Sarah O'Neill

The Platelet Integrin αIIbβ3 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 receptors tertiary structure and may be involved in activation-induced conformational changes. Here we demonstrate that the platelet-specific integrin, αIIbβ3, contains an endogenous thiol isomerase activity, predicted from the presence of the tetrapeptide motif, CXXC, in each of the cysteine-rich repeats of the β3 polypeptide. This motif comprises the active site in enzymes involved in disulfide exchange reactions, including protein-disulfide isomerase (EC 5.3.4.1) and thioredoxin. Intrinsic thiol isomerase activity is also observed in the related integrin, αvβ3, which shares a common β-subunit. Thiol isomerase activity within αIIbβ3 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 αIIbβ3 activation.

S-nitrosocysteine inhibition of human platelet secretion is correlated with increases in platelet cGMP levels.

Platelet inhibition by exogenous and endogenous nitrovasodilators has been shown to be associated with increases in cGMP, but proof of a role for cGMP in this process is lacking. We therefore studied the effects of cGMP and guanylate cyclase stimulation on human platelet secretion by pharmacologically modulating intraplatelet cGMP levels. The endothelium-derived relaxing factor (EDRF)-like activator of guanylate cyclase, S-nitrosocysteine (SNOC), led to a dose-dependent inhibition of secretion in intact human platelets (IC50 = 10(-6) M). The cGMP phosphodiesterase inhibitor M&B 22,948 augmented SNOC-induced inhibition of secretion through elevations in cGMP without affecting cAMP levels (from 50% to 81% inhibition versus control, p = 0.02). Methylene blue reversed the inhibitory effects of SNOC on platelet secretion (p = 0.03). Dibutyryl-cGMP and 8-bromo-cGMP also significantly inhibited secretion in this system. Incubation of platelets with exogenous cGMP to achieve intraplatelet cGMP levels comparable to those after SNOC treatment resulted in similar degrees of inhibition of secretion (32% inhibition versus control, p = 0.01) and was also potentiated by M&B 22,948 (from 32% to 68% inhibition, p = 0.003). In addition, a highly significant correlation between intraplatelet cGMP levels and the degree of inhibition of secretion was demonstrable in these studies (r = 0.94, p = 0.016). These data demonstrate that elevation of intraplatelet cGMP levels by the EDRF-like compound SNOC is correlated with inhibition of human platelet secretion.

Sibling relationships in adults who have siblings with or without intellectual disabilities

There is relatively little research on the relationships between adults with intellectual disability and their siblings, despite the potential importance of these relationships for either individuals psychological well-being and future care roles that might be adopted by adult siblings. In the present study, sibling relationships of adults with adult siblings with (N=63) and without (N=123) intellectual disability were explored. Contact, warmth, conflict, and rivalry were measured using questionnaires available as an on-line survey. Expressed emotion was measured using the Five Minute Speech Sample over the telephone to establish an independently coded measure of criticism from the participant towards their sibling. Overall, there were few group differences in contact and sibling relationship. There was less telephone contact in the intellectual disability group, and less reported warmth in the relationship with siblings with intellectual disability although this was mainly associated with severe/profound intellectual disability. Exploratory analyses were conducted of the correlates of sibling relationships in both the intellectual disability and control groups. These analyses revealed a small number of different associations especially for conflict, which was lower when either the participant or sibling was younger in the control group but associated with relative age in the intellectual disability group.

Signalling via CD28 of human naive neonatal T lymphocytes.

Accessory molecules play a crucial role in the development of the T cell response to antigenic challenge. We have examined the role of CD28 in modulating the‘naive’ neonatal T cell response to anti‐CD2‐mediated activation. To compare the role of CD28, neonatal and adult T cells were stimulated with a pair of mitogenic anti‐CD2 antibodies in the presence or absence or anti‐CD28 MoAb. With anti‐CD2 alone, neonatal T cells proliferated slightly but produced no detectable IL‐2, whereas adult T cells proliferated vigorously, with significant IL‐2 production. Costimulation with anti‐CD28 MoAb greatly enhanced the proliferative response of neonatal T cells to levels equivalent to those of adult T cells, whereas adult T cells showed only slight increases. Although IL‐2 secretion was increased in the presence of anti‐CD28 MoAb. neonatal T cell IL‐2 production remained lower than in adults. In contrast, enhancement of IL‐2 mRNA expression in neonates was similar to adult levels. Anti‐CD28 MoAb costimulation increased NFkB levels in neonates, albeit to levels lower than that of adults. The cellular mechanism governing the diminished proliferative response of neonatal T lymphocytes to anti‐CD2 may therefore be due to decreased NFkB induction, reduced IL‐2 mRNA expression and deficient IL‐2 production. Although anti‐CD28 MoAb costimulation enhances all of the above signals, NFkB and IL‐2 levels remain lower than in adults, suggesting the need for further activation requirements in the neonate.

Bacitracin reveals a role for multiple thiol isomerases in platelet function.

