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Biomedical subjects

N Moran

Publications and source records attributed to N Moran.

At least 19 recordsLinked to original sources
PubMed2006

Monitoring modulators of platelet aggregation in a microtiter plate assay.

Platelets play a central role in maintaining biological hemostasis. Inappropriate platelet activation is responsible for thrombotic diseases such as myocardial infarction and stroke. Therefore, novel agents that can inhibit platelet activation are necessary. However, assays that monitor platelet aggregation are generally time-consuming and require high volumes of blood and specialized equipment. Therefore, a medium- to high-throughput assay that can monitor platelet aggregation would be considered useful. Such an assay should be sensitive, comparable to the "gold standard" assay of platelet aggregometry, and able to monitor multiple samples simultaneously but with low assay volumes. We have developed such a microtiter assay. It can assay an average of 60 independent treatments per 60 ml blood donation and demonstrates greater sensitivity than the current gold standard assay, namely platelet aggregation in stirring conditions in a platelet aggregometer. The microtiter plate (MTP) assay can detect known inhibitors of platelet function such as indomethacin, aspirin, and ReoPro. It is highly reproducible when using standard doses of agonists such as thrombin receptor-activating peptide (20 microM) and collagen (0.19 mg/ml). Finally, the MTP assay is rapid and sensitive and can detect unknown platelet-modulating agents from a library of compounds.

Amino Acid Sequence↗
PubMed2006

Evidence for a differential functional regulation of the two beta(3)-integrins alpha(V)beta(3) and alpha(IIb)beta(3).

The functional regulation of integrins is a major determinant of cell adhesion, migration and tissue maintenance. The binding of cytoskeletal proteins to various sites of integrin cytoplasmic domains is a key mechanism of this functional regulation. Expression of recombinant integrin alpha(IIb)beta(3) and alpha(M)beta(2) lacking the GFFKR-region in CHO cells results in constitutively activated integrins. In contrast, CHO cells stably expressing either a GFFKR-deleted alpha(V(del))beta(3) or a FF to AA-substituted alpha(V(AA))beta(3) do not reveal a constitutively activated integrin. Adhesion to immobilized fibrinogen is strongly impaired in alpha(V(del))beta(3) or alpha(V(AA))beta(3)-expressing cells, whereas it is not impaired in alpha(IIb)beta(3) and alpha(M)beta(2), both lacking the GFFKR-region. In a parallel plate flow chamber assay, alpha(V)beta(3)-expressing cells adhere firmly to fibrinogen and spread even at shear rates of 15 to 20 dyn/cm(2), whereas alpha(V(del))beta(3) or alpha(V(AA))beta(3) cells are detached at 15 dyn/cm(2). Actin stress fiber formation and focal adhesion plaques containing alpha(V)beta(3) are observed in alpha(V)beta(3) cells but not in alpha(V(del))beta(3) or alpha(V(AA))beta(3)-expressing cells. As an additional manifestation of impaired outside-in signaling, phosphorylation of pp125(FAK) was reduced in these cells. In summary, we report that the GFFKR-region of the alpha(V)-cytoplasmic domain and in particular two phenylalanines are essential for integrin alpha(V)beta(3) function, especially for outside-in signaling. Our results suggest that the two beta(3)-integrins alpha(IIb)beta(3) and alpha(V)beta(3) are differentially regulated via their GFFKR-region.

Animals↗
PubMed2006

A novel functional role for the highly conserved alpha-subunit KVGFFKR motif distinct from integrin alphaIIbbeta3 activation processes.

