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Hemostasis and Thrombosis
C H A P T E R
49 Hemostasis and Thrombosis Antonio Capurso1, Cristiano Capurso2 1
Geriatrics and Internal Medicine, University of Bari Medical School, Bari, Italy 2Internal Medicine, University of Foggia, Foggia, Italy
HEMOSTASIS AND COAGULATION Coagulation of the blood safeguards against death by hemorrhage or against the undue loss of blood.
Primary and Secondary Hemostasis Hemostasis is the normal physiological response that prevents significant blood loss after vascular injury, whereas thrombosis is the pathological intravascular phenomenon that leads to the formation of a clot along the wall of a blood vessel, frequently causing occlusion of the vessel. Hemostasis depends on a complex series of events involving platelets and the activation of specific blood proteins, known as coagulation factors (Figs. 49.1e49.3). These coagulation factors are generally serine proteases (enzymes), with the exception of Factor V (FV) and FVIII, which are glycoproteins, and FXIII, which is a transglutaminase. They circulate as inactive zymogens (inactive enzyme precursors) and act by cleaving downstream proteins so that they become active enzymes. Coagulation begins almost instantly after injury to a blood vessel has damaged the endothelium lining the vessel. The exposure of blood to the space under the endothelium initiates two processes: (1) Primary hemostasis, in which platelets change their status and adhere to the site of injury to form a primary cloth. When the endothelium is damaged, the normally isolated, underlying collagen becomes exposed to circulating platelets that bind directly to collagen with collagen-specific glycoprotein Ia/IIa surface receptors. This adhesion is further strengthened by von Willebrand factor (vWF) released from the endothelium and platelets; vWF forms additional links between the platelets’ glycoprotein Ib/IX/V and the collagen fibrils. Through these mechanisms, platelets become Principles of Nutrigenetics and Nutrigenomics https://doi.org/10.1016/B978-0-12-804572-5.00049-5
activated and release the contents of stored granules into the blood plasma. The granules include adenosine diphosphate (ADP), serotonin, plateletactivating factor (PAF), vWF, platelet factor 4, and thromboxane A2 (TxA2), which in turn activate additional platelets that adhere to the site of injury. (2) Secondary hemostasis is a complex phenomenon consisting of the formation of insoluble, cross-linked fibrin through the action of activated coagulation factors, particularly thrombin. Fibrin stabilizes the primary platelet plug, particularly in larger blood vessels in which the platelet plug is insufficient in itself to stop hemorrhage.
The Secondary Hemostasis Pathways To stabilize the primary cloth, a secondary insoluble cloth made of cross-linked fibrin is created. This phenomenon represents the core of secondary hemostasis. Traditionally, secondary hemostasis has been divided into three distinct pathways: intrinsic, extrinsic, and common pathway (Fig. 49.3). Intrinsic pathway. The intrinsic pathway (Fig. 49.3) starts after a surface contact phenomenon and is sustained by coagulation factors FXII, FXI, and FIX, cofactor FVIII, Ca, and phosphatidylserine. The ultimate product of the intrinsic pathway is activated Factor IX (FIXa), which, with the aid of activated cofactor FVIIIa, activates FX. Extrinsic pathway. The extrinsic pathway (Fig. 49.3) is the coagulation phenomenon that starts when tissue damage takes place; it is called the tissue factor (TF)-activated extrinsic pathway. It starts when, in damaged blood vessels, circulating FVII comes into contact with subendothelial cell membranes with exposed TF, forming a TF-FVII complex (Fig. 49.1). The monomer FVII is then activated to the active dimer FVIIa in the presence of FXIIa, FXa, FIXa, and thrombin. The role of TF is to
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INTRINSIC
EXTRINSIC
XIIa XI
COMMON PATHWAY
FXIIa FXa FIXa thrombin
FVII
FVIIa Tissue Factor
XIa IX
PLATELET AND ENDOTHELIUM FUNCTIONS
tissue damage
surface contact XII
OXIDATIVE STRESS
IXa X
FVIIa +TF complex Xa
prothrombin
X thrombin fibrin
stable fibrin
FIGURE 49.1 The coagulation cascade of secondary hemostasis. Both intrinsic and exrinsic pathways converge in the common patway, where they activate FX which in turn activate the subsequent coagulation cascade leading to the thrombin formation and the fibrin cloth. F, factor; TF, tissue factor.
Human popliteal artery
CIRCULATING BLOOD
Damaged endothelial surface
FVII
Tissue Factor FVIIa
Platelet aggregation Expression of platelet adhesion molecules PAI-1 FVII NO
POLYPHENOLS INFLAMMATION
XIII XIIIa
fibrinogen
LDL oxidation DNA oxidation F2-isoprostane GSSG ROS GSH GSH-Px
FXIIa FXa FIXa thrombin
TF + FVIIa Complex FIGURE 49.2
A schematic representation of the extrinsic pathway of blood coagulation. The extrinsic pathway is activated when blood comes in contact with cell membranes with exposed tissue factor (TF). The monomer FVII in activated to the active dimer FVIIa in the presence of FXIIa, FXa, FIXa and thrombin. These sequential proteolytic activation take place on cell membrane surfaces of damaged vessels and leads to the formation of TF þ FVIIa complex. F, factor; TF, tissue factor.
enhance the activity of FVIIa, making it an efficient catalyst of FIX and FX activation. These sequential proteolytic activations take place on cell membrane surfaces and depend on phospholipids, mostly provided by activated platelets, but also endothelial cells and leukocytes. Platelets, which are attracted to the vessel wall by collagen, become activated and release several of their constituents, including fibrinogen, causing thrombosis in concert with activated coagulation and fibrin formation (Figs. 49.2 and 49.3).
TxB2 LTB4 CRP IL6 COX-1 COX-2
LIPID METABOLISM CHEMOPREVENTION Metabolism of carcinogens Induced cell death VEGF
Total cholesterol LDL cholesterol Triglycerides HDL cholesterol
FIGURE 49.3 COX-1, cyclooxygenase-1; CRP, C-reactive protein; FVII, Factor VII; GSH, glutathione; GSH-Px, glutathione peroxidase; GSSG, glutathione disulfide; HDL, high-density lipoprotein; IL6, interleukin 6; LDL, low-density lipoprotein; LTB4, leukotriene B4; NO, nitric oxide; PAI-1, plasminogen activator inhibitor 1; ROS, reactive oxygen species; TxB2, thromboxane B2; VEGF, vascular endothelial growth factor.
Common pathway. The extrinsic and intrinsic coagulation pathways converge on FX (Fig. 49.3), which is activated to FXa. FXa starts the final part of the coagulation process shared by intrinsic and extrinsic pathways (Fig. 49.3, area under the red line), leading to fibrin (clot) formation.
The Prominent Role of the Tissue FactoreActivated Extrinsic Pathway in the Coagulation Process and Human Pathology It was previously thought that the two pathways of coagulation cascade, the intrinsic (platelets) pathway and the extrinsic (FVII) pathway were equally important, but it is now known that the primary pathway for initiating blood coagulation is the TF-activated-extrinsic pathway. FVII has a pivotal role in activating the common pathway of the clotting cascade and thrombin formation. The main role of the TF pathway is to generate a thrombin burst, a process by which thrombin, an important constituent of the coagulation cascade, is rapidly released. The role of the TF pathway, and specifically of FVII, is particularly relevant in the thrombotic process associated with coronary heart disease (CHD). Numerous studies have determined the significant association between FVII and cardiovascular mortality. In a prospective cohort study (De Stavola and Meade, 2007) reporting mortality data after a follow-up of nearly 30 years, FVIIc was confirmed to be significantly related to CHD mortality as an independent risk factor. High FVII levels increase the risk of fatal CHD by increasing the probability of a life-threatening
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coronary thrombosis. The increase in circulating FVII thus appears to be one of the most important risk factors for CHD, equal to high cholesterol levels, hypertension, smoke, and diabetes.
The Circulating Level of Factor VII Is Influenced by Diet Coagulation FVII is a 50-kDa single-chain, vitamin Kedependent protease that has an important role in the extrinsic pathway of blood coagulation. FVII is synthesized principally in the liver and is secreted as an inactive single-chain glycoprotein. In the presence of TF, inactive FVII is converted by limited proteolysis to its fully activated two-chain form. Activation can be affected by a number of activated coagulation factors, including Xa, IXa, XIIa, and thrombin. After activation, FVIIa rapidly converts FIX and FX into their active forms, initiating the generation of thrombin and fibrin clot formation. Numerous studies demonstrated that (i) the levels of circulation FVII are influenced to some extent by diet; (ii) there is a substantial increase in FVII circulating levels in the postprandial phase; (iii) the total intake of dietary fat appears to be the main determinant of postprandial FVII plasma levels; and (iv) the ratio of saturated fatty acids (SFAs) to monounsaturated fatty acids (MUFAs) in the diet is crucial to postprandial levels of FVII.
Postprandial Increase in Factor VII FVIIc increases consistently in the postprandial phase. This FVII increase has been shown to be associated with postprandial triglyceride levels and takes place within 2e3 h after the intake of a fatty meal, persisting for several hours afterward. The maximum activation of FVIIa takes place 8 h after eating. Fat intake, rather than dietary energy intake, has been shown to be the primary determinant of the postprandial increase in FVIIc.
