104 Hemostasis

Overview of Hemostasis

Hemostasis is the natural process that stops blood loss when an injury occurs.

Hemostasis is the natural process in which blood flow slows and a clot forms to prevent blood loss during an injury, with hemo- meaning blood, and stasis meaning stopping. During hemostasis, blood changes from a fluid liquid to a gelatinous state.

Steps of Hemostasis

Hemostasis includes three steps that occur in a rapid sequence: (1) vascular spasm, or vasoconstriction, a brief and intense contraction of blood vessels; (2) formation of a platelet plug; and (3) blood clotting or coagulation, which reinforces the platelet plug with fibrin mesh that acts as a glue to hold the clot together. Once blood flow has ceased, tissue repair can begin.

Vasoconstriction

Intact blood vessels are central to moderating blood’s clotting tendency. The endothelial cells of intact vessels prevent clotting by expressing a fibrinolytic heparin molecule and thrombomodulin, which prevents platelet aggregation and stops the coagulation cascade with nitric oxide and prostacyclin. When endothelial injury occurs, the endothelial cells stop secretion of coagulation and aggregation inhibitors and instead secrete von Willebrand factor, which causes platelet adherence during the initial formation of a clot. The vasoconstriction that occurs during hemostasis is a brief reflexive contraction that causes a decrease in blood flow to the area.

Platelet Plug Formation

Platelets create the “platelet plug” that forms almost directly after a blood vessel has been ruptured. Within twenty seconds of an injury in which the blood vessel’s epithelial wall is disrupted, coagulation is initiated. It takes approximately sixty seconds until the first fibrin strands begin to intersperse among the wound. After several minutes, the platelet plug is completely formed by fibrin.

Contrary to popular belief, clotting of a skin injury is not caused by exposure to air, but by platelets adhering to and being activated by collagen in the blood vessels’ endothelium. The activated platelets then release the contents of their granules, which contain a variety of substances that stimulate further platelet activation and enhance the hemostatic process.

When the lining of a blood vessel breaks and endothelial cells are damaged, revealing subendothelial collagen proteins from the extracellular matrix, thromboxane causes platelets to swell, grow filaments, and start clumping together, or aggregating. Von Willebrand factor causes them to adhere to each other and the walls of the vessel. This continues as more platelets congregate and undergo these same transformations. This process results in a platelet plug that seals the injured area. If the injury is small, the platelet plug may be able to form within several seconds.

Coagulation Cascade

If the platelet plug is not enough to stop the bleeding, the third stage of hemostasis begins: the formation of a blood clot. Platelets contain secretory granules. When they stick to the proteins in the vessel walls, they degranulate, thus releasing their products, which include ADP (adenosine diphosphate), serotonin, and thromboxane A2 (which activates other platelets).

First, blood changes from a liquid to a gel. At least 12 substances called clotting factors or tissue factors take part in a cascade of chemical reactions that eventually create a mesh of fibrin within the blood. Each of the clotting factors has a very specific function. Prothrombin, thrombin, and fibrinogen are the main factors involved in the outcome of the coagulation cascade. Prothrombin and fibrinogen are proteins that are produced and deposited in the blood by the liver.

When blood vessels are damaged, vessels and nearby platelets are stimulated to release a substance called prothrombin activator, which in turn activates the conversion of prothrombin, a plasma protein, into an enzyme called thrombin. This reaction requires calcium ions. Thrombin facilitates the conversion of a soluble plasma protein called fibrinogen into long, insoluble fibers or threads of the protein, fibrin. Fibrin threads wind around the platelet plug at the damaged area of the blood vessel, forming an interlocking network of fibers and a framework for the clot. This net of fibers traps and helps hold platelets, blood cells, and other molecules tight to the site of injury, functioning as the initial clot. This temporary fibrin clot can form in less than a minute and slows blood flow before platelets attach.

Next, platelets in the clot begin to shrink, tightening the clot and drawing together the vessel walls to initiate the process of wound healing. Usually, the whole process of clot formation and tightening takes less than a half hour.

Vascular Spasm

Vasoconstriction is the narrowing of the blood vessels, which reduces blood loss during injury.

Vasoconstriction is the narrowing of the blood vessels resulting from contraction of the smooth muscle wall of the vessels, particularly in the large arteries and small arterioles. The process is the opposite of vasodilation, the dilation and expansion of blood vessels. During hemostasis, a brief spasm of vasoconstriction occurs, which slows blood flow into the injured area while the clot forms.

Mechanisms of Vasoconstriction

The vasoconstriction response is triggered by factors such as a direct injury to vascular smooth muscle, signaling molecules released by injured endothelial cells and activated platelets (such as thromboxane A₂), and nervous system reflexes initiated by local pain receptors. The spasm response becomes more effective as the amount of damage is increased. Vascular spasm is much more effective at slowing the flow of blood in smaller blood vessels. Vasoconstriction also causes an increase in blood pressure for affected blood vessels.

