Receptors In Thrombosis Research Articles

Roles of Platelet Toll-like Receptors in Thrombosis

Platelets are non-nuclear blood cells that are widely involved in physiological and pathological processes.Their main role is to participate in hemostasis and thrombosis.Toll-like receptors(TLRs)are innate immune receptors.Platelets express multiple TLRs and can promote thrombosis by recognizing ligand-induced platelet activation and aggregation.This article reviews the relationship between platelets/TLR and thrombosis and the roles of TLRs in the development of thrombotic diseases.

Platelet Purinergic Receptors in Thrombosis and Inflammation.

It took approximately 40 years from the seminal identification of adenosine diphosphate (ADP) as the factor R, an agent derived from red blood cells inducing platelet adhesion to glass, to the completion of the repertoire of its receptors on platelets and its importance in haemostasis and thrombosis. ADP, either derived from red blood cells or released by platelets themselves, stimulates platelets via two G protein-coupled receptors, P2Y1 and P2Y12. In addition, adenosine triphosphate, also contained in the platelet dense granules, activates the P2X1 cation channel. Each of these receptors plays a specific role during platelet activation and aggregation, with relevance to haemostasis, thrombosis and various inflammatory processes where platelets are involved including chronic responses such as atherosclerosis or acute responses such as sepsis, endotoxaemia or allergic asthma. Finally, platelets also express P2Y14, a receptor activated by released uridine diphosphate glucose. Although devoid of any known role in haemostasis, this receptor seems to play a specific role in neutrophil chemotaxis.

Role of P2Y12 Receptor in Thrombosis.

P2Y12 receptor is a 342 amino acid Gi-coupled receptor predominantly expressed on platelets. P2Y12 receptor is physiologically activated by ADP and inhibits adenyl cyclase (AC) to decrease cyclic AMP (cAMP) level, resulting in platelet aggregation. It also activates PI3 kinase (PI3K) pathway leading to fibrinogen receptor activation, and may protect platelets from apoptosis. Abnormalities of P2Y12 receptor include congenital deficiencies or high activity in diseases like diabetes mellitus (DM) and chronic kidney disease (CKD), exposing such patients to a prothrombotic condition. A series of clinical antiplatelet drugs, such as clopidogrel and ticagrelor, are designed as indirect or direct antagonists of P2Y12 receptor to reduce incidence of thrombosis mainly for patients of acute coronary syndrome (ACS) who are at high risk of thrombotic events. Studies on novel dual-/multi-target antiplatelet agents consider P2Y12 receptor as a promising part in combined targets. However, the clinical practical phenomena, such as "clopidogrel resistance" due to gene variations of cytochrome P450 or P2Y12 receptor constitutive activation, call for better antiplatelet agents. Researches also showed inverse agonist of P2Y12 receptor could play a better role over neutral antagonists. Personalized antiplatelet therapy is the most ideal destination for antiplatelet therapy in ACS patients with or without other underlying diseases like DM or CKD, however, there is still a long way to go.

Purinergic Receptors in Thrombosis and Inflammation.

Under various pathological conditions, including thrombosis and inflammation, extracellular nucleotide levels may increase because of both active release and passive leakage from damaged or dying cells. Once in the extracellular compartment, nucleotides interact with plasma membrane receptors belonging to the P2 purinergic family, which are expressed by virtually all circulating blood cells and in most blood vessels. In this review, we focus on the specific role of the 3 platelet P2 receptors P2Y1, P2Y12, and P2X1 in hemostasis and arterial thrombosis. Beyond platelets, these 3 receptors, along with the P2Y2, P2Y6, and P2X7 receptors, constitute the main P2 receptors mediating the proinflammatory effects of nucleotides, which play important roles in various functions of circulating blood cells and cells of the vessel wall. Each of these P2 receptor subtypes specifically contributes to chronic or acute vascular inflammation and related diseases, such as atherosclerosis, restenosis, endotoxemia, and sepsis. The potential for therapeutic targeting of these P2 receptor subtypes is also discussed.

