BLOOD PLATELETS
(THROMBOCYTES)
• Blood platelets, or thrombocytes, are nonnucleated cytoplasmic fragments derived from
megakaryocytes in the bone marrow.
• They have a discoid biconvex shape, 2- 4 µm
in diameter and 0.5-1.0 µm in thickness.
• The normal platelet count is 150- 400 x
103/mm3 of blood. They contain actin and
myosin filaments which could contract and
shrink
the
size
of
the
platelets.
Platelets (cntd)
• Platelets remain in the circulation for 810 days. They are removed by the
monocyte-macrophage
system,
particularly in the spleen.
• About one third of the platelets in the
body are found in the spleen.
Splenectomy increases the blood platelet
count.
Platelets (cntd)
• The production of blood platelets
(thrombocytopoiesis) is stimulated and
controlled by a group of growth factors
which
include
the
granulocyte
macrophage-colony stimulating factor
(GM-CSF), and a group of interleukins
(IL-1, 3, 4, 5, 6).
• These growth factors are produced by Tlymphocytes as well as other cells.
Functions of the platelets
. Haemostasis:
• Platelets are important for haemostasis.
This is because:
• They aggregate to form platelet plug,
which stops bleeding from small wounds.
• They release serotonin and thromboxane
A2 which constrict local blood vessels.
1
Functions of the platelets (cntd)
• They release platelet factor 3 (PL3) which
activates blood clotting factors X and II.
• They release von Willebrand factor which
regulates the circulating level of clotting
factor VIII.
• They release platelet factor 4 (PL4) which
neutralizes heparin.
• They release clotting factor V
Functions of the platelets (cntd)
• 2. Clot retraction: After fibrin is formed,
platelets get attached to fibrin fibers then
contract leading to shrinking of the clot.
• 3. Wound healing: Platelets release a
polypeptide called “Platelet derived growth
factor - PDGF”, which stimulates wound
healing. This substance is also released by
tissue macrophages and endothelial cells.
Abnormalities in platelet number
and function
An increase in the number of platelets is called
“thrombocytosis” or “thrombocythemia “ It
occurs in pregnancy, after hemorrhage, and
after surgical operations. The risk of
thrombosis is increased.
• A decrease in the number of platelets is called
“thrombocytopenia “. It occurs in cases of
aplastic anemia, pernicious anemia, and during
menstruation. The platelet haemostatic function is
defective. The condition could lead to
thrombocytopenic
Purpura.
• When the number is normal but the platelets are
unable to carry out their function properly the
condition is called “thrombasthenia”, It is a
congenital
condition
which
leads
to
thrombasthenic purpura
Haemostasis
• Haemostasis
involves
in sealing of
from
means stoppage of bleeding. It
all
reactions
involved
a wound to prevent loss of blood
the
cut
vessels.
Haemostatic reactions
• Injury of a blood vessel initiates the following
haemostatic
reactions:
1. Vascular reactions.
2. Platelet reactions.
3. Blood clotting (also called blood coagulation).
1. Vascular reactions
• Vascular reactions aim at constricting or even
obliterating the injured vessels. They include:
• (a) Inward rolling of the inner layers of the blood
vessel.
(b) Elastic retraction of the vascular wall.
• (c) Vasoconstriction, which is caused by:
i.
Reflex sympathetic discharge because
of pain.
ii.
Local myogenic contraction of the
vascular wall in response to trauma.
iii. Serotonin (5-hydroxytryptamine; 5-HT) and
thromboxane A2 which are released from the
platelets.
• These reactions are sometimes so powerful so as
to constrict a vessel as large as the radial artery
and obliterate it completely for several minutes
after vascular injury.
Haemostasis
• 2. Platelet reactions
• (a) Adhesion: Adhesion is the sticking of the
platelets to the exposed sub endothelial collagen
fibers.
This makes the platelet wall more
“sticky”.
• (b) -Release:
• After adhesion, the platelet membrane allows the
release of the following substances:
• Serotonin which potentiates vasoconstriction.
• Thromboxane A2 which potentiates the
vasoconstriction and platelet aggregation.
• ADP (adenosine diphosphate) which potentiates
platelet aggregation.
• Platelet factor 3 (PL3): a group of
phospholipids which provide a catalytic
surface for activation of the clotting factors X
and II.
• Platelet factor 4 (PL4): a heparin-neutralizing
factor.
• Clotting factor V.
