Hemostasis and Blood Coagulation part 1

Hemostasis and Blood Coagulation

Events in Hemostasis
 Hemostasis: prevention of blood loss whenever a vessel is severed or ruptured.
 Mechanisms:
(1) Vascular constriction
(2) Formation of a platelet plug
(3) Formation of a blood clot as a result of blood coagulation
(4) Eventual growth of fibrous tissue into the blood clot to close the hole in the vessel
permanently

(1) First mechanism: Vascular Constriction

 Ruptured of blood vessel (by trauma) causes vascular smooth muscle contraction (vascular
constriction)
 Aim: instantaneous reduction in blood flow from the ruptured vessel to allow blood coagulation.
 Stimulus for contraction:
(1) Local myogenic spasm (major) – by direct damage to the vascular wall
(2) Local autacoids from the traumatized tissues (PGI2) and blood platelets (TXA2)
(3) Nervous reflexes (initiated by sensory impulses (e.g.; pain) from the traumatized vessel or
nearby tissues.
 Duration: many minutes or even hours to allow for platelet plugging and blood coagulation

(2) Second mechanism: Formation of the Platelet Plug

 Very small cut in blood vessel is sealed by a platelet plug
 Larger cut in blood vessel is sealed by blood clot
 Many very small vascular holes do develop throughout the body each day

Physical and Chemical Characteristics of Platelets

[Megakaryocytes fragment into the minute platelets either in the bone marrow or soon after entering the blood,
especially as they squeeze through capillaries]

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Mechanism of the Platelet Plug

 Platelet Plug: aggregation of platelets in the site of vascular openings (vascular wall damage).
 Aim: repair vascular opening
 Mechanism:
 During vascular injury collagen fibers are exposed ____ vascular change
 Platelets’ surface glycoprotein coat come in contact with collagen fibers in the vascular wall
 Palatals change their characteristics____ platelets change
 Begin to swell
 Assume irregular forms
 Numerous irradiating pseudopods protruding from their surfaces
 Actin-myosin contract forcefully
 Release multiple granular active factors
 Become sticky
 Adhere to collagen fibers and von Willebrand factor (leaks from the plasma)
 Secrete large quantities of ADP
 Form thromboxane A2 (TXA2)
 ADP and TXA2 the membrane of nearby platelets sticky and adhere to original activated ones.

 Successively increase in the numbers of actively attracted platelets at the site of damaged
vascular wall forming a platelet plug.

Which activate the platelets?

Answer: Collagen fibers
What is the source of collagen fibers?
Answer: The damaged vascular wall
Is this plaque hard or loss?
Answer: Platelets plague alone is a loose plug, but it is usually successful in blocking blood loss if the
vascular opening is small.
When it will be hard?
Answer: During the process of blood coagulation
What make platelet plaque be hard?
Answer: Fibrin threads attach tightly to the platelets, thus constructing an unyielding plug.
Are these normal or pathological changes?
Answer: normal physiological changes
Does this underlie the pathogenesis of atherosclerosis?
Answer:__________________

Importance of the Platelet Mechanism for Closing Vascular Holes

Why a thrombocytopenic person (few blood platelets) develops small hemorrhagic areas under
the skin and throughout the internal tissues even with no obvious traumatic injury?
 Our body subjected daily to thousands of minute ruptures in very small blood vessels and to many
multiple small holes through the endothelial cells.
 Platelet-plugging is extremely important for closing such minute ruptures and small endothelial
holes [platelets fuse with the endothelial cells to form additional endothelial cell membrane]

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(3) Third mechanism: Blood Coagulation in the Ruptured Vessel

 Blood coagulation = formation of the blood clot
 Onset: development depends on the severity of the trauma to the vascular wall
 If sever: 15-20 seconds
 If minor: 1-2 minutes
 Participants
 Several of active substances adhere to the traumatized vascular wall and initiate clotting.
 Origin of activator substances:
 Traumatized vascular wall
 Platelets
 Blood proteins
 >13 proteins

 Numbered by Roman numbers

 Numbered by their order of discovery

 Duration - After minor rupture of a vessel (not too

large opening):
 Within 3-6 minutes the entire opening of the
vessel is filled with clot.
 After 20-60 minutes, the clot retracts
 Clot retraction is a function of platelets and it
closes the vessel still further.

