Coagulation

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The coagulation of blood is a complex process during which blood forms solid clots. It is an important part of hemostasis whereby a damaged blood vessel wall is covered by a fibrin clot to stop hemorrhage and aid repair of the damaged vessel. Disorders in coagulation can lead to increased hemorrhage and/or thrombosis and embolism.

Coagulation is similar in all mammals, but as most is known about the system in humans, this article focuses on human blood coagulation.

In brief

In a normal person coagulation is initiated within seconds after an injury occurs to the blood vessel endothelium; when platelets form a hemostatic plug at the site of injury. This is called primary hemostasis. Secondary hemostasis then follows when plasma components called clotting factors respond (in a complex cascade) to form fibrin strands which strengthen the platelet plug. Contrary to popular belief, coagulation from a cut on the skin is not initiated by air or drying out, but it is because platelets adhere to and are activated by collagen outside the blood vessel and the skin. The activated platelets then release granules that contain pro-coagulant proteins furthering clotting.

The use of adsorbent chemicals, such as zeolite, and other hemostatic agents is also being explored for use in sealing severe injuries quickly.

Primary hemostasis

Primary hemostasis is initiated when platelets adhere, using a specific platelet collagen receptor glycoprotein Ia/IIa, to collagen fibers in the vascular endothelium. This adhesion is mediated by von Willebrand factor (vWF), which forms links between the platelet glycoprotein Ib/IX/X and collagen fibrils.

The platelets are then activated and release the contents of their granules in to the plasma, this in turn activates other platelets and white blood cells. They undergo a change in their shape which exposes a phospholipid surface for those coagulation factors that require it. Fibrinogen links adjacent platelets by forming links via the glycoprotein IIb/IIIa. In addition, thrombin activates platelets.

Secondary hemostasis

The coagulation cascade

The coagulation cascade of secondary hemostasis has two pathways, the Contact Activation pathway (formally known as the Intrinsic Pathway) and the Tissue Factor pathway (formally known as the Extrinsic pathway) that lead to fibrin formation. It was previously thought that the coagulation cascade consisted of two pathways of equal importance joined to a common pathway. It is now known that the primary pathway for the initiation of blood coagulation is the Tissue Factor pathway. The pathways are a series of reactions, in which a zymogen of a serine protease and its glycoprotein co-factor are activated to become active components that then catalyze the next reaction in the cascade. Coagulation factors are generally indicated by Roman numerals, with a lowercase a appended to indicate an active form, ultimately resulting in cross-linked fibrin.

The coagulation factors are serine proteases (enzymes) except FVIII and FV which are glycoproteins. The serine proteases act by cleaving other proteins at specific sites. Factor XIII is a transglutaminase. Protein C is also a serine protease.

The coagulation cascade can be summarised as follows: -

1. Tissue Factor pathway: the main role of the tissue factor pathway is to generate a "thrombin burst". Thrombin being the single most important constituent of the coagulation cascade in terms of its feedback activation roles. FVIIa circulates in a higher amount than any other activated coagulation factor and following damage to the blood vessel endothelium Tissue Factor (TF) is released, this then forms a complex with FVIIa (TF-FVIIa) this activates FIX and FX. FVII itself is activated by thrombin, FXIa, plasmin, FXII and FXa. The activation of FXa by TF-FVIIa is almost immediately inhibited by tissue factor pathway inhibitor (TFPI). FXa and its co-factor FVa form the prothombinase complex which activates prothrombin to thrombin. Thrombin then activates other components of the coagulation cascade, including FV and FVII (which activates FXI which in turn activates FIX), and activates and releases FVIII from being bound to vWF. FVIIIa is the co-factor of FXIa and together they form the "tenase" complex which activates FX and so the cycle continues.
2. Contact Activation pathway: formation of the primary complex on collagen by high molecular weight kininogen (HMWK), prekallikrein and FXII (Hageman factor), prekallikrein is converted to kallikrein and FXII becomes FXIIa. FXIIa converts FXI into FXIa. FXI is also activated by FVIIa. Factor IX is in turn activated by FXIa which with its co-factor FVIIIa form the tenase complex which activates FX to FXa. The minor role that the contact activation pathway has in initiating clot formation can be illustrated by the fact that patients with severe deficiencies of FXII, HMWK and prekallikrein do not have a bleeding disorder.
3. Thrombin Thrombin has a large array of functions. Its primary role is the conversion of fibrinogen to fibrin, the building block of a haemostatic plug. In addition, it activates Factors VIII and V and their inhibitor protein C (in the presence of thrombomodulin), and it activates Factor XIII, which forms covalent bonds that crosslink the fibrin polymers that form from activated monomers.

