Fadi T. Khasawneh

Characterization of isoprostane signaling: Evidence for a unique coordination profile of 8-iso-PGF2α with the thromboxane A2 receptor, and activation of a separate cAMP-dependent inhibitory pathway in human platelets

Since isoprostanes are thought to participate in the pathogenesis of thrombosis, presumably through their interaction with thromboxane receptors (TPRs), we examined the ability of 8-iso-PGF(2alpha) to bind/signal through TPRs. Using TPR expressing HEK cells, it was found that 8-iso-PGF(2alpha) mobilized calcium and bound TPRs with a dissociation constant (K(d)) of 57 nM. Interestingly, site-directed-mutagenesis revealed that 8-iso-PGF(2alpha) has a unique coordination profile with TPRs. Thus, while Phe184 and Asp193 are shared by both 8-iso-PGF(2alpha) and classical TPR ligands, Phe196 was found to be required only for 8-iso-PGF(2alpha) binding. Functional studies also revealed interesting results. Namely, that 8-iso-PGF(2alpha) signals in human platelets through both a stimulatory (TPR-dependent) and an inhibitory (cAMP-dependent) pathway. Consistent with the existence of two signaling pathways, platelets were also found to possess two separate binding sites for 8-iso-PGF(2alpha). While the stimulatory site is represented by TPRs, the second cAMP inhibitory site is presently unidentified, but does not involve receptors for PGI(2), PGD(2) or PGE(2). In summary, these studies provide the first documentation that: (1) 8-iso-PGF(2alpha) coordinates with Phe184, Asp193 and Phe196 on platelet TPRs; (2) Phe196 serves as a unique TPR binding site for 8-iso-PGF(2alpha); (3) 8-iso-PGF(2alpha) signals through both stimulatory and inhibitory pathways in platelets; (4) 8-iso-PGF(2alpha) inhibits human platelet activation through a cAMP-dependent mechanism; (5) 8-iso-PGF(2alpha) interacts with platelets at two separate binding sites. Collectively, these results provide evidence for a novel isoprostane function in platelets which is mediated through a cAMP-coupled receptor.

The P2Y12 antagonists, 2-methylthioadenosine 5'-monophosphate triethylammonium salt and cangrelor (ARC69931MX), can inhibit human platelet aggregation through a Gi-independent increase in cAMP levels.

ADP plays an integral role in the process of hemostasis by signaling through two platelet G-protein-coupled receptors, P2Y1 and P2Y12. The recent use of antagonists against these two receptors has contributed a substantial body of data characterizing the ADP signaling pathways in human platelets. Specifically, the results have indicated that although P2Y1 receptors are involved in the initiation of platelet aggregation, P2Y12 receptor activation appears to account for the bulk of the ADP-mediated effects. Based on this consideration, emphasis has been placed on the development of a new class of P2Y12 antagonists (separate from clopidogrel and ticlopidine) as an approach to the treatment of thromboembolic disorders. The present work examined the molecular mechanisms by which two of these widely used adenosine-based P2Y12 antagonists (2-methylthioadenosine 5′-monophosphate triethylammonium salt (2MeSAMP) and ARC69931MX), inhibit human platelet activation. It was found that both of these compounds raise platelet cAMP to levels that substantially inhibit platelet aggregation. Furthermore, the results demonstrated that this elevation of cAMP did not require Gi signaling or functional P2Y12 receptors but was mediated through activation of a separate G protein-coupled pathway, presumably involving Gs. However, additional experiments revealed that neither 2MeSAMP nor ARC69931MX (cangrelor) increased cAMP through activation of A2a, IP, DP, or EP2 receptors, which are known to couple to Gs. Collectively, these findings indicate that 2MeSAMP and ARC69931MX interact with an unidentified platelet G protein-coupled receptor that stimulates cAMP-mediated inhibition of platelet function. This inhibition is in addition to that derived from antagonism of P2Y12 receptors.

Glutathione and Adaptive Immune Responses against Mycobacterium tuberculosis Infection in Healthy and HIV Infected Individuals

Glutathione (GSH), a tripeptide antioxidant, is essential for cellular homeostasis and plays a vital role in diverse cellular functions. Individuals who are infected with Human immuno deficiency virus (HIV) are known to be susceptible to Mycobacterium tuberculosis (M. tb) infection. We report that by enhancing GSH levels, T-cells are able to inhibit the growth of M. tb inside macrophages. In addition, those GSH-replenished T cell cultures produced increased levels of Interleukin-2 (IL-2), Interleukin-12 (IL-12), and Interferon-gamma (IFN-γ), cytokines, which are known to be crucial for the control of intracellular pathogens. Our study reveals that T lymphocytes that are derived from HIV infected individuals are deficient in GSH, and that this deficiency correlates with decreased levels of Th1 cytokines and enhanced growth of M. tb inside human macrophages.

