Memory-like response in platelet attenuates platelet hyperactivation in arterial thrombosis

Nitric oxide (NO) stimulates soluble guanylyl cyclase and, thus, enhances cyclic guanosine monophosphate (cGMP) levels. It is a currently prevailing concept that NO inhibits platelet activation. This concept, however, does not fully explain why platelet agonists stimulate NO production. Here we show that a major platelet NO synthase (NOS) isoform, NOS3, plays a stimulatory role in platelet secretion and aggregation induced by low doses of platelet agonists. Furthermore, we show that NOS3 promotes thrombosis in vivo. The stimulatory role of NOS is mediated by soluble guanylyl cyclase and results from a cGMP-dependent stimulation of platelet granule secretion. These findings delineate a novel signaling pathway in which agonists sequentially activate NOS3, elevate cGMP, and induce platelet secretion and aggregation. Our data also suggest that NO plays a biphasic role in platelet activation, a stimulatory role at low NO concentrations and an inhibitory role at high NO concentrations.

NK cells are cytotoxic lymphocytes that provide systemic defense against pathogens and malignancy. Although historically considered cells of the innate immune system, NK cells are now known to be capable of memory or memory-like immune responses in certain settings. Memory NK responses were initially reported over a decade ago in studies involving mouse models of cytomegalovirus infection and delayed-type hypersensitivity reactions to chemical haptens and viral antigens. Since then, a growing body of literature suggests that memory or memory-like NK cell responses may occur in a broader range of immunological settings, including in response to various viral and bacterial infections, and some immunization protocols. Memory-like NK cell responses have also now been reported in humans and non-human primates. Here, we summarize recent studies demonstrating memory or memory-like responses by NK cells in settings of infection and immunization against infectious agents.

After injury to the blood vessel, platelets adhere to the exposed subendothelium by a process (adhesion) that involves the interaction of a plasma protein, von Willebrand factor (vWF), and a specific protein on the platelet surface, glycoprotein Ib (GPIb; the Figure⇓). Adhesion is followed by recruitment of additional platelets that form clumps, a process called aggregation (cohesion). This involves binding of fibrinogen to specific platelet surface receptors—a complex comprising glycoproteins IIb-IIIa (GPIIb-IIIa). Activated platelets release the contents of their granules (secretion or release reaction), such as ADP and serotonin from dense granules, which subsequently cause recruitment of additional platelets. In addition, platelets play a major role in coagulation mechanisms; several key enzymatic reactions occur on the platelet membrane–lipoprotein surface. A number of physiological agonists interact with specific receptors on the platelet surface to induce responses, including a change in platelet shape from discoid to spherical, aggregation, secretion, and thromboxane A2 (TxA2) production. Other agonists such as prostacyclin inhibit these responses. Ligation of the platelet receptors initiates the production or release of several intracellular messenger molecules, including Ca2+ ions, products of phosphoinositide (PI) hydrolysis by phospholipase C (PLC; diacylglycerol [DG] and inositol 1,4,5-triphosphate [InsP3]), TxA2, and cyclic nucleotides (cAMP; the Figure⇓). These subsequently induce or modulate the various platelet responses of Ca2+ mobilization, protein phosphorylation, aggregation, secretion, and liberation of arachidonic acid. The interaction between the agonist receptors and the key intracellular effector enzymes (eg, PLA2, PLC, adenylyl cyclase) is mediated by a group of GTP-binding proteins that are modulated by GTP. As in most secretory cells, platelet activation results in …

Dendritic cells (DCs), a vital component of the innate immune system, are considered to lack antigen specificity and be devoid of immunological memory. Strategies that can induce memory-like responses from innate cells can be utilized to elicit protective immunity in immune deficient persons. Here we utilize an experimental immunization strategy to modulate DC inflammatory and memory-like responses against an opportunistic fungal pathogen that causes significant disease in immunocompromised individuals. Our results show that DCs isolated from protectively immunized mice exhibit enhanced transcriptional activation of interferon and immune signaling pathways. We also show long-term memory-like cytokine responses upon subsequent challenge with the fungal pathogen that are abrogated with inhibitors of specific histone modifications. Altogether, our study demonstrates that immunization strategies can be designed to elicit memory-like DC responses against infectious disease.

