The potential role of milk-derived peptides in cardiovascular disease

Abstract

Bioactive peptides derived from milk proteins are of particular interest to the food industry due to the potential functional and physiological roles that they demonstrate, particularly in relation to cardiovascular disease (CVD). By 2020 it is estimated that heart disease and stroke will become the leading cause of death and disability worldwide. Acute and chronic cardiovascular events may result from alterations in the activity of the renin-angiotensin aldosterone system and activation of the coagulation cascade and of platelets. Medications that inhibit angiotensin converting enzyme (ACE) are widely prescribed in the treatment and prevention of cardiovascular disease. ACE inhibitory peptides are of particular interest due to the presence of encrypted inhibitory peptide sequences. In particular, Ile-Pro-Pro and Val-Pro-Pro are fore runners in ACE inhibition, and have been incorporated into commercial products. Additionally, studies to identify additional novel peptides with similar bio-activity and the ability to withstand digestion during transit through the gastrointestinal tract are ongoing. The potential sources of such peptides in cheese and other dairy products are discussed. Challenges to the bio-availability of such peptides in the gastro intestinal tract are also reviewed. Activation of platelets and the coagulation cascade play a central role in the progression of cardiovascular disease. Platelets from such patients show spontaneous aggregation and an increased sensitivity to agonists which results in vascular damage and endothelial dysfunction associated with CVD. Peptide sequences exhibiting anti-thrombotic activity have been identified from fermented milk products. Studies on such peptides are reviewed and their effects on platelet function are discussed. Finally the ability of food derived peptides to decrease the formation of blood clots (thrombi) is reviewed. In conclusion, due to the widespread nature of cardiovascular disease, the identification of food derived compounds that exhibit a beneficial effect in such widespread areas of CVD regulation will have strong clinical potential. Due to the perception that food derived products have an acceptable risk profile they have the potential for widespread acceptance by the public. In this review, selected biological effects relating to CVD are discussed with a view to providing essential information to researchers.

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Martha Phelan

Martha Phelan received a BSc in Nutritional Science in 2006 and an MSc from University College Cork in 2009. She is currently working for Food for Health Ireland determining how milk bioactives can improve cardiovascular health.

David Kerins

David Kerins is a medical graduate of University College Cork. He completed fellowships in Clinical Pharmacology and in Clinical Cardiology at Vanderbilt University, Nashville TN. He was appointed to the faculty of Vanderbilt University Medical Center and staff physician at Nashville Veterans Affairs in 2003. He was subsequently promoted to become an Associate Professor of Medicine and Chief of the Cardiology Section at Nashville VA. He was appointed as Associate Professor of Therapeutics at University College Cork and Consultant Physician at Mercy University Hospital in 2006. He served as Dean of the School of Medicine at UCC from 2007–2010.

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1. Introduction

Bioactive peptides are described as ‘food components that in addition to their nutritional value, exert a physiological effect in the body’.¹ During the last decade, research has focused on bioactive or biogenic substances derived from foods with interest in introducing them as functional food ingredients.

Bovine milk naturally contains a selection of bioactivities such as lysozyme, lactoferrin, immunoglobulins, growth factors, and hormones, which are secreted in their active form by the mammary gland.² Additionally, many bioactivities in milk are encrypted within the primary structure of milk proteins, requiring proteolysis for their release from precursors.^3,4 Milk proteins consist of 80% casein and 20% whey. The principal casein fractions found in milk are αs₁-, αs₂- β- and κ-casein (45.0%, 12.0%, 35.0% and 8.0%, respectively).^5,6 The major whey proteins are in order of abundance β-lactoglobulin, α-lactalbumin, bovine serum albumin, immunoglobulins and proteose peptones.^7,8

The main milk proteins αs₁-casein and β-casein have the capacity to liberate about 20 000 peptides each.⁹ Liberation of peptides may occur in a number of ways: (a) during transit through the gastrointestinal tract while the proteins are exposed to the action of digestive enzymes,¹⁰ (b) during microbial fermentation when subjected to proteolytic splitting by the proteolytic system of these microorganisms,^3,11 or, (c) during proteolysis by enzymes derived from microorganisms or plants.¹² In many studies, combinations of (a), (b) and (c) are used for the generation of biofunctional peptides.¹³

Once liberated and absorbed these bioactive peptides may exert a physiological effect on the various systems of the body – for example the cardiovascular, digestive, endocrine, immune and nervous systems. Examples of milk derived bioactive peptides include caseinophosphopeptides that have a role in the transport and absorption of minerals,¹⁴ glycomacropeptides that bind toxins,¹⁵ casoxins and casomorphins that act as opioid antagonists and agonists,^16,17 isracidin that has immunomodulatory effects¹⁸ and casoplatelin which has antithrombotic effects.^19–21 Other activities identified include angiotensin converting enzyme (ACE) inhibition, anticarcinogenic,²² and antitumour effects.²³

Peptide activity is based on the inherent amino acid composition and sequence which may vary from two to 20 amino acid residues. These peptides may also have multifunctional properties i.e. specific peptide sequences having two or more different physiological activities.^3,24,25 These regions have been considered as ‘strategic zones’ that are partially protected from further proteolytic breakdown.²⁶ Therefore, for the above reasons milk-derived bioactive peptides are considered as prominent candidates for various health promoting functional foods.

Cardiovascular diseases (CVDs) are a group of disorders of the heart and blood vessels and include: coronary heart-, cerebrovascular-, peripheral arterial-, rheumatic heart-, and congenital heart-disease. Deep vein thrombosis and pulmonary embolism may also be classified as CVDs. The World Health Organisation estimates that by 2020 heart disease and stroke will have surpassed infectious diseases to become the leading cause of death and disability worldwide.²⁷ Hypertension is one controllable risk factor in the development of CVD.²⁸ There are 2 general classes of hypertension. Primary/essential hypertension which accounts for 95% of cases, for which there is no known cause,²⁹ and secondary hypertension³⁰ which results from several disease states including kidney disease.³¹ Haemostatsis is the process of arresting the escape of blood by either natural or artificial means. It is an integrated and localized interaction that occurs rapidly and involves four components: the blood vessels, the platelets, the coagulation cascade, and the fibrinolytic system. Thrombosis is a pathological condition in which improper activity of the haemostatic mechanism results in clot or thrombus formation in arteries, veins or the chambers of the heart. Platelet attachment, spreading, and aggregation on extracellular matrices are central events in thrombus formation. Additionally, abnormalities in platelet function may account for the pathogenesis and complications of thrombotic events associated with hypertension.^32–34 Fibrinolysis, the process by which fibrin clot is digested to release fibrin degradation products may be suppressed due to high plasma concentrations of plasminogen-activator inhibitor type 1 (PAI-1). This in turn is associated with an increased risk for myocardial infarction.^35–38

The anti-hypertensive and anti-thrombotic effects of milk-derived bioactive peptides are the focus of this review. Much research has been carried out in these areas and there seems to be an indication of ‘cardiovascular continuum’ i.e. cardiovascular risk factors such as hypertension leading to endothelial damage, coronary artery disease and ultimately heart failure and death.³⁹ Intervention at multiple sites along this continuum is necessary to dissipate the morbidity and mortality resulting from cardiovascular disease. Hence, identification of additional compounds, which could interfere in these processes, is desirable, as some of these selected bioactivities have functional food applications. A continued review of the literature focussing on these areas is therefore beneficial.