The P2Y2 Nucleotide Receptor Mediates Tissue Factor Expression in Human Coronary Artery Endothelial Cells

The discovery of the role of P2Y(12) receptor in platelet aggregation leads to a new anti-thrombotic drug Plavix; however, little is known about non-platelet P2Y receptors in thrombosis. This study tested the hypothesis that endothelial P2Y receptor(s) mediates up-regulation of tissue factor (TF), the initiator of coagulation cascade. Stimulation of human coronary artery endothelial cells (HCAEC) by UTP/ATP increased the mRNA level of TF but not of its counterpart-tissue factor pathway inhibitor, which was accompanied by up-regulation of TF protein and cell surface activity. RT-PCR revealed a selective expression of P2Y(2) and P2Y(11) receptors in HCAEC. Consistent with this, TF up-regulation was inhibited by suramin or by siRNA silencing of P2Y(2) receptor, but not by NF-157, a P2Y(11)-selective antagonist, suggesting a role for the P2Y(2) receptor. In addition, P2Y(2) receptor activated ERK1/2, JNK, and p38 MAPK pathways without affecting the positive NF-κB and negative AKT regulatory pathways of TF expression. Furthermore, TF up-regulation was abolished or partially suppressed by inhibition of p38 or JNK but not ERK1/2. Interestingly, blockade of the PLC/Ca(2+) pathway did not affect P2Y(2) receptor activation of p38, JNK, and TF induction. However, blockade of Src kinase reduced phosphorylation of p38 but not JNK, eliminating TF induction. In contrast, inhibition of Rho kinase reduced phosphorylation of JNK but not p38, decreasing TF expression. These findings demonstrate that P2Y(2) receptor mediates TF expression in HCAEC through new mechanisms involving Src/p38 and Rho/JNK pathways, possibly contributing to a pro-thrombotic status after vascular injury.

P2Y Nucleotide Receptor and Thrombosis: Insight beyond Plavix

The discovery of platelet P2Y12 receptor leads to a new anti-thrombotic drug Plavix; however little is known about non-platelet P2Y receptors in thrombosis. This study tested the hypothesis that endothelial P2Y receptor(s) induces tissue factor (TF) expression, the initiator of coagulation cascade. Simulation of human coronary artery endothelial cells (HCAEC) by UTP/ATP increased TF mRNA, protein and its cell surface activity. RT-PCR detected expression of P2Y2 and P2Y11 receptors in HCAEC. Consistent with this, TF upregulation was inhibited by suramin or by siRNA silencing of P2Y2 receptor, but not by NF-157, a P2Y11-selective antagonist, suggesting a role for P2Y2 receptor. In addition, P2Y2 receptor activated ERK1/2, JNK and p38 MAPK pathways, without affecting the NF-kB pathway. Furthermore, TF upregulation was suppressed by inhibition of p38, or JNK, but not ERK1/2. Interestingly, blockade of the PLC/Ca2+ pathway did not affect P2Y2 receptor activation of p38, JNK and TF induction. However, blockade of Src kinase reduced activation of p38 but not JNK, eliminating TF induction. In contrast, inhibition of Rho kinase reduced activation of JNK but not p38, attenuating TF expression. These findings demonstrate P2Y2 receptor mediates TF expression in HCAECs through new mechanisms involving Src/p38 and Rho/JNK pathways, highlighting non-platelet P2Y receptor may contribute to the initiation of thrombosis.

MRS2500 [2-Iodo-N6-methyl-(N)-methanocarba-2′-deoxyadenosine-3′,5′-bisphosphate], a Potent, Selective, and Stable Antagonist of the Platelet P2Y1Receptor with Strong Antithrombotic Activity in Mice

The platelet P2Y(1) ADP receptor is an attractive target for new antiplatelet drugs. However, because of the lack of strong and stable antagonists, only a few studies have suggested that pharmacological inhibition of the P2Y(1) receptor could efficiently inhibit experimental thrombosis in vivo. Our aim was to determine whether the newly described potent and selective P2Y(1) receptor antagonist MRS2500 [2-iodo-N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate] could inhibit platelet function ex vivo and experimental thrombosis in mice in vivo. MRS2500 was injected intravenously into mice, and its effect on ex vivo platelet aggregation and in several models of thrombosis in vivo was determined. MRS2500 displayed high potency and stable and selective P2Y(1) receptor inhibition ex vivo. Although MRS2500 injection resulted in only moderate prolongation of the bleeding time, it provided strong protection in systemic thromboembolism induced by infusion of a mixture of collagen and adrenaline. MRS2500 also potently inhibited localized arterial thrombosis in a model of laser-induced vessel wall injury with two degrees of severity. Moreover, combination of MRS2500 with clopidogrel, the irreversible inhibitor of the platelet P2Y(12) receptor for ADP, led to increased antithrombotic efficacy compared with each alone. These results add further evidence for a role of the P2Y(1) receptor in thrombosis and validate the concept that targeting the P2Y(1) receptor could be a relevant alternative or complement to current antiplatelet strategies.

