If blood does not clot in a timely manner, we can lose our lives due to excessive bleeding. When blood volume drops because of severe bleeding, blood pressure also drops, and oxygen and nutrients cannot reach vital organs, which can lead to death. However, the life-saving process of blood clotting can also pose a threat to us living in modern life style. This is because blood clots formed during coagulation may be generated excessively or in inappropriate situations unrelated to hemostasis. Factors like high blood pressure, diabetes, and obesity can cause blood clots to form inside blood vessels, leading to impaired blood circulation or ischemia, a condition where blood supply is reduced or blocked. If a clot detaches and blocks a vessel in the heart or brain, it can lead to serious diseases such as myocardial infarction or stroke. Many people, including my own family members, take anticoagulants daily to prevent further narrowing of blood vessels. Understanding the detailed process and mechanism of coagulation helps to grasp how various anticoagulant drugs work by targeting specific pathways in this process. This understanding may also help explain the pros and cons of different medications. While I initially began exploring the blood coagulation mechanism to understand and explain the functions of heparin and heparan sulfate, I became determined to delve deeper into the topic to better understand the problematic and covert relationship between blood clotting and inflammation, particularly chronic inflammation.
Key Player in Coagulation: Platelets
Platelets are disc-shaped, nucleus-free blood cells, with about 70% circulating in the blood and the remaining stored in the spleen. They have a lifespan of about 7-10 days, after which they are cleared by the liver and spleen.[1] Platelets possess various receptors on their surface and contain numerous α-granules and dense granules (δ-granules) in their cytoplasm, which constitute about 30-50% of total protein. These granules are central to platelet function. The α-granules include fibrinogen, von Willebrand factor (vWF), platelet-derived growth factor (PDGF), platelet factor IV (PF4), coagulation factors (e.g., factor XI and XIII), and plasminogen activation inhibitors (PAI). Dense granules contain abundant ADP, serotonin (5-hydroxytryptamine, 5-HT), polyphosphates, and calcium ions.[2] It's important to note that platelet granules are also involved in inflammation and immune responses, beyond hemostasis and thrombosis.
Integrity of the Vascular Wall
Fortunately, our blood vessels are designed to allow smooth blood flow under normal conditions. Endothelial cells line the interior of blood vessels, forming a boundary with surrounding tissues. In a healthy vascular wall, circulating platelets do not adhere or coagulate, and blood flows smoothly without obstruction. Both endothelial cells and platelets carry similar charges, causing them to repel each other. Endothelial cells also secrete substances like nitric oxide (NO) and prostacyclin (PGI₂) to keep the vessel wall smooth, relax blood vessels, and inhibit platelet aggregation. In addition, anticoagulant proteins such as protein C and S, as well as heparan sulfate (which activates antithrombin III), are synthesized to continuously assist anticoagulant activity and prevent thrombus formation. These substances not only inhibit platelet aggregation but also promote vasodilation, which increases blood flow and reduces the likelihood of clot formation due to less platelet and coagulation factor retention at the vessel wall. Moreover, endothelial CD39 hydrolyzes ADP released by activated platelets, preventing further platelet activation. However, when the vessel is ruptured and endothelial cells are damaged, the secretion of these anticoagulant substances is inhibited, prompting immediate pro-coagulant responses.
Let’s briefly examine the structure of blood vessels and the composition of vascular endothelial cells and extracellular matrix that are in direct contact with blood. Arteries, veins, and capillaries have distinct structures optimized for their respective roles in circulation. Arteries, which must withstand high pressure and fast flow, have thick and elastic walls with developed smooth muscle layers. Capillaries, with low pressure and slow flow, are thin and narrow, allowing close contact with surrounding tissue for efficient exchange of oxygen, nutrients, and waste. ECM composition around endothelial cells also varies depending on vascular function. Different types of collagen and more porous ECMs are found in capillaries to facilitate permeability. Let us explore the process of hemostasis and coagulation that occurs when endothelial cells are ruptured or damaged, imagining that the substances secreted by endothelial cells to maintain the integrity of the vascular wall are densely distributed around them, based on the structure of the endothelium.