2. Platelet function in hypertension

Hypertension is a significant health problem worldwide.⁴⁰ It is one of the major controllable risk factors associated with cardiovascular disease events such as myocardial infarction, heart failure and end-stage diabetes.^41–46 The control of blood pressure has been associated with many systems such as the renin angiotensin aldosterone system (RAAS). However, the renin angiotensin system (RAS) is not an exclusive regulator of blood pressure as the neutral endopeptidase system, the endothelin-converting enzyme system and the kinin-nitric oxide systems generate additional vaso-regulatory peptides independent of ACE.^47–50 These systems generate a variety of regulatory peptides that collectively modulate blood pressure, fluid and electrolyte balance via membrane bound receptors located on different tissues throughout the body.⁵¹ Central to RAS is an exopeptidase, angiotensin-converting enzyme I. ACE ([EC 3.4.15.1], peptidyl-dipeptidase A, peptidyl-dipeptidase I, dipeptidyl carboxypeptidase I (DCP1), peptidyldipeptidase I, kininase II, peptidase P, carboxycathepsin) is responsible for the conversion of angiotensin I, a decapeptide generated by the action of renin on the glycoprotein substrate known as angiotensinogen, to the vasoconstrictor octapeptide angiotensin II. In a separate parallel pathway ACE inactivates the vasodilatory peptide bradykinin.⁵²

Platelets are the smallest formed elements in the blood. They have a mean volume of about seven to nine femtolitre, a discoid shape and lack a nucleus. They are produced by fragmentation of megakaryocytes in the bone marrow, and are released into the circulation. Platelets play a crucial role not only in haemostasis but also in the development of CVD.^53,54 Under normal conditions, platelets circulate in the bloodstream without adhering to the endothelium of the vessel wall. However, when the endothelium is damaged, platelets adhere to the subendothelium by binding to exposed extracellular matrix proteins. They become activated, undergo shape change, and aggregate and in the process of haemostasis, a haemostatic plug stabilized by fibrin is formed at the site of vessel injury. This process is aided by glycoproteins (GPs) on their plasma membrane that bind specific adhesive proteins to promote platelet-to-surface interactions (adhesion) and platelet-to-platelet interactions (aggregation). Two adhesion receptors, glycoprotein GPIb-IX-V and GPVI, that bind von Willebrand factor (vWF) and collagen, respectively, are primarily responsible for regulating the initial platelet adhesion and activation in flowing blood.^55–60 Following adhesion, rapid signal transduction leads to platelet activation, and a sequence of events including cytoskeletal changes associated with shape change, spreading and secretion, and inside-out activation of integrins that support adhesion and aggregation. The major platelet integrin, α_IIbβ₃ (GPIIb/IIIa), binds vWF or fibrinogen to mediate platelet aggregation under conditions of increased shear.^55,61 Platelet activation involving GPIb-IX-V or GPVI also leads to secretion of platelet agonists, such as adenosine diphosphate (ADP), which acts via the G protein-coupled receptors (P2Y₁ and P2Y₁₂) to reinforce α_IIbβ₃-dependent platelet aggregation.^62,63 The activated platelet aggregate or thrombus in turn accelerates the coagulation cascade, leading to stabilization of the clot by fibrin and α_IIbβ₃-dependent contraction. Activated platelets also express surface P-selectin, a counter receptor for platelet GPIb-IX-V and leukocyte PSGL-1. Adhered activated platelets also interact with circulating leukocytes and facilitate platelet-leukocyte-endothelial cell adhesion. This involves receptors that also regulate thrombosis.⁶⁴ The interaction of inflammatory leukocytes with the vessel wall in atherothrombosis is promoted by platelets, initiating development of atherosclerotic plaque that may eventually lead to thrombotic events.^65–67

Although platelet activation and subsequent aggregation and the renin-angiotension systems are two individual pathways they have overlapping effects on each other. Platelets from hypertensive patients have several morphological and functional abnormalities compared to normal individuals. Hypertensive patients have an increased platelet size.⁶⁸ In addition they have a higher mean platelet volume (MPV)^69–71 and mean platelet mass,^69,70 and lower mean platelet granularity (MPG)⁶⁹ compared to controls. An increased MPV may be a marker of platelet activation,^72–74 in addition, lower MPG may be consistent with platelet activation.⁷⁵ High blood pressure promotes vascular damage and endothelial dysfunction, which enhance platelet activation and adhesion. Endothelial dysfunction is associated with reduced generation of anti-platelet agents such as nitric oxide (NO) and prostacyclin, and exaggerated production of endothelin 1 which enhance platelet activation.⁷⁶

In response to hypertension, platelets show a number of responses. Spontaneous aggregation⁷⁷ and increased sensitivity to agonists⁶⁸ has been identified in platelets from hypertensive individuals. Activated platelets in turn release different mediators, such as 5-hydroxytryptamine (5-HT or serotonin), ADP, adenosine triphosphate (ATP) and lysophosphatidic acid.⁷⁸ Some of these agents enhance the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in vascular smooth muscle cells (VSMC). An increase in cytosolic Ca²⁺ leads to aggregation, clot formation, and secretion of a variety of vasoactive and proaggregating substances, including thromboxane A2, serotonin and platelet derived growth factor (PDGF).^79,80,81,90 Furthermore, the number of platelet α-adrenergic receptors increases in hypertensive persons,⁸² which may promote catecholamine responses. Catecholamines, the β-adrenoceptor agonist isoprenaline and angiotensin II (Ang II) increase [Ca²⁺]_i and promote contraction of VSMC, platelet activation and aggregation⁸² which may in turn participate in the genesis and maintenance of hypertension. Angiotensin II has also been shown to increase [Ca²⁺]_i and pH in platelets from hypertensive patients, which may be associated with enhanced platelet aggregation.⁸³ Activated platelets release different growth factors e.g. PDGF and vascular derived growth factor (VEGF) that participate in the development of atherosclerosis by promoting VSMC proliferation.⁷⁸ Furthermore, platelets from hypertensive individuals release more β-thromboglobulin⁸⁴ and P-selectin.⁸⁵ Hypertension is also associated with oxidative stress and reduced antioxidant status.⁸⁶ Platelets from hypertensive patients, produce more reactive oxygen species (ROS) which enhance platelet activity by reducing the bioavailability of NO, increasing tyrosine phosphorylation, activation of platelet membrane GPIIb/IIIa, and enhancing [Ca²⁺]_i among others cellular effects.^87–89 Platelet endothelial NO synthase (eNOS) activity is also reduced⁹⁰ in patients with hypertension, which reduces NO synthesis. NO is an important vasodilator⁹¹ and inhibitor of platelet aggregation,⁹² secretion,⁹³ adhesion,⁹⁴ and fibrinogen binding to the platelet GPIIb/IIIa receptor.^95,96 Furthermore, NO interferes with Ca²⁺ mobilization in platelets by reducing both Ca²⁺ influx across the plasma membrane and Ca²⁺ release from internal stores.⁹⁷ Angiotensin II stimulates eNOS and NO release which by a negative feed-back inhibits ACE and decrease the number of Angiotensin II type 1 receptors (AT1).⁹⁸ Moreover, eNOS can reside in an uncoupled form and produce superoxide (O₂⁻) which account for further NO degradation and enhance platelet activation. Thus, as described different platelet signalling pathways are altered in hypertension and platelets might play a role in hypertension-associated angiogenesis. By understanding specific molecular mechanisms underlying platelet activation in hypertension, new targets may be identified for interventions to prevent and/or treat hypertension complications associated with platelet hyperactivity.

3. ACE inhibition

3.1. General introduction

Many potent synthetic ACE inhibitors such as captopril, enalapril, lisinopril, and ramipril have been widely used in the clinical treatment of hypertension and heart failure. However, ACE inhibitors can have side effects including hypotension, increased potassium levels, reduced renal function, cough, taste disturbances and skin rashes.^99–102 Therefore, interest has increased in identifying foods as natural sources of ACE inhibitors. It has been shown that certain foods reduce or prevent the risk of certain diseases,^103,104 one such food is milk. Milk contains natural compounds such as calcium and potassium which have been shown to have a beneficial effect on blood pressure.¹⁰⁴ Additionally, it is well recognized that apart from their basic nutritional role many milk proteins contain encrypted peptide sequences within their primary structures capable of modulating specific physiological functions.¹⁰⁵ In fact, there are numerous products on the market or under development containing bioactive peptides from milk which have ACE Inhibitor activity (Table 1).