The only reason P2Y12 inhibitors are administered in addition to aspirin is to improve the prevention of thrombosis. The clinical efficacy of adding clopidogrel to aspirin as a secondary prevention strategy in patients with high-risk coronary artery disease is well established.1 There are no effects of clopidogrel on any receptor other than P2Y12 to explain the magnitude of the clinical benefit. All of the established clinical effects are attributed to reduced platelet responsiveness to ADP.2 Therefore, the patient with inadequate P2Y12 inhibition determined by ex vivo testing logically has an increased risk for thrombosis. Persistent ischemic event occurrence and the irrefutable demonstration of clopidogrel antiplatelet response variability are 2 potent arguments against the widely practiced nonselective or one-size-fits-all strategy of administering clopidogrel therapy. Observational studies conducted in thousands of patients have led to an international consensus that high on-treatment platelet reactivity (HPR) to ADP is a major risk factor for post–percutaneous coronary intervention (PCI) ischemic event occurrence.3,4 Moreover, the recent 2011 American and European guidelines have given a Class IIb recommendation for platelet function testing or genotyping if the results of testing may alter management.5–7 Furthermore, the Society of Thoracic Surgeons gave a Class IIb recommendation for platelet function testing to determine the timing of surgery in patients on clopidogrel therapy (Level of Evidence C).8 These recommendations for personalizing antiplatelet therapy are unprecedented and acknowledge that a large body of data has accrued demonstrating the relation of HPR to ischemic risk in the PCI-treated patient. Finally, the evidence of diminished effectiveness of clopidogrel in poor metabolizers (those having 2 loss-of-function [LoF] cytochrome P450 [ CYP ] 2C19 alleles) has been recognized by the Food and Drug Administration (FDA) boxed warning about treatment with clopidogrel.9 Response by Krishna …

Thrombin 2 -adrenergic receptor (

Platelet activation and coagulation normally do not occur in an intact blood vessel. After blood vessel wall injury, platelet plug formation is initiated by the adherence of the platelets to subendothelial collagen [1,2]. In high shear arterial blood, platelets are first slowed down from their blood flow velocity by interacting with the collagen-bound von Willebrand factor and are subsequently stopped by binding directly to the collagen by their glycoprotein (GP) receptor complex [2,3]. The activation of these collagen receptors on platelets after their binding to the collagen activates phospholipase C-mediated cascades (Fig. 1) [1–3]. This results in the mobilization of calcium from the dense tubular system [4,5]. An increase in intracellular calcium is associated with the activation of several kinases necessary for morphologic change, the presentation of the procoagulant surface, the secretion of platelet granular content, the activation of GPs, and the activation of phospholipase A2 (see Fig. 1) [2,5–7]. The presentation of the procoagulant surface results in the colocalization of different coagulation factors on the surface of the activated platelet, which triggers a series of zymogen conversions, resulting in the release of active thrombin from prothrombin [8]. Adenosine diphosphate (ADP), adenosine triphosphate, and serotonin are released from the dense platelet granule. Activated phospholipase A2 enzymes release arachidonic acid (AA) by the cleaving of fatty acids, especially phosphatidylcholine and phosphatidylethanolamine, at their sn-2 position [9–11]. AA is a precursor for thromboxane A2 (TBXA2) synthesis. In the first step in platelets, prostaglandin (PG)-endoperoxide synthase 1 (PTGS1; also known as cyclooxygenase 1) catalyzes the transformation of AA into cyclic endoperoxide PG G2 and H2 [9]. In platelets, PGG2 and PGH2 are then mainly converted by TBXA synthase into TBXA2 [9]. Fig. 1 Effects of antiplatelet drugs in platelet aggregation pathway. (PA154444041; http://www.pharmgkb.org/do/serve?objId=PA154444041o cAMP, cyclic AMP; GNAS, guanine nucleotide binding protein a s; IP3, inositol ... The mechanism of action of aspirin is the inhibition of PTGS1, thereby preventing the production of PGs and, particularly in platelets, inhibiting TBXA2 production [10–12]. In ex vivo platelet aggregation testing, aspirin affects predominantly AA-stimulated platelet aggregation through a direct pathway, and also collagen-stimulated platelet aggregation through indirect pathways. A review by Lopez Farre et al. [12] discusses further mechanisms associated with platelet response to aspirin. The processes described above result in the local accumulation of molecules such as thrombin, TBXA2, and ADP, which are important for the further recruitment of platelets and the amplification of activation signals as described above. The secreted agonists activate their respective G protein-coupled receptors: coagulation factor II (thrombin) receptors (F2R also known as protease-activated receptor 1; F2RL3 also known as protease-activated receptor 4), TBXA2 receptor (TBXA2R), and ADP receptors (P2RY1 and P2RY12) [10,11,13–15]. The P2RY12 receptor couples to Gi, and when activated by ADP, inhibits adenylate cyclase [16]. This interaction counteracts the stimulation of cyclic AMP formation by endothelial-derived PGs, which alleviates the inhibitory effect of cyclic AMP on inositol 1,4,5-trisphosphate-mediated calcium release [14,16–20]. P2RY12 has a major role in arterial thrombosis and pharmacologic targeting of this receptor, which is an important strategy in the treatment of cardiovascular diseases [21]. Thienopyridines (ticlopidine, clopidogrel, prasugrel), a class of oral anti-platelet agents, permanently inhibit P2RY12 signaling by irreversibly binding the receptor and blocking ADP-induced platelet activation and aggregation [22]. F2R, TBXA2R, and P2RY1 couple to Gq-phospholipase C–inositol 1,4,5-trisphosphate–Ca2+ pathway, inducing shape change and platelet aggregation [14,23,24]. In addition, receptor signaling by G12/13 (F2R; TBXA2R) contributes to morphologic changes through the activation of kinases [23,24]. Platelet adhesion, cytoskeletal reorganization, secretion, and amplification loops are all different steps toward the formation of a platelet plug. These cascades finally result in the activation of the fibrinogen receptor (GPIIb/GPIIIa) expressed on platelet cells [14,25,26]. This activation results in the exposure of the binding sites for fibrinogen, which are not available in inactive platelets. The binding of fibrinogen results in the linkage of the activated platelets through fibrinogen bridges, thereby mediating aggregation [3]. The inhibition of this receptor by GPIIb/GPIIIa inhibitors blocks platelet aggregation induced by any agonist [27,28]. The individual platelet response is variable because of polymorphisms in genes involved in the activation and aggregation of platelets, in conjunction with environmental factors, and contributes to diseases such as arterial thrombosis ([29–33], and http://www.bloodomics.org/web/). In addition to the variation in platelet physiology, platelet sensitivity to drugs targeting platelet activation and aggregation is also influenced by gene polymorphisms and clinical and environmental variables [29,31,34].

