Differential roles of integrins alpha(2)beta(1), and alpha(IIb)beta(3) in collagen and CRP-induced platelet activation

Collagen and collagen-related peptide (CRP) activate platelets by interacting with glycoprotein (GP)VI. In addition, collagen binds to integrin alpha(2)beta(1) and possibly to other receptors. In this study, we have compared the role of integrins alpha(2)beta(1) and alpha(IIb)beta(3) in platelet activation induced by collagen and CRP. Inhibitors of ADP and thromboxane A(2) (TxA(2)) substantially attenuated collagen-induced platelet aggregation and dense granule release, whereas CRP-induced responses were only partially inhibited. Under these conditions, a proportion of platelets adhered to the collagen fibres resulting in dense granule release and alpha(IIb)beta(3) activation. This adhesion was substantially mediated by alpha(2)beta(1). The alpha(IIb)beta(3) antagonist lotrafiban potentiated CRP-induced dense granule release, suggesting that alpha(IIb)beta(3) outside-in signalling may attenuate GPVI signals. By contrast, lotrafiban inhibited collagen-induced dense granule release. These results emphasise the differential roles of alpha(2)beta(1) and alpha(IIb)beta(3) in platelet activation induced by collagen and CRP. Further, they show that although ADP and TxA(2) greatly facilitate collagen-induced platelet activation, collagen can induce full activation of those platelets to which it binds in the absence of these mediators, via a mechanism that is dependent on adhesion to alpha(2)beta(1).

Distinct roles of GPVI and integrin alpha(2)beta(1) in platelet shape change and aggregation induced by different collagens

1 Various platelet membrane glycoproteins have been proposed as receptors for collagen, in some cases as receptors For specific collagen types. In this study we have compared the ability of a range of collagen types to activate platelets.

Functional characterization of alpha-adrenoceptors mediating pupillary dilation in rats

Previously, we reported that the alpha(1A)-adrenoceptor, but not the alpha(1D)-adrenoceptor, mediates pupillary dilation elicited by sympathetic nerve stimulation in rats. This study was undertaken to further characterize the alpha-adrenoceptor subtypes mediating pupillary dilation in response to both neural and agonist activation. Pupillary dilator response curves were generated by intravenous injection of norepinephrine in pentobarbital-anesthetized rats. Involvement of alpha(1)-adrenoceptors was established as mydriatic responses were inhibited by systemic administration of nonselective alpha-adrenoceptor antagonists, phentolamine (0.3-3 mg/kg) and phenoxybenzamine (0.03-0.3 mg/kg), as well as by the selective alpha(1)-adrenoceptor antagonist, prazosin (0.3 mg/kg). The alpha(2)-adrenoceptor antagonist, rauwolscine (0.5 mg/kg), was without antagonistic effects. alpha(1A)-Adrenoceptor selective antagonists, 2-([2,6-dimethoxyphenoxyethyl]aminomethyl)-1,4-benzodioxane (WB-4101; 0.1-1 mg/kg) and 5-methylurapidil (0.1-1 mg/kg), the alpha(1B)-adrenoceptor selective antagonist, 4-amino-2-[4-[1-(benzyloxycarbonyl)-2(S)- [[(1,1-dimethylethyl)amino]carbonyl]-piperazinyl]-6,7-dimethoxyquinazoline (L-765314; 0.3-1 mg/kg), as well as the alpha(1D)-adrenoceptor selective antagonist, 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione (BMY-7378; 1 mg/kg), were used to delineate the adrenoceptor subtypes involved. Mydriatic responses to norepinephrine were significantly antagonized by intravenous administration of both WB-4101 and 5-methylurapidil, but neither by L-765314 nor by BMY-7378. L-765314 (0.3-3 mg/kg, i.v.) was also ineffective in inhibiting the mydriasis evoked by cervical sympathetic nerve stimulation. These results suggest that alpha(1B)-adrenoceptors do not mediate sympathetic mydriasis in rats, and that the alpha(1A)-adrenoceptor is the exclusive subtype mediating mydriatic responses in this species.

