Integrin adhesion receptors mediate the adhesion of blood cells, such as leukocytes, to other cells, such as endothelial cells. Integrins also are critical for anchorage of hematopoietic precursors to the extracellular matrix. Blood cells can dynamically regulate the affinities of integrins for their ligands ("activation"), an event central to their functions. Here we review recent progress in understanding the mechanisms of integrin activation with a focus on the functions of blood cells. We discuss how talin binding to the integrin b cytoplasmic domain, in conjunction with the plasma membrane, induces long-range allosteric rearrangements that lead to integrin activation. Second, we review our understanding of how signaling events, particularly those involving Rap1 small guanosine triphosphate (GTP) hydrolases, can regulate the talin-integrin interaction and resulting activation. Third, we review recent findings that highlight the role of the Rap1-GTP-interacting adapter molecule (RIAM), encoded by the APBB1IP gene, in leukocyte integrin activation and consequently in leukocyte trafficking. (Blood. 2016;128(4):479-487) Introduction Circulating blood cells patrol the body to guard against pathogens and maintain homeostasis. The adhesive interactions of leukocytes with the blood vessel walls are transient and dynamic and serve a wide range of immune functions.1 Similarly, platelets do not stably adhere to the intact vessel wall; however, when the endothelium is disrupted, a rapid interaction between circulating platelets and the vessel leads to the formation of a platelet-leukocyte-fibrin aggregate that mediates hemostasis.2 Members of the integrin adhesion receptor family play important roles in these processes. Integrins are type 1 transmembrane receptors that mediate cell-cell and cell-extracellular matrix adhesion. 3 Each integrin heterodimer contains 1 a and 1 b subunit. In vertebrates, 18 a and 8 b subunits can form 24 integrins with characteristic tissue distributions and distinct ligand binding specificities. 3,4 Leukocyte integrins include aLb2 (LFA-1, CD11a/CD18), a4b1 (VLA-4), and aMb2 (Mac-1, CD11b/CD18), 5,6 whereas integrin aIIbb3 (GPIIbIIIa) is by far the most abundant platelet integrin. 7 Integrins sense chemical and physical properties of extracellular matrix and control signal transduction pathways that regulate cell adhesion, proliferation, differentiation, and apoptosis. 3 Mutations of integrin genes can lead to blood diseases such as leukocyte adhesion deficiency I syndrome, 8,9 due to mutations in the gene encoding integrin b2, or Glanzmann thrombasthenia, 10 due to mutations in the genes encoding aIIb or b3. Integrins in blood cells are usually in a low-affinity state until agonist stimulation induces a high-affinity form, a process operationally defined as "integrin activation." 6,[11][12][13] On platelet stimulation by agonists such as thrombin or collagen, activated aIIbb3 binds fibrinogen, fibronectin, and von Willebrand factor to enable both stable adherence to the vessel wal...

Loss of Krit1 disables an angiogenic checkpoint by reducing TSP1 expression, thereby enabling cerebral cavernous malformation (CCM) formation. Lopez-Ramirez et al. propose that replacing TSP1 by TSP1 fragments or angiogenesis inhibitors may provide an approach to inhibit CCM development.

The leading edge of migrating cells contains rapidly translocating activated integrins associated with growing actin filaments that form "sticky fingers" that sense extracellular matrix and guide cell migration. Here we utilized indirect bimolecular fluorescence complementation (BiFC) to visualize a molecular complex containing an MRL protein (RIAM or lamellipodin), talin, and activated integrins in living cells. This complex localizes at the tips of growing actin filaments in lamellipodial and filopodial protrusions, thus corresponding to the tips of the "sticky fingers." Formation of the complex requires talin to form a bridge between the MRL protein and the integrins. Moreover, disruption of the MRL protein-integrin-talin (MIT) complex markedly impairs cell protrusion. These data reveal the molecular basis of the formation of "sticky fingers" at the leading edge of migrating cells and show that an MIT complex drives these protrusions.

The phosphoinositide metabolic pathway, which regulates cellular processes implicated in survival, motility, and trafficking, is often subverted by bacterial pathogens. Shigella flexneri, a bacterium that causes dysentery, injects IpgD, a phosphoinositide phosphatase that generates the lipid phosphatidylinositol 5-phosphate (PI5P), into host cells, thereby activating the phosphoinositide 3-kinase-Akt survival pathway. We show that epidermal growth factor receptor (EGFR) is required for PI5P-dependent activation of Akt in infected HeLa cells or cells ectopically expressing IpgD. Cells treated with PI5P had increased numbers of early endosomes with activated EGFR, no detectable EGFR in the late endosomal or lysosomal compartments, and prolonged EGFR signaling. Endosomal recycling and retrograde pathways were spared, indicating that the effect of PI5P on the degradative route to the late endocytic compartments was specific. Thus, we identified PI5P, which was enriched in endosomes, as a regulator of vesicular trafficking that alters growth factor receptor signaling by impairing lysosomal degradation, a property used by S. flexneri to favor survival of host cells.

