HEMOSTASIS AND BLOOD COAGULATION

If there’s a cut on:

 Arteries – spurting blood, pulsating flow, bright red color
 Veins – steady, slow flow, dark red color
 Capillaries – slow, even flow

HEMOSTASIS
 Derived from the Greek word meaning “stoppage of blood flow”
 Study dates back to the time of Aristotle and Plato
 Hemophilia – the 1st coagulation disorder recognized during 2nd century AD
- means love of hemorrhage by Schonlein
- first described as such by Hopff in 1828
 12th century – Moses Maimonides described 2 male children who died from excessive bleeding after
circumcision

VASCULAR
RESPONSE

BLEEDING

HEMOSTATIC ARREST PLATELET RELEASE OF TISSUE RELEASE OF ADP,

CONTROL BLEEDING ACTIVATION THROMBOPLAST CALCIUM
MECHANISM
EXPOSURE OF EPINEPHRINE
SUBENDOTHELIAL THROMBOXANE A2
COLLAGEN
COAGULATION
PATHWAY VASCULAR PLATELET EVENTS COAGULATION NORMAL
RESPONSE PATHWAY CONTROL
MECHANISMS
TO BLOOD
CLOTTING

VASOCONSTRICTION
 It is caused in part by neurogenic factors and in part by several regulatory substances that interact with
receptors on the surface of the cells of blood vessel wall.
 These include serotonin and thromboxane A2 (TXA2), both products of activated platelets, and
endothelin – 1 produced by endothelial cells.
 Endothelial cells produce vasoconstrictors such as angiotensin II and serotonin which helps in
vasoconstriction
 Activated platelets produce thromboxane A2 (TXA2) which is a potent vasoconstrictor.

HEMOSTASIS
3 BASIC COMPONENTS

1. EXTRAVASCULAR COMPONENT
 Involves in tissues surrounding the blood vessel
 Mechanism: to provide back pressure on the injured vessel thru swelling and the trapping of the
escaped blood
 Factors involved: 1) bulk of the surrounding tissue, 2) type of tissue surrounding the injured vessel, 3)
tone of the surrounding tissue

2. VASCULAR COMPONENT
 Involves the vessels thru which blood flows
 Role played by the vessel depends on their: 1) size, 2) amount of smooth muscle within their walls,
3) integrity of the endothelial cell lining

3. INTRAVASCULAR COMPONENT
 Platelets and other biochemicals (procoagulants) in the plasma
 Involved in 2 essential processes of hemostasis: 1) Coagulation (clot/thrombus formation) 2)
Fibrinolysis (clot/thrombus formation)
CONCEPTS OF NORMAL HEMOSTASIS, HYPOCOAGULATION & HYPERCOAGULATION

1. Normal Hemostatic Balance

 A complex interaction between blood vessels, platelets and biochemical reactants or factors in the
plasma
 These interactions not only create clots that stop bleeding thru the coagulation process but also
dissolve clots thru the fibrinolytic process as injured vessels are healed
 Hemorrhage – failure of hemostasis
 Thrombosis – failure to maintain fluidity of blood

2. Hypocoagulation
 Inherited or acquired conditions
with abnormal bleeding
 Several conditions are readily
diagnosed by lab tests
particularly coagulation factor
assays

Hemophilia
 Example of inherited
hypocoagulable disorder
 Classic hemophilia (type A)
 Factor VII deficiency
 DIC, liver and kidney
diseases
 May cause acquired
hypocoagulation

ACUTE COMPLICATIONS OF HEMOPHILIA

 Muscle hematoma (pseudotumor)
 Hemarthrosis (joint bleeding)

3. Hypercoagulation
 Associated with the inappropriate formation of thrombi in the vasculature that occlude normal blood flow
 Thrombi usually consists of leukocytes, platelets, and RBC held together by fibrin
 Thrombi can be painful and life threatening if the blood supply to a vital organ is cut off
 Caused by a defect in or lack of activation of the fibrinolytic system
 Most are associated with acquired diseases or altered physiologic states
 Most often, a malignancy or surgical procedure is the stimulus for hypercoagulation

PRIMARY AND SECONDARY HEMOSTASIS

 Hemostasis occurs in 2 phases: Primary and
Secondary
 Primary hemostasis – involves the vascular and
platelet response to vessel injury
 Secondary hemostasis – includes the response of
the coagulation process to such injury
 These processes ultimately lead to the formation of
a stable fibrin – platelet plug at the site of injury,
which permit vessel healing
 At the same time, fibrinolysis is initiated, allowing for
gradual clot dissolution