alpha(1A)-adrenoceptors mediate sympathetically evoked pupillary dilation in rats

Evidence suggests that in some species (cats, rabbits, and possibly humans) alpha-adrenoceptors in the iris dilator muscle are "atypical" in that they cannot be readily classified by conventional criteria. This study was undertaken in an attempt to characterize the alpha-adrenoceptor subtype(s) mediating sympathetically elicited mydriasis in rats. Frequency-response pupillary dilator curves were generated by stimulation of the preganglionic cervical sympathetic nerve (1-32 Hz) in pentobarbital-anesthetized rats. Evoked responses were inhibited by systemic administration of nonselective alpha-adrenergic antagonists, phentolamine (0.3-10 mg/kg) and phenoxybenzamine (0.03-1 mg/kg). The selective alpha(1)-adrenergic antagonist, prazosin (0.01-1 mg/kg), also was effective, although alpha(2)-adrenergic antagonism with rauwolscine (0.1-1 mg/kg) was not. alpha(1A)-Adrenoceptor-selective antagonists, 2-([2,6-dimethoxyphenoxyethyl]aminomethyl)-1,4-benzodioxane (WB-4101; 0.1-1 mg/kg) and 5-methylurapidil (0.1-1 mg/kg), as well as the alpha(1D)-adrenoceptor-selective antagonist 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione (BMY-7378; 1-3 mg/kg), were used to determine the subtype(s) involved. Evoked mydriasis was significantly antagonized by both WB-4101 and 5-methylurapidil but not by BMY-7378. These results suggest that, unlike some other species, adrenoceptors in the rat iris dilator mediating neurogenic mydriasis are "typical" and, in addition, can be characterized as being primarily of the alpha(1A)-adrenoceptor subtype.

Alpha-2 agonists for sedation of mechanically ventilated adults in intensive care units: a systematic review

BACKGROUND: Care of critically ill patients in intensive care units (ICUs) often requires potentially invasive or uncomfortable procedures, such as mechanical ventilation (MV). Sedation can alleviate pain and discomfort, provide protection from stressful or harmful events, prevent anxiety and promote sleep. Various sedative agents are available for use in ICUs. In the UK, the most commonly used sedatives are propofol (Diprivan(®), AstraZeneca), benzodiazepines [e.g. midazolam (Hypnovel(®), Roche) and lorazepam (Ativan(®), Pfizer)] and alpha-2 adrenergic receptor agonists [e.g. dexmedetomidine (Dexdor(®), Orion Corporation) and clonidine (Catapres(®), Boehringer Ingelheim)]. Sedative agents vary in onset/duration of effects and in their side effects. The pattern of sedation of alpha-2 agonists is quite different from that of other sedatives in that patients can be aroused readily and their cognitive performance on psychometric tests is usually preserved. Moreover, respiratory depression is less frequent after alpha-2 agonists than after other sedative agents.

OBJECTIVES: To conduct a systematic review to evaluate the comparative effects of alpha-2 agonists (dexmedetomidine and clonidine) and propofol or benzodiazepines (midazolam and lorazepam) in mechanically ventilated adults admitted to ICUs.

DATA SOURCES: We searched major electronic databases (e.g. MEDLINE without revisions, MEDLINE In-Process & Other Non-Indexed Citations, EMBASE and Cochrane Central Register of Controlled Trials) from 1999 to 2014.

METHODS: Evidence was considered from randomised controlled trials (RCTs) comparing dexmedetomidine with clonidine or dexmedetomidine or clonidine with propofol or benzodiazepines such as midazolam, lorazepam and diazepam (Diazemuls(®), Actavis UK Limited). Primary outcomes included mortality, duration of MV, length of ICU stay and adverse events. One reviewer extracted data and assessed the risk of bias of included trials. A second reviewer cross-checked all the data extracted. Random-effects meta-analyses were used for data synthesis.

