The microtubule targeting agent PBOX-15 inhibits integrin-mediated cell adhesion and induces apoptosis in acute lymphoblastic leukaemia cells

Although recent decades have seen an improved cure rate for newly diagnosed paediatric acute lymphoplastic leukaemia (ALL), the treatment options for adult ALL, T-cell ALL (T-ALL) and relapsed disease remain poor. We have developed a novel series of pyrrolo-1,5-benzoxazepine (PBOX) compounds and established their anticancer efficacy in a variety of human tumour cell types. Here, we demonstrate that PBOX-15 inhibits cell growth, and induces G2/M cell cycle arrest and apoptosis in both T-ALL and B-cell ALL (B-ALL) cells. In addition, prior to PBOX-15-induced apoptosis, PBOX-15 decreases ALL cell adhesion, spreading and migration. Concurrently, PBOX-15 differentially down-regulates β1-, β2- and α4-integrin expression in ALL cells and significantly decreases integrin-mediated cell attachment. PBOX-15 interferes with the lateral mobility and clustering of integrins in both B-ALL and T-ALL cells. These data suggest that PBOX-15 is not only effective in inducing apoptosis in ALL cells, but also has the potential to disrupt integrin-mediated adhesion of malignant lymphocytes, which represents a novel avenue for regulating leukaemic cell homing and migration.

Integrin genetic variants and stage-specific tumor recurrence in patients with stage II and III colon cancer

Integrins (ITGs) are key elements in cancer biology, regulating tumor growth, angiogenesis and lymphangiogenesis through interactions of the tumor cells with the microenvironment. Moving from the hypothesis that ITGs could have different effects in stage II and III colon cancer, we tested whether a comprehensive panel of germline single-nucleotide polymorphisms (SNPs) in ITG genes could predict stage-specific time to tumor recurrence (TTR). A total of 234 patients treated with 5-fluorouracil-based chemotherapy at the University of Southern California were included in this study. Whole-blood samples were analyzed for germline SNPs in ITG genes using PCR-restriction fragment length polymorphism or direct DNA sequencing. In the multivariable analysis, stage II colon cancer patients with at least one G allele for ITGB3 rs4642 had higher risk of recurrence (hazard ratio (HR)=4.027, 95% confidence interval (95% CI) 1.556-10.421, P=0.004). This association was also significant in the combined stage II-III cohort (HR=1.975, 95% CI 1.194-3.269, P=0.008). The predominant role of ITGB3 rs4642 in stage II diseases was confirmed using recursive partitioning, showing that ITGB3 rs4642 was the most important factor in stage II diseases. In contrast, in stage III diseases the combined analysis of ITGB1 rs2298141 and ITGA4 rs7562325 allowed to identify three distinct prognostic subgroups (P=0.009). The interaction between stage and the combined ITGB1 rs2298141 and ITGA4 rs7562325 on TTR was significant (P=0.025). This study identifies germline polymorphisms in ITG genes as independent stage-specific prognostic markers for stage II and III colon cancer. These data may help to select subgroups of patients who may benefit from ITG-targeted treatments.

The function of drosophila integrin adhesion complexes in pupal hemocyte migration in vivo

Tese de doutoramento, Ciências Biomédicas (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Medicina, 2014

The role of integrin avB3 in bone remodeling

This paper examines the role of the avB3 integrin in osteoclastic resorption using a model of a localized inflammatory bone loss.

A dual role for integrin-linked kinase in platelets: regulating integrin function and alpha-granule secretion

Integrin-linked kinase (ILK) has been implicated in the regulation of a range of fundamental biological processes such as cell survival, growth, differentiation, and adhesion. In platelets ILK associates with beta 1- and beta 3-containing integrins, which are of paramount importance for the function of platelets. Upon stimulation of platelets this association with the integrins is increased and ILK kinase activity is up-regulated, suggesting that ILK may be important for the coordination of platelet responses. In this study a conditional knockout mouse model was developed to examine the role of ILK in platelets. The ILK-deficient mice showed an increased bleeding time and volume, and despite normal ultrastructure the function of ILK-deficient platelets was decreased significantly. This included reduced aggregation, fibrinogen binding, and thrombus formation under arterial flow conditions. Furthermore, although early collagen stimulated signaling such as PLC gamma 2 phosphorylation and calcium mobilization were unaffected in ILK-deficient platelets, a selective defect in alpha-granule, but not dense-granule, secretion was observed. These results indicate that as well as involvement in the control of integrin affinity, ILK is required for alpha-granule secretion and therefore may play a central role in the regulation of platelet function. (Blood. 2008; 112: 4523-4531)

The regulation of integrin-linked kinase in human platelets: evidence for involvement in the regulation of integrin alpha(2)beta(1)

