Download Blood Clotting

Gihan Gawish.Dr

Dr. Gihan Gawish

Coagulation  Coagulation is a complex process by which blood forms clots.

 Coagulation begins almost instantly after an injury to the blood vessel has damaged the endothelium (lining of the vessel).

Gihan Gawish.Dr

Hemostasis 

Primary hemostasis: platelets form a plug at the site of injury



immediately

Secondary hemostasis: occurs simultaneously: 1. proteins in the blood plasma (clotting factors) respond in a complex cascade 2. to form fibrin strands 3. which strengthen the platelet plug Gihan Gawish.Dr

1. Primary hemostasis Platelet activation 1. Damage to blood vessel walls exposes sub endothelium proteins, most notably collagen, present under the endothelium. 2. Circulating platelets bind collagen with surface collagen-specific glycoprotein Ia/IIa receptors.

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3. The adhesion is strengthened by circulating proteins (vWF) Binding intermediaries von Willebrand factor vWF

BLOOD

PLATELETS ENDOTHELIAL CELL

vWF vWF

vWF

EXPOSED COLLAGEN Gihan Gawish.Dr

4. This adhesion activates the platelets. 5. Activated platelets release the contents of stored granules into the blood: 1. 2. 3. 4. 5. 6.

plasma ADP, serotonin, platelet activating factor (PAF), von Willebrand factor (vWF) , platelet factor 4 thromboxane A2 (TXA2)

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6. The granules' contents activate: Gq-linked protein receptor cascade

Increased calcium concentration in the platelets' cytosol Activates protein kinase C Activates phospholipase A2 (PLA2).

Modifies the integrin membrane glycoprotein Increasing its affinity to bind fibrinogen. Gihan Gawish.Dr

7. The activated platelets changed shape from spherical to stellate 8. The fibrinogen cross-links with glycoprotein aid in aggregation of adjacent platelets.

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2. Secondary hemostasis The coagulation cascade 

The coagulation cascade hemostasis has two pathways:

of

secondary

1. The contact activation pathway (formerly known as the intrinsic pathway) 2. The tissue factor pathway (formerly known as the extrinsic pathway)

That lead to fibrin formation. Gihan Gawish.Dr

The Clotting Cascade

Thrombin

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Fibrin polymers

Fibrin monomers

Fibrinogen

The biological of the coagulation factors  The coagulation factors are generally serine proteases (enzymes).  There are some exceptions. For example, FVIII and FV are glycoproteins  Factor XIII is a transglutaminase.  Serine proteases act by cleaving other proteins at specific sites.  The coagulation factors circulate as inactive zymogens. Gihan Gawish.Dr

The pathways are a series of reactions  zymogen + its glycoprotein co-factor are activated to become active components that then catalyze the next reaction in the cascade, ultimately resulting in cross-linked fibrin. zymogen is inactive enzyme precursor of a serine protease Gihan Gawish.Dr

 Coagulation factors are generally indicated by Roman numerals, with a lowercase a appended to indicate an active form.

Gihan Gawish.Dr

Generation of Thrombin  The prothrombin (Factor II) gene is located on the eleventh chromosome (11p11-q12(  Thrombin is produced by the enzymatic cleavage of two sites on prothrombin by activated Factor X (Xa).

 The activity of factor Xa is greatly enhanced by binding to activated Factor V (Va), termed the prothrombinase complex. Gihan Gawish.Dr

 Prothrombin is produced in the liver and is post-translationally modified in a vitamin Kdependent reaction that converts ten glutamic acids on prothrombin to gammacarboxyglutamic acid (Gla).  In the presence of calcium, the Gla residues promote the binding of thrombin to phospholipid bilayers

Gihan Gawish.Dr

 Deficiency of vitamin K or administration of the anticoagulant warfarin inhibits the production of gamma-carboxyglutamic acid residues, slowing the activation of the coagulation cascade.  In human beings the level of prothrombin in the blood stream increases after birth and typically peaks on the 8th day after which the prothrombin level lowers to normal levels Gihan Gawish.Dr

Action of Thrombin  Thrombin converts fibrinogen to an active form that assembles into fibrin.  Thrombin also activates factor XI ,factor V ,and factor VIII .This positive feedback accelerates the production of thrombin.  Factor XIII is also activated by thrombin. Factor XIIIa is a transglutaminase that catalyzes the formation of covalent bonds between lysine and glutamine residues in fibrin. The covalent bonds increase the stability of the fibrin clot. Gihan Gawish.Dr

Action of Thrombin In platelets  In addition to the thrombin activity in the coagulation cascades, thrombin also promotes platelet activation, via activation of protease-activated receptors on the platelet.

