hemostasis reviewer

8/12/2019 Hemostasis Reviewer

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4. factor X bind to its cofactor factor V it converts to prothrombin to

thrombin

*thrombin is an enzyme key for coagulation.

5. Thrombin splits into 2:

i. Fibrinogen- to produce fibrin

ii. Factor XIII- to cross-link and

stabilize the clot.

*activates platelet and feedback to activate

factors V, VIII, XI.

*Fibrin= necessary for control bleeding

*absence of single plasma procoagulant may

doom the individual to lifelong anatomic

hemorrhage.

Fibrinolysis:

Final event of hemostasis Plasminogen is bound to fibrin

during coagulation

o Activated by tissue plasminogen

activator (TPA) into the serine

protease.

o Plasmin slowly and

systematically degrades thefibrin degredation products

o Designed X, Y, D, E and D-

Dimer.

VASCULAR INTIMA IN HEMOSTASIS :

1. Endothelial cells Innermost lining of blood vessels

is a monolayer metabolic active Forms a smooth, unbroken

surface that promotes the fluid

passage of blood

It prevents turbulence

2. Smooth muscle cells in the arteries and arterioles

3. fibroblast occupy the connective tissue

layer and produce collagen.

4. Elastin-rich internal elastic lamina Supports endothelial cells.

ANTICOAGULANT PROPERTIES OF

INTACT VASCULAR INTIMA

Collagen promotes plateletactivation and adhesion

TF= promotes coagulation and

fibrin formation.

1. Prostacyclin Platelet inhibitor, is synthesized

through the eicosanoid pathway.

Prevents unnecessary orundesirable platelet activation in

intact vessels.

2. Nitric oxide


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Synthesized in endothelial cells,

vascular SMC, neutrophils and

macrophages

Counteracts vasoconstriction and

maintains healthy arterioles

3. Tissue factor pathway inhibitor

(TFPI)

Controls the TF or extrinsic

coagulation pathway.

4. Thrombomodulin Membrane protein that activates

protein C pathway.5. Heparin sulfate

Glycosaminoglycan the retards

coagulation by activating

antithrombin.

Coagulation regulatory protein.

PROCOAGULANT PROPERTIES OF

DAMAGED VASCULAR INTIMA

1. Smooth muscle cells in arterioles and

arteries

Induce vasoconstriction

2. Exposed subendothelial collagen Binds VWF and platelets

3. Damaged or activated endothelial

cells.

Secrete VWF Secrete adhesion molecules

a) P-selectin

b) Intra cellular adhesion molecules

(ICAMS)

c) Platelet endothelial adhesion

molecules (PECAMS)

d) Exposed smooth muscle cells and

fibroblast

Tissue factor exposed on cell

membranes.

e) Endothelial cells in inflammation Tissue factor is induced by

inflammation.

Fibrinolytic properties of vascular intima

During thrombus formation, both

TPA and plasminogen bind to

polymerized fibrin.

TPA activates fibrinolysis by

converting plasminogen to plasmin,

which slowly digest fibrin and

restores blood flow.

Endothelial cells may secrete

plasminogen activator Inhibitor -1

(PAI-1)

Thrombin bound to

thrombomodulin activates thrombin

activatable fibrinolysis inhibitor

(TAFI) w/ inc the tendency for

thrombus formation.

Platelet function:


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1. Adhesion:

-platelets roll and cling to nonplatelet

surfaces

2. Aggregation:

-platelet adhere to each other.

3. Secretion:

-platelets discharge the contents of

their granules

Characteristics:

- Irreversible, occurs during aggregation,

platelet contents are secreted, essential tocoagulation.

