Coagulation, also known as clotting, is the process by which blood changes from a liquid to a gel, forming a blood clot. This leads to hemostasis, the stopping of blood loss from a damaged vessel, followed by repair. The coagulation process involves the activation, adhesion, and aggregation of platelets, as well as the deposition and maturation of fibrin.

 

 

 

Coagulation begins almost immediately after injury to the endothelium that lines a blood vessel. Exposure of blood to the subendothelial space initiates two processes: changes in platelets and exposure of tissue factor in the subendothelial space to factor VII, ultimately leading to cross-linked fibrin formation. Platelets immediately form a plug at the site of injury, known as primary hemostasis. Secondary hemostasis occurs simultaneously: additional coagulation factors beyond factor VII (mentioned below) respond in a cascade, forming fibrin strands that reinforce the platelet plug.

Coagulation is highly conserved across biology. In all mammals, coagulation involves cellular components (platelets) and protein components (coagulation factors or clotting factors). This pathway has been extensively researched in humans and is best understood. Coagulation disorders can lead to bleeding problems, bruising, or thrombosis.

Physiology of Coagulation

The physiology of blood coagulation is based on hemostasis, the body’s natural process to stop bleeding. Coagulation is part of a series of hemostatic reactions involving plasma, platelets, and vascular components.

Hemostasis involves four main stages:

1. Vascular constriction (vasospasm): This refers to the contraction of smooth muscles in the tunica media of the endothelium (the wall of blood vessels).

2. Activation of platelets and formation of the platelet plug:

   • Platelet activation: Platelet activators, such as platelet-activating factor and thromboxane A2, activate platelets in the bloodstream, leading to the binding of platelet membrane receptors (e.g., glycoprotein IIb/IIIa) to extracellular matrix proteins, such as von Willebrand factor on the membrane of damaged endothelial cells and collagen at the injury site.
   • Formation of the platelet plug: Aggregated platelets form a temporary plug to stop bleeding. This process is often referred to as primary hemostasis.

3. Coagulation cascade: This is a series of enzymatic reactions that leads to the formation of a stable blood clot. Endothelial cells release substances like tissue factor that stimulate the extrinsic pathway of the coagulation cascade. This state is known as secondary hemostasis.

4. Formation of fibrin clot: Near the end of the extrinsic pathway, after thrombin converts fibrinogen to fibrin, factor XIIIa (plasma transglutaminase; the activated form of the fibrin-stabilizing factor) creates cross-links in fibrin, leading to the formation of a fibrin clot (the final blood clot) that temporarily seals the wound until it heals, while the interior is dissolved by fibrinolytic enzymes, and the exterior of the clot disintegrates.

After fibrin clot formation, clot retraction occurs, followed by clot resolution, and these two processes together are known as tertiary hemostasis. Activated platelets contract their internal actin and myosin filaments in their cytoskeleton, leading to a reduction in clot volume. Plasminogen activators, such as tissue-type plasminogen activator (t-PA), convert plasminogen to active plasmin, which lyses the fibrin clot. This restores blood flow in the damaged/blocked blood vessels.

Vascular Constriction

When a blood vessel is damaged, endothelial cells can release various vasoconstrictive substances such as endothelin and thromboxane to induce contraction of the smooth muscles in the vessel wall. This helps reduce blood flow at the site of injury and limits bleeding.

Platelet Activation and Formation

When the endothelium is damaged, the underlying collagen, normally isolated, is exposed to circulating platelets, which directly bind to collagen via specific glycoprotein Ia/IIa surface receptors. This adhesion is reinforced by von Willebrand factor (vWF), which is released from the endothelium and platelets. vWF forms additional links between glycoprotein Ib/IX/V and the platelet A1 domain. This localization of platelets in the extracellular matrix enhances collagen interaction with platelet glycoprotein VI. The binding of collagen to glycoprotein VI triggers a signaling cascade that leads to the activation of platelet integrins. The activated integrin mediates the firm adhesion of platelets to the extracellular matrix, anchoring them to the site of injury.

