Outside-In Signalling Generated by a Constitutively Activated Integrin αIIbβ3 Impairs Proplatelet Formation in Human Megakaryocytes

BACKGROUND: The interaction of megakaryocytes with matrix proteins of the osteoblastic and vascular niche is essential for megakaryocyte maturation and proplatelet formation. Fibrinogen is present in the vascular niche and the fibrinogen receptor α(IIb)β(3) is abundantly expressed on megakaryocytes, however the role of the interaction between fibrinogen and α(IIb)β(3) in proplatelet formation in humans is not yet fully understood. We have recently reported a novel congenital macrothrombocytopenia associated with a heterozygous mutation of the β(3) subunit of α(IIb)β(3). The origin of thrombocytopenia in this condition remains unclear and this may represent an interesting natural model to get further insight into the role of the megakaryocyte fibrinogen receptor in megakaryopoiesis. METHODOLOGY/PRINCIPAL FINDINGS: Patients' peripheral blood CD45+ cells in culture were differentiated into primary megakaryocytes and their maturation, spreading on different extracellular matrix proteins, and proplatelet formation were analyzed. Megakaryocyte maturation was normal but proplatelet formation was severely impaired, with tips decreased in number and larger in size than those of controls. Moreover, megakaryocyte spreading on fibrinogen was abnormal, with 50% of spread cells showing disordered actin distribution and more evident focal adhesion points than stress fibres. Integrin α(IIb)β(3) expression was reduced but the receptor was constitutively activated and a sustained, and substrate-independent, activation of proteins of the outside-in signalling was observed. In addition, platelet maturation from preplatelets was impaired. CONCLUSIONS/SIGNIFICANCE: Our data show that constitutive activation of α(IIb)β(3)-mediated outside-in signalling in human megakaryocytes negatively influences proplatelet formation, leading to macrothombocytopenia

The impact of digital health technologies on tuberculosis treatment : a systematic review

Digital technologies are increasingly harnessed to support treatment of persons with tuberculosis (TB). Since in-person directly observed treatment (DOT) can be resource intensive and challenging to implement, these technologies may have the potential to improve adherence and clinical outcomes. We reviewed the effect of these technologies on TB treatment adherence and patient outcomes. We searched several bibliographical databases for studies reporting the effect of digital interventions, including short message service (SMS), video-observed therapy (VOT) and medication monitors (MMs), to support treatment for active TB. Only studies with a control group and which reported effect estimates were included. Four trials showed no statistically significant effect on treatment completion when SMS was added to standard care. Two observational studies of VOT reported comparable treatment completion rates when compared with in-person DOT. MMs increased the probability of cure (RR 2.3, 95% CI 1.6-3.4) in one observational study, and one trial reported a statistically significant reduction in missed treatment doses relative to standard care (adjusted means ratio 0.58, 95% CI 0.42-0.79). Evidence of the effect of digital technologies to improve TB care remains limited. More studies of better quality are needed to determine how such technologies can enhance programme performance