BACKGROUND: The highly conserved integrin alpha-subunit membrane-proximal motif KVGFFKR plays a decisive role in modulating the activation of integrin alphaIIbbeta3. Previously, we have shown that a platelet permeable palmityl (pal)-peptide with this seven amino acid sequence can directly activate alphaIIbbeta3 leading to platelet aggregation. OBJECTIVES: To investigate further the role of the KVGFFKR motif in integrin alphaIIbbeta3 function. METHODS: We used two sequence-specific complementary model systems, palmityl pal-peptides in platelets, and mutant alphaIIbbeta3-expressing Chinese Hamster Ovary (CHO) cell lines. RESULTS: In platelets we show that the two phenylalanine amino acids in pal-KVGFFKR (pal-FF) peptide are critical for stimulating platelet aggregation. Pal-FF peptide treatment of platelets also gives rise to a tyrosine phosphorylation signal despite the presence of inhibitors of fibrinogen binding. In CHO cells, a double alanine substitution, alphaIIb(F992A, F993A)beta3, induces constitutive integrin activation but prevents actin stress fiber formation upon adhesion to fibrinogen, suggesting that alphaIIbbeta3-mediated cytoskeletal reorganization is also dependent on F992 and F993. This further highlights a critical role for the two phenylalanine residues in both of these alphaIIbbeta3-mediated processes. CONCLUSION: In addition to regulating integrin alphaIIbbeta3 activation state, the KVGFFKR motif also influences cytoskeletal reorganization. This activity is critically determined by F992 and F993 within the seven amino acid sequence.

Amino Acid Motifs↗
PubMed2003

A palmitylated peptide derived from the glycoprotein Ib beta cytoplasmic tail inhibits platelet activation.

The platelet receptor GPIb/IX/V mediates a crucial role in hemostasis, yet the signaling mechanisms involved are incompletely understood. The complex consists of four polypeptides GPIb alpha, GPIb beta, GPIX and GPV. We identified an amino acid sequence in the cytoplasmic tail of the GPIb beta subunit between residues R151 and A161 that is highly conserved across species and hypothesized that it has functional importance. To target this motif, we synthesized a corresponding cell-permeable palmitylated peptide (Pal-RRLRARARARA) and investigated its effect on platelet function. Pal-RRLRARARARA completely inhibited low dose thrombin- and ristocetin-induced aggregation in washed platelets but only partially inhibited collagen- and U46619-induced aggregation. Thromboxane production in platelets stimulated with thrombin was significantly reduced by Pal-RRLRARARARA compared with collagen. Activation of the integrin alpha IIb beta 3 in response to thrombin was significantly reduced when platelets were preincubated with Pal-RRLRARARARA. The adhesion of washed platelets to von Willebrand factor (VWF) under static conditions was significantly reduced by Pal-RRLRARARARA. Under conditions of high shear, the velocity of platelets rolling on VWF was significantly increased when platelets are preincubated with Pal-RRLRARARARA. This study defines a novel function for the RRLRARARARA motif of GPIb beta in platelet activation.

Blood Platelets↗
PubMed2001

A Val193Met mutation in GPIIIa results in a GPIIb/IIIa receptor with a constitutively high affinity for a small ligand.

We have identified a patient designated as (GTa) with Glanzmann's Thrombasthenia (GT) diagnosed on the basis of a prolonged bleeding time and failure of the patient's platelets to aggregate. The number of glycoprotein (GP)IIb/IIIa receptors on the platelet surface was 37% of normal and those receptors displayed a defect in soluble fibrinogen binding. Nevertheless, GTa platelets showed increased adhesion to solid-phase fibrinogen and binding affinity for the RGD-mimetic (3)H-SC52012, a non-peptide GPIIb/IIIa antagonist. Dithiothreitol (DTT) and ADP enhanced the affinity for [(3)H]-SC52012 in normal platelets, but had little effect in GTa platelets. These findings suggested that GTa platelets were locked in an altered affinity state. Genetic analysis showed that GTa was a compound heterozygote for the GPIIIa gene. One allele showed a deletion at the 3' end of exon 3 resulting in a premature stop codon. The second GPIIIa allele had a G to A transition at nucleotide 577, resulting in a Val193Met substitution. HEK 293T cells transfected with mutant GPIIb/IIIaV193M bound [(3)H]-SC52012 with a higher affinity than wild-type GPIIb/IIIa, and this was not increased by DTT. The mutant receptor distinguishes between platelet adhesion and aggregation, and demonstrates the phenotype that may be expected when platelet aggregation alone is inhibited.