 Steps of clotting:
1- Platelets agglutination
2- Fibrin formation
3- Clot formation
4- Clot retraction

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(4) Fourth mechanism: Fibrous Organization or Dissolution of the Blood Clot

What is the fate of blood clot?
Once a blood clot has formed, it can follow one of two courses:
(1) Fibrous Organization:
 Invasion of clot by fibroblast that form a connective tissue through the entire clot.
 Usual course for a clot that forms in a small hole of a vessel wall
 Begins within a few hours after the clot is formed
 Promoted at least partially by growth factor secreted by platelets
 Continues to complete organization of the clot into fibrous tissue within about 1-2 weeks

(2) Dissolution (dissolve)

 The clot digested by platelets enzymes within the clot and dissolved
 Large hole of a vessel wall causes excess blood be leak into the tissues and clot (unusual)

Mechanism of Blood Coagulation Basic Theory

 The blood and tissues contain > 50 important substances that cause or affect blood coagulation
 Some promote coagulation and called procoagulants – tissue factors
 Others inhibit coagulation and called anticoagulants – blood factors
 The balance between procoagulants and anticoagulants determine whether the
blood will coagulate or not
 Normally, circulating vascular blood does not coagulate because the
anticoagulants normally predominate
 When a vessel is ruptured, procoagulants from the area of tissue damage become “activated”
and override the anticoagulants and then a clot does develop.

General Mechanism

Clotting takes place in three essential steps:

(1) Initiation of Coagulation: Formation of Prothrombin Activator
 In blood, >12 coagulation factors are activated in a complex cascade of chemical reactions.
 This occur in response to rupture of the vessel (extrinsic pathway) or damage to the blood itself
(intrinsic pathway)
 The result is formation of a complex of activated substances collectively called prothrombin
activator.
 This is the rate-limiting step in causing blood coagulation and not subsequent (2) or (3) steps,
because these terminal steps normally occur rapidly to form the clot

(2) Conversion of Prothrombin to Thrombin

Prothrombin activator (in the presence of sufficient amounts of ionic Ca2+) catalyzes conversion
of prothrombin into thrombin.
(3) Conversion of Fibrinogen to Fibrin—Formation of the Clot
The thrombin acts as an enzyme to convert, (polymerize within 10-15sec), fibrinogen into fibrin
fibers that enmesh platelets, blood cells, and plasma to form the clot.

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Conversion of Prothrombin to Thrombin and Conversion of Fibrinogen to Fibrin

What is the role of platelets also in the conversion of prothrombin to thrombin?
 Platelets have prothrombin receptors as well as collagen receptors
 Much of the prothrombin first attaches to prothrombin receptors on the platelets already bound
to the damaged tissue
 Such binding hold prothrombin in place to eventually form the clot

What is prothrombin? And what is fibrinogen?

Prothrombin also:
 Continually synthesized by the liver
 Continually used throughout the body for blood clotting.
 A one day failure of synthesis  too low plasma conc.
 Liver disease or vitamin K deficiency  prevents normal prothrombin formation can decrease
the prothrombin level so low that a bleeding tendency results.
Fibrinogen also:
 Fibrinogen is one of the essential factors in the coagulation process (factor I)
 Fibrinogen is large Mwt protein and little normally leaks into the interstitial fluids
[Interstitial fluids ordinarily do not coagulate]
 During inflammation, the permeability of the capillaries increased and fibrinogen leaks into the
tissue fluids in sufficient quantities to allow clotting of these fluids “whaling-off” (similar to what
happen during blood clotting)

What is thrombin? And what is fibrin?