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Ca

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Electron Micrograph of Fibrin

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 A fibrin clot is formed by the interplay of the intrinsic, extrinsic, and final common pathways.  The intrinsic pathway begins with the activation of factor XII (Hageman factor) by contact with abnormal surfaces produced by injury.

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 The extrinsic pathway is triggered by trauma, which activates factor VII and releases a lipoprotein, called tissue factor, from blood vessels. Inactive forms of clotting factors are shown in red; their activated counterparts (indicated by the subscript "a") are in yellow.

 Stimulatory proteins that are not themselves enzymes are shown in blue.  A striking feature of this process is that the activated form of one clotting factor catalyzes the activation of the next factor. I. Gihan Gawish.Dr

Cofactors Various substances are required for the proper functioning of the coagulation cascade: 1. Calcium and phospholipids (a platelet membrane constituent)  They are required for the tenase and prothrombinase complexes to function. Gihan Gawish.Dr



Calcium mediates the binding of the complexes via the terminal gammacarboxyl residues on

1. FXa 2. FIXa (to the phospholipids surfaces expressed by platelets) Prothrombin binds calcium ions with the modified amino acid g-carboxyglutamate (red). Gihan Gawish.Dr

The CalciumBinding Region of Prothrombin

2. Vitamin K  It is an essential factor to a hepatic gammaglutamyl carboxylase that adds a carboxyl group to glutamic acid residues on:  Factor II,  Factor VII,  Factor IX  Factor X,  Protein S ,  Protein C Gihan Gawish.Dr

 In adding the gamma-carboxyl group to glutamate residues on the immature clotting factors Vitamin K is itself oxidized.

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 Another enzyme ,Vitamin K epoxide reductase VKORC reduces vitamin K back to its active form.  Vitamin K epoxide reductase is pharmacologically important as a target for anticoagulant (antagonists) drugs warfarin and related coumarins such as acenocoumarol , phenprocoumon and dicumarol . Gihan Gawish.Dr

 These drugs create a deficiency of reduced vitamin K by blocking VKORC, thereby inhibiting maturation of clotting factors.  Other deficiencies of vitamin K (e.g. in malabsorption ,)or disease( hepatocellular carcinoma impairs the function of the enzyme and leads to the formation of PIVKAs (proteins formed in vitamin K absence) this causes partial or non gamma carboxylation and affects the coagulation factors ability to bind to expressed phospholipids Gihan Gawish.Dr

The Clotting Process Must Be Precisely Regulated  There is a fine line between hemorrhage and thrombosis. Clots must form rapidly yet remain confined to the area of injury.  Activated factors are short-lived because they are diluted by blood flow, removed by the liver, and degraded by proteases.  For example, the stimulatory proteins factors Va and VIIIa are digested by protein C Gihan Gawish.Dr

 Protein C is switched on thrombin.

a protease that is by the action of

 Thus, thrombin has a dual function: it catalyzes the formation of fibrin and it initiates the deactivation of the clotting cascade.

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Fibrinolysis

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 Fibrinolysis is the process wherein a fibrin clot is broken down  Plasmin cuts the fibrin mesh at various places, leading to the production of circulating fragments that are cleared by other proteases or by the kidney and liver.

 Plasmin is produced in an inactive form, plasminogen, in the liver. Gihan Gawish.Dr

Coagulation factors and related substances  I fibrinogen Forms clot (fibrin (  (II( prothrombin Its active form (IIa) activates I, V, VII, VIII, XI, XIII, protein C, platelets  Tissue factor Co-factor of VIIa (formerly known as factor III)  Calcium Required for coagulation factors to bind to phospholipid (formerly known as factor IV  )V( proaccelerin, labile factor )Co-factor of X with which it forms the prothrombinase complex Gihan Gawish.Dr

