bluuebunny/crossref_metadata_2025 huggingface dataset:
Platelet tetraspanin complexes and their association with lipid rafts
Authors: McMillan-Ward Eileen , Israels Sara
Published: October 2007

Tetraspanins are a superfamily of integral membrane proteins that facilitate the organization of membrane and intracellular signaling molecules into dynamic signaling microdomains, tetraspaninenriched microdomains (TEMs). Four tetraspanin family members have been identified in platelets: CD9, CD151 and TSSC6, which are constitutively associated with αIIb3, and CD63, which is present on granule membranes in resting platelets and associates with αIIbβ3-CD9 following platelet activation. CD63 and CD9 associate with a type II phosphatidylinositol 4-kinase, PI4K55, in both resting and activated platelets. Immunoelectron microscopic studies showed co-localization of CD63 and PI4K55 on internal membranes of resting platelets and on the filopodia of thrombin-activated platelets. Because TEMs in malignant cell lines appear to be distinct from prototypic lipid rafts, this study examined whether CD63-PI4K55 and CD9-PI4K55 complexes were resident in platelet-lipid rafts, or formed distinct microdomains. CD63, CD9 and PI4K55 were recovered from low-density membrane fractions (LDMFs) of sucrose gradients following platelet lysis in Brij 35, but unlike lipidraft proteins were not insoluble in Triton X-100, being absent from LDMFs of platelets lysed with Triton. In cubation of platelets with methyl-β-cyclodextrin, to deplete cholesterol and disrupt lipid rafts, shifted the complexes to higher density sucrose gradient fractions, but did not disrupt the tetraspanin-PI4K55 complexes. These results demonstrate that tetraspanin complexes in platelets form cholesterol-associated microdomains that are distinct from lipid rafts. It is probable thatTEMs and lipid rafts associate under certain conditions, resulting in the close proximity of distinct sets of signaling molecules, facilitating signal transduction.
Residual platelet reactivity is an independent predictor of myocardial injury in acute myocardial infarction patients on antiaggregant therapy
Authors: Paniccia Rita , Antonucci Emilia , Poli Serena , Gori Anna Maria , Valente Serafina , Giglioli Cristina , Lazzeri Chiara , Prisco Domenico , Abbate Rosanna , Gensini Gian Franco , Marcucci Rossella
Published: October 2007

In this study we sought to evaluate if platelet function measured after percutaneous coronary intervention (PCI) affects the severity of myocardial infarction (MI), measured by markers of cardiac necrosis. We measured platelet function by both a point-of-care assay (PFA-100) and platelet-rich plasma aggregation by two agonists (arachidonic acid –AA- and 2 and 10 μM ADP) in 367 patients with MI after PCI (200 patients on dual antiplatelet agents – group A- and 167 on dual antiplatelet agents plus GpIIb/ IIIa inhibitors – group B). One hundred twenty-one (32.9%) patients were found to have a residual platelet reactivity (RPR) by PFA (CT/EPI <203 sec): 74/200 (37%) in group A and 47/167 (28.1%) in group B (p=0.07). In 129 (35.1%) patients we found a RPR by AA-PA: 80/200 (40%) in group A and 49/167 (29.3%) in group B (p<0.05). Seventeen out of 367 (4.6%) were found to have a RPR by ADP2-PA [15/200 (7.5%) in group A and 2/167 (1.2%) in group B; p<0.005] and 88/367 (23.9%) by ADP10-PA [64/200 (32%) in group A and 24/167 (14.4%) in group B, p<0.0001]. CK-MB and cTnI mean peak values were significantly higher in the first tertile of CT/ADP and CT/EPI distribution with respect to the other tertiles and they were significantly higher in patients with RPR by CT/EPI in both groupA and group B patients. CK-MB and cTnI peak values were significantly higher in the third tertile of AA-PA,ADP 2 μM-PA and ADP 10 μM-PA distribution with respect to the other tertiles and were significantly higher in patients with RPR by AA-PA and by ADP 10-PA in both group A and group B patients. Multivariate analysis revealed platelet function as an independent predictor of CK-MB and cTnI peak values in both groups of patients independently of clinical, laboratory ad procedural parameters. In conclusion, we found that the severity of MI in patients with MI undergoing primary PCI is influenced by a persistent platelet activation on multiple antiplatelet therapy.
Factor VIIa and tissue factor procoagulant activity in diabetes mellitus after acute ischemic stroke: Impact of hyperglycemia
Authors: Vaidyula Vijender , Kanamalla Uday , De Angelis Michael , Gaughan John , Gentile Nina , Rao Koneti
Published: October 2007

Alterations in blood coagulation may explain the poorer neurological outcome with diabetes mellitus and hyperglycemia after acute ischemic stroke. We studied the relationships between diabetes mellitus, hyperglycemia, whole blood tissue factor procoagulant activity (TF-PCA) and plasma Factor VIIa (FVIIa) in ten patients with type 2 diabetes mellitus and 11 non-diabetic patients at baseline and 6, 12, 24, and 48 hours (h) after presentation for acute stroke. In addition, we examined plasma prothrombin fragment 1+2 (F1.2) and thrombin-antithrombin complexes (TAT) as markers of thrombin generation. Stroke severity, assessed by National Institute of Health Stroke Scale (NIHSS), was similar at baseline (p=0.26) but worse in diabetic (8.20 ± 4.3) than nondiabetic patients (2.67 ± 2.1,p=0.023) at 48 h. At presentation, diabetic patients had higher FVIIa (p=0.004) and lower TF-PCA (p=0.027) than non-diabetic patients but both were higher than in normal control subjects. FVIIa levels remained higher in diabetic patients at 6, 12 and 24 h after stroke. In diabetic patients, FVIIa (r=0.40, p=0.02) and TF-PCA (r=0.50, p=0.02) correlated with blood glucose; and, FVIIa correlated with plasma F1.2 (r=0.34, p=0.002) and TAT levels (r=0.62, p<0.0001). In non-diabetic patients, TF-PCA, but not FVIIa, correlated with F1.2 (r=0.402, p=0.010) andTAT (r=0.39, p=0.011). Combining both groups, NIHSS scores were positively related to FVIIa levels (r=0.50,p=0.021) and inversely related toTF-PCA levels (r=-0.498, p=0.02). Acute ischemic stroke patients with diabetes and hyperglycemia have a more intense procoagulant state compared with nondiabetic patients. This is related to glucose levels and provides a potential mechanism for the observed worse prognosis in such patients after acute stroke.
Staphylococcal infections impair the mesothelial fibrinolytic system: The role of cell death and cytokine release
Authors: Brück Michaela , Grundmeier Matthias , Peters Georg , Sinha Bhanu , Haslinger-Löffler Bettina
Published: October 2007