Tuberculosis (TB), caused by M. tuberculosis (Mtb), remains the top killer among infectious diseases. The current TB vaccine, Bacille Calmette–Guérin (BCG), only protects young children from severe disseminated TB, but not effectively protects against pulmonary TB in adults. Improved TB vaccine or vaccination approach is needed for effective global prevention against TB. Our decade-long mechanistic studies in nonhuman primates (NHP) employed 2 innovative “gain-of-function” manipulations to generate Vγ2Vδ2 T effector cells in vivo for definition of their function and immunity against high-dose TB. These seminal studies established that HMBPP-specific Vγ2Vδ2 T effector cells are fast-acting, multi-functional and protective against high-dose TB in NHP. These novel findings prompted us to perform creative immunization of Vγ2Vδ2 T subset using our innovative vaccine vector Listeria ΔactA prfA*. Respiratory vaccination of macaques with an HMBPP-producing attenuated LM ΔactA prfA*, but not the control, caused prolonged expansion of HMBPP-specific Vγ2Vδ2 T cells in pulmonary and circulating compartments. After pulmonary Mtb challenge, macaques vaccinated with LM ΔactA prfA* exhibited rapid memory-like response of Vγ2Vδ2+ Th1 cells. The selective immunization of Vγ2Vδ2 T cells contained Mtb infection/dissemination and reduced lung TB pathology, enhancing the ability of tissue-resident Vγ2Vδ2 T cells to inhibit Mtb growth in macrophages. Thus, selective immunization of Vγ2Vδ2 T cells elicits fast-acting/durable memory-like responses, providing an approach to creating more effective TB vaccines.

