The Blood Clotting Cascade Berg, Tymoczko, Stryer (pages 293-297) The blood clotting cascade is an example of many

biochemical processes in the body. The principle players in the cascade are proteins. The interactions between the various proteins involved in the process are complex. So much so that there is no single chronology that will accurately model the normal function and pathologies of the process. The purpose of this essay is to give a qualitative snapshot of the blood clotting process, highlighting the importance of the process, the key players, and the risks associated with select malfunctioning or over-active proteins. Loss of blood, unchecked, can mean the loss of life. The body has mechanisms in place to prevent loss of blood in the event of damage to the vascular system. A successful clotting process involves an intricate balance in the timing and localization of clot formation, inhibition, and destruction. Hemophilia is a disorder occurring from excessive loss of blood in response to vascular system trauma. It can result from the failure of clot formation, or more realistically, the slow response of clot formation. At the other extreme is thrombophilia, a disorder resulting from over-aggressive clot formation. This can lead to the dislodging of sizeable pieces of clot (embolism). These are primary instigators of myocardial infarctions and strokes. Most of the proteins involved in the blood clotting process circulate in the blood as proproteins or proenzymes. They are always present but nonfunctional until the appropriate activation signals are activated. The clot itself is made by polymerizing the protein fibrin, although other components, for example platelet cells, will be trapped and incorporated into the final clot. Fibrin circulates in the blood in its soluble form, fibrinogen, a protein comprised of 2 copies each of 3 subunits (AB)2. The self attracting components of the fibrin structure are buried by excess polypeptide segments of fibrinogen labeled A and B in the representation above, part of the and chains respectively. Peptide segments A and B are called the activation peptides. These are the peptides that are excised by the serine protease thrombin, during clotting. Fibrinogen has a dumbbell-like structure with an extra spherical section in the middle of the bar. The peptides removed are highly negative and repel each other. Upon removal of the activation peptides the fibrin molecules self-associate to form a mesh-like structure of half-overlapping fibrin units. This is what is known as a soft clot, the interaction between fibrin units being entirely noncovalent. At this point blood clotting factor XIII (fibrin-stabilizing factor), performs its function as a transglutaminase, covalently cross-linking the Gln side chains in different units of the fibrin clot to form the hard clot. Factor XIII is also activated by thrombin. In addition to its clot forming role, fibrin also acts as a cofactor in platelet aggregation (via the chain), recruiting platelet cells to the site of clotting. Among other things the platelet aggregation assures that the clotting remains localized at the damaged tissue site. This is partly due to the fact that many of the clotting components are also anchored to membrane phospholipids of the damaged cells. The clot inhibiting capability of aspirin, which is heart friendly, but can also be an unwanted side effect of aspirin use is due to its blockage of platelet accumulation at clot sites. But clot formation is the middle chapter of the story that begins with tissue damage. If the vascular tissue damage is localized to the inside surface of a blood vessel, then only the intrinsic clotting pathway is activated. If a laceration of a blood vessel occurs, tissue factor (TF) proteins released from the extra-cellular matrix of the damaged cells activate both the extrinsic and intrinsic clotting pathways. (See the table at the end for a list of factors and the approximate order of the events leading to the formation of clotting). The released TF forms a localized, anchored complex with factor VII, which then plays a role in activating factors IX and X. In the meantime Kallikrein released by the intrinsic pathway, activates factor XII, but also binds to Kininogen, instigating a number of parallel reactions to vascular trauma including bradykinin release, inflammation mediation, increased vascular permeability (which can produce shock) etc.

Thus, tissue damage sets the blood clotting cascade in motion. The complexity of a cascade provides rapid amplification of the desired outcome, but also provides for checks and balances to limit the clotting result to the affected area. One of the downsides of cascade processes is that there are more potential components that can malfunction. Although the deficiency of factor VIII is the most famous genetic defect causing hemophilia, there are countless mutations that can lead to varying degrees of hemophilia or thrombophilia. In addition to fibrin which forms the actual clot material another key component of the process is thrombin. Thrombin is a serine protease which, like trypsin, is specific for cleaving after basic residues, particularly Arg. Unlike trypsin, which will cleave almost any Arg-X or Lys-X peptide bond in any protein, the thrombin targets seem to be limited to specific Arg-X connections rather than any Arg-X connection. It activates finbrinogen, and factors V, VII, VIII, XIII, and Protein C (which begins the termination of the blood clotting process). To be effectively activated, thrombin should be anchored to the surface of a phospholipid membrane. This is part of the mechanism that limits clotting to the site of the trauma. Thrombin has 13 Glu residues that are modified to an abnormal amino acid called -carboxyglutamic acid. This modification is carried out by a vitamin K dependent carboxylase. The resulting modified -carboxy Glu residues are very efficient Ca++ chelators, which in turn binds prothrombin to the phospholipid surface of traumatized tissue cells. The prothrombin is more readily activated in place at the damaged tissue surface, than floating free in the blood. This is why a vitamin K deficiency results in hemophilia symptoms. In addition, thrombin contains two Kringle domains that help anchor it to fibrinogen. Almost every protease in the body has a corresponding protein inhibitor. For thrombin this is the enzyme antithrombin III. During tissue damage, mast cells release heparin, a polysaccharide that enhances the activity of antithrombin III. However, it is not very effective against Fibrin bound thrombin, so its role is to limit thrombin activity away from the site of clotting. Another circulating serine protease inhibitor is -1-antitrypsin, which actually functions as an anti-elastase (with mild anti-thrombin capability). One known case of a hemophilic individual was due to a mutation of Met Arg at the active site of this enzyme making it an anti-thrombin more than an anti-elastase. Thus, proper regulation of serine proteases are critical to the balance in the blood clotting process. Medical intervention in the blood clotting process is important for surgery, chemotherapy, prolonged need for IV fluids, to name just a few.