RESULTS: Eighteen RCTs (2489 adult patients) were included. One trial at unclear risk of bias compared dexmedetomidine with clonidine and found that target sedation was achieved in a higher number of patients treated with dexmedetomidine with lesser need for additional sedation. The remaining 17 trials compared dexmedetomidine with propofol or benzodiazepines (midazolam or lorazepam). Trials varied considerably with regard to clinical population, type of comparators, dose of sedative agents, outcome measures and length of follow-up. Overall, risk of bias was generally high or unclear. In particular, few trials blinded outcome assessors. Compared with propofol or benzodiazepines (midazolam or lorazepam), dexmedetomidine had no significant effects on mortality [risk ratio (RR) 1.03, 95% confidence interval (CI) 0.85 to 1.24, I (2) = 0%; p = 0.78]. Length of ICU stay (mean difference -1.26 days, 95% CI -1.96 to -0.55 days, I (2) = 31%; p = 0.0004) and time to extubation (mean difference -1.85 days, 95% CI -2.61 to -1.09 days, I (2) = 0%; p < 0.00001) were significantly shorter among patients who received dexmedetomidine. No difference in time to target sedation range was observed between sedative interventions (I (2) = 0%; p = 0.14). Dexmedetomidine was associated with a higher risk of bradycardia (RR 1.88, 95% CI 1.28 to 2.77, I (2) = 46%; p = 0.001).

LIMITATIONS: Trials varied considerably with regard to participants, type of comparators, dose of sedative agents, outcome measures and length of follow-up. Overall, risk of bias was generally high or unclear. In particular, few trials blinded assessors.

CONCLUSIONS: Evidence on the use of clonidine in ICUs is very limited. Dexmedetomidine may be effective in reducing ICU length of stay and time to extubation in critically ill ICU patients. Risk of bradycardia but not of overall mortality is higher among patients treated with dexmedetomidine. Well-designed RCTs are needed to assess the use of clonidine in ICUs and identify subgroups of patients that are more likely to benefit from the use of dexmedetomidine.

STUDY REGISTRATION: This study is registered as PROSPERO CRD42014014101.

FUNDING: The National Institute for Health Research Health Technology Assessment programme. The Health Services Research Unit is core funded by the Chief Scientist Office of the Scottish Government Health and Social Care Directorates.

Recombinant alpha 2(IV)NC1 domain of type IV collagen is an effective regulator of retinal capillary endothelial cell proliferation and inhibits pre-retinal neovascularisation

Background A recombinant form of the alpha 2(IV)NC1 domain of type IV collagen has been shown to have potent anti-angiogenic activity although this peptide has not been studied in the context of proliferative retinopathies. In the current investigation we examined the potential for alpha 2(IV) NC1 to regulate retinal microvascular endothelial cell function using a range of in vitro and in vivo assay systems.

Use of synthetic peptides to locate novel integrin alpha(2)beta(1)-binding motifs in human collagen III

A set of 57 synthetic peptides encompassing the entire triple-helical domain of human collagen III was used to locate binding sites for the collagen-binding integrin alpha(2)beta(1). The capacity of the peptides to support Mg2+-dependent binding of several integrin preparations was examined. Wild-type integrins (recombinant alpha(2) I-domain, alpha(2)beta(1) purified from platelet membranes, and recombinant soluble alpha(2)beta(1) expressed as an alpha(2)-Fos/beta(1)-Jun heterodimer) bound well to only three peptides, two containing GXX'GER motifs (GROGER and GMOGER, where O is hydroxyproline) and one containing two adjacent GXX'GEN motifs (GLKGEN and GLOGEN). Two mutant alpha(2) I-domains were tested: the inactive T221A mutant, which recognized no peptides, and the constitutively active E318W mutant, which bound a larger subset of peptides. Adhesion of activated human platelets to GER-containing peptides was greater than that of resting platelets, and HT1080 cells bound well to more of the peptides compared with platelets. Binding of cells and recombinant proteins was abolished by anti-alpha(2) monoclonal antibody 6F1 and by chelation of Mg2+. We describe two novel high affinity integrin-binding motifs in human collagen III (GROGER and GLOGEN) and a third motif (GLKGEN) that displays intermediate activity. Each motif was verified using shorter synthetic peptides.

