Casein peptide release and passage to the blood in humans during digestion of milk or yogurt.

In adult humans, after milk or yogurt ingestion, many peptides derived from alpha s1-, beta- or kappa-caseins were detected in stomach, including the kappa-caseinoglycopeptide, an inhibitor of platelet aggregation. Smaller peptides derived from casein and lactoferrin were recovered from duodenum. Two long peptides, the kappa-caseinoglycopeptide and the N-terminal peptide of alpha s1-casein, were absorbed and detected in plasma. These results support the concept that food-born peptides could have physiological activities in man.

Measurement of platelet aggregation peptide inhibitors by ultrasonic interferometry.

Several peptide inhibitors of thrombin- or collagen-induced platelet aggregation and of the interaction between glycoprotein Ib and von Willebrand factor were studied by a new method--ultrasonic interferometry (Echo Cell). Inhibition of aggregate formation in a concentration-dependent manner was observed. The sensitivity of the method was 3 to 40 times higher than that of classical turbidimetry.

Binding of the bovine caseinoglycopeptide to the platelet membrane glycoprotein GPIb alpha.

The bovine caseinoglycopeptide (residues 106-169), the C-terminal part of kappa-casein, inhibited the von Willebrand factor-dependent platelet aggregation in a dose-dependent manner. An affinity matrix made of the caseinoglycopeptide selectively bound the platelet membrane glycoprotein GPIb alpha which contains the von Willebrand factor binding site. The amino acid residues of GPIb alpha participating in the caseinoglycopeptide binding were located after residue Glu 90.

Measurement of human platelet microaggregates by a new method: ultrasonic interferometry.

We have adapted the ultrasonic interferometry technique (Echo-Cell), which was initially designed to study red blood cell aggregation and agglutination, to the detection of human platelet microaggregates. The experimental parameter chosen was the slope of the signal over the first 5 minutes of sedimentation. We compared our new method with the conventional aggregometry for the measurement of aggregates after thrombin-, collagen-, and epinephrine-induced platelet activation. Under these conditions we demonstrated the particular sensibility of the present method in detecting small platelet aggregates induced in the first phase of aggregation and formed by low concentrations of agonists. Furthermore, as an illustration of this method, we showed an inhibition of the formation of thrombin-induced platelet aggregates in a concentration-dependent manner by the well known antagonist arginine-glycine-aspartic acid-serine with a median inhibitory concentration of 0.4 micromol/L, which is 30 times lower than the median inhibitory concentration found by aggregometry.

Effect of kappa-casein split peptides on platelet aggregation and on thrombus formation in the guinea-pig.

An undecapeptide (residues 106-116 of cow kappa-casein) is known to inhibit human platelet aggregation and fibrinogen binding through inhibition of the interaction between the fibrinogen gamma-chain C-terminus and alphaIIbbeta3. This was due to structural homologies with the fibrinogen gamma-chain C-terminal dodecapeptide. We have therefore compared in this work the in vitro anti-aggregating activity of kappa-casein split peptides and their in vivo potential antithrombotic activity in a model of arterial thrombosis triggered by laser-induced intimal injury in the guinea-pig. Caseinoglycopeptide (residues 106-169), the undecapeptide (residues 106-116) and the pentapeptide KNQDK (residues 112-116) from cow kappa-casein, were anti-aggregating peptides and exerted a significant antithrombotic activity in the guinea-pig. Caseinoglycopeptides from three species (cow, ewe and human) were also antithrombotic and the most potent being the human one. The antithrombotic activity was achieved in vivo for doses less than the one suspected from in vitro data and for which, ex vivo platelet aggregation was not decreased. In conclusion, the relative involvement of the fibrinogen gamma-chain C-terminal dodecapeptide could be much more important in in vivo thrombosis process than in in vitro platelet aggregation. Its specificity and activity in vivo unveiled an interesting potential way for inhibition of arterial thrombosis if alternative molecular presentation (i.e. peptidomimetics) and alternative route (i.e. per os) can be developed.

Presence of casein immunoreactive epitopes in molluscs, fish and frog.

The presence of a group of peptides derived from milk proteins (caseins) was examined by immunocytochemistry in various tissues from invertebrates and lower vertebrates. Phagocytic hemocytes from different species of molluscs, and cells located in the intestine wall or in related glands of invertebrates and lower vertebrates showed immunoreactivity to antibodies to whole casein and related fragments. Several functional tests (cell migration, inhibition test, phagocytosis) using these peptides were performed on the mollusc hemocytes. Only ovine caseinoglycopeptide was able to increase the phagocytic activity of the hemocytes towards bacteria.

Sheep kappa-casein peptides inhibit platelet aggregation.