1. Stages of hemostasis after vascular injury.

2. Bleeding occurs after an injury to blood vessel.
3. The hemostatic system is activated to prevent
excessive blood loss
4. Hemostasis occurs in two stages
a) Primary hemostasis – when the platelets
aggregate at the site of injury and form platelet plug
b) Secondary hemostasis – when fibrin develops to
strengthen the platelet plug forming the fibrin –
platelet plug (secondary hemostatic plug)
SEQUENCE OF EVENTS IN PRIMARY AND SECONDARY HEMOSTASIS AFTER VESSEL INJURY

STEPS EVENT COMMENTS

Controlled by vessel smooth muscle; enhanced by chemicals
1. Vasoconstriction secreted by platelets

Adhesion to exposed subendothelial connective tissue

2. Platelet Adhesion

Interaction and adhesion of platelets to one another to form

3. Platelet Aggregation initial plug at injury site

Coagulation factors interact on platelet surface to produce

4. Fibrin – platelet plug formation fibrin, fibrin platelet plug then forms at site of vessel injury

Fibrin clot must be stabilized by coagulation factor XIII

5. Fibrin stabilization

ROLE OF BLOOD VESSELS IN HEMOSTASIS

1. Intact Vessels (CAPPILARIES)

 Where metabolic exchange between the blood
and tissues take place
 Lined by as single continuous endothelial cell
layer that is attached to a supportive basement
membrane
 There are openings called “junctions” along
the capillary wall that allow passage of WBC,
O2, and nutrients into and out of the blood as
necessary
 RBC and platelets usually do not leave the
intravascular system
Pericytes (Rouget cells)
 Cells that lie beneath the endothelium of capillaries, arteries, and veins that may differentiate into
vessel related cells when needed
 Contractile cells that wrap around the endothelial cells of capillaries and venules throughout the
body
 Embedded in the BM where they communicate with endothelial cells through direct physical
contact and paracrine signaling
 2. Intact Vessels (ARTERIES AND VEINS)
3 Layers
a) Tunica intima (inner endothelial lining)
 Endothelial cells deposit von Willebrand
factor (vWF) in the subendothelial matrix
where vWF binds to collagen
 Separates the blood cells from a
subendothelium (BM, elastic connective
tissue, and collagen fibers)
b) Tunica media
 Composed of smooth muscle cells and
connective tissue
c) Tunica adventitia (outer part)
 Composed of connective tissue fibroblast and
collagen fibers

INTACT VESSELS: FUNCTION

 Vasoconstriction and vasodilation provide the means of control of blood flow rate and blood pressure
 Controlled by smooth muscles of tunica media
 Endothelial cells secrete several important substances that influence coagulation, fibrinolysis and
platelets

ANTITHROMBOTIC, FIBRINOLYTIC, AND COAGULANT SUBSTANCES REEASED FROM OR FOUND ON

THE SURFACE OF INTACT ENDOTHELIAL CELLS

SUBSTANCES ACTION HEMOSTATIC ROLE

Prostacyclin - Inhibits platelet activation Anticoagulant
(PGI2) - Stimulates vasodilation s blood flow

Adenosine (met Stimulates vasodilation s blood flow rate

product of ADP & ATP)

Thrombomodulin Endothelial surface receptor for Anticoagulant

thrombin; inactivates thrombin Fibrinolytic

Heparan sulfate Coats endothelial cell surface and Anticoagulant

weakly enhances activity of
antithrombin III, a plasma
anticoagulant
Tissue plasminogen Converts plasminogen to plasmin, Fibrinolytic
activator (tPA) which plays important role in
fibrinolysis
Von Willebrand Factor Required for platelet adhesion to site Coagulation
(vWF) of vessel injury

INTACT VESSELS
 The intact endothelial lining of blood vessels is antithrombotic
 It does not activate platelets or promote coagulation
 It will provide a smooth surface that facilitates blood flow and reduces turbulence (promotes
thrombosis)

DAMAGE VESSELS
 Vasoconstriction occurs as a neurogenic response (nerve reflex)
 Breaks through the smooth endothelial lining exposing collagen which causes adherence of platelets to
the area of injury
 Collagen exposure also initiates the contact phase of coagulation, which begins a series of
biochemical reactions known as intrinsic coagulation pathway
 Tissue thromboplastin is released from the injured vessel, which promotes coagulation thru a
different series of reactions known as extrinsic coagulation pathway
 VESSEL DAMAGE

initiates

VASOCONSTRICTION PLATELET COAGULATION FIBRINOLYSIS

ACTIVATION

Nervous system Platelet Plug Platelet surface Collagen Tissue Tissue

response forms becomes exposure thromboplastin Plaminogen
available releases Activator
for coagulation releases
activities