NUTRACEUTICAL EFFECTS OF DIET ON HEMOSTASIS AND THROMBOSIS The impact of diet and its components on blood coagulation and thrombosis have been known for a long time.
Factor VII: The Impact of Extra Virgin Olive Oil and Monounsaturated Fatty Acids on Postprandial Factor VII Levels Alimentary fats are involved in the activity of FVII. Although the results on FVII and SFAeunsaturated fatty acids have given conflicting results, more recent data
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showed that olive oil and its main fatty acid, MUFA oleic acid, have a favorable impact on the activity of FVII, particularly in the postprandial phase. Numerous studies demonstrated that during the postprandial state, a procoagulatory situation exists (an increase in thromboxanes and D-dimer and a decrease in tissue plasminogen activator). The type of fat consumed, both in an acute meal and during the previous weeks, is the main determinant of these changes. A sustained and remarkable increase in FVII antigen (FVIIag) and activity (FVIIa) also takes place in the postprandial phase. Postprandial activation of FVIIa is mainly driven by a diet rich in long-chain saturated fatty acids. A single fat-rich meal, irrespective of whether it is rich in SFAs or polyunsaturated fatty acids (PUFAs), induces an increase in FVIIa only in individuals with a background diet rich in long-chain SFAs, not in those whose usual diet is rich in unsaturated fatty acids (Roche et al., 1998). Of interest are data on the different postprandial responses to dietary fat in North Europe compared with South Europe. FVIIc was shown to be significantly greater 8 h after eating in Northern Europeans compared with Southern Europeans, two populations that follow different habitual diets; the northern population is rich in SFAs and the southern one is rich in MUFAs, particularly extravirgin olive oil (EVOO) (Zampelas et al., 1998). These data were further confirmed in a comparative study in which 40% of SFAs was replaced with MUFAs (Silva et al., 2003). Postprandial FVIIa and FVIIag were significantly lower after an MUFA-rich diet than an SFA-rich diet. Also, longterm dietary intervention studies (Roche et al., 1998) confirmed the favorable effect of MUFAs on FVII circulating levels. Compared with an SFA-rich diet, consumption of an MUFA-rich diet for 16 weeks was associated with significantly lower postprandial FVIIc and FVIIa levels. The beneficial effects of the MUFArich diet were sustained in the long term with no attenuation through adaptation. Taken together, these data demonstrate that diets rich in MUFAs, particularly from olive oil, are associated with a lower postprandial peak level of FVII, and likely explain the lower rates of CHD in countries where the diet is habitually rich in MUFAs, such as in Southern European countries.
Factor VII: Mechanism of Action of Monounsaturated Fatty Acids on Factor VII. The Role of Triglycerides The mechanism by which lipoproteins and fatty acids support the increase or reduction of FVII is not completely clear. However, some studies partially clarified these mechanisms.
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The particle size of triglyceride-rich lipoproteins (TRLs) (chylomicrons and very lowedensity lipoproteins) has been suggested to be a determinant of postprandial FVII activation. It is known that an SFA-rich diet determines the postprandial formation of a high number of triglyceride-rich chylomicrons and activation of FVII. Different from SFA, an MUFA-rich diet results in the postprandial formation of a smaller number of larger chylomicron particles and attenuation of postprandial FVII activation. Chronic exposure to an MUFA-rich diet increases the capacity of large TRL particles to transport lipids during the postprandial phase while reducing the absolute number of TRL particles (Duttaroy, 2005). FVII binds to the protein moiety of TRLs, prolonging the length of its stay in the bloodstream. By producing a smaller number of large TRL particles, the MUFA-rich diet makes less FVII bind to TRL particles. In turn, this determines less activation of FVII. Furthermore, the large TRL particles rich in MUFAs or n-3 fatty acids are more easily cleared from the plasma than are particles rich in SFAs, because of their conformational structure (Sirtori et al., 1986). The SFAs are located primarily in position s2 of the triacylglycerols, which interchange with greater difficulty from the surface of the chylomicrons than do fatty acids located in position s1 and s3 of the acylglycerols, occupied preferentially by MUFAs and n-3 fatty acids (Karupaiah and Sundram, 2007). The combination of these two factors (i.e., the smaller number of large TRL particles and the shorter stay in the bloodstream of MUFA-rich particles compared with SFA-rich particles) may explain the lower concentration of FVII after an MUFA-rich diet as well as differences between MUFA- and SFA-rich diets in the postprandial phase. Another suggested mechanism implicated in postprandial levels of FVII is the different affinity of fatty acids for peroxisome proliferator activated receptor a (PPARa), a nuclear hormone receptor that has a critical role in regulating lipid metabolism. PPARa activity could be part of a more complex mechanism responsible for the FVII activation, at least in the acute test meal situation.
Factor VII: High Carbohydrate Diet Affects Postprandial Factor VII Studies that compared the effects of a high-fat meal with those of a high-carbohydrate meal in healthy subjects showed that both meals increase postprandial levels of FVII and impaired the antiplatelet functions of the endothelium. However, the postprandial absolute increase in FVIIa was significantly lower after the highcarbohydrate meal than after the high-fat meal. In the high-carbohydrate meal, FVIIa returned to normal values after an L-arginine intravenous infusion increased NO bioavailability; this was different from the high-fat
meal, in which the normalizing effect of L-arginine did not occur. Thus, increased NO availability from L-arginine or other substrates, such as vegetable inorganic nitrate, resets the activated hemostasis. This putative effect appears to be seriously impaired after a high-fat meal.
Platelets: Effects of Extravirgin Olive Oil, Monounsaturated Fatty Acids, and n-3 Polyunsaturated Fatty Acids Platelets are small cell fragments produced by the breakdown of megakaryocytes, the large precursor cells found in bone marrow. Upon release into the circulation, they circulate for approximately 9e12 days. The normal platelet count varies from 140 to 400 109/L. As discussed, platelets are involved in primary hemostasis, with the formation of a white thrombus. Dietary fats, which are expected to modify the composition of the platelet membrane, would affect their function. In principle, diets high in saturated fat, particularly long-chain fatty acids, are associated with a greater incidence of thrombosis compared with when a diet high in monounsaturated or polyunsaturated fat is eaten. Studies in vitro and in vivo showed that long-chain saturated fatty acids increase platelet aggregation, contrary to unsaturated fatty acids, which inhibit it. Studies in humans largely demonstrated the antiplatelet aggregation effect of EVOO and MUFAs. In a study (Barradas et al., 1990), a 21-g/day supplementation of olive oil for 8 weeks reduced platelet aggregation induced by ADP and collagen. In a population study on young adults, consumption of an MUFA-rich diet, compared with an SFA-rich diet, resulted in a significant decrease in platelet aggregation in response to ADP, collagen, and arachidonic acid at 8 weeks; reduced platelet aggregation was maintained at 16 weeks (Smith et al., 2003). Despite the contrasting results of other studies, the weight of evidence suggests the significant beneficial effect of an MUFA-rich diet on platelet aggregation. Studies in rabbits also showed that oleic acid is a potent inhibitor of PAF-induced platelet aggregation. PAF is a platelet agonist, a powerful endogenous mediator of platelet aggregation made of a mixture of unsaturated free fatty acids (FFAs). The PAF effect on platelets results from its interaction with a specific membrane receptor that induces the degradation of platelet plasma membrane phosphatidylinositol. Oleic acid has been demonstrated to induce the inhibition of phosphatidylinositol synthesis. The effect of oleic acid on platelet activation was attributed to the beneficial effects of oleic acid and other unsaturated FFAs in thrombotic disease prevention (Nunez et al., 1990; Delgado-Lista et al., 2008). u3 (n-3) PUFA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) extracted from fish oil were
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demonstrated to reduce platelet aggregation actively. This effect results from several mechanisms: (i) competition with arachidonic acid, replacing active TxA2 with TxA3; (ii) inhibition of cyclooxygenase; and (iii) a direct antagonistic effect on TxA2eprostaglandin H2 receptor in human platelets. The EPA and DHA metabolic activity, however, is not confined to the platelet antiaggregation effects. Numerous studies and extensive reports showed the cardioprotective effects of dietary fish, fish oil, or a combination of EPA and DHA used in nutritional supplementation. Cardioprotection conferred by these PUFA species has been attributed to different mechanisms, including regulation of lipid metabolism and blood pressure, stabilization of atherosclerotic plaques, and antiarrhythmic and antiinflammatory actions. Individual gene variations in apolipoprotein (apo)AI, apoA5, apoE, tumor necrosis factor a (TNFa), PPARa, nitric oxide synthase 3 (NOS3), and arachidonate 5-lipoxygenase interact with n-3 PUFA intake modulating lipid metabolism and cardiovascular outcomes. EPA and DHA also have a lowering effect on triglyceride blood levels. n-3 PUFA interacts with triglyceride metabolism modulated by NOS and PPARa gene polymorphisms. Carriers of the minor allele for rs1799983 single nucleotide polymorphism (SNP) of NOS3 gene have shown a negative correlation between plasma TG concentrations and plasma n-3 PUFA levels. After n-3 PUFA supplementation, subjects with the minor allele had a better response to the change in plasma n-3 PUFA in reducing serum triglyceride concentration than did major allele homozygous carriers. Concerning PPARa gene polymorphisms, the minor allele Leu162Val variant was associated with higher triglycerides and apoC-III blood levels only in subjects consuming a low-PUFA diet. Conversely, high consumption of PUFAs with diet modulates the effect of this SNP on lipid metabolism and triglyceride blood levels.