Cerebral cavernous malformations (CCMs) are common brain vascular dysplasias that are prone to acute and chronic hemorrhage with significant clinical sequelae. The pathogenesis of recurrent bleeding in CCM is incompletely understood. Here, we show that central nervous system hemorrhage in CCMs is associated with locally elevated expression of the anticoagulant endothelial receptors thrombomodulin (TM) and endothelial protein C receptor (EPCR). TM levels are increased in human CCM lesions, as well as in the plasma of patients with CCMs. In mice, endothelial-specific genetic inactivation of Krit1 (Krit1ECKO) or Pdcd10 (Pdcd10ECKO), which cause CCM formation, results in increased levels of vascular TM and EPCR, as well as in enhanced generation of activated protein C (APC) on endothelial cells. Increased TM expression is due to upregulation of transcription factors KLF2 and KLF4 consequent to the loss of KRIT1 or PDCD10. Increased TM expression contributes to CCM hemorrhage, because genetic inactivation of 1 or 2 copies of the Thbd gene decreases brain hemorrhage in Pdcd10ECKO mice. Moreover, administration of blocking antibodies against TM and EPCR significantly reduced CCM hemorrhage in Pdcd10ECKO mice. Thus, a local increase in the endothelial cofactors that generate anticoagulant APC can contribute to bleeding in CCMs, and plasma soluble TM may represent a biomarker for hemorrhagic risk in CCMs.

Cerebral cavernous malformations (CCMs) are common neurovascular lesions caused by lossof-function mutations in one of three genes, including KRIT1 (CCM1), CCM2, and PDCD10 (CCM3), and generally regarded as an endothelial cell-autonomous disease. Here we reported that proliferative astrocytes played a critical role in CCM pathogenesis by serving as a major source of VEGF during CCM lesion formation. An increase in astrocyte VEGF synthesis is driven by endothelial nitric oxide (NO) generated as a consequence of KLF2 and KLF4-dependent elevation of eNOS in CCM endothelium. The increased brain endothelial production of NO stabilized HIF-1a in astrocytes, resulting in increased VEGF production and expression of a "hypoxic" program under normoxic conditions. We showed that the upregulation of cyclooxygenase-2 (COX-2), a direct HIF-1a target gene and a known component of the hypoxic program, contributed to the development of CCM lesions because the administration of a COX-2 inhibitor significantly prevented the progression of CCM lesions. Thus, non-cell-autonomous crosstalk between CCM endothelium and astrocytes propels vascular lesion development, and components of the hypoxic program represent potential therapeutic targets for CCMs.

PtdIns5P is a lipid messenger acting as a stress-response mediator in the nucleus, and known to maintain cell activation through traffic alterations upon bacterial infection. Here, we show that PtdIns5P regulates actin dynamics and invasion via recruitment and activation of the exchange factor Tiam1 and Rac1. Restricted Rac1 activation results from the binding of Tiam1 DH-PH domains to PtdIns5P. Using an assay that mimics Rac1 membrane anchoring by using Rac1-His and liposomes containing Ni 2 þ -NTA modified lipids, we demonstrate that intrinsic Tiam1 DH-PH activity increases when Rac1 is anchored in a PtdIns5P-enriched environment. This pathway appears to be general since it is valid in different pathophysiological models: receptor tyrosine kinase activation, bacterial phosphatase IpgD expression and the invasive NPM-ALK( þ ) lymphomas. The discovery that PtdIns5P could be a keystone of GTPases and cytoskeleton spatiotemporal regulation opens important research avenues towards unravelling new strategies counteracting cell invasion.

Rap1 is a major convergence point of the platelet signaling pathways that result in talin1 binding to the integrin b cytoplasmic domain and consequent integrin activation, platelet aggregation, and effective hemostasis. The nature of the connection between Rap1 and talin1 in integrin activation is an important remaining gap in our understanding of this process. Previous work identified a low affinity Rap1 binding site in talin1 F0 domain that makes a small contribution to integrin activation in platelets. We recently identified an additional Rap1 binding site in talin1 F1 domain that makes a greater contribution than F0 in model systems. Here we generated mice bearing point mutations, which block Rap1 binding without affecting talin1 expression, in either talin1 F1 domain (R118E) alone, which were viable, or both F0 and F1 domains (R35E,R118E), which were embryonic lethal. Loss of the Rap1-talin1 F1 interaction in platelets markedly decreases talin1-mediated activation of platelet b1- and b3-integrins. Integrin activation and platelet aggregation in mice whose platelets express only talin1(R35E,R118E) are even more impaired, resembling the defect seen in platelets lacking both Rap1a and Rap1b. Although Rap1 is important in thrombopoiesis, platelet secretion, and surface exposure of phosphatidylserine, loss of Rap1-talin interaction in talin1(R35E,R118E) platelets had little effect on these processes. These findings show that talin1 is the principal direct effector of Rap1 GTPases that regulates platelet integrin activation in hemostasis.