CD40 ligand shedding is regulated by interaction between matrix metalloproteinase‐2 and platelet integrin αIIbβ3

Summary. Background: CD40 ligand (CD40L, CD154) in the circulatory system is mainly contained in platelets, and surface‐expressed CD40L on activated platelets is subsequently cleaved by proteolytic activity to generate soluble CD40L (sCD40L). However, the enzyme responsible for the shedding of CD40L in activated platelets has not been clearly identified yet. We have recently found that molecular interaction of matrix metalloproteinase‐2 (MMP‐2) with integrin α_IIbβ₃ is required for the enhancement of platelet activation. Objectives: To elucidate the biochemical mechanism of MMP‐2‐associated sCD40L release. Methods: Localization of MMP‐2 and CD40L in platelets was analyzed by flow cytometry and fluorescence microscopy. The release of sCD40L from activated platelets was measured by enzyme‐linked immunosorbent assay. MMP‐2 binding to α_IIbβ₃ was analyzed by immunoprecipitation and western blotting. Recombinant hemopexin‐like domain and MMP‐2‐specific inhibitor were used to characterize the nature of MMP‐2 binding and catalytic activity. Results: It was revealed that interaction of MMP‐2 with α_IIbβ₃ is required for effective production of sCD40L in activated human platelets. Platelet activation and release of sCD40L were significantly affected by inhibition of platelet‐derived MMP‐2 activity or by inhibition of binding between the enzyme and the integrin. It was also found in platelet‐rich plasma that MMP‐2 activity is responsible for generating sCD40L. Conclusions: The results presented here strongly suggest that MMP‐2 interacts with α_IIbβ₃ to regulate the shedding of CD40L exposed on the surfaces of activated human platelets.     

Vicinal thiols are required for activation of the αIIbβ3 platelet integrin

Summary. Background: Closely spaced thiols in proteins that interconvert between the dithiol form and disulfide bonds are called vicinal thiols. These thiols provide a mechanism to regulate protein function. We previously found that thiols in both αIIb and β3 of the αIIbβ3 fibrinogen receptor were required for platelet aggregation. Methods and Results: Using p‐chloromercuribenzene sulfonate (pCMBS) we provide evidence that surface thiols in αIIbβ3 are exposed during platelet activation. Phenylarsine oxide (PAO), a reagent that binds vicinal thiols, inhibits platelet aggregation and labeling of sulfhydryls in both αIIb and β3. For the aggregation and labeling studies, binding of PAO to vicinal thiols was confirmed by reversal of PAO binding with the dithiol reagent 2,3‐Dimercapto‐1‐propanesulfonic acid (DMPS). In contrast, the monothiol β‐mercaptoethanol did not reverse the effects of PAO. Additionally, PAO did not inhibit sulfhydryl labeling of the monothiol protein albumin, confirming the specificity of PAO for vicinal thiols in αIIbβ3. As vicinal thiols represent redox sensitive sites that can be regulated by reducing equivalents from the extracellular or cytoplasmic environment, they are likely to be important in regulating activation of αIIbβ3. Additionally, when the labeled integrin was passed though a lectin column containing wheat germ agglutinin and lentil lectin a substantial amount of non‐labeled αIIbβ3 eluted separately from the labeled receptor. This suggests that two populations of integrin exist on platelets that can be distinguished by thiol labeling. Conclusion: A vicinal thiol‐containing population of αIIbβ3 provides redox sensitive sites for regulation of αIIbβ3.     

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.     

RGD‐ligand mimetic antagonists of integrin αIIbβ3 paradoxically enhance GPVI‐induced human platelet activation

Summary. Background: The integrin α_IIbβ₃ is the major mediator of platelet aggregation and has, therefore, become an important target of antithrombotic therapy. Antagonists of α_IIbβ₃, for example abciximab, tirofiban and eptifibatide, are used in the treatment of acute coronary syndromes. However, in addition to effective blockade of the integrin, binding of can induce conformational changes in the integrin and can also induce integrin clustering. This class effect of RGD‐ligand mimetics might, therefore, underlie paradoxical platelet activation and thrombosis previously reported. Objectives: To examine the components of signaling pathways and functional responses in platelets that may underlie this phenomenon of paradoxical platelet activation. Methods: We assessed the effect of lotrafiban, and other α_IIbβ₃ antagonists including the clinically used drug tirofiban, on tyrosine phosphorylation of key signaling proteins in platelets by immunoblotting and also platelet functional outputs such as cytosolic calcium responses, phosphatidylserine exposure (pro‐coagulant activity) and dense granule release. Results: In all cases, no effect of α_IIbβ₃ antagonists were observed on their own, but these integrin antagonists did lead to a marked potentiation of glycoprotein VI (GPVI)‐associated FcR γ‐chain phosphorylation, activation of Src family kinases and Syk kinase. This correlated with increased dense granule secretion, cytosolic calcium response and exposure of phosphatidylserine on the platelet surface. P2Y₁₂ antagonism abolished the potentiated phosphatidylserine exposure and dense granule secretion but not the cytosolic calcium response. Conclusions: These data provide a mechanism for enhancement of platelet activity by α_IIbβ₃ inhibitors, but also reveal a potentially important signaling pathway operating from the integrin to GPVI signaling.     