Smooth muscle in the vessel wall goes through intense contractions that constrict the vessel. If the vessels are small, spasms compress the inner walls together and may be able to stop the bleeding completely. If the vessels are medium to large-sized, the spasms slow down immediate outflow of blood, lessening the damage but still preparing the vessel for the later steps of hemostasis. The spasm response becomes stronger and lasts longer in more severe injuries. Vasoconstriction may be induced by drugs called vasopressins, which increase blood pressure and can help treat certain conditions.

Injury and Inflammation

During injury, vasoconstriction is brief, lasting only a few minutes while the platelet plug and coagulation cascade occur. This is because as tissues are damaged during an injury, inflammation occurs as a result of inflammatory mediator release from immune system cells (such as mast cells or NK cells) that receive cell stress cytokines from damaged enothelial cells or vasoactive amines (serotonin) that are secreted by activated platelets. During inflammation, vasodilation occur, along with increased vascular permeability and leukocyte chemotaxis, ending the spasm of vasoconstriction and hemostasis as wound healing begins.

Platelet Plug Formation

At the site of vessel injury, platelets stick together to create a plug, which is the beginning of blood clot formation.

The second critical step in hemostasis, which follows vasoconstriction, is platelet plug formation. The three steps to platelet plug formation are platelet adherence, activation, and aggregation.

Platelet Adherence

The other factors released during platelet activation perform other important functions. Thromboxane is an arachidonic acid derivative (similar to prostaglandins) that activates other platelets and maintains vasoconstriction. Serotonin is a short-lived inflammatory mediator with a vasoconstrictive effect that contributes to vascular changes associated with inflammation during an injury. PDGF and VEGF are involved in angiogenesis, the growth of new blood vessels and cell cycle proliferation (division) following injury. The coagulation factors include factor V and VIII, which are involved in the coagulation cascade that converts fibrinogen into fibrin mesh after platelet plug formation.

Platelet Aggregation

The final step of platelet plug formation is aggregation of the platelets into a barrier-like plug. Receptors on the platelet bind to VWF and fibrinogen molecules, which hold the platelets together. Platelets may also bind to subendothelial VWF to anchor them to the damaged endothelium. The completed plug will cover the damaged components of the endothelium and will stop blood from flowing out of it, but if the wound is large enough, blood will not coagulate until the fibrin mesh from the coagulation cascade is produced, which strengthens the platelet plug. If the wound is minor, the platelet plug may be enough to stop the bleeding without the coagulation cascade.

Coagulation

Coagulation is the process by which a blood clot forms to reduce blood loss after damage to a blood vessel.

Coagulation is the process by which a blood clot forms to reduce blood loss after damage to a blood vessel. Several components of the coagulation cascade, including both cellular (e.g. platelets) and protein (e.g. fibrin) components, are involved in blood vessel repair. The role of the cellular and protein components can be categorized as primary hemostasis (the platelet plug) and secondary hemostasis (the coagulation cascade). The coagulation cascade is classically divided into three pathways: the contact (also known as the intrinsic) pathway, the tissue factor (also known as the extrinsic pathway), and the common pathway. Both the contact pathway and the tissue factor feed into and activate the common pathway.

Intrinsic Pathway

The intrinsic pathway (contact activation pathway) occurs during exposure to negatively charged molecules, such as molecules on bacteria and various types of lipids. It begins with formation of the primary complex on collagen by high-molecular-weight kininogen (HMWK), prekallikrein, and factor XII (Hageman factor). This initiates a cascade in which factor XII is activated, which then activates factor XI, which activated factor IX, which along with factor VIII activates factor X in the common pathway.

Extrinsic Pathway

The main role of the extrinsic (tissue factor) pathway is to generate a “thrombin burst,” a process by which large amounts of thrombin, the final component that cleaves fibrinogen into fibrin, is released instantly. The extrinsic pathway occurs during tissue damage when damaged cells release tissue factor III. Tissue factor III acts on tissue factor VII in circulation and feeds into the final step of the common pathway, in which factor X causes thrombin to be created from prothrombin.

Common Pathway

In the final common pathway, prothrombin is converted to thrombin. When factor X is activated by either the intrinsic or extrinsic pathways, it activates prothrombin (also called factor II) and converts it into thrombin using factor V. Thrombin then  cleaves fibrinogen into fibrin, which forms the mesh that binds to and strengthens the platelet plug, finishing coagulation and thus hemostasis. It also activates more factor V, which later acts as an anticoagulant with inhibitor protein C, and factor XIII, which covalently bonds to fibrin to strengthen its attachment to the platelets.