Bacterial peritonitis is a serious complication of peritoneal dialysis patients and of patients after abdominal surgery. Especially episodes due to Staphylococcus aureus can harm the peritoneum severely, resulting in peritoneal fibrosis. Human peritoneal mesothelial cells play a critical role in maintaining the integrity of the peritoneum, as they release components of the fibrinolytic system and regulate the influx of immune cells by expressing chemokines and adhesion molecules. Using cultured human peritoneal mesothelial cells (HMCs) and blood mononuclear cells,we analyzed the effect of different staphylococcal strains on mesothelial fibrinolysis and on inflammatory reactions and show that only S. aureus strains with an invasive and hemolytic phenotype decrease the production of fibrinolytic system components, most likely via cell death induction. Furthermore, HMCs react to invading staphylococci by enhanced expression of chemokines and adhesion molecules. Mononuclear cells were activated by all staphylococcal strains tested, and their culture supernatants impaired mesothelial fibrinolysis. Simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, efficiently enhanced the mesothelial fibrinolytic capacity under these inflammatory conditions, but did not protect HMCs against S. aureus-induced cell death. We conclude that only selected S. aureus strains decrease the release of fibrinolytic system components and provoke a mesothelial inflammatory response. These factors most likely contribute to peritoneal fibrosis and might account for the severe clinical presentation of S. aureus peritonitis.
Elevated factor VIII in hereditary haemorrhagic telangiectasia (HHT): Association with venous thromboembolism
Authors: Sulaiman Laila , Govani Fatima , Jackson James , Begbie Megan , Shovlin Claire
Published: October 2007

Hereditary haemorrhagic telangiectasia (HHT) causes chronic nasal and gastrointestinal haemorrhage. Prothrombotic agents are commonly used for severe haemorrhage. Thrombotic risks have not been defined. In order to identify prothrombotic variables in HHT patients, and assess their potential functional significance, a pilot ELISA-based study comparing plasma proteins in healthy individuals with HHT to age/sex-matched non-HHT controls was validated in a full study of 309 consecutive HHTaffected individuals. In the pilot study, factor VIII (FVIII) and von Willebrand factor antigen concentrations were elevated in the HHT group compared to non-HHT controls (p<0.0013, Mann- Whitney). Service laboratory measurements confirmed high FVIII:Ag in 125 HHT-affected individuals with no recent illhealth, intervention or venous thromboemboli. FVIII:Ag levels increased with age. Logistic regression also suggested an age-independent association with HHT-associated pulmonary arteriovenous malformations (AVMs). No association was demonstrated between FVIII:Ag and acute phase response, disseminated intravascular coagulation, ABO group, pulmonary artery pressure, or markers of HHT haemorrhage. Elevated FVIII:Ag were associated with shortened activated partial thromboplastin times (APTTs), andVTE:VTE affected 20/309 (6.5%) HHT-affected individuals, at median age 61(36–71) years. Four VTE occurred in factorV Leiden heterozygotes in the months following PAVM-associated brain abscess. The strongest association with VTE was with log-transformed FVIII:Ag measured 10–132 months from VTE (odds ratio 2.41, 95% confidence intervals 1.254, 4.612, p=0.008). Age made no additional contribution to VTE risk once adjusted for FVIII:Ag. In conclusion, HHT-related elevation of FVIII:Ag levels may influence thrombotic risk in HHT. Individualised risk-benefit considerations may be helpful in HHT management.
Microparticles from apoptotic monocytes induce transient platelet recruitment and tissue factor expression by cultured human vascular endothelial cells via a redox-sensitive mechanism
Authors: Essayagh Sanah , Xuereb Jean-Marie , Terrisse Anne-Dominique , Tellier-Cirioni Lise , Pipy Bernard , Sié Pierre
Published: October 2007

Circulating microparticles derived from different types of blood cells have been reported to impair endothelial function and to induce pro-inflammatory and prothrombotic endothelial phenotypes. Although the number of monocyte-derived microparticles (M-MPs) is elevated in the blood of patients with various inflammatory conditions, their interaction with endothelial cells has been poorly investigated so far. In this study, we produced microparticles in vitro from apoptotic human monocytes and examined the effects of their interaction with cultured human umbilical vascular endothelial cells (HUVECs). We found that low concentrations of M-MPs induced the production of reactive oxygen species (ROS), mainly anion superoxide, by the endothelial cells. At sub-toxic concentrations, M-MPs induced a rapid expression of von Willebrand factor at the cell surface, which mediated the transient attachment of non-activated platelets to the endothelium in flow conditions. In parallel, M-MPs up-regulated the expression of functional tissue factor by the endothelial cells. ROS controlled these two major changes and the process involved the phosphorylation of p38 mitogen activated protein kinase. We conclude that M-MPs may contribute to thrombotic events by producing redox signalling in endothelial cells.
Role of murine integrin α2β1 in thrombus stabilization and embolization: Contribution of thromboxane A2
Authors: Pozgajova Miroslava , Cosemans Judith , Munnix Imke , Eckes Beate , Nieswandt Bernhard , Heemskerk Johan , Kuijpers Marijke
Published: October 2007

