Haemostasis by Means of Blood Coagulation:

An Analyzation of the Mechanism

By Joseph Leon Sparks;

April 30, 1994;

Dr. Michael C. Reed, Professor and Advisor

The survival of muticellular animals in a hostile environment depends on how they obtain food. When food is scarce, the animals will compete with one another for it. The competition, without doubt, will result in a physical battle, which increases the possibility of bodily injury. For simplicity’s sake, let us assume the animals are no different from you or me. They are not brutes or wild savages as the definition of "animal" implicates; yet, each can indeed cause harm when the opportunity arises. These animals are of the species homo sapiens, or human beings.

Human beings, of which each of us include our ownselves, are a highly developed animal species. Although we do not run the best, climb trees the best, or tear with our teeth that of which is quite delectable, we do have one important task which surpasses all other activities. That activity is thinking. Through thinking or planning, and then enacting our individual plans to receive results, we can accomplish almost anything. Indubitably, we do limit ourselves in the challenges we undertake especially if its a physical challenge. This is because our bodies are practically defenseless for preventing the loss of body fluids from superficial wounds and morbid wounds acquired through overplay, war, or some unpreventable accident. A mechanism for impeding the loss of bodily fluids, especially blood, had to be evolved. This is because blood connects and communicates with tissues throughout the body (Davis et al, 1985).

Over the years, observations have been made of the response of the body to injury. Reseachers know today that whenever a vessel severs and

ruptures, prevention of blood loss, or hemostasis is achieved by several different mechanisms. The mechanisms are: (1) vascular spasm, (2) formation of a platelet plug, (3) blood coagulation and eventual growth of a fibrous tissue into a blood clot to close the hole in the vessel

permanently (Guyton, 1991). Vascular spasm is the immediate response of a vessel when it is cut or ruptured. The vessel contracts after signals are sent by means of the nervous system that a potential life threatening trauma has occurred. The greater the degree of trauma, the greater the spasm or response.

When small rents occur in the vessel, a platelet plug stops the loss of blood. Platelets, normal circulating blood cells, undergo a surface change upon contact to damaged vascular surface. The purpose of the surface change, is to release substances which attract other platelets in the area of injury in order that these additional platelets can join in the formation of the platelet plug. This process is known as agglutination.

Assuming that the activity of the first two mechanisms is spent, it is the third mechanism that will be most critical for prevention of blood loss. Studies on blood coagulation of individual patients are important since they provide valuable information to surgeons on specific actions that must be taken in order to impede the flow of blood when a surgical procedure is necessary. Also, studies of the components of blood coagulation have been used to determine the interactions among the inactive components when plasma is pooled and stored. This supply of plasma can be used to correct patients’ bleeding tendencies from congenital deficiencies and acquired deficiencies (due to disease of the liver) of their coagulation components (Briggs & Rizza, 1984).

Although blood coagulates in almost the same way from one person to the next, the time required for the individual interactions to occur can vary infinitely. Although a diagram model of the interactions has been conceived, individual steps of the process are studied using enzyme kinetics and analyzed using the results graphed from bioassays. Bioassays test the potency of specific reactants involved in the process. Thus, the focus of the first portion of this research paper will be to inform the reader of significant historical developments that have lead to our present understanding of blood coagulation, as well as a description of the process of blood coagulation.

For more than 2000 years, philosophers of the phenomenon of blood coagulation have known that blood, when placed in a tube outside the body, forms a fibrin clot. It was not until the eighteenth and nineteenth centuries that researchers began to propose methods of how a fibrin clot was formed. The enzymatic or fibrin-ferment theory, one of the first theories, was proposed by Andrew Buchanan (Hougie, 1963). Through experimentation, he showed that serum, a yellowish fluid withdrawn from a clot after it contracted, could be reclotted on addition of a few washed clot fragments. He proposed that fibrin existed in a dissolved state in serum and was induced to clot by means of a catalyst (Hougie, 1963).