Coagulation Problems

While the coagulation cascade is critical for hemostasis and wound healing, it can also cause problems. An embolism is any thrombosis (blood clot)  that breaks off without being dissolved and travels through the bloodstream to another site. If it obstructs an artery that supplies blood to a tissue or organ, it can cause ischemia and infarcation to those tissues, leading to a pulmonary embolism, stroke, or heart attack).

Coagulation can occur even without injury, as blood pooling from prolonged immobility can cause clotting factors to accumulate and activate a coagulation cascade independently. Additionally, endothelial damage caused by immune system factors like inflammation or hypersensitivity may also cause unnecessary thrombosis and embolism. For example, during severe bacterial infections (septic shock), inflammation-induced tissue damage and the negatively charged molecules of bacteria activate both pathways of the coagulation cascade and cause disseminated intravascular coagulation (DIC), in which many clots form and break off, leading to massive organ failure.

Anticoagulants

Many anticoagulants prevent unnecessary coagulation, and those that genetically lack the ability to produce these molecules will be more susceptible to coagulation. These mechanisms include:

1. Protein C: a vitamin K-dependent serine protease enzyme that degrades Factor V and factor VIII.
2. Antithrombin: a serine protease inhibitor that degrades thrombin, Factor IXa, Factor Xa, Factor XIa, and Factor XIIa.
3. Tissue factor pathway inhibitor (TFPI): limits the action of tissue factor (TF) and the factors it produces.
4. Plasmin: generated by proteolytic cleavage of plasminogen, a potent fibrinolytic that degrades fibrin and destroys clots.
5. Prostacyclin (PGI2): released by the endothelium and inhibits platelet activation.
6. Thrombomodulin: released by the endothelium and converts thrombin into an inactive form.

Role of Vitamin K

Vitamin K is an essential factor of the coagulation cascade.

Coagulation is a complex cascade that requires many different cofactors and molecules to occur. Vitamin K, calcium, and phospholipids are necessary cofactors for proper coagulation, and people deficient in these substances will be more susceptible to uncontrolled bleeding.

Vitamin K

Vitamin K is a fat-soluble vitamin necessary for synthesis of coagulation factors involved in the coagulation cascade. Factors II, VII, IX, and X which are all important for the intrinsic and common pathways of coagulation. Vitamin K also synthesizes Protein C, Protein S, and Protein Z, anticoagulant proteins that degrade specific coagulation factors, preventing excessive thrombosis following the initial coagulation cascade.

Vitamin K can be inhibited by the anticoagulant drug warfarin, which acts as an antagonist for vitamin K. Warfarin is used in medicine for those at high risk of thromboembolism to prevent the coagulation cascade by reducing vitamin K dependent synthesis of coagulation factors. Warfarin’s effects can be overcome by ingesting more vitamin K to reactivate the coagulation factor synthesis pathway.

Vitamin K deficiency  is associated with impaired coagulation function and excessive bleeding and hemorrhage (internal bleeding, often severe). This can be caused by poor diet, malabsorption in the intestines, or liver failure. Those with vitamin K deficiency produce alternative proteins that improperly bind with phospholipids, which also contributes to the lack of coagulant function.

Calcium and Phospholipids

Calcium and phospholipids (a platelet membrane constituent) are required cofactors for prothrombin activation enzyme complexes to function. This enzyme is called tenase, and converts prothrombin to thrombin. Calcium mediates the binding of the tenase enzyme complexes (via the terminal gamma-carboxy residues on FXa and FIXa) to the phospholipid surfaces expressed by platelets, which in turn activates prothrombin to produce thrombin, which then produces fibrin from fibrinogen. Calcium acts as a catalyst for this reaction, speeding up the rate of the reaction to occur within the time frame of the factors involved in the coagulation cascade. Calcium is also required to to synthesize the anticoagulant Protein C (along with vitamin K).

Calcium deficiencies inhibit proper blood coagulation. This can be caused by a nutritional deficiency or acute problems in which calcium is allocated elsewhere in the blood. Phosopholipid deficiency is also associated with thrombocytopenia (platelet deficiency) because the phospholipids involved with clotting come from platelets. Thrombocytopenia causes more severe issues with blood clotting as the platelet plug will not be able to form or activate the coagulation cascade.

Clot Retraction and Repair

Clot retraction is the shrinking of a blood clot facilitated by thrombolytic agents.

The blood clots produced in hemostasis are merely the first step in repair and healing that occur after injury. Following a clot, inflammation draws leukocytes to the injury site to eliminate any pathogens that may have entered the body during the initial injury. Then, over the course of the next 24 hours, the clot retracts as tissue healing begins.

Clot Retraction

As the healing process occurs following blood clot formation, the clot must be destroyed in order to prevent thromboembolic events, in which clots break off from the endothelium and cause ischemic damage elsewhere in the body. By reducing the size of and breaking down the clot, the disease process can be arrested or the complications reduced.