The C-terminal part (residues 106-171) of sheep kappa-casein, called caseinoglycopeptide (CGP), inhibits thrombin- and collagen-induced platelet aggregation in a dose-dependent manner (mean inhibitory concentration (IC50) 215 microM and 100 microM, respectively). An enzymatic hydrolysate of CGP was fractionated by reverse phase high performance liquid chromatography: three peptides KDQDK (residues 112-116), TAQVTSTEV (residues 163-171) and QVTSTEV (residues 165-171) completely inhibited thrombin-induced platelet aggregation. CGP at a concentration near its IC50 had a very long life when incubated in human or guinea-pig plasma. An ex vivo experiment showed that 17% of CGP was found 60 min after its i.v. bolus injection in guinea-pig. By hydrophobic cluster analysis, human fibrinogen and sheep kappa-casein peptides, inhibitors of platelet aggregation, were compared and we observed similarities for their C-terminal parts and for their short peptides (RGDF and KDQDK).

Characterization of an antithrombotic peptide from kappa-casein in newborn plasma after milk ingestion.

Bovine and human kappa-caseinoglycopeptides, two antithrombotic peptides derived from the corresponding kappa-caseins, were detected in physiologically active concentrations in the plasma of 5-d-old newborn infants after ingestion of cow's-milk-based formula or human milk respectively. It is suggested that these two bioactive peptides are released from milk proteins during digestion.

Isolation and characterization of sheep lactoferrin, an inhibitor of platelet aggregation and comparison with human lactoferrin.

Highly purified sheep lactoferrin was isolated from ovine whey in a single chromatographic step (FPLC): it was characterized by electrophoresis, N-terminal sequence determination and compared with lactoferrins from other species. Sheep and human lactoferrins inhibited thrombin-induced platelet aggregation (median inhibitory concentration: IC50 5 and 4 microM, respectively). Pepsin hydrolysates of human and sheep lactoferrins were fractionated by reverse-phase high-performance liquid chromatography and only one peak was an inhibitor of platelet aggregation. The sheep or human lactoferrin binding to platelets was studied.

A conformational study of Lys-Arg-Asp-Ser and analogs, a series of potent antithrombotic peptides. An approach based on simulated annealing and 1H NMR.

Simulated annealing techniques were used to explore the conformational space of the potent antithrombotic peptide L.Lys-L.Arg-L.Asp-L.Ser (KRDS) and of two analogs: D.Lys-L.Arg-L.Asp-L.Ser (KDRDS), which is inactive, and L.Lys-L.Arg-L.Glu-L.Glu (KREE), which exhibits a strong biological activity. For each peptide, a set of initial conformations was generated and submitted to simulated annealing, including a heating to 1000 K followed by a cooling to 300 K. 200 resulting conformations of each compound were analyzed and classified according to the network of electrostatic interactions involving charged side chains and charged C- and N-terminal groups. A reduced number of conformational classes was obtained and conformations corresponding to predominant classes were found to be in qualitative agreement with structural parameters deduced from 1H NMR spectra. A comparison between the classes of the active and non active peptide was achieved. Some conformations were found to be specific of active peptides.

Biologically active peptides from milk proteins with emphasis on two examples concerning antithrombotic and immunomodulating activities.

The present paper is devoted to the study of short peptides derived from milk proteins with physiological activities. Some of them behaved as opioids, enzyme inhibitors that convert angiotensin I, peptides that enhance calcium absorption, antiaggregating and antithrombotic peptides, and immunomodulating peptides. Some possessed several physiological properties, such as the C-terminal part of bovine alpha s1-casein. A strategic zone, containing immunostimulating and opioid peptides, could be located in cow and human beta-caseins. Few of these peptides or precursor peptides have so far been characterized in vivo in blood or brain after ingestion of milk. If, in the future, some of the active peptides cannot be characterized in vivo, they can all nevertheless be synthesized and used either as food additives or in pharmacology.

KRDS, a new peptide derived from human lactotransferrin, inhibits platelet aggregation and release reaction.

KRDS (Lys-Arg-Asp-Ser), a tetrapeptide from human lactotransferrin, was tested in vitro on human platelet function, and its effects were compared to those of RGDS, a tetrapeptide from human fibrinogen. Both peptides had a high probability of initiating a beta-turn and were highly hydrophilic. KRDS inhibited ADP-induced platelet aggregation [median inhibitory concentration (IC50) 350 microM] and fibrinogen binding (IC50 360 microM) to a lesser extent than RGDS (IC50 75 microM and 20 microM, respectively). Different from RGDS, thrombin-induced serotonin release was inhibited by KRDS (750 microM) on normal platelets (55 +/- 10%) and type I Glanzmann's thrombasthenia platelets (43% +/- 1). However, KRDS had no effect on cytoplasmic Ca2+ mobilization, inositol phospholipid metabolism or protein phosphorylation (myosin light chain P20 and P43). In contrast to RGDS, KRDS does not inhibit the binding of monoclonal antibody PAC-1 to activated platelets. KRDS and RGDS inhibited 4 beta-phorbol-12-myristate-13-acetate (PMA)-induced aggregation and fibrinogen binding, while proteins were normally phosphorylated. Thus, the tetrapeptide KRDS is (a) an inhibitor of serotonin release by a mechanism independent of protein phosphorylation and (b) an inhibitor of fibrinogen binding and, hence, aggregation by a mechanism that may not necessarily involve its direct binding to the glycoprotein IIb-IIIa-complex.