• Von Willebrand factor which is important for
initiating the platelet aggregation process.
• (c) -Aggregation:
Aggregation is the
adhesion of platelets to each other.
• The aggregated platelets make a “platelet
plug” seals the injured vessel
3. Blood clotting (Blood Coagulation)
• There are 14 plasma factors plus one platelet
factor involved in the process of blood
clotting.
Factor
Names
I
Fibrinogen
II
Prothrombin
III
Tissue thromboplastin (TPL)
IV
Calcium
V
Labile factor, Proaccelerin
VII
Stable factor, proconvertin
VIII
Antihaemophilic globulin (AHG)
IX
X
Christmas factor
Stuart-Prower factor
XI
XII
Pl. thromboplastin antecedent
(PTA)
Hageman factor, contact factor
XIII
Fibrin-stabilizing factor
HMW-K
High-molecular-weight
kininogen
Kallilikrein
Platelet
factor
(phospholipoprotein)
Ka
PL3
3
• With the exception of factors III (tissue
thromboplastin) and IV (Ca2), all the
plasma clotting factors are proteins made
by the liver.
• The platelet factor (PL3) is mentioned
above. There is no factor VI. Blood
clotting occurs in three steps as follows.
• (i) Activation of factor x
• This step occurs through either of two
pathways. The “intrinsic pathway” is a
pathway where all the needed clotting factors
are found in the blood.
• They are found inactive and only need
activation .
• The “extrinsic pathway” is a pathway where
clotting is initiated by a factor not normally
found in the blood but provided by tissue
extract released by injured tissues.
• This factor is a protein-phospholipid mixture
called “tissue thromboplastin “.
• The process of activation of factor X proceeds
as shown below.
Stages of Blood Coagulation
a = activation
+ = thrombin induces formation of more thrombin
• The intrinsic mechanism starts with contact
activation of factor XII when it gets in contact
with the sub endothelial collagen of the injured
vessel.
• This contact activate factor XII to active factor
XII
(XIla).
Some proteolytic fragments of XIIa act as
prekallikrein activator which converts plasma
prekallikrein (a β-globulin made by the liver)
into active kallikrein.
• This step is catalyzed by high molecular
weight kininogen (HMWK, an α-globulin
made by the liver).
• kallikrein in the presence of HMWK,
catalyzes further activation of factor XII.
Factor XII and kallikrein go on auto
activating each other.
• In vitro, factor XII is activated by contact
with electronegative water- wettable
surfaces such as that of glass.
• When factor XIIa is formed, it starts a cascade
enzymatic reaction, where factor XIIa activates
factor XI to XIa, then factor XIa activates
factor
IX
to
IXa.
• Factor VIII is found in a complex with von
Willebrand
factor
(a
protein
made vascular endothelial cells and
megakaryocytes) in an inactive form.
• When it is released from that complex, it
becomes
activated
→
VIlla.
• Factor IXa releases factor VIII from the
complex.
• Next, Factors VIlla and IXa combine to make
a VIlla/IXa complex. This complex in the
presence of Ca2 and PL3 activates factor X to
Xa.
• The extrinsic mechanism starts when
tissue thromboplastin (TPL - Factor III) is
released in the juice of the injured tissues.
• TPL activates factor VII to VIla. Factor
VIla in the presence of Ca2, PL3, and
TPL activates factor X to Xa.
(ii). Activation of prothrombin to
thrombin
• Active factor X (Xa) converts the inactive
prothrornbin (factor II) into the active thrombin
(factor IIa).
• The reaction is accelerated 100,000 times in the
presence of PL3, Ca2, and active factor V (Va).
• When thrombin is formed it activates V to Va,
then Va activates more prothrombin to thrombin
by an autocatalytic mechanism.
• Thrombin also helps platelet aggregation,
activation of factor VIII and factor XIII.
(iii). Formation of fibrin clot
• Thrombin acts on fibrinogen (factor I) to form
fibrin monomer.
• The fibrin monomer makes a soft, unstable clot
which is soluble in urea solutions.
• The activated fibrin-stabilizing factor (XIIIa)
catalyzes the polymerization of the fibrin
monomer into a firm, stable clot which
is Insoluble in urea solutions.
Remember
• Blood clotting is a plasma function. Neither the
RBCs nor the WBCs are needed.