Significant progress on the theory was made after basic properties of the activating substances once other enthusiastic researchers began to characterize activating substances in the phenomenon. "Virchow, after careful chemical analysis, concluded that fibrin could not exist in a liquid state, but rather existed in a precursor state, which he called fibrinogen with entirely different characteristics from fibrin" (Hougie, 1963). Alexander Schmidt, the founder of a famous school of blood coagulation, proposed that the enzymatic agent in the washed clot was thrombin. He believed thrombin was brought into activation by a precursor, prothrombin, which was itself brought into activation by zymoplastic agents found in protoplasm and cells (Hougie 1963). These zymoplastic agents were termed thromboplastin or thrombokinase. His incredible hypothesis would have changed the theory on blood coagulation form one step to three steps. At any rate, the former theory, that coagulation was brought about by the "fermentative transformation of fibrinogen into fibrin", remained (Hougie, 1963).

At this point, reseachers believed that the true mechanism of blood coagulation had been uncovered. Based on Morawitz’s theory, Armand Quick designed the one-stage and two-stage prothrombin tests to measure the transformation of prothrombin to thrombin in order, perhaps, to model the whole coagulation process (Bloom et al. 1994). These test were informative, but only to a certain extent. This was due to the inexplicable, characteristic inconsistency in the measured time necessary for the transformation of blood into an insoluble clot exhibited by the two test when compared. This inconsistency, between the two test, produced a schism among the opinions of researchers. Some were supporters of the present theory while others began to envision a more complicated coagulation process. It should be noted here that the thrombokinase or thromboplastin was not a specific molecular entity, since it could not be isolated from tissues. A more accurate comparison of thrombokinase is to that of a "soup bowl," since it contained many inert components as well as inhibitors and activators of coagulation (Hougie, 1963).

Today over 50 inert components, some promoting coagulation, called procoagulants, and others inhibiting coagulation, called anticoagulatants, have been found existing in normal blood and tissues. Whether or not the blood will coagulate depends on the degree of balance between these two groups of substances. Normally anticoagulants predominate, but when a vessel is ruptured, procoagulants in the area of damage become "activated" and override the anticoagulants. Of particular interest are the activators of coagulation or procoagulants.

Essentially to highlight certain properties of the procoagulants, it is

helpful to arrange them in to three groups: the fibrinogen group, the prothrombin group, and the contact group (Harker, 1974). First, factors of the fibrinogen group (fibrinogen, proaccelerin, antihemaphillic factor, and fibrin stabilizing factor) are classified together based on the observation that their activity is destroyed during the coagulation process. In other words, they are present in plasma but not in serum. Second, factors of the prothrombin group (prothrombin, proconvertin, Christmas factor and Straurt factor) are dependent on vitamin K for their production. The factors in this group are not consumed during coagulation. Finally, the factors of the contact group (Hageman factor and tissue factor) are involved in the initial phase of intrinsic activation.

Although Harker (1974) groups the procoagulants according to certain properties shared by each, the shared physiological nature of all procoagulants place them into their present group. Researches have found that each procoagulant undergoes proteolytic processing which is the final step in its biosynthesis (Neurath, 1989). Initially, all the enzymes involved circulate in the blood as inactive precursors. When proteolytic processing occurs, specific peptide bonds cleave to the target enzyme to induce it to a biologically active state. The usual cleavage site is in relatively flexible interdomain segments or surface loops. This site is usually the N-terminal region of the precursor (Neurath, 1989). In order for zymogen activation to occur, reactants must be catalyzed with the aid of serine proteases (Neurath, 1989).

I will now describe the interaction of each factor in the pathways of blood coagulation.

The formation of prothrombin activator complex is most critical in the coagulation process because it is a rate limiting step (Guyton, 1991). Generally, an abnormal coagulation time is seen when coagulation factors are missing due to congenital defects, acquired diseases, or some inhibitor in the body, such as dicoumeral which competitively inhibits vitamin K, a necessary cofactor in the synthesis in the liver of procoagulants dependent on its activity (Suttie, 1980). When coagulation is uninhibited and all procoagulants are present, the formation of prothrombin activator complex is formed through a unique interaction among them. Two general pathways, the extrinsic pathway and the intrinsic pathway converge to form this complex.