Adenosine Diphosphate↗
PubMed2001

Potassium-efflux channels in extensor and flexor cells of the motor organ of Samanea saman are not identical. Effects of cytosolic calcium.

Leaflet movements in the mimosa-family tree Samanea saman stem from coordinated volume changes of cells in the leaf motor organs in the adaxial and abaxial motor cells ("flexors" and "extensors"). Shrinking, initiated by dissimilar light signals in extensors and in flexors, depends in both cell types on K(+) efflux via depolarization-dependent potassium (K(D)) channels. To compare between flexor and extensor K(D) channels and to test for a possible interaction of these channels with the Ca(2+)-mobilizing phosphoinositide cascade evoked in these motor cells by the "shrinking signals," we probed the channels with varying (5 nM-3 mM) cytosolic free-Ca(2+) concentration ([Ca(2+)](cyt)) in patch-clamped inside-out excised membrane patches. Ca(2+) was not required for K(D) channel activation. [Ca(2+)](cyt) of 600 nM decreased the mean number of open K(D) channels in flexors, as monitored at -30 mV. Detailed analysis revealed that in flexors millimolar [Ca(2+)](cyt) decreased the maximum number of open channels, but simultaneously increased K(D) channel opening probability by negatively shifting the half-maximum-activation voltage by 40 to 50 mV. Thus, the promoting and the inhibitory effects at millimolar [Ca(2+)](cyt) practically cancelled-out. In contrast to flexors, none of the gating parameters of the extensor K(D) channels were affected by [Ca(2+)](cyt). Irrespective of [Ca(2+)](cyt), the steady-state gating of extensor K(D) channels was slightly but significantly more voltage sensitive than that of flexors. The unitary conductances of flexor and extensor K(D) channels were similar and decreased by approximately 20% at millimolar [Ca(2+)](cyt). It is intriguing that the extensor K(D) channels were significantly less K(+) selective than those in flexors.

Calcium↗
PubMed2001

Extracellular protons inhibit the activity of inward-rectifying potassium channels in the motor cells of Samanea saman pulvini.

The intermittent influx of K+ into motor cells in motor organs (pulvini) is essential to the rhythmic movement of leaves and leaflets in various plants, but in contrast to the K+ influx channels in guard cells, those in pulvinar motor cells have not yet been characterized. We analyzed these channels in the plasma membrane of pulvinar cell protoplasts of the nyctinastic legume Samanea saman using the patch-clamp technique. Inward, hyperpolarization-activated currents were separated into two types: time dependent and instantaneous. These were attributed, respectively, to K+ -selective and distinctly voltage-dependent K(H) channels and to cation-selective voltage-independent leak channels. The pulvinar K(H) channels were inhibited by external acidification (pH 7.8-5), in contrast to their acidification-promoted counterparts in guard cells. The inhibitory pH effect was resolved into a reversible decline of the maximum conductance and an irreversible shift of the voltage dependence of K(H) channel gating. The leak appeared acidification insensitive. External Cs (10 mM in 200 mM external K+) blocked both current types almost completely, but external tetraethylammonium (10 mM in 200 mM external K+) did not. Although these results do not link these two channel types unequivocally, both likely serve as K+ influx pathways into swelling pulvinar motor cells. Our results emphasize the importance of studying multiple model systems.

Biological Transport↗
PubMed2000

The platelet integrin alpha IIbbeta 3 has an endogenous thiol isomerase activity.