A new optical imaging probe targeting αVβ3 integrin in glioblastoma xenografts

α_Vβ₃ Integrins are a widely recognized target for in vivo molecular imaging of pathological conditions such as inflammation, cancer and rheumatoid arthritis. We have evaluated the sensitivity of a new, near‐infrared fluorescence (NIRF), RGD cyclic probe (DA364) in noninvasive detection of α_Vβ₃ integrin‐overexpressing tumors. DA364's binding affinity for α_Vβ₃ integrin was first evaluated in vitro. Human α_Vβ₃ integrin‐positive, U‐87 MG glioblastoma cells were then xenografted in nude mice, and DA364 was injected intravenously (i.v.) to evaluate its in vivo distribution, specificity and sensitivity in comparison with a commercially available probe. DA364 bound α_Vβ₃ integrin on U‐87 MG cells with high affinity and specificity, both in vitro and in vivo. This binding specificity was corroborated by the strong inhibition of its tumor uptake induced by nonfluorescent, cyclic‐RGD peptides. Ex vivo analysis showed that DA364 accumulated at the tumor site, whereas very low levels were detected in liver and spleen. In conclusion, DA364 allows sensitive and specific detection of transplantable glioblastoma by NIRF imaging, and is thus a promising candidate for the elaboration of imaging and therapeutic probes for α_Vβ₃ integrin‐overexpressing tumors. Copyright © 2011 John Wiley & Sons, Ltd.     

A mutation in the β3 cytoplasmic tail causes variant Glanzmann thrombasthenia by abrogating transition of αIIbβ3 to an active state

Summary. Background: The cytoplasmic tails of α_IIb and β₃ regulate essential α_IIbβ₃ functions. We previously described a variant Glanzmann thrombasthenia mutation in the β₃ cytoplasmic tail, IVS14: ?3C>G, which causes a frameshift with an extension of β₃ by 40 residues. Objectives: The aim of this study was to characterize the mechanism by which the mutation abrogates transition of α_IIbβ₃ from a resting state to an active state. Methods: We expressed the natural mutation, termed 742ins, and three artificial mutations in baby hamster kidney (BHK) cells along with wild‐type (WT) α_IIb as follows: β₃‐742stop, a truncated mutant to evaluate the effect of deleted residues; β₃‐749stop, a truncated mutant that preserves the NPLY conserved sequence; and β₃‐749ins, in which the aberrant tail begins after the conserved sequence. Flow cytometry was used to determine ligand binding to BHK cells. Results and conclusions: Surface expression of α_IIbβ₃ of all four mutants was at least 60% of WT expression, but there was almost no binding of soluble fibrinogen following activation with activating antibodies (anti‐ligand‐induced‐binding‐site 6 [antiLIBS6] or PT25‐2). Activation of the α_IIbβ₃ mutants was only achieved when both PT25‐2 and antiLIBS6 were used together or following treatment with dithiothreitol. These data suggest that the ectodomain of the four mutants is tightly locked in a resting conformation but can be forced to become active by strong stimuli. These data and those of others indicate that the middle part of the β₃ tail is important for maintaining α_IIbβ₃ in a resting conformation.     

