The role of T-helper cells in atherosclerosis.

Atherosclerosis is rapidly gaining recognition as an inflammatory disease showing contribution from innate and adaptive immunity pathways towards disease initiation and progression. Components of adaptive immunity especially T cells, are shown to be involved in atherogenesis and subsets of T cells are known to drive/ dampen inflammatory processes in atherosclerosis. However, the regulatory balance between the T cell subsets remains unclear. In this review, we summarize the role of T helper cells Th1, 2, 3 and 17, and regulatory cells Treg in atherosclerosis by studying the cytokines involved in Th cell functioning. We further examine the diverse roles of T helper cells for regulating the progression of atherosclerosis.

Extended prophylaxis with bemiparin for the prevention of venous thromboembolism after abdominal or pelvic surgery for cancer: the CANBESURE randomized study.

BACKGROUND: There is not enough clinical evidence to make a strong recommendation on the optimal duration of thromboprophylaxis using low-molecular weight heparins (LMWH) in patients undergoing major cancer surgery. PATIENTS AND METHODS: CANBESURE is a randomized, double-blind study which enrolled patients admitted for abdominal or pelvic surgery for cancer. They received 3500 IU of bemiparin subcutaneously once daily for 8 days and were then randomized to receive either bemiparin or placebo for 20 additional days. Bilateral venography was performed after 20 days and evaluated blinded. The primary efficacy outcome was the composite of deep vein thrombosis (DVT), non-fatal pulmonary embolism (PE) and all-cause mortality at the end of double-blind period. Major venous thromboembolism (proximal deep-vein thrombosis, non-fatal pulmonary embolism and venous thromboembolism-related deaths) was also evaluated. The primary safety outcome was major bleeding. RESULTS: Six hundred and twenty-five and 488 patients were included in the safety and main efficacy analyzes, respectively. The primary efficacy outcome occurred in 25 out of 248 patients (10.1%) in the bemiparin group and 32 out of 240 (13.3%) in the placebo group (relative risk reduction 24.4%; 95% CI: -23.7-53.8%; P = 0.26). At the end of double-blind period, major venous thromboembolism occurred in 2 (0.8%) and 11 (4.6%) patients, respectively (relative risk reduction 82.4%; 95% CI: 21.5-96.1%; P = 0.010). No significant difference was found in major bleedings. CONCLUSIONS: Four weeks compared with 1 week of prophylaxis with bemiparin after abdominal or pelvic cancer surgery did not significantly reduce the primary efficacy outcome, but decreased major venous thromboembolism (VTE) without increasing hemorrhagic complications.

The role of integrin-mediated cell adhesion in atherosclerosis: pathophysiology and clinical opportunities.

Integrins have been reported to mediate cell survival, proliferation, differentiation, and migration programs. For this reason, the past few years have seen an increased interest in the implications of integrin receptors in atherosclerosis. This review considers the potential role of integrins in atherosclerosis and also addresses why integrins present attractive targets for drug design. It discusses the properties of the integrin-based chemotherapeutic agents currently under consideration clinically and endeavours to provide insights into development of cardiovascular drugs using integrins as targets.

Major joint replacement. A model for antithrombotic drug development: from proof-of-concept to clinical use.

AIM: Development of antithrombotic compounds has traditionally been performed in patients undergoing total hip and knee replacement surgery. A high number of asymptomatic deep-vein thromboses are radiologically detectable, and bleeding and other adverse events (AE) are easy to observe. However, standardization of study procedures and endpoints in early proof-of-concept studies and late pure clinical endpoint studies has been lacking. This has made comparison between studies difficult, economic analyses speculative and potential benefits of applying the drug regimen in non-selected patients uncertain. In this paper, the International Surgical Thrombosis Forum proposes a strategy for the clinical investigation of new pharmacological agents for the prophylaxis of postoperative thrombotic events. METHODS: First, dose titration safety studies of short duration, in highly selected patients using objective venographic endpoints are recommended. Bleeding should be divided into the quantified volume of surgical bleeding and other adjudicated clinical bleeding events. The number of AE should be described for each dose step and classified according to International Coding of Diagnoses (ICD). Second, a dose confirmatory study of moderate exposure period and sufficient follow-up time is recommended. The exclusion criteria should be restricted to contraindications of the compared drugs and technical procedure. RESULTS: The efficacy, bleeding and AE should be similar to those used in dose-titration studies. In addition, the failure rate of the drug to exert its effect and the net clinical benefit should be calculated. CONCLUSION: Finally, trials with simple clinical endpoints and long follow-up should be conducted to evaluate the potential benefits of the drug-regimen in non-selected populations.

