Arterial disease and thrombosis in the antiphospholipid syndrome (APS): A pathogenic role for endothelin-1 (ET-1)

Objective To explore a possible correlation between endothelin 1 (ET-1), the most potent endothelium-derived contracting factor that modulates vascular smooth muscle tone, and arterial disease in patients with the antiphospholipid syndrome (APS). Methods Plasma levels of ET-1 were measured in APS patients with (n = 16) and without (n = 11) arterial thrombosis and in non-APS patients with arterial thrombosis (n = 9). In addition, steady-state prepro-ET-1 messenger RNA (mRNA) levels were determined in endothelial cells treated with a range of human monoclonal anticardiolipin antibodies (aCL) (as anti-β2-glycoprotein I antibodies) by semiquantitative 32P-dCTP-labeled reverse transcription-polymerase chain reaction. Results Compared with healthy controls, markedly increased plasma levels of ET-1 were found in APS patients with arterial thrombosis (2.00 ± 0.87 versus 0.96 ± 0.37 pg/ml; P = 0.0001) but not in other groups. Three human monoclonal aCL induced prepro-ET-1 mRNA levels significantly more than did control monoclonal antibody lacking aCL activity. Conclusion Plasma ET-1 levels correlated significantly with a history of arterial thrombosis in patients with APS. Prepro-ET-1 mRNA was induced by human monoclonal aCL in the in vitro experimental system. The induction of ET-1 by antiphospholipid antibodies might contribute to increased arterial tone, leading to vasospasm and, ultimately, to arterial occlusion.

Gap junctions and connexin hemichannels underpin hemostasis and thrombosis

BACKGROUND: Connexins are a widespread family of membrane proteins that assemble into hexameric hemichannels, also known as connexons. Connexons regulate membrane permeability in individual cells or couple between adjacent cells to form gap junctions and thereby provide a pathway for regulated intercellular communication. We have now examined the role of connexins in platelets, blood cells that circulate in isolation, but upon tissue injury adhere to each other and the vessel wall to prevent blood loss and facilitate wound repair. METHODS AND RESULTS: We report the presence of connexins in platelets, notably connexin37, and that the formation of gap junctions within platelet thrombi is required for the control of clot retraction. Inhibition of connexin function modulated a range of platelet functional responses prior to platelet-platelet contact, and reduced laser induced thrombosis in vivo in mice. Deletion of the Cx37 gene (Gja4) in transgenic mice reduced platelet aggregation, fibrinogen binding, granule secretion and clot retraction indicating an important role for Cx37 hemichannels and gap junctions in platelet thrombus function. CONCLUSIONS: Together, these data demonstrate that platelet gap junctions and hemichannels underpin the control of haemostasis and thrombosis and represent potential therapeutic targets.

Gap junctions and connexin hemichannels in the regulation of haemostasis and thrombosis

Platelets are involved in the maintenance of haemostasis but their inappropriate activation leads to thrombosis, a principal trigger for heart attack and ischemic stroke. Although platelets circulate in isolation, upon activation they accumulate or aggregate together to form a thrombus, where they function in a coordinated manner to prevent loss of blood and control wound repair. Recent reports indicate that the stability and functions of a thrombus are maintained through sustained, contact dependent signalling between platelets. Given the role of gap junctions in the coordination of tissue responses, it was hypothesized that gap junctions may be present within a thrombus and mediate intercellular communication between platelets. Therefore studies were performed to explore the presence and functions of connexins in platelets. In this brief review, the roles of hemichannels and gap junctions in the control of thrombosis and haemostasis and the future directions for this research will be discussed.