Fibrin also:
 Fibrin monomer peptides are rapidly polymerized with another monomers
 The monomers held by weak hydrogen bonds
 The result is forming weak easily broken fibrin fibers (no cross-link formed yet)
 Next few minutes fibrin cross-linked and strengthen by the action of factor XIII

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Factor XIII also

 Called fibrin-stabilizing factor
 Released from platelets entrapped in the clot
 Present in small amount in plasma and leak also from plasma in tissue
 Require activation by thrombin to become as an enzyme
 It form covalent bonds between more and more of the fibrin monomer molecules
 It also form multiple cross-linkages between adjacent fibrin fibers
 The eventual effect is strengthening the three-dimensional (3D) fibrin meshwork.

Blood Clot
 Composed of a meshwork of fibrin fibers running in all directions (3D)
 Entrapping blood cells, platelets, and plasma.
 The fibrin fibers also adhere to damaged surfaces of blood vessels; therefore, the blood clot
becomes adherent to any vascular opening and thereby prevents further blood loss.

Clot Retraction _ Serum

 Retraction: expressing the fluid from the clot.
 Onset: few minutes after a clot is formed
 Duration: most of the fluid expressed from the clot within 20-60 minutes.
 Expressed fluid called serum [because all its fibrinogen and most of other clotting factors are removed]
 Serum = plasma – fibrinogen [serum cannot clot]

Role of entrapped platelets in clot retraction

(1) Thrombosthenin, actin and myosin of the entrapped platelets contract and hence contract the
overall clot by compress the fibrin meshwork into a smaller mass.
[Low blood platelet causes failure of clot retraction]
(2) Platelets become attached to and bond different fibers together
(3) Continue release procoagulants substances (factor XIII and other)
 Retraction is accelerated by thrombin and Ca+2
 Source of Ca+2: mitochondria, endoplasmic reticulum, and Golgi apparatus of the platelets.
 Clot retracts accompany by pulling the edges of the broken blood vessel thus contributing still
further to hemostasis.
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Positive Feedback of Clot Formation (Vicious Cycle of Clot Formation)

 Once a blood clot has started to develop, it normally extends within minutes into the
surrounding blood. [i.e., the clot itself initiates a positive feedback to promote more clotting]
 The blood clot continues to grow until blood leakage ceases.
 Causes:
 Thrombin act on fibrinogen
 Thrombin act to convert prothrombin into more thrombin
 Thrombin accelerate the action of factors VIII, IX, X, XI and XII in the formation of prothrombin
activator

Initiation of Coagulation: Formation of Prothrombin Activator

 Formation of prothrombin activator is triggered by intrinsic or extrinsic mechanism of activation
 These mechanisms are set into play by
 Initiating either mechanism causes activation of clotting factors that normally are inactive
proteolytic enzyme in the series
 Active clotting factor enzymatically cause successive cascading reactions of the clotting process.
[To indicate the activated form of the factor, a small letter “a” is added after the Roman numeral, such as
Factor VIIIa to indicate the activated state of Factor VIII]

Extrinsic Pathway for Initiating Clotting

 Begins with a traumatized vascular wall or traumatized extravascular tissues that come in
contact with the blood in the following steps:
1. Release of tissue thromboplastin (or called tissue factor) from traumatized tissue
 Membrane phospholipids + lipoprotein complex = tissue thromboplastin
 This factor functions mainly as a proteolytic enzyme
2. Activation of Factor X ______________ role of Factor VII and tissue factor.
 The lipoprotein complex of tissue factor further complexes with Factor VII
 The formed complex in the presence of Ca+2 ions acts enzymatically activate Factor X (Xa)
3. Effect of Xa to form prothrombin activator__________ role of Factor V
Factor Xa complex with phospholipids (from tissue or platelets) and with factor V to form the
prothrombin activator complex
[i.e. prothrombin activator complex = Xa + phospholipids + Factor V]

