Secondary Hemostasis: The Coagulation Cascade
In the primary hemostasis process, a platelet plug is formed by the rapid recruitment of platelets. To further stabilize this plug and promote robust clot formation, a fibrin mesh is generated. Let's explore the cascade of events that activate various coagulation factors to make this happen.
1. Extrinsic Pathway: Triggered by Tissue Factor (TF)
We begin with the extrinsic pathway. Referring again to the structure of vascular endothelial cells, direct injury can occur through rupture or tearing of blood vessels. Additionally, damage can happen to areas not usually exposed to the bloodstream, such as the smooth muscle in the tunica media or fibroblasts in the tunica externa. In the latter case, damaged cells begin to express a membrane protein called Tissue Factor (TF) on their surface. Because this expression of TF initiates the coagulation process, TF is regarded as the starting point of the extrinsic pathway.
However, tissue factor is not solely related to coagulation. As a cytokine receptor protein, TF is also closely linked to inflammatory responses. Pro-inflammatory cytokines such as TNF-α (Tumor Necrosis Factor-α), IL-1β (Interleukin-1β), and IL-6 promote the expression of TF, allowing the processes of blood clotting and immune defense to be integrated at sites of injury or infection. This co-activation of coagulation and immune response is quite logical: closing off the wound to prevent bacterial entry and triggering immune defense if bacteria have already infiltrated.
Tissue factor binds to factor 7,VII (FVII) as the first step in the exogenous coagulation process that we will now look at, and at the same time binds to protease-activated receptor (PAR) to regulate inflammatory responses such as increasing vascular permeability or promoting the migration of white blood cells through intracellular signaling. This means that if there is chronic inflammation in the body, TF can be a link that explains how chronic inflammation and coagulation are connected. Tissue factor is expressed by many cell types, including T cells and platelets, in various disease states such as acute inflammation, chronic inflammation, and cancer.
TF:VIIa Complex in Coagulation
Let’s now focus on the coagulation aspect of TF. In the bloodstream, FVII and its active form, FVIIa, circulate in small amounts (about 1%). When TF is expressed on the membrane of damaged cells, it binds circulating FVIIa to form the TF:VIIa complex. This complex increases the proteolytic activity of FVIIa by rearranging its active site, enabling it to cleave and activate the inactive Factor 10, X (FX) into its active form FXa.
Although initially, FXa generated here is weak in activity, it can activate more FVII into FVIIa, amplifying the process through positive feedback. However, if too much FXa accumulates, the TF:VIIa complex binds to Tissue Factor Pathway Inhibitor (TFPI), which then binds FXa to inactivate it. This self-limiting mechanism is vital, especially considering FXa is the immediate precursor to thrombin formation. These types of built-in brakes against excessive clotting appear repeatedly throughout the coagulation cascade.
In a related note, heparin is known to enhance the anticoagulant effect of Antithrombin III (AT III), a thrombin inhibitor, as discussed in previous article on heparin. Interestingly, in vitro studies show that heparin also accelerates TFPI activity, and in vivo, administration of heparin can increase circulating TFPI levels two to fourfold. [1]
TF:VIIa Complex involved in both Intrinsic and Extrinsic Pathway
The TF:VIIa complex also plays a major role in activating factor 9, IX (FIX), which belongs to the intrinsic pathway discussed next, by converting FIX into its active form, FIXa. FIX binds to the TF:VIIa complex under conditions rich in calcium ions and phospholipids (especially phosphatidylserine), and is cleaved by TF:VIIa to become FIXa. This active form of FIXa subsequently activates factor FX to FXa.
Therefore, if we literally define "ten-ase" as an enzyme that cleaves factor 10, X (FX), then the TF:VIIa complex functions as a tenase in both the intrinsic and extrinsic pathways. In the extrinsic pathway, it acts as the direct tenase that cleaves FX; in the intrinsic pathway, it activates FIXa, which is itself a tenase. For this reason, it can be said that the intrinsic pathway is dependent on the extrinsic pathway.
Although the amount is small, the presence of active FVIIa circulating in the blood under normal conditions enables the rapid initiation of the coagulation process, and this process also activates factors of the intrinsic pathway. Thus, the extrinsic pathway is considered the true starting point of the coagulation cascade. Another reason the intrinsic pathway is seen not as a starting point but rather as an amplification phase is that the intrinsic tenase complex activates FX and generates thrombin 50 times faster than the extrinsic tenase. [2]
However, the most important point is that FXa is generated by both pathways, meaning that both converge at a common point. Now, the time has come to assemble the complex that converts prothrombin into thrombin. But before that, let’s take a closer look at the intrinsic pathway, another initiating route in the coagulation cascade.
2. Intrinsic Pathway
The intrinsic, or contact-activated, pathway of blood coagulation activates factor X through a series of reactions that are independent of the extrinsic pathway and are not inhibited by TFPI (Tissue Factor Pathway Inhibitor). Although the extrinsic pathway is sufficient to initiate clot formation, the intrinsic pathway is required to sustain and amplify the coagulation cascade.