Platelet granules content:

1. Alpha- granules ( -granules) Large molecules B-thromboglobulin

Factor V Factor XI Protein S Fibrinogen VWF Platelet factor 4 Platelet-derived growth factor

2. Dense bodies Small molecules ADP (activates neighboring

platelets)

ATP Ca2+

Serotonin(vasoconstriction)

*Aspirin permanently inactivates

cyclooxygenase, blocking thromboxane A 2

production and causing impairment ofplatelet function (aspirin effect)

Role of rbc, wbc, TF-bearing cells in

hemostasis:

- Vessel injury exposes blood to the sub-

endothelial TF-bearing cells and leads toactivation of coagulation through factor

VIIa

RBC:

- Add bulk and structural integrity to the

fibrin clot.

Monocyte, Lymphocyte

- Provides surface borne TF that Triggers

coagulation.

WBC

- Have a series of membrane integrins and

selectins that bind adhesion molecule

and help stimulate the production of

inflammatory materials that promote

wound healing process.


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Coagulation system:

- 16 procoagulants also called

coagulation fa ctors or clotting factors.

- Synthesized in the liver o Few are made in monocyte,

endothelial cell and

megakaryocyte.

- 8 are enzymes that circulate in inactive

form called zymogens.

- In 1958 the International Committee for

the Standardization of the nomenclature

of the Blood clotting factors.

o Named by using roman numerals

*factor V used for the complexing the

procoagulant activation of factor VI.

*VonWillebrandFactor(VWF)synthesize

d in megakaryocyte and endothelial

cells .

Vitamin K-dependent Prothrombin

group:

- Prothrombin, factor VII, IX, X

- Protein C, S, Z o Structural resemblance to

prothrombin - All 7 proteins have 10-12 glutamic acid

- Vit-K is a quinone found in green leafy

vegetables, fish, liver.

o Produced by intestinal

organisms:

1. Bacteroides fragilis

2. Escherichia coli. (E-coli)

Posttranslational modification of the

prothrombin group:

- - carboxylation of A.A terminal

glutamic acid

- glutamic acid -carboxyglutamic acid

- 2nd carboxy grp added to gamma- carbon

- Negative charges w/ enables them to

bind ionic calcium (Ca 2+)

- Calcium permits Vit-K to bindnegatively charged phospholipids

*phospholipid binding is essential to

coagulation rection.

*Warfarin suppresses Epoxide reductase

In vitamin-K deficiency or intake of

warfarin:

1. Cant produce 2 nd carboxyl group to

bind gamma- carbon.

2. Called des --carboxyl proteins or

proteins in Vit-K antagonism

(PIVKAs)

VWF:Factor VIII complex:

- VWF is a multemric GP.

- Composed of multiple subunits 240000

D


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- Stored in alpha-granules in platelets and

storage sites c alled Weibel -Palade

Bodies in endothelial cells

- Conc 7-1- mcg/mL

VWF:Factor VIII complex circulates

covalently bound to VWF

- VWF provides 3 active receptor sites.

1. Platelet surface receptor (GP

Ib/IX/V)

2. Platelet integrin (GP IIb/IIIa) during

platelet aggregation3. Collagen binding site

4. Factor VIII

Factor VIII is produced by Hepatocytes and

other tissues.

Sex linked procoagulants

1. Factor VIII- 260000 D2. Factor IX- 57,000 D

Free factor VIII is unstable: except

1. During coagulation

2. Cannot be detected in plasma

*Factor VIII depend on VWF stability if

VWF have diminished VWF also

Diminished factor VIII activity levels.

Fibrinogen Structure and Fibrin

formation:

- Fibrinogen is the primary structure of

thrombin- Synthesized in liver

- Plasma conc of fibrinogen 200-400

mg/dL

o Highest concentration of all

plasma procoagulants

- Fibrinogen is mirror-image dimer.o Consisting of 3 non-identical

polypeptides

- Designated B and united by disulfide

bonds

- 6 terminal assemble to form bulky

central region called E domain

- Carboxy termini assemble at the 2 ends

of molecule to form 2 D domains.

- Thrombin cleaves fibrinopeptides A and

B

- Cleaved fibrinogen called fibrin

monomer

- Factor XIII catalyzes the formation of

cavalent bonds between carboxy termini

of -chains adjacent to D domains.