• The extrinsic pathway for activation of factor X is
a rapid one that takes 6-10 seconds. The intrinsic
pathway is a slow one that takes 6-10 minutes.
• Steps (ii) and (iii) are the same for intrinsic and
extrinsic pathways of clotting. They are rapid
reactions that take 10-12 seconds.
• Factors I, V, VIII and XIII are consumed during
clot formation, but other factors act only as
catalysts.
Which pathway is taken in vivo, intrinsic
or extrinsic?
• This depends on the type of wound.
• In clear cut wounds with minimal tissue
damage (e.g. surgical wounds), the intrinsic
mechanism predominates.
• In wounds with excessive tissue damage
(crush injuries), the extrinsic mechanism
predominates.
Clot retraction {serum formation}
• lf clotted plasma is left for few minutes it
retracts (shrinks), squeezing out a yellowish
clear fluid called “serum”.
• Serum is the remaining fluid of the plasma
after clotting.
• It is plasma without factors I (fibrinogen), V,
VIII, and XIII.
• Platelets are essential for clot retraction to
occur. Clot retraction is induced by platelets.
Failure of retraction is an indicator that the
number of platelets in the circulating blood is
low.
• The main function of clot retraction is to
reduce the size of any intravascular clot
(thrombus) to restore the patency of the blood
vessel which was blocked by the thrombus.
Fibrinolysis (clot lysis)
• Fibrinolysis (i.e. dissolution of the clot) starts
shortly after the blood clot is formed.
• Tissue plasminogen activator is a polypeptide
secreted by the endothelial cells.
• It converts the inactive circulating α-globulin
“plasminogen” (profibrinolysin) into the active
proteolytic enzyme “plasmin” (fibrinolysin,).
• Plasmin digests fibrin into smaller soluble
molecules called fibrin degradation products
(FDPs). These steps proceed as follows:
• During the formation of the clot, a large
amount of plasminogen is adsorbed to
fibrin and is entrapped inside the clot.
• Plasminogen activator diffuses into the
clot and activates plasminogen. Plasmin
digests the clot. FDPs inhibit thrombin,
thus preventing further blood clotting.
• The activity of the tissue plasminogen
activator (TPA) is controlled by a polypeptide
released by the endothelial cells called”TPA
inhibitor”.
• This inhibitor suppresses the activation of
plasminogen to plasmin and maintains the
normal coagulability of blood.
• Plasmin can also digest other plasma
clotting factors including factors V. VIII,
XII and prothrombin.
• The presence of free plasmin in the blood
could hinder the normal clotting system
of the blood leading to hypocoagulability.
Circulating plasmin is inactivated by
irreversible binding to α2-antiplasmin. a
circulating glycoprotein.
• The kidney produces a plasminogen
activator called “urokinase” which is
found in urine. It dissolves any clots that
may be formed in renal tubules and block
them.
• Streptokinase is another plasminogen
activator produced by streptococci
bacteria. It can be used clinically to
dissolve recently formed clots.
• Human TPA is produced now by
recombinant DNA techniques and is
available for clinical use.
• Importance of fibrinolysis: Fibrinolysis
dissolves minute clots which are frequently
formed in the small vessels. Without
fibrinolysis, a large number of small vessels
would have been occluded. In addition, after
sealing a wound, fibrinolysis slowly clears off
blood clots from the tissues to help wound
healing, and allow reopening of the blocked
vessels.
Some important factors in blood clotting
platelets
• Platelets
are
important for haemostasis
because:
(a) They form a platelet plug that seals the cut
vessel.
(b) They release vasoconstrictor substances
(serotonin and thromboxane A2).;
• (c) They release phospholipids (Platelet factor
3) which are essential for blood clotting.
Calcium ions
• Calcium ions are essential for blood clotting.
• Calcium concentration in plasma is 9 — 11
mg/dL. If it falls below 7 mg/dL, manifest tetany
occurs, which could be fatal.
• Blood clotting needs a calcium concentration of
only 4mg/dL.
• Therefore blood clotting cannot be prevented in
vivo by lowering plasma calcium level (e.g. by
injection of sodium citrate or EDTA), because the
patient would die of tetany before clotting is
prevented.
Vitamin k
• Vitamin K is essential for the formation of
clotting factors II, VII, IX and X by the liver.
• Vitamin K deficiency leads to deficiency of these
factors in blood and reduce blood coagulability.