Platelets stably interact with collagen via glycoprotein (GP)VI and α2β1 integrin. With α2-null mice, we investigated the role of α2β1 in thrombus formation and stability in vivo and in vitro. Using a FeCl3-induced thrombosis model, in arteries from α2-null mice smaller thrombi were formed with more embolization compared to vessels from wild-type mice. Aspirin treatment of wild-type mice causes similar effects, while the thromboxane A2 analogue U46619 was borderline effective in suppressing the embolisation in α2-null mice. In vitro, perfusion of α2-null blood over collagen resulted in formation of thrombi that were smaller and looser in appearance, regardless of the presence or absence of coagulation. Aspirin treatment or blockage of thromboxane receptors provoked embolus formation in wildtype blood, while U46619 normalized thrombus formation in blood from α2-null mice. We conclude that integrin α2β1 plays a role in stabilizing murine thrombi, likely by enhancing GPVI activation and thromboxane A2 release. The increased embolization in α2-null mice may argue against the use of α2β1 integrin inhibitors for antithrombotic therapy.
Platelet interaction with progenitor cells: Potential implications for regenerative medicine
Authors: Stellos Konstantinos , Gawaz Meinrad
Published: October 2007

Circulating endothelial progenitor cells have been shown to instigate new vessel formation via angiogenesis and neovascularisation and to induce ongoing vascular and tissue repair by domiciliation to sites of vascular or tissue damage. However, the mechanisms that recruit circulating endothelial progenitor cells towards vascular lesions and regulate repair mechanisms of ischemic peripheral organs are poorly described. Domiciliation of endothelial progenitor cells in peripheral tissue is a multi-step cascade including initial adhesion to subendothelial matrix or endothelium, transmigration and invasion of the target tissue. Platelets are the first circulating blood cells that interact with the injured vessel wall. They contain a number of growth factors, chemokines, cytokines and adhesive proteins that are released or surface-expressed upon platelet activation including adhesion. Recent studies suggest that platelet interaction with endothelial progenitor cells influences chemotaxis, adhesion, activation and differentiation of progenitor cells. Release of the chemokine SDF-1 from platelets enhances neovascularization through mobilization of progenitor cells. Adherent platelets recruit bone marrow-derived progenitor cells to arterial thrombi in vitroand in vivoand induce their subsequent differentiation towards an endothelial phenotype. Moreover, platelet accumulation in a co-culture system with CD34+ progenitor cells results in the differentiation of the latter to macrophages in vitro. Although further studies are needed to elucidate the mechanisms that platelets determine the fate of endothelial progenitor cells into vascular lesions, platelet interaction with progenitor cells seems to play a decisive role in vascular and tissue regeneration.
Fibrotic injury after experimental deep vein thrombosis is determined by the mechanism of thrombogenesis
Authors: Varma Manu , Moaveni Daria , Dewyer Nicholas , Moore Andrea , Lynch Erin , Longo Christopher , Deatrick Barry , Kunkel Steven , Upchurch Gilbert , Wakefield Thomas , Henke Peter
Published: October 2007

Vessel wall matrix changes occur after injury, although this has not been well studied in the venous system. This study tested the hypothesis that the thrombus dictates the vein wall response and vein wall damage is directly related to the duration of thrombus contact. To determine the injury response over time, rats underwent inferior vena cava (IVC) ligation to produce a stasis thrombus, with harvest at various time points to 28 days (d). Significant vein wall matrix changes occurred with biomechanical injury (stiffness) peaking at 7–14 d, with concurrent early reduction in total collagen, an increase in early matrix metalloproteinase (MMP)-9 and late MMP-2, and concomitant increase in tumor necrosis factor (TNF)α, monocyte chemoattractant( MCP)-1 and tumor growth factor (TGF)β (all P <0.05). To isolate the effect of the thrombus and its mechanism of genesis, rats underwent 7 d or limited stasis (24 hours), non-stasis thrombosis, or non-thrombotic IVC occlusion (Silicone plug). Vein wall stiffness was increased seven-fold, with a five-fold reduction in collagen, and 5.5- to seven-fold increase in TNFα, MCP-1, and TGFβ with 7 d stasis as compared with controls (all P <0.05). By Picosirus red staining analysis, collagenolysis was significantly greater with 7 d stasis injury (P = 0.01) but neither MMP-9 nor MMP-2 activity correlated with injury mechanism. In addition, vein wall cellular proliferation and uPA gene expression paralled the stasis thrombotic injury. Limited stasis, non-stasis thrombosis and non-thrombotic IVC occlusion showed a lesser inflammatory response. These data suggest both a static component and the thrombus directs vein wall injury via multiple mechanisms.
Matrix metalloproteinase (MMP) induction and inhibition at different doses of recombinant tissue plasminogen activator following experimental stroke
Authors: Burggraf Dorothe , Martens Helge , Dichgans Martin , Hamann Gerhard
Published: October 2007

