Platelet

Platelets or thrombocytes are minute discs 1-4 micrometer in diameter which are formed from
megakaryocytes in the bone marrow. They fragment into the minute platelets either in the bone
marrow or shortly after released into blood. Normally there is 150k-300k platelets in blood. Platelets
don’t have nuclei and can’t reproduce. In their cytoplasm there is actin, thrombosthenin and myosin
which are contractile proteins and can cause the platelet to contract. There’s also residual endoplasmic
reticulum and golgi apparatus which synthesizes various enzymes and store large quantity of calcium
ions. It also has a mitochondria to produce ADP and ATP, enzyme systems that produce prostaglandin
that causes vascular and local tissue reactions, fibrin-stablizing factor, and a growth factor that causes
many cells (vascular endothelial cells, vascular smooth muscle, fibroblasts) to multiply and grow which
helps repair damaged vessel walls.

On the surface of a platelet is a coat of glycoprotein that repulses adherence to normal endothelium but
causes adherence to injured areas of the vessel wall (especially injured endothelial cells and exposed
collagen from deep within the vessel wall). Platelet membrane also contain large amount of
phospholipid that activates multiple stages in blood clotting process. The platelet has a half life in blood
of 8-12 days, after it doesn’t function anymore it is eliminated from circulation by tissue macrophage
system (more than half occur in the spleen).

Hemostasis
Hemostasis is the prevention of blood loss by several mechanisms such as vasoconstriction, platelet plug
formation, blood clot formation, and then growth of fibrous tissue.

I. Vascular constriction

Immediately after blood vessel has been ruptured, the trauma causes the smooth muscle wall to
contract and vasoconstriction occurs. The constriction occurs from local myogenic spasm, local
autacoids factors from traumatized tissue and blood platelets, as well as nervous reflexes. The nervous
reflex is initiated by pain nerve impulses, but the constriction is mainly from local myogenic spasm. The
platelets release a vasoconstrictor substance called thromboxane A2 which is the main mechanism in
smaller vessels. The more severe the trauma, the greater the vascular spasm.

II. Formation of platelet plug

If the cut in the blood vessels is very small, then the cut is often sealed by a platelet plug rather than a
blood clot. When platelet come into contact with damaged vascular surface (especially collagen fibers),
they change their own characteristics rapidly. They begin to swell and assume irregular froms (with
pesudopods protruding from their surface). Their contractile proteins also contract forcefully which
causes release of granules that contain multiple active factors. They become sticky to the exposed
collagen in the tissues and to a protein called von willebrand factor that leaks into the traumatized
tissue from plasma. They also generate ADP and thromboxane A2, which act on nearby platelets to
activate them as well and these additional platelets then stick to the original activated platelets. They
continue to stick, creating the platelet plug. This plug is loose at first, but can block the blood loss if the
opening is small. After that begins the blood coagulation where fibrin threads form, which attach tightly
to the platelets and construct an unyielding plug. Ruptures in very small blood vessels happen thousands
of times daily, so this mechanism is very important

III. Blood coagulation

The blood clot develops in 15-20 seconds if the trauma to the vascular wall has been severe, and in 1-2
minutes if the trauma has been minor. Activator substances from the traumatized vessel wall, platelets,
and blood proteins adhering to traumatized vascular wall initiate the clotting process. Within 3-6
minutes the entire opening is filled with clot if it isn’t too large. After 20 minutes-1 hour, the clot retracts
which closes the vessel even further. Once a clot has been formed it can either be invaded by fibroblasts
(forming connective tissue all through the clot) or it can dissolve. When the clot forms in a small hole it
is usually invaded by fibroblasts a few hours after the clot is formed (promoted partially by growth
factor secreted by platelet). When excess blood has leaked into the tissues and they have formed where
they aren’t needed, special substances within the clot itself usually become activated and they function
as enzymes to dissolve the clot.
Mechanism of blood coagulation

More than 50 important substances that affect blood coagulation have been found in the blood, and
come of them promote coagulation (procoagulants) and some inhibit coagulation (anticoagulants).
Normally in the bloodstream anticoagulants predominate so blood doesn’t coagulate when it is
circulating. However when a vessel is injured procoagulants from the area of tissue damage become
activated and override the anticoagulants which causes the clot to form.