Platelets: Polyphenols and Platelet Aggregation Polyphenols are potent antioxidants present in several foods, particularly in fresh fruit, wine, and EVOO. The EVOO is particularly rich in phenolic compounds. The richness of EVOO in polyphenols depends on the mechanical procedures employed to obtain it, without the use of chemical solvents. Other olive oils (i.e., refined olive oils), obtained with solvents by pomace, contain no polyphenols because solvents do not allow any phenolic compound to be recovered by the pomace. The refining process serves to remove color, odor, and flavor from low-quality olive oil and leaves behind a pure form of olive oil that is tasteless, colorless, and odorless, with no bioactive compound. EVOO, on
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the contrary, contains large amounts of polyphenols, which, however, are only a tenth of the polyphenols contained in olives; in fact, most of these olives’ bioactive compounds are lost during the various processing stages, particularly in the mill wastewater. The major phenolic compounds in EVOO are oleuropein, tyrosol, hydroxytyrosol, and luteolin. These phenols, which represent almost 90% of the EVOO polyphenols, are only four of the almost 30 phenolic compounds present in EVOO. The polyphenols have been mostly investigated in CHD, in which they have demonstrated to reduce numerous cardiovascular risk factors significantly. EVOO polyphenols possess antioxidant properties and influence many biological activities that may account, at least partly, for the observed effects of olive oil on the cardiovascular system. Some of these effects include (i) inhibition of low-density lipoprotein oxidation, (ii) production of nitric oxide, and (iii) downregulation of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression in endothelial cells (Carluccio et al., 2003). EVOO with a high phenolic compound content (400 mg/kg) has also been demonstrated to inhibit plasminogen activator inhibitor-1 and FVII significantly. As far as platelet aggregation is concerned, EVOO polyphenols have been demonstrated to reduce platelet aggregability. The EVOO phenol component (2-[3,4-dihydroxyphenil]-ethanol) significantly decreases platelet aggregation induced in vitro by ADP and collagen, and thromboxane B2 (TxB2) production by collagen and thrombin-stimulated platelet-rich plasma (Carluccio et al., 2003). Two isochroman polyphenols present in EVOO (1-[30 -cyclic adenosine monophosphate (cAMP)methoxy-40 -hydroxy-phenyl]-6,7-dihydroxy-isochroman and 1-phenyl-6,7-dihydroxy-isochroman), inhibit in vitro platelet aggregation and thromboxane release induced by arachidonic acid and collagen (Togna et al., 2003). In vivo beneficial effects of EVOO polyphenols on hemostasis were also reported. In a study on volunteers, a significant reduction in plasma TxB2 concentration after an EVOO-rich diet, compared with a high oleic-sunflower diet, was observed; this effect was attributed to the greater number of polyphenols in the EVOO-rich diet (Oubi na et al., 2001). The molecular mechanisms of EVOO polyphenol antiplatelet activity were studied. Platelet activation is regulated by a number of physiological activators (TxA2, vasopressin, ADP, thrombin, and serotonin) and inhibitors (endothelium-derived relaxing factor and prostaglandin inhibitor-2). Platelet antagonists inhibit platelet function by increasing the intracellular levels of cyclic nucleotides cAMP and cyclic guanosine monophosphate by activating the respective cyclases. Cyclic nucleotide levels are downregulated by degradation
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through phosphodiesterases (PDE). Platelets contain mainly PDE3, which preferentially hydrolyzes cAMP as substrate. EVOO phenols, particularly luteolin, were demonstrated to act on the cAMP-PDE complex by inhibiting PDE activity, increasing intracellular levels of cyclic nucleotide cAMP (Dell’Agli et al., 2008). 3,30 ,40 ,5,7-Pentamethylquercetin (PMQ), a member of the polymethoxylated flavone family, is the methylated form of quercetin, a flavonol polyphenol found in many fruits and vegetables, teas, grains, and olive oil. PMQ is a potent antioxidant with anticarcinogenic activity and cardioprotective properties. In vitro studies showed that PMQ exerts a potent inhibitory effect on platelet function; in vivo, it inhibits thrombus formation in an acute animal model. PMQ has been shown to protect mice from death in the acute lung thromboembolism model, and from carotid artery injury induced by ferric chloride. Moreover, PMQ inhibits platelet aggregation induced by several agonists and regulates the functional response of platelets, including the release of adenosine triphosphate and Pselectin. The molecular mechanisms of the inhibitory effects of PMQ on platelet function were shown to be the suppression of the PI3K/Akt-GSK3b and Syk-PLCg2Erk signaling cascades (Liang et al., 2015). Finally, the peculiar combination of biologically active compounds in EVOO produces a milder activation of mechanisms of inflammation and coagulation during the postprandial phase, a situation that leads to reduced postprandial activation of nuclear factor-kB, an important cellular regulator that initiates the formation of procoagulant and proinflammatory signal peptides (Nunez et al., 1990).
NUTRIGENETICS, NUTRIGENOMICS, AND COAGULATION SYSTEM: THE ROLE OF DIET AND ITS COMPONENTS Polymorphisms of Factor VII Gene Plasma FVII levels vary significantly in the general population; they are influenced by environmental and genetic factors. Studies linked the presence of mutations in FVII gene with plasma FVII levels and activity. The FVII gene is 12.8 kb long; the mature FVII protein is encoded by exons 2e8. Several insertion-deletion and single-point mutations in the FVII gene were described in the promoter region (323P0/10), within the transcribed space (R353Q point mutation), and within intronic sequences (IVS7). These FVII gene polymorphisms may have opposite effects on FVII levels and were shown to modulate the risk for myocardial infarction (MI) in males with advanced coronary artery disease. R353Q is a polymorphism in the FVII gene closely associated with variations in plasma levels of FVII. It is
a single nucleotide polymorphism (point mutation in the transcribed region) characterized by the substitution of an arginine (R allele) with a glutamine (Q allele) in codon 353 of the transcribed protein. Lower postprandial levels of FVII associated with hypertriglyceridemia were found in carriers of the Q allele compared with individuals who were homozygous for the more common R allele. Compared with subjects with the R allele (Arg353), subjects with one or two Q alleles (Gln353) had levels of FVIIc that were 20% and 78% lower, respectively. FVII gene polymorphisms may explain up to about one-third of FVII-level variation in plasma. This indicates that to a major extent, plasma FVII levels are determined by genetic influences. Other FVII gene polymorphisms have been described: 323A1/A2, 401G/T, and 402G/A. 323A1/A2 is a polymorphism characterized by a 10ebase pair insertion in the FVII promoter region at position 323. Carriers of the A2 allele of the 323A1/ A2 polymorphism have been demonstrated to have a significant decrease in FVII circulating level (by 36% in males and 39% in females) and a lower risk for MI than noncarriers (odds ratio [OR] ¼ 0.40; 95% confidence interval [CI] ¼ 0.20e0.80). 401G/T and 402G/A are two novel polymorphisms found in the promoter region of FVII gene. They are associated with altered plasma concentrations of FVII in vivo in population-based healthy human volunteers. Together, the 401G/T and 402G/A polymorphisms account for 18% and 28% of variation in the plasma concentrations of total FVII and fully activated factor FVII (FVIIa). These two polymorphisms affect the binding of two hepatic nuclear proteins to the promoter of the FVII gene, with accompanying changes of FVII expression in hepatocytes and FVII secretion by the liver. This, in turn, results in decreased (401T allele) or increased (402A allele) plasma levels of FVII, respectively. Male carriers of the 402A allele were associated with a significantly increased risk for MI (OR ¼ 1.79; 95% CI ¼ 1.15e2.80). Among these FVII gene polymorphisms, R353Q SNP has been described as dominant over the other polymorphisms and as the major contributor to circulating levels of FVII.
Polymorphisms of Genes Involved in Homocysteine-Methionine Metabolism: Example of GeneeEnvironment Interaction Elevated levels of homocysteine in the blood represent a potential risk factor for cerebrovascular diseases such as stroke and a predisposition to thrombosis and other pathological conditions. The reason for an elevated homocysteine level in the blood may be inadequate nutrition or genetic variants that reduce the
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SUMMARY
activity of enzymes necessary to remove homocysteine efficiently from the blood. A diet containing vitamins B12 and B6 and folic acid helps lower homocysteine level in the blood. Several polymorphisms have been described in 5,10methylene tetrahydrofolate reductase (MTHFR) and methionine synthase (MS) genes. Some of these polymorphisms were found to have a crucial role in the thrombotic risk associated with homocysteine blood level and nutritional status. MTHFR is an enzyme involved in converting the amino acid homocysteine to another amino acid, methionine, providing the folate derivative for converting homocysteine to methionine. Another enzyme, MS, is responsible for regenerating methionine from homocysteine. Two polymorphisms of these genes hare crucial for thrombotic risk: 1 .The C677T polymorphism in exon 4 of the MTHFR gene, which leads to the amino acid substitution of alanine by valine at codon 222 (A222V), causing a thermolabile enzyme with lower activity; and 2 .2756 A / G of MS, which leads to amino acid substitution of aspartate by glycine. These two polymorphisms are associated with (i) decreased activity of the enzyme MTHFR; (ii) a relative deficiency in the remethylation process of homocysteine into methionine; and (iii) mild t -moderate hyperhomocysteinemia, a condition recognized as an independent risk factor for atherosclerosis and thrombosis. In studies on the interaction between these polymorphisms and B vitamin nutritional status in individuals affected by thromboembolic events, the genetic influence of the MTHFR polymorphism on homocysteine levels was not significant in individuals at high risk for thrombotic events who exhibited serum levels of folate and/or vitamin B12 above the 50th percentile of distribution in the general population.