Although recombinant tissue plasminogen activator (rt-PA) is successfully used for thrombolysis in human stroke, it may increase the risk of haemorrhagic complications. It was shown that the matrix metalloproteinase (MMP) system is critically involved in basal lamina degradation after middle cerebral artery occlusion and reperfusion following rt-PA administration. We describe the effects of different doses of rt-PA (saline, 0.9, 9, or 18 mg rt-PA/kg body weight) on the MMPs, their specific inhibitors (TIMPs), and also their inducer protein EMMPRIN following experimental cerebral ischemia (3 hours [h], 24 h reperfusion, suture model) in rats. The amount of MMP-2 and -9 was measured by gelatine zymography, TIMP-1 and -2 by reverse gelatine zymography, and the content of EMMPRIN and the basal lamina component collagen type IV by Western blotting. The amount of both MMPs steadily rose with increasing doses of rt- PA (p<0.05). In contrast, their endogenous inhibitors TIMPs decreased (p<0.001). A balance between the proteases and their inhibitors was achieved at the low dose of 0.9 mg/kg rt-PA in the rats, which significantly coincided with the demonstrated protection of collagen type IV degradation at this dose. The inducer protein EMMPRIN increased in parallel to its substrate MMP-2. Exogenous rt-PA leads to an increase of the MMP-inducing system by EMMPRIN, and a rise of the degrading MMPs follows. However, at low to moderate doses of rt-PA the microvascular basal lamina was protected, probably due to inhibition of MMP-2 and MMP-9 by the upregulation of their inhibitors. This strongly supports use of the lowest effective dosage of rt-PA available.
PSGL-1 regulates platelet P-selectin-mediated endothelial activation and shedding of P-selectin from activated platelets
Authors: Dole Vandana , Bergmeier Wolfgang , Patten Ian , Hirahashi Junichi , Mayadas Tanya , Wagner Denisa
Published: October 2007

We have previously shown that activated platelets in circulation stimulate release of endothelial Weibel-Palade bodies thus increasing leukocyte rolling in venules. P-selectin on the activated platelets mediates adhesion to leukocytes via PSGL-1 and is rapidly shed into plasma. We were interested in studying the role of PSGL-1 in regulating expression and function of platelet P-selectin. We show here that PSGL-1 is critical for the activation of endothelial cells in venules of mice infused with activated platelets. The interaction of platelet P-selectin with PSGL-1 is also required for P-selectin shedding, as P-selectin was retained significantly longer on the surface of activated platelets infused into PSGL-1-/- compared to wild-type mice. The leukocyte integrin αMβ2 (Mac-1) was not required for P-selectin shedding. In addition to shedding, P-selectin can be downregulated from the platelet surface through internalization and this is the predominant mechanism in the absence of PSGL-1. We demonstrate that leukocyte- neutrophil elastase,known to cleave P-selectin in vitro, is not the major sheddase for P-selectin in vivo. In conclusion, interaction of platelet P-selectin with PSGL-1 is crucial for activation of the endothelium andWeibel-Palade body secretion. The interaction with PSGL-1 also results in rapid shedding of P-selectin thus downregulating the inflammatory potential of the platelet.
The angiotensin-converting enzyme insertion/deletion polymorphism and serum levels of angiotensin-converting enzyme in venous thromboembolism
Authors: Ay Cihan , Bencur Peter , Vormittag Rainer , Sailer Thomas , Jungbauer Christof , Vukovich Thomas , Mannhalter Christine , Pabinger Ingrid
Published: October 2007

The angiotensin-converting enzyme (ACE) has been suggested to affect blood coagulation and fibrinolysis. Results from literature on the role of the frequent insertion/deletion (I/D) polymorphism in the ACE gene in venous thromboembolism (VTE) are controversial. Only limited data on ACE serum levels in VTE exist. We determined the ACE I/D polymorphism by genotyping and ACE serum levels by an enzymatic assay in 100 high-risk patients with objectively confirmed recurrent VTE and at least one event of an unprovoked deep venous thrombosis or pulmonary embolism. One hundred twenty-five age- and sex-matched healthy individuals served as controls. ACE genotype frequencies were not significantly different between patients (DD: 26.0%,ID: 52.0%, II: 22.0%) and controls (DD: 29.6%, ID: 44.8%, II: 25.6%; p=0.56). Neither individuals with ACE DD genotype nor those with ACE ID genotype had a higher risk for VTE in comparison to those with ACE II genotype (odds ratio and [95% confidence interval]: 1.0 [0.5–2.1] and 1.4 [0.7–2.6], respectively). Serum ACE levels (U/l) did not differ between patients (median = 25.25, 25th –75th percentile: 20.20–33.70) and controls (24.20, 17.85–34.50, p=0.49). In the total population involved in the study the ACE DD genotype (n=63: 36.00 [26.40–43.00]) was associated with higher ACE levels than the ACE ID genotype (n=108: 24.10 [19.80–31.48], p<0.001) and the ACE II genotype (n=54: 19.35 [15.00–22.95], p<0.001). In conclusion, we found a significant association of the ACE I/D polymorphism with ACE serum levels. However, neither the serum levels nor the I/D genotype were associated with VTE.
Venous thromboembolism (VTE) in Europe
Authors: Agnelli Giancarlo , Anderson Frederick , Arcelus Juan , Bergqvist David , Brecht Josef , Greer Ian , Heit John , Hutchinson Julia , Kakkar Ajay , Mottier Dominique , Oger Emmanuel , Samama Meyer-Michel , Spannagl Michael , Cohen Alexander ,   for the VTE Impact Assessment Group in Europe (VITAE)
Published: October 2007

