Coagulation Process and Related Laboratory Tests

Coagulation is the process through which liquid blood solidifies at the point of injury to prevent further loss of blood. It is a complex process facilitated by platelets and other clotting factors and is important to prevent a pathogenic invasion of the body as well as excessive loss of blood which otherwise would lead to the collapse of vital organs. This article discusses the three coagulation processes, the coagulation factors, the coagulation process regulation factors and the relevant coagulation tests to detect any pathologies relating to the body’s coagulation system.

Coagulation factors

1. Fibrinogen (Factor I)

This is a protein that is made in the liver and released into circulation to aid in the clotting process. During an injury, it is converted into fibrin mesh which traps cells and other blood products to form a blood-based occlusion or clot that seals the injured part of the blood vessel. Its other functions during an injury include mediation of platelet spreading, the formation of new blood vessels and proliferation of fibroblasts in the tissue. Due to genetic factors, some disease may result in defective fibrinogen formation. As well, liver problems may affect the formation of fibrinogen. This may result in diseases such as the production of low amounts or no fibrinogen, storage problems, dysfibrinogenemia, hypo dysfibrinogenemia and Fibrinogen Aa-chain amyloidosis.

2. Prothrombin (Factor II)

This is a proenzyme in the coagulation cascade that is dependent on vitamin K. when proteolytically cleaved, it forms thrombin which acts as a serine protease that activated fibrin from fibrinogen through polymerization. Its size is about 72 Kilodaltons and is synthesized in the liver. In the blood, it circulates at concentrations of 100 micrograms per millilitre which is normally a single chain of 579 glycoproteins. It binds to the fibrin upon contact at the Kringle domain. The Kringle domain is made up of a C-terminal serine protease and two Kringle structures. It can also exert anticoagulation by binding to the thrombomodulin on the surface of endothelial cells to activate protein C. this activity results in digestion of factor V which deprives prothrombinase from binding to its cofactor.

3. Thromboplastin (Factor III)

This is a mixture of tissue factor and phospholipids which aids in the coagulation process through activation of thrombin in the extrinsic pathway. A complete thromboplastin consists of tissue factor, phospholipids and calcium chloride.

4. Calcium (Factor IV)

This is an ion that mediates the formation of complexes by binding of activated factor ten and factor nine through terminal carboxy residues to the phospholipid surfaces expressed by platelets. It works together with phosphatidylserine protease to form a platform for clot formation through platelet membrane binding. The process of membrane binding involves vitamin K which facilitates the carboxylation of various clotting factors. Calcium also ensures the stability of the labile factor.

5. Labile Factor (Factor V)

Sometimes referred to as proaccelerin. It mostly functions as a cofactor and not enzymatically active as other coagulation factors. it is composed of heavy and light protein chains which are non-covalently bound through calcium ions upon activation. It interacts with activated factor ten to convert prothrombin to thrombin.

6. Stable Factor (Factor VII)

Stable factor or proconvertin is a vitamin K dependent glycoprotein serine protease formed in the kidney and liver. Its recombinant form is used to treat patients with severe bleeding. This factor is activated when it interacts with the tissue factor which is normally located outside the bloodstream. Its actions are regulated by the tissue factor pathway inhibitor which is released almost immediately to prevent an excessive chain reaction. Warfarin and similar anticoagulants function by interfering with its formation in the respective synthesis organs.

7. Antihemophilic Factor A (Factor VIII)

This is a clotting factor produced in the liver sinusoidal cells and the endothelial cells in the other parts of the body. It circulates in the body bound to the Von Willebrand Factor in its inactive form. Upon injury of a blood vessel, it separates from the VWF to form an active protein that which interacts with factor IX where it acts as a cofactor when activated. In the presence of calcium and phospholipids, it forms the protease that activates factor X. high levels of this factor in circulation increases the risk of deep vein thrombosis and pulmonary embolism. Copper regulates its activity as a cofactor and therefore the deficiency results in overactivity. Its deficiency is associated with haemophilia A and therefore the recombinant form is used in the treatment of such cases.

