Haemostasis-1 Dr. István Léránt

Sept 7, 2015

Department of Medical Biochemistry Semmelweis University

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Haemostasis

• Physiological process which causes bleeding to stop

• Vasoconstriction

• platelet plug

• formation of a clot

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Haemostasis

• Endothelium

• Blood coagulation

• Platelets

• Liver

• Fibrinolysis

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Clot formation Fibronolysis

Haemostasis

Disorders

• Hemophilia

• formation of unwanted blood clots

• Embolism

• heart attack

• pulmonary

• embolism

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Haemostasis

Disorders

• Atherosclerosis

• Diabetes mellitus

• Cancer

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Mortality in Hungary 2010 [%]

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search

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8 4 7

Circulatory system

Cancer

Digestive system

Respiratory system

Others

FG

sol

FN gel

D

E

D

FDP

Water insoluble

Ext. Int.

VIIa - TF XII

CLOTFORMATION

Plasminogen-

activators

FIBRINOLYSIS

CLOT FORMATION & FIBRINOLYSIS

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FIBRINOGEN-STRUCTURE - POLYMERISATION of FIBRIN

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MM: 340.000 Da

FIBRIN/OGEN - POLYMERSATION

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Thrombin OLIGOMER

Monomer

Polymer

FPA, FPB

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Factor XIII

Laki-Lóránd Factor

Fibrinoligase

Plasma Transglutaminase Fibrin stabilizing factor

E.C. 2.3.2.13)

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Factor XIII

Thrombin Mr = 4000 Peptide N-terminal

Arg36-Gly37 von A subunit.

Ca2+- dependent Dissociation of B subunits.

Maximal Activity Kd= 10-3 - Ca2+

Kd= 10-8 - Fibrinogen

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Carrier-subunits

Factor XIIIa Activity

Thrombin

• Hepatocytes

• Precursor : Prothrombin in Blood

• Protease Activity

• Signal functions

• Serin-protease

• Fibrinogen = Fibrin + Fibrinopeptids

• Substrat

– Factor V + feed back

– Factor VII

– Factor VIII + feed back

– Factor XI + feed back

– Factor XIII

• Thrombin + Thrombomodulin

• Protein Ca >>> Factor Va Factor VIIIa

- feed back

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Thrombin

• Hepatocytes

• Precursor : Prothrombin in Blood

• Protease Activity

• Signal functions

• Signal

• Protease-activated Rezeptors (PAR) – Platelets

• Plateletactivation

– Smooth muscle • Proliferation

• Migrantion

– Atherosclerosis

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Protrombin

• Synthesis:

– Liver

– Vitamin K dependent

– Gla-domain

– Kringle-domain

– Catalytic-domain

• Serin-protease

• catalytic triad

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PROTHROMBIN - TRHOMBIN

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N Arg273 Thr 274 Arg323 Ser324 C

S S

N Arg Thr Arg

AC Ser C

S

S

Fragment 1-2

Gla

Gla

Factor Xa

Thrombin

Thrombin

• Fg – Fn

• Factor XI – XIa

• Factor V – Va

• Factor VIII – VIIIa

• Factor XIII - XIIIa

• Platelet activation

• TM-PC

• Vasoconstriction

• Mitogen

• Cerebral Aneurysm – Thrombin – Stroke

• Thrombin-”cleavage site”

– Leu-Val-Pro-Arg-Gly-Ser

– Recombinant Fusion Protein

– „Purification tag”

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Eastern Brown Snake

(Pseudonaja textilis)

• Neurotoxin

• Prothrombin Activator

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MELILOTUS ALBA MEDICUS

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N

VITAMIN K & DICOUMAROL

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g-carboxylation

g-carboxylation of coagulation factors

CH2

CH2

C

O O

CH2

CH

C

O O

COO-

CO2

VITAMIN K(HYDROQUINONE)

Glutamate-seiteinkette

g-Karboxy-glutamate-seiteinkette

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Factor II, VII, IX, X, PROTEIN C, S, M, Z…