Venous thromboembolism (VTE) is often asymptomatic, misdiagnosed, and unrecognized at death, and there is a lack of routine postmortem examinations. These factors are thought to result in marked underestimates ofVTE incidence. The objective of our study was to estimate the total burden of VTE within the European Union (EU) per annum. An epidemiological model was constructed to estimate the number of community- and hospital-acquired incidents and recurrent cases (attack rate) of nonfatal VTE and VTE-related deaths, as well as incident and prevalent cases of post-thrombotic syndrome (PTS) and chronic thromboembolic pulmonary hypertension (PH) occurring in the EU per annum. Individual models were developed for six EU countries. The models were populated with data from published literature and, where necessary, expert opinions. The findings were tested using probabilistic sensitivity analyses. The esti mated total number of symptomaticVTE events (range based on probabilistic sensitivity analysis) per annum within the six EU countries was 465,715 (404,664–538,189) cases of deep-vein thrombosis, 295,982 (242,450–360,363) cases of pulmonary embolism (PE), and 370,012 (300,193–483,108) VTE-related deaths. Of these deaths, an estimated 27,473 (7%) were diagnosed as being antemortem; 126,145 (34%) were sudden fatal PE, and 217,394 (59%) followed undiagnosed PE. Almost threequarters of all VTE-related deaths were from hospital-acquired VTE.VTE is a major health problem in the EU, with over one million VTE events or deaths per annum in the six countries examined. Given the availability of effective VTE prophylaxis, many of these events and deaths could have been prevented. These results have important implications for the allocation of healthcare resources.
Spiral computed tomography for the diagnosis of acute pulmonary embolism
Authors: Kayali Fadi , Hull Russell , Stein Paul
Published: October 2007

The accuracy of computed tomography (CT) imaging for the diagnosis of acute pulmonary embolism (PE) was reviewed. Single detector CT, based on pooled data, showed a sensitivity of 73% and multidetector CT, mostly 4-slice, showed a sensitivity of 83%. Respective specificities were 87% and 96%. Among patients with suspected PE evaluated with single slice CT,20% of patients found to have venous thromboembolic disease were diagnosed on the basis of a positive CT venous phase venogram. With multislice CT, 14% were diagnosed on the basis of a positive CT venogram.The positive likelihood ratio with single detector CT was 5.7 and with multidetector CT it was 19.6. Respective negative likelihood ratios were 0.31 and 0.18. Calculations of post-test probability using pretest probability and likelihood ratios according to Bayes’ theorem showed that even with multidetector CT, false positive and false negative images are not uncommon when clinical assessment is discordant with the CT interpretation. Outcome studies showed recurrent PE in only 1.7% or fewer untreated patients with negative CT pulmonary angiograms.
Food deprivation induces adipose plasminogen activator inhibitor-1 (PAI-1) expression without accumulation of plasma PAI-1 in genetically obese and diabetic db/db mice
Authors: Oishi Katsutaka , Ohkura Naoki , Matsuda Juzo , Ishida Norio
Published: October 2007

Relationships between energy intake and fibrinolytic functions have been documented in detail. We evaluated food deprivation (FD) as a means of modulating fibrinolytic activity in genetically obese and diabetic db/db mice and in their lean counterparts. Twelve hours of FD induced considerable gene expression of plasminogen activator inhibitor-1 (PAI-1) in both epididymal (3.8-fold, p<0.05) and intestinal (2.4-fold, p<0.05) adipose tissues without affecting plasma PAI-1 levels in db/db mice, whereas the FD did not affect these parameters in wild-type mice. Importantly, 24 hours of FD increased the plasma PAI-1 content in wild-type (1.9-fold, p<0.01) but not in db/db mice, although adipose PAI-1 mRNA levels were significantly increased in db/db mice. The plasma PAI-1 content significantly correlated with hepatic PAI-1 mRNA levels in wild-type (r=0.84, p<0.01) and in db/db (r=0.63, p<0.01) mice. However, plasma PAI-1 did not correlate with adipose PAI-1 expression in db/db mice, although adipose tissue in general is thought to be the principal site of PAI-1 production in obesity. Hepatic PAI-1 expression was closely correlated with serum levels of free fatty acids in wild-type (r=0.72, p<0.01), but not in db/db mice. Adipose PAI-1 expression significantly correlated with serum corticosterone levels in both genotypes (wild-type, r=0.52, p<0.05; db/db, r=0.51, p<0.01), suggesting that adipose PAI-1 expression is up-regulated by fastinginduced glucocorticoids. The present findings suggested that fasting differentially affects fibrinolytic activity in obese and lean subjects and that PAI-1 expression in the liver as well as in adipose tissues comprises an important determinant of increased risk for cardiovascular disease in obesity.
Absolute annual incidences of first events of venous thromboembolism and arterial vascular events in individuals with elevated FVIII:c
Authors: van de Poel Marlène , Coppens Michiel , Hamulyák Karly , Prins Martin , van der Meer Jan , Veeger Nic , Büller Harry , Middeldorp Saskia , Bank Ivan
Published: October 2007

Elevated levels of factor VIII:c (elevated FVIII:c) are associated with an increased risk for venous thromboembolism (VTE) and arterial vascular events, and are at least in part determined genetically. We prospectively followed 192 asymptomatic individuals with elevated FVIII:c (>150%) and 340 with normal levels for an average duration of 31 months (range 7 to 56 months) to investigate the risk of VTE and arterial vascular events. Participants were first degree relatives of consecutive patients with elevated FVIII:c and VTE or arterial vascular events before the age of 50 years. The incidences ofVTE were 1.25% (0.46–2.73) per year in the subjects with elevated FVIII:c, versus 0.23% (0.03–0.82) in those with normal levels (OR 5.5 [1.1–27.3]). The annual incidences of arterial vascular events were 1.04% (0.34–2.42) and 0.23% (0.03–0.82) in relatives with and without elevated levels of FVIII:c, respectively (OR: 4.5 [0.9–23.5]). After adjustment for age, smoking, known diabetes mellitus, hyperlipidemia, and hypertension, the odds ratio for any event was 3.7 (1.1–13.1). In conclusion, asymptomatic individuals with elevated FVIII:c levels and a positive family history of VTE or arterial vascular events before the age of 50 appear to have a high annual incidence of first VTE and arterial vascular events. Elevated FVIII:c may be a common risk factor for both clinical entities.
Urotensin-II in the lung: A matter for vascular remodelling and pulmonary hypertension?
Authors: Djordjevic Talija , Görlach Agnes
Published: October 2007

