Glycoprotein VI but not alpha2beta1 integrin is essential for platelet interaction with collagen

Abstract

C.Brakebusch and W.Bergmeier contributed equally to this workPlatelet adhesion on and activation by components of the extracellular matrix are crucial to arrest posttraumatic bleeding, but can also harm tissue by occluding diseased vessels. Integrin a2b1 is thought to be essential for platelet adhesion to subendothelial collagens, facilitating subsequent interactions with the activating platelet collagen receptor, glycoprotein VI (GPVI). Here we show that Cre/loxP-mediated loss of b1 integrin on platelets has no signi®cant effect on the bleeding time in mice. Aggregation of b1-null platelets to native ®brillar collagen is delayed, but not reduced, whereas aggregation to enzymatically digested soluble collagen is abolished. Furthermore, b1-null platelets adhere to ®brillar, but not soluble collagen under static as well as low (150 s ±1 ) and high (1000 s ±1 ) shear ow conditions, probably through binding of aIIbb3 to von Willebrand factor. On the other hand, we show that platelets lacking GPVI can not activate integrins and consequently fail to adhere to and aggregate on ®brillar as well as soluble collagen. These data show that GPVI plays the central role in platelet±collagen interactions by activating different adhesive receptors, including a2b1 integrin, which strengthens adhesion without being essential. Keywords: collagen/Cre/loxP/GPVI/a2b1 integrin/ platelets IntroductionDamage to the integrity of the vessel wall results in exposure of the subendothelial extracellular matrix (ECM), which triggers adhesion and aggregation of platelets (Weiss, 1975). The consequence of this process is the formation of a thrombus, which prevents blood loss at sites of injury or leads to occlusion and irreversible tissue damage or infarction in diseased vessels. Integrins play a central role in adhesion and aggregation of platelets (Phillips et al., 1991;Shattil et al., 1998). Integrins are heterodimeric transmembrane receptors composed of an a and a b subunit. Resting platelets express their integrins in a low af®nity state. After activation, mediated by other platelet receptors, integrins shift to a high af®nity state and bind their ligands ef®ciently (Phillips et al., 1991;Shattil et al., 1998).The ECM of the vessel walls is rich in collagens, which play essential roles in thrombus formation by providing a substrate for platelet adhesion and by activating platelets (Baumgartner, 1977). Besides GPIb-V-IX and aIIbb3 integrin, which interact indirectly with collagen via von Willebrand factor (vWF) (Savage et al., 1998), a large number of collagen receptors have been identi®ed on platelets, including most importantly a2b1 integrin (Santoro, 1986) and glycoprotein VI (GPVI) (Moroi et al., 1989). A current model of platelet adhesion to collagen suggests that the GPIb±vWF interaction mediates initial tethering of platelets at high shear, followed by a2b1 integrin-mediated ®rm adhesion, which halts platelet translocation and allows collagen interactions with GPVI, ®nally resulting in platelet activation and thrombus growth (Sixma et...

CEACAM1 negatively regulates platelet-collagen interactions and thrombus growth in vitro and in vivo

“…37,38 In this model, integrin ␣2␤1 is now thought to have a supportive, rather than a primary role as the adhesive collagen receptor, while GPVI is a major platelet-collagen activating receptor that interacts with collagen fibrils via a triplicate GPO sequence (glycine, proline, hydroxyproline) and integrin ␣2␤1 interacts with collagen via GFOFER (glycine, phenylalanine, hydroxyproline, glutamic acid and arginine). [39][40][41] For in vitro studies, we used CRP as the GPVI-selective agonist because this peptide contains 10 GPO repeat sequences and serves as a potent activator of GPVImediated signaling and does not recognize integrin ␣2␤1 . 42 In this way, we can differentiate GPVI-mediated platelet responses from integrin ␣2␤1-mediated platelet responses.…”

Suboptimal Activation of Protease-activated Receptors Enhances α2β1 Integrin-mediated Platelet Adhesion to Collagen

“…There has been much debate regarding the roles of ␣ 2 ␤ 1 integrin and GPVI/FcR␥ complex in the two-step, two-site model (44,45). It has been suggested that engagement of GPVI may be required for activation of the ␣ 2 ␤ 1 integrin (29,44,45). We have previously shown that platelets from ␣ 2 ␤ 1 -deficient mice have delayed thrombus formation in vivo within the carotid artery (12), and that under flow conditions in vitro, GPVI/FcR␥ complex and ␣ 2 ␤ 1 integrin both play independent and vital roles in platelet adhesion and aggregation to collagen (46).…”

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

“…It also becomes activated through inside-out signaling by soluble agonists and by GPVI (27). The concept of a need for prior activation of ␣ 2 ␤ 1 for hemostatic function was furthered by murine studies, in which platelet adhesion was markedly attenuated by the absence of GPVI (8). Here, we show that although platelet binding to the lower affinity sequences does indeed require prior activation, binding to GFOGER (and to a lesser extent GLOGER) does not, which may allow platelets to interact with such sequence-containing collagens without a preceding switch in the integrin conformation.…”

“…Accordingly, the expression levels of ␣ 2 ␤ 1 have been associated with myocardial infarction and stroke (7). 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).…”