The intrinsic pathway is initiated directly when blood vessels are injured, more specifically when the negatively charged surface of a damaged vessel is exposed. In other words, coagulation begins when clotting factors come into contact with specific surfaces. This is why blood left in a glass tube clots within minutes—because the glass surface carries a negative charge. This phenomenon is known as the human contact system.
The human contact system is not only involved in blood coagulation but is also closely linked to inflammatory pathways. It can be triggered not only by negatively charged surfaces but also by artificial materials, misfolded proteins, or the surfaces of certain microorganisms. There are several reasons why the membrane of a damaged vessel carries a negative charge. Normally, negatively charged phospholipids like phosphatidylinositol (PI) are located on the inner side of the lipid bilayer in healthy cells. However, when a blood vessel ruptures, the inner side of the cell membrane is exposed to the outside.
Additionally, activated platelets that gather at the injury site express an enzyme called polyphosphate kinase, which attaches chains of phosphate groups (PO₄³⁻) onto their surface, resulting in an abundance of negatively charged polyphosphate on the platelet membrane. Due to these factors, when a negatively charged cell membrane interacts with coagulation factors, the human contact system is activated.
Human Contact System
The human contact system consists of High Molecular Weight Kininogen (HMWK), Prekallikrein (PK), and Factor 12, XII (FXII), all of which are synthesized in the liver. It's important to remember that activation of the contact system not only leads to coagulation but also triggers inflammation by generating bradykinin, a potent inflammatory mediator.
Let’s look at the process more closely: prekallikrein, the precursor of the protease kallikrein, circulates in the blood in a complex with HMWK. When FXII circulating in the blood encounters a negatively charged surface, it binds to it and undergoes autoactivation. Although the initial activity is low, it becomes activated to FXIIa. FXIIa binds non-covalently to the HMWK-PK complex, forming a ternary complex. In this state, FXIIa begins cleaving more circulating FXII, creating a positive feedback loop. Once sufficient FXII is activated, the cascade proceeds in two directions: FXIIa cleaves Factor 11, XI (FXI) to activate it into FXIa, thereby continuing the intrinsic coagulation pathway.
However, FXIIa also cleaves prekallikrein into kallikrein, which contributes to the inflammatory response. Kallikrein then cleaves high molecular weight kininogen to release bradykinin, a potent inflammatory peptide. Bradykinin plays multiple roles in inflammation, including vasodilation, regulation of blood flow, smooth muscle contraction, increased vascular permeability, blood pressure modulation, and pain sensation. This mechanism helps explain why the immune system triggers inflammation upon detecting components like lipopolysaccharides (LPS)—negatively charged molecules on the outer membranes of Gram-negative bacteria.
The fact that FXIIa simultaneously triggers coagulation and inflammation highlights the tight interconnection between these two processes. The diagram below illustrates the dual arms of the human contact system.
Let us now focus on the coagulation pathway. FXIIa cleaves factor XI (FXI) to activate it into FXIa, and FXIa in turn cleaves factor IX (FIX) to activate it into FIXa. As we saw earlier in the extrinsic pathway, the TF:VIIa complex also activates FIX, but whereas the TF:VIIa complex can be inhibited by the tissue factor pathway inhibitor (TFPI), FXIa can continue to activate FIX, thus amplifying the cascade.
Activated FIXa binds with factor 8, VIII (FVIII) to form a complex that activates factor X (FX). FVIII functions not as a serine protease but as a cofactor. Under normal conditions, FVIII circulates in an inactive form in the bloodstream bound to von Willebrand factor (vWF), which protects it from degradation. However, once thrombin is activated through the extrinsic pathway, it activates FVIII, causing FVIII to dissociate from vWF and bind to FIXa, forming a tenase complex that cleaves FX. Within the complex, FVIII regulates the rate at which FIXa cleaves FX. The fact that FVIII is activated by thrombin, which itself was activated via the extrinsic pathway, once again shows how the intrinsic and extrinsic pathways are not independent processes but are intricately intertwined and proceed in close coordination.
As in the activation of FXa in the extrinsic pathway, calcium ions and negatively charged phospholipids are required. Once FVIIIa is released from vWF and no longer protected, it becomes unstable and is easily inactivated by protein C.
One of the main reasons the intrinsic pathway is not considered the starting point of coagulation is that individuals with severe deficiencies in FXII, HMWK, or PK—components related to the human contact system that initiates the intrinsic pathway—do not show significant problems in hemostasis or coagulation. This has led to the widely accepted view that the intrinsic pathway serves more to amplify an already initiated coagulation process, rather than ‘kicks off’ it. Based on this understanding, the development of anticoagulants has progressed, aiming to regulate the coagulation response without interfering with normal hemostasis. Drugs targeting FVIIa are being developed to overcome the downside of bleeding complications that can occur with anticoagulants used to prevent various thrombotic diseases.