Management of venous thromboembolism.

INTRODUCTION: Venous Thromboembolism is an important healthcare problem the world over, resulting in significant morbidity, mortality and resource expenditure. The rationale for use of thromboprophylaxis is based on solid principles and scientific evidence. Indian perspective on this topic is lacking due to the non-availability of published Indian data. This document reviews the available International and Indian data and discusses the relevance of recommendations for prevention and management of Venous Thromboembolism (VTE) in the Indian context. MATERIALS AND METHODS: Meetings of various specialists from different Indian hospitals in the field of Gastrointestinal Surgery, General and Vascular Surgery, Hematology, Intensive Care, Obstetrics and Gynecology, Oncology and Orthopedics were held in the months of August 2005 to January 2006. The guidelines published by American College of Chest Physicians (ACCP), the International Union of Angiology (IUA), and the Royal College of Obstetricians and Gynecologists (RCOG), were discussed during these meetings. The relevance of these guidelines and the practical implications of following these in a developing country like India were also discussed. Any published data from India was collected from data base searches and the results, along with personal experiences of the participating specialists were discussed. The experiences and impressions of the experts during these meetings have been included in this document. Data from recent sources (International Union of Angiology and the National Comprehensive Cancer Network (NCCN) Practice guidelines in Oncology on Venous thromboembolic disease) was subsequently also included in this document. RESULTS: The suggestions formulated in this document are practical, and would intend to serve as a useful practical reference. CONCLUSIONS: A number of unanswered questions remain in the field of thromboprophylaxis, and carefully designed research protocols may help answer some of these. Implementation of the suggestions outlined in the document remains to be studied in the Indian context.

Structure-activity relationship studies on ADAM protein-integrin interactions.

The ADAM (a disintegrin and metalloprotease) family of proteins possess multi-domain structures composed of a signal peptide, a prodomain, a metalloprotease domain, a disintegrin-like domain, a cysteine rich domain, an epidermal growth factor-like domain, a transmembrane domain and cytoplasmic tail. The disintegrin-like domain shares sequence similarity with the soluble venom disintegrins, a family of proteins which are potent inhibitors of integrin-mediated platelet aggregation and cell adhesion. Several ADAMs have been reported to interact with integrins, and the disintegrin-like domain may be crucial part in this respect. A description of structure-activity relationship of ADAM proteins interacting with integrin is outlined in this review. The review highlights recent reports on potential integrin family for ADAMs and how ADAMs selectively modulate interaction for integrin mediated cell function. Lastly, it describes progress in understanding the structural features and functional roles of the ADAMs in normal and pathological conditions and how this insight may assist the development of new therapeutic approaches.

Antithrombotic therapy in patients with chronic heart failure: rationale, clinical evidence and practical implications.

Chronic heart failure (CHF) is traditionally associated with increased risk of thromboembolic complications. Key features of CHF pathophysiology, such as impairment of intracardiac hemodynamics, peripheral blood flow deceleration, neuroendocrine activation, chronic oxidative stress and proinflammatory changes, could explain the predisposition to thromboembolism. However, conclusive epidemiologic data on thromboembolic event incidence in CHF are lacking. Furthermore, the place of antithrombotic therapy in CHF management is still uncertain. Apart from established indications for warfarin (e.g. atrial fibrillation and previous embolic events), there is no robust evidence to support administration of vitamin K antagonists to other patients with CHF, particularly to patients in sinus rhythm. The role of aspirin in preventing thromboembolism in these patients is also controversial. Large randomized trial data on the effectiveness and risks of warfarin and aspirin use in CHF patients with sinus rhythm are forthcoming. This article provides a brief overview of the epidemiologic and pathobiological background of thromboembolism in CHF, and discusses the up-to-date clinical evidence on antithrombotic therapy in detail.

Integrins in drug targeting-RGD templates in toxins.

Integrins are a family of heterodimeric receptors, which modulate many cellular processes including: growth, death (apoptosis), adhesion, migration, and invasion by activating several signaling pathways. Integrin-binding RGD (arginine-glycine-aspartic acid) is found in several important extracellular matrix proteins which serve as adhesive integrin ligands. The RGD motif has also been found in many toxins from snake venom and other sources that specifically inhibit integrin binding function and serve as potent integrin antagonists, particularly of platelet aggregation and integrin-mediated cell adhesion. Many of these proteins have potential as therapeutic agents which can target integrins directly. Structural and functional studies of several RGD-containing toxins suggest that the inhibitory potency of these proteins lies in subtle positional requirements of the tripeptide RGD at the tip of a flexible loop, a structural feature for binding to integrins. In addition, amino acid residues in this loop in close vicinity to the RGD-motif determine the integrin-binding specificity and selectivity. This review will present a review of integrin structure and function, and of disintegrin structural features responsible for their activity as antagonists of integrin function. The use of integrins in drug targeting and integrins as targets for drug delivery by using the RGD as a template structure will also be discussed.

