Blood Clotting

When blood vessels are cut or damaged, the loss of blood from the system must be stopped before shock and possible death occur. This is accomplished by solidification of the blood, a process called coagulation or clotting.

• a plug of platelets enmeshed in a
• network of insoluble fibrin molecules.

• calcium ions (Ca²⁺)(which is why blood banks use a chelating agent to bind the calcium in donated blood so the blood will not clot in the bag).
• about a dozen other protein clotting factors. Most of these circulate in the blood as inactive precursors. They are activated by proteolytic cleavage becoming, in turn, active proteases for other factors in the system.

  By tradition, these factors are designated by Roman numerals. I find this somewhat confusing and will use Arabic numerals instead.

Initiating the Clotting Process

• Damaged cells display a surface protein called tissue factor (TF)
• Tissue factor binds to activated Factor 7.
• The TF-7 heterodimer is a protease with two substrates:
  • Factor 10 and
  • Factor 9
  • Let's follow Factor 10 first.
• Factor 10 binds and activates Factor 5. This heterodimer is called prothrombinase because it is a protease that converts prothrombin (also known as Factor II) to thrombin.
• Thrombin has several different activities. Two of them are:
  • proteolytic cleavage of fibrinogen (aka "Factor I") to form:
    • soluble molecules of fibrin and a collection of small
    • fibrinopeptides
  • activation of Factor 13 which forms covalent bonds between the soluble fibrin molecules converting them into an insoluble meshwork — the clot.

Amplifying the Clotting Process

• The TF-7 complex (which started the process) also activates Factor 9.
  • Factor 9 binds to Factor 8, a protein that circulates in the blood stabilized by another protein, von Willebrand Factor (vWF).
  • This complex activates more Factor 10.
• As thrombin is generated, it activates more
  • Factor 5
  • Factor 8, and
  • Factor 11 (all shown above with green arrows).
• Factor 11 amplifies the production of activated Factor 9.

Platelets

Blood normally contains 150,000 to 350,000 per microliter (µl). If this value should drop much below 50,000/µl, there is a danger of uncontrolled bleeding. This is because of the essential role that platelets have in blood clotting.

• von Willebrand factor links the collagen to platelets forming a plug of platelets there.
• The bound platelets release ADP and thromboxane A2 which recruit and activate still more platelets circulating in the blood.
• (This role of thromboxane accounts for the beneficial effect of low doses of aspirin — a cyclooxygenase inhibitor — in avoiding heart attacks.)

ReoPro® is a monoclonal antibody directed against platelet receptors. It inhibits platelet aggregation and appears to reduce the risk that "reamed out" coronary arteries (after coronary angioplasty) will plug up again.

Bleeding Disorders

The deficiency may arise because

• not enough of the factor is produced or
• a mutant version of the factor fails to perform properly.

• von Willebrand disease (the most common)
• hemophilia A for factor 8 deficiency
• hemophilia B for factor 9 deficiency.
• hemophilia C for factor 11 deficiency

In some cases of von Willebrand disease, either a deficient level or a mutant version of the factor eliminates its protective effect on factor 8. The resulting low level of factor 8 mimics hemophilia A.

• tissue factor or
• factor 7

Hemophilia A and B

The genes encoding factors 8 and 9 are on the X chromosome. Thus their inheritance is X-linked.

Like other X-linked disorders, hemophilia A and B are found almost exclusively in males because they inherit just a single X chromosome, and if the gene for factor 8 (or 9) on it is defective, they will suffer from the disease.

There are many different mutant versions of the genes for factors 8 and 9. Although some produce only a minor effect on the function of their protein, others fail to produce any functioning clotting factor.

Treating Hemophilia A and B

Factor 8 and 9 can be extracted from donated blood, usually pooled from several thousand donors, and purified. Injections of this material can halt episodes of bleeding in hemophiliacs and have allowed countless young men to live relatively normal lives.

However, in the early 1980s, blood contaminated with the human immunodeficiency virus (HIV) was unknowingly used to manufacture preparations of factors 8 and 9. In some areas, 90% or more of the hemophiliacs became infected by these contaminated preparations. Many have since died of AIDS.

• all donated blood is now tested to see if the donor has been infected with HIV (as well as hepatitis B and C);
• plasma-derived preparations of factors 8 and 9 are now treated with heat and/or solvents to destroy any viruses that might be present;
• recombinant factor 8 and recombinant factor 9 made by genetic engineering are now available.

These recombinant factors are made by inserting the DNA encoding the human protein into mammalian cells grown in culture [Discussion]. E. coli cannot be used because these factors are glycoproteins, and E. coli lacks the machinery to attach carbohydrate properly.

And the team that brought us Dolly reported in the 19 December 1997 issue of Science that they have succeeded in cloning female sheep transgenic for the human factor 9 gene. The human gene is coupled to the promoter for the ovine (sheep) milk protein beta-lactoglobulin. When the lambs mature, it is hoped that they will secrete large amounts of human factor 9 in their milk, which can then be purified for human therapy.

Several attempts have also been made to try to cure hemophilia by gene therapy. [Link]

It is difficult to see how even the most worried critics of genetic engineering can fail to approve its potential to save the lives of thousands of hemophiliacs in the years to come.

Liver Transplants

Controlling Clotting

Antithrombin III

It does so by binding to and thus inactivating:

• prothrombin
• factor 9
• factor 10

Heparin is a mixture of polysaccharides that bind to antithrombin III, inducing an allosteric change that greatly enhances its inhibition of thrombin synthesis.

Protein C

With its many clotting promoting activities, it is probably no accident that thrombin sits at the center of the control mechanism.

• Excess thrombin binds to cell-surface receptors called thrombomodulin.
• The resulting complex activates a plasma protein called Protein C and its cofactor Protein S.
• Together these inhibit further thrombin formation
  • directly — by inactivating Factor 5 and
  • indirectly — by inactivating Factor 8.

• inherited deficiency of Protein C or Protein S;
• inherited mutation in the Factor 5 gene producing a protein that no longer responds to the inhibitory effect of Protein C.

Recombinant Protein C is now available to treat people threatened with inappropriate clotting, e.g., as a result of widespread infection (sepsis).

Vitamin K

• factors 2 (prothrombin), 7, 9, and 10
• proteins C and S

Conversely, blocking the action of vitamin K helps to prevent inappropriate clotting.

Warfarin (aka coumadin) is sometimes prescribed as a "blood thinner" because it is an effective vitamin K antagonist. (Warfarin is also used as a rat poison because it can cause lethal (internal) bleeding when eaten.)

Dissolving clots

Plasma contains plasminogen, which binds to the fibrin molecules in a clot. Nearby healthy cells release tissue plasminogen activator (TPA), which also binds to fibrin and, as its name suggests, activates plasminogen forming plasmin. Plasmin (another serine protease) proceeds to digest fibrin, thus dissolving the clot.

Recombinant human TPA is now produced by recombinant DNA technology. Injected within the first hours after a heart attack, it has saved many lives by dissolving the clot blocking the coronary artery and restoring blood flow before the heart muscle becomes irreversibly damaged. It is also used for people who suffer an ischemic stroke; that is, a clot in the brain. (It must not, of course, be used for hemorrhagic strokes, that is, a burst blood vessel!)

Angiogenesis

Thrombin (as well as factors 7 and 10) promotes healing by stimulating the growth of new blood vessels at the site of damage. [Link to a discussion of angiogenesis.]