Enhancement of fibrinolysis by inhibiting enzymatic cleavage of precursor α2‐antiplasmin

Summary. Background and objective: Resistance of thrombi to plasmin digestion depends primarily on the amount of α₂‐antiplasmin (α₂AP) incorporated within fibrin. Circulating prolyl‐specific serine proteinase, antiplasmin‐cleaving enzyme (APCE), a homologue of fibroblast activation protein (FAP), cleaves precursor Met‐α₂AP between ‐Pro12‐Asn13‐ to yield Asn‐α₂AP, which is crosslinked to fibrin approximately 13× more rapidly than Met‐α₂AP and confers resistance to plasmin. We reasoned that an APCE inhibitor might decrease conversion of Met‐α₂AP to Asn‐α₂AP and thereby enhance endogenous fibrinolysis. Methods and results: We designed and synthesized several APCE inhibitors and assessed each vs. plasma dipeptidyl peptidase IV (DPPIV) and prolyl oligopeptidase (POP), which have amino acid sequence similarity with APCE. Acetyl‐Arg‐(8‐amino‐3,6‐dioxaoctanoic acid)‐d ‐Ala‐l ‐boroPro selectively inhibited APCE vs. DPPIV, with an apparent K_i of 5.7 nm vs. 6.1 μm , indicating that an approximately 1000‐fold greater inhibitor concentration is required for DPPIV than for APCE. An apparent K_i of 7.4 nm was found for POP inhibition, which is similar to 5.7 nm for APCE; however, the potential problem of overlapping FAP/APCE and POP inhibition was negated by our finding that normal human plasma lacks POP activity. The inhibitor construct caused a dose‐dependent decrease of APCE‐mediated Met‐α₂AP cleavage, which ultimately shortened plasminogen activator‐induced plasma clot lysis times. Incubation of the inhibitor with human plasma for 22 h did not lessen its APCE inhibitory activity, with its IC₅₀ value in plasma remaining comparable to that in phosphate buffer. Conclusion: These data establish that inhibition of APCE might represent a therapeutic approach for enhancing thrombolytic activity.     

Platelet hyperreactivity and a prothrombotic phenotype in mice with a gain‐of‐function mutation in phospholipase Cγ2

Summary. Background: Agonist‐induced platelet activation involves different signaling pathways leading to the activation of phospholipase C (PLC) β or PLCγ2. Activated PLC produces inositol 1,4,5‐trisphosphate and diacylglycerol, which trigger Ca²⁺ mobilization and the activation of protein kinase C, respectively. PLCβ is activated downstream of Gq‐coupled receptors for soluble agonists with only short interaction times in flowing blood. In contrast, PLCγ2 becomes activated downstream of receptors that interact with immobilized ligands such as the collagen receptor glycoprotein (GP) VI or activated integrins. Objective and methods: We speculated that PLCγ2 activity might be optimized for sustained but submaximal signaling to control relatively slow platelet responses. To test this hypothesis, we analyzed platelets from mice heterozygous for a gain‐of‐function mutation in the Plcg2 gene (Plcg2^Ali5/+). Results: Plcg2^Ali5/+ platelets showed enhanced Ca²⁺ mobilization, integrin activation, granule secretion and phosphatidylserine exposure upon GPVI or C‐type lectin‐like receptor‐2 stimulation. Furthermore, integrin α_IIbβ₃ outside‐in signaling was markedly enhanced in the mutant platelets, as shown by accelerated spreading on different matrices and faster clot retraction. These defects translated into virtually unlimited thrombus formation on collagen under flow in vitro and a prothrombotic phenotype in vivo. Conclusions: These results demonstrate that the enzymatic activity of PLCγ2 is tightly regulated to ensure efficient but limited platelet activation at sites of vascular injury.     

Collagen‐mimetic peptides mediate flow‐dependent thrombus formation by high‐ or low‐affinity binding of integrin α2β1 and glycoprotein VI

Summary. Background: Collagen acts as a potent surface for platelet adhesion and thrombus formation under conditions of blood flow. Studies using collagen‐derived triple‐helical peptides have identified the GXX’GER motif as an adhesive ligand for platelet integrin α2β1, and (GPO)_n as a binding sequence for the signaling collagen receptor, glycoprotein VI (GPVI). Objective: The potency was investigated of triple‐helical peptides, consisting of GXX’GER sequences within (GPO)_n or (GPP)_n motifs, to support flow‐dependent thrombus formation. Results: At a high‐shear rate, immobilized peptides containing both the high‐affinity α2β1‐binding motif GFOGER and the (GPO)_n motif supported platelet aggregation and procoagulant activity, even in the absence of von Willebrand factor (VWF). With peptides containing only one of these motifs, co‐immobilized VWF was needed for thrombus formation. The (GPO)_n but not the (GPP)_n sequence induced GPVI‐dependent platelet aggregation and procoagulant activity. Peptides with intermediate affinity (GLSGER, GMOGER) or low‐affinity (GASGER, GAOGER) α2β1‐binding motifs formed procoagulant thrombi only if both (GPO)_n and VWF were present. At a low‐shear rate, immobilized peptides with high‐ or low‐affinity α2β1‐binding motifs mediated formation of thrombi with procoagulant platelets only in combination with (GPO)_n. Conclusions: Triple‐helical peptides with specific receptor‐binding motifs mimic the properties of native collagen I in thrombus formation by binding to both platelet collagen receptors. At a high‐shear rate, either GPIb or high‐affinity (but not low‐affinity) GXX’GER mediates GPVI‐dependent formation of procoagulant thrombi. By extension, high‐affinity binding for α2β1 can control the overall platelet‐adhesive activity of native collagens.     

