Hemostasis
There are 3 mechanisms that work together to stop the flow of blood. They are

1. Vasoconstriction
2. Platelet plug formation
3. Clotting of blood

1. Vasoconstriction of a damaged blood vessel slows the flow of blood and thus helps to limit blood loss. This process is mediated by:

• Local controls. Vasoconstrictors such as thromboxane are released at the site of the injury.
• Systemic control. Epinephrine released by the adrenal glands stimulates general vasoconstriction.

2. Formation of a Platelet Plug.
Platelets, also called thrombocytes, are small cellular fragments that lack a nucleus and are derived from megakaryocytes. When a blood vessel is damaged, the blood is exposed to collagen fibers in the basement membrane of the vessel (shown in green below). Platelets stick to collagen and become activated. Activated platelets release chemicals such as ADP, and thromboxane, that cause the aggregation of more platelets to the site of injury. Platelet aggregation results in the formation of a platelet plug which acts to stem the flow of blood from the broken vessel. It is essential that platelets become activated only at the site of a broken vessel. Otherwise activated platelets would form plugs and induce clots in inapropriate places. Healthy vessels secrete an enzyme called prostacyclin that functions to inhibit platelet activation and aggregation.

Aspirin inhibits platelet activation by inhibiting the action of thromboxane. By inhibiting platelet activation, aspirin limits blood clotting in general. Aspirin is used clinically as a "blood thinner" in individuals that are at risk for developing life-threatning clots. Patients with advanced atherosclerosis take one baby aspirin per day to reduce the probability of heart attack and stroke.

Thrombocytopenia. Small tears of the capillaries and arterioles are happening all the time. Platelets are responsible for quickly sealing these tears before the slower process of clotting completes the job. In the absence of adequate numbers of platelets these micro-tears allow blood to seep into the tissues. This is evidenced by purple blotches (thrombocytopenia purpura) visible on the skin. Thrombocytopenia can be acute or chronic and has many causes. Severe, untreated cases result in death.

3. Clotting of Blood
The blood contains about a dozen clotting factors. These factors are proteins that exist in the blood in an inactive state, but can be called into action when tissues or blood vessels are damaged. The activation of clotting factors occurs in a sequential manner. The first factor in the sequence activates the second factor, which activates the third factor and so on. This series of reactions is called the clotting cascade.

Blood clotting is the transformation of liquid blood into a semisolid gel. Clots are made from fibers (polymers) of a protein called fibrin (see the diagram below). Fibrin monomers come from an inactive precursor called fibrinogen. The body of the fibrinogen molecule has caps on its ends that mask fibrin-to-fibrin binding sites. If the caps are removed then fibrin monomers polymerize to form fibrin polymers. This process requires thrombin, the enzyme that converts fibrinogen to fibrin. This process also requires calcium, which acts as a kind of glue to hold the fibrin monomers to each other to form the polymeric fiber. The fibrin fibers form a loose meshwork that is stabilized by clotting factor XIII. The stabilized meshwork of fibrin fibers traps erythrocytes, thus forming a clot that stops the flow of blood.

Control of the Clotting Cascade

From the diagram above we can see that thrombin is the key to the clotting mechanism. If thrombin is present then clotting will procede, but if thrombin is absent then clotting will not occur. How then is thrombin controlled? Thrombin is derived from an inactive precursor called prothrombin. There are two pathways that lead to the conversion of prothrombin to thrombin; (1) the intrinsic pathway and (2) the extrinsic pathway.

Intrinsic Pathway

The intrinsic pathway, which is triggered by elements that lie within the blood inself (intrinsic to the blood), occurs in the following way. Damage to the vessel wall stimulates the activation of a cascade of clotting factors (for the sake of simplicity we will not consider the individual factors). This cascade results in the activation of factor X. Activated factor X is an enzyme that converts prothrombin to thrombin. Thrombin converts fibrinogen to fibrin monomers, which then polymerize in fibrin fibers. Fibrin fibers form a loose meshwork that is stabilized by crosslinks created by factor XIII. The stabilzed meshwork of fibrin fibers is now a clot that traps red blood cells and platelets and thus stops the flow of blood.

Extrinsic Pathway

The extrinsic pathway is triggered by tissue damage outside of the blood vessel. This pathway acts to clot blood that has escaped from the vessel into the tissues. Damage to tissue stimulates the activation of tissue thromboplastin, an enzyme that catalyzes the activation of factor X. At this point the intrinsic and extrinsic pathways converge and the subsequent steps are the same as those described above.