Activation of GPVI by collagen is regulated by alpha(2)beta(1) and secondary mediators

We have compared the roles of adenosine diphosphate (ADP), thromboxanes and the integrin alpha(2)beta(1) in the activation of washed platelets by collagen in the presence of the alpha(IIb)beta3 antagonist lotrafiban. The stimulation of protein tyrosine phosphorylation by a collagen suspension is markedly delayed in the presence of the above inhibitors but shows substantial recovery with time. In comparison, activation of phospholipase C (PLC), Ca2+ elevation and dense granule secretion are more severely suppressed by the above inhibitors. blockade has a slightly greater inhibitory effect on all of the above responses than a combination of ADP receptor antagonists and cyclooxygenase inhibitor. Platelets exposed to a collagen monolayer show robust elevation of Ca2+ that is delayed in the presence of the above inhibitors and which is accompanied by of-granule secretion. These results demonstrate that secondary mediators and alpha(2)beta(1) modulate collagen-induced intracellular signaling but have negligible effect on GPVI signaling induced by the specific agonist convulxin. This work supports the postulate that the major role of of alpha(2)beta(1) is to increase the avidity of collagen for the platelet surface and by doing so enhance activation of GPVI. Therefore we propose an important role of secondary mediators in collagen-induced signaling is the indirect regulation of GPVI signaling via activation of alpha(2)beta(1).

Consequences of unlocking the cardiac myosin molecule in human myocarditis and cardiomyopathies

Myocarditis, often initiated by viral infection, may progress to autoimmune inflammatory heart disease, dilated cardiomyopathy and heart failure. Although cardiac myosin is a dominant autoantigen in animal models of myocarditis and is released from the heart during viral myocarditis, the characterization, role and significance of anti-cardiac myosin autoantibodies is poorly defined. In our study, we define the human cardiac myosin epitopes in human myocarditis and cardiomyopathies and establish a mechanism to explain how anti-cardiac myosin autoantibodies may contribute to heart disease. We show that autoantibodies to cardiac myosin in sera from myocarditis and dilated cardiomyopathies in humans targeted primarily epitopes in the S2 hinge region of cardiac myosin. In addition, anti-cardiac myosin antibodies in sera or purified IgG from myocarditis and cardiomyopathy targeted the beta-adrenergic receptor and induced antibody-mediated cAMP-dependent protein kinase A (PKA) cell signaling activity in heart cells. Antibody-mediated PKA activity in sera was abrogated by absorption with anti-human IgG. Antibody-mediated cell signaling of PKA was blocked by antigen-specific inhibition by human cardiac myosin or the beta-adrenergic receptor but not the alpha adrenergic receptor or bovine serum albumin. Propranolol, a beta blocker and inhibitor of the beta-adrenergic receptor pathway also blocked the antibody-mediated signaling of the beta-adrenergic receptor and PKA. The data suggest that IgG antibody against human cardiac myosin reacts with the beta-adrenergic receptor and triggers PKA signaling in heart cells. In summary, we have identified a new class of crossreactive autoantibodies against human cardiac myosin and the beta-adrenergic receptor in the heart. In addition, we have defined disease specific peptide epitopes in the human cardiac myosin rod S2 region in human myocarditis and cardiomyopathy as well as a mechanistic role of autoantibody in the pathogenesis of disease.