The novel suramin analogue NF864 selectively blocks P2X1 receptors in human platelets with potency in the low nanomolar range

The role of ATP-stimulated P2X1 receptors in human platelets is still unclear. They may act alone or in synergy with other pathways, such as P2Y1 or P2Y12 receptors, to accelerate and enhance calcium mobilisation, shape change and aggregation. To date very few pharmacological means of selectively inhibiting platelet P2X1 receptors have been described, although recent work has shown that suramin is a useful lead compound for the development of high-affinity P2X1 antagonists. We therefore investigated the effects of a series of bivalent and tetravalent suramin analogues on alphabeta meATP (P2X1 receptors)-induced or ADP (P2Y1 receptors)-induced intracellular calcium increases and shape change, as well as on ADP-induced aggregation (P2Y1 & P2Y12 receptors) in human platelets. Changes in intracellular calcium were measured using standard fluorescence techniques, while shape change and aggregation were determined by turbidimetry. The novel tetravalent compound NF864 (8,8',8'',8'''-(carbonylbis(imino-5,1,3-benzenetriyl-bis(carbonylimino)))tetrakis-naphthalene-1,3,5-trisulfonic acid-dodecasodium salt) proved to be the most potent platelet P2X1 antagonist reported to date, blocking alphabeta meATP-induced Ca2+ increases and shape change in a concentration-dependent manner, with a pA2 of 8.17 and 8.49, respectively. The ability to inhibit the platelet P2X1 receptor displayed the following order : NF864 > NF449 > or = NF110 > NF023 = MK-HU1 = suramin. A different antagonistic profile was observed for ADP-induced Ca2+ increases, shape change and aggregation; however, overall four compounds showed sufficient ability to selectively inhibit P2X1 responses, with the order NF110 > NF449 > or = NF864 > or = MK-HU1. Therefore, these compounds should prove useful tools for investigating the functional significance of platelet P2X1 receptors in thrombosis and haemostasis, NF864 being the most promising compound.

The Platelet P2 Receptors in Thrombosis

Adenosine diphosphate (ADP) and adenosine triphosphate (ATP) play a crucial role in hemostasis and thrombosis, and their receptors are potential targets for antithrombotic drugs. The ATP-gated channel P2X (1) and the two G protein-coupled P2Y (1) and P2Y (12) ADP receptors selectively contribute to platelet aggregation. Because of its central role in the formation and stabilization of a thrombus, the P2Y (12) receptor is a well-established target of antithrombotic drugs such as clopidogrel, which has proven efficacy in many clinical trials and experimental models of thrombosis. Competitive P2Y (12) antagonists have also been shown to be effective in experimental thrombosis as well as in several clinical trials. Studies in P2Y (1) and P2X (1) knockout mice and experimental thrombosis models using selective P2Y (1) and P2X (1) antagonists have shown that, depending on the conditions, these receptors could also be potential targets for new antithrombotic drugs. Because both P2X (1) and P2Y (1) receptor inhibition result in milder prolongation of the bleeding time as compared with P2Y (12) inhibition, the idea is put forward that combinations of P2 receptor antagonists could improve efficacy with diminished hemorrhagic risk. However, further studies with stronger and more selective P2 receptor antagonists are required to validate such a point of view.

Scientific and therapeutic insights into the role of the platelet P2Y12 receptor in thrombosis.

Platelets are important mediators of thrombosis in both healthy and diseased vessels. When platelets become activated by various soluble agonists or by adhesion to subendothelium under high shear, they release adenosine-5'-diphosphate that acts in a positive feedback mechanism on two different G-protein coupled receptors (P2Y(12), P2Y(1)) on platelets. This released adenosine-5'-diphosphate, acting through P2Y(12), is critical for sustained aggregation and stabilization of thrombi. P2Y(12) is the target of antithrombotic drugs (ticlopidine, clopidogrel), whereas the role of P2Y(1) in thrombosis remains to be fully established. Recent studies using either inhibitors of key components of signaling pathways or genetically engineered mice have contributed to our understanding of the signaling mechanisms in platelets mediated by adenosine-5'-diphosphate through the P2Y(12) receptor. Studies of patients with defective adenosine-5'-diphosphate mediated aggregation, as well as P2Y(12)-null mice, have revealed the importance of this receptor in mediating platelet activation and aggregation. Recent clinical trials using approved P2Y(12) blockers have extended the use of these drugs to additional patient populations. Recent data demonstrating the role of P2Y(12) in mediating platelet adhesion to thrombogenic surfaces (collagen, von Willebrand factor) provide further rationale as to the clinical efficacy of P2Y(12) blockers. P2Y(12) antagonists in combination with anticoagulants (thrombin inhibitors, factor Xa inhibitors) act synergistically in inhibiting thrombus formation (similar to aspirin) ex vivo. These findings suggest the potential for combination therapies (P2Y(12) antagonists with inhibitors of GPIIb-IIIa, thrombin or Factor Xa, etc.) to provide additional clinical benefit to patients with various cardiovascular diseases, especially those who may be aspirin-resistant.