Integrins are cysteine-rich heterodimeric cell-surface adhesion molecules that alter their affinity for ligands in response to cellular activation. The molecular mechanisms involved in this activation of integrins are not understood. Treatment with the thiol-reducing agent, dithiothreitol, can induce an activation-like state in many integrins suggesting that cysteine-cysteine dithiol bonds are important for the receptor's tertiary structure and may be involved in activation-induced conformational changes. Here we demonstrate that the platelet-specific integrin, alpha(IIb)beta(3), contains an endogenous thiol isomerase activity, predicted from the presence of the tetrapeptide motif, CXXC, in each of the cysteine-rich repeats of the beta(3) polypeptide. This motif comprises the active site in enzymes involved in disulfide exchange reactions, including protein-disulfide isomerase (EC ) and thioredoxin. Intrinsic thiol isomerase activity is also observed in the related integrin, alpha(v)beta(3), which shares a common beta-subunit. Thiol isomerase activity within alpha(IIb)beta(3) is time-dependent and saturable, and is inhibited by the protein-disulfide isomerase inhibitor, bacitracin. Furthermore, this activity is calcium-sensitive and is regulated in the EDTA-stabilized conformation of the integrin. This novel demonstration of an enzymatic activity associated with an integrin subunit suggests that altered thiol bonding within the integrin or its substrates may be locally modified during alpha(IIb)beta(3) activation.

Amino Acid Sequence↗
PubMed2000

A fast signal-induced activation of Poly(ADP-ribose) polymerase: a novel downstream target of phospholipase c.

We present the first evidence for a fast activation of the nuclear protein poly(ADP-ribose) polymerase (PARP) by signals evoked in the cell membrane, constituting a novel mode of signaling to the cell nucleus. PARP, an abundant, highly conserved, chromatin-bound protein found only in eukaryotes, exclusively catalyzes polyADP-ribosylation of DNA-binding proteins, thereby modulating their activity. Activation of PARP, reportedly induced by formation of DNA breaks, is involved in DNA transcription, replication, and repair. Our findings demonstrate an alternative mechanism: a fast activation of PARP, evoked by inositol 1,4,5,-trisphosphate-Ca(2+) mobilization, that does not involve DNA breaks. These findings identify PARP as a novel downstream target of phospholipase C, and unveil a novel fast signal-induced modification of DNA-binding proteins by polyADP-ribosylation.

Animals↗
PubMed2000

Surface expression of glycoprotein ib alpha is dependent on glycoprotein ib beta: evidence from a novel mutation causing Bernard-Soulier syndrome.

Bernard-Soulier syndrome is a rare bleeding disorder caused by a quantitative or qualitative defect in the platelet glycoprotein (GP) Ib-IX-V complex. The complex, which serves as a platelet receptor for von Willebrand factor, is composed of 4 subunits: GPIb alpha, GPIb beta, GPIX, and GPV. We here describe the molecular basis of a novel form of Bernard-Soulier syndrome in a patient in whom the components of the GPIb-IX-V complex were undetectable on the platelet surface. Although confocal imaging confirmed that GPIb alpha was not present on the platelet surface, GPIb alpha was readily detectable in the patient's platelets. Moreover, immunoprecipitation of plasma with specific monoclonal antibodies identified circulating, soluble GPIb alpha. DNA-sequence analysis revealed normal sequences for GPIb alpha and GPIX. There was a G to A substitution at position 159 of the gene encoding GPIb beta, resulting in a premature termination of translation at amino acid 21. Studies of transient coexpression of this mutant, W21stop-GPIb beta, together with wild-type GPIbalpha and GPIX, demonstrated a failure of GPIX expression on the surface of HEK 293T cells. Similar results were obtained with Chinese hamster ovary alpha IX cells, a stable cell line expressing GPIbalpha that retains the capacity to re-express GPIX. Thus, we found that GPIbbeta affects the surface expression of the GPIb-IX complex by failing to support the insertion of GPIb alpha and GPIX into the platelet membrane. (Blood. 2000;96:532-539)

Animals↗
PubMed2000

Imaging and radiological-pathological correlation in histologically proven cases of focal cortical dysplasia and other glial and neuronoglial malformative lesions in adults.