Studies of alpha-adrenoceptor antagonists on sympathetic mydriasis in rabbits

This study was undertaken to identify the alpha-adrenergic receptor type responsible for sympathetically evoked mydriasis in pentobarbital-anesthetized rabbits. Frequency-response curves of pupillary dilation were generated by stimulation of the preganglionic cervical sympathetic nerve (1-64 Hz). Evoked mydriatic responses were inhibited by systemic administration of nonselective alpha-adrenergic antagonists, phentolamine (0.3-10 mg/kg) and phenoxybenzamine (0.03-0.3 mg/kg), as well as the selective alpha(1)-adrenergic antagonist, prazosin (0.1-1 mg/kg). The alpha(2)-adrenergic antagonist, RS 79948 (0.3 mg/kg, i.v.) was without inhibitory effect, but potentiated the mydriatic response. In addition, the selective alpha(1A)-adrenoceptor antagonist, 5-methylurapidil (0.1-1 mg/kg, i.v.), antagonized the elicited mydriasis in a dose-dependent fashion. Unlike previous observations that prazosin does not block the adrenoceptor in rabbit iris dilator muscle, our results suggest that prazosin is effective in inhibiting neuronally elicited mydriasis in this species, and that alpha(1A)-adrenoceptors appear to mediate the response.

The effect of chronic captopril therapy on adrenergic receptors, plasma noradrenaline and the vascular responses to infused noradrenaline

Adrenergic receptors (alpha 2, beta 2), plasma noradrenaline, heart rate and the pressor responsiveness to infused noradrenaline were examined in ten healthy male volunteers before and after 2 weeks of placebo or captopril therapy in a double blind cross-over study. No significant differences in these measurements were observed between the captopril and placebo treated groups. The study shows that in sodium replete normotensive subjects, long-term angiotensin converting enzyme inhibition does not lead to changes in adrenoceptor density. There is also no alteration in plasma noradrenaline levels nor in the pressor responsiveness to infused noradrenaline. These data suggest that the known interaction between the renin-angiotensin system and the sympathetic nervous system observed in animals is probably of little significance in man.

Differential Modulation of Transcriptional Activity of Estrogen Receptors by Direct Protein-Protein Interactions with the T Cell Factor Family of Transcription Factors

Two major signaling pathways, those triggered by estrogen (E(2)) and by the Wnt family, interact in the breast to cause growth and differentiation. The estrogen receptors ER(alpha) and ER(beta) are activated by binding E(2) and act as ligand-dependent transcription factors. The effector for the Wnt family is the Tcf family of transcription factors. Both sets of transcription factors recognize discrete but different nucleotide sequences in the promoters of their target genes. By using transient transfections of reporter constructs for the osteopontin and thymidine kinase promoters in rat mammary cells, we show that Tcf-4 antagonizes and Tcf-1 stimulates the effects of activated ER/E(2). For mutants of the former promoter, the stimulatory effects of ER(alpha)/E(2) can be made to be dependent on Tcf-1, and for the latter promoter the effects of the T cell factors (TCFs) are dependent on ER/E(2). Direct interaction between ERs and Tcfs either at the Tcf/ER(alpha)-binding site on the DNA or in the absence of DNA is established by gel retardation assays or by coimmunoprecipitation/biosensor methods, respectively. These results show that the two sets of transcription factors can interact directly, the interaction between ERs and Tcf-4 being antagonistic and that between ERs and Tcf-1 being synergistic on the activity of the promoters employed. Since Tcf-4 is the major Tcf family member in the breast, it is suggested that the antagonistic interaction is normally dominant in vivo in this tissue.

Cell-collagen interactions: the use of peptide Toolkits to investigate collagen-receptor interactions

Fibrillar collagens provide the most fundamental platform in the vertebrate organism for the attachment of cells and matrix molecules. we have identified specific sites in collagens to which cells can attach, either directly or through protein intermediaries. Using Toolkits of triple-helical peptides, each peptide comprising 27 residues of collagen primary sequence and overlapping with its neighbours by nine amino acids, we have mapped the binding of receptors and other proteins on to collagens II or III. Integrin alpha 2 beta 1 binds to several GXX'GER motifs within the collagens, the affinities of which differ sufficiently to control cell adhesion and migration independently of the cellular regulation of the integrin. The platelet receptor, Gp (glycoprotein) VI binds well to GPO (where 0 is hydroxyproline)-containing model peptides, but to very few Toolkit peptides, suggesting that sequence in addition to GPO triplets is important in defining GpVI binding. The Toolkits have been applied to the plasma protein vWF (von Willebrand factor), which binds to only a single sequence, identified by truncation and amino acid substitution within Toolkit peptides, as GXRGQOGVMGFO in collagens II and III. Intriguingly, the receptor tyrosine kinase, DDR2 (discoidin domain receptor 2) recognizes three sites in collagen II, including its vWF-binding site, although the amino acids that support the interaction differ slightly within this motif. Furthermore, the secreted protein BM-40 (basement membrane protein 40) also binds well to this same region. Thus the availability of extracellular collagen-binding proteins may be important in regulating and facilitating direct collagen-receptor interaction.