Background: Activation of the platelet integrin alpha(2)beta(1) is closely regulated due to the high thrombogenicity of its ligand. As a beta(1) interacting kinase, ILK represents a candidate intracellular regulator of alpha(2)beta(1) in human platelets. Objectives We investigated the regulation of ILK in human platelets and the role of ILK in regulating alpha(2)beta(1) activation in HEL cells, a megakaryocytic cell line. Methods: An in-vitro kinase assay was used to determine the effect of platelet agonists on ILK kinase activity together with the contribution of PI3K and PKC on ILK activation. Interaction of ILK with beta(1)-integrin subunits was investigated by coimmunoprecipitation and the role of ILK in regulating alpha(2)beta(1) function assessed by overexpression studies in HEL cells. Results: We report that collagen and thrombin modulate ILK kinase activity in human platelets in an aggregation-independent manner. Furthermore, ILK activity is dually regulated by PI3K and PKC in thrombin-stimulated platelets and regulated by PI3K in collagen-stimulated cells. ILK associates with the beta(1)-integrin subunits immunoprecipitated from platelet cell lysates, an association which increased upon collagen stimulation. Overexpression of ILK in HEL cells enhanced alpha(2)beta(1)-mediated adhesion whereas overexpression of kinase-dead ILK reduced adhesion, indicating a role for this kinase in the positive regulation of alpha(2)beta(1). Conclusions: Our findings that ILK regulates alpha(2)beta(1) in HEL cells, is activated in platelets and associates with beta(1)-integrins, raise the possibility that it may play a key role in adhesion events upon agonist stimulation of platelets.

Platelet adhesion to collagen and collagen-related peptide under flow. Roles of the alpha(2)beta(1) integrin, GPVI, and Src tyrosine kinases

Objective - Platelet stimulation by collagen and collagen-related peptides (CRPs) is associated with activation of protein tyrosine kinases. In the present study, we investigated the role of Src family tyrosine kinases in the initial adhesion events of human platelets to collagen and cross-linked CRP. Methods and Results - Under arterial flow conditions, a glycoprotein VI - specific substrate, cross-linked CRP, caused rapid (<2 second) platelet retention and protein tyrosine phosphorylation that were markedly decreased by the Src family kinase inhibitor pyrozolopyrimidine (PP2) or by aggregation inhibitor GRGDSP. CRP-induced platelet retention was transient, and 90% of single platelets or aggregates detached within seconds. PP2, although having no effect on RGD peptide-binding to CRP, completely blocked aggregation and tyrosine phosphorylation of Syk and phospholipase Cγ2 (PLCγ2). In contrast, PP2 weakly (<30%) suppressed firm adhesion to collagen mediated primarily by the alpha(2)beta(1) integrin. Although PP2 prevented activation of Syk and PLCgamma2 in collagen-adherent platelets, tyrosine phosphorylation of several unidentified protein bands persisted, as did autophosphorylation of pp125(FAK). Conclusions - These findings indicate that activation of Src-tyrosine kinases Syk and PLCgamma2 is not required for the initial stable attachment of human platelets to collagen and for FAK autophosphorylation. However, Src-tyrosine kinases are critical for glycoprotein VI - mediated signaling leading to platelet aggregation.

Rhinocetin, a venom-derived integrin-specific antagonist inhibits collagen-induced platelet and endothelial cell functions

Snaclecs are small non-enzymatic proteins present in viper venoms reported to modulate haemostasis of victims through effects on platelets, vascular endothelial and smooth muscle cells. In this study, we have isolated and functionally characterised a snaclec which we named rhinocetin from the venom of West African gaboon viper, Bitis gabonica rhinoceros. Rhinocetin was shown to comprise α and β chains with the molecular masses of 13.5 and 13kDa respectively. Sequence and immunoblot analysis of rhinocetin confirmed this to be a novel snaclec. Rhinocetin inhibited collagen-stimulated activation of human platelets in dose dependent manner, but displayed no inhibitory effects on glycoprotein VI (collagen receptor) selective agonist, CRP-XL-, ADP- or thrombin-induced platelet activation. Rhinocetin antagonised the binding of monoclonal antibodies against the α2 subunit of integrin α2β1 to platelets and coimmunoprecipitation analysis confirmed integrin α2β1 as a target for this venom protein. Rhinocetin inhibited a range of collagen induced platelet functions such as fibrinogen binding, calcium mobilisation, granule secretion, aggregation and thrombus formation. It also inhibited integrin α2β1 dependent functions of human endothelial cells. Together, our data suggest rhinocetin to be a modulator of integrin α2β1 function and thus may provide valuable insights into the role of this integrin in physiological and pathophysiological scenarios including haemostasis, thrombosis and envenomation.

A collagen-like peptide stimulates tyrosine phosphorylation of syk and phospholipase C gamma2 in platelets independent of the integrin alpha2beta1.