Although antithrombin keeps the action of clot formation localized, there still must be a mechanism that signals the termination of clot formation, and another one to dissolve the clot during the time that the damaged tissue is being repaired. Thrombin initiates clot formation, but also activates Protein C, with the assistance of thrombomodulin. This is the initial step in dismantling the blood clotting cascade. Protein C is also a serine protease. Its targets are the nonenzymatic cofactors of the clotting cascade, factors V & VIII. By deactivating these cofactors the growth of new clot material is slowed and eventually stopped. Genetic defects in Protein C activity result in thrombosis. A severe, recessive form of the disease results in neonatal death. However, formed clots must eventually dissolve. I suspect the rate of this process is somewhat synchronized with new tissue growth, but have no knowledge of what the rates are and how accurately they are synchronized. However, if the clot dissolves before the tissue is repaired hemorrhaging would likely result. The key protein players in the dissolution of clots are plasmin, and Tissue Plasminogen activator (TPA). Plasmin is yet another serine protease. Its principle target in its role as a clot dissolver is a specific sequence in the rod section of the fibrin molecule. Proteolysis of the rod portion of the clot breaks it apart. I presume other proteases finish digesting the peptide fragments remaining. Plasmin activity is not restricted to only blood clotting. Its proteolytic reaction is also required in tissue remodeling (part of the related tissue repair process); tumor migration/invasion, which establishes a link between clotting and cancer; embryonic development (which is similar to metastasis in that it involves the migration of cells and establishment of turf); inflammation; ovulation, and other processes. Multiple uses of protein resources by the body poses problems to curing disease by inhibiting protein/enzyme activity, because we often cant limit the inhibitory activity to the desired function (location), the way the bodies natural regulatory mechanisms often can. Plasmin must also be activated from its proenzyme form, plasminogen. This is done by yet another serine protease, TPA. It has a strong fibrin binding affinity, an interaction that increases its catalytic activity between 100 to 1000 fold. Heparin binding also enhances its binding to plasmin. TPA is a critical regulatory agent in the clotting process. Increased activity leads to excessive bleeding. A decreased activity causes thrombosis. Clot restricted blood flow in the coronary arteries is a primary cause of myocardial infarctions (MI), and recombinant TPA is commonly used as an initial treatment for MI. It has also found favor as a treatment for stroke if the treatment can be started within hours after the stroke occurred. This minimizes stroke damage and makes resumption of all related brain activities much more likely. Hemophilia is a disease whose symptoms involve excessive bleeding due to failure or slow rate of clot formation following trauma. Severe cases are typically fatal in utero. Less severe cases can still be life-threatening to individuals if they suffer what would normally be a routine trauma. Although there are a many diseases as there are clotting factors the two most common forms encountered are hemophilia A (insufficient factor VIII about 1 in 10,000 males) and hemophilia B (insufficient factor IX about the amount of hemophilia A). The latter has been the source of a successful gene therapy trial to introduce the factor IX gene into afflicted individuals.

Factor V VII VIII IX X XI XII XIII III IV II I?

name proaccelerin Proconvectin Antihemophilic Factor Christmas Factor Stuart Factor Thromboplastin antecedent Hageman Factor Fibrin Stabilizing Factor Tissue Factor (TF) Calcium thrombin fibrin

role activates prothrombin (with X) activates X extrinsic (with Ca & TF) activates X intrinsic (with IX) activates X intrinsic (with VIII) activates prothrombin (with V) activates IX activates XI converts soft clots to hard clot by forming fibrin crosslinks released by external trauma activates X (with VII & Ca) activates X (with VII & TF) activates fibrinogen (also V, VII, VIII, XIII) forms soft clot then cross links to hard clot

Chronology 1. 2. 3.

4.

5. 6. 7. 8.

Intrinsic Extrinsic Inside surface of vessel Trauma/laceration of vessel damaged glands release Kallikrein (p) Tissue Factor (TF) (a lipoprotein) Kallikrein + Kininogen released from blood vessels activate XII: VII activated XII* activates XI XI* activates IX (thrombin + X* activate VIII) (thrombin activates V) VIII* + IX* activate X TF + VII* + Ca activates X V* + X* activate prothrombin thrombin thrombin activates fibrinogen fibrin (thrombin also activates V, VIII, XIII) XIII* + fibrin cross-linked fibrin = clot * = activated form; bold = nonenzymatic protein factor