Design and evaluation of potent peptide‐mimetic PAR1 antagonists

AbstractThe serine protease α‐thrombin is a powerful mediator of cellular responses, not only playing a key regulatory role in the maintenance of normal hemostasis, but also contributing more broadly to a range of physiological and potentially pathological conditions. Most of the cellular actions attributed to α‐thrombin have been linked to the activation of a family of seven‐transmembrane G‐protein‐coupled protease‐activated receptors, termed PARs. Of the four family members identified, PAR1 has received recognition as the most significant receptor for mediating thrombin's cellular actions. Thus, the development of a potent, selective antagonist of PAR1 would be an important and essential tool to probe the physiological and potential pathological roles of PAR1. In this review, we provide a synopsis of our de novo design approach to the discovery of selective PAR1 antagonists, exemplified by RWJ‐58259, along with the in vitro and in vivo studies performed to demonstrate the important role of this receptor in thrombosis and vascular injury. RWJ‐58259 demonstrated antithrombotic activity in a model of vascular injury‐induced thrombosis in cynomolgus monkeys and prevented neointimal proliferation in a rat balloon angioplasty model. These seminal studies clearly lay the groundwork for more detailed preclinical in vivo studies and ultimately, advancing PAR1 antagonists into human clinical studies. Expanded chemical synthetic efforts and other novel technologies targeted to PAR1 and its intracellular signaling pathways are already on the horizon in an effort to further modulate thrombin's biological actions. Drug Dev. Res. 59:355–366, 2003. © 2003 Wiley‐Liss, Inc.

Laboratory issues in diagnosing abnormalities of protein C, thrombomodulin, and endothelial cell protein C receptor.

To review the current understanding of the pathophysiology of protein C deficiency and its role in congenital thrombophilia. Recommendations for diagnostic testing for protein C function and concentration, derived from the medical literature and consensus opinions of recognized experts in the field, are included, specifying whom, how, and when to test. The role of related proteins, such as thrombomodulin and endothelial protein C receptor, is also reviewed. Data Sources.-Review of the published medical literature. A summary of the medical literature and proposed testing recommendations were prepared and presented at the College of American Pathologists Conference XXXVI: Diagnostic Issues in Thrombophilia. After discussion at the conference, consensus recommendations presented in this manuscript were accepted after a two-thirds majority vote by the participants. Protein C deficiency is an uncommon genetic abnormality that may be a contributing cause of thrombophilia, often in conjunction with other genetic or acquired risk factors. When assay of protein C plasma levels is included in the laboratory evaluation of thrombophilia, a functional amidolytic protein C assay should be used for initial testing. The diagnosis of protein C deficiency should be established only after other acquired causes of protein C deficiency are excluded. A low protein C level should be confirmed with a subsequent assay on a new specimen. Antigenic protein C assays may be of benefit in subclassification of the type of protein C deficiency. The role of thrombomodulin and endothelial cell protein C receptor in thrombosis has yet to be clearly established, and diagnostic testing is not recommended at this time.

Integrin alpha IIb beta 3 mediates binding of the Lyme disease agent Borrelia burgdorferi to human platelets.

Lyme disease is a chronic, multisystemic infection caused by the tick-borne spirochete Borrelia burgdorferi. Attachment of the spirochete to host cells via specific receptors is likely to be important in the establishment of infection. B. burgdorferi have previously been shown to bind to a variety of mammalian cells in vitro. Here we demonstrate that binding of B. burgdorferi to human platelets is mediated by the integrin alpha IIb beta 3 (glycoprotein IIb-IIIa), a critical receptor in thrombosis and hemostasis. Functional expression of this receptor requires platelet activation, and binding of the spirochete was observed only to activated platelets. Binding was inhibited by a synthetic Arg-Gly-Asp peptide that blocks ligand interaction with many integrins and by a synthetic peptide based on the gamma chain of fibrinogen that blocks binding to alpha IIb beta 3. In addition, attachment of the spirochete to platelets was inhibited by monoclonal antibodies directed against alpha IIb beta 3 that are known to block ligand-receptor interaction. No inhibition was seen with control peptides or with antibodies directed against other platelet receptors. B. burgdorferi bound efficiently to purified alpha IIb beta 3 but did not bind to platelets deficient in this integrin. Efficient platelet binding was displayed by a cloned, infectious B. burgdorferi strain, whereas a cloned noninfectious strain did not bind to platelets. Binding to integrins may be important for the ability of B. burgdorferi to establish infection in the diverse tissues affected by Lyme disease.