Formation and Regulation of the Hemostatic Plug

Amy S. Gewirtz, MD amy.gewirtz@osumc.edu

Block Objective
Describe the coagulation cascade and the processes of clot formation and fibrinolysis

Lecture Specific Goals and Objectives

Hemostasis
Coagulation Proteins Vascular Wall/Endothelium

Platelets The right amount of clot in the right place

Hemostatic Balance
Procoagulant

Regulatory

Overview of Hemostasis
Primary Hemostasis
Platelet plug formation

Secondary Hemostasis
Fibrin clot formation

Regulation of Hemostasis
Primary hemostasis Secondary hemostasis

Procoagulant Response in Hemostasis

Platelets
Adhesion Activation Aggregation

Coagulation
Activation Thrombin formation Fibrin formation, polymerization and stabilization

Platelet Adhesion Platelet interaction with a non platelet surface

Membrane receptor: Glycoprotein Ib/IX
Adhesive protein: Von Willebrand factor Appropriate surface: Subendothelial matrix (collagen)

Platelet Adhesion
GP Ib/IX

Platelet vWF

Sub-endothelial Matrix
Von Willebrand Factor is produced by endothelial cells and megakaryocytes

Platelet Activation
Agonist-Receptor Interaction
Signal transduction

Release of Second Messengers

Signal transduction

Biological Responses
Most agonists suppress levels of cAMP allowing for increased CA++ availability

Platelet Activation

Shape change

Secretion Activation of GP IIb/IIIa Phospholipid reorganization Contraction

Platelet Activation
Increase in cytoplasmic calcium
Internal storage sites; dense tubular system External environment; membrane calcium channels

Phospholipase A2 pathway Phospholipase C pathway Regulation of platelet activation

Prostacyclin (PGI2) Cyclic nucleotide system Decrease available calcium

Phospholipase A2 Pathway
R1

R2
P

PL A2
Ch

R1 OH
P

Platelet
Ch

+ Arachidonic acid
Prostacylcin (PGI2) (increases cAMP decreases Ca++ )

Cyclo-oxygenase

Cyclic endoperoxides
Thromboxane synthetase
(Activates phospholipase C pathway)

Endothelial cell

Thromboxane A2

Phospholipase A2

Releases arachadonic acid from membrane phospholipids Goal: Synthesis of thromboxane A2 Thromboxane A2 activates phospholipase C pathway Activates prostacyclin pathway

Phospholipase C
Converts phosphatidylinositol-biphosphate (PIP2) into diacylglycerol (DG) and inositol triphosphate (IP3) DG activates protein kinase C which phosphorylates substrate proteins, activates phospholipase A2 and activates GPIIb/IIIa IP3 releases calcium from storage sites Increased cytoplasmic calcium activates calmodulin dependent protein kinases resulting in platelet shape change and contraction

Phospholipase C Pathway
PI
R1

PIP
R1 R2
P

PIP2
R1 R2
P P P

R2
In

In

In

Phospholipase C
R1 R2
P

+
OH

In

Diacylglycerol

Inositol Trisphosphate

Activates protein kinase C phospholipase A2 activation and GPIIb/IIIa and calcium

Exposure of Phosphatidylserine Upon Platelet Activation

Outer
Inner Platelet activation

Exposure of GP IIb/IIIa

Platelet recovery

Phosphatidylserine

Platelet Aggregation
Platelet interaction with other platelets
Membrane receptor: Glycoprotein IIb/IIIa

Adhesive protein: Fibrinogen

Structure of Fibrinogen
B chain A chain

chain

D domain

E domain

D domain

Platelet Aggregation
Platelet
ADP

Fibrinogen

GP IIb/IIIa

Platelet

Secondary Hemostasis
Fibrin Clot Formation

Formation of thrombin

Formation of fibrin clot

Coagulation Factors
Most coagulation factors are designated by a Roman numeral Circulate as inactive proteins

Converted to active form by limited proteolytic cleavage Active proteins may function as: enzymes cofactors

Thrombin Formation
A sequence of enzyme reactions Cleavage of amino acid bonds Reactions occur on a phospholipid

surface Cofactors accelerate reactions

Intrinsic
Kal

Coagulation Cascade
PreK
HMWK

XII
XI IX

XIIa
HMWK

XIa
PL, Ca++ VIIIa, PL, Ca++

IXa Xa
Va, PL, Ca++

Extrinsic VIIa
TF, PL, Ca++

X Common II

X IIa (thrombin) Fibrin Fibrinn

Fibrinogen

Formation of Factor Xa
Factor X

Factor Xa

Factor VIIa

Tissue Factor

Ca++

Phospholipid Surface

Formation of Factor Xa
Factor X

Factor Xa

Factor IXa Factor VIIIa

Ca++

Phospholipid Surface

Intrinsic Kal XII

XI

Coagulation Cascade
PreK
HMWK

XIIa
HMWK

XIa
PL, Ca++

Extrinsic IXa Xa
Va, PL, Ca++

PT

IX PTT

VIIIa, PL,

Ca++

VIIa
TF, PL, Ca++

X II

X IIa Fibrin Fibrinn

Fibrinogen

Tissue Factor Pathway of Coagulation

IXa
VIIIa, PL, Ca++

IX
TF, PL, Ca++

VIIa
TF, PL, Ca++

VII

X II Fibrinogen Fibrin(n)

