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Haemostasis-3 Dr. Léránt István
Sept. 14, 2015.
Department of Medical Biochemistry Semmelweis University
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Fibrinolysis
• Dissolution of fibrin network
– Plasmin
• Fibrinpolymer FDP (Fibrin degradation produts)
• Dissolution of thrombus.
5
FG Water soluble
FN Water insoluble
D
E
D
FDP
Water insoluble
Ext. Int.
VIIa - TF XII
Blood coagulation
Plasminogen activators
Fibrinolysis
Fibrinolysis
Fibrinolysis
6
Plasminogen
Plasmin
Fibrin Fibrin degradation
products
t-PA
urokinase
PAI-1 -2
Thrombin TAFI
Factor XIIa, XIa,
Kallikrein
a2-antiplasmin
a2-macroglobulin
Streptokinase
11
Formation of blood clots
Thrombin
Factor XIII Factor XIIIa
Inh.
Inh.
FDP
Time
F
FxxF
FxxFxxInh
Plasmin
Inh.
Plasmin/ogen
• Liver
• Plasminogen - Plasmin
• t1/2 2,2 days
• [Plasminogen] 0,2 g/l
• Serinprotease
• Degradation of fibrin
• Kollagenase
• Mediators of complement
system
• ovulation
• fibronection
• Thombospondin
• Laminine
• Von-Willendbrand factor
• Monocytes
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Plasmin/ogen
• Activation
– t-PA
– Urokinase - uPA
– Streptokinase
– Fibrin
– Factor XII
• Inaktivation
– .a2-antiplasmin
– . a2-makroglobulin
– Aprotinin
– . eACA
– p-Aminomethylbensoic acid
• [plasmin]
– Thrombosis
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Glu-PLAsMINOGeN
K1
K2
K3
K4
K5
Lys77 Glu1
Arg561
S
S
Plasmin
Plasminogen Activator
Glu-PLASMIN
Lys-PLASMINOGEN
MINI-PLASMINOGEN
des-KRINGLE 1-4
ELASTASE
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Activation of plasminogen
Pln
PA
Glu-Plg
Proteolysis
Lys-Plg
Pln
PA
Conformational change
Time
Plasmin
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PLASMIN
• Primer function: – Fibrinonlysis
• t-PA – tissue Plasminogen Activator
• u-PA – urokinase
– Inhibitors • plasminogen activator inhibitor
(PAI)-1
• plasminogéen activator inhibitor PAI-2.
– The cell receptors of u-PA • Transmembran proteins,
• Signal transduction – nucleus
• Gene expression
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PLASMIN
• Activation of plasmin – Cell movement
– repair
– metastasis
• t-PA – Cognitive memory
– neurodegeneration
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Tissue-type plasminogen activator, t-PA
• Intrinzic activator of fibrinolysis
• serinprotease
• 527 amino acids
• Thrombolysis – Alteplase
– (rt-PA)
– Recombinant variants
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t-PA
Synthesis
Liver
Tumorcells
Active
cofactors
FDP
Myosin
Aktin
EC Membran
Polylysine
Denatured proteins
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Urokinase-Typ Plasminogen Aktivator (uPA)
• Peptidase activity • Treatment – myocardial infarction
• Pulmonary embolims
• Urine - isolation
• Plasminogen – plasmin – conversion in circulation
• Inhibition of urokinase by PAI-1, PAI-2
• Cancer – Level of urokinase and PAI-1 is significantly elevated
– Marker - prognosis
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Single-chain urokinase
scu-PA
Synthesis
EC
Tumorcells
K
K
S S
Pn
K
S
S
Two-chain urokinase
tcu-PA
UROKINASE
AC
Lys158-Ile159
Fibrinolysis – feed back reactions
Urokinase
(scuPA)
Endothelcells
Tumorcells
Urokinase
(tcuPA)
Plasminogen
Plasmin
Plazmin
+ feed back
Endothelcells
Tumorcells
tPA
(sctPA)
t-PA
(tctPA)
Plazmin
?