The platelet‐specific integrin αIIbβ3 has endogenous thiol isomerase activity associated with the CXXC motifs within the β subunit. Using a highly purified form of bacitracin, a thiol isomerase inhibitor, we now provide further evidence of the functional significance of this enzymatic activity in integrin activation. In addition, we demonstrate a role for multiple thiol isomerases in platelet function. This bacitracin prevented platelet aggregation to thrombin and collagen, and directly inhibited αIIbβ3 activation, as detected by PAC‐1 binding. In parallel, bacitracin inhibited the endogenous thiol isomerase activity of purified αIIbβ3 with a 50% inhibitory concentration of 15·5 μmol/l. In order to determine whether the effects of bacitracin are solely mediated by inhibition of integrin enzymatic activity, we examined integrin‐independent indices of platelet activation. We found bacitracin inhibited both platelet secretion (CD62P and CD63) and thromboxane (TxA2) production, with complete inhibition at different concentrations. Thus, we demonstrated a role for multiple thiol isomerases in platelet function. Taken together, these studies support a role for the endogenous integrin thiol isomerase activity in activation of αIIbβ3 and highlight the novel regulation of platelet function by other, as yet undefined thiol isomerases.

Annexin V binding to platelets is agonist, time and temperature dependent

Platelets bind annexin V when stimulated with combinations of platelet agonists such as collagen and thrombin. Previous studies have demonstrated significant heterogeneity of platelets binding annexin V. The relative role of the thrombin protease-activated receptors (PARs), PAR1 and PAR4, together with different methods of platelet preparation on annexin V binding to platelets is unclear. We therefore investigated the role of PAR1- and PAR4-activating peptides in combination with collagen-related peptide on annexin V binding. In diluted whole blood, PAR1- and PAR4-activating peptides were as effective as thrombin in inducing annexin V binding. However, in washed platelets, PAR-activating peptides were less potent than thrombin at inducing annexin V binding. This difference was more pronounced when experiments were performed at 37°C compared to room temperature. In studies of diluted whole blood, platelet rich plasma and washed platelets, platelets incubated at room temperature bound more annexin V than platelets incubated at 37°C. We also saw a significant effect of time on annexin V binding, in that more annexin V bound to platelets with longer incubation times. In conclusion, PAR1 and PAR4-activating peptides were as effective as thrombin in inducing annexin V binding in combination with collagen-related peptide in diluted whole blood and platelet rich plasma, but not in washed platelets. In addition, incubation temperature and time has a strong influence on annexin V binding to platelets. Thus variations in these conditions may explain the differences observed between previous studies.

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 Glanzmanns Thrombasthenia (GT) diagnosed on the basis of a prolonged bleeding time and failure of the patients 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 3H‐SC52012, a non‐peptide GPIIb/IIIa antagonist. Dithiothreitol (DTT) and ADP enhanced the affinity for [3H]‐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 [3H]‐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.

Integrin α(IIb)β₃ exists in an activated state in subjects with elevated plasma homocysteine levels.

Elevated levels of plasma homocysteine (Hcy) are an independent risk factor for cardiovascular disease and thrombosis. The molecular basis for this phenomenon is not known but may relate to modification of cell surface thiols. The platelet specific integrin αIIbβ3 is a cysteine-rich cell adhesion molecule that plays a critical role in platelet aggregation and adhesion in haemostasis and thrombosis. In this study, we looked for evidence of a homocysteine-induced modification of αIIbβ3 using a fluorescently labeled PAC-1 antibody that recognizes the activated conformation of the integrin on the platelet surface. We show that exogenous Hcy (10–100 µM) and homocysteine thiolactone (HcyTL) (10–100 µM) increased PAC-1 binding to platelets in a concentration dependent manner in vitro. In parallel, we show subjects with clinical hyperhomocysteinemia exhibit a greater degree of activation of αIIbβ3 compared to age-matched controls. These findings demonstrate that circulating Hcy can modulate the activation state of the platelet integrin αIIbβ3, a key player in platelet aggregation and thrombosis.

Novel Integrin-Specific Targets for Anti-Platelet Therapies

Extensive study in integrin research has seen the platelet specific receptor alpha(IIb)beta(3) (Glycoprotein GPIIb/IIIa) under much scrutiny, and provided vast information as to the workings of this integrin within the blood. Glanzmanns thrombasthenia, a rare autosomal recessive bleeding disorder, highlights the vital role played by this receptor in platelet function. Glanzmanns thrombasthenic platelets fail to aggregate due to a lack of surface expression of functional alpha(IIb) or beta(3) on the platelet surface. However, little is known about the precise molecular mechanisms involved in the operation of this receptor on the platelet surface. Clinical trials using intravenous antagonists to this receptor have shown them to be effective anti-thrombotics. However the recent observations that the oral alpha(IIb)beta(3)antagonists have failed to show benefits in the treatment of acute coronary syndromes, and in fact, increase mortality, underscores the necessity for a more complete understanding of alpha(IIb)beta(3) and its functions. A more profound knowledge of the precise nature of the platelet integrin-activation, along with an understanding of its interactions with cellular signaling proteins, will undoubtedly lead to the identification of novel strategies for the effective inhibition of platelet integrin function.