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.     

Redox control of β2‐glycoprotein I–von Willebrand factor interaction by thioredoxin‐1

Summary. Background: β₂‐Glycoprotein I (β₂GPI) is an abundant plasma protein that is closely linked to blood clotting, as it interacts with various protein and cellular components of the coagulation system. However, the role of β₂GPI in thrombus formation is unknown. We have recently shown that β₂GPI is susceptible to reduction by the thiol oxidoreductases thioredoxin‐1 and protein disulfide isomerase, and that reduction of β₂GPI can take place on the platelet surface. Methods: β₂GPI, reduced by thioredoxin‐1, was labeled with the selective sulfhydryl probe N^a‐(3‐maleimidylpropionyl)biocytin and subjected to mass spectrometry to identify the specific cysteines involved in the thiol exchange reaction. Binding assays were used to examine the affinity of reduced β₂GPI for von Willebrand factor (VWF) and the effect of reduced β2GPI on glycoprotein (GP)Ibα binding to VWF. Platelet adhesion to ristocetin‐activated VWF was studied in the presence of reduced β₂GPI. Results: We demonstrate that the Cys288–Cys326 disulfide in domain V of β₂GPI is the predominant disulfide reduced by thioredoxin‐1. Reduced β₂GPI in vitro displays increased binding to VWF that is dependent on disulfide bond formation. β₂GPI reduced by thioredoxin‐1, in comparison with non‐reduced β₂GPI, leads to increased binding of GPIbα to VWF and increased platelet adhesion to activated VWF. Conclusions: Given the importance of thiol oxidoreductases in thrombus formation, we provide preliminary evidence that the thiol‐dependent interaction of β₂GPI with VWF may contribute to the redox regulation of platelet adhesion.     

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.     

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.     

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.     

The glycoprotein Ibα–von Willebrand factor interaction induces platelet apoptosis

Summary. Background: The interaction of glycoprotein (GP) Ibα with von Willebrand factor (VWF) initiates platelet adhesion, and simultaneously triggers intracellular signaling cascades leading to platelet aggregation and thrombus formation. Some of the signaling events are similar to those occurring during apoptosis, however, it is still unclear whether platelet apoptosis is induced by the GPIbα–VWF interaction. Objectives: To investigate whether the GPIbα–VWF interaction induces platelet apoptosis and the role of 14‐3‐3ζ in apoptotic signaling. Methods: Apoptotic events were assessed in platelets or Chinese hamster ovary (CHO) cells expressing wild‐type (1b9) or mutant GPIb–IX interacting with VWF by flow cytometry or western blotting. Results: Ristocetin‐induced GPIbα–VWF interaction elicited apoptotic events in platelets, including phosphatidylserine exposure, elevations of Bax and Bak, gelsolin cleavage, and depolarization of mitochondrial inner transmembrane potential. Apoptotic events were also elicited in platelets exposed to pathologic shear stresses in the presence of VWF; however, the shear‐induced apoptosis was eliminated by the anti‐GPIbα antibody AK2. Furthermore, apoptotic events occurred in 1b9 cells stimulated with VWF and ristocetin, but were significantly diminished in two CHO cell lines expressing mutant GPIb–IX with GPIbα truncated at residue 551 or a serine‐to‐alanine mutation at the 14‐3‐3ζ‐binding site in GPIbα. Conclusions: This study demonstrates that the GPIbα–VWF interaction induces apoptotic events in platelets, and that the association of 14‐3‐3ζ with the cytoplasmic domain of GPIbα is essential for apoptotic signaling. This finding may suggest a novel mechanism for platelet clearance or some thrombocytopenic diseases.     

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 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.     

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.     

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.     

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.     

Improvements in endotoxemic syndromes using a disintegrin,rhodostomin, through integrin αvβ3‐dependent pathway

Summary. Background and objectives: Septic shock is a major cause of morbidity and mortality in intensive care units, but there is still no effective therapy for the patients. We evaluated the effects of rhodostomin (Rn), an Arg‐Gly‐Asp‐containing snake venom disintegrin, on lipopolysaccharide (LPS)‐activated phagocytes in vitro and LPS‐induced endotoxemia in vivo. Methods and results: Rn inhibited adhesion, migration, cytokine production and mitogen‐activated protein kinase (MAPK) activation of macrophage induced by LPS. Flow cytometric analysis revealed that Rn specifically blocked anti‐αv mAb binding to RAW264.7. Besides inhibiting MAPK activation of THP‐1, Rn bound to LPS‐activated THP‐1 and specifically blocked anti‐αvβ3 mAb binding to THP‐1. Binding assays proved that integrin αvβ3 was the binding site for rhodostomin on phagocytes. Rn reversed the enhancement of fibronectin and vitronectin on LPS‐induced monocyte adhesion and cytokine release. Transfection of integrin αv siRNA also inhibited LPS‐induced activation of monocyte, and Rn exerted no further inhibitory effect. Furthermore, Rn significantly decreased the production of tumor necrosis factor‐α (TNF‐a), interleukin (IL)‐6, ‐1β and ‐10 and attenuated cardiovascular dysfunction, including blood pressure and heart pulse, and thrombocytopenia in LPS‐induced endotoxemic mice. Rn also protected against tissue inflammation as evidenced by histological examination. Conclusions: Rn may interact with αvβ3 integrin of monocytes/macrophages leading to interfere with the activation of phagocytes triggered by LPS. These results suggest that the protective function of Rn in LPS‐induced endotoxemia may be attributed to its anti‐inflammation activities in vivo.     