- - amino acid of lysine moieties and the-amide group of glutamine units.

- Fibronectin, a plasma protein involve in

cell adhesion.

- 2-antiplasmin, rendering the fibrin

mesh resistant to fibrinolysis.


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- Plasminogen, the primary serine

protease of fibrinolytic system.

Coagulation pathway:

Tissue Factor Pathway:

- TF is a membrane receptor for factor

VIIa.

- Coagulation is triggered by the exposure

of TF to plasma proteins on injury.

- Factor VIIa binds to TF in the presenceof phospholipid and calcium and triggers

the activation of both factor IX and

factor X.

- Factor IXa binds factor VIIIa on platelet

phospholipid surfaces

o Thus complex activates factor X

- Factor Xa, activated by both TF:VIIa

and IX:VIIIa, binds factor Va on

phospholipid surfaces

- Xa:Va complex activates prothrombino Multistep hydrolytic process that

release peptide fragment from

prothrombin called prothrombin

1 and 2

- Activated prothrombin is termed

thrombin, thrombin cleaves

fibrinopeptides A and B from plasma

fibrinogen.

- Tissue factor pathway is characterized

by three multimolecular complex

formation

First complex:

Composed TF, factor VIIa,

phospholipid and Ca 2+on the TF-

bearing cell complex IX and X.

Second Complex:

Factor IXa, Factor VIIIa,

phospholipid and Ca 2+ called

Tenase

Also activates factor X.

Third complex:

Composed of factor Xa, Factor Va,

phospholipid and Ca 2+

Often called prothrombinase

This complex convert prothrombin to

thrombin.

Contact factors

Composed of Factor XIIa,HMWK,

Pre-K, activates Factor XI.

Factor XIIa transform pre-K, a GP

the circulates bound to HMWK, into

its active form.

Kallikrein

- Cleaves HMWK to form bradykinin.


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Laboratory test for screening thes

factors:

*APTT

Extrinsic pathway;

- Order of reaction

1. Factor VII

2. Factor X

3. Factor V

4. Prothrombin 5. fibrinogen

initiation of coagulation by:

TF:VIIa

o the principal mechanism in vivo for

generating thrombin.

Test used for Extrinsic pathway:

- prothrombin time (PT)

*factor IXa:VIIIa complex is activation of

factor X (tenase)

Hemophilia A= deficient factor VIII

Hemophilia B= deficient factor IX

- can cause severe hemorrhage.

Common pathway:

- Both extrinsic and intrinsic have the

some common factors

1. Factor X

2. Factor V

3. Prothrombin

4. Fibrinogen

Cell-based (In Vivo) coagulation

- A spectacular combination of cellular

and biochemical function in harmony to

keep blood liquid within the veins and

arteries

3 phases :

1. Initiation:

- Continuous low-level activation of the

TF pathway occurs on TF-bearin cells

outside the circulation

- 1% or less of factor VII is present

normally in activated form.

1. Factor VIIa bind to TF

2. TF:VIIa complex activates small amount of

IX and X

3. Xa:Va complexes to form prothrombinase

and generates small amount of thrombin

4. Xa:Va bound to the cell is protected for

inactivation by control proteins

- If it dissolves from the cell, it is rapidly

inactivated by the protease inhibitors

TFPI, antithrombin and protein Z-

dependent protease inhibitor (ZPI)


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- Xa:Va in the initiation phase does not

produce fibrin clot.

2. Amplification:

- Starts when theres injury and bleeding

occur.

- VIII:VWF spill into extravascular space

coat platelets

- Partially activated by collagen and

thrombin

- Have higher levels of procoagulant

activity and platelet exposed to collagen

alone.

- In thrombin,o Factor V release from a-arginine o Factor VIII dissociates it from

VWF

o It activates XI.

3. Propagation

- Reaction occurs on the surface of the

activated platelet

- Factor IXa from initiation phase binds to

VIIIa on the platelet.