The antithrombotic effect of KRDS, a lactotransferrin peptide, compared with RGDS.

Due to the functional homologies between milk and plasma coagulations and the molecular homologies between a plasma protein (human gamma-chain fibrinogen) and a milk protein (Kappa-casein), we thought to characterize a RGDS sequence in milk proteins. A KRDS sequence theoretically analogous to RGDS was found in human lactotransferrin. This study compares in 2 species (rat and guinea-pig) in vitro (on platelet aggregation) and in vivo (in an experimental model of arteriolar thrombosis), relative to RGDS, the effects of KRDS and of a modified sequence KRDR. In vitro, in the rat, while RGDS and KRDR did not affect significantly the platelet aggregation induced by ADP, KRDS had a significant inhibitory effect; on the guinea-pig platelets, KRDS and RGDS had an inhibitory effect of similar magnitude, and KRDR, a minimal effect. In vivo and in the two species, KRDS and RGDS had an antithrombotic activity much more pronounced than KRDR. Additionally, when KRDS and RGDS were studied together in vitro, no more than additive effect could be noticed in the guinea-pig. But in vivo, in the guinea-pig and even more pronounced in the rat, a potentiating activity was evidenced. These results indicate that KRDS, a peptide present in the human lactotransferrin sequence, has, in vitro, an anti-platelet activity and, in vivo, an anti-thrombotic activity. The mechanism of action is probably different from that of RGDS and is specific of the sequence since a similar KRDR sequence loses most of these actions.

Caseins of various origins and biologically active casein peptides and oligosaccharides: structural and physiological aspects.

The first part of the present review is focused on structural aspects concerning the so far studied casein fractions of various origins: they are compared to the four classical major bovine caseins (alpha s1-, alpha s2-, beta- and kappa). The calcium-sensitive casein fractions are always phosphorylated whereas kappa-caseins are glycosylated. The study of the casein genes showed that the calcium-sensitive caseins diverged from a common ancestral gene and during the evolution, intergenic and intragenic duplications occurred. The considerable conservation of the phosphorylation sites emphasizes the importance of phosphorylated residues for the function of caseins, i.e. the formation of micelles and the binding of Ca2+. In kappa-caseins all the prosthetic sugar groups are linked by O-glycosidic linkages: their number varies from 0 to 5 in bovine kappa-casein and up to 10 in human kappa-casein. The structures of the known kappa-casein carbohydrate moieties are described. Finally the milk clotting process (interaction kappa-casein/chymosin) is compared to the blood clotting process (interaction fibrinogen/thrombin): a large number of similarities could be noted between both clotting phenomena. The second part of the review is devoted to the study of short casein peptides endowed with various biological activities. Some of them behaved as immunomodulators or casomorphins or angiotensin I converting enzyme inhibitors; others demonstrated an effect on platelet functions. A 'strategic zone' containing immunostimulating and opioid peptides could be located in cow and human beta-caseins. Furthermore bitter peptides, emulsifying peptides, calcium absorption enhancing peptides, chymosin-inhibiting peptides, have also been described and several further properties have been attributed to the kappa-caseinoglycopeptide; two tetrasaccharides isolated from the latter possess blood group activities. In conclusion caseins, the main milk proteins, should not only be considered as a nutriment but as a possible source of biologically active components. If, in the future, some of the discussed active peptides cannot be characterized in vivo, they can all, nevertheless, be synthesized and used either as food additives or in pharmacology.

Biologically active peptides of casein and lactotransferrin implicated in platelet function.

Casein and other milk proteins in maternal colostrum and milk, the earliest food of the newborn, should not only be considered as a nutritional supply but also as a source of biologically active peptides. Some of them isolated from casein and lactotransferrin were active on platelet function. They inhibited both aggregation of ADP-treated platelets and binding of [125I]fibrinogen to ADP-treated platelets. Their behaviour was compared to that of fibrinogen peptides possessing similar effects: once more similarities between the milk and blood-clotting phenomena could be observed.

KRDS--a tetrapeptide derived from lactotransferrin--inhibits binding of monoclonal antibody against glycoprotein IIb-IIIa on ADP-stimulated platelets and megakaryocytes.