• In obstructive jaundice (post-hepatic jaundice),
bile salts do not reach the intestine. This reduces
the absorption of fat and fat-soluble vitamins.
Lack of vitamin K absorption leads to vitamin K
deficiency, and blood hypocoagulability. Also in
chronic diarrhea, fat absorption is impaired →
impaired vitamin K absorption → vitamin K
deficiency → blood hypocoagulability.
Prostaglandins
• Thromboxane A2 (TXA2): This is a
prostaglandin released from the platelets during
the release reaction.
• It induces vasoconstriction as well as further
platelet aggregation and release → platelet factor
3 which promotes blood clotting.
• Aspirin inhibits the formation of TXA2
• This reduces the coagulability of blood. Small
daily doses of aspirin are useful in reducing the
risk of coronary or cerebrovascular thrombosis.
• Prostacyclin (PGI2): This is a prostaglandin
produced by the endothelium and smooth
muscles of the vascular wall.
• It suppresses platelet aggregation and produces
local vasodilatation.
• This limits the clotting process and prevents its
spread.
Factors that prevent spontaneous blood
clotting in vivo
• The presence of intact, smooth, healthy
vascular endothelium.
• The presence of a monomolecular layer of
mucopolysaccharide on the inner surface of the
vascular endothelium which repels the clotting
factors and platelets and prevents their
activation.
• The rapid flow of blood inside the blood
vessels. Slow blood circulation increases the
risk of contact activation and thrombosis.
• The clotting factors are present in an inactive
form.
• The presence of α2-macroglobulin which
combines with circulating
active clotting
factors to make inactive complexes.
• Heparin. However, the physiological levels of
heparin in blood have
insignificant
anticoagulant effect.
Factors that limit blood clotting
when it occurs
• Prostacyclin: released from the endothelium and
smooth muscles of blood vessels near the clot. It
prevents platelet aggregation.
• Fibrinolysis
• Fibrin and fibrin degradation products
(FDPs): They inhibit the action of thrombin.
Thrombin is adsorbed to, and inactivated by,
fibrin.
• This action of fibrin is referred to as
‘antithrombin I”
• This complex activates protein C, a circulating
plasma proenzyme. Active protein C (protein
Ca) is a protease which enhances fibrinolysis.
• Also, protein Ca combines with a coenzyme
called protein S. The complex inactivates
factors Va and VIlla, thus inhibiting thrombin
formation.
• Activation of protein C: Thrombin makes a
complex with a glycoprotein on the endothelial
cells surface called “thrombomodulin”.
Activation of protein C
and its actions to limit
the clotting process.
The
process
of
thrombin inactivation
by adhesion to fibrin is
called “antithrombin
I”. Antithrombin Ill is
activated by combining
with heparin. Active
Antithrombin
Ill
inactivates
clotting
factors XIIa, XIa, IXa,
Xa, and thrombin.
Factors that promote clotting in vivo
• Roughening of the endothelial surface of blood
vessels (e.g. atherosclerosis).
• Slowing of blood flow inside the vessels.
• Thrombocythemia (increased number of
platelets in blood), e.g. after surgical
operations.
• Admission of tissue extract into the blood
stream (e.g. crush injuries).
Anticoagulants
• Anticoagulants are substances or methods that
prevent
blood
clotting.
They
are classified into two categories:
• Anticoagulants in vitro:
• These are substances or methods which
prevent blood clotting outside the body
• I. Inactivation of Ca2+: This may be done by
adding:
• 1. Sodium, potassium, or ammonium citrate
which converts
in Ca2+ to non-ionized
calcicitrate salts.
• This is the most common anticoagulant in
vitro.
• ii. Sodium or potassium ethylenediamine
tetraacetate (EDTA) which convert Ca2+ into
non-ionized calcium EDTA.
• iii. Sodium oxalate which precipitates Ca2+
insoluble calcium oxalate.
• iv. A mixture of citrate, dextrose, and
phosphate (CD?):
• This mixture is used as anticoagulant by blood
banks. It has the advantage of preventing the
drop in 2, 3-DPG level in the stored blood.
• V. Sodium fluoride: Fluoride makes a
weakly dissociated calcium compound.
At the same time it is an enzyme poison.
It inhibits the action of the glycolytic
enzymes. It is useful in delaying
glycolysis if the blood sample is taken for
determining the blood glucose level.
• 2. Defibrination: Removal of fibrin by
stirring the blood with feather.