Low molecular weight heparins in cardiovascular medicine.

Cardiovascular diseases are the major cause of morbidity and mortality in the Western countries and their prevalence is increasing in developing world. The final biological evolution of atherosclerotic process, supporting development and progression of cardiovascular diseases, is thrombosis. In the most recent years several clinical trails have established that low molecular weight heparins play a major role in the area of prevention and treatment of arterial and venous thrombosis. It is now established, that low molecular weight heparins are efficacious and safe anticoagulant options for patients with deep vein thrombosis, pulmonary embolism, unstable angina and non-ST-segment elevation myocardial infarction. In addition, low molecular weight heparins play a major role to prevent thromboembolic events in patients with chronic diseases (e.g. due to cerebrovascular ischemic events, cancer) and in patients undergoing surgical interventions. Clinical trials have also shown that low molecular weight heparins might play a role in the treatment of patients with ST-segment elevation acute myocardial infarction, in the prevention of thrombotic events in patients with congestive heart failure, and in patients undergoing percutaneous coronary interventions. The combined use of low molecular weight heparins with fibrinolysis and other antithrombotic agents has been also studies in a number of clinical trials. This review summarises the results of the most recent clinical studies regarding the use of low molecular weight heparins in prevention and treatment of cardiovascular diseases.

Snake venom metalloproteinase containing a disintegrin-like domain, its structure-activity relationships at interacting with integrins.

Snake venom disintegrins represent a family of RGD (Arg-Gly-Asp) or KGD (Lys-Gly-Asp)-containing proteins which have been reported to be unique and potentially useful tools not only for investigating integrin-ligand interactions, but also for the development of anti-thrombotic agents in terms of their anti-platelet activities. Snake venom proteins containing a disintegrin-like domain represent another super-family of proteins in which many of them have been demonstrated to have similar ability to inhibit platelet aggregation and integrin-mediated cell adhesion as the disintegrins. This super-family includes a large number of snake venom metalloproteinases and disintegrin related, RGD-containing snake venom proteins (disintegrin-like proteins) such as dendroaspin. Recently, a family of homologues of the snake venom metalloproteinases have been found in a wide variety of mammalian tissues as well as in other eukaryotic organisms termed ADAM (a disintegrin-like and metalloproteinase) proteins. ADAMs are members of the metazincins that also include the related matrix metalloprotease (MMPs). Some of ADAM proteins have now shown to interact with integrins, and the disintegrin-like domain may be crucial part in their function as proteases. A description of structure-activity relationships of snake venom proteins containing a disintegrin-like domain is outlined in this review, along with reports of the modulation of protein activity by recombinant mutation. Comparison is also made of the structural and functional features of the metalloproteinases in snakes compared with those from other species. The review is intended to provide insights in which may assist the development of new therapeutic approaches.

Chronic heart failure- potential for pharmacological intervention.

Heart failure is commonly associated with vascular diseases and a high rate of athero-thrombotic events, but the risks and benefits of antithrombotic therapy are unknown. The incidence of thromboembolism in heart failure patients (which may include stroke, peripheral embolism, pulmonary embolism) seems to be around 2%, based on the data available from several small studies. However, the incidence of thromboembolism should greatly depend upon what is being looked at in each of these studies, as it will (generally) not be individually categorised. There is very little true epidemiological data to base this figure. The pathophysiology of heart failure is complex. There are many well- recognised factors, which are associated with thrombosis in heart failure patients, such as vascular abnormalities, increased coagulability and impaired blood flow. In the past 50 years, many studies have been performed to find out if oral anticoagulation is of benefit for the prevention of thromboembolism in patients with heart failure. Expert therapeutic guidelines in the Europe and North America agree that there is insufficient evidence to recommend that antithrombotic therapy should be given to patients with heart failure, unless they have atrial fibrillation or, perhaps, a previous thrombo-embolic episode. There is a lack of evidence for any antithrombotic agent that is effective in patients with heart failure; therefore, randomised clinical trials need to be designed to test the hypothesis that patients with chronic heart failure would have benefit from anticoagulant therapy. This review summarises the incidence, potential mechanism and therapeutic approaches for the management of thromboembolism in heart failure.