Platelet function and Isoprostane biology. Should isoprostanes be the newest member of the orphan-ligand family?

While there have been many reports investigating the biological activity and signaling mechanisms of isoprostanes, their role in biology, particularly in platelets, appears to still be underestimated. Moreover, whether these lipids have their own receptors is still debated, despite multiple reports that discrete receptors for isporpstanes do exist on platelets, vascular tissues, amongst others. This paper provides a review of the important literature of isoprostanes and provides reasoning that isoprostanes should be classified as orphan ligands until their receptor(s) is/are identified.

Differential mapping of the amino acids mediating agonist and antagonist coordination with the human thromboxane A2 receptor protein.

Despite the well documented involvement of thromboxane A2 receptor (TPR) signaling in the pathogenesis of thrombotic diseases, there are currently no rationally designed antagonists available for clinical use. To a large extent, this derives from a lack of knowledge regarding the topography of the TPR ligand binding pocket. On this basis, the purpose of the current study was to identify the specific amino acid residues in the TPR protein that regulate ligand coordination and binding. The sites selected for mutation reside within or in close proximity to a region we previously defined as a TPR ligand binding region (i.e. the C terminus of the second extracellular loop and the leading edge of the fifth transmembrane domain). Mutation of these residues caused varying effects on the TPR-ligand coordination process. Specifically, the D193A, D193Q, and D193R mutants lost SQ29,548 (antagonist) binding and exhibited a dramatically reduced calcium response, which could not be restored by elevated U46619 (agonist) doses. The F184Y mutant lost SQ29,548 binding and exhibited a reduced calcium response (which could be restored by elevated U46619); and the T186A and S191T mutants lost SQ29,548 binding and retained a normal U46619-induced calcium response. Furthermore, these last three mutants also revealed a divergence in the binding of two structurally different antagonists, SQ29,548 and BM13.505. Two separate mutants that exhibited SQ29,548 binding yielded either a normal (F196Y) or reduced (S201T) U46619 response. Finally, mutation of other residues directly adjacent to those described above (e.g. E190A and F200A) produced no detectable effects on either SQ29,548 binding or the U46619-induced response. In summary, these results identify key amino acids (in particular Asp193) involved in TPR ligand coordination. These findings also demonstrate that TPR-specific ligands interact with different residues in the ligand-binding pocket.

The Antidepressant 5-HT2A Receptor Antagonists Pizotifen and Cyproheptadine Inhibit Serotonin-Enhanced Platelet Function

There is considerable interest in defining new agents or targets for antithrombotic purposes. The 5-HT2A receptor is a G-protein coupled receptor (GPCR) expressed on many cell types, and a known therapeutic target for many disease states. This serotonin receptor is also known to regulate platelet function. Thus, in our FDA-approved drug repurposing efforts, we investigated the antiplatelet activity of cyproheptadine and pizotifen, two antidepressant 5-HT2A Receptor antagonists. Our results revealed that cyproheptadine and pizotifen reversed serotonin-enhanced ADP-induced platelet aggregation in vitro and ex vivo. And the inhibitory effects of these two agents were found to be similar to that of EMD 281014, a 5-HT2A Receptor antagonist under development. In separate experiments, our studies revealed that these 5-HT2A receptor antagonists have the capacity to reduce serotonin-enhanced ADP-induced elevation in intracellular calcium levels and tyrosine phosphorylation. Using flow cytometry, we also observed that cyproheptadine, pizotifen, and EMD 281014 inhibited serotonin-enhanced ADP-induced phosphatidylserine (PS) exposure, P-selectin expression, and glycoprotein IIb-IIIa activation. Furthermore, using a carotid artery thrombosis model, these agents prolonged the time for thrombotic occlusion in mice in vivo. Finally, the tail-bleeding time was investigated to assess the effect of cyproheptadine and pizotifen on hemostasis. Our findings indicated prolonged bleeding time in both cyproheptadine- and pizotifen-treated mice. Notably, the increases in occlusion and bleeding times associated with these two agents were comparable to that of EMD 281014, and to clopidogrel, a commonly used antiplatelet drug, again, in a fashion comparable to clopidogrel and EMD 281014. Collectively, our data indicate that the antidepressant 5-HT2A antagonists, cyproheptadine and pizotifen do exert antiplatelet and thromboprotective effects, but similar to clopidogrel and EMD 281014, their use may interfere with normal hemostasis.

