HEMOSTASIS

Formation of Primary Hemostatic Plug/

Primary Hemostasis Run Down
1. Adhesion- platelets adhere to
exposed subendothelial surface
(collagen) in vivo, (glass) in vitro;
occurs with the presence of Von
Willebrand factor
a. Von Willebrand disease- lack
vWF
b. Bernard Soulier Syndromelack Gp 1B (receptor for vWF)
2. Activation- morphologic and
functional changes in platelets
a. Agonist- substance that
stimulate platelet activation
3. Secretion- release of platelet
activation
4. Aggregation- platelet attachment
to each other; require fibrinogen
and calcium
a. Glanzmanns
Thrombasthenia- lack Gp IIBIIIA complex (receptor for
fibrinogen)
Normal endothelium
- Provides a physical barrier
- Secretes substances which
promote normal blood flow
NO, PGI2, heparin sulfate,
thrombomodulin, TFPI
tPA, PAI-1 (plasminogen
activator inhibitor 1)
(fibrinolytic properties)
Products of Vascular Intima with
Anticoagulant Properties
1. NO (Nitric oxide)- counteracts
vasoconstriction and maintains
healthy arterioles
2. PGI2 (Prostaglandin I2/
Prostacyclin)- platelet activation
inhibitor
3. Heparan sulfate- activates
antithrombin

4. Thrombomodulin- activates the

protein C pathway (digests aFV &
aFVIII)
5. TFPI (Tissue Factor Pathway
Inhibitor)- inactivates coagulation
via aFVII
Products of Vascular Intima Theat
Promote Fibrinolytic Properties
1. tPA (Tissue Plasminogen
Activator)- activates plasminogen
into plasmin
2. vWF (von Willebrand Factor)
Vasoconstriction
- what happens when vessel wall in
injured?
- Transient, locally-induced
phenomenon
- Retards, extravascular blood loss
- Slows local blood flow
- Enhance the adherence of platelets
to exposed subendothelial surfaces
- vWF- facilitates platelet adherence
to exposed collagen in artioles
- P-selectin- adhesion molecules that
promotes platelet and leukocyte
binding
- Adhesion molecules- promote
leukocyte binding to endothelium
- Tissue factor- activates the
coagulation system
Formation of the primary platelet plug
- Platelet adhesion- property of
binding to nonplatelet surface
- Platelet activation- interplatelet
attachment
- Aggregation to form a platelet plug
Activation of the coagulation process
- Intrinsic pathway
- Extrinsic pathway
Both produce fibrin to form a
clot
Activation of fibrinolysis
- Slow digestion and removal of the
fibrin clot as healing occurs

Facilitated by the activation of

plasminogen into plasmin

Primary hemostasis
- Is defined as the primary platelet
plug
- Involves the blood vessel wall,
platelets and vWF
Abnormalities in Primary Hemostasis
- Result in:
Hemorrhage from mucosal
surfaces
Epistasis
Melena
Hematuria
Petechial rashes
Ecchymosis
Hemarthosis
Hematemesis
Hematoma
Menorrhagia
Prolonged bleeding after
venipuncture or wounds
Petechiae- is a small (1-2mm) red or
purple spot on the body, caused by a
minor hemorrhage (broken capillary
blood vessels)
Purpura- is the appearance of red or
purple discoloration on the skin that
do not blanch on applying pressure.
They are caused by bleeding
underneath the skin. Purpura measure
0.3-1 cm (3-10 mm)
Ecchymosis- subcutaneous bleeding
larger than 1 cm or a hematoma,
commonly called a bruise. It can be
located in the skin or in a mucous
membrane
Disorders of Primary Hemostasis
1. Hereditary Hemorrhagic
Telengiectasia (Osler Weber Rendu
Disease)

2. Congenital hemangiomata
(Kasabach Meritt Syndrome)
3. Ehlers Danlos Syndrome
4. Marfan Syndrome
5. Pseudoxanthoma elasticum
6. Senile purpura
7. Scurvy
8. Henoch- Schonlein purpura
Characteristics:
-

Highly complex, anucleated

fragment that is derived from bone
marrow megakaryocytes
Hemostasis and thrombosis:
Adhesion to subendothelium
Secretion of granule contents
Aggregation
Provision of membrane
surface for activation of
coagulation factors