Table 1 Products enriched with ACE-inhibitory peptides.^{51,120–1221}

Trade mark	Company	Peptide sequence
1 ND: not described.
Ameal S	Calpis Co, Japan	VPPIPP
Biozate	Davisco, USA	ND
Calpis	Calpis Co, Japan	VPPIPP
Casine DP	Kanebo Ltd., Japan	FFVAPFEVFGK
C12 peptide	DMV International, Holland	FFVAPFEVFGK
Danaten	Danone, France	ND
Evolus	Valio, Finland	VPPIPP

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Many ACE inhibitory peptides have been isolated from various dairy proteins,^106,107 cheese whey,^108–110 and fermented milk products.^111–113 In addition, many ACE inhibitory peptides have been discovered from enzymatic hydrolysates of casein,^12,114–117 and whey protein.^109,118 These ACE inhibitors have moderate inhibitory potencies, usually within an IC₅₀ range of 100–500 μmol L⁻¹; however, there are exceptions such as the casokinins αs₁-casein f(23–27) and β-casein f(177–183) with IC₅₀ values less than 20 μmol L⁻¹.¹¹⁹

3.2. Renin-angiotensin and Kallikenin-kinin system

The renin angiotensin system (RAS) is a regulator of blood pressure (Fig. 1). The RAS begins with an inactive precursor angiotensinogen. Renin liberates angiotensin-I from angiotensinogen. Angiotensin-I-converting enzyme (ACE) a zinc metallopeptidase exopeptidase removes the C-terminal tripeptide His-Leu from angiotensin I resulting in the formation of angiotensin II. Angiotensin II is a vasoconstrictor, which increases peripheral vascular resistance. In addition, it is involved in the release of sodium retaining steroid, aldosterone which has a role in increasing blood pressure through increased Na⁺ and water retention. A combination of increased peripheral vascular resistance, Na⁺, and water retention result in increased blood pressure.¹²³

Fig. 1 Renin-angiotensin and Kallikenin-kinin system. ACE: angiotensin converting enzyme, ARB: angiotensin II receptor blockers, ACE I: angiotensin converting enzyme I, NO: nitric oxide.

ACE is a multifunctional enzyme and is also involved in the Kallikrein-kinin system. In the Kallikrein-kinin system, bradykinin is formed through an intermediatory kallidin by the action of kallikrein from kininogen.¹²⁴ In the Kallikrein-kinin system ACE removes the C-terminal dipeptide from the vasodilator nonapeptide bradykinin resulting in the formation of inactive fragments.¹²⁵ Bradykinin controls blood pressure by increasing prostaglandin and nitric oxide (NO) synthesis, which results in vasodilation, decreased peripheral vascular resistance and hence a decrease in blood pressure.¹²⁶

At present targets against the renin-angiotensin system are direct renin inhibitors, ACE inhibitors, angiotensin II receptor blockers (ARB) and aldosterone antagonists (for review see ref. 127). Direct renin inhibitors such as Aliskiren target the RAS by decreasing plasma renin activity and inhibiting the conversion of angiotensinogen to angiotensin I. ACE inhibitors target ACE and inhibit angiotensin II formation, examples include captopril and enalapril. Angiotensin II receptor blockers including losartan and candesartan, block the action of angiotensin II hence preventing angiotensin II from binding to angiotensin II receptors on blood vessels. As a result, blood vessels dilate and blood pressure is reduced. Aldosterone antagonists such as spironolactone act through competitive binding of receptors at the aldosterone-dependent sodium-potassium exchange site in the distal convoluted renal tubule. Spironolactone causes increased amounts of sodium and water to be excreted, while potassium is retained. In the kallikrein-kinin system ACE inhibitors are one current target for control. ACE inhibitors increase bradykinin by inhibiting its degradation to inactive fragments, hence through the action of prostaglandin and NO there is a decrease in blood pressure.¹²⁸

3.3. Structure of ACE

There are two isoforms of human ACE, somatic (sACE) and germinal/testicular (gACE) forms,¹²⁹ encoded by a single gene located on chromosome 17 at q23. Human sACE is a type-I membrane bound protein that consists of many domains, a 28-residue C-terminal cytosolic domain, a 22-residue hydrophobic transmembrane domain and a 1227-residue extracellular domain that is heavily glycosylated and further divided into a 612- residue N-terminal domain, linked by a 15-residue sequence to a 600-residue C-terminal domain.¹³⁰ The extracellular C-terminal domain and N-terminal domain contain a HEXXH sequence, which serves as the zinc binding ligand.¹³⁰ In sACE, the C-terminal domain is primarily involved in blood pressure regulation, while the N-terminal domain is involved in the control of hematopoietic stem cell differentiation and proliferation.^131–133 Human gACE corresponds to the C-terminal domain of sACE.^130,134

ACE inhibitors are thought to be competitive substrates for ACE. ACE-inhibitory peptides usually contain 2–12 amino acids, although active peptides with up to 27 amino acids have been identified.^12,135

While the structure activity relationship for food derived ACE inhibitors has not been fully established many things are known. ACE is responsible for the conversion of angiotensin I to the vasoconstrictor octapeptide angiotensin II. The C-domain catalytic site of the somatic form of ACE consists of three subsites, S′, S1′ and S2′. These accommodate the three hydrophobic C-terminal residues of the substrate angiotensin I, i.e., Phe-His-Leu.^52,136 Accordingly, the C-terminal of the inhibitory peptides is very important for binding to the ACE enzyme and for their inhibitory activity as ACE cleaves dipeptides from the C-terminal of the substrate peptides.^120,123 Evidence has shown that peptides with hydrophobic residues at each of the three C-terminal positions have the highest inhibitory activity^120,123 with Tryptophan (Trp), Tyrosine (Tyr), Phenalanine (Phe) and especially Proline (Pro) being most effective at the ultimate C-terminal of ACE-inhibitory peptides.^114,137

Otte et al.¹³⁸ showed that two peptides with the highest ACE-Inhibitory activity in the thermolytic β-casein hydrolysate had the same C terminal sequence as one of the two well-known antihypertensive peptides, Valine-Proline-Proline (Val-Pro-Pro) and Isoleucine-Proline-Proline (Ile-Pro-Pro).^139–142 The Ile-Pro-Pro sequence identified contains three C-terminal hydrophobic amino acid residues, in addition to a proline residue at the ultimate C-terminal, explaining their high ACE-inhibitory activity. A proline residue at the penultimate position is very unfavourable¹³⁷ whereas it has been found to be present at the antepenultimate position in several inhibitors.^143–145 Additonally, Otte et al.¹³⁸ identified a peptide with a lower ACE-inhibitory activity f(59–81), and having the C-terminal sequence Glutamine-Threonine-Proline (Glu-Thr-Pro). The hydrophobic nature of these residues and the ultimate position of Pro probably results in good binding to ACE and explains the inhibitory activity of this peptide.

Peptides with an aromatic residue at C-terminal position are better inhibitors of ACE than those with a basic one.¹³⁷ Nevertheless, numerous peptides resulting from specific tryptic activity have been identified and total chymotryptic hydrolysates do not appear to be more active than tryptic ones.¹⁴⁶

ACE inhibitor interactions may involve more than the three C-terminal residues. Residues from the N-terminal side of the cleaved bond could interact with critical subsites of ACE.¹⁴⁷ N-terminal extension can increase the inhibition potency as shown by Kohmura et al.¹⁴⁸ who found that IC₅₀ values decreased upon addition of amino acids to the N terminus (IC₅₀ values: Ile-Tyr-Pro (61 μmol), Leu-Ile-Tyr-Pro (10 μmol), Pro-Leu-Ile-Tyr-Pro (4.4 μmol), Leu-ProLeu-Pro (720 μmol), His-Leu-Pro-Leu-Pro (41 μmol) and Leu-His-Leu-Pro-Leu-Pro (2.9 μmol)). The ACE inhibitory mechanism is quite complex and relates to the assortment of the inhibitors and the absence of known amino acid sequence recognised by ACE.