Nitric oxide inhibits von Willebrand factor‐mediated platelet adhesion and spreading through regulation of integrin αIIbβ3 and myosin light chain

Summary. Background: von Willebrand factor (VWF)‐mediated platelet adhesion and spreading at sites of vascular injury is a critical step in hemostasis. This process requires two individual receptors: glycoprotein Ib (GPIb)‐V‐IX and integrin α_IIbβ₃. However, little is known about the negative regulation of these events. Objectives: To examine if the endogenous platelet inhibitor nitric oxide (NO) has differential effects on adhesion, spreading and aggregation induced by immobilized VWF. Results: S‐nitrosoglutathione (GSNO) inhibited platelet aggregation on immobilized VWF under static and flow conditions, but had no effect on platelet adhesion. Primary signaling events underpinning the actions of NO required cyclic GMP but not protein kinase A. Dissecting the roles of GPIb and integrin α_IIbβ₃ demonstrated that NO targeted α_IIbβ₃‐mediated aggregation and spreading, but did not significantly influence GPIb‐mediated adhesion. To understand the relationship between the effects of NO on adhesion and subsequent aggregation, we evaluated the activation of α_IIbβ₃ on adherent platelets. NO reduced the phosphorylation of extracellular stimuli‐responsive kinase (ERK) and p38, required for integrin activation resulting in reduced binding of the activated α_IIbβ₃‐specific antibody PAC‐1 on adherent platelets. Detailed analysis of platelet spreading initiated by VWF demonstrated key roles for integrin α_IIbβ₃ and myosin light chain (MLC) phosphorylation. NO targeted both of these pathways by directly modulating integrin affinity and activating MLC phosphatase. Conclusion: These data demonstrate that initial activation‐independent platelet adhesion to VWF via GPIb is resistant to NO, however, NO inhibits GPIb‐mediated activation of α_IIbβ₃ and MLC leading to reduced platelet spreading and aggregation.     

An αIIb mutation in patients with Glanzmann thrombasthenia located in the N‐terminus of blade 1 of the β‐propeller (Asn2Asp) disrupts a calcium binding site in blade 6

Summary. Background: Studies of Glanzmann thrombasthenia (GT)‐causing mutations has generated invaluable information on the formation and function of integrin αIIbβ₃. Objective: To characterize the mutation in four siblings of an Israeli Arab family affected by GT, and to analyze the relationships between the mutant protein structure and its function using artificial mutations. Methods and Results: Sequencing disclosed a new A97G transversion in the αIIb gene predicting Asn2Asp substitution at blade 1 of the β‐propeller. Alignment with other integrin α subunits revealed that Asn2 is highly conserved. No surface expression of αIIbβ₃ was found in patients’ platelets and baby hamster kidney (BHK) cells transfected with mutated αIIb and WT β₃. Although the αIIbβ₃ was formed, the mutation impaired its intracellular trafficking. Molecular dynamics simulations and modeling of the αIIbβ₃ crystal indicated that the Asn2Asp mutation disrupts a hydrogen bond between Asn2 and Leu366 of a calcium binding domain in blade 6, thereby impairing calcium binding that is essential for intracellular trafficking of αIIbβ₃. Substitution of Asn2 to uncharged Ala or Gln partially decreased αIIbβ₃ surface expression, while substitution by negatively or positively charged residues completely abolished surface expression. Unlike αIIbβ₃, αVβ₃ harboring the Asn2Asp mutation was surface expressed by transfected BHK cells, which is consistent with the known lower sensitivity of αVβ₃ to calcium chelation compared with αIIbβ₃. Conclusion: The new GT causing mutation highlights the importance of calcium binding domains in the β‐propeller for intracellular trafficking of αIIbβ₃. The mechanism by which the mutation exerts its deleterious effect was elucidated by molecular dynamics.     