Transcriptomic Effects of Adhesion to Mediterranean Diet The TCF7L2 gene has been shown to have the strongest association with type 2 diabetes. The effect of adhering to the Mediterranean diet (MeD) on the incidence of diabetes and cardiovascular risk in subjects with the TCF7L2 gene polymorphism was investigated in the PREDIMED study (Corella and Ordovas, 2014). The rs7903146 (C > T) SNP in intron 4 of the TCF7L2 gene was the most important one associated with type 2 diabetes. In the PREDIMED study, the strong association between risk allele (T) and the incidence of type 2 diabetes mellitus, high glucose, high cholesterol, high triglycerides, and high risk for cardiovascular disease was completely reversed in subjects with a high level
of adherence to the MeD. These effects appeared to result from a synergistic action of different components of the MeD, particularly MUFA, PUFA, and polyphenols. The intake of polyphenols was calculated as 1 g/ day, 54% of which were flavonoids, 37% phenolic acids, and 9% other polyphenols. These high polyphenol intakes derived mostly from EVOO and fresh fruit. When the transcriptomic profile in peripheral blood mononuclear cells was evaluated in the PREDIMED study, a weak thrombin signaling pathway modulation was found in subjects following the MeD supplemented with EVOO or nuts. The authors concluded that one mechanism by which MeD supplemented with EVOO can exert health benefits is through changes in the transcriptomic response of genes related to cardiovascular risk. In a meta-analysis summarizing current knowledge concerning nutrigenomic studies on MeD and olive oil interventions, significant changes in proatherothrombotic (TF and TFP1), inflammation (TNFa and monocyte chemoattractant protein 1), and oxidative stress-related gene expression were found in subjects following a MeD pattern compared with control subjects (Konstantinidou et al., 2013). TF and TF pathway inhibitor (TFPI) genes were downregulated and upregulated, respectively, by the MeD. The TNFa gene acts as an activator of a cascade of inflammatory cytokine production; TNFa itself is considered to be a crucial proinflammatory cytokines. The TNFa gene is upregulated after consumption of a butter-rich meal, but not after an EVOO-rich meal or diet.
SUMMARY Hemostasis is a complex phenomenon involving numerous coagulation factors. These factors circulate as inactive zymogens (inactive enzyme precursor) and act by cleaving downstream proteins so that they become active enzymes (Figs. 49.1 and 49.2). The role of FVII in secondary hemostasis while forming a complex with TF is of primary importance. It was thought that the two pathways of coagulation cascade, the intrinsic (platelet) pathway and the extrinsic (FVII) pathway were equally important, but it is now known that the primary pathway for the initiation of blood coagulation is the TF-activated extrinsic pathway. FVII has a pivotal role in activating the common pathway of the clotting cascade and thrombin formation (Fig. 49.3). The MeD, EVOO, and polyphenols have been shown to affect several coagulation factors. The circulating level of FVII is deeply influenced by diet. There is a substantial increase in FVII circulating levels in the postprandial phase. The intake of dietary fat is the main determinant of the postprandial FVII plasma level. The ratio of SFAs to MUFAs is crucial to
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postprandial levels of FVII. Diets rich in MUFA (i.e., olive oil, are associated with a significantly lower postprandial peak level of FVII and likely explain the lower rates of CHD in countries in which the diet is habitually rich in MUFAs, such as the Southern European countries. Gene polymorphisms in the FVII promoter region modulate FVII circulating levels. Some of these polymorphisms are associated with lower levels of circulating FVII. Also, platelet activity is influenced by diet. The n-3 PUFAs EPA and DHA actively reduce platelet aggregation. This effect results from several mechanisms (i) competition with arachidonic acid, replacing active TxA2 with TxA3; (ii) inhibition of cyclooxygenase; and TABLE 49.1
Protective Effects of Eicosapentaenoic Acid/Docosahexaenoic Acid on Cardiovascular Disease Risk Factors.
Effect
Proposed Mechanism
Reduced platelet aggregation
Reduction in prothrombotic prostanoids through competition with arachidonic acid
Antiarrhythmic effect
Modulation of electrophysiological properties of cardiac myocytes
Serum triglycerides reduction
Reduction in hepatic triglyceride production and lipoprotein assembly
Lowered blood pressure
Improved endothelial function, vascular relaxation, and arterial compliance
Decreasing inflammation
Reduced leukotriene production and signaling through competition with arachidonic acid and leukotriene receptor antagonism
TABLE 49.2
The most important phenolic compounds of the extra virgin olive oil (EVOO).
Phenolic Acids Verbascoside Caffeic acid p-Hydroxybenzoic acid Protocatechuic acid Vanillic acid Coumaric acid Ferulic acid
Secoiridoids Oleuropein glucoside Oleuropein aglycone Ligstroside
Anthocyanin Cyanidin glucoside Cyanidin rutinoside Glucosyl rutinoside Delphinidin rhamnosyl
Simple Phenols Hydroxytyrosol Tyrosol
Flavanols Quercetin rutinoside Flavones Luteolin 5-glucoside apigenin 5-glucoside
(iii) a direct antagonistic effect on the TxA2e prostaglandin H2 receptor in human platelets. Polyphenols of EVOO, particularly luteolin, also reduce platelet aggregability, acting as an inhibitor of platelet PDE3 (Table 49.1). Probably the most exciting data are those concerning the effects of the MeD in modulating gene expression. Dietary interventions have been demonstrated to modulate the expression of pro-atherothrombotic and inflammation genes actively even in high-risk populations. SFAs upregulate both proinflammatory and proatherothrombotic genes, whereas the MeD, EVOO, and polyphenols downregulate the expression of these genes. The MeD, which is rich in olive oil, MUFAs, and polyphenols were demonstrated to exert a modulatory effect toward a protective mode on genes related to chronic degenerative diseases, oxidation, inflammation, and thrombosis (modulating the activity of TF, TFP1, and thrombin). The phenolic compounds present in EVOO appear to be responsible for the transcriptomic effects, as demonstrated in randomized, controlled human studies in which similar olive oils, but with different phenolic contents, were tested (Table 49.2).
References Barradas, M.A., Chrirtofides, J.A., Jeremy, J.Y., Mikhailidis, D.P., Fry, D.E., Dandona, P., 1990. The effect of olive oil supplementation on human platelet function, serum cholesterol related variables and plasma fibrinogen concentration: a pilot study. Nutr Res 10, 403e411. Carluccio, M.A., Siculella, L., Ancora, M.A., Massaro, M., Scoditti, E., Storelli, C., Visioli, F., Distante, A., De Caterina, R., 2003. Olive oil and red wine antioxidant polyphenols inhibit endothelial activation: antiatherogenic properties of Mediterranean diet phytochemicals. Arterioscl Thromb Vasc Biol 23, 622e629. Corella, D., Ordovas, J.M., 2014. How does Mediterranean diet promote cardiovascular health? Current progress toward molecular mechanism gene-diet interactions at the genomic, transcriptomic, and epigenomic levels provide novel insight into new mechanisms. Bioessays 36, 526e537. De Stavola, B.L., Meade, T.W., 2007. Long term effects of hemostatic variables of fatal coronary heart disease. 30-years results from the first prospective Northwick Park Heart Study (NPHS-I). J Thromb Haemost 5, 461e471. Delgado-Lista, J., Lopez-Miranda, J., Cortes, B., Perez-Martinez, P., Lozano, A., Gomez-Luna, R., Gomez, P., Gomez, M.J., Fuentes, F., Perez-Jimenez, F., 2008. Chronic dietary fat intake modifies the postprandial response of hemostatic markers to a single fatty test meal. Am J Clin Nutr 87, 317e322. Dell’Agli, M., Maschi, O., Galli, G.V., Fagnani, R., Dal Cero, E., Caruso, D., Bosisio, E., 2008. Inhibition of platelet function by olive oil phenols via cAMP-phosphodiesterase. Br J Nutr 99, 945e951. Duttaroy, A.K., 2005. Postprandial activation of hemostatic factors: role of dietary fatty acids. Prostaglandins Leucot Essent Fatty Acids 72, 381e391. Karupaiah, T., Sundram, K., 2007. Effects of stereospecific positioning of fatty acids in triacylglycerol structures in native and randomized fats: a review of their nutritional implications. Nutr Metab 4, 16.