Activated platelets release the contents of stored granules into the plasma. These granules contain ADP, serotonin, platelet-activating factor (PAF), vWF, platelet factor 4, and thromboxane A2 (TXA2), which, in turn, activate additional platelets. The contents of the granules activate a Gq-coupled receptor cascade, resulting in an increased calcium concentration in the cytosol of platelets. Calcium activates protein kinase C, which subsequently activates phospholipase A2 (PLA2). PLA2 then modifies the integrin glycoprotein IIb/IIIa membrane, increasing its affinity for binding fibrinogen. Activated platelets change from a discoid to a stellate shape, and fibrinogen binds to glycoprotein IIb/IIIa to facilitate the aggregation of adjacent platelets, forming the platelet plug and completing primary hemostasis.

Coagulation Cascade

The coagulation cascade of secondary hemostasis has two primary pathways that lead to fibrin formation. These are the contact activation pathway (intrinsic pathway) and the tissue factor pathway (extrinsic pathway), both of which lead to the same essential downstream reactions that produce fibrin. It was previously thought that the two coagulation cascade pathways were of equal importance, but it is now established that the primary pathway for initiating blood coagulation is the tissue factor (extrinsic) pathway. The pathways consist of a series of reactions in which a zymogen (inactive enzyme precursor) of a serine protease and its glycoprotein cofactor are activated to convert into active components that subsequently catalyze the next reaction in the cascade, ultimately leading to cross-linked fibrin formation. Coagulation factors are generally denoted by Roman numerals, with a lowercase "a" added to indicate the active form.

Coagulation factors are generally enzymes known as serine proteases that act by cleaving downstream proteins. Exceptions include tissue factor, FV, FVIII, and FXIII. Tissue factor, FV, and FVIII are glycoproteins, and factor XIII is a transglutaminase. Coagulation factors circulate as inactive zymogens. Therefore, the coagulation cascade is traditionally divided into three pathways. Tissue factor and contact activation pathways both activate the "common final pathway" factors X, thrombin, and fibrin.

 

 

 

Tissue Factor Pathway (Extrinsic)

The main role of the tissue factor (TF) pathway is to create a "thrombin burst," a process in which thrombin, the most critical component of the coagulation cascade in terms of its feedback activation roles, is rapidly released. FVIIa is present in larger quantities than other circulating active coagulation factors. This process involves the following steps:

Following damage to a blood vessel, FVII leaves circulation and interacts with tissue factor expressed on tissue factor-bearing cells (stromal fibroblasts and leukocytes), forming an activated complex (TF-FVIIa). TF-FVIIa activates FIX and FX. FVII itself is activated by thrombin, FXIa, FXII, and FXa. Activation of FX (to form FXa) is almost immediately inhibited by tissue factor pathway inhibitor (TFPI). FXa and its cofactor FVa form the prothrombinase complex that converts prothrombin to active thrombin. Thrombin then activates other components of the coagulation cascade, including FV and FVIII (which forms a complex with FIX), and releases FVIII from its association with vWF. FVIIIa is a cofactor for FIXa and together they form the "tenase" complex that activates FX. Thus, the cycle continues. ("Tenase" is a contraction of "ten" and the suffix "-ase" used for enzymes.)

Contact Activation Pathway (Intrinsic)

The contact activation pathway begins with the formation of an initial complex on collagen by high molecular weight kininogen (HMWK), prekallikrein, and FXII (Hageman factor). Prekallikrein is converted to kallikrein, and FXII is converted to FXIIa. FXIIa converts FXI to FXIa. Factor XIa activates FIX, which forms the tenase complex with factor FVIIIa that activates FX to FXa. The small role that the contact activation pathway plays in initiating blood clot formation is highlighted by the fact that individuals with severe deficiencies in FXII, HMWK, and prekallikrein do not exhibit bleeding disorders. Instead, the contact activation system appears to play a larger role in inflammation and innate immunity. Nevertheless, interference with this pathway may provide protection against thrombosis without significant bleeding risk.