Ibuprofen inhibits migration and proliferation of human coronary artery smooth muscle cells by inducing a differentiated phenotype: role of peroxisome proliferator-activated receptor y

Objectives: The search for agents that are capable of preventing restenosis and reduce the risk of late thrombosis is of utmost importance. In this study we aim to evaluate the in vitro effects of ibuprofen on proliferation and migration of human coronary artery smooth muscle cells (HCASMCs) and on human coronary artery endothelial cells (HCAECs) migration. Methods: Cell proliferation was evaluated by direct cell counting using trypan blue exclusion. Cell migration was assessed by wound healing “scratch” assay and by time lapse video-microscopy. Protein expression was assessed by immunoblotting, and morphological changes were studied by immunocytochemistry. The involvement of the PPARγ pathway was studied with the selective agonist troglitazone, and the use of highly selective antagonists of PPARγ such as PGF2α and GW9662. Results: We demonstrate that ibuprofen inhibits proliferation and migration of HCASMCs and induces a switch in HCASMCs towards a differentiated and contractile phenotype, and that these effects are mediated through the PPARγ pathway. Importantly we also show that the effects of ibuprofen are cell type specific as it does not affect migration and proliferation of endothelial cells. Conclusions: Taken together, our results suggest that ibuprofen could be an effective drug for the development of novel drug eluting stents, which could lead reduced rates of restenosis and potentially other complications of DES stent implantation.

SLAP/SLAP2 prevent excessive platelet (hem)ITAM signaling in thrombosis and ischemic stroke in mice

Glycoprotein VI and C-type lectin-like receptor 2 are essential platelet activating receptors in hemostasis and thrombo-inflammatory disease, which signal through a (hem)immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathway. The adapter molecules Src-like adapter proteins (SLAP and SLAP2) are involved in the regulation of immune cell surface expression and signaling, but their function in platelets is unknown. In this study, we show that platelets expressed both SLAP isoforms and that overexpression of either protein in a heterologous cell line almost completely inhibited glycoprotein VI and C-type lectin-like receptor 2 signaling. In mice, single deficiency of SLAP or SLAP2 had only moderate effects on platelet function, whereas double deficiency of both adapters resulted in markedly increased signal transduction, integrin activation, granule release, aggregation, procoagulant activity, and thrombin generation in response to (hem)ITAM-coupled, but not G protein-coupled, receptor activation. In vivo, constitutive SLAP/SLAP2 knockout mice displayed accelerated occlusive arterial thrombus formation and a dramatically worsened outcome after focal cerebral ischemia. This was attributed to the absence of both adapter proteins in platelets, as demonstrated by adoptive transfer of Slap(-/-)/Slap2(-/-) platelets into wild-type mice. Our results establish SLAP and SLAP2 as critical inhibitors of platelet (hem)ITAM signaling in the setting of arterial thrombosis and ischemic stroke.

Official warnings on thromboembolism risk with oral contraceptives fail to inform users adequately

Following the 1995 “pill scare” relating to the risk of venous thrombosis from taking second- or third-generation oral contraceptives, the Committee on Safety of Medicines (CSM) withdrew their earlier recommended restrictions on the use of third-generation pills and published recommended wording to be used in patient information leaflets. However, the effectiveness of this wording has not been tested. An empirical study (with 186 pill users, past users, and non-users) was conducted to assess understanding, based on this wording, of the absolute and relative risk of thrombosis in pill users and in pregnancy. The results showed that less than 12% of women in the (higher education) group fully understood the absolute levels of risk from taking the pill and from being pregnant. Relative risk was also poorly understood, with less than 40% of participants showing full understanding, and 20% showing no understanding. We recommend that the CSM revisit the wording currently provided to millions of women in the UK.

Omega-3 fatty acids, cardiovascular disease and stability of atherosclerotic plaques

Long-chain n-3 polyunsaturated fatty acids are found in oily fish and in fish oils and similar preparations. Substantial evidence from epidemiological and case-control studies indicates that consumption of fish, oily fish and long-chain n-3 fatty acids reduces risk of cardiovascular mortality. Secondary prevention studies using long-chain n-3 fatty acids in patients post-myocardial infarction have shown a reduction in total and cardiovascular mortality with an especially potent effect on sudden death. Long-chain n-3 fatty acids have been shown to beneficially modify a range of cardiovascular risk factors, which may result in primary cardiovascular prevention. However, reduced non-fatal and fatal events and a reduction in sudden death probably involve other mechanisms. Reduced thrombosis following long-chain n-3 fatty acids may play a role. A decrease in arrhythmias is a favoured mechanism of action of long-chain n-3 fatty acids and is supported by cell culture and animal studies. However human trials using implantable cardiac defibrillators have produced inconsistent findings and a recent meta-analysis does not support this mechanism of action. An alternative mechanism of action may be stabilisation of atherosclerotic plaques by long-chain n-3 fatty acids. This is suggested by one published human study which showed that incorporation of long-chain n-3 fatty acids into plaques collected at carotid endarterectomy resulted in fewer macrophages in the plaque and a morphology indicative of increased stability. These findings are supported from observations in an animal model and suggest that the primary effect of long-chain n-3 fatty acids might be on macrophages within the plaque.

