Background

Integrins are type I transmambrane glycoprotein receptors that mediate cell-cell and cell-matrix adhesion interactions, signaling and communication. All integrins are heterodimers of non-covalently associated α and β subunits. In humans integrin heterodimers are formed from 9 types of β subunits and 24 types of α subunits. This diversity is further increased by alternative splicing of some integrin RNAs. Each heterodimer consists of a large extracellular domain which binds proteins in the extracellular environment, a single transmembrane domain, and an intracellular cytoplasmic tail domain. The largest integrin subfamily is composed by integrin β₁ (CD29) that is able to associates with 12 different α subunits (α_1-11 and α_v). Integrin β₁ together with integrin chains α₄, α₅, α₆ and α₉ are expressed by murine hematopoietic stem and progenitor cells (HSPCs) and play important roles in regulating their BM retention, migration and development.

Integrins, like other transmembrane receptors, display an ‘outside-in signaling’, i.e., to transduce the signal intracellularly after the binding with their ligand. Moreover, integrins have a peculiar feature: they are able to shift between high- and low-affinity conformation states for ligand binding (‘inside-out’ signaling) (Takagi and Springer, 2002). According to the cell type, integrins can be either basically activated or basally inactive. In the inactive state, the integrin extracellular domains are not bounded to the ligands, and are in a bent conformation. Following intracellular activation signals, the extracellular domain is straightened, stabilizing the extended active conformation. Thus, the external ligand binding site, is now exposed to the ligand binding, allowing the transmission of the signals from the outside to the inside (Luo et al., 2007).

Very Late Antigen-4 (VLA-4, also known as CD49d/CD29 or α₄β₁) is a member of the integrin α₄ family together with α₄β₇. Within the integrin family, VLA-4 has some unique features. In contrast to related members of β₁ subfamily, VLA-4 is predominantly expressed on hematopoietic lineage cells (Hemler, 1990) and is functionally involved in both cell-cell and cell-ECM adhesive interactions. Moreover, despite sequence homology with other integrin α subunits, the α₄ strand because of the lack of the inserted I-domain doesn’t undergo post-translational cleavage near the transmembrane region. Finally, the α₄ chain contains a trypsin-like cleavage site, constitutively expressed on most leukocytes and on hematopoietic stem and progenitor cells (Hynes, 1992).

VLA-4 plays a major role in the regulation of immune cell recruitment to inflamed endothelia and sites of inflammation through its interactions with two alternative ligands, vascular cell adhesion molecule-1 (VCAM-1) and the alternatively spliced connecting segment 1 (CS-1) of fibronectin (Hemler, 1990; Papayannopoulou et al., 1998). It also participates in many cellular events and is crucial for BM retention and mobilization of immature stem and progenitors cells from the bone marrow (Lapidot and Petit, 2002; Peled et al., 2000).

Migration of hematopoietic stem cells to the bone marrow is a regulated multistep process that requires precise regulation and activation of various molecules including chemoattractants, selectins and integrins. While the initial steps of hematopoietic stem and progenitor cells tethering and rolling along BM blood vessel endothelium are primarily regulated by selectins, various integrins have been shown to be involved in the next stages of this process. VLA-4 plays an important role in homing, lodgment and retention of HSCs within the marrow microenvironment (Rettig et al., 2012). Previous studies demonstrate that treatment of donor BM cells with a neutralizing anti α₄ integrin antibody before injection into lethally irradiated recipients, inhibits their homing to the femurs of recipient mice, increasing the number of HSPCs in the peripheral blood and spleen. Moreover, recipient mice pretreated with neutralizing antibodies against VCAM-1 gave similar results, adding evidences to the important role of the VLA-4/VCAM-1 axis in HSPC homing to the bone marrow (Papayannopoulou et al., 1995).

Recently, factors traditionally related to coagulation and inflammation have been shown to independently control long term (LT) HSCs retention in the bone marrow and their recruitment to the blood (Aronovich et al., 2013; Gur-Cohen et al., 2015). Adult murine BM LT-HSCs, endowed with the highest repopulation and self-renewal potential, express endothelial protein C receptor (EPCR) which is used as a marker to isolate them (Balazs et al., 2006).

Protease-activated receptor-1 (PAR1) is functionally expressed by bone marrow stromal and endothelial cells as well as HSC and immature and maturing leukocytes (Gur-Cohen et al., 2016). Activated protein C ([aPC], the major ligand for EPCR)-EPCR/PAR1 signaling facilitate LT-HSC BM repopulation, retention, survival, and chemotherapy resistance by restricting nitric oxide (NO) production. Inhibition of NO generation by aPC/EPCR/PAR1 signaling on LT-HSC, inhibits downstream CDC42 activity and induces CDC42 polarity, as well as increasing VLA4 expression, affinity and adhesion. Conversely, acute stress and clinical mobilization up-regulate thrombin generation and activate different PAR1 signaling leading to NO generation that overcomes BM EPCR⁺LT-HSC retention, inducing TACE mediated EPCR and VLA-4 shedding, up-regulation of CXCR4 and PAR1 on LT-HSC, stromal PAR1 mediated CXCL12 secretion, resulting in stem and progenitor cell recruitment to the blood stream (Gur-Cohen et al., 2015). VLA-4 is expressed at a higher level by bone marrow EPCR⁺LT-HSC together with higher affinity to its ligands, inducing their BM retention and protection from DNA damaging agents. The restriction of NO by EPCR/PAR1 signaling increase the affinity of VLA-4 regulating anchorage and bone marrow retention of LT-HSC and chemotherapy resistant (Gur-Cohen et al., 2015).

Multiple small molecules have been developed in an attempt to regulate integrin dependent adhesion. The affinity states of human VLA-4 can be recognized by monoclonal antibodies sensitive to its molecular conformation (Masumoto and Hemler, 1993). Importantly, changes in VLA-4 affinity can be detected in real-time and on a physiologically relevant time frame using a ligand mimicking LDV-containing fluorescent small molecule (LDV-FITC) by FACS (Chigaev et al., 2001). VLA-4 recognize with high affinity a peptide sequence within fibronectin, which comprises 25 amino acid, termed CS-1 (Hynes, 1992). LDV (leu-asp-val) is the tripeptide identified as the minimal sequence for specific VLA-4 recognition of CS-1 segment of fibronectin. Here we describe a method to detect VLA-4 affinity monitoring mean fluorescent intensity through flow cytometry using LDV-FITC.