Final Common Pathway

The coagulation cascade is divided into two pathways, arising from laboratory experiments where coagulation is measured either at the time of clotting or after clotting has begun by glass, which initiates the intrinsic pathway, or by thromboplastin (a combination of tissue factor and phospholipids), which initiates the extrinsic pathway.

Additionally, the design of the common final pathway indicates that prothrombin is converted to thrombin only through the intrinsic or extrinsic pathways, which is an oversimplification. In fact, thrombin is produced by activated platelets at the start of platelet aggregation, which in turn causes further activation of platelets. Thrombin not only acts to convert fibrinogen to fibrin but also activates factors VIII and V, as well as their inhibitor protein C (in the presence of thrombomodulin). By activating factor XIII, covalent bonds are formed that link fibrin polymers formed from active monomers together, stabilizing the fibrin mesh.

The coagulation cascade is sustained by the continuous activation of FVIII and FIX in a prothrombotic state to form the tenase complex until regulated by anticoagulant pathways.

Cell-Based Coagulation Model

A newer model of the coagulation mechanism describes a complex interplay of cellular and biochemical events occurring during the coagulation process within the body. Along with plasma pre-coagulation and anticoagulant proteins, normal physiological coagulation requires the presence of two types of cells to form coagulation complexes: cells expressing tissue factor (usually extravascular) and platelets.

The coagulation process occurs in two stages. The first stage is initiation, which occurs in tissue factor-expressing cells. This is followed by the amplification phase occurring on activated platelets. The initiation phase progresses through the classic extrinsic pathway mediated by exposure to tissue factor and contributes about 5% to thrombin production. The enhanced thrombin production occurs through the classic intrinsic pathway during the propagation phase. Approximately 95% of thrombin produced will occur in this second phase.

Fibrinolysis

Ultimately, blood clots are reorganized and absorbed through a process called fibrinolysis. The main enzyme responsible for this process is plasmin, which is regulated by plasmin activators and inhibitors.

Role in the Immune System

The coagulation system overlaps with the immune system. Coagulation can physically trap invading microbes in blood clots. Additionally, some products of the coagulation system can aid the innate immune system with their ability to increase vascular permeability and act as chemotactic agents for phagocytic cells. Furthermore, some coagulation system products are directly antimicrobial. For example, beta-lysin, an amino acid produced by platelets during coagulation, can lyse many Gram-positive bacteria by acting as a cationic detergent. Many acute phase proteins of inflammation play roles in the coagulation system. Additionally, pathogenic bacteria may secrete factors that alter the coagulation system, e.g., coagulase and streptokinase.

Immunohemostasis integrates the activation of the immune system with clot formation. Immunothrombosis is a pathological result of the interplay between immunity, inflammation, and coagulation. Mediators of this process include damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) recognized by pattern recognition receptors (PRRs), triggering pro-coagulation and pro-inflammatory responses such as the formation of neutrophil extracellular traps (NETs).

Cofactors

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

Calcium and Phospholipids

Calcium and phospholipids (components of platelet membranes) are required for the function of tenase and prothrombinase complexes. Calcium mediates the binding of complexes through the gamma-carboxy terminal residues of factor Xa and factor IXa to phospholipid surfaces expressed by platelets, as well as to pro-coagulant microparticles or microvesicles shed from them. Calcium is also needed at other points in the coagulation cascade. Calcium ions play a major role in regulating the coagulation cascade, which is critical for maintaining homeostasis. Besides platelet activation, calcium ions are responsible for the full activation of several coagulation factors, including coagulation factor XIII.