A novel mechanism of cytokine release in phagocytes induced by aggretin,a snake venom C‐type lectin protein,through CLEC‐2 ligation

Summary Background: Macrophages are major immune cells and play an important role in modulating homeostasis and the immune defense mechanism. In inflammatory responses to the infection of pathogens, macrophages are activated, producing various inflammatory mediators. Snake venom C‐type lectin proteins (snaclecs) have diverse targets, including platelet GPVI, GPIb, integrin α2β1 or CLEC‐2 expressed in platelets, endothelial cells or myeloid cells. Methods: In this study, murine macrophages (RAW 264.7 cells) and human monocytes (THP‐1) were treated with different snaclecs, including aggretin, gramicetin, trowaglerix and convulxin, in the absence or presence of LPS for 24 h. Results: The production of cytokines, such as tumor necrosis factor‐α (TNF‐α) and interleukin‐6 (IL‐6), in supernatants was measured by ELISA. Aggretin increased the production of TNF‐α and IL‐6 in both RAW264.7 and THP‐1 cells; however, the other snaclecs did not. Aggretin induced extracellular signal‐regulated kinase 1/2 (ERK1/2) and c‐Jun N‐terminal kinase (JNK) tyrosine phosphorylation of RAW264.7 cells. Pretreatments with inhibitor of ERK, JNK, p38 or NF‐κB abolished cytokine release caused by aggretin. Aggretin bound to THP‐1 cells in a concentration‐dependent manner and it displaced the CLEC‐2 mAb binding to THP‐1 cells and the immobilized aggretin selectively bound to CLEC‐2 of both platelets and THP‐1 cell lysates. Furthermore, aggretin elevated the plasma level of IL‐6 in ICR mice as it was administered intramuscularly. Conclusion: These results indicate that aggretin may induce cytokine TNF‐α/IL‐6 release via interacting with CLEC‐2 receptor and the subsequent MAPK and NF‐κB activation in monocytes/macrophages.     

Triple heterozygosity in the integrin αIIb subunit in a patient with Glanzmann's thrombasthenia

Summary. We report triple heterozygosity in the integrin α_IIb subunit in a 5‐year‐old Canadian girl with Glanzmann's thrombasthenia. The patient has a severe bleeding history possibly aggravated by low VWF suggestive of associated type 1 von Willebrand's disease. Platelet aggregation was absent or severely reduced for all physiologic agonists. Flow cytometry showed an ～ 4% residual surface expression of α_IIbβ₃. Western blotting confirmed a low platelet expression of both subunits. PCR‐SSCP and direct sequencing showed no abnormalities in the β₃ gene, but revealed a G→A transition at a splice site [IVS 19 (+1)] of exon 19 in the α_IIb gene. Of maternal inheritance, the splice site mutation was associated with intermediate levels of α_IIbβ₃ in carriers. Unexpectedly, two G→A transitions were detected in exon 29 of the α_IIb gene and led to V⁹⁵¹→M and A⁹⁵⁸→T amino acid substitutions. Family studies using restriction enzymes showed that both exon 29 mutations were paternal in origin and cosegregated across three generations. Transient expression in which mutated α_IIb was cotransfected with wild‐type β₃ in COS‐7 cells showed that V⁹⁵¹→M gave a much reduced surface expression of α_IIbβ₃ and a block in the maturation of pro‐α_IIb. In contrast, the A⁹⁵⁸ substitution appeared to be a novel polymorphism. Our studies highlight an unusual mixture of defects giving rise to severe bleeding in a child and describe the first pathological missense mutation affecting a C‐terminal residue of the calf‐2 domain of α_IIb.     

An αIIbβ3 antagonist prevents thrombosis without causing Fc receptor γ‐chain IIa‐mediated thrombocytopenia

Essentials

• FcγRIIa‐mediated thrombocytopenia is associated with drug‐dependent antibodies (DDAbs).
• We investigated the correlation between α_IIbβ₃ binding epitopes and induction of DDAbs.
• An FcγRIIa‐transgenic mouse model was used to evaluate thrombocytopenia among anti‐thrombotics.
• An antithrombotic with binding motif toward α_IIbβ‐propeller domain has less bleeding tendency.

Summary

Background

Thrombocytopenia, a common side effect of Arg‐Gly‐Asp‐mimetic antiplatelet drugs, is associated with drug‐dependent antibodies (DDAbs) that recognize conformation‐altered integrin α_IIbβ₃.

Objective

To explore the correlation between α_IIbβ₃ binding epitopes and induction of DDAb binding to conformation‐altered α_IIbβ₃, we examined whether two purified disintegrins, TMV‐2 and TMV‐7, with distinct binding motifs have different effects on induction of α_IIbβ₃ conformational change and platelet aggregation in the presence of AP2, an IgG₁ inhibitory mAb raised against α_IIbβ₃.