 XII (Hageman factor) Activates factor XI and prekallikrein

 XIII (fibrin-stabilizing factor) Cross links fibrin  von Willebrand factor Binds to VIII, mediates platelet adhesion  Prekallikrein Activates cleaves HMWK

XII

and

prekallikrein;

 high molecular weight kininogen (HMWK) Supports reciprocal activation of XII, XI, and prekallikrein Gihan Gawish.Dr

 antithrombinIII Inhibits IIa, Xa, and other proteases  heparin cofactor II Inhibits IIa, cofactor for heparin  protein C Inactivates Va and VIIIa  protein S Cofactor for activated protein C Gihan Gawish.Dr

Specific inhibitors of clotting factors 1.Antithrombin III is the most important one,  It is a plasma protein that inactivates thrombin by forming an irreversible complex with it.  It resembles alpha 1-antitrypsin except that it inhibits thrombin much more strongly than it inhibits elastase.  Also, it blocks other serine proteases in the clotting cascade namely, factors XIIa, XIa, IXa, and Xa. Gihan Gawish.Dr

2.Heparin  The inhibitory action enhanced by heparin

of

antithrombin

III

is

 It is a negatively charged polysaccharide found in mast cells near the walls of blood vessels and on the surfaces of endothelial cells  Heparin acts as an anticoagulant by increasing the rate of formation of irreversible complexes between antithrombin III and the serine protease clotting factors.  Antitrypsin and antithrombin are serpins, a family of serine protease inhibitors. Gihan Gawish.Dr

Electron Micrograph of a Mast Cell. Heparin and other molecules in the dense granules are released into the extracellular space when the cell is triggered to secrete. Gihan Gawish.Dr

3. Alpha 1-antitrypsin  which normally inhibits elastase  alpha 1-Antitrypsin activity normally increases markedly after injury to counteract excess elastase arising from stimulated neutrophils.  The mutant a 1-antitrypsin caused the patient's thrombin activity to drop to such a low level that hemorrhage ensued. Gihan Gawish.Dr

Disease and clinical significance of thrombosis 1.

Hemophilias

are the coagulation factor disorders.

best-known

The three main forms are:

hemophilia A (factor VIII deficiency)

hemophilia B (factor IX deficiency or "Christmas disease")

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hemophilia C (factor XI deficiency, mild bleeding tendency).

 2. von Willebrand disease  It is the most common hereditary bleeding disorder and is characterized as being inherited autosomal recessive or dominant.  In this disease there is a defect in von Willebrand factor (vWF) which mediates the binding of glycoprotein Ib (GPIb) to collagen.

 This binding helps mediate the activation of platelets and formation of primary hemostasis. Gihan Gawish.Dr

3. Deficiency of Vitamin K  It may also contribute to bleeding disorders because clotting factor maturation depends on Vitamin K.

4. Liver diseases:  Some clotting factors; II, IX, VII, X are synthesized in liver  Liver diseases deficiency of these factors bleeding disorders. Gihan Gawish.Dr

Coagulation Tests

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Coagulation Cascade PT

PTT

VIIIa

Heparin

Hirudin, Argatroban

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Coagulation and Fibrinolysis

Gihan Gawish.Dr

Coagulation Tests Screen test Bleeding Time (Duke method, Template method), Thrombin Time, PT, PTT

Other coagulation inhibitor Study, VIII, XI, XII DIC profile Fibrinogen, FDP, 3P Test, D-dimer

Antiphospholipid syndrome Dilute Russell Viper Venom Time (dRVVT), Anti-Cardiolipin Ab, ACA, IgG, Anti-Phospholipid Ab, APA, IgG, Anti-Cardiolipin Ab, ACA, IgM

Fibrinolysis Euglobulin clot lysis time, Plasminogen activator inhibitor, Alpha2-antiplasmin

Coagulation factor Factor I (Fibrinogen), II, V, VII, VWF, VIII, IX, X, Urea solubility test,

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PLT function Platelet aggregation Thrombosis APC Resistance, Protein S, Antithrombin III, Protein C, Plasminogen

1. Bleeding time  Done with a template.  Time taken for the blood to stop

 Normal range 2-10mts  Prolonged in plt disorders, low plts, severe anemia, Vwf, collagen vascular disease  Great variability in results, unreliable, invasive, insensitive Gihan Gawish.Dr