Peptide conformation is also important when determining activity. The superiority of proline as the C-terminal residue is probably due to a rigid ring structure of this amino acid. It may lock the carboxyl group into a conformation favourable for interaction with the positively charged residue at the active site of the enzyme.¹⁴⁹ Research has shown that peptides containing the trans-Pro conformer were better substrates for ACE than those carrying cis-Pro.^150,151 Gómez-Ruiz et al.¹⁵² showed that the trans-Pro conformer found in the β-casein f(47–51) peptide DKIHP had higher ACE inhibitor activity compared to the cis-Pro form. The reason for this difference in activity is due to inhibitor structure and interaction with the active site. The change of trans-Pro to its cis-Pro results in the displacement of the CO group of the inhibitor, to the opposite side of the peptide chain, losing its original H-bonding with the enzyme. This leads to the loss of interactions with the active site and in turn to a decrease in binding to the enzyme and inhibitory activity.¹⁵²

Additionally, Otte et al.¹³⁸ demonstrated that all of the ACE-inhibitory peptides identified from the α-Lactoalbumin (α-la) contained the same sequence at the C-terminal (Proline-Glutamic Acid-Tryptophan; Pro-Glu-Trp). Glutamic acid is a negatively charged amino acid and its presence at the C-terminal would be expected to weaken the binding and thus lower the inhibitory potential of these peptides.¹²³ The combination of two hydrophobic and one negatively charged residues in the peptides from α-La would cause an incorrect orientation of the peptides into the active site with respect to the bond to be cleaved. Since the subsite S1′, normally accommodating the penultimate amino acid residue, is a large subsite containing a glutamic acid and an aspartic acid residue¹⁵³ it is unlikely that it can accommodate the glutamic acid residue in the α-La derived peptides. A wrong fit into the subsites of the active site of ACE and thus lack of cleavage combined with a high binding affinity could explain the high inhibitory activity of these peptides. Perhaps the C-terminal tryptophan in the peptides from α-La would be oriented into the S1′ subsite and proline into the S2′ subsite, which is a small hydrophobic subsite with tyrosine and two phenylanaine residues at the bottom.¹⁵³ This would leave glutamic acid out of the subsites, and probably bound to the Zn²⁺ atom which is essential for the catalytic action. This arrangement is similar to that seen for the ACE inhibitory drug lisinopril.^136,153

In summary, aliphatic (Valine, Isoleucine, Alanine), basic (Arginine) and aromatic residues (Tyrosine, Phenylalanine) are preferred in the penultimate positions, while tyrosine, phenylalanine and tryptophan, proline and aliphatic residues (Isoleucine, Alanine, Leucine and Methionine) are preferred in ultimate positions. The positive charge of Arganine at the C terminus has also been shown to contribute to the ACE-Inhibitory potential of several peptides.^144,154,155 Also, a C-terminal Lysine with a positive charge on the Guanidino or ε-amino group contributes substantially to the ACE-inhibitory potential.^119,137

3.4. Source of ACE-inhibitory peptide

Food products containing antihypertensive peptides are highly relevant in the treatment of mild and moderate hypertension since the risk of developing cardiovascular diseases is directly proportional to the level of the blood pressure. When blood pressure is reduced by 5 mmHg the risk is reduced by 16%.⁵¹ ACE-inhibitory peptides have been isolated from many fermented milks and dairy commercial products, through the action of lactic acid bacteria (LAB) or their proteinases.^{12,51,112,113,142,156–162} Strain selection is one of the main factors that influences their release in fermented foods^163,164 Among LAB, Lactobacillus helveticus has been shown to exhibit strong proteolytic activity in milk-based media. The proteolytic systems of Lb. helveticus are composed of a cell-envelope proteinase and more than 10 intracellular peptidases.^165,166 The cell-envelope proteinase is a key enzyme in the proteolytic system. It catalyzes the initial steps in the degradation of protein into different oligopeptides.¹⁶⁷ Intracellular peptideases including endopeptidases, aminopeptidases and X-prolyldipeptidyl aminopeptidase, may then act to form short chain peptides and amino acids.¹⁶⁸ Proteinases of lactic acid bacteria may hydrolyze more than 40% of the peptide bonds of αs₁- and β-caseins, producing oligopeptides of 4 to 40 amino acid residues.¹⁶⁹

Two of the most well known ACE inhibitory peptides Val-Pro-Pro and Ile-Pro-Pro were derived from casein in milk fermented with a starter containing Lb. helveticus¹⁴² and Saccharomyces cerevisiae.¹⁶¹ Takano,¹⁷⁰ reported that these peptides contribute to most of the inhibitory activity in Calpis sour milk. Minervini et al.¹⁶¹ identified additional peptide fractions in bovine milk hydrolysed by Lb. helveticus PR4 with ACE inhibitory activity. These included peptide fractions αs₁-casein f(24–47), αs₁-casein f(169–193) and β-casein f(58–76).

Additionally, other LAB including Lactocbacillus delbrueckii subsp. bulgaricus SS1, Lactococcus lactis subsp. cremoris FT4, Lactocbacillus acidophilus, Lactobacillus animalis, Lactobacillus casei, bifidobacteria, Lactobacillus jensenii, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus lactic ssp. lactis, Lactococcus raffinolactis, Streptococcus thermophilus, Leuconostoc mesenteroides ssp. cremoris and Enterococcus faecalis,^{111,161,168,171–176} have been shown to produce ACE-Inhibitory peptides. LAB have been shown to produce ACE-Inhibitory peptides in other milks such as buffalo (β-casein; f58–66), ovine (αs₁-casein; f1–6, αs₂-casein; f182–185, f186–188), and caprine (β-casein f58–65 and αs₂-casein f182–187).¹⁶¹

ACE inhibitors have also been reported in several cheeses including Cheddar,¹⁷⁷ Parmigiano-Reggiano¹⁷⁸ and Gouda,^111,179 Italico and Gorgonzola¹⁸⁰ and ripened or cooked cheeses such as Comté.^111,181 Many factors determine their presence however. The appearance and subsequent disappearance of ACE-inhibitory peptides is influenced by the degree of proteolysis and ripening. Anti-hypertensive peptides from αs-casein found in 6-month-old Parmigiano-Reggiano cheese disappeared following 15 months of ripening.¹⁸² Additionally, peptides with ACE-inhibitory activity found in “Festivo” a low-fat cheese after maturation for 13 weeks, decreased when proteolysis exceeded a certain level during storage for 20 weeks.¹⁸³ Nonetheless, the presence of ACE-inhibitory peptides in several ripened cheeses was reported, with the most potent inhibitory activities detected in short to medium-ripened cheeses such as Gouda.¹⁸⁴

Generally, ACE inhibitory peptides identified in cheese correspond to ACE-inhibitory peptide fractions from αs-casein and β-casein. Numerous peptides have been identified to date. ACE-inhibitory peptides FFVAPFPEVFGK and FFVAP, which correspond to αs₁-casein f(23–34), and αs₁-casein f(23–27) respectively,¹⁴³ were identified in Crescenza cheese.¹⁷⁵ DKIHP a fragment from β-casein was identified in Manchego cheese.¹⁸⁵ The antihypertensive peptides β-lactoglobulin f(147–148), lactoferrin f(288–289) and lactoferrin f(319–320) were isolated from mature and vintage Cheddar and Feta cheese, respectively.¹⁸⁶ Moreover, ACE inhibitors with the amino acid sequences LTLTDVE corresponding to β-casein f(125–131), YPQRDMPIQ corresponding to β-casein f(180–197), PGPIP corresponding to β-casein f(63–67) and PKHKEMPFPPKYPVEPFT corresponding to β-casein f(104–120)¹⁸⁷ were found in enzyme-modified cheese obtained by hydrolysis of pasteurized cheese homogenized with Neutrase® and an enzyme preparation from Lactobacillus casei. Two novel ACE-inhibiting peptides PPEIN (κ-casein; f156–160) and PLPLL (β-casein; f136–140), were identified in Yak (Bos grunniens) a milk casein derived from Qula, a kind of acid curd cheese after hydrolyses with alcalase.¹⁸⁸

Bioactive peptides can be generated not only during the manufacture of cheese, yogurt¹⁸⁶ and other dairy products, but also by in vitro hydrolysis of milk proteins using enzymes. Enzymes have been sourced from animals, plants, and microbes including cell-wall proteases from LAB.^{51,108,116,120,138,189,190} These enzymes include pepsin, trypsin, chymotrypsin, alcalase, proteinase K, actinase E and thermolysin.^{108,115,147,188,191–193}

Examples of such bioactives are the dodecapeptide (C12; αs₁-casein f23–34) enriched hydrolysate produced by tryptic hydrolysis of casein, and Biozate, a whey protein hydrolysate, for which the enzyme used was not reported.^51,120 Additionally, lactokinin (ALPMHIR) derived from a tryptic digest of the whey protein β-lacotglobulin has an ACE-inhibitory activity 100 times lower than captopril.¹⁹⁴ Otte et al.¹³⁸ found that thermolysin was a good enzyme for the release of ACE-inhibitory peptides from both caseins and whey proteins. In the study five bacterial and digestive enzymes were used for the hydrolysis of nine milk protein preparations. The hydrolysate made from purified α-lactalbumin by the action of thermolysin had the highest in vitro ACE inhibitory activity.