Humoral Factors that Regulate

Megakaryocyte Production and
Maturation
- MK-CSF- induces committed cells
to proliferate
- Thrombopoietin- promotes
differentiation and maturation of
megakaryocytes
Stages of Megakaryocytic Maturation
Endomitosis- characteristic
megakaryocytic development, that
involves nuclear division without
cytoplasmic division, generating
giant multinucleated or polyploid
cells
1. Megakaryoblast
- Cell size: 6-24 um
- Morphology: overlapping nuclear
lobes (compact, multiple
nucleoli) and a small amount of
basophilic cytoplasm
- Demarcation membranes:
present (by EM)
- Granules: absent (single
nucleus, fine chromatin)

- N/C ratio: 10:1

2. Promegakaryocyte
- Cell size: 14-30 um
- Morphology: Horseshoe shape
nuclear lobe that increases and
spreads out; fused pink granules
become visible, first in the
center of the cell
- Demarcation membrane:
proliferating to the center of the
cell
- Granules: starting to increase
(double nucleus)
- N/C Ratio: 4:1 to 7:1
3. Granular megakaryocyte
- Cell size: 16-56 um in diameter
- Morpho: multilobulated nucleus
and pink granules increasingly
spreads out over bluish
cytoplasm
- Demarc. Membrane: extensive
but assymetric; two or more
nuclei
- Granules: numerous
4. Mature megakaryocyte
(Metamegakaryocyte)
- Ultimate producer of
thrombocytes to platelets
- Cell size: 20-50 um in diameter
- Morpho: more compact highly
lobulated nucleus and wholly
eosinophilic cytoplasm with
clustered granules
- Demark. Membrane: evenly
distributed
- Granules: aggregated into
Platelet fields; N/C ratio is less
than 1:1
- Disintegrated cell surrounded by
platelets checkpoints:
Endomitosis- nuclear division
w/o cytoplasmic division
Each megakaryocyte
produces 2000 platelets
Lifespan: 10 days
Circulating platelets
represents 2/3 (67%); and

the remaining 1/3 (33%) are

stored in the spleen
Morphology: Smallest Discoid Shape,
Non Nucleated Cell
- 2-4 um in diameter, originate from
polyploid megakaryocyte (largest
cell) in B<
- Stain as violet purple granule like
appearance
- Small fragments of megakaryocyte
cytoplasm; life span of 9-11 days
(4-5 days on marrow as
megakaryocytes)
- May appear as satellites around the
cytoplasm of neutrophils
- Major function:
Maintains vascular integrity
Hemostasis by forming plugs
to stop blood loss, hence,
promoting blood coagulation
- Quantity: 200,000- 400,000/mm3
- Life span: 10 days

The IgG antibody is directed

against the glycoprotein IIb/IIIa
complex on the platelet
membrane. As the antibody coats
the platelets, the platelets rosette
around segmented neutrophils,
bands, and sometimes around
monocytes
Remedy: recollect using sodium
citrate, multiply the platelet count
by 1.1

Platelet morphology:
o Peripheral Zone
- Composition:
o Glycocalyx
- rich in glycoproteins which is
important in platelet
adhesion and aggregation
- Provides adherence surface
for coagulation
- All glycoproteins and
coagulation factors from the

prothrombinase complex (VA,

XA, and Calcium) for the
conversion of prothrombin to
thrombin.
- 1. GP IIB/IIIA Receptor
complex- the most abundant
GP with approx. 50,000
copies per platelet;
Aggregation and Adhesion
- 2. GP IB/IX Receptor
Complex- next abundant with
25,000 copies per platelet;
GP V forms a tight, non
covalent association with GPs
IB and IX; Adhesion
o Trilaminar plasma membrane
- Rich in lipoproteins
(phosphatidylserine,
phosphatidylcholine,
phosphatidylinositol)
- Maintains ionic gradient by
the Sodium/Potassium
ATPase ionic pump
- Site of platelet factor VIII that
hastens blood coagulation
and platelet adhesion
- Stains with Ruthenium Red
o Submembrane area
- Contains specialized
submembrane filaments
which:
Form the wall to
separate organelles
from the membrane
May help regulate
normal platelet discoid
shape
Acts as a Base for
Pseudopod Formation
Modulate platelet
adhesion and clot
retraction after
activation