• 3. Prevention of contact activation: This is
done by collecting the blood in a siliconized,
or paraffin-lined container. This provides a
non-wettable, positively charged surface which
prevents contact activation of factor XII and
platelets. However, this anticoagulant process
is a temporary one that lasts only for about one
hour.
4.
Heparin: This is used as an
anticoagulant in vitro as well as in vivo.
5. Rapid cooling of the blood: This inhibits
all the enzymatic reactions of clotting.
Anticoagulants in vivo
•
•
•
•
(1).
Heparin:
taken as injection.
(2). Vitamin k antagonists:
These are derivatives of coumarin which are
taken orally. E.G. dicumarol and warfarin.
Heparin
• Heparin is a naturally occurring anticoagulant
found in the granules of the circulating
basophil leukocytes and mast cells which are
found in most tissues, particularly the lungs,
from which it extracted commercially.
• Its clinical importance is due to its use as an
anticoagulant drug.
• Heparin is used as an anticoagulant in vivo and
in vitro.
• The anticoagulant effect appears as soon as
heparin reaches the blood.
• Heparin is administered by injections
intravenously or subcutaneously, but never
intramuscularly because of the frequent
formation of hematomas at the site of
injection.
Abnormalities of haemostasis
hemophilia
Haemophilia is a hereditary X-linked, recessive
disease characterized by blood hypocoagulability
due to lack of certain clotting factors. A small
wound may result in excessive, serious bleeding.
The disease is transmitted by females to males,
but females do not suffer from it.
• So, females are the carriers but the males are the
sufferers of the disease.
• Females carrying the abnormal X-chromosome
transmit the disease to half their sons, while
half their daughters will be carriers.
• A hemophilic father will have normal sons but
carrier daughters.
• If a haemophilic man marries a haemophilic
carrier, which is a very rare coincidence, it is
possible in this case to have haemophilic
daughters.
Types of haemophilia
• Hemophilia A: Due to lack of factor VIII.
• Haemophilia B (Christmas disease): Due to
lack of factor IX.
• Parahaemophilia: Due to lack of factor V
Treatment of hemophilia:
• Hemophilia can not be cured, but it can be
temporarily corrected by transfusion of
purified preparations of the defective factor.
• In this case, normal coagulability is restored
for several days after each treatment.
Purpura
• Purpura is a disease characterized by easy
bruisability
and
spontaneous
small
hemorrhages (petichae).
• It is due to lack of platelet activity.
• The haemostatic platelet reaction is defective
• The skin of the patient shows many small
purplish blotches, caused by subcutaneous
small hemorrhages giving the disease the name
“purpura “.
• Blood clots are soft and poorly retracted.
There are two types of purpura:
• Thrombocytopenic purpura: It is due to a
decrease in the number of platelets in the
blood.
• Thrombasthenic purpura: It is due to
abnormally weak platelets.
• Platelet
number
could
be
normal
Tests of haemostasis
• bleeding time
• This is the time taken to stop bleeding from small
wounds without clotting. It tests the efficiency of
the vascular and platelet haemostatic reactions.
• A sharp, pointed scalpel is used to pierce the lobe
of the ear. The oozing blood is wiped off by a
filter paper every few seconds. The end point is
when bleeding stops.
• The normal bleeding time is 1-3 minutes. It is
prolonged in purpura, but is normal in
haemophilia.
• Clotting time
• This is the time taken by a blood sample to clot
in vitro. It is a test of the activity of the blood
factors involved in the intrinsic clotting
mechanism.
• A blood sample is taken in a chemically clean
glass test tube.
• The tube is then tilted back and forth every 30
seconds in a water bath at 37oC.
• The end point is when the blood clots. The
normal clotting time is 6-10 minutes. Clotting
time is markedly prolonged in hemophilia (112 hrs). It is also prolonged in vitamin K
deficiency, in thrombocytopenia and in
thrombasthenia.
• Prothrombin time
• This is the time taken by blood to clot by the
extrinsic mechanism. It is a test of the
efficiency of factors I, II, III, V, VII, X.
• A blood sample is collected on sodium oxalate.
Calcium chloride and tissue extract are added
to the oxalated blood, and then time is
measured until clotting occurs. The normal
prothrombin time is 15-20 seconds.
• Prothrombin time is prolonged in vitamin K
deficiency,
thrombocytopenia,
and
in
Parahaemophilia. It is normal in hemophilia.
THE END