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REFERENCES
Konstantinidou, V., Covas, M.I., Sola, R., Fito`, M., 2013. Up-to date knowledge on the in vivo transcriptomic effect of the Mediterranean diet in humans. Mol Nutr Food Res 57, 772e783. Liang, M.L., Da, X.W., He, A.D., Yao, G.Q., Xie, W., Liu, G., Xiang, J.Z., Ming, Z.Y., 2015. Pentamethylquercitin (PMQ) reduces thrombus formation by inhibiting platelet function. Sci Rep 5, 11142. Nunez, D., Randon, J., Gandhi, C., Siafaka-Kapadai, A., Olson, M.S., Hanahan, D.J., 1990. The inhibition of platelet-activating factorinduced platelet activation by oleic acid is associated with a decrease in polyphosphoinositide metabolism. J Biol Chem 265, 18330e18338. Oubi na, P., Sa`ncez-Muniz, F.J., Ro`denas, S., Cuesta, C., 2001. Eicosanoid production, Thrombogenic ratio, and serum LDL peroxides in Normo- and hypercholesterolaemic post-menopausal women consuming two oleic acid-rich diets with different content of minor components. Br J Nutr 85, 41e47. Roche, H.M., Zampelas, A., Knapper, J.M., Webb, D., Brooks, C., Jackson, K.G., Wright, J.W., Gould, B.J., Kafatos, A., Gibney, M.J., Williams, C.M., 1998. Effect of long-term olive oil dietary intervention on postprandial triacylglycerol and factor VII metabolism. Am J Clin Nutr 68, 552e560.
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Silva, K.D., Kelly, C.N., Jones, A.E., Smith, R.D., Wootton, S.A., Miller, G.J., Williams, C.M., 2003. Chylomicron particle size and number, factor VII activation and dietary monounsaturated fatty acids. Atherosclerosis 166, 73e84. Sirtori, C.R., Tremoli, E., Gatti, E., Montanari, G., Sirtori, M., Colli, S., Gianfranceschi, G., Maderna, P., Dentone, C.Z., et al., 1986. Controlled evaluation of fat intake in the Mediterranean diet: comparative activities of olive oil on plasma lipids and platelets in high-rick patients. Am J Clin Nutr 44, 635e642. Smith, R.D., Kelly, C.N., Fielding, B.A., Hauton, D., Silva, K.D., Nydahl, Miller, G.J., Williams, C.M., 2003. Long-term monounsaturated fatty acids reduce platelet aggregation in healthy young subjects. Br J Nutr 90, 597e606. Togna, G.I., Togna, A.R., Franconi, M., Marra, C., Guiso, M., 2003. Olive oil isochromans inhibit human platelet reactivity. J Nutr 23, 622e629. Zampelas, A., Roche, H., Knapper, J.M., Jackson, K.G., Tornaritis, M., Gibney, M.J., Kafatos, A., Gould, B.J., Wright, J., Williams, C.M., 1998. Differences in postprandial lipaemic response between Northern and Southern Europeans. Atherosclerosis 139, 83e93.
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49 Hemostasis and Thrombosis Antonio Capurso1, Cristiano Capurso2 1
Geriatrics and Internal Medicine, University of Bari Medical School, Bari, Italy 2Internal Medicine, University of Foggia, Foggia, Italy
HEMOSTASIS AND COAGULATION Coagulation of the blood safeguards against death by hemorrhage or against the undue loss of blood.
Primary and Secondary Hemostasis Hemostasis is the normal physiological response that prevents significant blood loss after vascular injury, whereas thrombosis is the pathological intravascular phenomenon that leads to the formation of a clot along the wall of a blood vessel, frequently causing occlusion of the vessel. Hemostasis depends on a complex series of events involving platelets and the activation of specific blood proteins, known as coagulation factors (Figs. 49.1e49.3). These coagulation factors are generally serine proteases (enzymes), with the exception of Factor V (FV) and FVIII, which are glycoproteins, and FXIII, which is a transglutaminase. They circulate as inactive zymogens (inactive enzyme precursors) and act by cleaving downstream proteins so that they become active enzymes. Coagulation begins almost instantly after injury to a blood vessel has damaged the endothelium lining the vessel. The exposure of blood to the space under the endothelium initiates two processes: (1) Primary hemostasis, in which platelets change their status and adhere to the site of injury to form a primary cloth. When the endothelium is damaged, the normally isolated, underlying collagen becomes exposed to circulating platelets that bind directly to collagen with collagen-specific glycoprotein Ia/IIa surface receptors. This adhesion is further strengthened by von Willebrand factor (vWF) released from the endothelium and platelets; vWF forms additional links between the platelets’ glycoprotein Ib/IX/V and the collagen fibrils. Through these mechanisms, platelets become Principles of Nutrigenetics and Nutrigenomics https://doi.org/10.1016/B978-0-12-804572-5.00049-5
activated and release the contents of stored granules into the blood plasma. The granules include adenosine diphosphate (ADP), serotonin, plateletactivating factor (PAF), vWF, platelet factor 4, and thromboxane A2 (TxA2), which in turn activate additional platelets that adhere to the site of injury. (2) Secondary hemostasis is a complex phenomenon consisting of the formation of insoluble, cross-linked fibrin through the action of activated coagulation factors, particularly thrombin. Fibrin stabilizes the primary platelet plug, particularly in larger blood vessels in which the platelet plug is insufficient in itself to stop hemorrhage.
The Secondary Hemostasis Pathways To stabilize the primary cloth, a secondary insoluble cloth made of cross-linked fibrin is created. This phenomenon represents the core of secondary hemostasis. Traditionally, secondary hemostasis has been divided into three distinct pathways: intrinsic, extrinsic, and common pathway (Fig. 49.3). Intrinsic pathway. The intrinsic pathway (Fig. 49.3) starts after a surface contact phenomenon and is sustained by coagulation factors FXII, FXI, and FIX, cofactor FVIII, Ca, and phosphatidylserine. The ultimate product of the intrinsic pathway is activated Factor IX (FIXa), which, with the aid of activated cofactor FVIIIa, activates FX. Extrinsic pathway. The extrinsic pathway (Fig. 49.3) is the coagulation phenomenon that starts when tissue damage takes place; it is called the tissue factor (TF)-activated extrinsic pathway. It starts when, in damaged blood vessels, circulating FVII comes into contact with subendothelial cell membranes with exposed TF, forming a TF-FVII complex (Fig. 49.1). The monomer FVII is then activated to the active dimer FVIIa in the presence of FXIIa, FXa, FIXa, and thrombin. The role of TF is to
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Copyright © 2020 Elsevier Inc. All rights reserved.
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INTRINSIC
EXTRINSIC
XIIa XI
COMMON PATHWAY
FXIIa FXa FIXa thrombin
FVII
FVIIa Tissue Factor
XIa IX
PLATELET AND ENDOTHELIUM FUNCTIONS
tissue damage
surface contact XII
OXIDATIVE STRESS
IXa X
FVIIa +TF complex Xa
prothrombin
X thrombin fibrin
stable fibrin
FIGURE 49.1 The coagulation cascade of secondary hemostasis. Both intrinsic and exrinsic pathways converge in the common patway, where they activate FX which in turn activate the subsequent coagulation cascade leading to the thrombin formation and the fibrin cloth. F, factor; TF, tissue factor.
Human popliteal artery
CIRCULATING BLOOD
Damaged endothelial surface
FVII
Tissue Factor FVIIa
Platelet aggregation Expression of platelet adhesion molecules PAI-1 FVII NO
POLYPHENOLS INFLAMMATION
XIII XIIIa
fibrinogen
LDL oxidation DNA oxidation F2-isoprostane GSSG ROS GSH GSH-Px
FXIIa FXa FIXa thrombin
TF + FVIIa Complex FIGURE 49.2
A schematic representation of the extrinsic pathway of blood coagulation. The extrinsic pathway is activated when blood comes in contact with cell membranes with exposed tissue factor (TF). The monomer FVII in activated to the active dimer FVIIa in the presence of FXIIa, FXa, FIXa and thrombin. These sequential proteolytic activation take place on cell membrane surfaces of damaged vessels and leads to the formation of TF þ FVIIa complex. F, factor; TF, tissue factor.
enhance the activity of FVIIa, making it an efficient catalyst of FIX and FX activation. These sequential proteolytic activations take place on cell membrane surfaces and depend on phospholipids, mostly provided by activated platelets, but also endothelial cells and leukocytes. Platelets, which are attracted to the vessel wall by collagen, become activated and release several of their constituents, including fibrinogen, causing thrombosis in concert with activated coagulation and fibrin formation (Figs. 49.2 and 49.3).
TxB2 LTB4 CRP IL6 COX-1 COX-2
LIPID METABOLISM CHEMOPREVENTION Metabolism of carcinogens Induced cell death VEGF
Total cholesterol LDL cholesterol Triglycerides HDL cholesterol
FIGURE 49.3 COX-1, cyclooxygenase-1; CRP, C-reactive protein; FVII, Factor VII; GSH, glutathione; GSH-Px, glutathione peroxidase; GSSG, glutathione disulfide; HDL, high-density lipoprotein; IL6, interleukin 6; LDL, low-density lipoprotein; LTB4, leukotriene B4; NO, nitric oxide; PAI-1, plasminogen activator inhibitor 1; ROS, reactive oxygen species; TxB2, thromboxane B2; VEGF, vascular endothelial growth factor.
Common pathway. The extrinsic and intrinsic coagulation pathways converge on FX (Fig. 49.3), which is activated to FXa. FXa starts the final part of the coagulation process shared by intrinsic and extrinsic pathways (Fig. 49.3, area under the red line), leading to fibrin (clot) formation.