- For IX:VIIIa complex (tenase)o It activates Xa

- Factor Xa is complex to Va it will

activate prothrombin and generate a

burst of thrombin which cleaves

fibrinogen into fibrin clot.

- Thrombin activates also factor XIII to

stabilize clot

o It binds to thrombomodulin to

activate protein C control

pathway and activates TAFI to

inhibit fibrinolysis.

Coagulation Regulatory Mechanisms

- Serine proteases and cofactor in

coagulation system are regulated by

inhibitors, cofactor and feedback loops

to maintain complex and delicate

balance between thrombosis and

abnormal bleeding.- Principal regulator

1. TFPI

2. Antithrombin

3. Protein C pathway

- They prevent excessive clot formation or

thrombosis.

Tissue factor Pathway inhibitor

(TFPI)

- TFPI is synthesized by endothelial cells

- 80% bound to the vessel wall

- 20% circulates in the plasma

- 2% is the full-length free form that

effective inhibitor of tissue factor

pathway.

TFPI inhibit coagulation in 2 step process:

1. Inactivating Xa


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2. Binding TF:VIIa to prevent it from

inactivating additional Xa.

- TFPI provides feedback inhibitiono Its not functional until X is

activated.

Protein S

- Cofactor of activated protein C (APC)

- Also cofactor of TFPI

- Stimulates Xa inhibition by TFPI

- It synthesized in the liver.

- It circulates in the plasma in 2 forms:

1. Free Protein S2. Covalently bound to the complement

control protein C4b-binding protein

(C4bBP)

- Only free plasma protein S can serve

as APC cofactor.

- Protein S-C4bBP binding is particular

interest in inflammatory conditions

Protein C regulatory system

-thrombin binds to EC membrane protein

thrombomodulin and trigger an essential

coagulation regulatory system called Protein

C system.

-

thrombin-thrombomodulin complexactivates zymogen protein C to serine

protease.

- APC binds its cofactor protein S

o Stabilized APC-protein S

complex.

- The APC-protein S complex hydrolyzes

and inactivates factor Va and VIIIa

o It slowing or blocking

coagulation.

Thromboembolic disease:

- Chronic, acquired or inherited: o Protein C deficiency o Protein S deficiency

- Or mutations of Protein C and S and

Factor V

o It compromised downregulation of Va and VIIIa.

Serine protease inhibitors (serpins)

- Antithrombin the first of the

coagulation regulatory proteins

- The first to be assayed routinely in the

clinical hemostasis laboratory

Other members of the serpin family:

Heparin cofactor II

ZPI

Protein C inhibitor

A1-protease inhibitor (alpha-antitrypsin)

Alpha2-macroglobulin

Alpha2-antiplasmin

PAI-1

Antithrombin


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- It binds and neutralize all serine

proteases except factor VIIa.

- Neutralize and bind also the plasmin.

Heparin cofactor II

- Inactivates primarily thrombin

- Both antithrombin and heparin cofactor

II require heparin for effective

anticoagulant activity.

Unfractionated heparin:

- A heterogenous glycosaminoglycancomposed of 15-30 saccharide unit

- It increase the ability of antithrombin to

neutralize thrombin and factor IX, Xa,

Xia, and XIIa 1000-fold.

Firbrinolysis

- Final stage of coagulation

- Plasminogen, plasmin, TPA and PAI-1

are incorporated into the fibrin clot by

binding to lysine through kringle

loops

- Systematic, accelerating hydrolysis of

fibrin by bound plasmin cleaves peptide

bond at arginine and lysine moieties in

fibrins D and E domains.

- TPA and Urokinase activate fibrin-

bound plasminogen several hours after

thrombus formation.

Plasminogen and plasmin

- Plasminogen is a 92,000 D plasma

zymogen produced in liver

- Plasminogen is a single chain protein possessing five glycosylated loops

termed kringles.

- Kringles enables plasminogen to bind

fibrin lysine residue during

polymerization.

- Plasminogen binds plasma alpha2-

antiplasmin the major fibrinolysis

control protein.