Vitamin K

Vitamin K is an essential factor for hepatic gamma-glutamyl carboxylase, which adds a carboxyl group to glutamic acid residues on factors II, VII, IX, and X, as well as proteins S, C, and Z. By adding a gamma carboxyl group to the glutamate residue on immature coagulation factors, vitamin K itself is oxidized. Another enzyme, vitamin K epoxide reductase (VKORC), reduces vitamin K to its active form. Vitamin K epoxide reductase is pharmacologically important as a target for anticoagulant drugs like warfarin and related coumarins such as acenocoumarol, fenprocoumon, and dicoumarol. These drugs create a state of reduced vitamin K by blocking VKORC, thus inhibiting the maturation of clotting factors. Vitamin K deficiency due to other reasons (such as malabsorption) or disorders in vitamin K metabolism in disease (e.g., liver failure) leads to the formation of PIVKAs (proteins formed in the absence of vitamin K), which are partially or completely under gamma-carboxylated, affecting the ability of coagulation factors to bind to phospholipids.

Regulators

Multiple mechanisms control platelet activation and the coagulation cascade. Abnormalities can lead to an increased tendency towards thrombosis:

Protein C and Protein S

Protein C is a major physiological anticoagulant. It is a vitamin K-dependent serine protease that is activated by thrombin to activated protein C (APC). Protein C is activated in a sequence that begins with the binding of protein C and thrombin to thrombomodulin, a cell surface protein. Thrombomodulin binds these proteins in a way that activates protein C. The activated form, along with protein S and a phospholipid as a cofactor, degrades FVa and FVIIIa. A quantitative or qualitative deficiency of either (protein C or protein S) may lead to thrombophilia (a tendency to develop thrombosis). Dysfunction of protein C (resistance to activated protein C), for example, by having the "Leiden" variant of factor V or high levels of FVIII, may also lead to a thrombotic tendency.

Antithrombin
Antithrombin is a serine protease inhibitor (serpin) that degrades serine proteases: thrombin, FIXa, FXa, FXIa, and FXIIa. It is continuously active, but its binding to these factors is enhanced in the presence of heparan sulfate (a glycosaminoglycan) or by the administration of heparins (various heparinoids preferentially enhance FXa, thrombin, or both). A deficiency in antithrombin (either congenital or acquired, for example, in proteinuria) leads to thrombophilia.

Tissue Factor Pathway Inhibitor (TFPI)
The tissue factor pathway inhibitor (TFPI) limits the action of tissue factor (TF). It also prevents excessive activation of FVII and FX mediated by TF.

Plasmin
Plasmin is produced through the proteolytic cleavage of plasminogen, a plasma protein synthesized in the liver. This cleavage is catalyzed by tissue plasminogen activator (t-PA), which is synthesized and secreted by the endothelium. Plasmin proteolytically cleaves fibrin into fibrin degradation products, preventing excessive fibrin formation.

Prostacyclin
Prostacyclin (PGI2) is released by the endothelium and activates platelet Gs protein-coupled receptors. This, in turn, activates adenylate cyclase, which synthesizes cAMP. cAMP prevents platelet activation by reducing cytosolic calcium levels, thereby inhibiting the release of granules that would lead to the activation of additional platelets and the coagulation cascade.

Medical Evaluation
Numerous medical tests are used to assess the function of the coagulation system:
Common tests: aPTT, PT (also used to determine INR), fibrinogen test (often by Clauss fibrinogen assay), platelet count, platelet function test (often by PFA-100), thromboelastography.
Other tests: TCT, bleeding time, mixing study (whether a deficiency is corrected when the patient's plasma is mixed with normal plasma), factor assay, antiphospholipid antibodies, D-dimer, genetic tests (such as factor V Leiden, prothrombin G20210A mutation), dilute Russell viper venom time (dRVVT), various platelet function tests, thromboelastography (TEG or Sonoclot), and euglobulin lysis time (ELT).

The intrinsic activation pathway begins with the activation of the contact activation system and can be measured by the activated partial thromboplastin time (aPTT) test.

The tissue factor pathway (extrinsic) begins with the release of tissue factor (a specific cellular lipoprotein) and can be measured by the prothrombin time (PT) test. PT results are often reported as a ratio (the INR value) to monitor the dose of oral anticoagulants such as warfarin.

Fibrinogen screening, both quantitatively and qualitatively, is measured with the thrombin clotting time (TCT). The precise measurement of fibrinogen present in the blood is generally performed using the Clauss fibrinogen assay. Many analyzers can measure "fibrinogen derived" levels from prothrombin time clotting charts.