In the in vitro hydrolysis approach challenges arise however and it is important to choose protein and enzyme combinations that give rise to a high yield of the bioactive peptides, and perhaps to enrich for these. Abubakar et al.¹⁰⁸ observed that whey protein hydrolysed by thermolysin and proteinase K resulted in products with higher ACE inhibitory activity compared to actinase K.

Da Costa et al.¹⁹⁵ and Mullally et al.¹⁴⁶ showed that α-chymotrypsin, which preferentially cleaves the C-terminal phenylalanine, tyrosine and tryptophan residues,¹⁹⁶ produced hydrolysates with the highest ACE inhibitory activity, followed by Alcalase, which shows low specificity but preferentially cleaves C-terminal hydrophobic residues. In view of the specificity of these enzymes, it is evident that the structural changes in proteins caused by different treatments, aggregate formation by hydrophobic interactions, or the presence of a compact structure with hidden hydrophobic sites, have great impact on their activity. Da Costa et al.¹⁹⁵ also showed that hydrolysates obtained with Proteomix had the lowest activity which may be due to the combined action of the main enzymes present, trypsin and α-chymotrypsin, which possibly fail to release peptides with an appropriate structure for inhibiting ACE.

When considering ACE inhibitory activity in vitro it is important to consider whether this effect is also seen in vivo. In vitro studies have identified potent milk-derived ACE inhibitory peptides whose activity is not seen once they are tested in vivo.^108,111

Abubakar et al.¹⁰⁸ reported strong in vitro ACE inhibitory activity and in vivo antihypertensive activity for five short peptides less than six residues in length but no antihypertensive activity in rats by an undecapeptide ACE inhibitor (β-casein; f80–90). The authors concluded that the undecapeptide was probably degraded by digestive enzymes while the short peptides were more resistant to enzyme attack. Additionally, Saito et al.¹¹¹ showed that two nonapeptides (αs₁-casein; f1–9 and β-casein; f60–68) failed to show any antihypertensive activity following oral ingestion, although they had potent ACE inhibitory activity.

Contrarily, Maeno et al.¹¹⁶ reported that the heptapeptide (β-casein; f169–175; KVLPVQ), which had low in vitro ACE inhibitory activity (1000 μmol L⁻¹) had strong antihypertensive activity in rats. It was found that this peptide is hydrolysed by in vitro digestion with pancreatin and carboxypeptidase A. The hydrolysis treatment led to the release of the C terminal glutamine residue from the heptapeptide producing a hexapeptide with stronger antihypertensive and ACE inhibitory activities (5 μmol L⁻¹). In this specific case, gastrointestinal digestion was beneficial for the release of more potent molecules. Two β-casein peptide fragments of previously identified ACE-inhibitory peptide LLYQQPVLGPVRGPFPIIV,¹⁶⁰ have been reported in two Spanish commercially available fermented milks following simulated gastrointestinal digestion.¹⁰ Oligopeptide sequences containing encrypted bioactive peptides may also be activated and the potency of these oligopeptides increased, due to in vivo proteinase and peptidase activities.

These results suggest that some bioactive peptides may need protection against gastric or intestinal degradation to exert their physiological effects in vivo. Some work on the specificity of enzymes has been carried out to date. The ACE-inhibitory peptide ALPMHIR derived from tryptic digest of whey protein β-lactoglobulin, is fairly resistant to hydrolysis by pepsin and chymotrypsin.¹⁴⁶ Pepsin preferentially cleaves at Phe, Tyr and Leu at C- and N-terminal position of the cleaved bond.¹⁹⁷ Roufik et al.¹⁹⁸ studied the in vitro digestibility of three bioactive peptides from β-lactoglobulin (β-Lg) containing theoretical cleavage sites for pepsin and chymotrypsin. When incubated with pepsin, β-Lg f(142–148) remained intact whereas β-Lg f(15–20) and f(102–105) were weakly hydrolysed. β-Lg f(9–20), β-Lg f(92–105) and β-Lg f(139–148) contain, respectively, four, eight, and two theoretical cleavage sites for pepsin. The high resistance to pepsin attack of bioactive peptides in this case could be due to their short chains (4–7 amino acid residues). Additionally, pepsin is an endoprotease and the hydrolysis of scissile bonds in short peptides adjacent to N- or C-terminal residues would probably be hindered in part due to the ionized state of the end amino acid groups. In relation to chymotrypsin, only β-Lg f(142–148) was strongly hydrolysed. The degree of hydrolysis was found to be influenced by both the length of the peptide chain and the nature of the peptide. Furthermore, higher degrees of hydrolysis of a β-Lg f(102–105)/f(92–105) mixture were obtained with both enzymes than with each peptide treated separately.

In summary, these studies support the view that in vivo analysis is essential to validate the physiological effects of bioactive peptides. In addition, the theoretical specificity of proteases and in vitro digestion experiments cannot be used to safely predict the fate of bioactive peptides during the gastrointestinal process, since the degree of hydrolysis can vary depending on the peptide chain length, the nature of the peptide, and the presence of other peptides in the medium. Short chain peptides may be preserved during the gastrointestinal process whereas long-chain bioactive peptides require protection from gastrointestinal enzymes when orally administered in order to exert their physiological effects in the organism.

3.5. Preparation of peptides

When developed as food ingredients, the processing of these antihypertensive peptides is vital to their activity. In order to obtain peptides with high activity a number of factors need to be considered. Maximum biological activity and a limitation to the development of bitter flavour may be obtained by appropriate selection of enzymes for proteolysis. Trypsin may be used to improve protein digestibility and to decrease protein allergenicity.¹⁴⁶ Consideration of appropriate hydrolysis time is important^114,188 as is the use of high pressure and filter sizes. Knudsen et al.¹⁹⁹ showed that the use of high pressure to partially unfold whey proteins, before or during proteolysis, might increase the rate of proteolysis and alter the relative proportion of peptides formed. In addition, since the size of most ACE-inhibitory peptides is less than 3 kilodaltons (kDa), ultrafiltration with a cut-off of 3 or 5 kDa may be essential.^51,114 With a membrane of 1 kDa, some of the active peptides may be lost.¹⁹¹

Heat treatments will have negative effects on the bioavailability of whey peptides, so carefully monitoring during production is important. Moreover, it has been shown that adequate combination of heat treatment, protein source and type of enzyme is of great importance when obtaining high activity hydrolysates. Heat treatments and mechanical damage can drastically reduce the biological activity of food proteins,²⁰⁰ as they may alter the profile of peptides released during gastro-intestinal digestion, since enzymes could hydrolyze parts of the protein that were previously inaccessible to enzymatic action.²⁰¹ Da Costa et al.¹⁹⁵ found that the best ACE inhibitors were obtained from isolates previously treated at 65 °C, compared to isolates previously treated at 95 °C. This difference in activity can be explained by the structural changes induced in the WPI by different heat treatments. Heating at 65 °C possibly induces a partially unfolded conformation in whey proteins, known as molten globule state, characterized by exposition of hydrophobic clusters.²⁰² This temperature allows greater access of the enzymes to certain sites previously inaccessible to enzyme action, resulting in peptides different from untreated control, which has a more compact structure that may have hindered enzyme access to the same sites.¹⁹⁵ This fact may have special impact on the action of enzymes such as α-chymotrypsin and Alcalase that preferentially cleave peptide bonds involving hydrophobic amino acids. Additionally, when the temperature of the heat treatment exceeds 90 °C, the formation of aggregates occurs, mainly due to hydrophobic interaction, resulting in insoluble aggregates of high molecular mass.²⁰³