3 essential steps:

 In response to rupture of the vessel, a complex cascade of chemical reactions occur in the blood
involving several coagulation factors. The result is formation of a complex of activated
substances collectively called prothrombin activator.
 The prothrombin activator catalyzes conversion of prothrombin into thrombin
 Thrombin acts as an enzyme to convert fibrinogen into fibrin fibers that enmesh platelets, blood
cells, and plasma to form the clot.

II. Clotting

A. Conversion of prothrombin to thrombin

First, prothrombin activator is formed as a result of

rupture of the blood vessel or damage to special
substances in the blood. Second, the prothrombin
activator in presence of sufficient amount of calcium
ion causes conversion of prothrombin to thrombin.
Third, thrombin causes polymerization of fibrinogen
molecules into fibrin fibers within 10-15 seconds. The
limiting factr in blood coagulation rate is usually the
prothrombin activator and not the reactions after that
because they can occur rapidly. Platelets also play
important role because prothrombin first attaches to
prothrombin receptors on the platelets bound to
damaged tissue.

Prothrombin is a plasma protein (α2 globulin)that’s present in a concentration of 15 mg/dL. It’s an

unstable protein that can split easily into smaller compounds, one of which is thrombin. Prothrombin is
formed continually by the liver, continuously used. If the liver fails to produce prothrombin, in around a
day the prothrombin in blood will be too low to start normal blood coagulation. Vitamin K is required by
the liver for normal activation of prothrombin and other factors. So, lack of vit K or liver disease can
decrease prothrombin.
B. Conversion of fibrinogen to fibrin

Fibrinogen is a protein that’s formed in the liver, and liver disease can decrease the concentration of
circulating fibrinogen. It usually doesn’t leak from the blood vessel so tissue doesn’t coagulate. But,
when there’s a pathologically increased permeability of the capillary fibrinogen can leak causing clotting
of fluid in tissues. Thrombin acts on fibrinogen to remove four low-molecular-weight-peptides from each
fibrinogen molecule, creating a fibrin monomer that has the capability to polymerize with other fibrin
monomers to form fibrin fibers. These fibers constitute the reticulum of the blood clot. In early stages of
polymerization the fibrin monomers are held together by weak noncovalent hydrogen bonding, and
newly formed fibers cross-link with one another so the clot is still fragile. Fibrin-stabilizing-factor is
present in plasma globulins and released from platelets entrapped in the clot. It must first be activated
before it can work on the fibrin. After activated, it operates as an enzyme to cause covalent bonds
between the fibrin monomers, thus strengthening the meshwork.

The clot is composed of a meshwork of fibrin fibers running in all directions and entrapping blood cells,
platelets, plasma. The fibers also adhere to damaged surfaces of the blood vessel. After a few minutes,
the clot begins to contract and expresses most of the fluid within the clot in 20-60 minutes. The fluid
expressed is called serum (there’s no fibrinogen and most other clotting factors). Platelets are needed
for clot retraction, so if it occurs we can assume that the platelet number might be low. Platelet also
contribute to clot contraction by activating its contractile proteins (activated and accelerated by
thrombin as well as Ca ions). A blood clot has a positive feedback on its surroundings to create more
clots as it usually extends within minutes. The reason is the proteolytic action of thrombin, which can
convert more prothrombin into thrombin,, accelerate action of factors, and aggregation of platelets.
Thus the blood clot continues to grow until blood leakage ceases.

I. Initiation of coagulation (formation of prothrombin activator)

Damage to vascular wall, adjacent tissues, and blood itself causes the formation of prothrombin
activator which eventually initiates blood coagulation. Prothrombin activator is formed by the extrinsic
pathway that begins with trauma to the vascular walls and surrounding tissue, and the intrinsic pathway
that begins in the blood. In both pathways different plasma proteins called blood-clotting factors play a
major role. Most of these proteins are inactive forms of proteolytic enzymes. When they are activated,
they cause the enzymatic reactions for the coagulation cascade to occur.