Focal cortical dysplasia (FCD) is a pathological entity first described in 1971. Other more subtle cortical malformations found in patients with epilepsy include microdysgenesis (MD), and glioneuronal hamartias. Although these glial and neuronoglial malformations have distinct histological features, there is terminological confusion in the radiological literature. Few cases have been reported in adults with both imaging and histology. We address these issues, giving a radiological-pathological correlation of histologically proven cortical malformations in adults. We describe clinical, radiological and histological features of 12 cases (five FCD, five MD with glioneuronal hamartias, and two hamartomas), unassociated with other conditions, and discuss them in the light of the literature. FCD is usually seen on MRI as cortical thickening, with or without signal change, which may extend into the adjacent white matter. On histology, abnormal neurons and/or glial cells, blurring of the grey-white matter interface, myelin pallor, demyelination, and gliosis may be found. Glioneuronal hamartias and hamartomas usually appear as complex masses on MRI. FCD and hamartias may be associated, and a combination of imaging findings may be seen on MRI. Atrophy of the ipsilateral hippocampus may be present on MRI in patients with hamartias, and minor cell loss on histology, but not definitive hippocampal sclerosis. Although the imaging findings of cortical malformations are protean, some characteristic MRI features, with histological correlates, may be found. The relevance of most of these observations remains unclear.

Adult↗
PubMed2000

Patients' perspectives on services for epilepsy: a survey of patient satisfaction, preferences and information provision in 2394 people with epilepsy.

The objectives of this study were to provide a comprehensive survey of satisfaction with care, care preferences and information provision for patients with epilepsy, and to formulate recommendations for the development of epilepsy services based on the findings. A questionnaire was distributed to 4620 patients who were currently receiving antiepileptic drugs for epilepsy, regardless of aetiology, duration or severity. Two different samples of patients with epilepsy were questioned: the first an unselected sample drawn from primary care, and the second consisting of consecutive patients drawn from hospital clinics. There were 2394 responses to the questionnaire. Satisfaction with primary and hospital care was high, both overall and for specific aspects. However, two major shortcomings were identified. First, few respondents felt that their care was shared between hospital and GP. Secondly, provision of information about epilepsy was perceived to be poor, particularly by the elderly. Younger patients and patients with severe epilepsy had a higher satisfaction with and preference for hospital care, whereas older age groups were more satisfied with and preferred primary care. Patients' main reasons for preferring primary care were that it was more personal and the GP was more familiar with them, and secondary care was preferred because the hospital doctor knew more about epilepsy. In conclusion, we have conducted the largest representative UK survey of patients' perceptions and views of the care available for epilepsy. Although patient satisfaction was high, information provision is poor and the shared care model is not operating effectively. We recommend that an emphasis be placed on methods for improving the interface between primary and secondary care. The setting up of hospital epilepsy centres, as recommended by the recently published Clinical Standards Advisory Group report on epilepsy, would provide a focus for these efforts and for information provision.

Adolescent↗
PubMed2000

NHS services for epilepsy from the patient's perspective: a survey of primary, secondary and tertiary care access throughout the UK.

The aims of this study were to estimate the proportion of patients with epilepsy who made primary care and/or hospital outpatient medical consultations within 1 year; to formulate a model of the explanatory variables that influence whether patients consult or not; and to estimate the frequency of referral to, and waiting time for, hospital outpatient clinics in patients with new-onset seizures. Suggestions are offered for improvement of epilepsy services based on the findings. A questionnaire was distributed to 3455 unselected patients identified at population level from primary care practices in all NHS regions of the UK. There were 1652 respondents with epilepsy of all types, irrespective of aetiology, duration or severity. Fifty-two per cent of the whole sample made at least one medical consultation of any type specifically for epilepsy (42.0% primary care, 30.5% hospital, 20.4% both). Most patients with controlled epilepsy (74.5%) had no consultations. Of patients with severe epilepsy, 27.5% made no primary care consultations, 43.4% no hospital consultations and 14.1% no consultations of either type. Gender did not influence the likelihood of either GP or hospital consultations in patients with either controlled or active epilepsy. Increasing seizure frequency was associated with a greater likelihood of one or more hospital consultations for epilepsy, whereas increasing duration of epilepsy was associated with a decreased likelihood of either type of consultation. Age affected consultation rates: of those patients over the age of 65 years, only 29.9% made a medical consultation for epilepsy, compared to 53.8% of young adults. Patients under the age of 17 years were less likely to have consulted a GP and more likely to have consulted a hospital doctor. Ninety percent of new-onset patients had been referred to a hospital doctor, and the mean wait was 6.5 weeks. In conclusion, many patients with epilepsy, including severe epilepsy, are not receiving specialist input, and a significant proportion are receiving no medical supervision. The elderly are over-represented in this group. Care tends to be polarized between hospital or primary care, falling short of the ideal of shared care. It will be important to address the influences on consultation seeking in epilepsy, particularly for those patients currently under no medical supervision.

