Xfm

XIIIa

Fibrinogen

Fibrin

Stable fibrin clot

Figure 1 The tissue factor pathway and contact activation pathways of blood coagulation. PL, platelet phospholipids; TF, tissue factor.

produce TF are endothelial cells and monocytes when the proinflammatory cytokines and bacterial endotoxins stimulate these cells to express this cell membrane-bound protein. The presence of a NFkB binding site in the promoter region of the tissue factor gene on chromosome 1 further indicates involvement of hemostasis in inflammatory processes.

TF is an integral membrane protein of 263 amino acids and molecular weight 47000. It has been assigned CD142 by the VIth International Workshop and Conference on Human Leukocyte Differentiation Antigens held in Kobe, Japan, in 1996. The ligand for this cell surface protein is factor VII or factor VIIa. There are relatively small amounts of factor VIIa constantly present in normal blood. However, only when factor VIIa binds to TF does it become significantly potent as an activator of blood coagulation. A TF:Ca2+:factor VIIa complex is formed and this activates both factor X to factor Xa and factor IX to factor IXa, which forms a complex with factor VIIa, Ca2+, and platelet phos-pholipids to activate further molecules of factor X. The activated factor X forms a factor Xa:Ca2+:fac-tor Va complex, which converts prothrombin to thrombin. Thrombin acts on fibrinogen molecules to convert them to fibrin monomers. These monomers form an instantaneous clot by associating via noncovalent bonds. The clot is then stabilized in a reaction catalyzed by factor XIIIa by crosslinking the fibrin molecule by covalent bonds between glu-tamic acid and lysine residues of adjacent fibrin monomers. Thrombin plays a central role in hemos-tasis in that it not only converts fibrinogen to fibrin but also activates other key players in the pathways; in particular, factor VII, factor XI, and the copper-dependent factors V and VIII. The major physiological activator of factor VII remains unidentified but, in addition to thrombin; factor Xa, factor XIa, and factor XIIa are capable of converting factor VII to factor VIIa.

TF is inhibited by a specific inhibitor of the TF:Ca2+:factor VIIa complex (Figure 3). This inhibitor, designated tissue factor pathway inhibitor (TFPI), is a 276 amino acid polypeptide that has three Kunitz-like regions. Therefore, this belongs to the Kunitz-type protease inhibitors whilst most other inhibitors of blood coagulation are serpins. In peripheral blood this inhibitor is associated with the lipid fractions and is, like tissue factor, synthesized by activated endothelial cells and monocytes. The TFPI remains inactive until sufficient amounts of activated factor X (i.e., factor Xa) can bind. The TFPI:factor Xa complex then inactivates the TF:Ca2+:factor VIIa complex.

Figure 2 The action of tissue factor pathway inhibitor (TFPI). TF, tissue factor.

In the second mechanism of initiation, the contact activation pathway (Figure 1 and Figure 2), blood coagulation may be triggered by activation upon contact with collagen and other highly charged surfaces, including glass or plastic test tubes. Contact with collagen leads to activation of factor XII and, in turn, factor XIIa activates factor XI. Factor XIa activates factor IX, which together with Ca2+, platelet phospholipid, and factor VIIIa activates factor X to factor Xa. The contact activation pathway merges with the tissue factor pathway

Figure 3 Contact activation pathway. PL, platelet phospholipids.

at this point and, as before, the factor Xa, together with Ca2+, platelet phospholipid, and factor Va activates prothrombin to thrombin, which converts fibrinogen to fibrin. The existence of this second pathway means that fibrin production remains switched on as long as the blood remains in contact with external surfaces despite the fact that the tissue factor pathway may, at this point, be closed down by the action of TFPI and factor Xa. The formation of factor XIIa has important consequences for other processes involved in the response of the body to injury. For example; high-molecular-weight kininogen and prekallikrein are activated to the kinins and to kallikrein, mediators of inflammation that can activate the contact pathway (Figure 3). In addition, fibrinolysis and the complement pathway are both activated by factor XIIa. It is probable that the physiological role of factor XIIa is to act as a mediator in many of the processes involved in the defense of the body during trauma and its role in the activation of blood coagulation may be of minor importance.

