Platelets and Hemostasis

Learning objectives: Platelets and Hemostasis

Define hemostasis and enumerate the phases of
hemostasis
Describe the process of vascular spasm
Explain the roles of platelets, vWF and endothelial
cells in hemostasis
List the contents of the alpha and dense granules of
platelets
Discuss the processes of platelet adhesion,
aggregation, and release and the factors involved in
the regulation of platelet aggregation

Learning objectives: Platelets and Hemostasis

Explain the classical pathway of coagulation
Enumerate the natural inhibitors of
coagulation and mechanism of action of each
Explain the different functions of thrombin
Discuss the process of fibrinolysis
Differentiate between a thrombus and
embolus

Learning objectives:Platelets and Hemostasis

Discuss the clinical manifestations of bleeding and
the criteria that prompt its clinical investigation
Explain the laboratory investigation of bleeding and
coagulation disorders
Discuss the role of vitamin K and the liver in
hemostasis
Discuss the clinical features and pathogenesis of
disorders of coagulation--hemophilia, vWD and DIC

Hemostasis
Orderly and well regulated process to stop
bleeding in response to vessel injury with
dissolution of the blood clot following repair
of injured tissue
Blood clotting is achieved normally in 3-6
minutes
Clot remains while the endothelium
regenerates and is broken down after tissue
repair occurs

Hemostasis
Four phases of hemostasis:
1) Vascular spasm
2) Platelet plug formation
3) Coagulation
4) Fibrinolysis

Phases of hemostasis

Blood vessel

Vascular spasm
Once vessel is damaged, tissue factor
(TF) is released with exposure of collagen
Supporting structures of vessel wall
(media and adventitia) allow vessel to
constrict following injury limiting blood
flow to the area (local vasoconstriction)
Vasoconstriction can last from 30
minutes to a few hours

Vascular spasm
Disruption of the endothelium occurs and
platelets bind to the exposed collagen
Adhesive proteins in the subendothelium
such as elastin,collagen, thrombospondin, and
fibronectin provide binding sites for platelets

Vascular spasm
Exposed collagen triggers platelet
activation
Thrombaxane A2, vasoconstrictor
released from activated platelets is
responsible for much of vasoconstriction

Role of endothelial cell in hemostasis

Release of vasodilators :nitric acid, prostacyclin
During injury, releases tissue factor (TF) which binds
to FVII initiating the coagulation cascade
Slows bleeding by secreting vasoconstrictors:
endothelin and platelet activating factor (PAF)
Endothelin stimulates smooth muscle contraction
and cell division of endothelial cells, smooth muscle
cells and fibroblasts to repair the site of damage

Role of endothelial cell in hemostasis

Activates fibrinolysis by releasing tissue
plasminogen activator(TPA) and facilitating
release of inhibitor to TPA which is
plasminogen activator inhibitor (PAI-1)
Secretes heparin and heparan sulfate
(anticoagulants)
Site of conversion of protein C activation by
thrombin

Tissue factor
(Thromboplastin/ Factor III)

Major initiator of coagulation

Transmembrane glycoprotein expressed
by fibroblasts and smooth muscle cells of
BV wall
Expressed in many tissues such as brain,
heart, lungs kidneys,testis and placenta

Platelets
(Thrombocytes)

Anucleate fragments of megakaryocytes

Mitochondria produce ATP for energy
Calcium ions are stored in the Golgi
apparatus
Life span: 70% circulate in blood for 7 to
10 days; 30% stored in the spleen

Platelets
(Thrombocytes)
Thrombopoietin produced by the liver (main
site) and kidneys is the major regulator of
platelet production
Glycoprotein membrane promotes adhesion
to collagen and damaged endothelium
Normal platelet count : 150,000-400,000/ul

Platelet role in hemostasis

Important role of platelets in hemostasis
involves:
1) Maintenance of vascular integrity
2) Formation of platelet plug for initial arrest
of bleeding
3) Contribution to formation of fibrin and
stabilization of the hemostatic plug

Thrombopoiesis

Megakaryocyte

Platelets (peripheral smear)

Platelet Structure

Platelet structure and function

Glycocalyx: mucopolysaccharide coat with adhesive
membrane glycoproteins involved with interaction
of platelets with each other and with other
aggregating agents
Microtubules: form a marginal band which
maintains the discoid shape of non-activated or
resting platelets

Platelet Structure and Function

Alpha granules: contain proteins which regulate clot
formation and blood vessel formation
-fibrinogen
-factors V,XI
-vWF (von Willebrand factor)
-PDGF (platelet derived growth factor)
-VEGF (vascular endothelial growth factor)

Platelet structure
Dense granules:contain substances that
contribute to platelet recruitment and
thrombus stabilization at the site of injury
-ADP/ATP
-Serotonin
-Calcium