Methods

We investigated the possible mechanisms of intrinsic platelet activation of TMV‐2 and TMV‐7 in the presence of AP2 by examining the signal cascade, tail bleeding time and immune thrombocytopenia in Fc receptor γ‐chain IIa (FcγRIIa) transgenic mice.

Results

TMV‐7 has a binding motif that recognizes the α_IIb β‐propeller domain of α_IIbβ₃, unlike that of TMV‐2. TMV‐7 neither primed the platelets to bind ligand, nor caused a conformational change of α_IIbβ₃ as identified with the ligand‐induced binding site mAb AP5. In contrast to eptifibatide and TMV‐2, cotreatment of TMV‐7 with AP2 did not induce FcγRIIa‐mediated platelet aggregation and the downstream activation cascade. Both TMV‐2 and TMV‐7 efficaciously prevented occlusive thrombosis in vivo. Notably, both eptifibatide and TMV‐2 caused severe thrombocytopenia mediated by FcγRIIa, prolonged tail bleeding time in vivo, and repressed human whole blood coagulation indexes, whereas TMV‐7 did not impair hemostatic capacity.

Conclusions

TMV‐7 shows antiplatelet and antithrombotic activities resulting from a mechanism different from that of all other tested α_IIbβ₃ antagonists, and may offer advantages as a therapeutic agent with a better safety profile.     

Rap1b is critical for glycoprotein VI-mediated but not ADP receptor-mediated α2β1 activation

Summary. Background: The platelet α₂β₁ integrin functions as both an adhesion and signaling receptor upon exposure to collagen. Recent studies have indicated that α₂β₁ function can be activated via inside‐out signaling, similar to the prototypical platelet integrin α_IIbβ₃. However, signaling molecules that regulate α₂β₁ activation in platelets are not well defined. A strong candidate molecule is the small GTPase Rap1b, the dominant platelet isoform of Rap1, which regulates α_IIbβ₃ activation. Objectives: We hypothesized that Rap1b positively regulates α₂β₁ during agonist‐induced platelet activation. Methods: To test whether Rap1b activates α₂β₁ downstream of glycoprotein (GP)VI or other platelet receptors, we stimulated platelets purified from Rap1b^?/? or wild‐type mice with diverse agonists and measured α₂β₁ activation using fluorescein isothiocyanate‐labeled monomeric collagen. We also examined the role of Rap1b in outside‐in signaling pathways by analyzing adhesion and spreading of Rap1b^?/? or wild‐type platelets on monomeric, immobilized collagen. Finally, we monitored the activation status of related Rap GTPases to detect changes in signaling pathways potentially associated with Rap1b‐mediated events. Results: Rap1b^?/? platelets displayed comparable ADP‐induced or thrombin‐induced α₂β₁ activation as wild‐type platelets, but reduced convulxin‐dependent α₂β₁ activation. Rap1b^?/? platelets exhibited increased spreading on immobilized collagen but similar adhesion to immobilized collagen compared to wild‐type platelets. Rap1b^?/? platelets also showed Rap1a and Rap2 activation upon agonist stimulation, possibly revealing functional compensation among Rap family members. Conclusions: Rap1b is required for maximal GPVI‐induced but not ADP‐induced activation of α₂β₁ in murine platelets.     

Activation of integrin αIIbβ3 in the glycoprotein Ib-high population of a megakaryocytic cell line, CMK, by inside-out signaling

Summary. Affinity/avidity state of integrin α_IIbβ₃ is regulated by intracellular inside‐out signaling. Although several megakaryocytic cell lines have been established, soluble ligand binding to α_IIbβ₃ expressed in these cells by cellular agonists has not been demonstrated. We have re‐examined agonist‐induced α_IIbβ₃ activation on megakaryocytic cell lines with a marker of the late stage of megakaryocytic differentiation, glycoprotein Ib (GPIb). Activation of α_IIbβ₃ was assessed by PAC1 and soluble fibrinogen binding to the cells. We found that α_IIbβ₃ expressed in CMK cells with high GPIb expression was activated by a phorbor ester, phorbol myristate acetate (PMA). Although the population of the GPIb^high cells was <0.5% of the total cells, incubation with a nucleoside analog, ribavirin, efficiently increased the PMA‐reactive GPIb^high cells. Not only PMA but also a calcium ionophore, A23187, induced α_IIbβ₃ activation, and PMA and A23187 had an additive effect on α_IIbβ₃ activation. Ligand binding to the activated α_IIbβ₃ in the GPIb^high CMK cells is totally abolished by an α_IIbβ₃‐specific antagonist, and inhibited by wortmannin, cytochalasin‐D and prostaglandin E₁, and the effects of these inhibitors on α_IIbβ₃ activation in the GPIb^high CMK cells were compatible with those in platelets. We have also demonstrated that the ribavirin‐treated CMK cells express PKC‐α, ‐β, ‐δ and ‐θ, and suggested that PKC‐α and/or ‐β appear to be responsible for PMA‐induced activation of α_IIbβ₃ in CMK cells.     