- Fibrin-bound plasminogen converted

into 2-chain active plasmin.

o When cleaved between

arginine at position 561 and

valine position 562 by

neighboring bound TPA or

Urokinase

Plasmin:

- Serine protease that systematically

digest fibrin polymer by hydrolysis of

arginine and lysine peptide bonds.

- Fibrin becomes digested the lysine

residue, bind additional plasmin.- Plasmin is capable of digesting fluid-

phase fibrinogen and factor V and VIII

- Plasma alpha2-antiplasmin rapidly

binds and inactivates free plasmin.


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Plasminogen activation and Control:

1. Tissue Plasminogen Activator

(TPA)

- Secreted by endothelial cells- Hydrolyzes fibrin-bound plasminogen

and initiates fibrinolysis.

- Have 2 glycosylated kringle regions

form covalent lysine bonds w/fibrin

during polymerization

- Segregate the surface of the thrombus

w/ plasminogen.

- Free TPA circulates bound to inhibitor

such as PAI-1 and is cleaved from

plasma.

Urokinase

- Urinary tract epithelial cells, monocytes

and macrophages

- Circulates in plasma at a concentration2-4mg/mL

- Incorporated into the fibrin-bound

plasminogen and TPA at the time of

thrombus formation

- It has only one kringle region

- Does not bind to fibrin

-

Purified urokinase preparation areused to dissolve thrombi in myocardial

infarction, stroke and deep thrombosis.

- It activates plasminogen

Plasminogen Activator Inhibitor 1(PAI-1)

- Inhibits TPA and urokinase from

activating fluid-phase plasminogen.

- Sources: Endothelium, adipocytes,megakaryocytes and hepatocytes.

- PAI-1 is present in excess of TPA conc.

- Circulating TPA normally bound to

PAI-1

- PAI-1 deficiency associated w/chronic

mild bleeding due to increase

fibrinolysis

- Excess PAI-1 may be associated with

thrombosis due to reduction in

fibrinolysis.

Alpha2-antiplasmin

- Synthesized in liver

- Primary inhibitor of plasma

- Bound plasmin digest clots and restores blood vessel patency

- Free plasmin in the circulation digests

plasma fibrinogen, factor V, factor VIII

and fibronectin causing potentially fatal

primary fibrinolysis.

- A2-antiplasmin is a plasma protein that

rapidly and irreversibly binds free plasmin.

- Also become cross-linked through

lysine bonds to fibrin kringles during

polymerization

- It slows fibrin digestion by plasmin.


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Thrombin-Activatable Fibrinolysis

Inhibitor (TAFI)

- A plasma procarboxypeptide that

become activated by the thrombin-thrombomodulin complex.

- It inhibit fibrinolysis.o By cleaving exposed

carboxy-terminal lysine

residue from partially

degraded fibrin that prevents

binding of TPA and

plasminogen

o Blocking formation of

plasmin.

*decrease thrombin production may

reduce the activation of TAFI, resulting

increase the fibrinolysis.

*inc thrombin, Inc activation of TAFI it

will decrease fibrinolysis that may

contribute further to thrombosis.

- TAFI play a role in regulating

inflammation and wound healing.

Fibrin degradation products and D-

dimer.

- Fibrinolysis produces a series of

identifiable fibrin fragments X, Y, D, E

and D-dimer.

Fibrin X

- Described as central E domain w/2

Domains.

- Some peptide cleaved by plasmin

Fragment Y

- E domain after cleavage of one D

domain (D-E)

- Further digested to individual D and E

domains.

D-D fragment

- Also called D-dimer - Two D domains from separate fibrin

molecules cross-linked by the action of

factor XIIIa

- Fragment X,Y, D, E are produced by

digestion of either fibrin or fibrinogen

by plasmin, but D-dimer is a product of

digestion of fibrin.

- D-dimer immunoassay is used to

identify chronic and acute DIC and to

rule out venous thromboembolism is

suspected cases of deep venous

thrombosis of pulmonary embolism

hemostasis reviewer

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