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Intrinsic Pathway for Initiating Clotting

 Begins with trauma to the blood or exposure of the blood to collagen from a traumatized blood
vessel wall in the following steps:
1. Blood trauma or expose of blood to vascular wall collagen causes
(1) Activation of Factor XII
(2) Release of platelet phospholipids
 Collagen and wettable surfaces (glass) disturb Factor XII and platelets
 Factor XII take a new configuration and converted to a proteolytic enzyme (XIIa)
 Damages the platelets and releases platelet factor 3 (a lipoprotein)
2. Activation of Factor XI.
 XIIa enzymatically activate Factor XI (XIa)
 This reaction requires HMW (high-molecular weight) kininogen and is accelerated by prekallikrein
3. Activation of Factor IX by activated Factor XI.
 XIa then enzymatically activate Factor IX (IXa)
4. Activation of Factor X______________role of Factor VIII
 IXa + activated Factor VIII (VIIIa) + platelet phospholipids + platelet factor 3 activates Factor X (Xa)
 Factor VIII also called antihemophilic factor
 Factor VIII is missing in a person who has classic hemophilia
 Deficiency of either Factor VIII (hemophilia) or low blood platelets (thrombocytopenia)  deficient
step 4.
5. Effect of Xa to form prothrombin activator___________role of Factor V.
 Factor Xa complex with phospholipids (from tissue or platelets) and with factor V to form the
prothrombin activator complex
[i.e. prothrombin activator complex = phospholipids + Factor V]
 Prothrombin activator in presence of Ca+2 (Factor IV) splits prothrombin to form thrombin, and the
clotting process proceeds as already explained

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In summary

Common step after intrinsic or extrinsic pathway

 Prothrombin activator in presence of Ca+2 (Factor IV) splits prothrombin to form thrombin, and the
clotting process proceeds as already explained

Note:
 Recall: prothrombin activator complex = phospholipids + Factor V
 At first, the Factor V in the prothrombin activator complex is inactive
 Formed thrombin activates Factor V “ proteolytic action  Va”
 Va accelerates prothrombin activation
 Xa is the actual protease that causes splitting of prothrombin to form thrombin
 Va greatly accelerates Xa protease activity, and platelet phospholipids act as a vehicle that
further accelerates the process.
 The positive feedback effect of thrombin is the acting through Factor V to accelerate the entire
process once it begins.

Role of Ca+2 in the Intrinsic and Extrinsic Pathways

 Ca+2 are required for promotion or acceleration of all the blood-clotting reactions, except for the
activation of XII & XI in the intrinsic pathway (the first 2
steps)
 In the absence of Ca+2 blood clotting by either pathway
does not occur.
 In the living body, the Ca+2 conc. seldom falls low enough
to significantly affect the kinetics of blood clotting.
 Outside the body (in collecting blood tubes), clotting can be
prevented by reducing the Ca+2 conc. below the threshold
level for clotting
 Ca+2can be removed by:
 Deionization (reaction with citrate ion)
 Precipitation (reaction with oxalate ion)
 Chelation (reaction with EDTA)

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Interaction between the Extrinsic and Intrinsic Pathways—Summary of Blood-Clotting

Initiation
 It is clear from the schemas of the intrinsic and extrinsic systems that after blood vessels rupture,
clotting occurs by both pathways simultaneously.
 Tissue factor initiates the extrinsic pathway
 Contact of Factor XII and platelets with collagen in the vascular wall initiates the intrinsic
pathway.
 Important difference
 Extrinsic pathway can be explosive
 Once initiated, its speed of completion to the final clot
 Limitation:
 The amount of tissue factor released from the damaged tissues
 The quantities of Factors X, VII, and V in the blood.
 With severe tissue trauma, clotting can occur in as little as 15 seconds.
 The intrinsic pathway is much slower to proceed, usually requiring 1-6 minutes to cause
clotting.

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