To understand the clotting process, it is helpful to divide it into stages. Although these processes often are intertwined and occur simultaneously, since the roles and mechanisms at each stage are different, understanding them separately seems to be able to provide a clearer understanding of the major parties involved. Broadly, coagulation can be divided into primary and secondary hemostasis. Primary hemostasis involves platelet recruitment, while secondary hemostasis involves activation of various coagulation factors to form a stable clot. Secondary hemostasis is further subdivided into the intrinsic and extrinsic pathways, depending on how the coagulation cascade is triggered. These pathways merge into a common pathway that results in the formation of a fibrin mesh. Finally, as healing progresses, the fibrinolytic system gradually breaks down clots to restore blood flow. However, the very first reaction is vasoconstriction.
First Step of Hemostasis – Vasoconstriction
When a blood vessel is ruptured or damaged, the initial response is vasoconstriction. Nerve receptors in the vessel wall stimulate smooth muscle in the tunica media to contract, narrowing the vessel almost reflexively. This rapid reaction is likely one of the most instinctive responses. Vasoconstriction is further enhanced by substances released by platelets that rush to the site. Smooth muscle contraction is closely tied to calcium (Ca²⁺). An increase in calcium concentration induces contraction, while a decrease promotes relaxation, making calcium regulation crucial in signal transduction. For example, serotonin (5-HT), found in dense granules of platelets, binds to 5-HT₂A receptors on smooth muscle, triggering the IP₃ (Inositol Triphosphate) pathway to increase the amount of Ca²⁺ in the cytoplasm, causing smooth muscles to contract. It is through this same principle that organs such as the heart contract and relax through calcium.
Primary Hemostasis: Platelet Plug Formation
Initial Platelet Binding
When the endothelial cells of blood vessels are damaged, collagen in the extracellular matrix of the endothelial cells and von Willebrand factor (vWF), a glycoprotein that promotes platelet adhesion, are exposed. In normal blood vessels, these substances never come into contact with platelets. Upon exposure, the nearest platelets quickly bind to them. vWF is particularly attractive to platelets. Platelets bind to collagen via GPVI receptors and to vWF via the GPIb-V-IX receptor complex. These early interactions do not require prior activation and represent the swiftest step in coagulation. Even if the blood flow is rather fast, platelets can stably bind to these receptors, but if there is an abnormality in von Willebrand factor and they cannot bind to the receptors normally, a coagulation disorder called von Willebrand disease (VWD) may occur and bleeding may increase.
Formation of Platelet Plug and Activation Cascade
Platelets bound to collagen and vWF immediately eject granules prepared in the cytoplasm to recruit more platelets. You need to quickly fill in the holes in your blood vessels to prevent blood loss. Just as putting a rubber stopper in a hole in a sink prevents water from flowing out, when platelets gather at a high speed and clump together, they can quickly block a hole in a blood vessel like a plug. The key here is to attract rapidly as many platelets as possible. Activated platelets release more granules to continuously activate nearby platelets, in a positive feedback loop (like zombies infecting others, though maybe not the best analogy...). Platelets also change shape to increase surface area and better cover the damage. The entire plug formation process is driven by intracellular signaling cascades initiated by binding to surface receptors. Remember, the main goal at this stage is to recruit more platelets.
ADP: Intracellular Signaling for Platelet Recruitment
First, ADP initially secreted from granules binds to the P2Y₁ receptor on other surrounding platelets. These receptors are G-protein-coupled receptors (GPCR) that bind to Gq protein and activate the Phospholipase C (PLC) pathway. PLC is a phospholipase that hydrolyzes PIP₂ (Phosphatidylinositol 4,5-bisphosphate), a phospholipid of the cell membrane, into IP₃ (Inositol trisphosphate) and DAG (Diacylglycerol). IP₃ opens calcium channels in the endoplasmic reticulum (ER) in the cytoplasm, increasing Ca²⁺ levels. When the concentration of calcium increases, platelets secrete more granules, and fibrinogen, von Willebrand factor, ADP, and platelet-derived growth factor (PDGF) are secreted from the granules, causing positive feedback that activates other platelets around them. ADP secreted from platelets activates other platelets, causing them to secrete ADP again.