Intrinsic Extrinsic
Coagulation Coagulation Plasminogen
Pathway Pathway

Plasmin

Vessel Muscular Platelet-fibrin Stable fibrin Clot dissolution

response clot formation clot formation as vessel heals

ROLE OF PLATELETS IN HEMOSTASIS

History
 1842 – platelets were first discussed
 Later, it was discovered that platelets originated from megakaryocytes in the bone marrow
 1940 – platelet structure was studied using E/M
 1950 – a method for counting platelets was described that utilized phase contrast microscopy
 Today – platelets are routinely counted using automated methods
 Qualitative (functional) platelet evaluation was first available with the introduction of the bleeding time
by Duke in the early 1900s
 A prolonged BT can indicate either a thrombocytopathy
 BT is still the best screening test for platelet function
 More specific tests such as platelet aggregation studies are now available

Platelet Morphology and Function in Hemostasis

 Platelets play a central and immediate role in the response to vessel injury
 Smallest microscopically visible element in the peripheral blood film at 2-4 um and have a discoid
shape
 Wright stain: purple granular appearance and look like specks of dust
 Small fragments of megakaryocyte cytoplasm
 Life span: 9-10 days

Platelets play the following roles in hemostasis:

 They adhere to the injured vessels
 They aggregate at the injury site
 They promote coagulation on their phospholipid surface
 They release important chemicals for hemostasis
 They induced clot retraction

PLATELET ADHESION
 If vascular injury exposes the endothelial surface and
underlying collagen, platelets adhere to the
subendothelial collagen fibers, spread pseudopods along
the surface and clump together (aggregate).
 Platelet adhesion to the subendothelial connective
tissues, especially collagen, occurs in 1-2 minutes after
break in endothelium
 Epinephrine and serotonin promote vasoconstriction
PLATELET AGGREGATION
 The attachment of platelets to one another
 Newly arriving platelets flowing into the area become activated by contact with agonists such as ADP
and TXA2, products from the damage tissue and endothelial cells
 With activation, the new platelets undergo shape change

ACTIVE FACTORS IN PLATELET CYTOPLASM

 Actin and myosin molecules
 Residuals of endoplasmic reticulum and Golgi apparatus
 Mitochondria and enzyme systems
 Enzyme systems that synthesizes prostaglandins
 Fibrin stabilizing factor
 Growth factor – causes vascular endothelial cells, vascular smooth muscle cells, and fibroblast to
multiply and grow = helps repair damaged vascular walls
 Glycoproteins on platelet cell membrane – repulses adherence to normal endothelium, but adherence
to injured areas
 Membrane contains large phospholipids – activate multiple stages in blood clotting process
 Half-life in blood – 8 – 12 days
 Eliminated from circulation by tissue macrophages system

IMPORTANT SUBSTANCES SECRETED BY PLATELETES

ROLE IN HEMOSTASIS SUBSTANCE SOURCE COMMENTS

HMWK α granules Contact activation of
intrinsic coagulation
pathway
Fibrinogen α granules Converted to fibrin for
Promote coagulation clot formation
Factor V α granules Cofactor in fibrin clot
formation
Factor VIII - vWF α granules Assists platelet adhesion
to subendothelium
ADP Dense bodies Promote platelet
aggregation
Calcium Dense bodies Promote platelet
Promote aggregation aggregation
Platelet Factor 4 α granules Promote platelet
aggregation
Thrombospondin α granules Promote platelet
aggregation
Serotonin Dense bodies Promotes
vasoconstriction at injury
Promote vasoconstriction site
Thromboxane A2 Membrane phospholipids Promotes
vasoconstriction at injury
site
Platelet – derived growth α granules Promote smooth muscle
factor growth for vessel repair
Promote vascular repair Beta thromboglobulin α granules Chemotactic for
fibroblast to help in
vessel repair
Plasminogen α granules Precursor to plasmin
which induces clot lysis
Other systems affected α2 Antiplasmin α granules Plasmin inhibitor
C1 esterase inhibitor α granules Complement system
inhibitor
PLATELET INDUCTION OF CLOT RETRACTION
 The last act of platelets within a platelet – fibrin clot is contraction of the clot
 Requires calcium and lots of energy (ATP)
 Observed in vitro when blood clots in a test tube (indicative of normal platelet function)
 The retraction process requires stabilization of platelets and platelet – fibrin elements
 The pulling factors are provided by contractile platelet elements in a process similar to muscle
contraction
 Exact purpose of clot retraction is unclear

ROLE OF COAGULATION IN HEMOSTASIS

 Process whereby on vessel injury, plasma proteins, tissue factors, and calcium interact on the surface
of platelets to form a clot
 Platelets not only provides surface for the coagulation reaction but also interact with fibrin to form a
stable platelet – fibrin clot

COAGULANT FACTOR NOMENCLATURES

FACTORS

I Fibrinogen IX Plasma thromboplastin (PTC), antihemophilic

factor Christmas factor
II Prothrombin X Stuart – Prower factor

III Tissue Factor XI Plasma thromboplastin antecedent

IV Calcium XII Hageman factor

V Proaccelerin, labile factor XIII Fibrin stabilizing factor

VII Proconvertin, stable factor High-molecular-weight kininogen (HMWK)