Urotensin-II (UII) is an evolutionary conserved peptide which has been initially discovered in the urophysis of the fish goby regulating body fluid composition and vascular tone. Mammalian UII has gained increasing interest since it has been considered as an even more potent vasoconstrictor than endothelin-1, although its efficiency is greatly variable throughout species and vascular beds. More recently, it has been shown that UII, which mediates its action via binding to the G-protein coupled urotensin-II receptor, is not only involved in the regulation of the vascular tone but can also stimulate a variety of signaling cascades in different cells and organs in the body including generation of reactive oxygen species and nitric oxide, activation of MAP kinases, and modulation of gene expression. Indeed, UII can stimulate proliferative processes, affect the extracellular matrix and may even add to a prothrombotic state. Such vascular remodelling processes are, in conjunction with enhanced vasoconstriction, involved in the pathogenesis of pulmonary hypertension, suggesting that UII may play a novel role in the pathogenesis of this disorder.
ADP-induced platelet aggregation and platelet reactivity index VASP are good predictive markers for clinical outcomes in non-ST elevation acute coronary syndrome
Authors: Frere Corinne , Cuisset Thomas , Quilici Jacques , Camoin Laurence , Carvajal Joseph , Morange Pierre Emmanuel , Lambert Marc , Juhan-Vague Irène , Bonnet Jean-Louis , Alessi Marie-Christine
Published: October 2007

Clopidogrel responsiveness has been proposed to be involved in recurrent ischemic events after stenting for non-ST elevation acute coronary syndromes (NSTE ACS). However, its biological definition is not consensual. We assess the value ofADP-induced platelet aggregation (ADP-Ag) and platelet reactivity indexVASP (PRI VASP) in predicting recurrent ischemic events in patients with NSTE ACS undergoing percutaneous coronary intervention (PCI). We studied 195 consecutive NSTE ACS patients undergoing PCI after a 600 mg loading dose of clopidogrel. ADPAg and PRI VASP were analysed. The primary end-point was recurrent ischemic events within 30 days of PCI. It occurred in 14 patients (7%). Construction of ROC curves to examine the value of predictive models showed that sensitivity and specificity for primary endpoint were 79% and 76%, respectively, for a maximal intensity of ADP-Ag ≥70%, 93% and 50% for PRIVASP> 53%. The positive and negative predictive values were 21% and 98%, respectively, for ADP-Ag ≥70%, 12% and 99% for PRIVASP> 53%. In patients with NSTE ACS undergoing PCI, ADP-Ag and PRI VASP identify low responders to clopidogrel with an increased risk of recurrent ischemic events with respective cut-off values of 70% and 53%.
The risk of bleeding with warfarin: A systematic review and performance analysis of clinical prediction rules
Authors: Dahri Karen , Loewen Peter
Published: October 2007

It was the objective of this article to qualitatively review and evaluate the clinical prediction rules (CPRs) available for estimating bleeding risk in patients commencing warfarin therapy. A systematic review of PubMed (1949 to December 2006), MEDLINE (1966 to December 2006); EMBASE (1980 to December 2006), Cochrane Database of Systematic Reviews (to December 2006), and International Pharmaceutical Abstracts (1970 to Demember 2006) was conducted. Seven studies were found that detailed CPRs used to assess risk of bleeding prior to commencing warfarin therapy. Four studies described distinct CPRs. The remaining three studies were further validations of one of the CPRs, the Outpatient Bleeding Risk Index. The Outpatient Bleeding Risk Index was classified as being of Level 2 evidence while the remaining three indices were classified as being of Level 4 evidence. In no case did the CPRs exhibit performance characteristics that would indicate“strong” ability to predict the presence of absence of major bleeding among warfarin recipients. The modified Outpatient Bleeding Risk Index exhibited moderate predictive ability for major bleeding in two studies, although pooling of all studies of this CPR did not reveal moderate or better performance. None of the CPRs identified “any bleeding” with moderate or strong predictive ability. None of the available CPRs exhibit sufficient predictive accuracy or have trials evaluating the impact of their use on patient outcomes. Hence, no existing CPR can be recommended for widespread use in practice at present.
Low protein Z levels in patients with peripheral arterial disease
Authors: Cesari Francesca , Pratesi Giovanni , Cellai Anna Paola , Pulli Raffaele , Pratesi Carlo , Gensini Gian Franco , Abbate Rosanna , Fedi Sandra , Sofi Francesco
Published: October 2007