8. Christmas Factor/ Antihemophilic Factor B (Factor IX)

This factor was discovered and named after Stephen Christmas who was lacking it and therefore had haemophilia B. it is produced as a zymogen which is activated by removal of the signal peptide, glycosylation and cleavage by activated factor XI or factor VII in the intrinsic and extrinsic pathways respectively. On activation, it forms two chains which are linked by a disulphide bond. It functions to hydrolyze the arginine-isoleucine bond in factor X in the presence of calcium, phospholipids and factor VIII as a cofactor thus activating it to factor Xa.

9. Stuart- Power Factor (Factor X)

This is a vitamin K serine endopeptidase which is synthesized in the liver. Its activation is the convergent point of the intrinsic and extrinsic pathways. It was discovered in a patient named Stuart who had its deficiency. It was given a central position in the coagulation pathway after Russel’s-viper venom experiment which was found to able to activate it and in turn its active form able to convert prothrombin to thrombin. Complete lack of this factor from the body correlates with death.

10. Plasma Thromboplastin antecedent (Factor XI)

This is a dimeric serine protease which is synthesized independently of vitamin K in the liver. it contributes to the process of coagulation through activation of factor IX. It circulates in the blood as a high molecular weight kininogen. Its half-life in circulation is about 2 and a half days. The deficiency of factor XI results in haemophilia C.

11. Hageman Factor (Factor XII)

This is a 596-protein serine protease in the class of endopeptidase consisting of a heavy and light chain connected through a disulphide bond. It is activated by contact to surfaces that are negatively charged and therefore can be used to start the coagulation cascade in the laboratory. It was discovered in 1918 from a blood sample for a routine preoperative screening of John Hageman who was found to have a remarkably prolonged clotting time without hemorrhagic symptoms.

12. Fibrin Stabilizing Factor (Factor XIII)

This is a clotting factor that is activated by thrombin from its zymogen form to facilitate the crosslinking and stabilization of the blood clots (fibrin strands). It is a transglutaminase that exists in the circulation as a heterotetramer. It needs calcium ions to form isopeptide bonds with glutamines and lysines in the fibrins thus resulting in cross-linkage and stability of the formed clot.

Of note, factor VI was eliminated following rigorous research which concluded that it had no role in blood coagulation. It is referred to as accelerin

Coagulation Processes

The process of coagulation is aimed at maintaining a stable state in the human body as many other physiological processes. As a process, it has components that regulate it, act as building blocks and others that facilitate it. Some of these components include platelets, enzymes and coagulation proteins. The process of coagulation happens through the intrinsic and extrinsic pathways and a common pathway. The intrinsic pathway is called so because it is initiated when the endothelial collagen is exposed, the extrinsic pathway, on the other hand, is initiated when by the tissue factor released by endothelia cells following external damage. The two pathways meet and form a common pathway which leads to the formation of the fibrin which eventually becomes the basis for clot formation. In simple terms, the process of coagulation has a goal of forming the fibrin clot which involves the formation of a tenase, through the two pathways, followed by a common pathway that converts fibrinogen to fibrin through then enzyme thrombin.

The Intrinsic Pathway

The intrinsic pathway involves the coagulation factors such as factor I, II, IX, X, XI and XII; Fibrinogen, prothrombin, Christmas factor, Stuart power factor, thromboplastin and Hageman factor respectively. The process begins by activating factor XII following exposure to the endothelial collagen when damage is incurred. The activated factor XII then activates factor XI. Activated factor XI then activates factor IX. Activated factor IX, in turn, activates factor X. as this happens, the concentration of the activated factors increase in the blood and more zymogens are activated. The activated factor X then gets into the common pathway.

The extrinsic and the common pathway

The extrinsic pathway, however, involves factors such as the tissue factor (factor III), Factor VII and factor X. the exposure of the endothelial cells makes them release-activated factor II which activates factor VII. The activated factor VII activates factor X which joins the common pathway.

The common pathway involves activated factor X, Tissue factor (factor 3), factor VII, Calcium ions, cofactor VIII, activated factor IX and phospholipids. Factor X is activated by tenase which is formed by the two pathways. In the intrinsic pathway, the tenase has composed the cofactors VIII, activated factor IX, calcium ions and a phospholipid; in the extrinsic pathway, however, it is composed of the tissue factor, factor VII and calcium ions. The activated factor X activates factor II. Activated factor II then activates factor VIII and V which are cofactors with activated factor X.

