Platelet interaction with CNBr peptides from type II collagen via integrin α2β1

Abstract

Adhesion of blood platelets to fibrillar collagens plays a crucial role in haemostasis. Collagen type II is a homotrimeric member of the fibrillar collagen family, whose ability to interact with platelets has been poorly investigated. In this work, we analysed platelet adhesion to the whole collagen type II molecule, as well as to its CNBr peptides. We found that collagen type II is as efficient as collagen type I in supporting platelet adhesion. Platelet binding sites on collagen type II were identified in two different CNBr-derived peptides, CB8 and CB11. The ability of these peptides to support platelet adhesion required the triple helical conformation. Interaction of platelets with CB8 and CB11 peptides was totally dependent on the presence of Mg(2+) ions, and was completely inhibited by the anti-integrin alpha(2)beta(1) antibody P1E6. Upon adhesion to CB8 and CB11, a significant increase in intracellular protein tyrosine phosphorylation was observed. The pattern of tyrosine phosphorylated proteins in CB8- and CB11-adherent platelets was very similar to that observed in platelets adherent to the whole collagen molecule. By immunoprecipitation experiments, we identified two substrates that were tyrosine phosphorylated in adherent platelets as the tyrosine kinase Syk and the PLCgamma2 isozyme. By contrast, platelet adhesion to CB8 and CB11 did not promote tyrosine phosphorylation of FcR gamma-chain. Finally, we found that collagen type II, but not the CNBr-derived peptides, was able to induce cell aggregation associated to protein tyrosine phosphorylation when added to a platelet suspension. These results identify the CNBr peptides from collagen type II CB8 and CB11 as ligands for platelet integrin alpha(2)beta(1), and recognise their ability to support platelet adhesion and activation.

Role and regulation of phosphatidylinositol 3-kinase β in platelet integrin α2β1 signaling

“…4,24,29 However, stimulation of PI3K␤ activity by integrin ␣2␤1 was also confirmed using a different specific ligand, the collagen-related peptide GFOGER. Moreover, although the majority of the experiments reported in this study were performed using type I monomeric collagen as an integrin ␣2␤1 ligand, many of the results have been confirmed in experiments with the GFOGER peptide (data not shown).…”

“…on May 11, 2018. by guest www.bloodjournal.org From Monomeric type I collagen has been mainly used in this study as a reliable, cheap, and easy-to-obtain ligand for integrin ␣2␤1, because previous studies have clearly demonstrated that under these conditions no activation of GPVI occurs. 4,24,29 However, stimulation of PI3K␤ activity by integrin ␣2␤1 was also confirmed using a different specific ligand, the collagen-related peptide GFOGER. Moreover, although the majority of the experiments reported in this study were performed using type I monomeric collagen as an integrin ␣2␤1 ligand, many of the results have been confirmed in experiments with the GFOGER peptide (data not shown).…”

“…We demonstrated previously that, under the conditions of this assay, monomeric type I collagen promotes platelet adhesion exclusively through integrin ␣2␤1 and does not lead to GPVI stimulation. 4,24,29 GFOGER peptide is a wellcharacterized specific ligand for integrin ␣2␤1. 30 Adherent platelets were lysed and Akt phosphorylation on Ser473 was evaluated by immunoblotting with a phosphospecific Ab.…”

“…20,21 It has also been shown that integrin ␣2␤1 stimulates tyrosine kinases, including Src and Syk, as well as small GTPases such as Rac and Rap1b. [20][21][22][23][24] Based on the observation that the PI3K inhibitor wortmannin affects some platelet responses, 22,23 the stimulation of PI3K by integrin ␣2␤1 has been hypothesized, but so far this has not been demonstrated directly.…”

Integrin Activation State Determines Selectivity for Novel Recognition Sites in Fibrillar Collagens

“…In contrast, the phenylalanine may be less important, since two other collagen type I ␣ 1 chain sequences, GLOGER and GASGER, were found to bind ␣ 1 I and ␣ 2 I, when I-domain-interacting areas were mapped within collagen by rotary shadowing (16). No other recognition sequences have been unequivocally identified hitherto, although collagens I and III contain 11 and 14 GER triple helical motifs respectively some of which occur within cyanogen bromide-cleaved peptides, which support integrin binding (17)(18)(19)(20)(21). One conserved sequence, GMOGER, is cleaved at methionine by cyanogen bromide, and so is not present in such peptides.…”