Tuberculosis (TB) remains a leading killer among infectious diseases, and a better TB vaccine is urgently needed. The critical components and mechanisms of vaccine-induced protection against Mycobacterium tuberculosis (Mtb) remain incompletely defined. Our previous studies demonstrate that Vγ2Vδ2 T cells specific for (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) phosphoantigen are unique in primates as multifunctional effectors of immune protection against TB infection. Here, we selectively immunized Vγ2Vδ2 T cells and assessed the effect on infection in a rhesus TB model. A single respiratory vaccination of macaques with an HMBPP-producing attenuated Listeria monocytogenes (Lm ΔactA prfA*) caused prolonged expansion of HMBPP-specific Vγ2Vδ2 T cells in circulating and pulmonary compartments. This did not occur in animals similarly immunized with an Lm ΔgcpE strain, which did not produce HMBPP. Lm ΔactA prfA* vaccination elicited increases in Th1-like Vγ2Vδ2 T cells in the airway, and induced containment of TB infection after pulmonary challenge. The selective immunization of Vγ2Vδ2 T cells reduced lung pathology and mycobacterial dissemination to extrapulmonary organs. Vaccine effects coincided with the fast-acting memory-like response of Th1-like Vγ2Vδ2 T cells and tissue-resident Vγ2Vδ2 effector T cells that produced both IFN-γ and perforin and inhibited intracellular Mtb growth. Furthermore, selective immunization of Vγ2Vδ2 T cells enabled CD4+ and CD8+ T cells to mount earlier pulmonary Th1 responses to TB challenge. Our findings show that selective immunization of Vγ2Vδ2 T cells can elicit fast-acting and durable memory-like responses that amplify responses of other T cell subsets, and provide an approach to creating more effective TB vaccines.

Impact of Anemia on Platelet Response to Clopidogrel in Patients Undergoing Percutaneous Coronary Stenting

Platelet aggregation, in fundamental terms, is considered a biological end point that contributes to the occurrence of clinical events among patients with advanced atherosclerotic coronary artery disease. Acute coronary syndrome, including non–ST elevation myocardial infarction (NSTEMI) and ST elevation myocardial infarction (STEMI), accounts for upward of 733 000 hospital admissions yearly in the United States.1 The primary pathophysiological mechanism responsible for the majority of acute coronary syndromes is endothelial plaque disruption with and subsequent platelet adhesion, activation, and thrombus formation.2 The end result is formation of thrombus within a coronary artery, leading to subtotal vessel occlusion with NSTEMI and complete occlusion of the artery with STEMI. In this review, we provide a contemporary view of platelet adhesion as a highly coordinated and teleologically conserved process achieved by surface receptors, protein ligands, and matrix proteins operating at the platelet–subendothelium interface. We also discuss drugs in development, including monoclonal antibodies, inhibitory peptides, and oligonucleotides; preclinical data; and, where available, clinical trial results, highlighting the potential translation of fundamental constructs in platelet biology to patient care. Platelets are derived from a hematopoietic bone marrow stem cell precursor.3 Through a highly conserved program of cellular differentiation, this stem cell precursor becomes a megakaryocyte at a rate of approximately 100 000 000 megakaryocytes produced per day.3 Subsequently, each individual megakaryocyte will give rise to approximately 500 platelets via additional developmental steps within the bone marrow that involve a proplatelet intermediate stage.3 Once released into circulation, a mature platelet has an expected life span of 7 to 10 days.3 Platelet maturation and development involve the expression of receptors on the platelet cell surface. These receptors facilitate platelet adhesion and activation, and they promote thrombus development through receptor–ligand interactions, with several ligands expressed on the surface of endothelial cells, within the …

Impact of Obesity and the Metabolic Syndrome on Response to Clopidogrel or Prasugrel and Bleeding Risk in Patients Treated After Coronary Stenting

12-LOX (12-lipoxygenase) produces a number of bioactive lipids including 12(S)-HETE that are involved in inflammation and platelet reactivity. The GPR31 (G-protein-coupled receptor 31) is the proposed receptor of 12(S)-HETE; however, it is not known whether the 12(S)-HETE-GPR31 signaling axis serves to enhance or inhibit platelet activity. Approach and Results: Using pepducin technology and biochemical approaches, we provide evidence that 12(S)-HETE-GPR31 signals through Gi to enhance PAR (protease-activated receptor)-4-mediated platelet activation and arterial thrombosis using both human platelets and mouse carotid artery injury models. 12(S)-HETE suppressed AC (adenylyl cyclase) activity through GPR31 and resulted in Rap1 (Ras-related protein 1) and p38 activation and low but detectable calcium flux but did not induce platelet aggregation. A GPR31 third intracellular (i3) loop-derived pepducin, GPR310 (G-protein-coupled receptor 310), significantly inhibited platelet aggregation in response to thrombin, collagen, and PAR4 agonist, AYPGKF, in human and mouse platelets but relative sparing of PAR1 agonist SFLLRN in human platelets. GPR310 treatment gave a highly significant 80% protection (P=0.0018) against ferric chloride-induced carotid artery injury in mice by extending occlusion time, without any effect on tail bleeding. PAR4-mediated dense granule secretion and calcium flux were both attenuated by GPR310. Consistent with these results, GPR310 inhibited 12(S)-HETE-mediated and PAR4-mediated Rap1-GTP and RASA3 translocation to the plasma membrane and attenuated PAR4-Akt and ERK activation. GPR310 caused a right shift in thrombin-mediated human platelet aggregation, comparable to the effects of inhibition of the Gi-coupled P2Y12 receptor. Co-immunoprecipitation studies revealed that GPR31 and PAR4 form a heterodimeric complex in recombinant systems. The 12-LOX product 12(S)-HETE stimulates GPR31-Gi-signaling pathways, which enhance thrombin-PAR4 platelet activation and arterial thrombosis in human platelets and mouse models. Suppression of this bioactive lipid pathway, as exemplified by a GPR31 pepducin antagonist, may provide beneficial protective effects against platelet aggregation and arterial thrombosis with minimal effect on hemostasis.