Strain selection is also an important factor to consider. Pihlanto et al.¹⁷¹ found that generally Lactococcus lactis strains did not produce ACE inhibition, whereas almost all of the Lactobacillus strains tested did. Additionally, it is accepted that strong proteolytic activity is required to produce ACE inhibitory and antihypertensive peptides. However, studies have shown that commercial starter strains, with low proteolytic activity, cannot generate ACE inhibitory peptides, although fermented products manufactured using theses strain can act as precursors of active peptides prior to digestion with gastrointestinal enzyme.^10,115

As previously mentioned another critical factor in bioactive peptide production is the adequate match of the enzyme and protein sources. In silico calculations using dedicated software may be of value in the initial selection of protein substrate and enzyme combinations.^154,204

In order to use protein hydrolysates as a functional ingredient, it is not sufficient for them to have a high ACE inhibitory potential; they must also show an in vivo hypotensive effect. Da Costa et al.¹⁹⁵ showed that the whey protein hydrolysate displaying the highest ACE-inhibitory activity, did not reduce arterial blood pressure when orally administered to spontaneous hypertensive rats, whereas hydrolysates showing lower activity, were the only ones to induce a significant arterial blood pressure reduction in the animals. Studies have shown that there are disagreements between peptide bioactivity as determined in vitro and in vivo since ACE inhibitory peptides may not show hypotensive effect or vice versa.^205–207 Even when a high ACE inhibitory activity is present, blood pressure reduction may not occur, either because the peptide or hydrolysate active fraction is not absorbed intact, or because it does not reach the target site or organ. Alterations in the activity of these peptides are attributed to gastric and pancreatic enzymes and brush border peptidases. To perform their biological activity, the peptides need to be absorbed efficiently and be resistant to degradation by serum peptidases in order to reach the target organ.⁵¹ Several strategies for increasing the bioavailability of ACE-inhibitory peptides have been described.^11,154 ACE-inhibitory peptides can be chemically modified to increase their stability by using techniques such as lipidization, glycosilation, cationization, microencapsulation, or nutrient transport which results in greater permeability, absorption or stability of the peptides.^208,209 These strategies have many disadvantages however such as a reduction of membrane permeability, receptor union decline in receptor affinity, greater joining of plasmatic proteins, or more rapid elimination. In addition, genetic engineering techniques allow the synthesis of specific ACE-inhibitory peptides, or the microbial production and delivery of bioactive peptides in situvia the enteric microflora as an alternative approach for delivery of bioactive peptides.¹¹

The use of bioinformatics could hasten the screening for high-potential sources of ACE inhibitory peptides. Databases containing bioactive peptides can be compared to amino acid sequences of proteins of interest.²⁰⁴ Furthermore, it is possible to predict the theoretical release of bioactive peptides using in silico digestion processes.

Finally, it is important to study the functional properties of hydrolysates. Peptides produced with different proteolytic enzymes can increase solubility and change gelling properties. Some enzymes can induce gelation following whey protein hydrolysis, whereas others impair gelling properties.^210–212 Ferreira et al.²¹³ found that gelation times and the strength of the final gels was highly dependent on the degree of hydrolysis. Smaller peptides liberated by hydrolysis contributed to the inability of whey protein hydrolysates to gel. Therefore, modulation of the gelling properties of whey proteins by enzymatic hydrolysis allows the incorporation of these ingredients, with ACE inhibitory peptides in liquid and semi solid foods with high protein content. Gels confer structure, texture and stability to food products; they also allow the retention of large quantities of water and other small molecules inside the food matrix.

3.6. Conclusion

Antihypertensive drugs play an important role in blood pressure regulation, however, for many patients targeted blood pressure is not achieved due to reasons such as insufficient medication, true drug resistance and non compliance.¹²⁷ Therefore there is a market for alternative solutions to the problem. Naturally occurring ACE inhibitory peptides derived from food proteins have been proven to be effective as in vivo antihypertensive agents.²¹⁴ Although these peptides have been found to be less active than the synthetic ACE inhibitors; they might play an important role as antihypertensive agents as they can be part of the daily diet in the form of functional foods and are perceived as natural and safe by the consumer.²¹⁵ Many factors need to be considered in the preparation of ACE inhibitory peptides however principles of the techniques are characterised but may need to be fine tuned.

4. Anti-thrombotic effects

4.1. General introduction

Bi-functional casein peptides have been reported to influence the cardiovascular system due to anti-thrombotic,²¹⁶ anti-hypertensive,^217,218 and anti-obesity effects.^219,220 Thrombosis is a pathological condition in which improper activity of the haemostatic mechanism results in clot or thrombus formation in arteries, veins or the chambers of the heart. There are three types of thrombi: the white thrombus, seen in arteries which consist mainly of platelets; the red thrombus, which is composed mainly of fibrin and red cells; and the mixed thrombus, which consists of both platelets and fibrin. Thrombus composition is influenced by the velocity of blood flow at the site of formation. White platelet-rich thrombi are found in high flow systems. The high shear rate in arteries prevents the accumulation of coagulation intermediates, however platelets have the capacity to form thrombi to the area of damage via vWF.^221,222 Arterial thrombosis is seen predominantly as myocardial infarction, cerebrovascular arterial thrombosis (both caused by thrombosis in an artery), peripheral arterial thrombosis and ischaemic stroke and is also associated with existing vessel wall disease, i.e., atherosclerosis. Red thrombi form in regions of stasis. On the venous side of circulation, thrombin can accumulate due to the slower flow rate and platelets play only a minor role. The most common forms of venous thrombosis are deep vein thrombosis of the leg and pulmonary embolism, with stasis and abnormalities in blood plasma (hypercoagulability) being the main causes.²²¹

Platelet attachment, spreading, and aggregation on extracellular matrices are central events in thrombus formation. These events can be regulated by a family of platelet adhesive glycoproteins such as fibronectin, vWF and fibrinogen. Fibronectin supports platelet attachment and spreading reactions,^223–225 while vWF is important in platelet attachment to and spreading on subendothelial matrices.^225–227 Fibrinogen has a bi-functional role, it participates in both platelet aggregation and in fibrin formation.^228–230 In platelet aggregation, two binding sites have been identified on the fibrinogen molecule: the C-terminal sequence of the fibrinogen γ-chain HHLGGAKQAGDV, f(400–411) and one (or two) tetrapeptdie sequence(s) (RGDS/F, f(572–575) or f(95–98)) of fibrinogen α-chain.²³¹

RGDF and the γ f(400–411) sequence are both accessible within the fibrinogen molecule and are both implicated in ligand binding and cell-cell interaction.²³² These peptides inhibit platelet aggregation and fibrinogen binding to ADP activated platelets.²³² Upon platelet activation, the integrin α_IIbβ₃ on the platelet surface interacts with fibrinogen and induces aggregation, a primary event in thrombus formation. Inhibition of platelet aggregation is therefore an essential requirement for effective antithrombotic therapy and vascular reocclusion prevention. A new and promising way of obtaining inhibition of platelet aggregation is hence, to impair the binding of fibrinogen to platelet membrane α_IIbβ₃ which forms the main molecular link between platelets in the aggregate.

There are several similarities between human and bovine fibrinogen, which supports the case for identifying natural agents from milk in the treatment of thrombsis. Functional and sequential homologies have been identified between the human fibrinogen γ-chain and bovine κ-casein or its glycomacropeptide. In fact Jolles et al.²³³ hypothesized that fibrinogen γ-chain and κ-casein may have evolved from a common ancestor during the past 450 million years. There are structural and functional similarities between the fibrinogen γ-chain C-terminal dodecapeptide f(400–411; ⁴⁰⁰HHLGGAKQAGDV⁴¹¹), which is involved in binding to platelet receptors, and various peptides from the f(106–116;¹⁰⁶MAIPPKKNQ-DK¹¹⁶) region of bovine κ-casein, which are termed casoplatelins.²³⁴ Similarities also exist between the fibrinogen α-chain tetrapeptide (RGDX) and human lactoferrin, KRDS (f39–42). Both have a high probability of initiating a β-turn and are highly hydrophobic.