Adolescent↗
PubMed2000

Blue light activates potassium-efflux channels in flexor cells from Samanea saman motor organs via two mechanisms.

Light-induced leaflet movement of Samanea saman depends on the regulation of membrane transporters in motor cells. Blue light (BL) stimulates leaflet opening by inducing K(+) release from the flexor motor cells. To elucidate the mechanism of K(+)-efflux (K(D))-channel regulation by light, flexor motor cell protoplasts were patch-clamped in a cell-attached configuration during varying illumination. Depolarization elicited outward currents through single open K(D) channels. Changes in cell membrane potential (E(M)) were estimated by applying voltage ramps and tracking the change of the apparent reversal potential of K(D)-channel current. BL shifted E(M) in a positive direction (i.e. depolarized the cell) by about 10 mV. Subsequent red light pulse followed by darkness shifted E(M) oppositely (i.e. hyperpolarized the cell). The BL-induced shifts of E(M) were not observed in cells pretreated with a hydrogen-pump inhibitor, suggesting a contribution by hydrogen-pump to the shift. BL also increased K(D)-channel activity in a voltage-independent manner as reflected in the increase of the mean net steady-state patch conductance at a depolarization of 40 mV relative to the apparent reversal potential (G(@40)). G(@40) increased by approximately 12 pS without a change of the single-channel conductance, possibly by increasing the probability of channel opening. Subsequent red-light and darkness reversed the change in G(@40). Thus, K(+) efflux, a determining factor for the cell-volume decrease of flexor cells, is regulated by BL in a dual manner via membrane potential and by an independent signaling pathway.

Cell Membrane↗
PubMed2000

Potassium-efflux channels in extensor and flexor cells of the motor organ of Samanea saman are not identical. Effects of cytosolic calcium.

Leaflet movements in the mimosa-family tree Samanea saman stem from coordinated volume changes of cells in the leaf motor organs in the adaxial and abaxial motor cells ("flexors" and "extensors"). Shrinking, initiated by dissimilar light signals in extensors and in flexors, depends in both cell types on K(+) efflux via depolarization-dependent potassium (K(D)) channels. To compare between flexor and extensor K(D) channels and to test for a possible interaction of these channels with the Ca(2+)-mobilizing phosphoinositide cascade evoked in these motor cells by the "shrinking signals," we probed the channels with varying (5 nM-3 mM) cytosolic free-Ca(2+) concentration ([Ca(2+)](cyt)) in patch-clamped inside-out excised membrane patches. Ca(2+) was not required for K(D) channel activation. [Ca(2+)](cyt) of 600 nM decreased the mean number of open K(D) channels in flexors, as monitored at -30 mV. Detailed analysis revealed that in flexors millimolar [Ca(2+)](cyt) decreased the maximum number of open channels, but simultaneously increased K(D) channel opening probability by negatively shifting the half-maximum-activation voltage by 40 to 50 mV. Thus, the promoting and the inhibitory effects at millimolar [Ca(2+)](cyt) practically cancelled-out. In contrast to flexors, none of the gating parameters of the extensor K(D) channels were affected by [Ca(2+)](cyt). Irrespective of [Ca(2+)](cyt), the steady-state gating of extensor K(D) channels was slightly but significantly more voltage sensitive than that of flexors. The unitary conductances of flexor and extensor K(D) channels were similar and decreased by approximately 20% at millimolar [Ca(2+)](cyt). It is intriguing that the extensor K(D) channels were significantly less K(+) selective than those in flexors.