ADP also binds to P2Y₁₂ receptors on other platelets, activating inhibitory Gi proteins, which suppress adenylate cyclase (AC). Normally, AC increases cAMP, which activates PKA. PKA then phosphorylates IP₃ receptors, inhibiting their activity. Inhibiting AC thus enhances IP₃ signaling and increases Ca²⁺, promoting platelet activation. Therefore, both P2Y₁ and P2Y₁₂ receptors contribute to elevated calcium levels and platelet activation.
Increased calcium also enhances the affinity of the fibrinogen receptor GP2b/IIIa on platelets, facilitating strong binding to fibrinogen. Fibrinogen connects aggregated platelets. In the final coagulation stage, fibrinogen is cleaved by thrombin into fibrin, forming a mesh that tightly holds the platelets like a spider web. At this stage, fibrinogen helps platelets adhere and solidify the plug. Calcium is a key second messenger in all coagulation-related signaling pathways and is essential for physiological functions such as heartbeat regulation, neuronal protection, hormone secretion, and kidney water reabsorption.
Meanwhile, unlike IP₃, which is hydrolyzed from PIP₂ in the above PLC pathway and then goes to the cytoplasm to open a calcium channel, DAG from the PLC pathway remains in the membrane and activates PKC (protein kinase C), which further activates GP2b/IIIa receptors and supports fibrinogen binding, as well as thromboxane A2 synthesis.[3]
Thromboxane A₂: A Potent Platelet Activator
The substances that play the greatest role in enabling platelets to recruit more platelets are ADP and thromboxane A₂. Let's take a look at how thromboxane A₂ is produced. As we saw earlier, when the intracellular calcium concentration increases, the calcium-dependent phospholipase A₂ (PLA₂)—which is activated by calcium—binds to calcium and becomes active. The activated PLA₂ breaks down fatty acids from phospholipids in the cell membrane, releasing arachidonic acid. When arachidonic acid encounters the enzyme COX (cyclooxygenase), it is converted into prostaglandin G₂ (PGG₂), which is then transformed into PGH₂ (prostaglandin H₂). PGH₂ is ultimately secreted in different tissues as a variety of physiologically active substances. For instance, in the stomach, it increases mucus secretion to protect the gastric lining, and in the kidneys, it helps maintain blood flow.
A particularly interesting aspect in relation to blood clotting is that PGH₂ is converted into prostacyclin (PGI₂) in vascular endothelial cells, while in platelets, it is converted into thromboxane A₂ (TXA₂) by thromboxane synthase. This means that the same precursor substance is synthesized into final products that have completely opposite functions. As mentioned earlier, prostacyclin is a potent anticoagulant continuously secreted by vascular endothelial cells to relax blood vessels and prevent platelets from adhering to the vessel walls. In contrast, thromboxane A₂ is a powerful vasoconstrictor and pro-coagulant that is produced and secreted by platelets when blood vessels are damaged. It stimulates and activates nearby platelets, promoting their aggregation. The human body cleverly converts arachidonic acid into the appropriate substance based on the situation—a truly remarkable system.
As a side note, aspirin is sometimes prescribed as an anticoagulant because it inhibits the COX pathway that generates thromboxane A₂, thereby preventing coagulation. Aspirin belongs to the class of drugs known as non-steroidal anti-inflammatory drugs (NSAIDs). This reflects that, unlike steroids, which block the entire inflammatory pathway by inhibiting PLA2 and preventing the release of arachidonic acid from the beginning, aspirin directly inhibits the COX pathway and blocks only the specific pathway where prostaglandins are synthesized.