The coagulation process is inhibited by nonspecific mechanisms such as blood flow and by the presence in plasma of general serine protease inhibitors such as alpha2-macroglobulin. In addition, unique molecular systems will specifically inhibit blood coagulation and one of these involves heparin, a sul-fated glycosaminoglycan. Heparin combines with antithrombin III in a one to one molar ratio and the resultant complex inhibits factor Xa and thrombin. Another specific control mechanism involves the vitamin K-dependent proteins, protein S and protein C, which combine with each other in molar ratios to form a complex with cellular membranes that inhibits the activity of factors Va and VIIIa. The complex also inhibits the action of PAI-1 thereby promoting fibrinolysis. Interestingly, a normal factor V molecule is essential for the inhibitory properties of this complex since mutation of factor V will cause a malfunction of the complex and result in a tendency to form clots (thrombophilia). This is the basis of the action of factor V Leiden, a mutant of factor V that causes familial thrombophilia.

Fibrinolysis, Fibrinogen, and the Acute Phase Response

The breakdown of the fibrin clot is initiated by activators of plasminogen, mainly tissue plasminogen activator (tPA) (Figure 4). This protein is produced by endothelial cells and activates plas-minogen by converting it to plasmin. The plasmin then acts on fibrin to form the fibrin split

Tissue plasminogen activator

Plasminogen ■

Plasmin

Plasmin

Fibrinogen

2 x Fragment D 1 x Fragment E

Fibrin

1 x D-dimer 1 x Fragment E

Figure 4 Fibrinolysis and fibrinogenolysis.

Figure 5 The role of interleukin-6 in fibrinogen synthesis.

products, a D-dimer and a fragment E from each fibrin monomer, and on fibrinogen to form the fibrinogen degradation products, two molecules of fragment D and one of fragment E. These breakdown products act as inhibitors of thrombin. They also stimulate the release of interleukin-6 (IL-6) from blood monocytes; the IL-6 then acts on the liver parenchymal cell to stimulate the synthesis of fibrinogen. The presence of this regulatory loop demonstrates the direct link between a proinflammatory cytokine, interleukin-6, and the production of a protein (fibrinogen) of the so-called acute phase response (Figure 5). Fibri-nolysis is inhibited by PAI-1 and alpha2-antiplasmin.

Thrombosis

Thrombosis is a major cause of mortality and morbidity in Western societies. Deep venous thrombosis, myocardial infarction, pulmonary embolism, and acute thromboembolic stroke are, probably, all consequences of inappropriate blood coagulation or fibrinolysis. Fibrin formation is instantaneous and therefore clots can quickly occlude an artery and thereby precipitate an acute cardiac or cerebral event. This usually, but not exclusively, occurs in atherosclerotic blood vessels where plaques may rupture and cause thrombus formation.

The expression of tissue factor on cells and their subsequent exposure to blood is probably the major initiator of thrombus formation in vivo. It has been demonstrated that the blood monocytes from unstable angina and myocardial infarction patients express increased amounts of tissue factor when compared to healthy controls. Furthermore, it has been shown that monocytes from atherosclerotic plaques from unstable angina patients express increased amounts of tissue factor when compared to controls, thereby further implicating plaque rupture in the pathology of heart disease.

Hemostatic Factors and Risk of Coronary Heart Disease

Elevated plasma coagulation factor levels are risk factors for coronary heart disease. Fibrinogen synthesis in the liver is stimulated by the proin-flammatory cytokine interleukin-6 and, therefore, elevated levels are found during the acute phase response. It has been argued that it may be difficult to assign elevated plasma fibrinogen as a definitive risk factor since the pathology of CHD involves inflammation and the acute phase response, which will lead to increased fibrinogen anyway. The same argument has been used in the case of elevated white cell counts, which is also a risk factor for coronary events. However, it has been demonstrated that if fibrinogen and/or white blood cell count remain high after a vascular event then there is greater risk of subsequent events. Therefore, increased plasma fibrinogen and elevated white cell count are now considered a major risk factor for CHD.