TC21/RRas2 regulates glycoprotein VI–FcRγ‐mediated platelet activation and thrombus stability

Essentials

• RAS proteins are expressed in platelets but their functions are largely uncharacterized.
• TC21/RRas2 is required for glycoprotein VI‐induced platelet responses and for thrombus stability in vivo.
• TC21 regulates platelet aggregation by control of α_IIbβ₃ integrin activation, via crosstalk with Rap1b.
• This is the first indication of functional importance of a proto‐oncogenic RAS protein in platelets.

Summary

Background

Many RAS family small GTPases are expressed in platelets, including RAC, RHOA, RAP, and HRAS/NRAS/RRAS1, but most of their signaling and cellular functions remain poorly understood. Like RRAS1, TC21/RRAS2 reverses HRAS‐induced suppression of integrin activation in CHO cells. However, a role for TC21 in platelets has not been explored.

Objectives

To determine TC21 expression in platelets, TC21 activation in response to platelet agonists, and roles of TC21 in platelet function in in vitro and in vivo thrombosis.

Results

We demonstrate that TC21 is expressed in human and murine platelets, and is activated in response to agonists for the glycoprotein (GP) VI–FcRγ immunoreceptor tyrosine‐based activation motif (ITAM)‐containing collagen receptor, in an Src‐dependent manner. GPVI‐induced platelet aggregation, integrin α_IIbβ₃ activation, and α‐granule and dense granule secretion, as well as phosphorylation of Syk, phospholipase Cγ2, AKT, and extracellular signal‐regulated kinase, were inhibited in TC21‐deficient platelets ex vivo. In contrast, these responses were normal in TC21‐deficient platelets following stimulation with P2Y, protease‐activated receptor 4 and C‐type lectin receptor 2 receptor agonists, indicating that the function of TC21 in platelets is GPVI–FcRγ‐ITAM‐specific. TC21 was required for GPVI‐induced activation of Rap1b. TC21‐deficient mice did not show a significant delay in injury‐induced thrombosis as compared with wild‐type controls; however, thrombi were unstable. Hemostatic responses showed similar effects.

Conclusions

TC21 is essential for GPVI–FcRγ‐mediated platelet activation and for thrombus stability in vivo via control of Rap1b and integrins.

     

Human target validation of phosphoinositide 3‐kinase (PI3K)β: effects on platelets and insulin sensitivity,using AZD6482 a novel PI3Kβ inhibitor

Nylander S, Kull B, Björkman JA, Ulvinge JC, Oakes N, Emanuelsson BM, Andersson M, Skärby T, Inghardt T, Fjellström O, Gustafsson D. Human target validation of phosphoinositide 3‐kinase (PI3K)β:effects on platelets and insulin sensitivity, using AZD6482 a novel PI3Kβ inhibitor. J Thromb Haemost 2012; 10: 2127–36. See also Jackson SP, Schoenwaelder SM. Antithrombotic phosphoinositide 3‐kinase β inhibitors in humans – a ‘shear’ delight! This issue, pp 2123–6. Summary. Background: Based on in vitro and animal data, PI3Kβ is given an important role in platelet adhesion and aggregation but its role in insulin signaling is unclear. Objective: To strengthen the PI3Kβ target validation using the novel, short‐acting inhibitor AZD6482. Methods and results: AZD6482 is a potent, selective and ATP competitive PI3Kβ inhibitor (IC₅₀ 0.01 μm ). A maximal anti‐platelet effect was achieved at 1 μm in the in vitro and ex vivo tests both in dog and in man. In dog, in vivo AZD6482 produced a complete anti‐thrombotic effect without an increased bleeding time or blood loss. AZD6482 was well tolerated in healthy volunteers during a 3‐h infusion. The ex vivo anti‐platelet effect and minimal bleeding time prolongation in the dog model translated well to data obtained in healthy volunteers. AZD6482 inhibited insulin‐induced human adipocyte glucose uptake in vitro (IC₅₀ of 4.4 μm ). In the euglycemic hyperinsulinemic clamp model, in rats, glucose infusion rate was not affected at 2.3 μm but reduced by about 60% at a plasma exposure of 27 μm . In man, the homeostasis model analysis (HOMA) index increased by about 10–20% at the highest plasma concentration of 5.3 μm . Conclusions: This is the first human target validation for PI3Kβ inhibition as anti‐platelet therapy showing a mild and generalized antiplatelet effect attenuating but not completely inhibiting multiple signaling pathways with an impressive separation towards primary hemostasis. AZD6482 at ‘supratherapeutic’ plasma concentrations may attenuate insulin signaling, most likely through PI3Kα inhibition.     