Conflicting findings regarding the association between protein Z and atherosclerotic disease have been reported. The aim of this case-control study was to evaluate the role of protein Z in a peripheral localization of atherosclerosis. We studied protein Z levels in 120 patients (102 male, 18 female; median age: 75 years) admitted to the Unit of Vascular Surgery of the University of Florence with a clinical manifestation of peripheral arterial disease (PAD), and in 360 healthy subjects selected to be comparable to the patients group in terms of age and gender. Protein Z levels were found to be significantly (p < 0.05) lower in PAD patients [1,594 (89–3,635) ng/ml] compared to the healthy control group [1,728 (300–3,736) ng/ml].A logistic regression analysis showed, at univariate analysis, a significantly increased risk of PAD in patients with low levels of protein Z (<5th percentile of our control group: < 601 ng/ml) (OR: 5.72, 95%CI 3.07–10.66; p < 0.0001). After adjustment for age, gender and traditional cardiovascular risk factors the association was confirmed (OR: 5.83, 95%CI 2.83–12.01; p < 0.0001). Moreover, a significant association between low protein Z levels and clinical severity of the disease, evaluated through Fontaine’s stages, was reported after adjustment for age, gender, and traditional cardiovascular risk factors (general linear model, p for trend: 0.03). In conclusion, our data shows an association between low protein Z levels and the occurrence of PAD. These findings provide evidence for the role of protein Z in the pathogenesis of the atherosclerotic disease.
Blood coagulation at the site of microvascular injury in healthy and coumadin-treated subjects heterogenous for factor V Leiden mutation
Authors: Undas Anetta , Brzezinska-Kolarz Beata , Orfeo Tom , Brummel-Ziedins Kathleen , Musial Jacek , Szczeklik Andrew , Mann Kenneth
Published: October 2007

There is little knowledge regarding the in-vivo course of coagulation reactions in subjects with factor (F)V Leiden (G1691A, R506Q). The aim of the current study was to evaluate the effect of FV Leiden on coagulant reactions triggered by vascular injury. At the site of microvascular injury, prothrombin activation and FVa formation and inactivation have been evaluated in 16 apparently healthy subjects and 16 patients on long-term anticoagulation with acenocoumarol, with eight heterogenous carriers of FV Leiden in the healthy and in the anticoagulated group. Thrombin formation, measured by levels of thrombin-antithrombin complexes (TAT) and thrombin B-chain, proceeded at similar rates in the bleeding-time blood in carriers and non-carriers of FV Leiden. The onset and rate of FV activation did not differ significantly among the four groups. When healthy carriers of FV Leiden were compared to healthy non-carriers, the onset of FVa inactivation by activated protein C (APC) was delayed by 60 to 90 seconds (p=0.01). Anticoagulated individuals also showed the same pattern of FV Leiden associated differences in the time of occurrence of the 30 kD FVa heavy-chain fragment (p=0.021). Our results indicate that when blood clotting is triggered by microvascular injury, the heterozygous expression of FV Leiden has no effect on thrombin generation in healthy or coumadintreated subjects. Inactivation of FVa by theAPC mechanism is attenuated significantly in carriers of FV Leiden but the magnitude of this effect is smaller than that observed in most purified systems.
New functional aspects of the L-arginine-nitric oxide metabolism within the circulating blood
Authors: Keymel Stefanie , Kelm Malte , Kleinbongard Petra
Published: October 2007

Nitric oxide (NO) is a signaling molecule of major importance modulating not only the function of the vascular wall but also that of blood cells, such as platelets and leukocytes. The synthesis of NO in the circulation has been attributed mainly to the vascular endothelium. Red blood cells (RBC) have been demonstrated to carry a non-functional NOS and – due to their huge haemoglobin content – have been assumed to metabolize large quantities of NO. More recently, however, RBC have been identified to reversibly bind, transport, and release NO within the cardiovascular system. We provide evidence that RBC from humans express an active and functional endothelial type NOS. RBC NOS activity may regulate deformability of RBC, and inhibits activation of platelets. This review aims to discuss the potential role of RBC NOS in the circulation and new concepts of NO research in the microcirculation.
Factor V Leiden mutation is associated with enhanced arterial thrombotic tendency in lean but not in obese mice
Authors: Nagai Nobuo , Cleuren Audrey , Rosendaal Frits , Van Hoef Berthe , Hoylaerts Marc , Van Vlijmen Bart , Lijnen Roger
Published: October 2007

The homozygous factorV Leiden mutation is associated with enhanced venous thrombotic risk. Obesity is a major risk factor for development of thrombotic cardiovascular disease. It was the objective of this study to investigate whether obesity affects the thrombotic risk associated with the mutation. Male mice with homozygous factor V Leiden mutation (Arg 504 to Gln) (FVQ/Q) and corresponding wild-type (WT) mice were kept on a standard fat diet (SFD) or high fat diet (HFD) for 14 weeks, and femoral artery thrombosis was induced by FeCl3 treatment. As compared to SFD, HFD feeding for 14 weeks resulted in significantly higher body weight and fat mass associated with adipocyte hypertrophy, which were, however, similar for both geno types. In the FeCl3-induced arterial thrombosis model, FVQ/Q mice kept on SFD had a 40% shorter occlusion time (p = 0.015) and 40% lower blood flow (p = 0.03), as compared to WT mice. However, on HFD the occlusion time and blood flow were not significantly different for both genotypes. This finding could not be explained by differential changes of coagulation factors in either genotype fed on SFD or HFD. In conclusion, on SFD, but not on HFD, the factorV Leiden mutation is associated with enhanced thrombotic tendency after FeCl3 injury of the femoral artery, suggesting that in this model obesity rescues the increased thrombotic risk associated with the factorV Leiden mutation.
Antithrombotic effects of factor Xa inhibition with DU-176b: Phase-I study of an oral, direct factor Xa inhibitor using an ex-vivo flow chamber
Authors: Zafar Mohammad Urooj , Vorchheimer David , Gaztanaga Juan , Velez Mauricio , Yadegar Daniel , Moreno Pedro , Kunitada Satoshi , Pagan Juan , Fuster Valentin , Badimon Juan
Published: October 2007

