examination of coagulation and fibrinolysis
DESCRIPTION
EXAMINATION OF COAGULATION AND FIBRINOLYSIS. MUDr. Pavel Maruna Dept. of Pathological Physiology 1st Faculty of Medicine. I. Physiology. Hemostasis. = The physiologic process protecting the integrity of the vascular system after tissue injury. Bleeding is halted to minimize blood loss. - PowerPoint PPT PresentationTRANSCRIPT
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EXAMINATIONOF COAGULATION AND FIBRINOLYSIS
MUDr. Pavel MarunaDept. of Pathological Physiology1st Faculty of Medicine
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I.Physiology
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Hemostasis= The physiologic process protecting the integrity of the vascular system after tissue injury.Bleeding is halted to minimize blood loss.
The hemostatic mechanisms include following steps:
1. Rest phase - To maintain blood in a fluid state while circulating within the vascular system
2. After injury - To arrest bleeding at the site of injury by formation of hemostatic plug
3. Restitution - To ensure the removal of the hemostatic plug when healing is complete
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Hemostasis
Hemostasis is an integral part of
• stress reaction• inflammatory response
Protective role Pathophysiol. roleX
non-specific defense mechanism
thrombosis / embolism
atherosclerosis
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Hemostasis as a physiological process must be:
1. Rapid
2. Localized
3. Reversible
Inappropriate hemostasis:
- Thrombosis / embolism
- DIC
- bleeding / blood loss
Hemostasis
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Hemostasis
Plasmacoagulation
system
Vessel wall
Platelets
Endothelium
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Antithrombotic Properties
Anti-platelet activities:
– Covers highly thrombogenic basement membrane
– Uninjured endothelium does not bind platelets
– PGI2 and NO from endothelium inhibit platelet binding
– ADPase counters the platelet aggregating effects of
ADP
Endothelium
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Anticoagulant activities:
– Heparin-like molecules ... activate anti-thrombin III
(inactivates active proteases)
– Thrombomodulin ... changes specificity of
thrombin (activates protein C , which inactivates factors
Va and VIIIa
– tPA ... activates fibrinolysis via plasminogen to plasmin
Antithrombotic Properties
Endothelium
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• Synthesis of von Willebrand factor
• Release of tissue factor
• Production of PAI (plasminogen activator inhibitors)
• Membrane phospholipids bind and facilitate activation of clotting factors via Ca bridges
Endothelium
Prothrombotic Properties
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Antithrombogenic Thrombogenic
Vessel injury
(Favors fluid blood) (Favors clotting)
Endothelium
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Vasoconstriction
Primary hemostasis
Secondary hemostasis
Fibrinolysis
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Fibrinogen FibrinThrombin
Prothrombin
XaVa
VIIa
TF
Extrinsic Pathway
IXa
VIIIa
XIa
XIIa
Intrinsic pathway
XIIIa
Soft clot
Fibrin
Hard clot
V
VIII
Coagulation
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• Enzymatic cascade (amplification)
• Several serine proteases
• Produced by liver (most)
• Require vitamin K (several)
• 3 protein cofactors (not enzymes)
• Requires Ca 2+
• Reversible (via production of plasmin)
Coagulation
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Coagulation
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Coagulation
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Fibrinogen Fibrin
Thrombin
Coagulation
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Fibrinogen Fibrin
Thrombin
Prothrombin
XaVa
Coagulation
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Fibrinogen Fibrin
Thrombin
Prothrombin
XaVa
VIIa
TF
Coagulation
Extrinsic Pathway
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Fibrinogen Fibrin
Thrombin
Prothrombin
XaVa
VIIa
TF
IXa
VIIIa
XIa
XIIa
Extrinsic Pathway
Intrinsic pathway
Coagulation
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Fibrinogen FibrinThrombin
Prothrombin
XaVa
VIIa
TF
IXa
VIIIa
XIa
XIIa
XIIIa
Soft clot
Fibrin
Hard clot
Extrinsic Pathway
Intrinsic pathway
Coagulation
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Fibrinogen FibrinThrombin
Prothrombin
XaVa
VIIa
TF
IXa
VIIIa
XIa
XIIa
XIIIa
Soft clot
Fibrin
Hard clot
V
VIII
Extrinsic Pathway
Intrinsic pathway
Coagulation
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Thrombin (IIa)
Prothrombin (II)
Xa
VIIa
TF
IXa
Revised tissue factor pathway
IX
New: Production of IXaInteraction of intrinsic and extrinsic pathways
Extrinsic Pathway
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Thrombin (IIa)
Prothrombin (II)
Xa
VIIa
TF
IXa
New: TFPI = Tissue Factor Pathway Inhibitor... inhibition of Xa and VIIa
IX
TFITFPI
Revised tissue factor pathway
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Prothrombin (II)
Xa
VIIa
TF
IXa
New: TFPI = Tissue Factor Pathway Inhibitor... inhibition of Xa and VIIa
IX
TFITFPI
Revised tissue factor pathway
Kunitz-type protease inhibitor (kringles)34 and 41 kD forms in plasma (C-term truncation)
Activities:- direct inhibition of Xa- inhibition VIIa-TF complex in a [Xa]-dependent manner- bounding to LDL, HDL and Lp (a)
~10% present in platelets (endothelium also)
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Production of IXa
Production of small amounts of thrombin (IIa)
Net results:
No or only little fibrin formed!