Ineffective
+ Fibrin, Miosine, Actin, denatured proteins
Effective activation of plasminogen Endothelcells
Surface
tPA+ Plg
Fibrinolysis
• Initiation:
– t-PA – Plasminogen-Plasmin
• Amplification
– Prourokinase – uPA – Plasminogen-Plasmin
• PLASMIN
• Dissolution of Fibrinogen
– t-PA: cofactor – is dissolved
• Plasmin + inhibitors
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Inhibitors of fibrinolysis activators
• Inhibitors
• Plasminogenactivator inhibitors – PAI-1
– PAI-2
– PAI-3
– PAI-4
• PAI-1 – „main” inhibtoros
• Inhibition of t-PA and urokinase
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Inhibitors of fibrinolysis activators
• Activation of trombocytes
• PAI-1 release
• Thrombus - increase in level of PAI-1
• Enhancement of stability of thrombus
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PLAsMINOGeN-AcTIVaTOR-INHIBITOR-1 (PAI-1)
• SERPIN family
– MM.: 52.000 Da
– glycoprotein
– 379 Amino acids
• Inhibition of t-PA-t & tcu-
PA-t
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PLAsMINOGeN-AcTIVaTOR-INHIBITOR-1 (PAI-1)
• Secretion – hormons
– Cytokines
• 3 forms • Aktive
• Inaktive
• Latent
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PLAsMINOGeN-AcTIVaTOR-INHIBITOR-1 (PAI-1)
• SZINTesis
• Endothel cells
• megakariocytes
• adipocytes
• Thrombocytes • 80 % of the blood PAI-1
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PLAsMINOGeN-AcTIVaTOR-INHIBITOR-1 (PAI-1)
• [PAI-1]
– In obesity
– DM
– Atherosklerosis
• Risk of thrombus formation
• Embolism
• Infarktion
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a2-PLASMIN INHIBITOR
• Liver
– MM.:70000 Glycoprotein
– 13 % carbohydrates
– 452 Aminoacids.
• Plasmaconcentration 1M
• Inactivation of Plasmin
• k= 400*104M-1sec-1
• Inactivation of Plasmin
• (Fibrin)
– k= 1*104M-1sec-1 Plazmin
a2-antiplasmin
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a2-PLAZMIN INHIBITOR
• Complex of Plasmin- a2-
PLAsMIN INHIBITOR
• Inhibiton of adsorption
of plasminogen on
fibrin
• In Fibrinnetwork:
FxxFxxaAP
Plasmin
a2-antiplasmin
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a2-PLAZMIN INHIBITOR
• a2-Plazmin Inhibitor
– C-terminale Lys452
– reversible
– Plasmin LBS-1 Domain
• Inhibitors
– Lysin
– eACA
• Ser-OH
• Acyl-enzyme intermedier
Plasmin
a2 - Antiplasmin
452
Lys
SerOH
Arg354-Met355
eACA
Fibrinolysis & aPC + TM
Trombin
Trombin+TM Trombinként
inaktiív
Trombin+TM
Protein C/Ca
Faktor Va
Faktor VIIIa
TAFI Inhibition of
clot dissolution
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Fibrinolysis & aPC + TM
FIBRINOGEN
FAKTOR XIII
PLATELETS
COFACTOR V
COFACTOR VIII
FACTOR XI
THROMBINF
PROTEIN C
ENDOTHELSEJTEK THROMBOMODULIN
THROMBINB
COFAKTOR Va
COFAKTOR VIIIa
a
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TAFI
Thrombin activated fibrinolysis
inhibitor
Fibrinolysis & aPC + TM
a2 – Macroglobulin
• 3 M
• liver
• 4 subunits
• -S-S- bridges
• Protease –
• „Bait” regio
• Protease+a2M
• makrofag receptors
47
Antitrombin/heparin ( 4 M)
• Fibrinolysis Faktoren
• Fibrinolysis
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