Activation of platelets by collagen is mediated through a tyrosine kinase-dependent pathway that is associated with phosphorylation of the Fc receptor gamma chain, the tyrosine kinase syk, and phospholipase C gamma2 (PLC gamma2). We recently described a collagen-related triple-helical peptide (CRP) with the sequence GCP*(GPP*)GCP*G (single letter amino acid code: P* = hydroxyproline; Morton et al, Biochem J306:337, 1995). The cross-linked peptide is a potent stimulus of platelet activation but, unlike collagen, does not support alpha2beta1-mediated, Mg2+-dependent adhesion, suggesting that its action is independent of the integrin alpha2beta1. This finding suggests the existence of a platelet receptor other than alpha2beta1 that underlies activation. In the present study, we show that CRP stimulates tyrosine phosphorylation of the same pattern of proteins in platelets as collagen, including syk and PLC gamma2. Protein tyrosine phosphorylation induced by CRP is not altered in the absence of Mg2+ or the presence of monoclonal antibodies (MoAbs) to the integrin alpha2beta1 (MoAb 6F1 and MoAb 13), conditions that prevent the interaction of collagen with the integrin. In contrast, phosphorylation of syk and PLC gamma2 by collagen is partially reduced by MoAb 6F1 and MoAb 13 or by removal of Mg2+. This may reflect a direct role of alpha2beta1 in collagen-induced signaling events or an indirect role in which the integrin facilitates the binding of collagen to its signaling receptor. The results show an alpha2beta1-independent pathway of platelet activation by CRP that involves phosphorylation of syk and PLC gamma2. This pathway appears to contribute to platelet activation by collagen.

Genetic evidence for a predominant role of PI3Kβ catalytic activity in ITAM- and integrin-mediated signaling in platelets.

Phosphatidylinositol 3-kinase (PI3K) isoforms PI3Kbeta and PI3Kgamma are implicated in platelet adhesion, activation, and aggregation, but their relative contribution is still unclear or controversial. Here, we report the first comparative functional analysis of platelets from mice expressing a catalytically inactive form of PI3Kbeta or PI3Kgamma. We demonstrate that both isoforms were similarly required for maximal activation of the small GTPase Rap1b and for complete platelet aggregation upon stimulation of G protein-coupled receptors for adenosine 5'-diphosphate (ADP) or U46619. Their contribution to these events, however, was largely redundant and dispensable. However, PI3Kbeta, but not PI3Kgamma, enzymatic activity was absolutely required for Akt phosphorylation, Rap1 activation, and platelet aggregation downstream of the immunoreceptor tyrosine-based activation motif (ITAM)-bearing receptor glycoprotein VI (GPVI). Moreover, PI3Kbeta was a major essential regulator of platelet adhesion to fibrinogen and of integrin alpha(IIb)beta(3)-mediated spreading. These results provide genetic evidence for a crucial and selective role of PI3Kbeta in signaling through GPVI and integrin alpha(IIb)beta(3).

Platelet endothelial cell adhesion molecule-1 inhibits platelet response to thrombin and von Willebrand factor by regulating the internalization of glycoprotein Ib via AKT/glycogen synthase kinase-3/dynamin and integrin αIIbβ3

OBJECTIVE: Platelet endothelial cell adhesion molecule-1 (PECAM-1) regulates platelet response to multiple agonists. How this immunoreceptor tyrosine-based inhibitory motif-containing receptor inhibits G protein-coupled receptor-mediated thrombin-induced activation of platelets is unknown. APPROACH AND RESULTS: Here, we show that the activation of PECAM-1 inhibits fibrinogen binding to integrin αIIbβ3 and P-selectin surface expression in response to thrombin (0.1-3 U/mL) but not thrombin receptor-activating peptides SFLLRN (3×10(-7)-1×10(-5) mol/L) and GYPGQV (3×10(-6)-1×10(-4) mol/L). We hypothesized a role for PECAM-1 in reducing the tethering of thrombin to glycoprotein Ibα (GPIbα) on the platelet surface. We show that PECAM-1 signaling regulates the binding of fluorescein isothiocyanate-labeled thrombin to the platelet surface and reduces the levels of cell surface GPIbα by promoting its internalization, while concomitantly reducing the binding of platelets to von Willebrand factor under flow in vitro. PECAM-1-mediated internalization of GPIbα was reduced in the presence of both EGTA and cytochalasin D or latrunculin, but not either individually, and was reduced in mice in which tyrosines 747 and 759 of the cytoplasmic tail of β3 integrin were mutated to phenylalanine. Furthermore, PECAM-1 cross-linking led to a significant reduction in the phosphorylation of glycogen synthase kinase-3β Ser(9), but interestingly an increase in glycogen synthase kinase-3α pSer(21). PECAM-1-mediated internalization of GPIbα was reduced by inhibitors of dynamin (Dynasore) and glycogen synthase kinase-3 (CHIR99021), an effect that was enhanced in the presence of EGTA. CONCLUSIONS: PECAM-1 mediates internalization of GPIbα in platelets through dual AKT/protein kinase B/glycogen synthase kinase-3/dynamin-dependent and αIIbβ3-dependent mechanisms. These findings expand our understanding of how PECAM-1 regulates nonimmunoreceptor signaling pathways and helps to explains how PECAM-1 regulates thrombosis.