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.     

A two‐step coagulation test to identify antiβ2‐glycoprotein I lupus anticoagulants

Summary. Lupus anticoagulants (LA) are immunoglobulins which inhibit phospholipid (PL)‐dependent coagulation tests. LA are not specific, as they may reflect the presence of antibodies to human prothrombin, human β₂‐Glycoprotein I (β₂GPI), an association of previous antibodies or other antibodies. Antibodies to human β₂GPI act as in vitro anticoagulants by enhancing the binding of β₂GPI to PL, and this binding may be influenced by calcium ion concentration. A reduction in final calcium concentration, from 10 mm to 5 mm , increased coagulation times in both dilute Russell Viper Venom Time (dRVVT) and dilute Prothrombin Time (dPT) when plasmas of patients with antiβ₂GPI antibodies were used. Ten LA patients showed increased dRVVT and dPT ratios from means of 1.5 to 1.7 (P < 0.001) and 2.4 to 4.3 (P = 0.002), respectively. Instead, all LA‐positive antiβ₂GPI antibody‐negative patients showed decreased coagulation times from mean ratios of 1.5 to 1.3 (P = 0.004) in dRVVT and from 2.0 to 1.5 (P = 0.01) in dPT. These results are confirmed by running dRVVT of normal plasma spiked with affinity purified IgG antiβ₂GPI antibodies. Therefore, when a PL–dependent coagulation test is run twice, at different final calcium concentrations, antiβ₂GPI LA can be identified.     

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.     

Effects of variation in the human α2A‐ and α2C‐adrenoceptor genes on cognitive tasks and pain perception

Background: The mechanisms underlying interindividual variability in pain perception and cognitive responses are undefined but highly heritable. α_2C‐ and α_2A‐adrenergic receptors regulate noradrenergic activity and are important mediators of pain perception and analgesia. We hypothesized that common genetic variants in these genes, particularly the ADRA2C 322–325 deletion variant, affect pain perception or cognitive responses. Methods: We studied 73 healthy subjects (37 Caucasians and 36 African–Americans) aged 25.4 ± 4.6 years. Pain response to a cold pressor test was measured using a 10 cm visual analog scale and again on the next day, after three infusions of the selective α₂‐agonist dexmedetomidine. Standardized cognitive tests were administered at baseline and after each infusion. The contribution of ADRA2C deletion genotype, dexmedetomidine concentration, and other covariates to pain perception and cognitive responses was determined using multiple linear regression models. Secondary analysis examined the effects of ADRA2A and other ADRA2C variants on pain perception. Results: ADRA2C Del homozygotes had higher pain scores in response to cold at baseline (6.3 ± 1.8 cm) and after dexmedetomidine (5.6 ± 2.2 cm) than insertion allele carriers (4.6 ± 2.1 cm [baseline] and 3.8 ± 1.9 cm [after dexmedetomidine]; adjusted P‐values = 0.019 and 0.004, respectively). Cognitive responses were unrelated to ADRA2C Ins/Del genotype. None of the other ADRA2A and ADRA2C variants was significantly related to cold pain sensitivity before dexmedetomidine; after dexmedetomidine, ADRA2A rs1800038 was marginally associated (P = 0.03). Conclusion: The common ADRA2C del322–325 variant affected pain perception before and after dexmedetomidine but did not affect other cognitive responses, suggesting that it contributes to interindividual variability in pain perception.     

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.

     

Induction of anti‐β2‐glycoprotein I autoantibodies in mice by protein H of Streptococcus pyogenes

Summary. Background: The antiphospholipid syndrome (APS) is characterized by the persistent presence of anti‐β₂‐glycoprotein I (β₂‐GPI) autoantibodies. β₂‐GPI can exist in two conformations. In plasma it is a circular protein, whereas it adopts a fish‐hook conformation after binding to phospholipids. Only the latter conformation is recognized by patient antibodies. β₂‐GPI has been shown to interact with Streptococcus pyogenes. Objective: To evaluate the potential of S. pyogenes‐derived proteins to induce anti‐β₂‐GPI autoantibodies. Methods and results: Four S. pyogenes surface proteins (M1 protein, protein H, streptococcal collagen‐like protein A [SclA], and streptococcal collagen‐like protein B [SclB]) were found to interact with β₂‐GPI. Only binding to protein H induces a conformational change in β₂‐GPI, thereby exposing a cryptic epitope for APS‐related autoantibodies. Mice were injected with the four proteins. Only mice injected with protein H developed antibodies against the patient antibody‐related epitope in domain I of β₂‐GPI. Patients with pharyngotonsillitis caused by S. pyogenes who developed anti‐protein H antibodies also generated anti‐β₂‐GPI antibodies. Conclusions: Our study has demonstrated that a bacterial protein can induce a conformational change in β₂‐GPI, resulting in the formation of antiβ₂‐GPI autoantibodies. This constitutes a novel mechanism for the formation of anti‐β₂‐GPI autoantibodies.