VITAMIN K CYCLUS

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Coagulation factors

g-Carboxylated

coagulation

factors

Vit-KH2

Vit-K-2,3-Epoxyd

Vit-K

Vitamin K

Reductase

Vitamin K

Epoxyd-

reductase

g-Glutamyl

carboxylase

N

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Purified Components Relative Rate of

Thrombin Generation

(1) Prothrombin, Xa, Ca++ 1 (35 days)

(2) Prothrombin, Xa, PL, Ca++ 50 (17 hours)

(3) Prothrombin, Xa, Va, Ca++ 350 (2.4 hours)

(4) Prothrombin*, Xa*, Va, PL, Ca++ <1000 (>50 min)

(5) Prothrombin, Xa, Va, PL, Ca++ 19,000 (2.5 min)

(6) Prothrombin, Xa, Va, platelets, Ca++ 300,000 (10 sec)

Prothrombin actication

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Activation of coagulation factors

ACTIVATION OF PROTHROMBIN

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Factor VII

– ‘proconvertin’

– serum prothrombin conversion accelerator

(SPCA)

– stabiler Factor,

– Prothrombinogen

• Serine Protease

• Hepatocytes

• Vitamin K dependent

• Gla-Domain

• Tissue Factor

• MM 59000 Da

• ß-Globulin

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Signalpeptide

Propeptide

Gla-domains

EGF-domains

VII-VIIa

Factor VII

• Activation of blood coagulation

• Damage of vascular endothelium

• Thromboplastin - Blut + Ca2+

• + Factor VII – Factor VIIa

• Factor VIIa + PL + Ca2+

• Factor X – Factor Xa

• Factor IX – Factor IXa

• Extrinsic pathway

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VIIa Xa T

VIIGla ProTGla XGla TF

IXGla

IXa

XIa

XI

XIIa

FN

FG

In vitro

Tissue factor pathway inhibitor (TFPI)

• MM 34000-40000

• Structure

– N-Terminal – negative

charges

– Kunitz-Typ Domain

– C-Terminal + charges

– Bovine pancreas trypsin

inhibitor Homology

• TFPI + Factor Xa

• TFPI-Factor Xa Complex –

Factor VIIa

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Tissue factor pathway inhibitor (TFPI)

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VIIa Xa T

VIIGla ProTGla XGla TF

IXGla

IXa

XIa

XI

XIIa

FN

FG

TFPI ?

Xa

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Intrinsic pathway

Intrinsic way(Kontakt Aktivation)

• F XII (Hagemann Factor)

• Prekallikrein (Fletcher Factor)

• F XI (plasma thromboplastin antecedent)

• HMWK (Fritzgerald Factor)

• C1-INH

• Limited Proteolysis – -Ser-OH

– CoFactor

– Inhibitor

• Factordeficiency: – FXII

– Precallikrein

– HMWK

– F XI Bleeding

• C1-INH – Hereditary angioedema

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Factor XII

• Hageman-Factor

• 15-45 mg/l – Serinprotease

– β-Globuline

– Monomer / Bloodplasma

– MM 80 kDa.

• Hepatocytes

• Cleavage of Propeptid / Zymogen (Plasma)

• Negatively charged surface

• Collagen,

• cell fragments,

• bacterial Endotoxins

• in vitro: Glass, Kaolin,

• preactivation. – Prekallikrein - Kallikrein.

– HMWK (Fitzgerald-Factor) - Kinin

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Factor XI

• Factor XI

– Plasma Thromboplasmin Antecedent (PTA)

– Rosenthal-Factor,

• Serinproteinase.

• Liver

• Homo-Dimer (Disulfidbridges)

• MM 80 kDa.

• [Plasma] 5 mg/l

• Half life~52 hours

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Factor XI

• Factor XI is activated by

– Factor XIIa

– Thrombin activated Factor XIa

– Intrinsic Pathway

• Factor XIa

– Factor IX - Factor IXa

– Cleavage of Arg-Ala- und eine Arg-Val-Peptide bonds.