Evaluation of fibrinogen self‐assembly: role of its αC region

Summary. Background: Exposure of cryptic, functional sites on fibrinogen upon its adsorption to hydrophobic surfaces of biomaterials has been linked to an inflammatory response and fibrosis. Such adsorption also induces ordered fibrinogen aggregation which is poorly understood. Objective: To investigate hydrophobic surface‐induced fibrinogen aggregation. Methods: Contact and lateral force scanning probe microscopy, yielding topography, image dimensions and fiber elastic modulus measurements were used along with transmission and scanning electron microscopy. Fibrinogen aggregation was induced under non‐enzymatic conditions by adsorption on a trioctyl‐surface monolayer (trioctylmethylamine) grafted onto silica clay plates. Results: A more than one molecule thick coating was generated by adsorption on the plate from 100 to 200 μg mL^?1 fibrinogen solutions, and three‐dimensional networks formed from 4 mg mL^?1 fibrinogen incubated with uncoated or fibrinogen‐coated plates. Fibrils appeared laterally assembled into branching and overlapping fibers whose heights from the surface ranged from approximately 3 to 740 nm. The elastic modulus of fibrinogen fibers was 1.55 MPa. No fibrils formed when fibrinogen lacking αC‐domains was used as a coating or was incubated with intact fibrinogen‐coated plates, or when the latter plates were sequentially incubated with anti‐Aα529–539 mAb and intact fibrinogen. When an anti‐Aα241–476 mAb was used instead, fine, long fibers formed. Similarly, sequential incubations of fibrinogen‐coated plates with recombinant αC‐domain (Aα392–610 fragment) or αC‐connector (Aα221–372 fragment) and fibrinogen resulted in distinctly fine fiber networks. Conclusions: Adsorption‐induced fibrinogen self‐assembly is initiated by a more than one molecule‐thick surface layer and eventuates in three‐dimensional networks whose formation requires fibrinogen with intact αC‐domains.     

The interaction between β1 integrins and ERK1/2 in osteogenic differentiation of human mesenchymal stem cells under fluid shear stress modelled by a perfusion system

Fluid shear stress (FSS) is an important biomechanical factor regulating the osteogenic differentiation of human mesenchymal stem cells (hMSCs) and is therefore widely used in bone tissue engineering. However, the mechanotransduction of FSS in hMSCs remains largely unknown. As β1 integrins are considered to be important mechanoreceptors in other cells, we suspect that β1 integrins should also be important for hMSCs to sense the stimulation of FSS. We used a perfusion culture system to produce FSS loading on hMSCs seeded in PLGA three‐dimensional (3D) scaffolds and investigated the roles of β1 integrins, FAK and ERK1/2 in FSS‐induced osteogenic differentiation of hMSCs. Our results showed that FSS not only markedly increased ALP activity and the expression of ALP, OCN, Runx2 and COLIα genes but also significantly enhanced the phosphorylation of ERK1/2, Runx2 and FAK. FSS‐induced activation of ERK1/2 and FAK was inhibited by blockade of the connection between β1 integrins and ECM with RGDS peptide and integrins β1 monoclonal antibody. Our study also found that FSS could upregulate the expression level of β1 integrins and that this upregulation could be abolished by PD98059. Further investigation indicated that FSS‐activated ERK1/2 led to the phosphorylation of IκBα and NFκB p65. The activation of NFκB p65 resulted in the upregulation of β1 integrin expression. Therefore, it could be inferred that β1 integrins should sense the stimulation of FSS and thus activate ERK1/2 through activating of FAK, and FSS‐activated ERK1/2 feedback to upregulate the expression of β1 integrins through activating NFκB. Copyright © 2012 John Wiley & Sons, Ltd.