If a coagulation factor is part of the contact activation or tissue factor pathways, the deficiency of that factor will affect only one of the tests: thus, hemophilia A, which is a deficiency of factor VIII, which is part of the contact activation pathway, results in an abnormal aPTT but a normal PT. Deficiencies of common pathway factors prothrombin, fibrinogen, FX, and FV prolong both aPTT and PT. If there is an abnormal PT or aPTT, additional testing is performed to determine which factor (if any) is present in abnormal concentrations. Fibrinogen deficiency (either quantitative or qualitative) prolongs PT, aPTT, thrombin time, and reptilase time.

Role in Disease
Coagulation defects can lead to bleeding or thrombosis, depending on the nature of the defect, and sometimes both.

Platelet Disorders
Platelet disorders can be congenital or acquired. Examples of congenital platelet disorders include: Glanzmann's thrombasthenia, Bernard-Soulier syndrome (an abnormal glycoprotein complex Ib-IX-V), gray platelet syndrome (alpha granule deficiency), and delta storage pool deficiency (deficiency of dense granules). Most are rare and predispose to bleeding. Von Willebrand disease is due to a deficiency or abnormal function of von Willebrand factor and leads to a similar bleeding pattern. Milder forms are relatively common.

Reduced platelet counts (thrombocytopenia) can be due to insufficient production (e.g., myelodysplastic syndrome or other bone marrow disorders), destruction by the immune system (immune thrombocytopenic purpura), or consumption (e.g., thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, disseminated intravascular coagulation, heparin-induced thrombocytopenia). Increased platelet counts are referred to as thrombocytosis, which may lead to thromboembolic formation. However, thrombocytosis may be associated with an increased risk of thrombosis or bleeding in patients with myeloproliferative neoplasms.

Coagulation Factor Disorders
The most well-known coagulation factor disorders are hemophilias. The three main forms are hemophilia A (factor VIII deficiency), hemophilia B (factor IX deficiency or "Christmas disease"), and hemophilia C (factor XI deficiency, with a tendency for mild bleeding).

Von Willebrand disease (which behaves more like a platelet disorder except in severe cases) is the most common inherited bleeding disorder and is known to be inherited in an autosomal recessive or dominant manner. In this condition, there is a deficiency of von Willebrand factor (vWF), which mediates the binding of glycoprotein Ib (GPIb) to collagen. This binding facilitates platelet activation and primary hemostasis.

In acute or chronic liver failure, there is inadequate production of coagulation factors, which likely increases the risk of bleeding during surgery.

Thrombosis is the pathological development of blood clots. These clots may dislodge and move to create emboli or grow large enough to occlude the vessel in which they formed. An embolus is said to occur when a thrombus (blood clot) becomes an embolus that migrates to another part of the body, disrupting circulation and thus impairing the function of downstream organs. This leads to ischemia and often results in ischemic tissue necrosis. Most cases of venous thrombosis are due to acquired conditions (advanced age, surgery, cancer, immobility). Unprovoked venous thrombosis may relate to hereditary thrombophilias (such as factor V Leiden, antithrombin deficiency, and other deficiencies or different genetic types), especially in younger patients with a family history of thrombosis. However, thrombotic events are more likely when acquired risk factors overlay a hereditary state.

Pharmacology

Procoagulants
The use of absorbent chemicals such as zeolites and other hemostatic agents is also employed for quickly sealing severe injuries (such as traumatic bleeding secondary to gunshot wounds). Thrombin and fibrin glue are used surgically to treat bleeding and thrombosis of aneurysms. The hemostatic powder spray TC-325 is used for the treatment of gastrointestinal bleeding.
Desmopressin is used to enhance platelet function by activating the 1A receptor of arginine vasopressin.
Concentrates of clotting factors are used to treat hemophilia, to reverse the effects of anticoagulant drugs, and to treat bleeding in individuals with impaired synthesis or increased consumption of clotting factors. Prothrombin complex concentrates, cryoprecipitate, and fresh frozen plasma are commonly used from clotting factor products. Activated human factor VII is sometimes used in the treatment of major bleeding.
Tranexamic acid and aminocaproic acid inhibit fibrinolysis, leading to a reduction in bleeding rates. Prior to its withdrawal, aprotinin was used in some types of major surgery to reduce the risk of bleeding and the need for blood products.