Platelet Structure

Activated Platelets

vWF (von Willebrand factor)

Large adhesive glycoprotein made up of multimers
synthesized by megakaryocytes and endothelial cells
Storage: alpha granules of platelets and Weibel Palade bodies
of endothelial cells
Functions: 1) carrier protein for FVIII
2) platelet adhesion-- binds with GPIb on
platelets surface and attaches platelet to
collagen and the underlying subendothelium

Functions of Platelets
Adhesion : initiated by exposure to collagen or
subendothelial connective tissue; depends on
vWF
Aggregation: platelets adhere to damaged
endothelium and aggregate in response to
stimuli (ADP, thrombin and TXA2)
Release: alpha granules and dense granules
release their contents

Platelet-BV wall interaction

Platelet glycoproteins
Function as receptors for platelet adhesion to
collagen in the subendothelial matrix
Important for platelet-platelet aggregation
GP Ib receptor binds to vWF
GP IIb/IIIa receptor links platelets to other
platelets via fibrinogen leading to further
aggregation of platelets

Platelet adhesion, aggregation and release

Platelets initially attach to exposed collagen by the
binding to vWF to platelet membrane GPIb
receptors
Platelets become activated, change shape and
release the contents of their granules
Released thrombaxane A2 and ADP lead to further
platelet aggregation by binding of fibrinogen to
platelet membrane GP IIb-IIIa ( major receptor on
the platelet surface); platelet plug is formed

Regulation of platelet aggregation

Phases of hemostasis

Blood clot

Blood coagulation

Coagulation factors

Coagulation Cascade
Involves sequential activation of proenzymes
to enzymes ultimately generating fibrin
Amplification of each step ensures rapid
response for effective hemostasis
Three pathways invoved: extrinsic,intrinsic,
and common pathways

Intrinsic Pathway
Activated when blood is exposed to collagen or
negatively charged surfaces
XII is activated to XIIa by collagen exposure
XIIa activates XI to XIa
XIa activates IX to IXa and simultaneously cofactor
VIII becomes activated to VIIIa
IXa with the help of phospholipids (PF3) and Ca++
combines with VIIIa to form the intrinsic tenase
complex (factor X activating complex)

Extrinsic Pathway
Damaged endothelial cells
release tissue factor (Factor III)
Tissue factor activates VII to VIIa

Common Pathway
X is activated by VIIa or by tenase to Xa
Xa with cofactor Va and Ca++
(prothrombinase) activates prothrombin
(II) into thrombin
Thrombin activates fibrinogen to fibrin
XIII stabilizes the fibrin clot

Coagulation cofactors
Tissue factor: extrinsic tenase (TF-VIIIa and
calcium)
Factor V : prothrombinase complex (Va,Xa,
calcium and phospholipid)
Factor VIII: intrinsic tenase complex with VIIIa,
Xa, calcium and phospholipid
Phospholipid :platelet membrane constituent
Calcium: found in platelet dense granules
Vitamin K: II,VII,IX,X are vitamin K dependent

Coagulation in vivo

Natural Inhibitors of Coagulation

Natural Inhibitors of Coagulation

TFPI (tissue factor pathway

inhibitor):once coagulation is
activated, FXa binds to TFPI
and inhibits FVIIa-TF

Tissue factor pathway inhibitor

Natural Inhibitors of Coagulation

AT III (antithrombin III): major
inhibitor of thrombin and FXa;
also inhibits FIXa, FXIa and XIIa;
heparin increases inhibitory
effect of AT III

Antithrombin III

Natural Inhibitors of Coagulation

PC (protein C): inhibitor of FVIIIa and FVa with
PS (protein S) as cofactor; activated by
thrombin which generates low levels of PC
T-TM (thrombin-thrombomodulin) complex
binding on endothelial surface enhances
activation of PC and PS

Natural inhibitors of coagulation

Fibrinolysis

Fibrinolysis

Process of clot breakdown which may be

primary (normal body process) or secondary
to disease (usually DIC), drugs or other causes
Plasminogen is activated to plasmin
(proteolytic enzyme that breaks down
fibrin)by tissue plasminogen activator (t-PA)
and urokinase
Plasmin cleaves fibrin in several places leading
to production of fibrin degradation products
(FDPs) which are cleared by the kidneys and
liver

Fibrinolysis
Alpha 2 antiplasmin inactivates plasmin
PAI-1 and 2 (plasminogen activator inhibitor 1
and 2) inhibit urokinase and t-PA
TAFI (thrombin activatable fibrinolysis
inhibitor) is activated by thrombin-anti
thrombin complex and inhibits fibrinolysis