Returning to the main topic, thromboxane A₂ operates in a manner very similar to ADP. When thromboxane A₂ binds to its receptor, the TP receptor, it activates the Gq protein → activates the PLC (phospholipase C) pathway → breaks down membrane phospholipid PIP₂ into IP₃ and DAG → IP₃ increases intracellular Ca²⁺ concentration → causes granule release → activates additional platelets by binding to their receptors. Like ADP, thromboxane A₂ plays a key role in accelerating platelet aggregation through a positive feedback loop.
The process in which rapidly recruited platelets, linked together by fibrinogen, adhere to exposed collagen at the injury site is called platelet plug formation. A simplified illustration of this process is shown below. While in reality, many more signaling cascades are involved, this diagram captures the most essential reactions.
Secondary Hemostasis: The Coagulation Cascade
If primary hemostasis involves the aggregation of platelets connected by fibrinogen to form a plug at the injury site, secondary hemostasis involves tightening that plug into a solid thrombus using an insoluble fibrin mesh. The key to secondary hemostasis is the generation of thrombin, which converts fibrinogen into fibrin—but this requires a multi-step process. Considering the serious diseases that can arise from excessive or inappropriate clot formation, the human body’s approach to thrombosis is clearly extremely cautious. This caution is reflected in the many activation steps that must be passed before forming the sticky fibrin mesh.
The secondary hemostasis process begins via two pathways, but both ultimately converge at the crucial point of thrombin synthesis.
Intrinsic Pathway vs. Extrinsic Pathway
The coagulation cascade of secondary hemostasis is divided into two pathways based on the trigger that initiates the activation of clotting factors: the intrinsic pathway and the extrinsic pathway. The naming implies whether the trigger is internal or external. If activation is initiated by internal factors such as collagen exposure or the activation of factor XII within blood vessels, it's classified as intrinsic. On the other hand, if activation is triggered by tissue factor (TF) released from surrounding tissues—not directly part of the vascular system—it’s called the extrinsic pathway. However, both pathways ultimately merge at the point of thrombin generation.
The extrinsic pathway involves fewer steps to reach thrombin synthesis compared to the intrinsic pathway—meaning it acts more quickly. The prevailing view is that the extrinsic pathway initiates coagulation rapidly, while the intrinsic pathway amplifies the response through positive feedback by activating a larger number of clotting factors. In this division of labor, the extrinsic pathway gets the process started swiftly, and the intrinsic pathway, although slower, reinforces the process by amplifying the coagulation factors.
Most of the factors involved in the coagulation cascade are zymogens—proteolytic enzymes that remain inactive under normal conditions, similar to those in the complement system. When activated, they trigger the activation of the next factor in line through proteolytic cleavage, following a cascade mechanism.
Coagulation Factors
The coagulation factors that appear in both pathways of the coagulation cascade are primarily synthesized in the liver and circulate in the bloodstream. Therefore, liver health is crucial for blood clotting. Although these factors are designated by Roman numerals, many also have specific names, often derived from the names of patients in whom deficiencies were first observed. However, using Roman numerals remains the standard convention. For example, fibrinogen is Factor I (FI), tissue factor is Factor III (FIII), and prothrombin is Factor II (FII). It's also important to note that activated forms of clotting factors are denoted by appending an “a” to their names (e.g., Factor Xa for activated Factor X).
Most coagulation factors are serine proteases, but some are glycoprotein precursors that function as cofactors to stabilize or enhance the action of enzymes. These factors don’t act independently but form complexes to work efficiently in a short time frame. Also, some clotting factors require vitamin K to function properly, and calcium ions (Ca²⁺) as well as negatively charged phospholipids (from platelets) are essential for the coagulation process.
In the next part, we’ll delve deeper into the detailed steps of secondary hemostasis.
[References]
https://doi.org/10.3390/ijms151017901
[2] The life cycle of platelet granules
https://doi.org/10.12688/f1000research.13283.1
[3] Essential Role of Protein Kinase Cδ in Platelet Signaling, αIIbβ3 Activation, and Thromboxane A2 Release
https://www.jbc.org/article/S0021-9258(19)33908-0/fulltext