Following activation by the contact factor or tissue factor pathways the coagulation cascade is maintained in a prothrombotic state by the continued activation of FVIII and FIX to form the tenase complex, until it is down regulated by the anticoagulant pathways.

Cofactors and inhibitors

Various substances are required for the proper functioning of the coagulation cascade:

• Calcium and phospholipid (a cell membrane constituent) are required for the tenase and prothrombinase complexes. Calcium mediates the binding of the complexes via the terminal gamma-carboxy residues on FXa and FIXa to the phospholipid surfaces expressed by platelets. Calcium is also required at other points in the coagulation cascade.
• Vitamin K is an essential factor to a hepatic gamma-glutamyl decarboxylase that adds a second carboxyl group to glutamic acid residues on factors II, VII, IX and X, as well as Protein S, Protein C and Protein Z. Deficiency of vitamin K (e.g. in malabsorption), use of inhibiting anticoagulants (warfarin, acenocoumarol and phenprocoumon) or disease (cirrhosis, hepatocellular carcinoma) impairs the function of the enzyme and leads to the formation of PIVKA's (proteins formed in vitamin K absence) this causes partial or non gamma carboxylation and affects the coagulation factors ability to bind to expressed phospholipid.

Three mechanisms keep the coagulation cascade in check. Abnormalities can lead to an increased tendency toward thrombosis:

• Protein C is an important co-factor inhibitor, which degrades the co-factors FVa and FVIIIa. It is activated by thrombin with thrombomodulin and requires its co-enzyme Protein S to function. Quantitative or qualitative deficiency of either may lead to thrombophilia (a tendency to develop thrombosis). Impaired action of Protein C (activated Protein C resistance), for example by Factor V, "Leiden" variant or high levels of FVIII also may lead to a thrombotic tendency.
• Antithrombin is a serine protease inhibitor (serpin) that degrades the serine proteases; thrombin and FXa, as well as FXIIa, and FXIa. It is constantly active, but its adhesion to these factors is increased by the presence of heparan sulfate (a glycosaminoglycan) or the administration of heparins (different heparinoids increase affinity to F Xa, F IIa, or both). Quantitative or qualitative deficiency of antithrombin (inborn or acquired, e.g. in proteinuria) leads to thrombophilia.
• Tissue factor pathway inhibitor (TFPI) inhibits F VIIa-related activation of F IX and F X after its original initiation.

Testing of coagulation

Numerous tests are used to assess the function of the coagulation system:

• Common: aPTT, INR (PT), TCT, bleeding time, D-dimer
• Other: mixing test (whether an abnormality corrects if the patient's plasma is mixed with normal plasma), antiphosholipid antibodies, coagulation factor assays, genetic tests (eg. factor V Leiden, prothrombin mutation G20210A), dilute Russell viper venom test (dRVVT), platelet function tests

The contact factor pathway is initiated by activation of contact factors of plasma, and can be measured by the activated partial thromboplastin time (aPTT) test.

The Tissue factor pathway is initiated by exposure of blood to "tissue factor" (a specific cellular lipoprotein), and can be measured by the prothrombin time (PT) test. This is reported as an INR value when used for the dosing of oral anticoagulants such as warfarin.

The quantatative and qualitative screening of fibrinogen is measured by the thrombin time (TCT). Measurement of the exact amount of fibrinogen present in the blood is generally done using the Clauss method for fibrinogen testing.

If a coagulation factor is part of the contact or tissue factor pathway, a deficiency of that factor will affect only one of the tests: thus hemophilia A, a deficiency of factor VIII, which is part of the contact factor pathway, results in an abnormally prolonged aPTT test but a normal PT test. The exceptions are prothrombin, fibrinogen and some variants of FX which can only be detected by either aPTT or PT.