The fibrinogen cleavage actions of the blood clotting enzyme thrombin and the κ-casein cleavage actions of the milk clotting enzyme chymosin also bear some similarities.^26,233 Both the blood and milk clotting processes involve limited proteolysis.²³⁴ Thrombin cleaves two R–G bonds to produce fibrin and fibrinopeptides,²³⁵ whereas chymosin cleaves a single unique F-M bond to form an insoluble part, para-κ-casein (N-terminal moiety of κ-casein), and a soluble fraction, the κ caseinoglycopeptide (CGP), also called κ-casein glycomacropeptide (GMP).^236,237 The latter is a hydrophilic glycopeptide of the C-terminal part of cow κ-casein f(106–169). It plays a major role in the stabilisation of casein micelles.²³⁸ Additionally, short soluble peptides (fibrinopeptides and caseinoglycopeptides) are released during both blood and milk coagulation processes. Although both of the released peptides are highly variable in sequence they maintain a net negative charge, and neither contains cysteine or tryptophan residues. The 1-amino groups of lysine appear to be involved in the polymerisation of both fibrin and casein, and calcium is important in both processes, accelerating the second phase of milk clotting and the aggregation of fibrin monomers. Prosthetic sugar groups do not play an important role in the clotting processes, however they retard the rate of chymosin or thrombin action.²³⁹

4.2. Peptides shown to affect platelet aggregation

Peptides from various sources have been shown to have an effect on platelet aggregation.²⁴⁰ Several peptides derived from κ-casein and lactoferrin have been shown to be inhibitors of platelet aggregation and to display anti-thrombotic activity.^241,242 The inhibitory effect of human lactoferrin on platelet aggregation was found to be 5 nmol.²⁴³ This concentration is not far from the physiological level in blood (1–3 nmol). It is thought that milk protein-derived anti-thrombotic peptides are absorbed into the bloodstream, as two peptides from human and bovine κ-caseinoglycopeptide respectively, have been identified in the plasma of five-day-old newborns following ingestion of a cow milk-based formula.²¹⁶ Peptides exhibiting anti-thrombotic activity have been observed in natural food such as in fermented milk products such as yoghurt (κ-casein; f113–116), and water soluble extract of two commercially available Spanish fermented milk drinks (κ-casein; f109–111, PPK).^10,186 Hence, a milk diet or nutritional supplement could have important antithrombotic properties.

Whole κ-casein has been shown to inhibit thrombin-induced platelet aggregation and thrombin-induced secretion of serotonin in vitro.²⁴⁴ In contrast para-κ-casein was shown to be inactive in all assay systems. Caseinoglycopeptide from bovine, caprine and ovine sources have been shown to inhibit platelet aggregation and hence thrombus formation.^{233,240,241,245} In fact, the enzymatic hydrolysate of sheep CGP with trypsin and chymotrypsin, was shown to be a better inhibitor of platelet aggregation induced by thrombin and collagen than the entire CGP, suggesting that the liberation of peptides may enhance the effect of inhibition.²⁴⁰ They found that the C-terminal part f(106–171) of sheep κ-casein, inhibited thrombin- and collagen-induced platelet aggregation in a dose-dependent manner. In 1991, Vu et al.²⁴⁶ cloned and directly expressed a functional human thrombin receptor. Thrombin binds to this receptor and proteolytically generates a neoaminoterminus by cleaving the receptor after the amino acid residue Arg-41. The newly generated N-terminal segment activates the receptor. This sequence SFFLRN, the tethered ligand, is followed by the YEPFWEDEEKNES region interacting with thrombin's anion-binding exosite. This proposed model by Vu et al.²⁴⁶ predicts that a peptide mimicking this receptor region would bind thrombin and inhibit its function. Sheep CGP and its three inhibitor thrombin-induced platelet aggregation peptides are highly acidic and perhaps mimick this thrombin receptor region. Additionally, Léonil and Mollé,²⁴² showed that the pentapeptide f(112–116) strongly inhibited ADP induced platelet aggregation, while the f(113–116) fragment was less active. The reason for the differences in activity relates to the lack of the N-terminal Lys residue.²⁴⁷

Qian et al.²⁴⁰ also studied enzymatic hydrolysates of CGP fractionated by reverse phase high performance liquid chromatography. The three peptides KDQDK f(112–116), TAQVTSTEV f(163–171) and QVTSTEV f(165–171) completely inhibited thrombin-induced platelet aggregation. The two active peptides TAQVTSTEV f(163–171) and QVTSTEV f(165–171) are situated at the C-terminal end of sheep CGP and κ-casein. No bovine peptide situated in this area of κ-casein has so far been described for inhibiting platelet aggregation. The other peptide KDQDK f(112–116) known as casoplatelin is similar to the corresponding bovine one KNQDK, which is also a platelet aggregation inhibitor.²⁴⁸ The difference between the residues Asp and Asn did not seem to affect the inhibition of platelet aggregation. The peptide KDQDK is a fragment of the main anti-thrombotic peptide isolated from bovine κ-casein corresponding to f(106–116, MAIPPKKNQDK). It is also structurally and functionally very similar to the C-terminal dodecapeptide of human fibrinogen γ-chain f(400–411) corresponding to the sequence HHLGGAKQAGDV. The amino acids, Ile108, Lys112 and Asp115 of κ-casein are in homologous positions as compared with γ-chain sequence of human fibrinogen. These three residues of κ-casein are important for inhibition due to the competition between the antithrombotic κ-casein peptide and the γ-chain for the platelet receptors.²⁴⁹ Jolles et al.²³³ showed that the natural or synthetic cow κ-casein undecapeptide (MAIPPKKNQDK) inhibited both aggregation and ‘I-fibrinogen’ binding to ADP-stimulated human washed platelets, confirming an inhibitory effect on a common pathway and probably on fibrinogen binding to α_IIbβ₃.²⁴¹ Furthermore, this inhibition was even greater than that induced by the dodecapeptide (IIHLGGAKQAGDV) from human fibrinogen γ-chain.

As described previously, similarities exist between the fibrinogen α-chain tetrapeptide with the amino acid sequence RGDX, and human lactoferrin with the amino acid sequence KRDS corresponding to lactoferrin f(39–42).²³⁹ Qian et al.²⁴⁰ compared the effects of human and sheep lactoferrin and some peptides on platelet function in vitro and found that sheep and human lactoferrins inhibited thrombin-induced platelet aggregation (IC₅₀ 5 and 4 μmol, respectively). Investigations into the mechanism of the platelet inhibition by human lactoferrin or by human lactoferrin peptides found that the inhibition mechanism of platelet aggregation by KRDS did not involve α_IIbβ₃.²⁵⁰ KRDS inhibition of thrombin-induced platelet aggregation has been associated with an inhibition of the release of the dense granule protein serotonin, but RGDS has no effect on the release.²⁴⁴ Synthetic peptides which contain the RGD sequence however, inhibit fibrinogen binding and platelet aggregation.²⁵¹ Moreover, RGDS has been found to induce detachment of endothelial cells in vitro and serious concerns exist relating to the toxicity of this sequence in vivo, although the sequence KRDS, found in human lactoferrin is not thought to have the potential detrimental effects of RGDX.²³⁹

Fiat et al.²⁶ showed that the tripeptide GLF, isolated from human α-lactalbumin f(51–53), specifically inhibits collagen-induced human platelet aggregation and serotonin release in a dose-dependent manner.²⁶ When combined with RGD it forms the chimeric peptide RGDGLF which plays the role of ligand sequence for integrin receptors.²⁵² Chabance et al.²⁵³ studied the effect of RGDS, GLF, RGDGLF, and the caseinoglycopeptide on platelet in human washed platelets. They showed that RGDS is essential for binding to many integrins, particularly GPIIb/IIIa, but not to GPIb. GLF was a specific inhibitor of collagen-induced human platelet aggregation and probably bound to a collagen platelet receptor. Of the three peptides studied, RGDS was the best inhibitor of platelet aggregate formation induced by collagen or thrombin. GLF was implicated only in collagen-induced platelet aggregation. The sequence RGD added on the N-terminal side of GLF removed this specificity, and the chimeric peptide RGDGLF was less inhibiting than GLF. Bovine caseinoglycopeptide inhibited bovine and human thrombin- and collagen-induced platelet aggregation and also inhibited the aggregation of human washed platelets by ristocetin in the presence of human vWF in a dose-dependent manner.