Integrin-linked kinase regulates the rate of platelet activation and is essential for the formation of stable thrombi

BACKGROUND: Integrin-linked kinase (ILK) and its associated complex of proteins are involved in many cellular activation processes, including cell adhesion and integrin signaling. We have previously demonstrated that mice with induced platelet ILK deficiency show reduced platelet activation and aggregation, but only a minor bleeding defect. Here, we explore this apparent disparity between the cellular and hemostatic phenotypes. METHODS: The impact of ILK inhibition on integrin αII b β3 activation and degranulation was assessed with the ILK-specific inhibitor QLT0267, and a conditional ILK-deficient mouse model was used to assess the impact of ILK deficiency on in vivo platelet aggregation and thrombus formation. RESULTS: Inhibition of ILK reduced the rate of both fibrinogen binding and α-granule secretion, but was accompanied by only a moderate reduction in the maximum extent of platelet activation or aggregation in vitro. The reduction in the rate of fibrinogen binding occurred prior to degranulation or translocation of αII b β3 to the platelet surface. The change in the rate of platelet activation in the absence of functional ILK led to a reduction in platelet aggregation in vivo, but did not change the size of thrombi formed following laser injury of the cremaster arteriole wall in ILK-deficient mice. It did, however, result in a marked decrease in the stability of thrombi formed in ILK-deficient mice. CONCLUSION: Taken together, the findings of this study indicate that, although ILK is not essential for platelet activation, it plays a critical role in facilitating rapid platelet activation, which is essential for stable thrombus formation.

The N-terminal SH2 domain of Syk is required for (hem)ITAM, but not integrin, signaling in mouse platelets

We have used a novel knockin mouse to investigate the effect of disruption of phosphotyrosine binding of the N-terminal SH2 domain of Syk on platelet activation by GPVI, CLEC-2, and integrin αIIbβ3. The Syk(R41Afl/fl) mouse was crossed to a PF4-Cre(+) mouse to induce expression of the Syk mutant in the megakaryocyte/platelet lineage. Syk(R41Afl/fl;PF4-Cre) mice are born at approximately 50% of the expected frequency and have a similar phenotype to Syk(fl/fl;PF4-Cre) mice, including blood-lymphatic mixing and chyloascites. Anastomosis of the venous and lymphatic vasculatures can be seen in the mesenteric circulation accounting for rapid and continuous mixing of the 2 vasculatures. Platelet activation by CLEC-2 and GPVI is abolished in Syk(R41Afl/fl;PF4-Cre) platelets. Syk phosphorylation on Tyr519/20 is blocked in CLEC-2-stimulated platelets, suggesting a model in which binding of Syk via its N-terminal SH2 domain regulates autophosphorylation. In contrast, outside-in signaling by integrin αIIbβ3 is not altered, but it is inhibited in the presence of inhibitors of Src and Syk tyrosine kinases. These results demonstrate that αIIbβ3 regulates Syk through an ITAM-independent pathway in mice and provide novel insight into the course of events underlying Syk activation and hemITAM phosphorylation by CLEC-2.

A role for adhesion and degranulation-promoting adapter protein in collagen-induced platelet activation mediated via integrin α(2) β(1)

Platelet aggregation and phosphorylation of phospholipase Cγ2 induced by collagen were attenuated in ADAP(-/-) platelets. However, aggregation and signaling induced by collagen-related peptide (CRP), a GPVI-selective agonist, were largely unaffected. Platelet adhesion to CRP was also unaffected by ADAP deficiency. Adhesion to the α(2) β(1) -selective ligand GFOGER and to a peptide (III-04), which supports adhesion that is dependent on both GPVI and α(2) β(1), was reduced in ADAP(-/-) platelets. An impedance-based label-free detection technique, which measures adhesion and spreading of platelets, indicated that, in the absence of ADAP, spreading on GFOGER was also reduced. This was confirmed with non-fluorescent differential-interference contrast microscopy, which revealed reduced filpodia formation in ADAP(-/-) platelets adherent to GFOGER. This indicates that ADAP plays a role in mediating platelet activation via the collagen-binding integrin α(2) β(1). In addition, we found that ADAP(-/-) mice, which are mildly thrombocytopenic, have enlarged spleens as compared with wild-type animals. This may reflect increased removal of platelets from the circulation.