If left unchecked, the outcome can be catastrophic. Excessive blood clotting can result in multiple organ failure and damages. This is because the clots will block blood vessels to these organs making them suffocate and the cells that make the tissues will die from intoxication and suffocation. Additionally, it will deplete some important elements needed by the body which will either be trapped in the blood clots or used in the process of coagulation. Such elements include iron and calcium.


Waterfall Illustration of The Coagulation Cascade

Regulation of the coagulation process

1. Protein C

The protein C system restricts the activity of activated factor VIII, V, cofactors in the activation of factor ten and prothrombin. The protein C complex circulates as an inactive form of a serine protease which is activated when it interacts with the thrombin bound to the surface of cells by thrombomodulin. When activated, together with cofactor protein S, they degrade the activated factor eight and five on the surface of a negatively charged phospholipid.

2. Antithrombin

This is a small protein consisting of 42 amino acids whose activity deactivates many key players in the process of coagulation. Antithrombin is a serine protease inhibitor (serpin) and targets the protease in the intrinsic pathways namely factor X, IX, XI, XII and II. It also inhibits kallikrein and plasmin. It functions by occupying the substrate sites of the enzymes making them inaccessible to the normal substrate thus limiting their enzymatic functions in the coagulation process. This is done by first forming a complex with thrombin which when attacked by the protease enzymes, they get trapped and their function ceases. Some protease, however, dissociates the complex faster including bacterial enzyme thymolysin and neutrophil elastase.

3. Tissue factor pathway inhibitor

The TFPI exists in two isoforms which have tissue-specific expression patterns and anticoagulant activity. They inhibit the activity of the factor VIIa-tissue factor complex dependent activated factor X generation. Presence of protein S enhances the inhibition of activated factor X. as well it inhibits prothrombinase activity at the beginning of the coagulation process.

4. Plasmin

This is an enzyme in blood circulation that degrades most proteins including fibrin clots a process called fibrinolysis. As a serine protease, it cleaves fibronectin, thrombospondin, laminin and von Willebrand factor. In circulation, it assumes a closed nonconvertible form while when it binds to a clot it assumes an open form that can be converted to an active form or deactivated.

5. Prostacyclin

This is a potent vasodilator and an inhibitor of platelet aggregation as a way of regulating the coagulation process. It is a prostaglandin member of the eicosanoid family of lipids and has been considered for certain antithrombotic therapies despite its instability.

Coagulation tests

1. Prothrombin time (PT)

This is a blood clotting assay that evaluates the extrinsic and the common pathway of the coagulation system. It evaluates the activity of factor I, II, V, VII and X. the reference ranges for this test mostly depend on the laboratory but averages between 12 and 13 seconds. It is performed by automated instruments which are set at 370C. blood is drawn into sodium citrate containing container which acts as an anticoagulant. The plasma is then extracted and mixed with calcium in excess to reverse the effect of citrate. Factor III (tissue factor) is then added and left to clot. The time of clotting is measured optically and recorded. Lipemic and icteric samples may give inaccurate results.

2. Activated partial thromboplastin time (aPTT)

It was formally known as the Kaolin-cephalin clotting time as kaolin and cephalin were the materials used in the test. It measures the rate of clot formation through the two pathways and therefore gauges the functionality of clotting factors such as fibrinogen, prothrombin, antihemophilic factor, Stuart power factor, plasma thromboplastin antecedent and Hageman factor. The reference range is 30 to 40 seconds when the activator is added and 60 to 70 seconds without an activator. It is used to monitor response to heparin therapy.

3. Thrombin Time (TT)

This is a coagulation test that gauges the amount of time it takes for a clot to form in the plasma following the addition of an excess amount of thrombin. It is used to assess the effectiveness of fibrinolytic therapy. It tests both qualitative and quantitative defects that might be present in the plasm coagulation process. When the sample contains heparin, a snake venom called batroxobin (reptilase) is used in place of thrombin. Normal values with thrombin range from 12 to 14 seconds while with the snake venom, it ranges from 15 to 20 seconds.

Possible errors

1. Artificially prolonged coagulation time

2. Artificially shortened coagulation time

International Normalized Ratio (INR)

The International Normalized Ration is a system that was established by the World Health Organization and the International Committee on Thrombosis and Hemostasis to create a standard for reporting results of blood clotting time.


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