The thrombotic response elicited by arterial injury or atherosclerosis varies widely between individuals; in some, diffuse coronary artery disease never triggers thrombotic occlusion, whereas others with limited disease experience myocardial infarction (MI) due to arterial thrombosis. Understanding the predictors for arterial thrombosis will improve tailored therapy for preventing and treating the complications of atherosclerosis and vascular injury. Extensive effort has been invested in developing accurate tools for risk stratification based on clinical features, but interactions between genetic and environmental factors will also influence an individual's risk for thrombosis. We are only beginning to appreciate how genetic factors may have an impact on the function of platelets, which play a key role in arterial thrombosis by forming an initial plug at sites of arterial injury or atherosclerotic plaque rupture or erosion. Ex vivo assays of platelet function reveal substantial interindividual heterogeneity, suggesting the hypothesis that intrinsic platelet reactivity may predict propensity to thrombosis. In animal models, the targeted deletion of any one of a number of proteins involved in platelet activation and/or aggregation protects from experimental thrombosis. Likewise, in the case of pharmacologically targeting P2Y12 receptors, more potent antagonists reduce clinical outcomes such as acute stent thrombosis. However, whether heightened platelet reactivity, in the absence of antiplatelet therapy, is causally associated with arterial thrombosis remains unknown. This is due, in part, to a lack of understanding of the pathways that may normally check platelet activation. In the current issue of Circulation , Angelillo-Scherrer et al 1 add connexin 37 (Cx37) to the small, but growing, list on membrane proteins that negatively regulate platelet function and implicate the Cx37 genotype as a risk marker for thrombosis. Article see p 930 The findings of Angelillo-Scherrer et al are important for …

A series of reports in 2007 established that single nucleotide polymorphisms (SNPs) in the 9p21.3 locus are associated with coronary heart disease (CHD), both in patients with acute myocardial infarction and chronic atherosclerosis.1,–,4 Subsequently, many additional studies used a candidate locus approach to replicate the 9p21.3-CHD association. Intriguingly, other phenotypes have also been found to be associated with this genomic region, including type II diabetes, stroke, malignant melanoma, aortic aneurysms, cerebral aneurysms, and periodontitis. Unfortunately, neither the causative variants nor genes have been established. Although genetic associations may permit better disease risk stratification, without the knowledge of the genes and variants we lose opportunities to improve our basic understanding of the disease process and hence the potential for targeted therapies. Article see p 445 Many cellular pathways in multiple tissues contribute to the pathogenic processes resulting in CHD. There is value in using intermediate phenotypes as outcomes in genetic association studies because there is enhanced power to detect gene associations when the number of genes potentially responsible for the phenotype is reduced, thereby increasing the fraction of the variance explained by any single factor or gene. In addition, intermediate traits are usually easier to define (have less heterogeneity) than clinical disease. Somewhat surprisingly, 9p21.3 has not been associated with measures believed to represent the chronic process of atherosclerosis or endothelial cell reactivity,5 suggesting that this locus may contribute to CHD by an alternate pathophysiologic mechanism. There is abundant pathological and clinical evidence demonstrating the vital role played by blood platelets in acute coronary syndromes, which result from the formation of occlusive platelet thrombi in coronary arteries at the sites of ruptured atherosclerotic plaques,6,7 and antiplatelet agents have become a mainstay of therapy in patients with acute coronary syndromes. In addition to …