The Prominent Role of the Tissue FactoreActivated Extrinsic Pathway in the Coagulation Process and Human Pathology It was previously thought that the two pathways of coagulation cascade, the intrinsic (platelets) pathway and the extrinsic (FVII) pathway were equally important, but it is now known that the primary pathway for initiating blood coagulation is the TF-activated-extrinsic pathway. FVII has a pivotal role in activating the common pathway of the clotting cascade and thrombin formation. The main role of the TF pathway is to generate a thrombin burst, a process by which thrombin, an important constituent of the coagulation cascade, is rapidly released. The role of the TF pathway, and specifically of FVII, is particularly relevant in the thrombotic process associated with coronary heart disease (CHD). Numerous studies have determined the significant association between FVII and cardiovascular mortality. In a prospective cohort study (De Stavola and Meade, 2007) reporting mortality data after a follow-up of nearly 30 years, FVIIc was confirmed to be significantly related to CHD mortality as an independent risk factor. High FVII levels increase the risk of fatal CHD by increasing the probability of a life-threatening
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coronary thrombosis. The increase in circulating FVII thus appears to be one of the most important risk factors for CHD, equal to high cholesterol levels, hypertension, smoke, and diabetes.
The Circulating Level of Factor VII Is Influenced by Diet Coagulation FVII is a 50-kDa single-chain, vitamin Kedependent protease that has an important role in the extrinsic pathway of blood coagulation. FVII is synthesized principally in the liver and is secreted as an inactive single-chain glycoprotein. In the presence of TF, inactive FVII is converted by limited proteolysis to its fully activated two-chain form. Activation can be affected by a number of activated coagulation factors, including Xa, IXa, XIIa, and thrombin. After activation, FVIIa rapidly converts FIX and FX into their active forms, initiating the generation of thrombin and fibrin clot formation. Numerous studies demonstrated that (i) the levels of circulation FVII are influenced to some extent by diet; (ii) there is a substantial increase in FVII circulating levels in the postprandial phase; (iii) the total intake of dietary fat appears to be the main determinant of postprandial FVII plasma levels; and (iv) the ratio of saturated fatty acids (SFAs) to monounsaturated fatty acids (MUFAs) in the diet is crucial to postprandial levels of FVII.
Postprandial Increase in Factor VII FVIIc increases consistently in the postprandial phase. This FVII increase has been shown to be associated with postprandial triglyceride levels and takes place within 2e3 h after the intake of a fatty meal, persisting for several hours afterward. The maximum activation of FVIIa takes place 8 h after eating. Fat intake, rather than dietary energy intake, has been shown to be the primary determinant of the postprandial increase in FVIIc.
NUTRACEUTICAL EFFECTS OF DIET ON HEMOSTASIS AND THROMBOSIS The impact of diet and its components on blood coagulation and thrombosis have been known for a long time.
Factor VII: The Impact of Extra Virgin Olive Oil and Monounsaturated Fatty Acids on Postprandial Factor VII Levels Alimentary fats are involved in the activity of FVII. Although the results on FVII and SFAeunsaturated fatty acids have given conflicting results, more recent data
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showed that olive oil and its main fatty acid, MUFA oleic acid, have a favorable impact on the activity of FVII, particularly in the postprandial phase. Numerous studies demonstrated that during the postprandial state, a procoagulatory situation exists (an increase in thromboxanes and D-dimer and a decrease in tissue plasminogen activator). The type of fat consumed, both in an acute meal and during the previous weeks, is the main determinant of these changes. A sustained and remarkable increase in FVII antigen (FVIIag) and activity (FVIIa) also takes place in the postprandial phase. Postprandial activation of FVIIa is mainly driven by a diet rich in long-chain saturated fatty acids. A single fat-rich meal, irrespective of whether it is rich in SFAs or polyunsaturated fatty acids (PUFAs), induces an increase in FVIIa only in individuals with a background diet rich in long-chain SFAs, not in those whose usual diet is rich in unsaturated fatty acids (Roche et al., 1998). Of interest are data on the different postprandial responses to dietary fat in North Europe compared with South Europe. FVIIc was shown to be significantly greater 8 h after eating in Northern Europeans compared with Southern Europeans, two populations that follow different habitual diets; the northern population is rich in SFAs and the southern one is rich in MUFAs, particularly extravirgin olive oil (EVOO) (Zampelas et al., 1998). These data were further confirmed in a comparative study in which 40% of SFAs was replaced with MUFAs (Silva et al., 2003). Postprandial FVIIa and FVIIag were significantly lower after an MUFA-rich diet than an SFA-rich diet. Also, longterm dietary intervention studies (Roche et al., 1998) confirmed the favorable effect of MUFAs on FVII circulating levels. Compared with an SFA-rich diet, consumption of an MUFA-rich diet for 16 weeks was associated with significantly lower postprandial FVIIc and FVIIa levels. The beneficial effects of the MUFArich diet were sustained in the long term with no attenuation through adaptation. Taken together, these data demonstrate that diets rich in MUFAs, particularly from olive oil, are associated with a lower postprandial peak level of FVII, and likely explain the lower rates of CHD in countries where the diet is habitually rich in MUFAs, such as in Southern European countries.
Factor VII: Mechanism of Action of Monounsaturated Fatty Acids on Factor VII. The Role of Triglycerides The mechanism by which lipoproteins and fatty acids support the increase or reduction of FVII is not completely clear. However, some studies partially clarified these mechanisms.
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The particle size of triglyceride-rich lipoproteins (TRLs) (chylomicrons and very lowedensity lipoproteins) has been suggested to be a determinant of postprandial FVII activation. It is known that an SFA-rich diet determines the postprandial formation of a high number of triglyceride-rich chylomicrons and activation of FVII. Different from SFA, an MUFA-rich diet results in the postprandial formation of a smaller number of larger chylomicron particles and attenuation of postprandial FVII activation. Chronic exposure to an MUFA-rich diet increases the capacity of large TRL particles to transport lipids during the postprandial phase while reducing the absolute number of TRL particles (Duttaroy, 2005). FVII binds to the protein moiety of TRLs, prolonging the length of its stay in the bloodstream. By producing a smaller number of large TRL particles, the MUFA-rich diet makes less FVII bind to TRL particles. In turn, this determines less activation of FVII. Furthermore, the large TRL particles rich in MUFAs or n-3 fatty acids are more easily cleared from the plasma than are particles rich in SFAs, because of their conformational structure (Sirtori et al., 1986). The SFAs are located primarily in position s2 of the triacylglycerols, which interchange with greater difficulty from the surface of the chylomicrons than do fatty acids located in position s1 and s3 of the acylglycerols, occupied preferentially by MUFAs and n-3 fatty acids (Karupaiah and Sundram, 2007). The combination of these two factors (i.e., the smaller number of large TRL particles and the shorter stay in the bloodstream of MUFA-rich particles compared with SFA-rich particles) may explain the lower concentration of FVII after an MUFA-rich diet as well as differences between MUFA- and SFA-rich diets in the postprandial phase. Another suggested mechanism implicated in postprandial levels of FVII is the different affinity of fatty acids for peroxisome proliferator activated receptor a (PPARa), a nuclear hormone receptor that has a critical role in regulating lipid metabolism. PPARa activity could be part of a more complex mechanism responsible for the FVII activation, at least in the acute test meal situation.
Factor VII: High Carbohydrate Diet Affects Postprandial Factor VII Studies that compared the effects of a high-fat meal with those of a high-carbohydrate meal in healthy subjects showed that both meals increase postprandial levels of FVII and impaired the antiplatelet functions of the endothelium. However, the postprandial absolute increase in FVIIa was significantly lower after the highcarbohydrate meal than after the high-fat meal. In the high-carbohydrate meal, FVIIa returned to normal values after an L-arginine intravenous infusion increased NO bioavailability; this was different from the high-fat
meal, in which the normalizing effect of L-arginine did not occur. Thus, increased NO availability from L-arginine or other substrates, such as vegetable inorganic nitrate, resets the activated hemostasis. This putative effect appears to be seriously impaired after a high-fat meal.