o Sol Gel Zone (Matrix of

platelet interior)
- A gel matrix that stabilizes the
organelles and mircotubular
systems within the resting platelet
a. Microfilaments (actin-myosin)provides contractile factors
after platelet activation to
maintain the discoid shape
b. Microtubules- compose of
protein tubulin which contribute
to the cytoskeletal support
system for the normally discoid
cell
c. Glycogen
o Organelle zone
A. Dense core (Bulls eye) granules
o ADP- to promote platelet
aggregation
o ATP
o Calcium- activates membrane
phospholipases that liberates
arachidonic acid metabolite,
TXA2
o 5-hydroxytryptamine (5-HT)
o Serotonin- promotes further
vasoconstriction
o Magnesium ions
B. Alpha-granules
a. Fibrinogen- coagulation,
adhesion and aggregation
b. Factor V- prothrombinase
complex
c. Platelet-derived growth factor
(PDGF)- smooth muscle cell
proliferation
d. vWF
e. Beta-thromboglobulin- inhibits
heparin
f. Heparin- neutralizing platelet
factor 4 (PF4)- inhibits heparin
g. P-selectin- for platelet
leukocyte interactions
C. Lysosomes
a. Acid hydrolases increase clot
lysis; digest endocytosed
materials

o Platelet membrane
system
o Open canalicular system
- Serves as routes of delivery
or release of substances in
and out of the platelet
- Easily visible during platelet
shape change, contraction,
adhesion and aggregation
o Dense tubular system
- Derived from SER
- Shows positive staining for
platelet peroxidase activity in
accord with its role as a site
for arachidonic acid
metabolism within the
platelet
- Also functions as a calcium
sequestering pump,
providing low levels of
cytoplasmic calcium in the
resting platelet
- Contains thrombosthenin
(actin-myosin) that maintains
platelet shape
- Site of platelet prostaglandin
synthesis
- Control system for platelet
activation
- Calcium sequestering pump
(maintains low level Ca in
resting platelet); contains
phospholipase A2,
cyclooxygenase,
thromboxane synthase,
phospholipase C
Functions in Hemostasis
A. Platelet adhesion
- Stimuli for adhesion:
a.
Exposed
subendothelium
b.
Collagen
- requires factor VIII;
vWF for adhesion to
specific glycogen

receptor Ib and
subendothelium
o Types I and III- promote
adhesion, and facilitate
aggregation and release
o Types IV and V- promote
adhesion only
Abnormalities:

Bernard- Soulier Syndromeabsence of glycoprotein

receptor Ib
Von Willebrands Diseasedefective or absence of vWF
glycoprotein
o Fibrinectin
o Basement membrane
B. Platelet release reaction
1. Sources of constituents being
released:
a. Alpha Granules (releases its
constituents even with
relatively weak stimuli)
b. Dense Granules
c. Lysosomes
2. Stimuli that cause platelets to
release their granular contents
a. Collagen
Epinephrine
b. Thrombin

c.
d. Thromboxane

A2

C. Platelet Aggregation
1. Stimuli that promote
aggregation
a. ADP
b. Thrombin
c. Thromboxane A2
d. Collagen
e. Epinephrine
2. Morphologic changes in
platelets upon activation:
a. Change in shape

b. Extend pseudopods
c. undergo internal contraction
(centralization) of alpha and
dense granules, and release
their constituents
d. Alteration and formation of
membrane glycoprotein
receptors for fibrinogen and
vWF
3. Three important mediators of
aggregation:
a. ADP- released from dense
grabules atimulated by
collagen, apinephrine and
TXA2
- causes secondary,
irreversible platelet
aggregation
- depends on specific
glycoprotein receptor IIb-IIIa
for aggregation

Glanzzmans thrombastheniaabsence of glycoprotein receptor

IIb-IIIa

b. Thrombin- (1) Increase ADP

release; (2) activates platelet
phospholipases to increase
TXA2 sythesis; (3) increase
coagulation by platelet
activation
c. Thromboxane A2 (TXA2)promotes secondary platelet
aggregation; potent
vasoconstrictor
Platelet Function
o Adhesion
o Activation
o Secretion
o Aggregation
I.

Platelet Adhesion
High shear rate (in the
microvasculature):

minor injuries (sloughing of

senescent endothelium)
- Occurs in capillaries and
arterioles
- site of injury is carpeted by von
Willebrand factor
- GP In/IX/V receptor of platelet
binds to vWF
- vWF binds platelet to the
subendothelium
Low shear rate (eg Aorta)
- Vessel damage is extensive
- Mediated by fibrinogen
Binds platelets to the other
platelets
Attaches to GpIIb/IIIa
(integrin receptor)
Adhesion to ECM is mediated
primarily by von Willebrand factor
(vwf)
vWF bridges platelets & ECM
platelet glycoprotein Ib also
IIb/IIIa
von Willebrand Factor
large complex multimetric
protein
synthesized by endothelial
cells and MKs
- Weibel-Palade bodies in EC
- Alpha granules in platelets
Carries coagulation factor VIII
-

II.