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Factor XI

• Hemofilia C

– 8 % Ashkenazi Jews

– spontaneous Bleedings,

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Bleeding tendency

F-IX F-VII

F-XIa F-XI

F-XII

K PK

F-XIIa

surface

HMWK

Factor XII

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Bleeding tendency

F-IX F-VII

F-XIa F-XI

F-XII

K PK

F-XIIa

Complement

Plasminogen

Fibrinolyse

Prorenin

Angiotensin Kininogen

Bradykinin

Plasminogen Activators

surface

HMWK

Factor XIIa

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Factor IX

• Factor IX – Christmas-Factor

• ß-Globulin

• Liver

• Factor IXa – Serinprotease

• Factor IX-Gen – X Chromosome

N

Gla EGF Katalytische Dom. EGF

TF Bindung?

Platelet binding

Tenase-Complex-Binding

Activation by

Factor XIa

Factor VIIa

Factor IX

• Factor IXa

• Substrat: Factor X

– Ca2+

– PL

– Factor VIIIa

• Hydrolyses of an Arg-Ile bond in Factor X

N

Gla EGF Katalytische Dom. EGF

TF Binding?

Platelet aggregation

Tenase-Complex-Binding

Aktivation:

Factor XIa

Factor VIIa

Factor IX

• Factor IX-Gen

– X Chromosome

• Deficiency of Factor IX – Hemophilia B

• Acquired deficiency of Factor IX

– Vit. K deficiency

– Lupus • Antibodies / Factor IX

Factor VIII

• Factor VIII • anti-hemophilic factor (AHF)

• Glycoprotein

• Synthesis – Liver / Sinusoids

– Vaskulare Endothelium

• Degradation: – Liver

– Kidneys

Factor VIII

• Factor IXa

• Substrat: Factor X

– Ca2+

– PL

– Factor VIIIa

• Hydrolysis of an Arg-Ile bondi in Factor X

Factor X

• Thrombokinase

• Stuart-Prower-Factor

• Vitamin K

• Liver

• Alpha-Globuline

• Factor Xa

• Serin protease

Factor X

• Activation by – Factor VIIa + TF

– Factor IXa – Complex

• Substrate – Prothrombin

• Arg-Thr

• Arg-Ile

• Inactivation of Factor Xa – Prot Z dep Protease Inhibitor

– Deficiency of Prot Z

Thrombin

• Fg – Fn

• Factor XI – XIa

• Factor V – Va

• Factor VIII – VIIIa

• Factor XIII - XIIIa

• Platelet activation

• TM-PC

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Feed back mechanisms

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XIIa

XI XIa

IXGla IXa

XGla Xa

ProTGla

VIIGla

VIIa

V

VIII

T

TF

Va

VIIIa

THROMBIN

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FIBRINOGEN

Factor XIII

Platelets

Cofactor V

Cofactor VIII

Factor XI

THROMBIN

PROTEIN C

Thrombomodulin

• Thrombin-Bindins

• T+TM – „Inactivation” of Thrombin /

– Activation of Protein C/Protein S

– Negativ feed back reationts

• TAFI

– Inhibition of fibrinolysis

THROMBIN

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FIBRINOGEN

Factor XIII

Platelets

Cofactor V

Cofactor VIII

Factor XI

THROMBINF

PROTEIN C

ENDOTHELIAL CELLS THROMBOMODULIN

THROMBINB Cofactor Va

Cofactor VIIIa

a

PROTEIN C AKTIVATION

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Zeit

[aPC] TH+TM

TH

PROTEIN C SYSTEME

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TM

THROMBINfree THROMBINbound

V VIII

Va VIIIa

Vi VIIIi

Protein C Protein Ca

?

aPC RESISTENCE

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TM

THROMBINfree THROMBINbound

V VIII

Va VIIIa

Vi VIIIi

Protein C Protein Ca

?

Cofactor V – Arg 506 / Gln

Prevalence of APC-Resistence ~ 5 %

Risk for venous Thrombosis