GPVI levels in platelets: relationship to platelet function at high shear

We have investigated the density of the collagen receptors glycoprotein VI (GPVI) and alpha(2)beta(1) on human platelets and their relationship to polymorphisms within the GPVI gene. GPVI levels varied 1.5-fold and showed a weak correlation (r = 0.35) with the levels of alpha(2)beta(1), which varied 3-fold. GPVI genotype had a significant effect on receptor levels with carriers of the proline 219 allele (approximately 22% of the population) having 10% lower GPVI levels than the more common serine homozygotes. GPVI and alpha(2)beta(1) levels were found to be significantly decreased on platelets from patients with myeloproliferative disorders (MPDs). In both the MPD and the control group, GPVI levels were found not to affect platelet function under high shear in whole blood. Similarly murine platelets that express up to 5-fold lower levels of GPVI showed no significant difference than controls in thrombus formation on a high-density collagen-coated surface. However platelets lacking the GPVI/Fc receptor gamma-chain (FcR gamma-chain) complex or a functional FcR gamma-chain (immunoreceptor tyrosine-based activation motif [ITAM] point mutant) exhibited severely abrogated thrombus formation at 800 s(-1) and 1500 s(-1). These results demonstrate that GPVI levels are tightly controlled and play a critical role in thrombus formation on collagen; nevertheless, a range of receptor densities can support platelet function under high shear. (C) 2003 by The American Society of Hematology.

Evidence for the presence of G-proteins, adenylyl cyclase and phospholipase C activities in lymphatic smooth muscle cell membranes

In plasma membranes derived from bovine mesenteric lymphatic smooth muscle cells, guanine nucleotide and forskolin stimulated adenylyl cyclase (AC) activity in a concentration-dependent manner, indicative of the presence of the stimulatory G-protein G(s) linked to AC. There was no significant enzyme inhibition by low concentrations of guanine nucleotide and no effect on basal or guanine nucleotide-stimulated activity following pertussis toxin treatment of cells, suggesting the absence of G(1) linked to inhibition of AC. Furthermore, there was no effect of adrenaline, isoprenaline or clonidine on basal or forskolin-stimulated activities, nor was there any specific binding of the beta-adrenoceptor ligand [I-125]cyanopindolol to membranes, suggesting that cate-cholamine receptors do not modulate AC activity in these membranes. Pertussis toxin-mediated ADP ribosylation of membrane proteins and Western immunoblotting analysis revealed the presence of G-protein subunits G(alpha l2), G(alpha q), G(alpha 11) and G(beta 1). In experiments designed to identify a possible effector enzyme for these G-proteins, membranes were screened with a range of antibodies raised against phospholipase C (PLC) beta, gamma and delta isozymes. Though no evidence was obtained by Western blotting for any of these proteins, PLC activity was concentration-dependently stimulated by Ca2+, but not by AlF4-, GTP[S], or purified G(beta gamma) subunits. Finally, no specific binding to membranes of the alpha(1)-adrenoceptor ligand [H-3]prazosin or the alpha(2)-adrenoceptor ligand [H-3]yohimbine was obtained. In conclusion, this study provides evidence for a G(s)-dependent stimulation of AC, and for the presence of G(2) and G(q11), which do not appear to regulate a PLC activity also identified in lymphatic smooth muscle cell membranes. Furthermore, neither AC nor PLC appear to be associated with catecholamine receptors. Copyright(C) 1996 Elsevier Science Inc.