Hemophilia A and B
The intrinsic pathway is also associated with hemophilia. Hemophilia A occurs when FVIII is not produced in sufficient amounts due to a genetic mutation, while hemophilia B involves a deficiency in FIX. There is also a rare form called hemophilia C, caused by low levels of FXI, but hemophilia A accounts for 80% of cases. Hemophilia A and B are inherited through the X chromosome, making them maternally inherited diseases. In hemophilia, the intrinsic pathway does not function properly, increasing the risk associated with bleeding.
3. Common Pathway
Both the intrinsic and extrinsic pathways generate tenase that activates FX into FXa. At this point, both pathways are now ready to use FXa to activate thrombin. The common pathway refers to the process by which thrombin leads to fibrin formation and the subsequent generation of a fibrin mesh, ultimately resulting in the formation of a stable blood clot.
Prothrombinase: FVa + FXa
FXa binds to its protein cofactor FVa to form an enzyme complex called the prothrombinase complex, which cleaves prothrombin(FII) into thrombin(FIIa). This complex assembles on cell membranes containing negatively charged phospholipids—such as those of damaged endothelial cells or activated platelets—through calcium ion mediation. It then starts cleaving and activating a large amount of surrounding prothrombin into thrombin. As mentioned earlier, anionic phospholipids (e.g., phosphatidylinositol, phosphatidylserine), which are typically located on the cytoplasmic side of the membrane, become exposed to the extracellular space when blood vessels are ruptured. For reference, prefixes such as "pro-" or suffixes like "-ogen" in enzyme names indicate inactive precursors (zymogens).
Calcium ions (Ca²⁺) are essential for certain coagulation factors. When calcium binds to the γ-carboxyglutamic acid (Gla) domains of these factors, their structure changes to better interact with membrane phospholipids. Both the phospholipid membrane and Gla domains carry negative charges, and the repulsion between them is neutralized by the positively charged Ca²⁺, enabling the binding. Factors FII, FVII, FIX, and FX all require Ca²⁺, and without it, the critical enzymes—tenase (FVIIIa + FIXa) and prothrombinase (FVa + FXa)—cannot function.
Thrombin (Factor IIa)
Thrombin, the central enzyme of the coagulation process, is now generated. Activated thrombin (FIIa) plays multiple roles. First, it can travel backward along the cascade pathways and activate upstream factors V, VIII, and XI, thereby enabling further thrombin generation and amplifying the coagulation response. This is a form of positive feedback, where thrombin enhances its own production by activating its precursors. In addition, as seen during primary hemostasis, thrombin cleaves the terminal end of PAR-1 and PAR-2 receptors on platelets, initiating signal transduction pathways. Activation of these receptors leads to Gq protein activation and the PLC pathway, which in turn stimulates further granule release and platelet recruitment, promoting platelet aggregation. Refer to the platelet aggregation diagram in the previous article.
However, thrombin’s most critical function is cleaving fibrinogen to form fibrin, which physically links the aggregated platelets like a net. Thrombin converts soluble fibrinogen into insoluble fibrin, forming a gel-like fibrin network that traps red and white blood cells together with the platelets. To complete the process, thrombin activates Factor 13, XIII (FXIII), which cross-links fibrin strands, pulling the edges of the platelet plug tightly together—like tightening shoelaces—and stabilizing the clot.
Fibrinogen → Fibrin → Fibrin Mesh
Fibrinogen (Factor I), the first factor in the coagulation cascade, is a large glycoprotein composed of three peptide chains: α, β, and γ. These chains are coiled together and have a structure with an E domain in the center and D domains on both sides. Thrombin cleaves two sites within the E domain—fibrinopeptides A (FpA) and B (FpB). The exposure of these cleavage sites creates new binding sites that allow the D domains of other fibrin monomers to bind via non-covalent interactions, forming a fibrin polymer. Initially, these interactions are reversible and non-covalent. When thrombin activates Factor XIII to FXIIIa, this enzyme catalyzes covalent cross-linking between neighboring D domains. One E domain can cross-link with the D domains of two separate fibrin monomers. Through fibrin cross-linking, a dense and stable fibrin mesh is formed. This is why FXIIIa is also known as the fibrin-stabilizing factor. The resulting mesh acts like a tightened net, securing the platelet plug. A thrombus is now formed.
The entire coagulation cascade—including the intrinsic, extrinsic, and the converging common pathways—can be illustrated schematically like below.
Both the intrinsic and extrinsic pathways contain multiple amplification mechanisms, including positive feedback loops. Among these, thrombin-mediated amplification is particularly prominent.
[References]
[1] The Tissue Factor Pathway in Cancer: Overview and Role of Heparan Sulfate Proteoglycanshttps://doi.org/10.3390/cancers15051524
[2] Back to basics: the coagulation pathway. Blood Res. 59, 35 (2024)
https://doi.org/10.1007/s44313-024-00040-8
https://reactome.org/PathwayBrowser/#/R-HSA-140877&SEL=R-HSA-140834&PATH=R-HSA-109582
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