A. Extrinsic pathway for initiating clotting

It begins with a traumatized vascular wall or traumatized extravascular tissues that come in contact with
blood.

 Traumatized tissue releases several factors called tissue factor or tissue thromboplastin. This
factor is composed mainly of phospholipids from the membranes of tissue plus a lipoprotein
complex that functions mainly as a proteolytic enzyme
 The lipoprotein complex of tissue factor further complexes with factor VII into factor VIIa, which
in the presence of calcium ions activates factor X into activated factor X (Xa)
  The activated factor Xa combines with tissue phospholipids which are part of tissue factor or
with additional phospholipids released from platelets and also factor V to form the complex
called prothrombin activator. In a few seconds, in the presence of calcium ions prothrombin is
split to form thrombin. At first the factor V in prothrombin activator isn’t activated yet, but
activated factor X is the actual protease that causes splitting of prothrombin to form thrombin.
Proteolytic action of thrombin then activates the factor V, which greatly accelerates the
protease activity of prothrombin activator. Platelet phospholipids also act as a vehicle that
speeds up the process even more. So, there is clearly a positive feedback mechanism with factor
V.

B. Intrinsic Pathway

This process begins with trauma to the blood or exposure of blood to collagen from traumatized
vascular wall.

 Blood trauma causes activation of factor XII and release of platelet phospholipids. When factor
XII is disturbed, such as by coming into contact with collagen or other things, it takes on a new
molecular configuration into its activated form (XIIa).The blood trauma also damages platelets
because of adherence to collagen and this releases platelet phospholipids that contain the
lipoprotein called platelet factor 3
 Factor XIIa acts enzymatically on factor XI to activate this factor into factor XIa. This reaction
requires high-molecular-weight kininogen and is accelerated by prekallikrein
 Factor Xia acts enzymatically on factor IX to activate it into factor IXa
 Factor IXa works with factor VIIa, platelet phospholipids, and factor III from traumatized
platelets to activate factor X. When factor VIII (antihemophilic factor) is deficient, then this step
is also deficient (hemophilia).Obviously when there’s thrombocytopenia, then there’s also a
deficiency of factor III.
 The same as the extrinsic pathway, where factor Xa combines with factor V and phospholipids to
form the complex called prothrombin activator.
Role of calcium ions in intrinsic and extrinsic pathways is required in the first two steps of the
intrinsic pathway and also for promotion or acceleration of all the blood clotting reactions. So, in the
absence of calcium ions, blood clotting by either pathways don’t occur. In the living body the
calcium ion concentration seldom falls low enough for there to be a problem with coagulation
significantly, but when blood is taken from a person usually the calcium ions are reduced to prevent
clotting like by causing it to react to substances such as citrate ion or oxalate ion.

Fibrinolysis
Plasma protein contain an euglobulin called
plasminogen (profibrinolysin) that when activated
becomes a substance called plasmin (fibrinolysin).
Plasmin is a proteolytic enzyme resembling trypsin.
Plasmin digests fibrin fibers and other protein
coagulants such as fibrinogen, factor V, factor VIII,
prothrombin, and factor XII. So, when plasmin is
formed it can cause lysis of the clot, sometimes even
causing hypercoagulability in the blood. When a clot
is formed, large amount of plasminogen is trapped in
the clot along other plasma proteins. The injured
tissues and vascular endothelium slowly release
 tissue plasminogen activator (t-PA). A few days after the clot has stopped the bleeding, t-PA
converts plasminogen into plasmin which in turn removes the unnecessary blood clot.