Xa
Va, PL, Ca++

IIa Fibrin

Conversion of Fibrinogen to Fibrin

B chain
IIa IIa

A chain

chain

IIa

IIa

Fibrin Monomer

Conversion of Fibrinogen to Fibrin

D
E D

Thrombin
Fbp A Fbp B

D Polymerization Factor XIIIa

Fbp A Fbp B

D E D

E D

D D E

Hemostatic Balance
Procoagulant

Regulatory

Regulation of Hemostasis
Blood Flow

Platelet Response
Fibrin Clot Formation

Regulation of Hemostasis Blood Flow Volume

Dilution and removal of activated

enzymes

Laminar flow
Keeps platelets away from vessel wall

Regulation of Primary Hemostasis

Platelet Response/Endothelial Cell
Prostacyclin (PGI2)
A potent inhibitor of platelet

aggregation from phospholipase A2 pathway

ADPase Breaks down ADP Nitric oxide Inhibits activation and adhesion

Regulation of Platelet Activation

Limit the amount of available calcium
Increase available cAMP

Phospholipase A2 Pathway
R1

R2
P

PL A2
Ch

R1 OH
P

Ch

+ Arachidonic acid
Prostacylcin (PGI2) (increases cAMP decreases Ca++ )

Cyclo-oxygenase

Cyclic endoperoxides
Thromboxane synthetase
(Activates phospholipase C pathway)

Thromboxane A2

Regulation of Primary Hemostasis

Effect of Prostacyclin on Platelets
Formed from cyclo-oxygenase acting on
arachidonic acid present within the vessel wall

A potent platelet function inhibitor and

vasodilator

Stimulates adenylate cyclase Results in increased platelet c-AMP Increased c-AMP results in:
Decreased cytoplasmic calcium Inhibition of myosin light chain kinase Inhibition of phospholipase C pathway

Cyclic Nucleotide System

cAMP
Formed by adenylate cyclase Increased levels suppress platelet responsiveness by limiting the amount of available Ca++ Decreased levels result in increased calcium availability and platelet contraction Down regulates the phospholipase C system

Regulation of Primary Hemostasis

ADPase

Produced in endothelium Degrades ADP

Regulation of Primary Hemostasis

Nitric Oxide

Short-lived vasodilator Increased c-GMP inhibits platelet adhesion

and aggregation.

Effect is synergistic with prostacyclin

Coagulation Cascade
Kal PreK
HMWK

XII
XI IX

XIIa
HMWK

XIa
PL, Ca++ VIIIa, PL, Ca++

IXa Xa
Va, PL, Ca++

VIIa
TF, PL, Ca++

X II

X IIa Fibrin Fibrinn

Fibrinogen

Regulation of Secondary Hemostasis

Direct coagulation protein inhibitors

Antithrombin
Protein C system Tissue factor pathway inhibitor Fibrinolytic system

Anticoagulant Activity of Antithrombin

AT IIa

AT

IIa

AT

IIi

Anticoagulant Activity of Antithrombin

AT

Xa

AT

Xa

AT

Xi

Anticoagulant Activity of Antithrombin

AT IIa

AT

IIa

Heparin

AT

IIi

Heparin

Protein C System Cofactor (factors Va and VIIIa)Destruction Major Components

Thrombin Thrombomodulin Protein C Protein S (cofactor) Factor Va Factor VIIIa

Formation of Thrombin
Prothrombin + F1.2 Factor Xa Factor Va Ca++ Thrombin

Phospholipid Surface

Activation of Protein C
Protein C Activated Protein C

Thrombin Thrombomodulin Endothelial Cell Surface

Function of Activated Protein C

Factor Vi APC Factor Va Phospholipid Surface

Protein S

Tissue Factor Pathway of Coagulation

IXa
VIIIa, PL, Ca++

IX
TF, PL, Ca++

VIIa
TF, PL, Ca++

VII

X II Fibrinogen Fibrin(n)

Xa
Va, PL, Ca++

IIa Fibrin

Tissue Factor Pathway Inhibitor

Xa TFPI

TF
VIIa Xa

PL membrane
TFPI

Xa

TFPI VIIa

TF

PL membrane

Regulation of Hemostasis Fibrinolysis Clot Destruction Multi component system

Fibrin clot Plasminogen/Plasmin Tissue plasminogen activator (tPA) Urokinase

Plasmin
Plasmin degrades fibrin
to D-dimer units Activated from plasminogen

tPA
Activates plasminogen by cleaving a
specific peptide bond Produced by endothelial cells tPA and plasminogen bind to fibrin In the absence of fibrin the activation of plasminogen by tPA is very slow

Activation of Plasminogen
Plasminogen

tPA
Fibrin

Plasmin
Pla

D-dimer

Inhibition of Plasminogen
PAI-1
Acute phase reactant Increased PAI-1 and decreased fibrinolytic activity

Alpha-2-antiplasmin
Inhibitor of free plasmin but not plasmin bound to clot This inhibitor helps localize fibrinolysis to sites of clot formation

Effect of 2-Antiplasmin
Pla
AP Pla

AP

+
Pla AP

Pla

AP

Pla

AP

Plasma

Fibrin Clot

Regulation of Fibrinolytic System

PAI-1

Plasminogen

tPA Fibrin

Plasmin
2-antiplasmin

Fibrin(n)

D-dimers

Regulation of Coagulation
Antithrombin: Inhibits serine proteases Protein C: Inhibits cofactors TFPI: Inhibits TF-VIIa activation

Plasmin: Removes excess clot

Serine Protease Inhibitors

Antithrombin
Protein C inhibitor Alpha2-antiplasmin PAI-1

Regulation of Coagulation
IXa
APC VIIIa, PL, Ca++ VIIIi PS TFPI

IX
TF, PL, Ca++

VIIa
TF, PL, Ca++

VII

X
AT

Xa
Va, PL, Ca++

II
AT

IIa Fibrin Fibrin(n)

Pla

APC Vi PS

Fibrinogen

D-dimer

Hemostatic Balance
Procoagulant

Regulatory

Thrombosis

Bleeding