Modulation of integrin-binding selectivity by mutation within the RGD-loop of snake venom proteins: a novel drug development approach.

Integrins are a family of heterodimeric class I transmembrane receptors, many of which bind to the RGD sequence in adhesive proteins and mediate the adhesive interactions of a variety of cells. The RGD motif has also been found in snake venom proteins that specifically inhibit integrin binding function and serve as potent integrin antagonists. The majority of these proteins interact with beta1 and beta3 associated integrins and their potency is at least 500-2000 times higher than short RGD peptides. Structural and functional studies suggest that the inhibitory potency of these proteins lies in subtle positional requirements of the tripeptide RGD that is harboured in a defined flexible loop. The integrin-binding specificity and selectivity of each of the proteins is controlled by amino acid residues in this loop in close vicinity to the RGD-motif. The review includes an overview of the structure and function of snake-venom integrin antagonists. The ability of these proteins to control platelet aggregation, cell adhesion and ligand binding is compared to that of short linear, cyclic RGD-peptides and RGD-containing proteins and the influence of modulation of amino acid residues flanking the RGD motif is also considered. The review is intended to provide insight into the development of novel inhibitors as drugs.

Inhibition of human alpha-thrombin by a phosphonate tripeptide proceeds via a metastable pentacoordinated phosphorus intermediate.

X-ray crystallographic studies of human alpha-thrombin with a novel synthetic inhibitor, an acyl (alpha-aminoalkyl)phosphonate, reveal the existence of a pentacovalent phosphorus intermediate state. Crystal structures of the complex of alpha-thrombin with the phosphonate compound were determined independently using crystals of different ages. The first structure, solved from a crystal less than seven days old, showed a pentacoordinated phosphorus moiety. The second structure, determined from a crystal that was 12 weeks old, showed a tetracoordinated phosphorus moiety. In the first structure, a water molecule, made nucleophilic by coordination to His57 of alpha-thrombin, is bonded to the pentacoordinated phosphorus atom. Its position is approximately equivalent to that occupied by the water molecule responsible for hydrolytic deacylation during normal hydrolysis. The pentacoordinated phosphorus adduct collapses to give the expected pseudo tetrahedral complex, where the phosphorus atom is covalently bonded to Ser195 O(gamma). The crystallographic data presented here therefore suggest that the covalent bond formed between the inhibitor's phosphorus atom and O(gamma) of Ser195 proceeds via an addition-elimination mechanism, which involves the formation of a pentacoordinate intermediate.

Effects of a low-molecular-weight heparin on thrombus regression and recurrent thromboembolism in patients with deep-vein thrombosis.

BACKGROUND: Low-molecular-weight heparins are frequently used to treat venous thromboembolism, but optimal dosing regimens and clinical outcomes need further definition. METHODS: In this multicenter, open-label study with blinded adjudication of end points, we randomly assigned patients with acute deep-vein thrombosis to one of three treatment regimens: intravenous administration of unfractionated heparin; subcutaneous administration of a low-molecular-weight heparin, reviparin, twice a day for one week; or subcutaneous administration of reviparin once a day for four weeks. The primary end point was evidence of regression of the thrombus on venography on day 21; secondary end points were recurrent venous thromboembolism, major bleeding within 90 days after enrollment, and death. RESULTS: Of the patients receiving unfractionated heparin, 40.2 percent (129 of 321) had thrombus regression, as compared with 53.4 percent (175 of 328) of patients receiving reviparin twice daily and 53.5 percent (167 of 312) of the patients receiving reviparin once daily. With regard to thrombus regression, reviparin administered twice daily was significantly more effective than unfractionated heparin (relative likelihood of thrombus regression, 1.28; 97.5 percent confidence interval, 1.08 to 1.52), as was reviparin administered once daily (relative likelihood, 1.29; 97.5 percent confidence interval, 1.08 to 1.53). Mortality and the frequency of episodes of major bleeding were similar in the three groups. CONCLUSIONS: In acute deep-vein thrombosis, reviparin regimens are more effective than unfractionated heparin in reducing the size of the thrombus. Reviparin is also more effective than unfractionated heparin for the prevention of recurrent thromboembolism and equally safe.

Bemiparin and fluid flow modulate the expression, activity and release of tissue factor pathway inhibitor in human endothelial cells in vitro.