Intravascular antigoagulants
 Endothelial surface factors  smoothness of endothelial cell surface (prevents contact
activation of intrinsic pathway), layer of glycocalyx on endothelium (repels platelets and
clotting factors), thrombomodulin (protein bound with endothelial membrane), which binds
thrombin. This slows the clotting process by removing thrombin, and the thrombomodulin-
thrombin complex also activates protein C (a plasma protein that inactivates factor V and
factor VIII)
 Antithrombin action of fibrin (during a clot formation, around 85-90 percent of the thrombin
becomes absorbed to the fibrin fibers which prevent excessive spread of the clot) and
antithrombin III (antithrombin-heparin cofactor). The remaining thrombin that isn’t
absorbed by the fibrin fibers then combines with antithrombin III
 Heparin is used widely as a pharmacological agent to prevent intravascular clotting.
Normally its concentration is so low that it only affects coagulation significantly in special
physiological conditions. By itself, it has little or no anticoagulant properties. When
combined with antithrombin III, the effectiveness for removing thrombin increases a
hundredfold to a thousandfold, ths having an anticoagulant effect. The complex also
removes other coagulation factors such as factor XII, XI, X, and IX. It’s produced by
basophilic mast cells in the pericapillary connective tissue throughout the body (mainly
lungs and to a lesser extent the liver).

Conditions that cause excessive bleeding

1. Vitamin K deficiency

Almost all the blood clotting factor are formed by the liver. So, diseases of the liver can depress the
clotting system so greatly that a patient has a tendency to bleed. Another cause of depressed clotting
factor formation by the liver is vit k deficiency. Vit k is an essential factor to a liver carboxylase that adds
a carboxyl group to glutamic acid residue on prothrombin, factor VII, factor IX< factor X, and protein C.
In absence of vit k the production of these factors is insufficient. In GIT disease there might be
abnormality to vit K absorption (it’s a fat so there is abnormal fat absorption). Failure of liver to secrete
bile can also cause its deficiency, because it prevents adequate fat digestion and absorption

2. Hemophilia

IT occurs almost exclusively in males, and 85% of cases there is a deficiency or abnormality in factor VIII
(hemophilia A/classic hemophilia) while the rest there is deficiency in factor IX (hemophilia B). This
disease is passed down by the female X gene. There are various degrees of severity, and usually bleeding
doesn’t occur except after trauma.

3. Thrombocytopenia
The presence of low platelets number in circulating blood, causing a tendency to bleed. Usually bleeding
is from smaller vessels unlike hemophilia, with petechia and purpura as manifestations. The name of the
disease is thrombocytopenic purpura. Usually bleeding won’t occur until platelet number fall below
50000.

Thromboembolic conditions
An abnormal clot that develops in a blood vessel is called a thrombus. Once it’s formed, continued blood
flow past the clot is likely to break it away from attachment and cause it to block a smaller vessel,
named emboli. The cause can be a roughened endothelial surface of a vessel because of atherosclerosis,
infection, trauma so it’s likely to initiate clotting, or the blood flow might be flowing very slowly causing
it to form clots within. Rt-PA is usually used to break a thrombus or emboli.

1. Femoral venous thrombosis and massive pulmonary embolism

Clotting almost always occurs when blood flow is blocked for any hours , so the immobility of a patient
confined to a bed or propping the knees with pillows often cause intravascular clotting. The clot also
slowly grows long the leg vein flow direction. About every 1 out of every 10 times a large part of the clot
disengages and flows freely, which might block the pulmonary arteries causing massive pulmonary
embolism.

2. Disseminated intravascular coagulation

Clotting mechanism occasionally becomes activated in widespread areas of the circulation, giving rise to
DIC. This condition can often result from large amount of dying or traumatized tissue in body, releasing a
lot of tissue factor into the blood. The clots are usually small but numerous. The process can especially
occur in a person with sepsis.

Anticoagulants
1. Heparin is extracted from animal tissues and causes blood clotting time to increase from normally 6
minutes to 30 or more minutes. This clotting time change occurs instantly, and the action lasts 1.5-4
hours. The injected heparin is destroyed by heparinase enzyme in the blood

2. Coumarin (warfarin) can drop levels of active prothrombin, factor VII, factor IX, factor X which are
produced in the liver.Warfarin inhibits enzyme VKOR c1( vitamin K epoxide reductase complex 1). This
enzyme converts the inactive oxidized form of vit k into its active reduced form. The factors continue to
be produced but with greatly reduced coagulant ability as they are in their inactive forms. After
administration the coagulation ability drops to about 50% by 12 houurs, and to about 20 % in 24 hours.
The coagulation process isn’t immediately blocked but must wait the degradation of active prothrombin
and other factors already in the plasma. They usually return to normal in 1-3 days after discontinuing
coumarin therapy.