Minimizing cardiovascular adverse effects of atypical antipsychotic drugs in patients with schizophrenia.

The use of atypical antipsychotic agents has rapidly increased in the United States and worldwide in the last decade. Nonetheless, many health care practitioners do not appreciate the significance of the cardiovascular side effects that may be associated with their use and the means to minimize them. Thus, atypical antipsychotic medications can cause cardiovascular side effects such as arrhythmias and deviations in blood pressure. In rare cases, they may also cause congestive heart failure, myocarditis, and sudden death. Patients with schizophrenia have a higher risk of cardiovascular mortality than healthy individuals, possibly because of excessive smoking, the underlying disorder itself, or a combination of both factors. Increased awareness of these potential complications can allow pharmacists and physicians to better manage and monitor high risk patients. Accurate assessments are very important to avoid medications from being given to patients inappropriately. Additionally, monitoring patients regularly via blood draws and checking blood pressure, heart rate, and electrocardiogram can help catch any clinical problems and prevent further complications. Finally, patient and family-member education, which pharmacists in particular can play key roles in, is central for the management and prevention of side effects, which is known to reflect positively on morbidity and mortality in these patients.

Mouse transient receptor potential channel 6: role in hemostasis and thrombogenesis.

Although changes in the intracellular levels of calcium (Ca(2+)) are a central step in platelet activation, the underlying mechanism of Ca(2+) entry is still unclear. Previous studies have demonstrated that TRPC6, a member of the canonical transient receptor potential channel (TRPC) family is expressed in platelets in a significant amount, and is predominantly found on the plasma membrane. Based on these considerations, we hypothesized that TRPC6 plays a critical role in platelet function. To characterize the role of TRPC6 in platelet function in vivo, we employed a genetic approach, subjecting TRPC6 knockout mice to the tail bleeding time test and a carotid artery injury thrombosis model. We found that TRPC6-deficient animals displayed a prolonged bleeding time, and an increased time for occlusion of the injured carotid artery, compared to their wild-type littermates. Taken together, our data demonstrate for the first time, that TRPC6 deletion in mice results in defects in hemostasis and protection against thrombogenesis, suggesting a vital role in platelet function. Furthermore, TRPC6 may define a new therapeutic target for managing multiple thrombosis-based disorders.

Thromboxane A2 Receptor Biology and Function of a Peculiar Receptor that Remains Resistant for Therapeutic Targeting

While blood platelets express several G-protein-coupled receptors (GPCRs) that play pivotal roles in their activation, several diseases, for example thrombotic disorders, may develop if these receptors are inappropriately activated. Thus, these receptors have been the subject of investigations to design therapeutic interventions for managing multiple thrombosis-based disease states. One such GPCR, the thromboxane A2 receptor (TPR), remains resistant to such interventions. The present review provides a critical examination of the binding, structural biology, and signaling of TPRs. The review also provides a rationale for using principles of “drug rediscovery” as an alternative/viable approach for the therapeutic targeting of TPRs. To this end, it is noteworthy that many US Food and Drug Administration (FDA)–approved drugs have been found to selectively (and nonselectively) block TPR-mediated functional responses, for example platelet aggregation, as described in this review. Therefore, while none of the antagonists, thus far developed for targeting TPRs, have made it into clinical use, this peculiar receptor can be antagonized by a large number of drugs used for indications unrelated to thrombosis.

Third-hand Smoke: Impact on Hemostasis and Thrombogenesis.

Abstract: Cigarette smoking is a major risk factor for acute coronary thrombosis. In fact, both active/first-hand smoke and passive/second-hand smoke exposure are known to increase the risk of coronary thrombosis. Although recently a new risk has been identified and termed third-hand smoke (THS), which is the residual tobacco smoke contaminant that remains after a cigarette is extinguished, it remains to be determined whether it can also enhance the risk of thrombogenesis, much like first-hand smoke and second-hand smoke. Therefore, the present studies investigated the impact of THS exposure in the context of platelet biology and related disease states. It was found that THS-exposed mice exhibited an enhanced platelet aggregation and secretion responses as well as enhanced integrin GPIIb-IIIa activation. Furthermore, it was found that THS exposure shortens the tail bleeding time and the occlusion time in a model of thrombosis. Thus, our data demonstrate for the first time (at least in mice) that THS exposure increases the risk of thrombosis-based disease states, which is attributed, at least in part, to their hyperactive platelets.