Anticoagulants
Anticoagulants and antiplatelet drugs (collectively referred to as "antithrombotics") are among the most commonly used medications. Antiplatelet agents include aspirin, dipyridamole, ticlopidine, clopidogrel, ticagrelor, and prasugrel. Injectable glycoprotein IIb/IIIa inhibitors are used during angioplasty. Among anticoagulants, warfarin (and related coumarins) and heparin are the most frequently used. Warfarin affects vitamin K-dependent clotting factors (II, VII, IX, X) and proteins C and S, while heparin and related compounds enhance the antithrombin effect on thrombin and factor Xa. A new class of drugs, direct thrombin inhibitors, is currently under development. Some members are already in clinical use (such as lepirudin, argatroban, bivalirudin, and dabigatran). There are also other small molecule compounds in clinical use that directly interfere with the enzymatic function of specific clotting factors (oral anticoagulants with direct action: dabigatran, rivaroxaban, apixaban, and edoxaban).

Early Discoveries
Theories regarding blood coagulation have existed since ancient times. The physiologist Johannes Müller (1801-1858) described fibrin, the thrombus material. Thus, its soluble precursor, fibrinogen, was named by Rudolf Virchow (1821-1902) and chemically isolated by Prosper Sylvain Denis (1799-1863). Alexander Schmidt proposed that the conversion of fibrinogen to fibrin is the result of an enzymatic process, naming the hypothetical enzyme "thrombin" and its precursor "prothrombin." Arthus discovered in 1890 that calcium is essential for coagulation. Platelets were identified in 1865, and their function was clarified by Giulio Bizzozero in 1882.
The theory that thrombin is produced in the presence of tissue factor was established by Paul Morawitz in 1905. At this stage, it became clear that thromboplastin (factor III) is released by damaged tissues and reacts with prothrombin (II), which, along with calcium (IV), forms thrombin that converts fibrinogen to fibrin (I).

Clotting Factors
The remaining biochemical factors in the coagulation process were largely discovered in the twentieth century.
The first clue to the true complexity of the coagulation system was the discovery of proaccelerin (initially and later named factor V) by Paul Orén (1905-1990) in 1947. He also identified its function as the generation of accelerin (factor VI), which was later found to be the active form of V (or Va). Hence, factor VI is no longer actively used.
Factor VII (also known as serum prothrombin converting accelerator or proconvertin, which precipitates with barium sulfate) was discovered in a young female patient in the years 1949 and 1951 by different groups.
Factor VIII is deficient in hemophilia A, which is clinically recognized but elusive in its cause. It was identified in the 1950s and named antihemophilic globulin due to its ability to correct hemophilia A.
Factor IX was discovered in 1952 in a young patient with hemophilia B. Its deficiency was identified by Dr. Rosemary Biggs and Professor R.G. Macfarlane in Oxford, England. Therefore, this factor is referred to as Christmas factor.
Hageman factor, now recognized as factor XII, was identified in 1955 in an asymptomatic patient with a prolonged bleeding time named John Hageman. Factor X or Stuart-Prower factor was followed in 1956. This protein was identified in Mrs. Audrey Prower from London, who had a lifelong tendency to bleed. In 1957, an American group identified the same factor in Mr. Rufus Stuart. Factors XI and XIII were identified in 1953 and 1961, respectively.

Other Species
All mammals have a coagulation process that is very similar, utilizing a combined cellular protease and serine protease process. A non-mammalian animal that uses serine proteases for blood coagulation is the horseshoe crab. The horseshoe crab, which shows a close link between coagulation and inflammation, has a primitive response to injury that is carried out by cells called amoebocytes (or hemocytes), which perform both hemostatic and immune functions.