Fitzgerald factor
VIII Antihemophilic factor A (AHE) Prekallikrein
Fletcher factor

 These coagulation factors (except calcium and tissue thromboplastin) normally circulate in the plasma
as inactive proteins
 On activation, some factors form enzymatic proteins known as “serine proteases” that activate other
specific factors in the coagulation sequence
 Zymogens are substrates that have no biologic activity until it converted by enzymes called serine
proteases, which have exposed, serine-rich, active enzyme sites
 Serine proteases selectively hydrolyzed arginine or lysine-containing peptide bonds of other zymogens,
thus converting them to serine proteases
 The zymogens are factors II, VII, IX, X, XII and prekallikrein
 The co – factors are factors V, VIII, Tissue factor, and HMWK
 3 interrelated pathways of coagulation, each, representing a unique series of biochemical reactions;
Intrinsic pathway, Extrinsic pathway, common pathway

COAGULATION CASCADE

COAGULATION
PATHWAY

EXTRINSIC PATHWAY INTRINSIC PATHWAY

Activator Tissue In vivo activator In vitro activator

thromboplastin Collagen, ADP, negative charged
Thrombin glass surface
Kaolin, ellagic acid
The Extrinsic Pathway
Activated by:
 The release of tissue thromboplastin into the plasma from the injured tissue cells
 Tissue thromboplastin activates factor VII to the serine protease factor VIIa
 Factor VII with calcium and platelet phospholipid (PL) activates factor X to factor Xa in the common
pathway

Extrinsic Coagulation

COAGULATION MECHANISM
EXTRINSIC PATHWAY

ACTIVATOR
Tissue thromboplastin

FACTOR VII

ACTIVATED FACTOR VII

FACTOR VII COMPLEX

Activated Factor VII Tissue Thromboplastin Calcium phospholipid

The Intrinsic Pathway

 Activated by exposure of the subendothelial BM and collagen.
 When the subendothelial surface is contacted by the coagulation contact factors XII, XI, HMWK, and
prekallikrein, the contact activation of the pathway is begun
 Factors XII and XI are converted to the serine proteases XIIa and Xia
 Factor Xia with calcium in turn converts factor IX to the serine protease IXa
 Factor IXa with platelet PL, calcium and a cofactor, factor VIIIa converts factor X to the serine protease
factor Xa in the common pathway

COAGULATION MECHANISM
INTRINSIC PATHWAY

ACTIVATORS
Collagen, Glass, etc.

F XII – F XIIa

F XI – F XIa

F IX – F IXa

F VIII COMPLEX

Calcium Phospholipid F VIIIa F IXa

The Common Pathway

 Begins with activation of factor X to factor Xa by either the intrinsic or extrinsic pathway
 Factor Xa with co factor, Factor V converts factor II, prothrombin to factor IIa, thrombin
 Factor IIa converts factor I, fibrinogen to fibrin

FACTOR V COMPLEX
V, Xa, Ca phospholipid FACTOR XIII

COMMON FACTOR X – X Prothrombin - Fibrinogen - Fibrin

PATHWAY ACTIVATED Thrombin Fibrin Polymer
FIBRINOLYSIS IN HEMOSTASIS
 1700’s - John Hunter reported the unexplained finding that blood from people who had died did not
clot
 1937- MacFariane reported that damaged tissues release a substance (plasminogen activator) that
activates the inert precursor called plasminogen to its active form, plasmin
 Plasmin - a nonspecific proteolytic enzyme capable of degrading fibrin as well as fibrinogen and factors
V and VIII

Function of Fibrinolysis in Hemostasis

 Fibrinolysis - system where the temporary fibrin clot is systematically and gradually dissolved as the
vessel heals in order to restore normal blood flow

 Plasmin - proteolytic enzyme that is the primary substance responsible for fibrinolysis

 Tissue plasminogen activator - released from injured vessel walls convert plasminogen to plasmin

 Plasmin is trapped within the clot, and clot lysis begins slowly as soon as the clot is formed, with fibrin
degradation (split) products (FDP or FSP) being released in the plasma

 Lysis is slow because of fibrin clot stabilization by factor XIII, fibrin stabilizing factor

 Protein C and S are 2 hemostatic substances that further fibrinolysis and inactivate tPA inhibitors

Blood Coagulation Test

Bleeding Time

 When a sharp pointed knife is used to pierce the tip of the finger or lobe of the ear, bleeding ordinarily
lasts for 1 to 6 minutes

 The time depends largely on the depth of the wound and the degree of hyperemia in the finger or ear
lobe at the time of the test

 Lack of any one of several of the clotting factors can prolong the bleeding time but it is usually
prolonged by lack of platelets