ADAPtation of Platelet Integrin αIIbβ3 to Inside-Out Activation Signals

Abstract Abstract 188 Platelets respond to excitatory agonists by binding adhesive ligands, such as fibrinogen, and by aggregating during hemostasis and thrombosis. These processes require the activation of αIIbβ3 from a low- to a high-affinity state. Activation of αIIbβ3 is mediated by inside-out signaling and ultimately by interactions of the molecular adaptors, talin and kindlin-3, with the β3 cytoplasmic domain. While certain key elements in the circuitry connecting agonist receptors and αIIbβ3 have been identified, some critical aspects of inside-out signaling remain undefined. The role of the hematopoietic adapter protein, ADAP, is a case in point. Platelets deficient in ADAP exhibit decreased αIIbβ3 activation in response to several agonists and reduced thrombus formation in vivo. How ADAP interfaces with the signaling machinery of platelets to regulate αIIbβ3 is unknown. Promotion of integrin adhesive function in lymphocytes by ADAP requires SKAP1, an ADAP-binding partner that is not expressed in platelets. While platelets express the SKAP1 homologue, SKAP2, the latter is dispensable for αIIbβ3 activation since SKAP2-null platelets are normal. We hypothesized that ADAP modulates αIIbβ3 function through interactions with talin and/or kindlin-3. Immunoprecipitation of ADAP from human or mouse platelets showed that ADAP was associated with a pool of talin and kindlin-3. To evaluate the proximity of this association in intact platelets, a proximity ligation assay was employed whereby antibody and DNA amplification techniques are combined to identify protein-protein interactions at the single cell level that occur at distances &lt;40 nm. In human platelets spread on fibrinogen, a 16-fold increase in the number of proximity ligation signals per platelet was detected when antibodies to ADAP and talin were employed in the assay compared to when control antibodies were used (P &lt; 0.01). In addition, ADAP partially co-localized with talin and kindlin-3 in platelets as determined by fluorescence deconvolution microscopy. In order to exclude a role for SKAP2 in these ADAP interactions, we turned to CHO cells that stably express αIIbβ3, a model cell system that normally lacks SKAP-2 and ADAP but can be used to reconstitute inside-out αIIbβ3 signaling by expression of relevant proteins. Following transient transfection of ADAP into αIIbβ3-CHO cells, ADAP was found to specifically co-immunoprecipitate with endogenous talin and ectopically-expressed kindlin-3. When these cells were further engineered to conditionally express the platelet PAR1 thrombin receptor and increased levels of talin, the presence of ADAP significantly enhanced PAR-1-mediated αIIbβ3 activation, as detected with antibody PAC-1 (P &lt; 0.05). Although overexpression of kindlin-3 in these cells failed to enhance PAR1-mediated, talin-dependent PAC-1 binding, αIIbβ3 activation was moderately enhanced when kindlin-3 was co-expressed with ADAP (P &lt; 0.05). Altogether, these studies establish that ADAP functions in close proximity to talin and kindlin-3 to promote agonist-dependent αIIbβ3 activation in platelets in a manner independent of SKAP2. Therefore, the mechanism whereby ADAP modulates integrin activation in platelets is subtly, if not fundamentally, different than in lymphocytes. This raises the potential of selectively targeting ADAP's integrin-activating function in platelets or lymphocytes in thrombotic or immune disorders, respectively, while leaving integrin function in the other cell type unaffected. Disclosures: No relevant conflicts of interest to declare. </jats:sec

Positive and negative regulation by SLP-76/ADAP and Pyk2 of chemokine-stimulated T-lymphocyte adhesion mediated by integrin α4β1

Stimulation by chemokines of integrin α4β1-dependent T-lymphocyte adhesion is a crucial step for lymphocyte trafficking. The adaptor Vav1 is required for chemokine-activated T-cell adhesion mediated by α4β1. Conceivably, proteins associating with Vav1 could potentially modulate this adhesion. Correlating with activation by the chemokine CXCL12 of T-lymphocyte attachment to α4β1 ligands, a transient stimulation in the association of Vav1 with SLP-76, Pyk2, and ADAP was observed. Using T-cells depleted for SLP-76, ADAP, or Pyk2, or expressing Pyk2 kinase-inactive forms, we show that SLP-76 and ADAP stimulate chemokine-activated, α4β1-mediated adhesion, whereas Pyk2 opposes T-cell attachment. While CXCL12-promoted generation of high-affinity α4β1 is independent of SLP-76, ADAP, and Pyk2, the strength of α4β1-VCAM-1 interaction and cell spreading on VCAM-1 are targets of regulation by these three proteins. GTPase assays, expression of activated or dominant-negative Rac1, or combined ADAP and Pyk2 silencing indicated that Rac1 activation by CXCL12 is a common mediator response in SLP-76-, ADAP-, and Pyk2-regulated cell adhesion involving α4β1. Our data strongly suggest that chemokine-stimulated associations between Vav1, SLP-76, and ADAP facilitate Rac1 activation and α4β1-mediated adhesion, whereas Pyk2 opposes this adhesion by limiting Rac1 activation.This work was supported by grants SAF2011-24022 from Ministerio de Economía y Competitividad, RD12/0036/0061, and S2010/BMD-2314 from Comunidad de Madrid to J.T.Peer Reviewe