Signal-transducing Mechanisms Involved in Activation of the Platelet Collagen Receptor Integrin α2β1

Abstract

Evidence was obtained about the mechanism responsible for platelet integrin ␣ 2 ␤ activation by determining effects of various inhibitors on soluble collagen binding, a parameter to assess integrin ␣ 2 ␤ 1 activation, in stimulated platelets. Agonists that can also activate platelet glycoprotein IIb/IIIa are able to activate integrin ␣ 2 ␤ 1 , but those operating via glycoprotein Ib cannot. Activation of ␣ 2 ␤ 1 induced by low thrombin or collagen-related peptide concentrations was almost completely inhibited by apyrase, and the inhibitors wortmannin, 4-amino-5-(chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, bisindolylmaleimide I, and SQ29548 significantly inhibited it. Activation induced by high thrombin or collagen-related peptide concentrations was far less sensitive to these inhibitors. However, only wortmannin markedly inhibited ADP-induced integrin ␣ 2 ␤ 1 activation, and this was not ADP concentration-dependent. These results suggest that at the low agonist concentrations, the released ADP would be a primary inducer of integrin ␣ 2 ␤ 1 activation, while at the high agonist concentrations, there would be several pathways through which integrin ␣ 2 ␤ 1 activation can be induced. Kinetic analyses revealed that ADP-induced platelets had about the same number of binding sites (B max ) as thrombin-induced platelets, but their affinity (K d ) for soluble collagen was 3.7-12.7-fold lower, suggesting that activated integrin ␣ 2 ␤ 1 induced by ADP is different from that induced by thrombin. The data are consistent with an activation mechanism involving released ADP and in which there exists two different states of activated integrin ␣ 2 ␤ 1 ; these activated forms of integrin ␣ 2 ␤ 1 would have different conformations that determine their ligand affinity.Integrins comprise a family of heterodimeric cell surface proteins that mediate intracellular and cell-to-extracellular interactions. In humans, at least 15 different ␣-subunits and eight different ␤-subunits have been identified to date. The various permutations of the ␣-and ␤-subunit complexes yield integrin dimers with diverse ligand specificities and biological activities. There is tissue-specific expression of each type of integrin; some integrins are only expressed in a certain tissue, while others are more universal.Integrin ␣ IIb ␤ 3 (platelet glycoprotein (GP) 1 IIb/IIIa) is only expressed in platelets and megakaryocytes, but integrin ␣ 2 ␤ 1 (platelet GP Ia/IIa) is known to be present in many cell types (1). The GP IIb/IIIa complex is present as a nonactive heterodimer in resting platelets and becomes activated when platelets are induced by agonists (2-4); activated GP IIb/IIIa possesses high affinity for its ligand, fibrinogen. GP IIb/IIIa is one of the most abundant proteins in the platelet membrane, and its binding reaction with fibrinogen was shown to be one of the most important reactions in platelet aggregation. On the other hand, although integrin ␣ 2 ␤ 1 was indicated to be a receptor for collagen from studies on a patient's platelets lacking...

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

“…2A). Adhesion to (GPP) 10 was significantly lower than to each GXXGER sequence. In separate experiments, the binding of HT1080 to the polar GQRGER motif was found to be within the same range as to GAO/GAS (data not shown).…”

“…Peptides containing these novel sequences were synthesized in addition to the other previously identified integrin recognition sequences using two different hosts, (GPP) 5 and (GPO) 3 (Table I). As controls, (GPP) 10 , or the GPPGPP sequence in a (GPO) 3 host were used. All the peptides had stable triple helices at room temperature with GER peptides lacking hydroxyproline as their third amino acid exhibiting lower melting points, demonstrating its important role in stabilizing the peptide helix.…”

“…Recently, knockout studies suggested that the platelet activatory collagen receptor glycoprotein VI (GPVI) is mandatory for the initiation of integrin-mediated adhesion (8), but this concept was challenged by further mouse studies (9). Moreover, in vitro studies suggest that soluble collagen binding to ␣ 2 ␤ 1 requires a change in the conformation of the integrin (10). Therefore the mechanism by which ␣ 2 ␤ 1 acts as a primary adhesive receptor and yet is subject to affinity modulation remains to be reconciled.…”

In vivo β1 integrin function requires phosphorylation-independent regulation by cytoplasmic tyrosines

“…Mouse and human platelets express the ␣2␤1 integrin, which functions as a receptor for vessel wall collagen. The ␣2␤1 integrin is essential for the adhesion of flowing platelets to exposed collagen under arterial levels of hemodynamic shear (Sarratt et al 2005) and is believed to require inside-out activation similar to that described for ␣IIb␤3 (Jung and Moroi 2000). To address the role of ␤1 cytoplasmic tyrosines in the context of defined inside-out and outside-in integrin signaling, we Figure 1 reveals loss of the wildtype 13-kb band and exclusive detection of the 5-kb targeted band in homozygous knock-in cells.…”

The Tetraspanin CD9 Localizes to Platelet-Platelet Contacts and Regulates Thrombus Stability

“…This integrin binds collagen types I-VI and XI (Kehrel 1995), leading to ADP release (Atkinson 2003, Jung 2000 and phosphorylation of Src kinases, SLP-76, Syk, and PLC (Inoue 2003). Its absence leads to a bleeding disorder and reduced platelet response to collagen, although GPVI appears to be most important in collagenmediated platelet activation (Kehrel 1995, Nieuwenhuis 1985.…”