2. Activated Partial Thromboplastin Time (aPTT)  It is a performance indicator measuring the efficancy of both the "intrinsic" and the common coagulation pathways.  It is also used to monitor the treatment effects with heparin.  It is used in conjunction with the prothrombin time (PT) which measures the extrinsic pathway. Gihan Gawish.Dr

Methodology (aPTT)  Container: blue top (3.2% citrate) tube

 Collection: Deliver tubes immediately to the laboratory  In order to activate the intrinsic pathway, phospholipid, an activator (such as silica, celite, kaolin, ellagic acid), and calcium (to reverse the anticoagulant effect of the citrate) are mixed into the plasma sample .  The time is measured until a thrombus (clot) forms.  The test is termed "partial" due to the absence of tissue factor from the reaction mixture. Gihan Gawish.Dr

Performing APTT      

At 37 ̊C Plasma Add kaolin/elgaic acid Phospholipid source Calcium Time the appearance of clot

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Activated Partial Thromboplastin Time (aPTT)  Causes for Rejection: Specimen received more than 4 hours after collection, tubes not filled, clotted specimens, visible hemolysis

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Activated Partial Thromboplastin Time (aPTT)  Reference Interval: 20-25 to 32-39 seconds. Prolong in newborns  However, newborns and infants do not normally experience bleeding, because a balance between procoagulants and natural anticoagulants is maintained.  Critical Values: >100-150 seconds

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PTT in clinical states  PTT prolonged in

 PTT shortened in

1. Congenital or acquired def of intrinsic pathway factors

1. Pregnancy

2. Heparin 3. Lupus AC (antiphospholipid

antibody)

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2. In conditions causing activation of factors

3. Prothrombin Time (PT)  Clotting time from factor VII to fibrin clot  PT↑: fibrinogen or factors II, V, VII, or X deficiency, therapeutic anticoagulants (heparin, hirudin, or argatroban)

 Container: Blue top (3.2% sodium citrate) tube Gihan Gawish.Dr

Prothrombin Time (PT)  heparin prolongs the PT to a lesser extent than PTT. Hirudin and argatroban prolong the PT and PTT. ► Collection: directly from a peripheral vein

Causes for Rejection: Specimen received more than 24 hours after collection, tube not filled, clotted specimen, visible hemolysis Gihan Gawish.Dr

Prothrombin Time (PT)  Reference Interval: 10-12 to 12-14 seconds. Prolong in newborns. Up to 16 seconds at birth, and gradually shortens into the adult normal range by the age of 6 months.  Critical Values: >30 seconds

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Prothrombin Time (PT)  Methodology: Reagent called thromboplastin (phospholipid with tissue factor and calcium) added, measure clot formation time.  Vitamin K trial may be performed with an unexplained PT prolongation. If vitamin K deficiency, the PT becomes normal or significantly shorter within 12-24 hours after vitamin K administration.

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Prothrombin Time (PT)  Monitoring warfarin: international normalized ratio (INR), therapeutic goal is an INR of 2-3. ► INR = [patient PT/normal PT]ISI ► international sensitivity index (ISI), varies in reagents The ISI is usually between 1.0 and 1.4. Gihan Gawish.Dr

Effects of Factor Deficiencies on PT and PTT  PTT Prolonged, PT Normal: Deficiencies of factor VIII, IX, XI, and/or XII (intrinsic pathway)  PT Prolonged, PTT Normal: Deficiency of factor VII (extrinsic pathway), mild-to-moderate deficiencies of factor II, V, X, and/or fibrinogen (common pathway)  Both PT and PTT Prolonged: Deficiencies of factor II, V, X, and/or fibrinogen (common pathway), Multiple factor deficiencies Gihan Gawish.Dr

4. Thrombin clotting time  TT is a coagulation assay which is usually performed in order to detect for the therapeutic level of the anticoagulant Heparin.  It is also sensitive in detecting the presence of a fibrinogen abnormality.

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Methodology of TT  After liberating the plasma from the whole blood by centrifugation ,bovine Thrombin is added to the sample of plasma.  The clot is formed and is detected optically or mechanically by a coagulation instrument.  The time between the addition of the thrombin and the clot formation is recorded as the thrombin clotting time Gihan Gawish.Dr

Reference Interval TT  The reference interval of the Thrombin Clotting time is generally