PECAM‐1 functions as a negative regulator of laminin‐induced platelet activation

Summary. Background: Interaction of resting platelets with exposed components of the subendothelial matrix is an important early activating event that takes place at sites of vascular injury. Platelet responses to collagen are mediated by integrin α₂β₁ and the glycoprotein (GP)VI–Fc receptor (FcR) γ‐chain complex, whereas platelet activation by laminin is mediated by the related integrin, α₆β₁, and similarly requires signaling through GPVI–FcR γ‐chain. Objective: Because the cell adhesion and signaling receptor PECAM‐1 has previously been shown to dampen collagen‐induced platelet activation, we sought to determine whether PECAM‐1 might similarly regulate platelet activation by laminin. Methods/Results: We found that PECAM‐1 became tyrosine phosphorylated on its cytoplasmic immunoreceptor tyrosine‐based inhibitory motifs following adhesion of either human or murine platelets to immobilized laminin. Whereas the presence or absence of PECAM‐1 had no effect on either the rate or extent of platelet adhesion or spreading on laminin, PECAM‐1 inhibited laminin‐induced phosphorylation of GPVI–FcR γ‐chain immunoreceptor tyrosine‐based activation motifs (ITAMs) and activation of its downstream effector, Syk kinase, and suppressed granule secretion. Conclusions: Taken together, these data are consistent with previous findings in platelets and other blood and vascular cells that PECAM‐1 functions by modulating ITAM‐mediated signaling pathways that amplify cellular activation.     

A role for adhesion and degranulation‐promoting adapter protein in collagen‐induced platelet activation mediated via integrin α2β1

Summary. Background: Collagen‐induced platelet activation is a key step in the development of arterial thrombosis via its interaction with the receptors glycoprotein (GP)VI and integrin α₂β₁. Adhesion and degranulation‐promoting adapter protein (ADAP) regulates α_IIbβ₃ in platelets and α_Lβ₂ in T cells, and is phosphorylated in GPVI‐deficient platelets activated by collagen. Objectives: To determine whether ADAP plays a role in collagen‐induced platelet activation and in the regulation and function of α₂β₁. Methods: Using ADAP^?/? mice and synthetic collagen peptides, we investigated the role of ADAP in platelet aggregation, adhesion, spreading, thromboxane synthesis, and tyrosine phosphorylation. Results and Conclusions: 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 α₂β₁‐selective ligand GFOGER and to a peptide (III‐04), which supports adhesion that is dependent on both GPVI and α₂β₁, 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 α₂β₁. 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.     

The PKB inhibitor Akti‐1/2 potentiates PAR‐1‐mediated platelet function independently of its ability to block PKB

Summary. Background: The role of PKB in platelet function is poorly defined due to the lack of genuinely selective small‐molecule inhibitors and limiting genetic models. Recently, a selective, non‐ATP‐competitive PKB inhibitor, Akti‐1/2 has been reported, but the efficacy and specificity against PKB activation in platelet function is unknown. Objective and methods: To determine the effect of the PKB inhibitor Akti‐1/2 on PKB activation and platelet function by Western blotting and aggregometry/flow cytometry, respectively. Results: Akti‐1/2 potently inhibited thrombin‐mediated PKB phosphorylation on Thr³⁰⁸ and Ser⁴⁷³ and phosphorylation of its downstream substrate GSK3β, with a negligible effect on the phosphorylation of pleckstrin, p38, ERK and JNK. Surprisingly, Akti‐1/2 strongly potentiated PAR‐1‐mediated platelet aggregation. This effect persisted in the presence of PI3 kinase inhibitors, indicating a mechanism of action that is independent of PKB. Potentiation of aggregation by Akti‐1/2 was associated with increased [Ca⁺⁺]_i, PKC activation and degranulation and was ablated by agents that antagonized this pathway. Conclusions: The PKB inhibitor Akti‐1/2 increases PAR‐1‐mediated platelet responses in a PKB‐independent, Ca⁺⁺/PKC‐dependent manner. This effect is strong and rapid and may impact on the therapeutic application of Akti‐1/2 and structurally related compounds.     

Small molecule targeting the Rac1‐NOX2 interaction prevents collagen‐related peptide and thrombin‐induced reactive oxygen species generation and platelet activation

Essentials

• Reactive oxygen species (ROS) generation by NOX2 plays a critical role in platelet activation.
• Rac1 regulation of NOX2 is important for ROS generation.
• Small molecule inhibitor of the Rac1‐p67^phox interaction prevents platelet activation.
• Pharmacologic targeting of Rac1‐NOX2 axis can be a viable approach for antithrombotic therapy.

Summary

Background

Platelets from patients with X‐linked chronic granulomatous disease or mice deficient in nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidase isoform NOX2 exhibit diminished reactive oxygen species (ROS) generation and platelet activation. Binding of Rac1 GTPase to p67^phox plays a critical role in NOX2 activation by facilitating the assembly of the NOX2 enzyme complex.

Objective

We tested the hypothesis that Phox‐I, a rationally designed small molecule inhibitor of Rac–p67^phox interaction, may serve as an antithrombosis agent by suppressing ROS production and platelet activation.