The extrinsic pathway occurs whenever traumatized tissue releases tissue thromboplastin, a complex composed of phospholipids, and a lipoprotein complex containing an important glycoprotein that functions as a proteolytic enzyme (Guyton, 1991). The lipoprotein complexes with proconvertin (Factor VII). Factor VII, containing 10 gamma carboxyglutamaic acid residues which are modified by a carboxylase enzyme requiring Vitamin K as a cofactor for its synthesis, requires calcium ions in order to bind to tissue factor in a 1:1 stoichiometric fashion (Bloom et al., 1994). The formation of this complex is critical since it enhances the activity of tissue factor several thousand-fold against its physiological substrates, Christmas factor (factor IX) and Straurt factor (factor X) (Bloom et al. 1994).

Factor X, composed of a heavy chain (MW=59 000) and a light chain (MW=16 000) held together by a disulfide bond, is the preferred recipient of this enzymatic activity. Following the release of a glycopeptide from the N-Terminal end of its heavy chain, factor X becomes activated. Activated factor X then complexes immediately with phospholipids and calcium ions which form the prothrombin activator complex (Bloom et. al 1994). This final complex catalyzes the splitting of prothrombin into its active form, thrombin (Bloom et al. 1994).

Unlike the extrinsic pathway, the intrinsic pathway would be activated if one of several events were to happen: (1) contact of the proteins to an unsmooth surface in the endothelium, (2) damage of the glycocalyx membrane and thrombomodulin membrane, or (3) the presence of foreign material circulating in the blood (Guyton, 1991) . Whichever event initiates the pathway, trauma to the blood or exposure of blood to vascular wall collagen alters Hageman factor (factor XII), a zymogen of a serine protease with MW= 80 000. When one of the above agents disturbs Factor XII normally bound to the wall of endothelium, kallikrein enzymatically converts it to activated Factor XII by splitting it into a two-chain enzyme in which the heavy chain, MW= 52 000 links via disulphide-bridge to the light chain, MW = 28 000.

Next, the modified Factor XII, requiring the cofactor high molecular weight kininogen (HMWK) accelerated itself by the cofactor prekallikrein, acts enzymatically on factor XI. Factor XI is a homodimer composed of two identical polypeptide chains held together by a disulfide bond. Activated factor XI has its same two chains linked by a disulphide-bond. The key to its activity is one chain has binding capabilities to HMWK, a necessary cofactor for increased activity, while the other possesses catalyzing capabilities.

The activated factor XI, now a serine protease, can act enzymatically on factor IX to activate this factor in the presence of calcium ions. The resulting enzyme-substrate interaction is unique in that it is one of the few interactions that do not require a protein cofactor or phospholipid surface for activation. (Bloom et al., 1994). Quickly following its proteolytic activation, activated factor IX, "acting in concert" with antihemophillic factor (factor VIII) and with platelet factor 3 (released initially from traumatized platelets) activates Factor X. Factor VIII, a glycoprotein with a molecular weight of approximately, 1.2 million, is composed of a number of similar or identical subunits held together by disulfide binds. This glycoprotein serves as the regulatory protein in the conversion of factor X to activated factor X, perhaps, by optimally binding to factor X substrate for activated factor IX proteolysis (Harker, 1974). Finally, activated factor X complexes with platelet phospholipid to induce splitting of prothrombin to thrombin (Guyton, 1991).

After formation of biologically active thrombin via either pathway, thrombin greatly catalyzes the coagulation process by activating proconvertin (factor V), which amplifies its own rate of formation. The increased amount of thrombin, as previously stated, catalyzes the proteolytic activation of fibrinogen to fibrin monomers. These monomers are formed when thrombin removes four low molecular peptides from fibrinogen. The monomers then polymerize within 10 to 15 seconds into long fibrin threads (Guyton, 1991).

In the initial stages of polymerization, the fibrin network is not strong. This is because the network, held together by hydrogen bonds, exhibits weak bonding. As soon as platelets become trapped in the clot, they are induced by thrombin to release fibrin-stabilizing factor (factor XIII). This factor promotes covalent bonding between the adjacent fibrin threads. The resulting configuration is a strong meshwork prohibiting the movement of platelets, plasma, and blood cells in all directions (Guyton, 1991).