Fibrinolysis

Fibrinolysis

D-dimer

Phases of hemostasis

Thrombus

Embolus

Coronary artery with recent thrombus

Cerebrovascular accident

Bleeding

Manifestations of bleeding disorders

Coagulation screening tests

Laboratory Evaluation of Coagulation System

CBC: complete blood count (includes platelet count)
PT: prothrombin time: extrinsic and common
pathways
PTT/APTT( activated partial thromboplastin time):
intrinsic and common pathways
TT (thrombin time): quantitative /qualitative
fibrinogen deficiency
Mixing studies: inhibitor vs.factor deficiency
Factor assays: factor deficiency
FDP (fibrin degradation products): fibrinolytic system

APTT

PT

Thrombin time

FDP:D-Dimer

Vitamin K
Essential factor that activates multiple
coagulation factors by addition of a carboxyl
group (II,VII, IX, X and proteins C and S) that
allows for binding of calcium to phospholipids
and in forming tenase and prothrombinase
complexes
Major source of vitamin K (K1/phylloquinone)
is from yellow or green plants; bacterial and
animal source form of Vit K (K2/menaquinone)
is less readily absorbed

Vitamin K
Warfarin blocks recycling of Vit K so active Vit
K levels are decreased; most common
deficiency of Vit K is due to warfarin
anticoagulation
Vitamin K deficiency results from liver
dysfunction due to impaired metabolism of Vit
K and from malabsorption syndromes

Vit K cycle and role in production of carboxylated clotting factors

Vitamin K deficiciency

Hemostatic defects in liver disease

Decreased synthesis of clotting factors (except
VIII and vWF)
Vitamin K deficiency
Thrombocytopenia(decreased production and
hypersplenism)
Platelet function defect(increased fibrin/FDPs
and decreased adhesion to GPIb)
Disseminated intravascular coagulation (DIC)

Hemostatic defects in liver disease

Systemic fibrinolysis (decreased alpha 2
antiplasmin and decreased clearance of t-PA)
Increased risk of thrombosis (increased acute
phase reactants VIII and vWF; decreased
levels of ATIII, protein C and S; decreased
clearance of activated coagulation factors)
Dysfibrinogenemia(abnormal fibrinogen)

Disorders of Coagulation
Hereditary coagulation disorders: vWD,
hemophilia
Acquired coagulation disorders: DIC
Platelet-vessel interaction disorders: vWD,
platelet function defects, thrombocytopenia

Hemophilia A (classical hemophilia)

X-linked recessive: congenital deficiency of FVIII resulting in
decreased or absent FVIII activity and lifelong bleeding
Incidence: 1 in 5000 male births
Clinically indistinguishable from hemophilia B (FIX deficiency)
Severity of bleeding depends on circulating clotting factor
activity: mild (>5-40% activity, moderate (1-5%) and severe
(<1%); usually delayed type of bleeding with hemarthroses
and muscle bleeding
Diagnosis: x- linked inheritance pattern, elevated PTT and
decreased plasma FVIII assay

Bleeding in hemophilia A

vWF (von Willebrand factor)

Large adhesive glycoprotein made up of multimers
synthesized by megakaryocytes and endothelial cells
Storage: alpha granules of platelets and Weibel Palade bodies
of endothelial cells
Functions: 1) carrier protein for FVIII which stabilizes FVIII
2) platelet adhesion-- forms link with GPIb on
platelet surface with collagen and underlying
subendothelial matrix

vWD

Most common inherited bleeding disorder

Due to deficiency of vWF
Incidence: 0.1-1.0% of general population
Varied symptoms but usually mucosal bleeding and
bleeding following invasive procedures or surgery
Lab findings: normal platelet count, increased
bleeding time, normal PT; normal or prolonged PTT

Types of vWD

DIC

Therapeutic anticoagulants

Therapeutic anticoagulants

Anti platelet drugs

Learning objectives: Platelets and Hemostasis

Define hemostasis and enumerate the phases of

hemostasis
Describe the process of vascular spasm
Explain the roles of platelets, vWF and endothelial
cells in hemostasis
List the contents of the alpha and dense granules of
platelets
Discuss the processes of platelet adhesion,
aggregation, release and the factors involved in the
regulation of platelet aggregation

Review of Learning objectives: Platelets and

Hemostasis
Explain the classical pathway of coagulation
Enumerate the natural inhibitors of
coagulation and mechanism of action of each
Explain the different functions of thrombin
Discuss the process of fibrinolysis
Differentiate between a thrombus and
embolus

Review of Learning objectives:Platelets and

Hemostasis
Discuss the clinical manifestations of bleeding and
the criteria that prompt its clinical investigation
Explain the laboratory investigation of bleeding and
coagulation disorders
Discuss the role of vitamin K and the liver in
hemostasis
Discuss the clinical features and pathogenesis of
disorders of coagulation--hemophilia, vWD and DIC