Results

Collagen‐related peptide (CRP) induced ROS generation in a time‐dependent manner. Platelets from Rac1^?/? mice or human platelets treated with NSC23766, a specific Rac inhibitor, produced significantly less ROS in response to CRP. Treatment of platelets with Phox‐I inhibited diverse CRP‐induced responses, including: (i) ROS generation; (ii) release of P‐selectin; (iii) secretion of ATP; (iv) platelet aggregation; and (v) phosphorylation of Akt. Similarly, incubation of platelets with Phox‐I inhibited thrombin‐induced: (i) secretion of ATP; (ii) platelet aggregation; (iii) rise in cytosolic calcium; and (iv) phosphorylation of Akt. In mouse models, intraperitoneal administration of Phox‐I inhibited: (i) collagen‐induced platelet aggregation without affecting the tail bleeding time and (ii) in vivo platelet adhesion/accumulation at the laser injury sites on the saphenous vein without affecting the time for complete cessation of blood loss.

Conclusions

Small molecule targeting of the Rac1–p67^phox interaction may present an antithrombosis regimen by preventing GPVI‐ and non‐GPVI‐mediated NOX2 activation, ROS generation and platelet function without affecting the bleeding time.

     

Detection of vascular cell adhesion molecule‐1 expression with USPIO‐enhanced molecular MRI in a mouse model of cerebral ischemia

Vascular damage plays a critical role after stroke, leading notably to edema, hemorrhages and stroke recurrence. Tools to characterize the vascular lesion are thus a real medical need. In this context, the specific nanoparticular contrast agent P03011, an USPIO (ultrasmall superparamagnetic iron oxide) conjugated to a peptide that targets VCAM‐1 (vascular cell adhesion molecule‐1), was developed to detect this major component of the vascular inflammatory response. This study aimed to make the proof of concept of the capacity of this targeted USPIO to detect VCAM‐1 with MRI after cerebral ischemia in mouse. The time course of VCAM‐1 expression was first examined by immunohistochemistry in our model of cerebral ischemia–reperfusion. Secondly, P03011 or nontargeted USPIO P03007 were injected 5 h after ischemia (100 µmol iron kg^?1; i.v.) and in vivo and ex vivo MRI were performed 24 h after ischemia onset. Double labeling immunofluorescence was then performed on brain slices in order to detect both USPIO and VCAM‐1. VCAM‐1 expression was significantly up‐regulated 24 h after ischemia in our model. In animals receiving P03011, both in vivo and ex vivo MRI performed 24 h after ischemia onset showed hypointense foci which could correspond to iron particles. Histological analysis showed a co‐localization of the targeted USPIO and VCAM‐1. This study demonstrates that VCAM‐1 detection is possible with the USPIO P03011 in a model of cerebral ischemia. This kind of contrast agent could be an interesting clinical tool to characterize ischemic lesions in terms of vascular damage. Copyright © 2012 John Wiley & Sons, Ltd.     

Functional association of phosphoinositide‐3‐kinase with platelet glycoprotein Ibα, the major ligand‐binding subunit of the glycoprotein Ib–IX–V complex

Summary. Background: The adhesion receptor glycoprotein (GP)Ib–IX–V, which binds von Willebrand factor (VWF) and other ligands, initiates platelet activation and thrombus formation at arterial shear rates, and may control other vascular processes, such as coagulation, inflammation, and platelet‐mediated tumor metastasis. The cytoplasmic C‐terminal domain of the ligand‐binding GPIbα subunit contains binding sites for filamin (residues 561–572, critically Phe568/Trp570), 14‐3‐3ζ (involving phosphorylation sites Ser587/590 and Ser609), and the phosphoinositide‐3‐kinase (PI3‐kinase) regulatory subunit, p85. Objectives: We previously showed that, as compared with wild‐type receptor, deleting the contiguous sequence 580–590 or 591–610, but not upstream sequences, of GPIbα expressed as a GPIb–IX complex in Chinese hamster ovary cells inhibited VWF‐dependent Akt phosphorylation, which is used as a read‐out for PI3‐kinase activity. Pulldown experiments using glutathione‐S‐transferase (GST)–p85 or GST–14‐3‐3ζ constructs, and competitive inhibitors of 14‐3‐3ζ binding, suggested an independent association of 14‐3‐3ζ and PI3‐kinase with GPIbα. The objective of this study was to analyze a further panel of GPIbα deletion mutations within residues 580–610. Results: We identified a novel deletion mutant, Δ591–595, that uniquely disrupts 14‐3‐3ζ binding but retains the functional p85/PI3‐kinase association. Deletion of other sequences within the 580–610 region were less discriminatory, and either partially affected p85/PI3‐kinase and 14‐3‐3ζ binding (Δ580–585, Δ586–590, Δ596–600, Δ601–605), or strongly inhibited binding of both proteins (Δ606–610). Conclusions: Together, these findings have significant implications for interpreting the functional role of p85 and/or 14‐3‐3ζ in GPIb‐dependent signaling or platelet functional studies involving truncation of the C‐terminal residues in cell‐based assays and mouse models. The Δ591–595 mutation provides another strategy for determining the function of GPIbα‐associated 14‐3‐3ζ by selective disruption of 14‐3‐3ζ but not p85/PI3‐kinase binding.     