In summary, there is an abundance of studies on peptides derived from milk displaying anti-thrombotic effects. Some peptides are currently used therapeutically to treat or prevent thrombosis one such peptide is GMP.²⁴⁴ When given intravenously the recommended adult dose level is 15–30 mg kg⁻¹ twice daily for prophylaxis. For oral administration, an adult dose of 50–100 mg kg⁻¹ twice daily has been recommended.²⁴⁴ Since GMP has no toxic effect, the dose rates can be varied depending on the sensitivity and/or tolerance of the patient. There has been considerable research activity on the antithrombotic action of the RGDX sequences of fibrinogen α-chain. However, the RGDS sequence has been found to induce detachment of endothelial cells in vitro, therefore serious concerns exist about general toxicity from the injection of these sequences in vivo. The related sequence that occurs in human lactoferrin KRDS is not thought to have the potentially detrimental effects of RGDX. KRDS is also antithrombotic, but its mechanism of action and/or its binding site may be different from RGDX. Thus, although it was identified by sequence homology studies between fibrinogen and milk proteins, it has novel antithrombotic activity. All research on this peptide has been based on in vitro and/or in vivo intravenous treatment in animal models. It is therefore not known if KRDS would be effective when administered orally, so more work needs to be done on this peptide before further recommendations are be made.

These milk peptides could be considered as possible ingredients in functional foods; however, peptides must be stable to exert activity in vivo. Synthetic linear peptides based on the RGD template have relatively little activity and poor stability in plasma.²⁵⁴ Whereas cyclic RGD peptides are more resistant to enzymatic breakdown and have a higher potency.^254,255 RGD peptides can be modified in many ways to improve their activity. For example, integrelin (COR Therapeutics, South San Francisco), a cyclic peptide based on a Lys-Gly-Asp sequence rather than an RGD sequence, may be a more specific inhibitor of glycoprotein IIb/IIIa receptors than RGD-containing peptides.²⁵⁶ Replacement of the Arg group in the RGD sequence with an amidinoor benzamidino-containing group and the use of D-amino acids increase the resistance of these compounds to enzymatic degradation.²⁵⁴ Additionally engineering proteins by grafting RGD and KGD peptides on to micro-proteins EETII (a trypsin inhibitor) and the melanorortin receptor binding domain of the human agouti-related protein AGRP can result in increased inhibition of platelet aggregation through an enhanced inhibition of binding of fibrinogen to its α_IIbβ₃ receptor.²⁵⁷ Hence by considering structural scaffold and neighbouring amino acids during peptide manufacturing, peptides with increased activity may be obtained.²⁵⁸

4.3. Conclusion

There is evidence of products containing natural compounds on the market. In 2009, Provexis Natural Products Limited submitted an application to EFSA for a food constituent that ‘helps maintain normal platelet aggregation’.²⁶¹ The food constituent in question was water soluble tomato concentrate (WSTC) in two variant forms WSTC I and its low-sugar derivative WSTC II. Both constituents are derived from ripe tomatoes, Lycopersicon esculentum and intended for use in fruit juices, fruit flavoured drinks and yogurt drinks or, powdered single-serve sachets, tablets, and capsules.²⁶²

Substantiation of the claim was based on eight human^263–270 and seven non-human studies.^{268,271–275} A cause and effect relationship was established between the consumption of WSTC and the reduction in platelet aggregation. The health claim on the product ‘water-soluble tomato concentrate (WSTC) I and II helps maintain normal platelet aggregation, which contributes to healthy blood flow’²⁷⁶ was approved by EFSA. Additionally, in order to achieve the claimed affect, 3 g WSTC I or 150 mg WSTC II in up to 250 ml of either fruit juices, flavoured drinks or yogurt drinks (unless heavily pasteurised) or, 3 g WSTC I or 150 mg WSTC II as powder, tablets or easily dissolved capsules with at least 200 ml of liquid should be consumed daily, with a target population of adults between 35 and 70 years of age.

Hence, there is potential to develop functional foods with anti-platelet agents as ingredients, as shown by the WSTC extract above. Therapeutic drugs currently play an important role in the regulation of the disease^259,260 however; many factors need to be taken into account when assessing the benefits and risks of these anti-platelet treatments. Bleeding is a major risk, as it is an independent predictor of poor prognosis in acute coronary syndrome patients. However, new reversible P2Y₁₂ antagonists offer the potential to discontinue anti-platelet therapy closer to invasive procedures compared with the thienopyridines, thus potentially reducing both procedure-related bleeding rates and duration of exposure to athero-thrombotic risk prior to procedures.²⁵⁹ Resistance to anti-platelet agents is another topic of concern however; dosage adjustments and the use of newer anti-platelet agents with less variability in patient response may help overcome this resistance.²⁷⁷ Overall, the future of anti-platelet drugs warrants large-scale Phase 3 trials that would provide important information on whether the strategies of achieving higher levels of P2Y₁₂ inhibition and using reversible inhibitors can improve anti-platelet therapy.²⁵⁹ Therefore, identification of further compounds from natural sources including milk could be beneficial and provide possible alternatives to current strategies used to treat the disease.

5. General conclusion

The possibility of designing new dairy products with health-promoting benefits looks promising and offers a perspective for consumers and producers. CVD is a major health problem in both the developed and developing world and identification of any compound that would alter the CV risk in any way would be beneficial. Milk and hydrolysates of milk are an important source of these compounds. Humans are continuously exposed to milk protein hydrolysates without experiencing undesirable effects. The Life Science Research Office of the Federation of American Societies for Experimental Biology (FASEB) concluded that there are no suspicious hazards related to protein hydrolysates as food ingredients for public health aspects.^278–280 Furthermore, the FDA lists protein hydrolysates as ‘generally recognized as safe’ (GRAS) under 21CFR 102.22.²⁸¹ Protein hydrolysates are described as a variable mixture of polypeptides, oligopeptides and amino acids derived from among others casein. Casein hydrolysates have been on the European market as an integral part of fermented milk products and as a source of protein in hypo-allergenic infant formula for more than a decade.²⁸² Hence there is potential for the use of milk peptides as ingredients in functional foods with an aim in reducing CV risk.

A review of the literature available to date on ACE inhibitors shows that they are important functional food ingredients. There certainly is a market for them, although there are many restrictions that must be over come in order to introduce them into foods. Recurring issues include the stability of the peptides when ingested, in addition to, a loss in biological activity when studied in vivo. It is essential that effects be seen in vivo, but very few milk peptides displaying activity in vitro retain their activity when tested in vivo.^283,284 Specific milk protein hydrolysates or fermented dairy products have shown moderate or significant reduction of blood pressure in human studies however.^285–301

It is now being considered a flaw in most research strategies that peptides are first screened in vitro against potential targets and then in vivo to confirm efficacy. Foltz et al.³⁰² believes that it is only valid to propose efficacy when the peptide exhibits reasonable proteolytic stability and physiologically relevant absorption, distribution, metabolism and excretion profiles.

In relation to anti-thrombotic effects several peptides have been shown to be inhibitors of platelet aggregation. EFSA have published one claim to date in the area of platelet aggregation. With the identification of a novel compounds and the correct substantiation of claims additional products may be introduced onto the market as an additional strategy to the treatment of the disease.

Important future research topics should focus on the bioavailability and safety of bioactive peptides. Furthermore, molecular studies are needed to assess the mechanisms by which bioactive peptides exert their activities. It may be is necessary to employ new nutrigenomic techniques, for example proteomics and metabolomics.³⁰³ By developing such novel facilities it will be possible to study the impact of proteins and peptides on the expression of genes and hence optimize the nutritional and health effects of these compounds. Also, bio-safety aspects of novel peptides generated by new technologies have to be considered before entering into proper product formulation and marketing efforts. Further challenges with functional food products containing novel bioactive peptides are associated with regulatory issues if any health-related claims will be attached to the products. Currently, health claim regulations seem to vary greatly in different countries but regional and continental harmonization processes are in progress.

6. References

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3. M. Gobbetti, L. Stepaniak, M. De Angelis, A. Corsetti and R. Di Cagno, Crit. Rev. Food Sci. Nutr., 2002, 42, 223–239.
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