Microenvironmental Changes in Platelet Membranes Induced by the Interaction of Fibrinogen‐Derived Peptide Ligands with Platelet Integrins

Abstract

A few studies have confirmed the influence of peptides containing either the RGD or dodecapeptide H-I 2-V (HHLGGAKQAGDV) sequence on cell membrane structure and function. In order to consider previous findings and to explore microenvironmental changes associated with the interaction of these two fibrinogen-derived peptides with platelet membranes, we employed fluorescence quenching and electron paramagnetic resonance techniques to monitor the possible alterations in platelet membrane dynamics induced by RGDS and H-12-V. The interaction of RGDS with platelet membranes resulted in reduced values of the h+,/h,, parameter in both 5-doxylstearic acid and 16-doxylstearic acid spectra indicating a significant rigidification of the membrane lipid bilayer. Otherwise, the fibrinogen-derived peptide that contained the y chain C-terminal sequence H-12-V had a fluidizing effect on the platelet membrane lipid bilayer. The labelling of platelet membranes with 1 -anilino-8-naphthalenesulphonate (ANS) enabled us to estimate the energy transfer efficiency and the apparent interchromophore distance between membrane protein tryptophan and ANS embedded into the membrane lipid bilayer. As RGDS interacts with platelet membrane this distance decreases, resulting in the relevant increase of energy transfer efficiency. The opposite alterations were recorded upon interaction of platelet membranes with H-I 2-V. Furthermore, a small shift towards longer wavelengths, which accompanies the spectra of ANS in control platelet membranes, vanishes during the interaction with the peptide H-12-V. This observation can be accounted for by a decrease in the polarity of the ANS environment, and may suggest an enhanced contact of the membrane tryptophan with phospholipid fatty acids. Thus, the data indicate that after the action of H-I 2-V on platelet membrane receptors, the membrane tryptophan residues become exposed to the external environment and the quenchable fraction of membrane tryptophan becomes smaller. The increase (a) in the relative rotational correlation time (z,) of 4-(ethoxyfluorophosphinyloxy)-2,2,6,6-tetramethylpiperidine-I -oxyl (ethoxyfluorophosphinyloxy-TEMPO) and (b) in the hJh, ratio in the spectra of 4-maleimido-2,2,6,6-tctramethylpiperidine-l -oxyl (maleimido-TEMPO) indicate that under these conditions there is an effective immobilization of some domains located on the hydrated surface of membrane proteins and mobilization of those domains buried inside the membrane protein molecules. The interaction of RGDS with platelet membrane integrins resulted in contrary effects, as compared to H-12-V. In conclusion, our spectroscopic data indicate that these two fibrinogen-derived peptides induce opposite effects in the dynamics of platelet membrane components. hexapeptide Gly-Arg-Gly-Glu-Ser-Pro; H-12-V, dodecapeptidc His-HisLeu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-~~1; h , ,/h,,, the ratio of lowfield to mid-field line heights in 5/16-DOXYL-Ste spectra; 12, and /I,, low-field line heights corresponding to weakly and strongly immobilized male...

Peptide Ligands Can Bind to Distinct Sites in Integrin αIIbβ3 and Elicit Different Functional Responses

“…Interaction of cRGD with platelets reduced the h ϩ1 /h 0 ratio, indicating a significant rigidification of membrane lipid bilayer, whereas cHarGD increased the h ϩ1 /h 0 ratio indicating an increase in membrane fluidity. This differential effect is consistent with previous studies in which linear RGD and ␥-chain peptides were used in ESR studies (31) and supports the hypothesis that cRGD and cHarGD are the respective high affinity analogs of the linear peptides. With the differential transmission of signals from sites A and B, which are likely to reside in the amino-terminal aspects of ␣ IIb ␤ 3 , to the membrane proximal LIBS epitopes and then further into the membrane bilayer, it is not unreasonable to suggest that these peptides also would induce different outside-in signaling events.…”

“…However, the changes were induced by very low concentrations of the cyclic peptides, substantially lower than their K d value for ␣ IIb ␤ 3 . This was also observed with the linear ligand peptides (31) and indicates that only limited occupancy of ␣ IIb ␤ 3 is sufficient to cause changes in membrane fluidity as detectable by ESR. …”

Merocyanine 540 as a fluorescent probe of altered membrane phospholipid asymmetry in activated whole blood platelets

“…Spin‐labeled PL mixtures containing increasing proportions of PS were used for the preparation of liposome suspensions, as described above. ESR measurements were performed in a Brüker SX‐300E spectrometer as described by Watala et al (24).…”

Blood Platelet Reactivity and its Pharmacological Modulation in (People with) Diabetes Mellitus

“…Associated with such changes in membrane lipid composition, blood platelets have been demonstrated to become more sensitive to aggregation induced by adrenalin or ADP [58][59][60][61][62]. On the other hand, there are reports showing that the interaction of protein and peptide ligands with platelet membrane receptors leads to the changes in the organisation of membrane components, and such displacements in membrane protein projection could be mediated by the alterations of membrane lipid fluidity [43,54,59,[63][64][65][66].…”