The profibrotic cytokine transforming growth factor‐β1 increases endothelial progenitor cell angiogenic properties

Summary. Background: Transforming growth factor‐β1 (TGF‐β1) is a profibrotic cytokine that plays a major role in vascular biology, and is known to regulate the phenotype and activity of various vascular cell populations. Because most fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF), are associated with vascular remodeling, and as endothelial progenitor cells (EPCs) may be involved in this process, we investigated the impact of TGF‐β1 modulation of EPC angiogenic properties. Methods: TGF‐β1 plasma levels were determined in 64 patients with IPF and compared with those in controls. The effect of TGF‐β1 on angiogenesis was studied in vivo in a Matrigel plug model and in vitro on endothelial colony‐forming cells (ECFCs). We studied the effects of inhibiting the expression of the three main receptors of TGF‐β1 in ECFCs by using short interfering RNA. Results: Total TGF‐β1 plasma levels were significantly increased in patients with IPF as compared with controls (P < 0.0001). TGF‐β1 had proangiogenic effects in vivo by increasing hemoglobin content and blood vessel formation in Matrigel plugs implanted in C57/Bl6 mice, and in vitro by enhancing ECFC viability and migration. The effects were abolished by silencing the three main TGF‐β1 receptors. Conclusions: TGF‐β1 is proangiogenic in vivo and induces ECFC angiogenic properties in vitro, suggesting that TGF‐β1 may play a role during vascular remodeling in fibrotic disease states via EPCs.     

INS‐1 cell glucose‐stimulated insulin secretion is reduced by the downregulation of the 67 kDa laminin receptor

Understanding β cell–extracellular matrix (ECM) interactions can advance our knowledge of the mechanisms that control glucose homeostasis and improve culture methods used in islet transplantation for the treatment of diabetes. Laminin is the main constituent of the basement membrane and is involved in pancreatic β cell survival and function, even enhancing glucose‐stimulated insulin secretion. Most of the studies on cell responses towards laminin have focused on integrin‐mediated interactions, while much less attention has been paid on non‐integrin receptors, such as the 67 kDa laminin receptor (67LR). The specificity of the receptor‐ligand interaction through the adhesion of INS‐1 cells (a rat insulinoma cell line) to CDPGYIGSR‐, GRGDSPC‐ or CDPGYIGSR + GRGDSPC‐covered surfaces was evaluated. Also, the effects of the 67LR knocking down over glucose‐stimulated insulin secretion were investigated. Culture of the INS‐1 cells on the bioactive surfaces was improved compared to the low‐fouling carboxymethyl dextran (CMD) surfaces, while downregulation of the 67LR resulted in reduced cell adhesion to surfaces bearing the CDPGYIGSR peptide. Glucose‐stimulated insulin secretion was hindered by downregulation of the 67LR, regardless of the biological motif available on the biomimetic surfaces on which the cells were cultured. This finding illustrates the importance of the 67LR in glucose‐stimulated insulin secretion and points to a possible role of the 67LR in the mechanisms of insulin secretion. Copyright © 2013 John Wiley & Sons, Ltd.     

Vascular defects in gain‐of‐function fps/fes transgenic mice correlate with PDGF‐ and VEGF‐induced activation of mutant Fps/Fes kinase in endothelial cells

Summary. Background: Fps/Fes is a cytoplasmic tyrosine kinase that is abundantly expressed in the myeloid, endothelial, epithelial, neuronal and platelet lineages. Genetic manipulation in mice has uncovered potential roles for this kinase in hematopoiesis, innate immunity, inflammation and angiogenesis. Objective: We have utilized a genetic approach to explore the role of Fps/Fes in angiogenesis. Methods: A hypervascular line of mice generated by expression of a ‘gain‐of‐function’ human fps/fes transgene (fps^MF) encoding a myristoylated variant of Fps (MFps) was used in these studies. The hypervascular phenotype of this line was extensively characterized by intravital microscopy and biochemical approaches. Results: fps^MF mice exhibited 1.6–1.7‐fold increases in vascularity which was attributable to increases in the number of secondary vessels. Vessels were larger, exhibited varicosities and disorganized patterning, and were found to have defects in histamine‐induced permeability. Biochemical characterization of endothelial cell (EC) lines derived from fps^MF mice revealed that MFps was hypersensitive to activation by vascular endothelial growth factor (VEGF) and platelet‐derived growth factor (PDGF). Conclusions: MFps mediates enhanced sensitization to VEGF and PDGF signaling in ECs. We propose that this hypersensitization contributes to excessive angiogenic signaling and that this underlies the observed hypervascular phenotype of fps^MF mice. These phenotypes recapitulate important aspects of the vascular defects observed in both VEGF and angiopoietin‐1 transgenic mice. The fps/fes proto‐oncogene product therefore represents a novel player in the regulation of angiogenesis, and the fps^MF line of mice constitutes a unique new murine model for the study of this process.