Direct and specific inhibition of factor Xa is an emerging therapeutic strategy for atherothrombotic disease. Parenteral factor Xa inhibitors promise efficacy comparable to standard therapies, which could be extended to ambulatory patients with oral agents. We evaluated the antithrombotic effect of the oral, direct factor Xa inhibitor DU-176b in a phase-I study. Healthy subjects (n=12) received a single, 60 mg dose of DU-176b. Antithrombotic effects were assessed by comparing ex-vivo thrombus formation at 1.5, 5, and 12 hours post-dose versus baseline, along with factor Xa activity, thrombin generation and clotting parameters. Under venous flow after 1.5 and 5 hours, the thrombus was 28% and 21% smaller versus baseline, respectively (p<0.05). Under arterial condition, the reduction was 26% and 17% (p<0.05). Thrombin generation decreased by 28% at 1.5 hours and 10% at 5 hours. Changes in PT and INR correlated well with plasma drug concentrations (R2=0.79 and 0.78). Direct and specific inhibition of factor Xa by DU-176b significantly reduced ex-vivo thrombus formation at both venous and arterial rheologies, up to 5 hours post-dose. The effects mirrored changes in clotting parameters, suggesting their potential usefulness for monitoring in a clinical setting.
Potential implications of vascular wall resident endothelial progenitor cells
Authors: Tilki Derya , Hohn Hans-Peter , Gehling Ursula , Kilic Nerbil , Ergün Süleyman
Published: October 2007

A rapidly increasing body of data suggests an essential role of endothelial progenitor cells (EPCs) in vascular regeneration, formation of new vessels in cardiovascular diseases and also in tumor vasculogenesis. Moreover, recent data obtained from clinical studies with anti-angiogenic drugs in tumor therapy or with pro-angiogenic stimuli in ischemic disorders implicate a predictive role of the number of EPCs circulating in the peripheral blood in monitoring of these diseases. However, there is still some controversial data regarding the relevance of the EPCs in vascular formation depending on models used and diseases studied. One of the essential prerequisites for a better understanding of the whole contribution of EPCs to vascular formation in adult, a process called postnatal vasculogenesis, is to identify their exact sources. We could recently discover the existence of EPCs in a distinct zone of the vascular wall of large and middle sized adult blood vessels and showed that these cells are capable to differentiate into mature endothelial cells, to form capillary sprouts in arterial ring assay and to build vasa vasorumlike structures within the vascular wall. They also can be mobilized very rapidly from the vascular wall by tumor cells. This review will discuss the functional implications of these vascular wall resident endothelial progenitor cells (VW-EPCs) in relation to those of EPCs circulating in peripheral blood or derived from the bone marrow in cardiovascular and neoplastic diseases.
Factor Xa-driven thrombin generation in plasma: Dependency on the aminophospholipid density of membranes and inhibition by phospholipid-binding proteins
Authors: Wielders Simone , Ungethüm Lisette , Reutelingsperger Chris , Bevers Edouard , Lindhout Theo
Published: October 2007

Phosphatidylserine (PS) externalization of activated platelets plays a pivotal role in haemostasis and thrombosis. In the present study we have explored the relationship between the PS density of membranes and the rate of thrombin generation in plasma. Factor (F)Xa-initiated thrombin generation was measured in platelet-free plasma (PFP) containing either phospholipid vesicles of varying PS-content or non-stimulated platelets (reconstituted PRP). The duration of the initiation phase of FXa-driven thrombin generation decreased dramatically with increasing PS density. Concomitantly, the maximal rate of thrombin generation during the propagation phase (maxR) increased non-linearly, with the steepest incline between 5 and 10 mol% PS. Titration of FVa into plasma containing 2 mol% PS increased maxR proportionally and diminished the lag phase. In contrast, platelet-dependent thrombin generation was not influenced by addition of FVa. With increasing platelet concentration, the duration of the initiation phase drastically decreased, and maxR increased proportionally. At a physiologically relevant platelet concentration, maxR corresponded with the maxR found with 2 µM of 10 mol% PS. Annexin A5 (AnxA5) and lactadherin appeared to be powerful inhibitors of in-situ thrombin generation under all conditions examined, with AnxA5 being three- to fourfold more potent than lactadherin. In conclusion, maximal thrombin generation in plasma requires membranes with a density of 10–20 mol% PS. Our data further indicate that thrombin formed in situ induces externalization of PS to approx 10 mol% in a substantial platelet subpopulation.
Immunogenicity of novel recombinant human activated factor VII analogues on factor VII neonatally-tolerized rats
Authors: Jørgensen Peer Norbert , Clausen Jes Thorn , Salanti Zaki , Jensen Lisbeth Bjerring , Sommer Christian
Published: October 2007

Recombinant activated factorVII (rFVIIa; NovoSeven®) has been widely used to treat bleeding in patients with haemophilia with inhibitors. To increase the intrinsic activity, analogues of rFVIIa (rFVIIaQ, rFVIIaDVQ, and rFVIIaDVQA) with altered amino acid sequence at or near the active centre have been developed. The immunogenicity of these analogues was tested in a rat immune tolerance model.Neonatal rats received rFVIIa intraperitoneally on post-natal Day 1 and were subsequently challenged with rFVIIa in Freunds Incomplete Adjuvant subcutaneously on Days 10 and 24. Rats were tested for tolerance on Day 32; the tolerant cohort and a parallel cohort of untreated control rats were challenged with rFVIIa, rFVIIaQ, rFVIIaDVQ, or rFVIIaDVQA on Days 46 and 76. Immune responses determined by enzymelinked immunosorbent assay (ELISA) on Day 84 showed no statistically significant difference between the responses in the four control cohorts. Immune responses were higher in the control than in the tolerant cohort. Compared with rFVIIa (4/16), there was no difference in the proportion of rats that broke tolerance following challenge with rFVIIaDVQ (3/16) and rFVIIaDVQA (7/16), whereas a statistically significant greater proportion broke tolerance after challenge with rFVIIaQ (11/16). Therefore, in this model rFVIIaDVQ or rFVIIa DVQA were not more immunogenic than rFVIIa.