After the meshwork of fibrin strands has been formed, and hemostatsis has been achieved, the body’s inhibitory system is activated. This system involves natural anticoagulants such as antithrombin III and heparin to inhibit the activity of thrombin. As the fibrin threads are developing, they impede mainly the activity of thrombin by absorbing it. In fact, these threads absorb nearly 80 to 90 per cent of all thrombin (Guyton, 1991). The thrombomodulin membrane also binds circulating thrombin which activates protein C, a plasma protein, to surpress the activity of factor V and factor VIII.

Several days after inhibition of thrombin and other activated procoagulants, the plasmin system or fibrinolytic system is activated. As the name implies, this system digests all participating substances, including the fibrin clot. It is activated after surrounding tissues and endothelium release a surplus of tissue plasminogen activator (TPA). TPA converts plasminogen to plasmin, a proteolytic enzyme resembling the digestive enzyme trypsin. If its activity were not controlled, it would produce hypocoagulability of the blood, since it digests at random participating substances in the coagulation process (Guyton, 1991).

In sum, the above presentation of the coagulation process was extended to account for its activation and deactivation. Both pathways converge to factor X (see Appendix 3). Factor X then forms a complex with phospholipid, calcium and factor VIII (if present) that enzymtically catalyzes the activation of thrombin from prothrombin. Thrombin then promotes increased thrombin formation (by activating factor V, a cofactor), in order to polymerize fibrin monomers and to induce platelets to release factor XIII which stabilizes the clot. Thrombin formation is eventually inhibited by the fibrin threads and the natural anticoagulants. Its inhibition triggers the inhibition of circulating activated procoagulants and complexes. Finally, the clot and participating reactants are digested upon activation of the plasmin system.

Due to a recent influx of information, proposals for the mechanism by which fibrin is formed and the role of thrombin has been modified. Present evidence suggests that the extrinsic pathway is critical to the initiation of fibrin formation, whereas the intrinsic pathway plays a role in the continued formation of fibrin (Hathaway 1993). Incidentally, the activation of one pathway indirectly activates the other, which shows a dependency of the process on both pathways for complete hemostatic response (Guyton 1991). Thrombin is now accepted as the central bioregulatory enzyme in hemostasis. This is not surprising since it was originally postulated as the principle enzyme in the reaction according to Alexander Smith over 100 years ago! Despite this, the need for a control on its activity is noted by the following:

Physiology imposes significant constraints on thrombin formation. This is due to the fact that circulating thrombin can initiate spontaneous clots that will occlude blood vessels leading ultimately to cardiac arrest. This is why the mechanism of blood coagulation, as already mentioned, must inhibit it from propagating beyond the site of which it is necessary. An observation whenever researchers study the process of blood coagulation, is that the formation of thrombin is quicker if all reactants are present (Bloom et al. 1994).

Due to the time lag characteristic of the one-stage and two-stage prothrombin tests, the full coagulation process discussed so far cannot be operative from time zero. This is because blood, immediately after the coagulation reaction has been triggered, does not contain activated proaccelerin (factor Va), activated antihemophillic factor (factor VIIIa) and sufficient phospholipid. These have to be provided by thrombin-dependent feedback reactions. So in order to start the full-blown thrombin formation, incomplete, partial mechanisms have to form small amounts of prothrombin first. The general formation through the intrinsic or the extrinsic pathway is the same, except the amount of reactants used are quite limited (Bloom et al., 1994) .

Now that an idea of how the coagulation mechanism behaves, as well as a brief summation of the interactions among the components has been stated, the remainder of this paper will explain how mathematical models and mathematical techniques are used to study specific mechanisms of the extrinsic pathway.

Hypothetically if one were to model the partial mechanism for thrombin formation via the extrinsic pathway, how would they go about setting up the model and possibly solving it? One would have to apply "mass action" kinetics to the model. "Mass action" is the accepted model for the describing kinetics at sufficiently low concentration of reactants. When this model is applied to the simplest case of enzyme action there results an analytically unsolvable system of equations. (See Appendix 1).

