Hemostasis includes 3 steps in a rapid sequences :

1) 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.

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 A 2), 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.

2) 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.

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.

a. Platelet Adherence

Platelets: A blood slide of platelets aggregating or clumping together. The platelets are small, bright purple
fragments. Normally, the endothelial cells express molecules that inhibit platelet adherence and activation while
platelets circulate through the blood vessels. These molecules include nitric oxide, prostacylcine (PGI 2) and
endothelial ADP-ase.

During an injury, subendothelial collagen from the extracellular matrix beneath the endothelial cells is exposed on
the epithelium as the normal epithelial cells are damaged and removed, which releases von Willebrand Factor
(VWF). VWF causes the platelets to change form with adhesive filaments (extensions) that adhere to the
subendothelial collagen on the endothelial wall.

b. Platelet Activation

After platelet adherence occurs, the subendothelial collagen binds to receptors on the platelet, which activates it.
During platelet activation, the platelet releases a number of important cytokines and chemical mediators via
degranulation. The released chemicals include ADP, VWF, thromboxane A2, platelet-derived growth factor (PDGF),
vascular endothelial growth factor (VEGF), serotonin, and coagulation factors. The extra ADP and VWF is especially
important because it causes nearby platelets to adhere and activate, as well as release more ADP, VWF, and other
chemicals. Platelet plug formation is considered a positive feedback process because ADP and VWF levels are
successively increased as more and more platelets activate to form the plug.

ADP: The chemical structure of ADP, a molecule that causes platelet activation and is involved in the positive
feedback component of platelet activation.

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.

c. 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.
 3) Coagulation

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.

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.

Secondary Hemostasis

Hemostasis can either be primary or secondary. Primary hemostasis refers to platelet plug formation, which forms
the primary clot. Secondary hemostasis refers to the coagulation cascade, which produces a fibrin mesh to
strengthen the platelet plug. Secondary hemostasis occurs simultaneously with primary hemostasis, but generally
finishes after it. The coagulation factors circulate as inactive enzyme precursors, which, upon activation, take part
in the series of reactions that make up the coagulation cascade. The coagulation factors are generally serine
proteases (enzymes).

a. 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.

b. 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.

c. 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.

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.

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.

a. 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.

Fibrinolysis : Blue arrows denote stimulation and red arrows inhibition.

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.

b. 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.