We investigated the localisation, gene expression, and activity of tissue factor pathway inhibitor (TFPI) in endothelial cells (EC) grown in static conditions or under shear stress, in the presence of unfractionated heparin (UFH) and two low-molecular-weight heparins (LMWHs). dalteparin and bemiparin (a second generation of LMWHs). All three preparations induced increased release, cellular redistribution, and enhanced activity of TFPI on the cell surface in static EC. In EC grown under shear stress (0.27, 4.1 and 19 dyne/cm2) and incubated with each heparin for 24 h, the release of TFPI was significantly correlated with the level of flow for bemiparin and dalteparin, but not for UFH. For all three levels of flow tested, bemiparin induced the highest secretion and increase of both cellular TFPI and cell surface activity of the inhibitor. The expression of TFPI mRNA, determined by Northern blotting, was specifically modulated by heparins. All three preparations increased the expression of TFPI by 60 to 120% in EC under minimal flow, but only bemiparin enhanced TFPI mRNA in EC under the arterial flow. Immunogold electron microscopy revealed that EC exhibited strong cellular labelling for TFPI when grown under arterial flow in the presence of bemiparin. We conclude that in EC subjected to shear stress in vitro bemiparin is more efficient than UFH or dalteparin in modulating the expression. release and activity of TFPI. We therefore suggest that bemiparin may be superior over the conventional heparins in maintaining the anticoagulant properties of the endothelium.

In situ analysis of tissue factor-dependent thrombin generation in human atherosclerotic vessels.

Tissue factor (TF) is expressed in human atherosclerotic plaques where it may contribute to the thrombogenicity of the lesions and their progression toward unstable syndromes and acute myocardial infarction. In this study we tested the hypothesis that thrombin generation takes place in the lesion. Localisation of TF, factor VII (FVII), factor X/Xa (FX/Xa), thrombin, thrombin receptor PAR-1 and FXa receptor EPR-1 was done by immunostaining, ligand binding, or immunogold electron microscopy. Quantitation of TF antigen was done using a modified ELISA on fixed tissue sections. The amount of antigen was correlated with the pattern and intensity of immunostaining as detected on consecutive sections using confocal microscopy. TF-dependent generation of FXa on cryosections was used to assess the functional activity of TF. Active thrombin was detected using hirudin as a specific probe, followed by anti-hirudin IgG. Our light microscopy and immunogold electron microscopy results showed that the factors involved in TF-dependent coagulation are localised in atherosclerotic plaques in close proximity and colocalise with active thrombin and fibrin deposits. We have detected 3 to 7-fold increase of TF antigen and TF-dependent FXa generation in atherosclerotic vessels as compared with controls. Hirudin binding proved that active thrombin is present within the lesions. In conclusion, our data show that active coagulation factors are generated within atherosclerotic lesions and co-localise with their cellular receptors. These findings may suggest possible roles of the TF-dependent coagulation pathway in the intramural fibrin deposition and the progression of the atherosclerotic lesions.

Fluid flow induces upregulation of synthesis and release of tissue factor pathway inhibitor in vitro.

Fluid flow modulates the synthesis and secretion by endothelial cells (ECs) of several proteins that control the hemostatic properties of the vessel wall. Tissue factor pathway inhibitor (TFPI), also synthesized by ECs, is the main downregulator of tissue factor-dependent procoagulant activity. In the present study, we investigated the effect of physiological shear stress on the expression, distribution, and release of TFPI in cultured ECs. The EA.hy926 cell line was grown in a hollow-fiber perfusion system and exposed for variable times to different shear values: 0.27 dyne/cm(2) (minimal flow), 4.1 dyne/cm(2) (venous flow), and 19 dyne/cm(2) (moderate arterial flow). Step increase of the shear stress from 0.27 to 19 dyne/cm(2) induced a sharp increase of TFPI released into the medium and a parallel decrease and redistribution of cell-associated TFPI, which suggests that an acute release of TFPI occurred from the cellular pools. During 24 hours of high shear stress, cell-associated TFPI antigen and mRNA increased time-dependently. Subjecting ECs to steady shear stress for 72 hours also upregulated the expression and production of TFPI, in direct correlation with the degree of the shear. The secretion of TFPI was enhanced 1.9-fold under venous flow and 2.4-fold under arterial flow compared with minimal flow. Equally, cell-associated TFPI antigen and cell surface TFPI activity increased proportionally with the shear stress. The expression of TFPI mRNA, as determined by Northern blotting, increased up to 2-fold in ECs under venous flow and up to 3-fold under arterial flow. These results suggest that shear forces regulate TFPI by modulating its release and gene expression in ECs in vitro.