Tweaking the gain on platelet regulation: The tachykinin connection

Soluble factors such as ADP and thromboxane (TX) A(2) that are secreted or released by platelets at sites of tissue injury, mediate autocrine and paracrine regulation of platelet function, resulting in rapid localised thrombus formation. The suppression of platelet function, particularly through targeting such secondary regulatory mechanisms, that serve to 'fine-tune' the platelet response, has proven effective in the prevention of inappropriate platelet activation that results in thrombosis. The most commonly used anti-platelet approaches (ADP receptor antagonism or inhibition of TXA(2) synthesis), however, lack efficacy in many patients, suggesting the existence of additional uncharacterised mechanisms for the regulation of platelet function. Recent data, which form a focus of this review, have identified peripheral tachykinin peptide family members, such as substance P and the newly identified endokinins, as physiologically important positive feedback regulators of platelet function. The actions of tachykinins that are released from platelets during activation are mediated by the neurokinin-1 receptor. Initial analysis of the role of this receptor in platelet thrombus formation, and thrombosis in the mouse, indicate this to be a promising new target for the development of anti-thrombotic drugs. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

A functional proteomic method for the enrichment of peripheral membrane proteins reveals the collagen binding protein Hsp47 is exposed on the surface of activated human platelets

Platelets are small blood cells vital for hemostasis. Following vascular damage, platelets adhere to collagens and activate, forming a thrombus that plugs the wound and prevents blood loss. Stimulation of the platelet collagen receptor glycoprotein VI (GPVI) allows recruitment of proteins to receptor-proximal signaling complexes on the inner-leaflet of the plasma membrane. These proteins are often present at low concentrations; therefore, signaling-complex characterization using mass spectrometry is limited due to high sample complexity. We describe a method that facilitates detection of signaling proteins concentrated on membranes. Peripheral membrane proteins (reversibly associated with membranes) were eluted from human platelets with alkaline sodium carbonate. Liquid-phase isoelectric focusing and gel electrophoresis were used to identify proteins that changed in levels on membranes from GPVI-stimulated platelets. Immunoblot analysis verified protein recruitment to platelet membranes and subsequent protein phosphorylation was preserved. Hsp47, a collagen binding protein, was among the proteins identified and found to be exposed on the surface of GPVI-activated platelets. Inhibition of Hsp47 abolished platelet aggregation in response to collagen, while only partially reducing aggregation in response to other platelet agonists. We propose that Hsp47 may therefore play a role in hemostasis and thrombosis.

Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins

In this study, we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell, the megakaryocyte, together with nucleated blood cell elements, endothelial cells, and erythroblasts, identified novel platelet membrane proteins with hitherto unknown roles in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)–based knockdown of 5 of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet glycoprotein (GP) IIb and the coagulation protein factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of 4 of the 5 novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis.

Proteomic analysis of resting and thrombin-stimulated platelets reveals the translocation and functional relevance of HIP-55 in platelets

The platelet surface is a dynamic interface that changes rapidly in response to stimuli to coordinate the formation of thrombi at sites of vascular injury. Tight control is essential as loss of organisation may result in the inappropriate formation of thrombi (thrombosis) or excessive bleeding. In this paper we describe the comparative analysis of resting and thrombin-stimulated platelet membrane proteomes and associated proteins to identify proteins important to platelet function. Surface proteins were labelled using a biotin tag and isolated by NeurtrAvidin affinity chromatography. Liquid phase IEF and SDS-PAGE were used to separate proteins, and bands of increased intensity in the stimulated platelet fractions were digested and identified by FT-ICR mass spectrometry. Novel proteins were identified along with proteins known to be translocated to the platelet surface. Furthermore, many platelet proteins revealed changes in location associated with function, including G6B and Hip-55. HIP-55 is an SH3-binding protein important in T-cell receptor signalling. Further analysis of HIP-55 revealed that this adaptor protein becomes increasingly associated with both Syk and integrin beta 3 upon platelet activation. Analysis of HIP-55 deficient platelets revealed reduced fibrinogen binding upon thrombin stimulation, suggesting HIP-55 to be an important regulator of platelet function.

