• Dissolving the
Dissolving the
•• Platelet aggregation
Platelet aggregation minutes
minutes thrombus
thrombus
takes minutes
takes minutes requires hours
requires hours
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56. Bleeding: Balance is Disrupted
Presence of Inhibitors*
•Pharmacologic (warfarin, heparin)
•Allo or Auto-antibodies to Factors
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57. Thrombosis: Balance is Disrupted
Inhibitors*
• Activated Protein C
• Protein S
• Antithrombin
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58. Conclusion
Primary hemostasis, a platelet-dependent process, forms
hemostatic plugs when a vessel is injured
Secondary hemostasis, a coagulation factor-dependent
process, begins with Tissue Factor exposure
– Small amounts of thrombin are generated via FXa
formation by the TF:FVIIa complex (“Extrinsic Pathway”)
– Sustained thrombin generation depends on FXa
formation via FIXa and FVIIIa-mediated complexes on an
activated platelet surface
– Amount of thrombin generated dictates bleeding or
thrombotic risk
The clinical history is the best “test” for hemostasis
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Crowther MA, et al. Practical aspects of anticoagulant therapy (Chapter 89). In: Colman RW, ed. Hemostasis and
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