Amino Acid Sequence↗
PubMed1999

A randomized, placebo-controlled, crossover study of E5510 and aspirin in healthy volunteers.

Platelet inhibition significantly reduces the risk of cardiovascular mortality and morbidity. However, current antiplatelet therapies have limitations, and more efficacious agents are needed. E5510 is a novel compound that has multiple platelet inhibitory effects in in vitro studies. We compared the in vivo, pharmacodynamic effects of maximal antiplatelet doses of E5510 (20 mg) with 300 mg aspirin in a placebo-controlled, triple crossover trial in nine healthy volunteers. Collagen-induced platelet aggregation and serum thromboxane B2 (TxB2) were similarly inhibited by both compounds in the first 12 h but showed recovery at 24 h in the E5510 group only (p < 0.05). Thrombin and U46619-induced platelet aggregation, as well as basal and prostaglandin E2 (PGE2)-stimulated platelet cyclic adenosine monophosphate (cAMP) levels were unchanged after ingestion of either agent. E5510 and aspirin reduced systemic thromboxane formation without affecting prostacyclin biosynthesis. Neither E5510 nor aspirin inhibited the excretion of 8-epi PGF2alpha and 5,6-DHET, two indices of cyclooxygenase-independent arachidonate metabolism. In conclusion, (a) E55 10 in vivo most likely induces a reversible inhibition of cyclooxygenase, without affecting thromboxane synthetase, phosphodiesterase, thrombin, or thromboxane receptor-mediated signaling; (b) single doses of aspirin or E5510 affect thromboxane/prostacyclin profiles favorably, supporting their use in acute coronary syndromes. This study outlines a comprehensive and minimally invasive approach for the assessment of the in vivo mechanism of action of novel antiplatelet agents.

Adult↗
PubMed1998

A sequence within the cytoplasmic tail of GpIIb independently activates platelet aggregation and thromboxane synthesis.

All integrin alpha subunits contain a highly conserved KXGFFKR motif in their cytoplasmic domains that plays a crucial role in the regulation of integrin affinity for their ligands. We show that a lipid-modified peptide corresponding to the cytoplasmic region, 989-995, of the platelet integrin subunit glycoprotein GpIIb (alphaIIb), palmitoyl-KVGFFKR (Ppep; 10 microM), but not a similarly modified scrambled peptide (palmitoyl-FKFVRGK), can specifically induce platelet activation and aggregation equivalent to that of strong agonists such as thrombin. Ppep-induced aggregation is also associated with indices of platelet activation including thromboxane A2 (TXA2) synthesis (EC50 = 45 +/- 5 microM), secretion of alpha-granules detected as enhanced surface expression of P-selectin (EC50 = 52 +/- 8 microM), and conformational changes in GpIIb/IIIa measured by the monoclonal antibody, PAC-1 (EC50 = 3.7 +/- 1 microM). The TXA2 receptor antagonist, SQ29548, PGE1, and the ADP scavenger, apyrase, differentially inhibit the aggregation response and TXA2 synthesis in response to Ppep. Similarly, GpIIb/IIIa antagonists (RO-449883 and integrelin), which inhibit aggregation by greater than 90%, have little effect on peptide-induced TXA2 synthesis, suggesting that this event is independent of fibrinogen binding to GpIIb/IIIa. Alanine-stepping of the Ppep sequence identifies GFFK(991-994) as the critical residues in all peptide-mediated events. We conclude that this peptide can imitate the cytoplasmic domain of GpIIb and initiate parallel but independent signaling pathways, one leading to ligand binding and platelet aggregation and the other to intracellular signaling events such as TXA2 synthesis and secretion.

Acetates↗