Future innovations in anti-platelet therapies

Platelets have long been recognized to be of central importance in haemostasis, but their participation in pathological conditions such as thrombosis, atherosclerosis and inflammation is now also well established. The platelet has therefore become a key target in therapies to combat cardiovascular disease. Anti-platelet therapies are used widely, but current approaches lack efficacy in a proportion of patients, and are associated with side effects including problem bleeding. In the last decade, substantial progress has been made in understanding the regulation of platelet function, including the characterization of new ligands, platelet-specific receptors and cell signalling pathways. It is anticipated this progress will impact positively on the future innovations towards more effective and safer anti-platelet agents. In this review, the mechanisms of platelet regulation and current anti-platelet therapies are introduced, and strong, and some more speculative, potential candidate target molecules for future anti-platelet drug development are discussed.

The neurokinin 1 receptor: a potential new target for anti-platelet therapy?

The important role of platelets in the development of arterial thrombosis and cardiovascular disease is well established. Current treatments for arterial thrombosis include anti-platelet agents such as aspirin, thienopyridines and glycoprotein IIb-IIIa inhibitors. Despite these drugs being effective there remains a substantial unmet clinical demand for more effective therapeutic approaches, which may reflect the existence of alternative underlying regulatory mechanisms to those already targeted. Recent publications have demonstrated a key role for tachykinins in the positive feedback regulation of platelet aggregation and thrombus formation. The pro-thrombotic effects of tachykinins on platelets are mediated through the neurokinin 1 receptor, which may therefore offer a novel therapeutic drug target in the prevention and the treatment of arterial thrombosis.

Peripheral tachykinins and the neurokinin receptor NK1 are required for platelet thrombus formation

Platelets play an important role in hemostasis, with inappropriate platelet activation being a major contributor to debilitating and often fatal thrombosis by causing myocardial infarction and stroke. Although current antithrombotic treatment is generally well tolerated and effective, many patients still experience cardiovascular problems, which may reflect the existence of alternative underlying regulatory mechanisms in platelets to those targeted by existing drugs. In this study, we define a role for peripherally distributed members of the tachykinin family of peptides, namely substance P and the newly discovered endokinins A and B that are present in platelets, in the activation of platelet function and thrombus formation. We have reported previously that the preferred pharmacologically characterized receptor for these peptides, the NK1 receptor, is present on platelets. Inhibition or deficiency of the NK1 receptor, or SP agonist activity, resulted in substantially reduced thrombus formation in vitro under arterial flow conditions, increased bleeding time in mice, and a decrease in experimentally induced thromboembolism. Inhibition of the NK1 receptor may therefore provide benefit in patients vulnerable to thrombosis and may offer an alternative therapeutic target.

Platelet PECAM-1 inhibits thrombus formation in vivo

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a cell surface glycoprotein receptor expressed on a range of blood cells, including platelets, and on vascular endothelial cells. PECAM-1 possesses adhesive and signaling properties, the latter being mediated by immunoreceptor tyrosine-based inhibitory motifs present on the cytoplasmic tail of the protein. Recent studies in vitro have demonstrated that PECAM-1 signaling inhibits the aggregation of platelets. In the present study we have used PECAM-1-deficient mice and radiation chimeras to investigate the function of this receptor in the regulation of thrombus formation. Using intravital microscopy and laser-induced injury to cremaster muscle arterioles, we show that thrombi formed in PECAM-1-deficient mice were larger, formed more rapidly than in control mice, and were more stable. Larger thrombi were also formed in control mice that received transplants of PECAM-1-deficient bone marrow, in comparison to mice that received control transplants. A ferric chloride model of thrombosis was used to investigate thrombus formation in carotid arteries. In PECAM-1-deficient mice the time to 75% vessel occlusion was significantly shorter than in control mice. These data provide evidence for the involvement of platelet PECAM-1 in the negative regulation of thrombus formation.