Revised tissue factor pathway
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• VIIa forms via binding of VII to TF• VIIa activates some XXa• Xa converts a small amount of II to IIa; this thrombin is
used to produce small amts of VIIIa and Va• As the concentration of TF-VIIa-Xa-IIa increases, TFPI
inactivates this complex stopping further production of thrombin.
• IXa, with VIIIa (produced as above), produces Xa; this Xa with Va produces new thrombin; this thrombin produces more VIIIa and Va and now we get lots of thrombin and fibrin.
Revised tissue factor pathway
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Fibrinogen FibrinThrombin
Prothrombin
XaVa
VIIa
TF
IXa
VIIIa
XIIIa
Soft clot
Fibrin
Hard clot
V
VIII
IX
Revised tissue factor pathway
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Thrombin (IIa)
VIII
VIIIa
V
Va
Revised tissue factor pathway
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Role of vitamin K
Factors II, VII, IX, X, proteins C and Srequire a post-translationalmodification before their activation
This PTM requires vitamin K
This PTM involves the addition of a COO- to certain Glu residues in the clotting factors
resulting in the formation of several g-carboxy glutamates
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Role of vitamin K
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Physiologic inhibitors of coagulation
• Antithrombin III – SERPIN
• Activated Protein C + protein S– Inactivates Va and VIIIa (via proteolysis)– mutation: Factor V Leiden (APC resistance)
• Thrombomodulin– Binds to thrombin– Decreases ability to produce fibrin– Increases ability to activate Protein C
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• Vitamin K antagonists(in vivo only)
• Ca chelators(in vitro only)– EDTA– Citrate– Oxalate
• Heparin
(in vivo and in vitro)
Non-physiologic inhibitors of coagulation
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Fibrinolysis
... Clot removal
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Fibrin Fibrin Split Products (FSP)Plasmin
Fibrinolysis
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Fibrin Fibrin Split Products (FSP)Plasmin
Plasminogen
tPA
Fibrinolysis
uPAbacterial enzymes
(streptokinase)
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Fibrin Fibrin Split Products (FSP)Plasmin
Plasminogen
tPA
Fibrinolysis
Inhibitors of fibrinolysis
PAI
a2-antiplasmin
... SERPINs
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Fibrinolysis
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II.Pathology
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Coagulopathies
AcquiredCongenital
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Coagulopathies
AcquiredCongenital
Hemophilia A ... f VIIIHemophilia B ... f IXHemophilia C ... f XIDys- / A- fibrinogenemiaF V defic. (parahemophilia)F XIII defic.APC resistance
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Coagulopathies
AcquiredCongenital
Liver proteosynthesisVitamin K defic.
- obstructive icterus- intestin. resorption
Anticoagulant therapy- Dicumarol- Heparin
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Vasculopathies
AcquiredCongenital
Purpura Henoch-SchönleinScorbutSteroid purpuraPurpura simplex and senilis
Mb. Rendu-Osler-Weber= hereditary hemorrhagic teleangiectasiaAD, TGFbeta1 rec.