Platelet Activation
Adhesion process activates
platelet
Cause platelets to change
shape
Extend pseudopods
Undergo internal contraction
Results in centralization of
alpha and delta granules
Activated platelets
- Release potent platelet
aggregating agonist and
vasoconstriction

Thromboxane A2
(TXA2), platelet
activating factor (PAF)
Platelet activation pathway
(picture)
Platelets- release reaction
- Initiated by agonist binding
to platelet surface receptors
followed by intracellular
phosphorylation
- Contents of granules are
secreted
ADP mediates
aggregation
Ca++ important for
coagulation
- Synthesis of thromboxane A2

III.

Platelet Aggregation
Occurs through activation and
recruitment of additional
platelets
- Thromboxane A2
- PAF
- ADP
- Serotonin
Enhanced by the generation of
thrombin (platelet agonist)
Primarily mediated by
fibrinogen
- Glycoprotein IIb/IIa
Formation of the primary
platelet plug
Stabilized by the formation of
fibrin
Platelets- aggregation
- ADP and Thromboxane A2
Autocatalytic reaction
Enlarging platelet
aggregate
- Primary hemostatic plug
- Platelet contraction
Secondary hemostatic
plug

Arachidonic Acid Metabolites

(Eicosanoids)

Cyclooxygenase (COX) Pathway

o TXA2
Major product in
platelet
- Vasoconstriction
- Platelet
aggregation
o PGI2
Vasodilation
Inhibit platelet
aggregation
o PGD2, PGE2, PGF2alpha
Vasodilation
Potentiates edema
formation
o PGE2
Augments pain
sensitivity
Fever production
Lipoxygenase pathway
o LTB4
Potent chemotactic
agent
Neutrophil aggregation
o LTC4, D4, E4
Vasoconstriction
Bronchospasm
Inc. vascular
permeability

Lipoxins
- Transcellular biosynthesis
- Natural andogenous negative
regulator of leukotriene actions
- LXA4, LXB4
- Actions:
o Vasodilation
o Antagonize LTC4-stimulated
vasoconstriction
o Inhibit neutrophil chemotaxis
and adhesion
o Stimulate monocyte adhesion
Regulators of AA metabolism:
NSAIDs and Aspirin
- Inhibit all cyclooxygenase
(COX-1 and COX-2) activity

COX-1 expressed in gastric

mucosa protects against
acid-induced damage
inhibition predispose to
gastric ulceration
Glucocorticoids
- Inhibit phospholipase A2
-

o Clot
-

Cell Damage

Cell Membrane
Phospholipids

5Cell
Lipoxygenas
e

Arachidonic
Acid

Laboratory evaluation of Platelets

A. Platelet Estimate (Peripheral Blood)
- Approx. 10-40 RCBS/Platelet
- 100 RBC/ 3-10 Platelets (OIO)
- 140-400x10 9/L
B. Manual Platelet counts
- Tocantins method (light
microscopy)
Rees-Ecker dil. Fluidcitrate formaldehyde
buffer with brilliant
cresyl blue stain
- Phase-contrast microscopy
method
1% ammonium oxalate
dil fluid
C. Automated method

Platelet adhesion
o Bleeding time
- Sensitive to
abnormalities of
platelet numbers and
function, to plasma
VIII:vWF def, to

abnormalities of vessel
wall comp.
Reference range: 2-9
minutes
retraction
Within 1 hr @ 37 C clot
will begin to shrink and
retract from walls of
the tube
Maximal at 24 hrs
Process dependent on:
number of platelets,
presence of
Steroi
Ca and ATP,
ds
normal conc.
of
Cyclooxygen
ase Cell

NSAID
Leukotrienes Prostaglandi
LTC4, D4, E4 ns
Prostacyclins

fibrinogen and normal

fibrin
- Abnormal in
thrombocytopenia, low
or abnormal
fibrinogens,
paraproteinemias,
Glanzmanns
thrombsthenia
Platelet aggregation
o Platelet aggregomentry
- Aggregating agents
added to PRP induce a
shape change and
aggregation of platelets
- PRP changes from
turbid suspension to
one the transmits more
light as aggregates are
formed

Platelet aggregometer
measures and records
change in light
transmission
o Aggregating agents;
- ADP
- Epinephrine
- Collagen
- Thrombin
- Ristocetin
- Arachidonic acid
o Von Willebrand Factor assay
-

Agglutination of fixed
platelets in response to
ristocetin depends on
the presence of vWF in
plpasma
The rate of
agglutination is
proportional to the
amount of vWF
present.