Lab Test
1. Bleeding time is done when a sharp pointed knife is used to pierce tip of fingeror lobe of the ear,
where the bleeding normally lasts for 1-6 minutes. The time depends on depth of the wound and degree
of hyperemia. IT’s especially prolonged in lack of platelets

2. Clotting time is done by collecting blood in a chemically clean glass test tube and then tip it back and
forth every 30 seconds until the blood has clotted. Normally the clotting time is 6-10 minutes.

3. Prothrombin time and international normalized ratio

Prothrombin time gives an indication to concentration of prothrombin in the blood. The blood removed
from the patient is immediately oxalated so that none of the prothrombin can change to thrombin.
Large excess of calcium ions and tissue factor is then mixed with the blood, nullifying effect of oxalate
and causing the prothrombin to turn into thrombin by means of the extrinsic pathway.The normal
prothrombin time is about 12 seconds.

Prolonged in :
a. Levels of factors V, VII, X falls below 50 % and when
prothrombin level are less than 30 % or normal.
b.The fibrinogen level is less than 100 mg / dl.
c.Severe hepato cellular disease
d.DIC
e. Vit K deficiency
f. Excess Warfarin therapy

This also depends on the type of tissue factor used, so a INR was devised to standardize measurements
of prothrombin time by international sensitivity index (ISI) provided by the manufacturer. Normally INR
in healthy person is 0,9-1,3. High inr (4-5) indicates high risk of bleeding, while a low one(e.g: 0.5)
suggests that there’s a chance of having a clot. Patients with warfarin therapy usually have inr 2-3.

𝑃𝑇 𝑡𝑒𝑠𝑡 𝐼𝑆𝐼
𝐼𝑁𝑅 = ( )
𝑃𝑇 𝑛𝑜𝑟𝑚𝑎𝑙

4. Platelet count  normally 150k-300k

Spontaneous bleeding rare in platelet count above 20k, surgical bleeding can happen if platelet count is
40k-70k

5.ACTIVATED PARTIAL THROMBOPLASTIN TIME

- Measured the integruty of the entire intrinsic pathway of coagulation --- Sensitive to
deficiency of all factors other than VII AND XIII
- Prolong if any of intrinsic or common path way factors decrease.
- Also prolong in presence of certain anticoagulants and is used to monitoring heparin
therapy.

-aPTT + PT = clotting time

6. Fibrinogen level --> decreased in DIC and severe liver disease

7. Rumpel-Leede test or Hess test or Torniquet test or

Percobaan Pembedungan

Used to assess capillary fragility.

- Pressure is applied to the forearm with a blood pressure cuff inflated to
between systolic and diastolic blood pressure for 10 minutes.
- After removing the cuff, the number of petechiae in a 5cm diameter circle of
the area under pressure is counted.
- Normally less than 10 petechiae are seen.
- Ten or more petechiae indicate capillary fragility, which occurs due to poor
platelet function, bleeding diathesis or thrombocytopenia, and can be seen
in cases of scurvy, and Dengue fever.

8. Thrombin Time (TT) or Thrombin Clotting Time (TCT).

Test use to examine time the conversion of fibrinogen to fibrin but is also sensitive to the presence of
inhibitors that may be present in the plasma e.g. heparin.
Pro long TT :
- Fibrinogen level less than 100mg/ml
- Abnormal fibrinogen function
- Present innhibitor thrombin ( For example : Heparin or FDP )

The thrombin time is used to diagnose bleeding disorders and to assess the effectiveness of fibrinolytic
therapy.
Reference values for thrombin time are 10 to 15 seconds or within 5 seconds of the control.

Clinical approach