Further studies have indicated that other blood clotting factors may act as risk factors for CHD. For example, a prospective study, The Northwick Park Heart Study, identified factor VII as a risk factor for CHD and showed that plasma levels of factor VII were predictive of CHD in a dose-dependent manner. Another study has shown that factor VIII may be a risk factor for cardiovascular disease. Increased levels of PAI-1 and decreased plasma levels of plasminogen activators have also been identified as risk factors for coronary heart disease.

Dietary Effects on Hemostatic Function

Dietary vitamin K profoundly affects the activity of the blood clotting factors II, VII, IX, and X and the inhibitory molecules, protein C and protein S. These proteins are synthesized in the liver and contain a region or module of the protein that contains modified glutamic acid residues (gamma carboxy glutamic acid) (Gla). Gla formation requires vitamin K and so a deficiency state will cause abnormal bleeding. A dietary deficiency of vitamin K is rare and nearly always occurs in the neonate leading to hemorrhagic disease of the newborn. However, the oral anticoagulant drugs based on warfarin are vitamin K analogs and thus prevent the synthesis of a biologically active coagulation protein.

Fibrinogen levels are elevated in obese individuals although many studies have failed to demonstrate any relationship between plasma fibrinogen levels and dietary fat. Active men have lower fibri-nogen levels and a number of studies have demonstrated an inverse relationship between alcohol consumption and plasma fibrinogen. A high-fiber diet was also shown to correlate negatively with plasma fibrinogen. A few studies on the effects of antioxidant vitamins show that they may lower fibrinogen. For example, the Swedish MONItoring CArdiovascular disease (MONICA) study found that high levels of plasma retinol were associated with lower fibrinogen levels. However, plasma tPA levels were also lowered indicating that the fibri-nolytic pathway was compromised in these subjects. Fish oil was shown to lower fibrinogen levels when the oil contained vitamin E.

Dietary fat profoundly affects the activity of factor VII. A high fat intake is associated with increased amounts of factor VIIa. Indeed, the postprandial levels of this active blood coagulation factor have been shown to be elevated after a high-fat meal. If exposed to tissue factor, these increased levels of factor VIIa would have major consequences for thrombogenesis. Platelet aggregation is often affected by diet and fish oils can reduce platelet aggregation.

The influence of diet on the components of the coagulation pathway is well recognized by the requirement of vitamin K. However, the role that diet plays in modulating the levels of coagulation factors that are, or may be, risk factors for coronary heart disease is unclear and the results are often conflicting. For example, in intervention studies examining the effects of fish oils on monocyte TF production, one study reported a decrease in expression whilst other studies showed no effect. The study where the positive effects were reported was carried out in Italy and it has been suggested that the Mediterranean diet of the Italians was a contributory factor in these findings. Obviously, more work is required to establish the effects of fish oils on TF expression.

Hemostasis is an exciting and important area of medicine and our knowledge of the molecular and cellular interactions that bring about clot formation is now at an advanced stage. Many of the studies on diet and hemostasis may have used inappropriate laboratory techniques. For example, blood platelets are easily activated making aggregation data difficult to interpret. The advent of automated coagul-ometers means that the clotting factors may be easily measured. There are commercially available immunoassays for many of the components of hemostasis described in this article. Using flow cyto-metry, TF can be measured on monocytes and P-selectin may be detected on activated platelets. Since thrombosis is a major cause of acute coronary events the rewards of studies on diet and hemostasis may be great.

See also: Cholesterol: Sources, Absorption, Function and Metabolism; Factors Determining Blood Levels. Coronary Heart Disease: Lipid Theory; Prevention. Vitamin K.

Further Reading

Hutton RA, Laffan MA, and Tuddenham EGD (1999) Normal haemostasis. In: Hoffbrand AV, Lewis SM, and Tuddenham EGD (eds.) Postgraduate Haematology, pp. 550-580. Oxford: Butterworth Heinemann. Laffin MA and Manning RA (2001) Investigation of haemostasis. In: Lewis SM, Bain BJ, and Bates I (eds.) Practical Haemato-logy, pp. 339-413. London: Churchill Livingstone. Ross R (1993) The pathogenesis of atherosclerosis: a perspective for the 1990's. Nature 362: 801-809.

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