Deficiencies of fibrinogen (quantitative or qualitative) will affect all screening tests.

Disorders of hemostasis

• disorders of the platelet and vessel wall
  • immune thrombocytopenic purpura (ITP)
  • thrombotic thrombocytopenic purpura (TTP)
  • hemolytic-uremic syndrome (HUS)
  • Glanzmann's thrombasthenia
  • Bernard-Soulier syndrome (abnormal glycoprotein Ib-IX-V complex)
  • storage pool disorders
  • paroxysmal nocturnal hemoglobinuria
  • gray platelet syndrome: deficient alpha granules.
  • delta storage pool deficiency: deficient dense granules.
• disorders of coagulation and thrombosis
  • disseminated intravascular coagulation
  • factor deficiencies
    • hemophilia A
    • hemophilia B ("Christmas disease")
    • hemophilia C (Factor XI deficiency, mild bleeding tendency)
    • Von Willebrand disease (the commonest bleeding disorder)
  • factor inhibitors
• disorders predisposing to thrombosis
  • heparin-induced thrombocytopenia and thrombosis("white clot syndrome")
  • antiphospholipid syndrome
    • Lupus anticoagulant
    • anticardiolipin antibody
  • factor V Leiden
  • prothrombin mutation
  • protein C deficiency
  • protein S deficiency
  • antithrombin deficiency

Coagulation factors

History

The exact process of coagulation was largely elucidated in the 20th century. At the end of the 19th century, it was presumed that the coagulation system consisted of four factors (Giangrande 2003): thrombokinase/thromboplastin (III, released by damaged tissues) - this reacted with prothrombin (II), which, together with calcium (IV), formed thrombin, which converted fibrinogen into fibrin (I).

A first clue as to the complexity of the system of coagulation was the discovery of proaccelerin (initially and later called Factor V) by Paul Owren (1905-1990) in 1947. He also postulated that its function was the generation of accelerin (Factor VI), which later turned out to be the activated form of V (or Va); hence, VI is not now in active use.

Factor VII (also known as serum prothrombin conversion accelerator or proconvertin, precipitated by barium sulfate) was discovered in a young female patient in 1949 and 1951 by different groups. Factor VIII turned out to be deficient in the clinically recognised but etiologically elusive hemophilia A; it was identified in the 1950s and is alternatively called antihemophilic globulin due to its capability to correct hemophilia A.

Factor IX was discovered in 1952 in a young patient with hemophilia B by the name of Stephen Christmas (1947-1993). His deficiency was described by Dr Rosemary Biggs and Professor R.G. MacFarlane in Oxford, UK. The factor is hence called Christmas Factor or Christmas Eve Factor. Christmas lived in Canada, and campaigned for blood transfusion safety until succumbing to transfusion-related AIDS at age 46. An alternative name for the factor is plasma thromboplastin component, given by an independent group in California.

Hageman factor, now known as factor XII, was identified in 1955 in an asymptomatic patient with a prolonged bleeding time by the name of John Hageman.

Factor X followed, in 1956. This protein was identified in a Ms Audrey Prower of London, who had a lifelong bleeding tendency. In 1957, an American group identified the same factor in a Mr Rufus Stuart. The factor is therefore termed Stuart-Prower factor.

Factors XI and XIII were identified in 1953 and 1961, respectively.

The usage of Roman numerals rather than eponyms or systematic names was agreed during various annual conferences (starting in 1955) of hemostasis experts. This committee evolved into the present-day International Committee on Thrombosis and Hemostasis (ICTH). Assignment of numerals ceased in 1963 after the naming of Factor XIII, but the names Fletcher Factor and Fitzgerald Factor were given to further coagulation-related proteins, namely prekallikrein and high molecular weight kininogen respectively.

Factors III and VI are unassigned, as thromboplastin was never identified and actually turned out to consist of ten further factors and accelerin was simply activated Factor V.

References

• Giangrande PL. Six characters in search of an author: the history of the nomenclature of coagulation factors. Br J Haematol 2003;121:703-12. PMID 12780784.

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