Clot retraction refers to a regression in size of the blood clot over a number of days. During this process, the edges of the endothelium at the point of injury are slowly brought together again to repair the damage. Clot retraction is dependent on the release of multiple coagulation factors released at the end of the coagulation cascade, most notably factor XIIIa crosslinks. These factors cause the fibrin mesh to contract by forming twists and knots that condense the size of the clot. Clot retraction generally occurs within 24 hours of initial clot formation and decreases the size of the clot by 90%. Following clot retraction, a separate process called fibrinolysis occurs which degrades the fibrin of the clot while macrophages consume the expended platelets, thus preventing possible thromboembolism.

Wound Healing

While the clot retracts, the wound begins to heal. The first step of wound healing is epithelial cell migration, which forms a scab before the clot retracts. This occurs due to the stimulus of platelet-derived growth factor (PDGF). After clot retraction, true repair begins as tissue proliferation starts and collagen from the extracellular matrix is deposited in the wound while granulation tissue forms. Then new blood vessels grow into the healing tissue in a process called angiogenesis, which is stimulated by vascular endothelial growth factor (VEGF). The wound itself contracts, reducing in size. After these steps occur, new epithelial cells grow to cover the wound. If the wound was severe or unevenly shaped, or if healing takes too long, scarring may occur from collagen deposition. Most scarring on the skin is benign, but scarring inside the tissues of organs such as the heart or the lungs can cause health problems.

Fibrinolysis

Fibrinolysis is a process of breaking down clots in order to prevent them from growing and becoming problematic.

Fibrinolysis is a process that removes clots following hemostasis and clot retraction, preventing uncontrolled thrombosis and embolism. There are two types of fibrinolysis: primary fibrinolysis and secondary fibrinolysis. Primary fibrinolysis is a normal body process, whereas secondary fibrinolysis is the breakdown of clots due to a medication, medical disorder, or other cause.

Mechanisms of Primary Fibrinolysis

Primary fibrinolysis normally occurs following clot retraction, in which the clot has already condensed considerably in size. The main enzyme in primary fibrinolysis is plasmin, a proteolytic enzyme that degrades fibrin mesh. Plasmin cleaves fibrin at various places, leading to the production of circulating fragments that are cleared by other proteases or by the kidneys and liver.

Plasmin is produced in an inactive form, plasminogen, in the liver. Plasminogen cannot cleave fibrin and circulates in the bloodstream. Instead, it is incorporated into the clot when it is formed and then activated into plasmin later. Plasminogen is activated to plasmin by tissue plasminogen activator (t-PA) and urokinase, an enzyme found in the urine.

T-PA is released into the blood very slowly by the damaged endothelium of the blood vessels. T-PA and urokinase are themselves inhibited by plasminogen activator inhibitor-1 and plasminogen activator inhibitor-2 (PAI-1 and PAI-2). In contrast, plasmin further stimulates plasmin generation by producing more active forms of both tissue plasminogen activator (tPA) and urokinase. Following fibrin degradation by plasmin, old activated platelets from the platelet plug are phagocytized and destroyed by macrophages.

Alpha 2-antiplasmin and alpha 2-macroglobulin inactivate plasmin. Plasmin activity is also reduced by thrombin -activatable fibrinolysis inhibitor (TAFI), which modifies fibrin to make it more resistant to the tPA-mediated plasminogen. Plasmin operates on a negative feedback process because it is reduced when the fibrin clot is fully degraded.

Mechanisms of Secondary Fibrinolysis

Secondary fibrinolysis generally refers to treatment of pathological thromboembolism. If blood clots embolize to different parts of the body, they can cause tissue death by blocking off blood flow to those tissues. This is a common cause of heart attacks, pulmonary embolism, and strokes. Several medications exist to help treat and prevent these conditions.

Fibrinolytic drugs include synthesized tissue plasminogen activator and streptokinase, a bacterial enzyme that has degrades fibrin directly. Clots may also be prevented or kept from worsening through the use of blood thinners ( anticoagulants ). Aspirin has anticoagulant properties because it inhibits cyoclo-oxygenase dependent pathways of platelet activation, which can prevent clotting from worsening. Heparin is a fast-acting anticoagulant produced by the body and used as a drug which inhibits the activity of thrombin. Warfarin inhibits vitamin K cofactor activation during the coagulation cascade, and citrates chelate calcium to prevent prothrombin activation into thrombin.

All of these treatments have been shown to have tremendous therapeutic benefit in treating those with thromboembolic diseases; however, they can make injury much more difficult to treat by disrupting the clotting process. For example, patients thought to be suffering from a stroke (obstructed artery in the brain ) must be screened through imaging before given aspirin or a fibrinolytic drug, because if they have an aneurysm or hemorrhage (burst blood vessel or bleeding in the brain), administering fibrinolytic treatment would make their condition worse and possibly fatal by inhibiting the clotting that could save their lives.