Short peptides isolated from fibrinogen and K-casein have been shown to inhibit platelet aggregation and fibrinogen binding to stimulated platelets. We studied the effects of synthetic peptides occurring in milk proteins (bovine K-casein, KNQDK, and human lactotransferrin, KRDS) and in fibrinogen (RGDS and L10) on subsequent binding of monoclonal antibodies (MoAb) against the glycoprotein (GP) IIb-IIIa complex (AP2 and P2) on adenosine diphosphate (ADP)-stimulated and unstimulated human platelets and megakaryocytes (MKs) by using an immunoperoxidase method to visualize antibody binding. Only KRDS (900 mumol/L) inhibited the binding of AP2 and P2 on ADP (5 mumol/L)-stimulated platelets, but not on unstimulated platelets. However, the binding of P2 was considerably more inhibited than that of AP2 as judged by immunoperoxidase intensity. Radiolabeled AP2 binding was inhibited by 30% with KRDS on ADP-stimulated platelets as compared with platelets incubated in the absence of ADP. KRDS did not inhibit the binding of MoAbs against GP IIIa (SZ 21), GP IIb (SZ 22), and GP Ib (SZ 2) on ADP-stimulated human platelets. Inhibition of P2 binding by KRDS was also observed in a section of MKs isolated from human bone marrow and stimulated by 15 or 20 micron ADP. A lower concentration of ADP (5 or 10 mumol/L) failed to produce any inhibition of binding. This indicates that MKs may not be equally responsive to agonists as platelets. Moreover, P2 binding inhibition was observed in a larger (P less than .001) percentage of mature MKs (29%) as compared with younger, maturing MKs (11%). The observations suggested that a functional ability possessed by platelets, namely, agonist-induced exposure of the site of interaction of KRDS, may occur at a late stage of MK development.

Structural variability of the neutral carbohydrate moiety of cow colostrum kappa-casein as a function of time after parturition. Identification of a tetrasaccharide with blood group I specificity.

New neutral oligosaccharides from cow colostrum kappa-casein were identified and characterized by 500-MHz 1H-NMR spectroscopy. Their structures are Gal beta(1----3)GalNAc-ol, Gal beta(1----3)[GlcNAc beta(1----6)]GalNAc-ol, Gal beta(1----3)[Gal beta(1----4)GlcNAc beta(1----6)]GalNAc-ol, Gal beta(1----3)[Fuc alpha(1----3)[Gal beta(1----4)]GlcNAc beta(1----6)]GalNAc-ol. The tetrasaccharide and the cow colostrum kappa-caseinoglycopeptide which contains this oligosaccharide inhibit the hemagglutination of blood group I human erythrocytes. In cow mature milk only the disaccharide is characterized. The variability of these neutral oligosaccharides in cow kappa-casein as a function of time after calving is studied.

Analogy between fibrinogen and casein. Effect of an undecapeptide isolated from kappa-casein on platelet function.

A large number of similarities have previously been noted between the blood and milk clotting phenomena [Jollès, P. (1975) Mol. Cell. Biochem. 7, 73-85; Jollès, P. & Henschen, A. (1982) Trends Biochem. Sci. 7, 325-328]: some analogous features have also been found between fibrinogen and kappa-casein. In this connection, the effect of a natural and a synthetic peptide derived from kappa-casein on platelet function was studied: the undecapeptide Met-Ala-Ile-Pro-Pro-Lys-Lys-Asn-Gln-Asp-Lys (residues 106----116 of cow kappa-casein) inhibited both aggregation of ADP-treated platelets and binding of 125I-fibrinogen to ADP-treated platelets: its behaviour was similar to that of the structurally related C-terminal dodecapeptide of human fibrinogen gamma-chain.

Isolation and structural characterization of the smaller-size oligosaccharides from desialylated human kappa-casein. Establishment of a novel type of core for a mucin-type carbohydrate chain.

Alkaline borohydride reductive cleavage (beta-elimination) of desialylated human kappa-caseinoglycopeptide resulted in the release of a series of oligosaccharides. The smaller-size compounds among them were purified to virtual homogeneity by gel filtration followed by high-performance liquid chromatography. The structures of 9 oligosaccharides were determined by 1H-NMR spectroscopy in conjunction with sugar analysis. The tetrasaccharide Gal beta(1----3)[Gal beta(1----4)GlcNAc beta(1----6)] GalNAc-ol and various partial structures thereof were characterized. Notably, the disaccharide GlcNAc beta(1----6)GalNAc-ol and the trisaccharide Gal beta(1----4)GlcNAc beta(1----6)GalNAc-ol were identified; they represent a novel type of core structure for mucin-type carbohydrate chains, namely a peptide-linked GalNAc that is mono-substituted at C-6. In addition, some oligosaccharides ending in GlcNAc-ol could be characterized. Their possible origin is discussed.