Ehlers-Danlos Sy.= defects in collagen synthesis
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Genetic examination
Hemophilia A X-linked recessive
1 : 10 000
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Clinical signs
Hemophilia Large hemorrhage after a small injury
Arthral hemorrhage
Secondary arthropathy
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Clinical signs
Thrombocytopenia Petechiae, pigmentation
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Clinical signs
Henoch-Schonlein
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Clinical signs
F XIII deficiency Late bleeding
Keloid scarring
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Clinical signs
Deep venous thrombosis
Pulmonary embolism
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III.Diagnostics and
monitoring
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Prothrombin Time (Quick test)
Principle: Stimulation of extrinsic (main) coag. system
Citrate plasma ... add TF (in excesive amount) + CaCl2 ... fibrin fibre
Normal: PT = 12 - 15 sINR = (PTP / PTN)ISI
ISI = international index of sensitivity of used thromboplastin (commonly > 1)
Prolongation: defic. vit. K dep. FII, VII, X, FbgUsage: screening, monitoring of oral anticoagulants,
liver proteosynthesis
Normal range INR 0,8 - 1,2Therapeutic range INR = 2,5 - 4,5Surgery INR < 1,6
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APTT, Activated partial thromboplastin time
Principle: Stimulation of intrinsic (contact) way of coag. system
Citrate plasma ... add contact activator (e. g. kaolin) + CaCl2 ... fibrin fibre
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APTT, Activated partial thromboplastin time
Principle: Stimulation of intrinsic (contact) way of coag. system
Citrate plasma ... add contact activator (e. g. kaolin) + CaCl2 ... fibrin fibre
Normal: APTT = 27 - 35 s
Prolongation: defic. of VII, V, X, XII, VIII, XI, IX (hemophilia A,B,C), Fbg, FDP
Shortening: prothrombotic statusUsage: screening, diagnostics of coagul. deficits,
monitoring of heparin therapy
Therapeutic range 1,2 - 2,5 x
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Lee-White test
Cloting time of whole blood
Whole blood without anticoagulants (CaCl2) ... polystyrene or glass tube, 37°C ... spontaneous stimulation of intrinsic
Normal: 4 - 10 min.
Usage: Basic, rough orientation in acute status
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Thrombin Time
Whole blood without anticoagulants (CaCl2) ... add thrombin in standard amount, 37°C ... fibrin fibre
Normal: 12 - 14 s
Prolongation: Fbg (acute stage of DIC)antithrombinsfibrinolysis
Usage: DICmonitoring of fibrinolytic therapy
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Fibrinogen, Fbg
Normal plasma levels = 2 - 4 g /lFunctional of immunological detection
High: InflammationDMSmoking
Low: Low synthesis (congenital or liver function)Consumption (DIC)
HypofibrinogenemiaDysfibrinogenemia
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FDP
Total degradation products of fibrin(-ogen)
ELISA or aglutination semiquantitative methods
High: Recent coagulation activity(thrombo/embolism, bleeding, surgery, DIC ...)
High senzitivity, low specificity
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Paracoagulation tests (Ethanol, Protamin)
Principle: Ethanol catalyzes conversion of fibrin monomers + PDP fibrin polymers
Low senzitivity and specificity
Usage: 1st stage of DIC
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Duke test
Duke, 1910Estimation of bleeding timeTime of spontaneous cutoff of bleeding after
standard puncture to auricle of ear
Limits: 2 - 5 min., or 4 - 8 min. (depends od methods)
Prolongation - Disturbance of primary hemostasis:Plt < 20 000 or Plt dysfunction, vW
disease
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Rumpel - Leede test
Capillary resistance
Number of petechia on forearm (area 4 x 4 cm) after a standard pressure (ruff 10,5 kPa for 10 min.) or after underpressure (Brown, 1949)
Limits: > 5 petechia ... higher capillary fragility(e.g. hereditary purpura Weber-Rendu-Osler)
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Presumable results
Diagnosis Plt Duke APTT Quick TT
Thrombocytopenia N N N
Hemophilia A N N N N
Hemophilia B N N N N
Hemophilia C N N N N
vWd N N / N N
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Presumable results
Diagnosis Plt Duke APTT Quick TT
F V defic. N N N
F II defic. N N N N
F VII defic. N N N N
Warfarin / vit. K def. N N N
Heparin i. v. N N / N /
Heparin s. c. N N N N N
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Presumable results
Diagnosis Plt Ethan APTT Quick TT
DIC 1st stage + N
DIC 2nd stage -
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Quick time, INR 0,8 - 1,2
APTT 27-35 s
Thrombin time 12 - 14 s
Fibrinogen 2 - 4 g/l
Antithrombin III > 70%
Ethanol test neg.
D-dimers (FDP) neg.
Quick time, INR 0,8 - 1,2
APTT 27-35 s
Thrombin time 12 - 14 s
Fibrinogen 2 - 4 g/l
Antithrombin III > 70%
Ethanol test neg.
D-dimers (FDP) neg.
Standard tests in General Faculty Hospital