Platelets: Effects of Extravirgin Olive Oil, Monounsaturated Fatty Acids, and n-3 Polyunsaturated Fatty Acids Platelets are small cell fragments produced by the breakdown of megakaryocytes, the large precursor cells found in bone marrow. Upon release into the circulation, they circulate for approximately 9e12 days. The normal platelet count varies from 140 to 400 109/L. As discussed, platelets are involved in primary hemostasis, with the formation of a white thrombus. Dietary fats, which are expected to modify the composition of the platelet membrane, would affect their function. In principle, diets high in saturated fat, particularly long-chain fatty acids, are associated with a greater incidence of thrombosis compared with when a diet high in monounsaturated or polyunsaturated fat is eaten. Studies in vitro and in vivo showed that long-chain saturated fatty acids increase platelet aggregation, contrary to unsaturated fatty acids, which inhibit it. Studies in humans largely demonstrated the antiplatelet aggregation effect of EVOO and MUFAs. In a study (Barradas et al., 1990), a 21-g/day supplementation of olive oil for 8 weeks reduced platelet aggregation induced by ADP and collagen. In a population study on young adults, consumption of an MUFA-rich diet, compared with an SFA-rich diet, resulted in a significant decrease in platelet aggregation in response to ADP, collagen, and arachidonic acid at 8 weeks; reduced platelet aggregation was maintained at 16 weeks (Smith et al., 2003). Despite the contrasting results of other studies, the weight of evidence suggests the significant beneficial effect of an MUFA-rich diet on platelet aggregation. Studies in rabbits also showed that oleic acid is a potent inhibitor of PAF-induced platelet aggregation. PAF is a platelet agonist, a powerful endogenous mediator of platelet aggregation made of a mixture of unsaturated free fatty acids (FFAs). The PAF effect on platelets results from its interaction with a specific membrane receptor that induces the degradation of platelet plasma membrane phosphatidylinositol. Oleic acid has been demonstrated to induce the inhibition of phosphatidylinositol synthesis. The effect of oleic acid on platelet activation was attributed to the beneficial effects of oleic acid and other unsaturated FFAs in thrombotic disease prevention (Nunez et al., 1990; Delgado-Lista et al., 2008). u3 (n-3) PUFA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) extracted from fish oil were
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demonstrated to reduce platelet aggregation actively. This effect results from several mechanisms: (i) competition with arachidonic acid, replacing active TxA2 with TxA3; (ii) inhibition of cyclooxygenase; and (iii) a direct antagonistic effect on TxA2eprostaglandin H2 receptor in human platelets. The EPA and DHA metabolic activity, however, is not confined to the platelet antiaggregation effects. Numerous studies and extensive reports showed the cardioprotective effects of dietary fish, fish oil, or a combination of EPA and DHA used in nutritional supplementation. Cardioprotection conferred by these PUFA species has been attributed to different mechanisms, including regulation of lipid metabolism and blood pressure, stabilization of atherosclerotic plaques, and antiarrhythmic and antiinflammatory actions. Individual gene variations in apolipoprotein (apo)AI, apoA5, apoE, tumor necrosis factor a (TNFa), PPARa, nitric oxide synthase 3 (NOS3), and arachidonate 5-lipoxygenase interact with n-3 PUFA intake modulating lipid metabolism and cardiovascular outcomes. EPA and DHA also have a lowering effect on triglyceride blood levels. n-3 PUFA interacts with triglyceride metabolism modulated by NOS and PPARa gene polymorphisms. Carriers of the minor allele for rs1799983 single nucleotide polymorphism (SNP) of NOS3 gene have shown a negative correlation between plasma TG concentrations and plasma n-3 PUFA levels. After n-3 PUFA supplementation, subjects with the minor allele had a better response to the change in plasma n-3 PUFA in reducing serum triglyceride concentration than did major allele homozygous carriers. Concerning PPARa gene polymorphisms, the minor allele Leu162Val variant was associated with higher triglycerides and apoC-III blood levels only in subjects consuming a low-PUFA diet. Conversely, high consumption of PUFAs with diet modulates the effect of this SNP on lipid metabolism and triglyceride blood levels.
Platelets: Polyphenols and Platelet Aggregation Polyphenols are potent antioxidants present in several foods, particularly in fresh fruit, wine, and EVOO. The EVOO is particularly rich in phenolic compounds. The richness of EVOO in polyphenols depends on the mechanical procedures employed to obtain it, without the use of chemical solvents. Other olive oils (i.e., refined olive oils), obtained with solvents by pomace, contain no polyphenols because solvents do not allow any phenolic compound to be recovered by the pomace. The refining process serves to remove color, odor, and flavor from low-quality olive oil and leaves behind a pure form of olive oil that is tasteless, colorless, and odorless, with no bioactive compound. EVOO, on
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the contrary, contains large amounts of polyphenols, which, however, are only a tenth of the polyphenols contained in olives; in fact, most of these olives’ bioactive compounds are lost during the various processing stages, particularly in the mill wastewater. The major phenolic compounds in EVOO are oleuropein, tyrosol, hydroxytyrosol, and luteolin. These phenols, which represent almost 90% of the EVOO polyphenols, are only four of the almost 30 phenolic compounds present in EVOO. The polyphenols have been mostly investigated in CHD, in which they have demonstrated to reduce numerous cardiovascular risk factors significantly. EVOO polyphenols possess antioxidant properties and influence many biological activities that may account, at least partly, for the observed effects of olive oil on the cardiovascular system. Some of these effects include (i) inhibition of low-density lipoprotein oxidation, (ii) production of nitric oxide, and (iii) downregulation of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression in endothelial cells (Carluccio et al., 2003). EVOO with a high phenolic compound content (400 mg/kg) has also been demonstrated to inhibit plasminogen activator inhibitor-1 and FVII significantly. As far as platelet aggregation is concerned, EVOO polyphenols have been demonstrated to reduce platelet aggregability. The EVOO phenol component (2-[3,4-dihydroxyphenil]-ethanol) significantly decreases platelet aggregation induced in vitro by ADP and collagen, and thromboxane B2 (TxB2) production by collagen and thrombin-stimulated platelet-rich plasma (Carluccio et al., 2003). Two isochroman polyphenols present in EVOO (1-[30 -cyclic adenosine monophosphate (cAMP)methoxy-40 -hydroxy-phenyl]-6,7-dihydroxy-isochroman and 1-phenyl-6,7-dihydroxy-isochroman), inhibit in vitro platelet aggregation and thromboxane release induced by arachidonic acid and collagen (Togna et al., 2003). In vivo beneficial effects of EVOO polyphenols on hemostasis were also reported. In a study on volunteers, a significant reduction in plasma TxB2 concentration after an EVOO-rich diet, compared with a high oleic-sunflower diet, was observed; this effect was attributed to the greater number of polyphenols in the EVOO-rich diet (Oubi na et al., 2001). The molecular mechanisms of EVOO polyphenol antiplatelet activity were studied. Platelet activation is regulated by a number of physiological activators (TxA2, vasopressin, ADP, thrombin, and serotonin) and inhibitors (endothelium-derived relaxing factor and prostaglandin inhibitor-2). Platelet antagonists inhibit platelet function by increasing the intracellular levels of cyclic nucleotides cAMP and cyclic guanosine monophosphate by activating the respective cyclases. Cyclic nucleotide levels are downregulated by degradation
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through phosphodiesterases (PDE). Platelets contain mainly PDE3, which preferentially hydrolyzes cAMP as substrate. EVOO phenols, particularly luteolin, were demonstrated to act on the cAMP-PDE complex by inhibiting PDE activity, increasing intracellular levels of cyclic nucleotide cAMP (Dell’Agli et al., 2008). 3,30 ,40 ,5,7-Pentamethylquercetin (PMQ), a member of the polymethoxylated flavone family, is the methylated form of quercetin, a flavonol polyphenol found in many fruits and vegetables, teas, grains, and olive oil. PMQ is a potent antioxidant with anticarcinogenic activity and cardioprotective properties. In vitro studies showed that PMQ exerts a potent inhibitory effect on platelet function; in vivo, it inhibits thrombus formation in an acute animal model. PMQ has been shown to protect mice from death in the acute lung thromboembolism model, and from carotid artery injury induced by ferric chloride. Moreover, PMQ inhibits platelet aggregation induced by several agonists and regulates the functional response of platelets, including the release of adenosine triphosphate and Pselectin. The molecular mechanisms of the inhibitory effects of PMQ on platelet function were shown to be the suppression of the PI3K/Akt-GSK3b and Syk-PLCg2Erk signaling cascades (Liang et al., 2015). Finally, the peculiar combination of biologically active compounds in EVOO produces a milder activation of mechanisms of inflammation and coagulation during the postprandial phase, a situation that leads to reduced postprandial activation of nuclear factor-kB, an important cellular regulator that initiates the formation of procoagulant and proinflammatory signal peptides (Nunez et al., 1990).
NUTRIGENETICS, NUTRIGENOMICS, AND COAGULATION SYSTEM: THE ROLE OF DIET AND ITS COMPONENTS Polymorphisms of Factor VII Gene Plasma FVII levels vary significantly in the general population; they are influenced by environmental and genetic factors. Studies linked the presence of mutations in FVII gene with plasma FVII levels and activity. The FVII gene is 12.8 kb long; the mature FVII protein is encoded by exons 2e8. Several insertion-deletion and single-point mutations in the FVII gene were described in the promoter region (323P0/10), within the transcribed space (R353Q point mutation), and within intronic sequences (IVS7). These FVII gene polymorphisms may have opposite effects on FVII levels and were shown to modulate the risk for myocardial infarction (MI) in males with advanced coronary artery disease. R353Q is a polymorphism in the FVII gene closely associated with variations in plasma levels of FVII. It is
a single nucleotide polymorphism (point mutation in the transcribed region) characterized by the substitution of an arginine (R allele) with a glutamine (Q allele) in codon 353 of the transcribed protein. Lower postprandial levels of FVII associated with hypertriglyceridemia were found in carriers of the Q allele compared with individuals who were homozygous for the more common R allele. Compared with subjects with the R allele (Arg353), subjects with one or two Q alleles (Gln353) had levels of FVIIc that were 20% and 78% lower, respectively. FVII gene polymorphisms may explain up to about one-third of FVII-level variation in plasma. This indicates that to a major extent, plasma FVII levels are determined by genetic influences. Other FVII gene polymorphisms have been described: 323A1/A2, 401G/T, and 402G/A. 323A1/A2 is a polymorphism characterized by a 10ebase pair insertion in the FVII promoter region at position 323. Carriers of the A2 allele of the 323A1/ A2 polymorphism have been demonstrated to have a significant decrease in FVII circulating level (by 36% in males and 39% in females) and a lower risk for MI than noncarriers (odds ratio [OR] ¼ 0.40; 95% confidence interval [CI] ¼ 0.20e0.80). 401G/T and 402G/A are two novel polymorphisms found in the promoter region of FVII gene. They are associated with altered plasma concentrations of FVII in vivo in population-based healthy human volunteers. Together, the 401G/T and 402G/A polymorphisms account for 18% and 28% of variation in the plasma concentrations of total FVII and fully activated factor FVII (FVIIa). These two polymorphisms affect the binding of two hepatic nuclear proteins to the promoter of the FVII gene, with accompanying changes of FVII expression in hepatocytes and FVII secretion by the liver. This, in turn, results in decreased (401T allele) or increased (402A allele) plasma levels of FVII, respectively. Male carriers of the 402A allele were associated with a significantly increased risk for MI (OR ¼ 1.79; 95% CI ¼ 1.15e2.80). Among these FVII gene polymorphisms, R353Q SNP has been described as dominant over the other polymorphisms and as the major contributor to circulating levels of FVII.