To circumvent unnecessary calculations that result, one could make assumptions about the reaction and apply some simpler technique of solving the resulting equations. One simpler technique used, and based on the principle of chemical relaxation, is the near-equilibrium approximation. This technique linearizes the resulting equations (see Appendix 1). The principle of chemical relaxation quantitatively describes the tendency of chemical reactions of whatever order (first, second, etc.), upon perturbation, to settle back to a state of equilibrium between reactants and products (Fleck, 1971). But in the case of blood coagulation in the body, equilibrium among reactants and products would result in circulating substances that have the potential of activating precursors to form clots which will occlude blood vessels, leading to cardiac arrest.

As previously stated, due to the dynamic nature of blood, thrombin must be limited to the site of injury after it is initiated by the partial mechanisms. This is achieved mainly with the fibrin threads. But additional thrombin has the potential being formed from prothrombin activator complex if tissue factor complex is allowed to circulate. Tissue factor complex will continue to activate factor X, since it does not form a complex with this factor. Factor X, as previously mentioned when activated, complexes with factor VIII, phosholipids and calcium ions to form prothrombin activator complex.

An important regulatory agent inhibiting the action of the tissue factor complex beyond the site of injury is tissue factor pathway inhibitor (TFPI). TFPI is a member of the Kunitz family serine protease inhibitors. In kinetic terms the Kunitz-type inhibitors typically produce slow, tight-binding, competitive, and reversible inhibition of the form:

E + I <====> EI <====> EI*

where E= Enzyme (tissue factor complex), I = inhibitor (TFIP), EI is the initial collision complex , and EI* the final inhibited complex (Bloom et al., 1994).

The reaction above can be modeled, in general, based on a enzyme kinetic study given by Dawes (1965) on competitive inhibition. Since the reaction is inhibiting tissue factor complex produced via the extrinsic coagulation pathway with factor X, a representation of the interaction of the complex and substrate are as follows:

E + S <====> E---S ----> E + P

where E = Enzyme (tissue factor complex), S = substrate (factor X), and P = (activated factor X). For details of the calculations of the above interaction of a competitive inhibitor and a enzyme, See Appendix 2.

Measurement of individual components of the blood coagulation system

also plays a central role in the understanding of normal hemostasis. Since each factor in the mechanism is part of an intricate network of inter-related enzyme-substrate-inhibitor interactions, its importance to the mechanism can be determined. Although many of the activated components of the coagulation system are enzymes, the sequential nature of the reactions, and the frequent requirement for one or more cofactors to accelerate the process, renders the application of classical enzyme kinetics both complex and inexact (See Appendix 1 and 2).

Most of the information gathered on the various interactions has

come from in vitro experiments concerning bioassays. A bioassay is the measurement of a substance in terms of its biological activity. In the case of a coagulation factor, the biological response is the time taken for the formation of a fibrin clot, in a reaction system where the concentration of the factor is made rate limiting (Briggs and Rizza, 1984) .

The central element of a bioassay is the establishment of an empirical dose-response relationship. For varying concentrations of the factor to be assayed, a progressive change in the biological response must be observed. This provides the means of converting responses measured for

preparations of unknown concentration to potencies, on a scale defined by reference preparation (Briggs and Rizza, 1984).

In order for a bioassay to be valid, a standard or reference for dose-response relationship must be prepared. The standard is constructed by testing it over an extensive series of dilutions. To use the standard curve subsequently, a sample plasma is tested at a given dilution, I / d, and the resultant clotting time, t, is noted. This clotting time is equated by means of the standard curve, to a concentration, say z, of the standard material. The concentration of the clotting factor in the is determined as: x = z x d

X is determined uniquely provide the standard curve is strictly monotonous (Briggs and Rizza, 1984).

There are two major drawbacks to the standard curve method. The standard cannot be relied upon to give the same dose-response relationship from one occasion of testing to the next because significant potency error can result. A second drawback is that the plasma sample can behave quantitatively different from the standard material due to some abnormality. Since the potency estimate will depend on the dilution at which it happens to be tested, it cannot be valid.

