Negative regulation of activated α2 integrins during thrombopoiesis

Abstract

Circulating platelets exhibit rapid signaling and adhesive responses to collagen that facilitate hemostasis at sites of vessel injury. Because platelets are anuclear, their collagen receptors must be expressed by megakaryocytes, platelet precursors that arise in the collagen-rich environment of the bone marrow. Whether and how megakaryocytes regulate collagen adhesion during their development in the bone marrow are unknown. We find that surface expression of activated, but not wild-type, ␣2 integrins in hematopoietic cells in vivo results in the generation of platelets that lack surface ␣2 receptors. Culture of hematopoietic progenitor cells ex vivo reveals that surface levels of activated, but not wild-type, ␣2 integrin receptors are rapidly down-regulated during cell growth on collagen but reach wild-type levels when cells are grown in the absence of collagen. Progenitor cells that express activated ␣2 integrins are normally distributed in the bone marrow in vivo and exhibit normal migration across a collagen-coated membrane ex vivo. This migration is accompanied by rapid down-regulation of activated surface integrins. These studies identify ligand-dependent removal of activated ␣2 receptors from the cell surface as a mechanism by which integrin function can be negatively regulated in hematopoi IntroductionHematopoietic cells develop as adherent cells in contact with matrix proteins in the bone marrow until they are released as nonadherent cells into the circulating blood. Circulating platelets use collagen receptors to mediate platelet activation and firm adhesion in response to exposed vessel wall collagen as a primary hemostatic response. 1,2 A critical platelet adhesive receptor for collagen that is expressed by mature megakaryocytes and platelets is integrin ␣2␤1. Like other platelet integrins, ␣2␤1 is held in a low-affinity conformation and unable to bind ligand until activating receptors on the platelet surface generate the "inside-out" intracellular signals that allosterically expose the ligand-binding ␣2 I domain. 3,4 Platelet activation signals are generated either by soluble ligands such as thrombin or ADP or by the glycoprotein VI (GPVI) receptor, an immune receptor homolog that activates platelets directly in response to collagen. [5][6][7] Affinity modulation of integrins is mediated by the interaction of talin with the integrin ␤ subunit and subsequent induction of a conformational change in the ␣ subunit from a low-to a high-affinity state. Once activated, integrin ␣2␤1 mediates firm adhesion of circulating platelets to exposed vessel wall matrix and provides a foundation for platelet thrombus formation at sites of vessel injury. [8][9][10] An outstanding question regarding platelet collagen responses is how platelets can be programmed to rapidly respond to minute amounts of collagen yet arise from megakaryocytes in the collagen-rich environment of the bone marrow. Megakaryocyte maturation is thought to require migration within the bone marrow from the osteoblastic stem cell niche to the bone ...

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

“…In the presence of the inhibitory antibody, adhesion to collagen I and ␣2-CRP was largely attenuated. We also determined ␣ 2 ␤ 1 integrin binding specificity by using a purified activated mutant ␣ 2 integrin inserted-domain (E318A ␣ 2 I-domain) (26). 3 When Mg 2ϩ was present, the E318A ␣ 2 I-domain bound to both collagen I and ␣2-CRP but not to BSA (Fig.…”

Proplatelet formation is selectively inhibited by collagen type I through Syk-independent GPVI signaling

“…However, the GFOGER peptide does not fully reflect the composition of collagen type IV (Knight et al, 1998) and difference between mouse and human cells, as well as experimental conditions like the presence of bivalent cations, might contribute to the different findings. In addition, the activation status of the α2-integrin plays an important role in thrombopoiesis (Zou et al, 2009), raising an important mode of feedback where GPVI activation increases the fraction of integrins being in the 'on' state. Using our system of mixing collagen I with type IV or laminin isoforms for analysis of PPF, we could unambiguously show that collagen I mediates the inhibitory signal by GPVI, as it was completely abrogated in Gp6 −/− mice or upon GPVI blockade (Fig.…”

Reconstruction of integrin activation

“…The idea that integrins are regulated from inside-out was suggested in mutational studies where mutation of the conserved ␣IIb cytoplasmic tail GFFKR motif strongly activated ␣IIb␤3, 96 ␣2␤1, 97 and ␣L␤2 98 ( Figure 4B). Chimeric integrins, such as ␣IIb␣5␤3, where ␣IIb cytoplasmic tail has been replaced by that of ␣5, are also constitutively active 96,99 in certain cells.…”

“…Mutation F302W in ␣M I domain stabilized the open conformation of I domain and conferred ligand-binding activity to ␣M␤2, 110 although the degree of activation was debated. 111 The corresponding mutation in ␣2 I domain, E318W 112 or E318A, 97 also increased the activation of ␣2␤1-integrin. Others have successfully used computational methods to design mutations that can stabilize the active open conformation of ␣M I-domain or ␣IIb␤3 head piece.…”