Polymorphisms of Genes Involved in Homocysteine-Methionine Metabolism: Example of GeneeEnvironment Interaction Elevated levels of homocysteine in the blood represent a potential risk factor for cerebrovascular diseases such as stroke and a predisposition to thrombosis and other pathological conditions. The reason for an elevated homocysteine level in the blood may be inadequate nutrition or genetic variants that reduce the
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activity of enzymes necessary to remove homocysteine efficiently from the blood. A diet containing vitamins B12 and B6 and folic acid helps lower homocysteine level in the blood. Several polymorphisms have been described in 5,10methylene tetrahydrofolate reductase (MTHFR) and methionine synthase (MS) genes. Some of these polymorphisms were found to have a crucial role in the thrombotic risk associated with homocysteine blood level and nutritional status. MTHFR is an enzyme involved in converting the amino acid homocysteine to another amino acid, methionine, providing the folate derivative for converting homocysteine to methionine. Another enzyme, MS, is responsible for regenerating methionine from homocysteine. Two polymorphisms of these genes hare crucial for thrombotic risk: 1 .The C677T polymorphism in exon 4 of the MTHFR gene, which leads to the amino acid substitution of alanine by valine at codon 222 (A222V), causing a thermolabile enzyme with lower activity; and 2 .2756 A / G of MS, which leads to amino acid substitution of aspartate by glycine. These two polymorphisms are associated with (i) decreased activity of the enzyme MTHFR; (ii) a relative deficiency in the remethylation process of homocysteine into methionine; and (iii) mild t -moderate hyperhomocysteinemia, a condition recognized as an independent risk factor for atherosclerosis and thrombosis. In studies on the interaction between these polymorphisms and B vitamin nutritional status in individuals affected by thromboembolic events, the genetic influence of the MTHFR polymorphism on homocysteine levels was not significant in individuals at high risk for thrombotic events who exhibited serum levels of folate and/or vitamin B12 above the 50th percentile of distribution in the general population.
Transcriptomic Effects of Adhesion to Mediterranean Diet The TCF7L2 gene has been shown to have the strongest association with type 2 diabetes. The effect of adhering to the Mediterranean diet (MeD) on the incidence of diabetes and cardiovascular risk in subjects with the TCF7L2 gene polymorphism was investigated in the PREDIMED study (Corella and Ordovas, 2014). The rs7903146 (C > T) SNP in intron 4 of the TCF7L2 gene was the most important one associated with type 2 diabetes. In the PREDIMED study, the strong association between risk allele (T) and the incidence of type 2 diabetes mellitus, high glucose, high cholesterol, high triglycerides, and high risk for cardiovascular disease was completely reversed in subjects with a high level
of adherence to the MeD. These effects appeared to result from a synergistic action of different components of the MeD, particularly MUFA, PUFA, and polyphenols. The intake of polyphenols was calculated as 1 g/ day, 54% of which were flavonoids, 37% phenolic acids, and 9% other polyphenols. These high polyphenol intakes derived mostly from EVOO and fresh fruit. When the transcriptomic profile in peripheral blood mononuclear cells was evaluated in the PREDIMED study, a weak thrombin signaling pathway modulation was found in subjects following the MeD supplemented with EVOO or nuts. The authors concluded that one mechanism by which MeD supplemented with EVOO can exert health benefits is through changes in the transcriptomic response of genes related to cardiovascular risk. In a meta-analysis summarizing current knowledge concerning nutrigenomic studies on MeD and olive oil interventions, significant changes in proatherothrombotic (TF and TFP1), inflammation (TNFa and monocyte chemoattractant protein 1), and oxidative stress-related gene expression were found in subjects following a MeD pattern compared with control subjects (Konstantinidou et al., 2013). TF and TF pathway inhibitor (TFPI) genes were downregulated and upregulated, respectively, by the MeD. The TNFa gene acts as an activator of a cascade of inflammatory cytokine production; TNFa itself is considered to be a crucial proinflammatory cytokines. The TNFa gene is upregulated after consumption of a butter-rich meal, but not after an EVOO-rich meal or diet.
SUMMARY Hemostasis is a complex phenomenon involving numerous coagulation factors. These factors circulate as inactive zymogens (inactive enzyme precursor) and act by cleaving downstream proteins so that they become active enzymes (Figs. 49.1 and 49.2). The role of FVII in secondary hemostasis while forming a complex with TF is of primary importance. It was thought that the two pathways of coagulation cascade, the intrinsic (platelet) pathway and the extrinsic (FVII) pathway were equally important, but it is now known that the primary pathway for the initiation of blood coagulation is the TF-activated extrinsic pathway. FVII has a pivotal role in activating the common pathway of the clotting cascade and thrombin formation (Fig. 49.3). The MeD, EVOO, and polyphenols have been shown to affect several coagulation factors. The circulating level of FVII is deeply influenced by diet. There is a substantial increase in FVII circulating levels in the postprandial phase. The intake of dietary fat is the main determinant of the postprandial FVII plasma level. The ratio of SFAs to MUFAs is crucial to
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postprandial levels of FVII. Diets rich in MUFA (i.e., olive oil, are associated with a significantly lower postprandial peak level of FVII and likely explain the lower rates of CHD in countries in which the diet is habitually rich in MUFAs, such as the Southern European countries. Gene polymorphisms in the FVII promoter region modulate FVII circulating levels. Some of these polymorphisms are associated with lower levels of circulating FVII. Also, platelet activity is influenced by diet. The n-3 PUFAs EPA and DHA actively reduce platelet aggregation. This effect results from several mechanisms (i) competition with arachidonic acid, replacing active TxA2 with TxA3; (ii) inhibition of cyclooxygenase; and TABLE 49.1
Protective Effects of Eicosapentaenoic Acid/Docosahexaenoic Acid on Cardiovascular Disease Risk Factors.
Effect
Proposed Mechanism
Reduced platelet aggregation
Reduction in prothrombotic prostanoids through competition with arachidonic acid
Antiarrhythmic effect
Modulation of electrophysiological properties of cardiac myocytes
Serum triglycerides reduction
Reduction in hepatic triglyceride production and lipoprotein assembly
Lowered blood pressure
Improved endothelial function, vascular relaxation, and arterial compliance
Decreasing inflammation
Reduced leukotriene production and signaling through competition with arachidonic acid and leukotriene receptor antagonism
TABLE 49.2
The most important phenolic compounds of the extra virgin olive oil (EVOO).
Phenolic Acids Verbascoside Caffeic acid p-Hydroxybenzoic acid Protocatechuic acid Vanillic acid Coumaric acid Ferulic acid
Secoiridoids Oleuropein glucoside Oleuropein aglycone Ligstroside
Anthocyanin Cyanidin glucoside Cyanidin rutinoside Glucosyl rutinoside Delphinidin rhamnosyl
Simple Phenols Hydroxytyrosol Tyrosol
Flavanols Quercetin rutinoside Flavones Luteolin 5-glucoside apigenin 5-glucoside
(iii) a direct antagonistic effect on the TxA2e prostaglandin H2 receptor in human platelets. Polyphenols of EVOO, particularly luteolin, also reduce platelet aggregability, acting as an inhibitor of platelet PDE3 (Table 49.1). Probably the most exciting data are those concerning the effects of the MeD in modulating gene expression. Dietary interventions have been demonstrated to modulate the expression of pro-atherothrombotic and inflammation genes actively even in high-risk populations. SFAs upregulate both proinflammatory and proatherothrombotic genes, whereas the MeD, EVOO, and polyphenols downregulate the expression of these genes. The MeD, which is rich in olive oil, MUFAs, and polyphenols were demonstrated to exert a modulatory effect toward a protective mode on genes related to chronic degenerative diseases, oxidation, inflammation, and thrombosis (modulating the activity of TF, TFP1, and thrombin). The phenolic compounds present in EVOO appear to be responsible for the transcriptomic effects, as demonstrated in randomized, controlled human studies in which similar olive oils, but with different phenolic contents, were tested (Table 49.2).
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