It would be dangerous if blood clotting were to continue to expand beyond the boundaries of the injury. Excessive clotting is inhibited because:

1. Clotting factors are rapidly inactivated. There are enzymes in the blood that function to inactivate clotting factors. As clotting factors are taken away from the site of injury by the blood stream, they become inactivated by these enzymes. This ensures that clotting will only occur at the site of injury and not progress steadily down the vessel.
2. Fibrin fibers inhibit the activity of thrombin. Thrombin acts to convert fibrinogen to fibrin, yet fibrin fibers have an inhibitory effect on the activity of thrombin. As the clot grows this inhibition intensifies. This constitutes a negative feedback loop, where the product of thrombin activity (fibrin) feeds back to shut off thrombin.

Blood clots are designed to be temporary. After the clot has formed, the process of vessel repair begins. Epithelial cells at the margin on the injury undergo cell division. These new cells eventually fill the gap in the vessel created by the injury. Also, cells called fibroblasts are recruited to the area. Fibroblasts form connective tissue that repairs the basement membrane of the vessel (fibroblasts also form scar tissue that may or may not be removed over time). At this point the vessel is healed and the blood clot is no longer needed. The clot is removed in the following way:

• The clot itself stimulates the secretion of tissue plasminogen activator (TPA) from the surrounding vascular epithelium. TPA is an enzyme that catalyzes the conversion of plasminogen to plasmin. Plasminogen is an inactive precursor molecule found in the blood, but plasmin is an enzyme that dissolves clots. Plasmin levels are not very high so clot removal is a slow process. By the time the clot has been completely dissolved by plasmin, the vessel has had a chance to heal itself. In summary, the clot, which forms rapidly, calls for its own destruction by initiating the activation of plasmin.

Clot Busting Drugs

Blood clots can be life-threatening if they form inappropriately in critical locations. Clots that block coronary arteries cause heart attacks, while clots that block arteries in the brain cause stroke. Drugs that can mediate the removal of clots, "clot busters", are used in cases of heart attack and stroke to decrease the damage caused by the clot. Drugs used clinically to remov clots include:

1. Tissue plasminogen activator (TPA) was recently cloned and is now produced in mass quantities by the biotech firm, Amgen. It is used clinically to dissolve clots in coronary arteries following a heart attack. It is also used to dissolve clots in the brain following stroke.
2. Streptokinase is an enzyme that directly dissolves blood clots. It is produced by streptococcus bacteria. The bacteria use streptokinase to dissolve clots that negatively effects their growth in the human host. This clot dissolving enzyme is apparently as effective as recombinant TPA.

Streptokinase cost $2 dollars per dose while TPA costs $2000 dollars per dose. Based on economic concerns, streptokinase is the drug of choice. However, streptokinase is not a human enzyme, therefore the immune system sees it as a foreign molecule that should be destroyed. The immune response increases with repeated use of the drug. This limits the effectiveness of the drug over time. TPA, on-the-other-hand is a human molecule which the immune system does not destroy.

Anticoagulants are substances that inhibit the process of clotting.

1. Heparin
   • produced primarily in the liver and lung (usually obtained from pigs or cows)
   • acts as an anticoagulant by inhibiting the activity of thrombin
   • Used clinically for acute problems, also used to prevent clotting in IVs (heplock)
   • fast acting, but short-lived drug. It must be injected to be effective.
2. Coumadin (dicoumarol, warfarin)
   • taken orally in small doses for long-term control of blood clotting
   • acts as an anticoagulant by inhibiting the processing of vitamin K, which is required for the synthesis of several clotting factors including prothrombin.
   • It is slow acting, requiring days to have an effect.
3. Citrates
   • Calcium is required for polymerization of fibrin.
   • Citrates bind up (chelate) calcium and thus inhibit the formation of clots
   • Citrates are used in long term blood storage.
   • Transfusion of large amounts of citrate-containing blood can be dangerous. The citrates chelate calcium in the body and thus disrupt processes such as nerve transmission and muscle contraction.

Blood Clotting Disorders

Hemophilia
Individuals with this inherited disorder bleed excessively due to an inability to rapidly form blood clots. This defect could be caused by a deficiency of any of the clotting factors, however 80% of all hemophilics are deficient in factor VIII.

Vitamin K deficiency
Vitamin K is essential to the maturation of several clotting factors including factor X and prothrombin. In the absence of vitamin K these clotting factors are defective and thus inhibit the clotting mechanism. People with a Vitamin K deficiency experience excessive bleeding.