To avoid such drawbacks, comparative bioassays are used. The basic idea of a comparative bioassay is that if two preparations are truly comparable, and they are tested together on the same occasion, their dose-response relationships should be identical apart form an adjustment to take account of any difference in potency between them. As a result, one preparation can be described in terms of another based on their potency ratio (Briggs and Rizza, 1984).

Although, two curves, the standard and the unknown, are eventually graphed based on this idea, there are several different ways to graph the curves. In the first method the response time (clotting time, t) is plotted directly against the concentration c ( 1/d, where d is the dilution factor).

The result is two diverging curves of the same shape, apart from a scaling factor in the horizontal displacement (See Appendix 3). In the second method the response is plotted against the logarithm of the concentration c (see Appendix 3). The result yields a distinguishable horizontal displacement through a distance equal to log R. A third method, which is also the most widely used, is the straight line method. It is derived form the latter curve by converting the y-axis logarithmically, a technique called transformation (see Appendix 3). Since the potency ratio depends only on horizontal displacement, anything done to the vertical axis does not affect the outcome (Briggs and Rizza, 1984).

In closing, modeling of the mechanism of blood coagulation is not an easy task. Various assumptions are usually made to simplify the work. Assumptions are made also so that the model can account for a wide range of data. Equilibrium in the blood is established among procoagulants and anticoagulants, not reactants and products. If this were the case, clotting would be spontaneous and cardiac arrest would result.

Enzyme kinetic studies are important for studying specific interactions in the process, since the complete process of blood coagulation is an enzymatically controlled process. Because thrombin is the bioregulatory enzyme of hemostasis, it is necessary to inhibit its activity. If one considers the essential step needed for the activation of thrombin of the extrinsic pathway which is tissue activator complex, then one can see that by inhibiting its activity, the possibility of unwanted clots, resulting form thrombin interaction, is reduced significantly.

Due to the complex physiological nature of the body, and the fact that physiology varies from one person to the next, bioassays are only good for analyzing graphically or mathematically the potency of coagulation factors of a particular individual. The major drawback, is that this type of modeling can not be used to account for specific coagulation phenomenon of different individuals, let alone the same individual from one occasion to the next. This is because significant potency error will result.

Works Cited

Bloom, Arthur L., Forbes, Charles D., Thomas, Duncan P., Tuddenham, Edward, G.D. (Eds.) Haemostatis and Thrombosis (3rd ed.). Churchhill Livingstone: New York; 1994.

Briggs, Rosemary, Rizza, C. R (Eds.). Human Blood Coagulation, Haemostasis and Thrombosis (3rd ed.). Blackwell Scientific Publications: Boston; 1984.

Davis, O. Bowman, PH.D., Holtz, Noel, M.D., Davis, Judith C., M.A., R.N.

Conceptual Human Physiology. Merril: Columbus; 1985.

Dawes, Edwin A. Quanitative Problems In Biochemistry (3rd ed.). Williams and Wilkins Company: Baltimore; 1965.

Fleck, George M. Chemical Reaction Mechanisms. Holt, Rinehart, and Winston, Inc.: New York; 1971.

Guyton, Arthur C. Textbook of Medical Physiology. Harcourt Brace Jovannovich: Philadelphia; 1991.

Harker, Laurence A., M.D. Hemostasis Manuel (2nd ed.). F.A. Davis Company: Philadelphia; 1974.

Hathaway, William E., Goodnight, Scott, H. Jr. Disorders of Hemostasis and Thrombosis. McGraw-Hill: New York; 1993.

Hougie, Cecil, M.D. Fundamentals of Blood Coagulation in Clinical Medicine. New York: Mc Graw Hill; 1963.

Neurath, Hans. (1989). Proteolytic processing and physiological regulation. Trends in Biochemical Sciences. 14. 268-270.

Suttie, J.W. (1980). Vitamin K-dependent carboxylation. Trends in Biochemical Sciences. 9. 100-102.