Thrombosis Research

Editorial Board

2010-03-01

“Coagulation Proteins with Uncertain Function”

Frank C. Church — 2010-03-01

“To raise new questions, new possibilities, to regard old problems from a new angle, requires creative imagination and marks real advance in science.” Albert Einstein (1879-1955)

The role of inflammation in regulating platelet production and function: Toll-like receptors in platelets and megakaryocytes

Lea M. Beaulieu, Jane E. Freedman — 2010-03-01

Abstract: Platelets have been extensively studied as hemostatic regulators, stopping uncontrolled flow of blood from an injured vessel and allowing for repair. However, multiple studies have shown that platelets can interact with bacterial proteins, particularly seen during sepsis and inflammation. Immune cells recognize pathogens through Toll-like Receptors (TLRs). These same receptors allow platelets to recognize bacterial proteins and regulate platelet immunity and function. This review examines the TLRs expressed on platelets and megakaryocytes and how these receptors affect the function of these cells. Through TLRs, platelets go beyond hemostatic regulation and play a pivotal role in inflammation and infection.

Factor XII: What does it contribute to our understanding of the physiology and pathophysiology of hemostasis & thrombosis

Evi Stavrou, Alvin H. Schmaier — 2010-03-01

Abstract: Factor XII (FXII) is a coagulation protein that is essential for surface-activated blood coagulation tests but whose deficiency is not associated with bleeding. For over forty years, investigators in hemostasis have not considered FXII important because its deficiency is not associated with bleeding. It is because there is a dichotomy between abnormal laboratory assay findings due to FXII deficiency and clinical hemostasis that investigators sought explanations for physiologic hemostasis independent of FXII. FXII is a multidomain protein that contains two fibronectin binding consensual sequences, two epidermal growth factor regions, a kringle region, a proline-rich domain, and a catalytic domain that when proteolyzed turns into a plasma serine protease. Recent investigations with FXII deleted mice that are protected from thrombosis indicate that it contributes to the extent of developing thrombus in the intravascular compartment. These findings suggest that it has a role in thrombus formation without influencing hemostasis. Last, FXII has been newly appreciated to be a growth factor that may influence tissue injury repair and angiogenesis. These combined studies suggest that FXII may become a pharmacologic target to reduce arterial thrombosis risk and promote cell repair after injury, without influencing hemostasis.

The relationship between monoclonal gammopathy of undetermined significance and venous thromboembolic disease

Adam L. Cohen, Rami Sarid — 2010-03-01

Abstract: Introduction: Monoclonal gammopathy of undetermined significance (MGUS) has been proposed to be a risk factor for venous thromboembolic disease (VTE). However, no series published to date has been population-based or included a control group with similar comorbidities to people with MGUS.Patients/Methods: We reviewed the records of all the male veterans in a single VA healthcare system with MGUS between January 1, 1996 and December 31, 2005. We compared the rate of VTE in 166 patients with MGUS with the rate of VTE in an age-matched control group of 465 patients who had tested negative for monoclonal gammopathy by serum protein electrophoresis (SPEP).Results: The VTE rate in the MGUS group was 2.2 per 100 person-years, which was not significantly different from the rate in the control group, 1.4 per 100 person-years (HR 1.38, CI 0.63-3.01, p=0.42). Most VTE events occurred within 4 months of the diagnosis of MGUS. In univariate analysis, albumin level (HR 0.21, CI 0.1-0.41, p<0.001), abnormal leukocyte count (HR 2.53, CI 1.09-5.86, p=0.03), and history of prior VTE (HR 4.41, CI 1.69-11.54, p=0.003) were associated with increased risk of VTE. On multivariate analysis, albumin level and history of prior VTE remained significant, but presence of MGUS was still not significantly associated with VTE risk.Conclusion: Our results suggest that the increased rate of VTE in people with MGUS may be primarily due to other underlying conditions that led to testing for a monoclonal gammopathy rather than to the monoclonal gammopathy itself.

Weight-based dosing of enoxaparin for VTE prophylaxis in morbidly obese, medically-Ill patients

2010-03-01

Abstract: Introduction: In clinical trials, fixed-dose enoxaparin (40 mg once daily) reduces the risk of venous thromboembolism (VTE) in medically-ill patients. However, morbidly obese patients were under-represented in these trials and using fixed-dose enoxaparin in obese patients may be inadequate. We completed a pharmacokinetic study in morbidly obese, medically-ill patients to determine if weight-based dosing of enoxaparin for VTE prophylaxis was feasible, without excessive levels of anticoagulation, as determined by peak anti-Xa levels.Materials and Methods: Twenty eight morbidly obese (BMI≥35 kg/m2) patients were enrolled and completed the study protocol. Enoxaparin 0.5 mg/kg was administered once daily subcutaneously and peak anti-Xa levels were measured approximately 4-6 hours after the enoxaparin dose.Results and Conclusions: Overall, 46% of patients were female, the average age (±SD) was 54 (±11) years, and the average weight and BMI were 135.6 kg (±25.3) and 48.1 kg/m2 (±11.1), respectively. The average daily dose of enoxaparin was 67 mg (±12). The average peak anti-Xa level was 0.25 (SD±0.11, range 0.08 to 0.59) units/mL. Peak anti-Xa levels did not significantly correlate with weight or BMI. There were no bleeding events, symptomatic VTE, or significant thrombocytopenia.In morbidly obese, medically-ill patients, use of weight-based enoxaparin dosed at 0.5 mg/kg once daily is feasible and results in peak anti-Xa levels within or near recommended range for thromboprophylaxis, without any evidence of excessive anti-Xa activity. These data suggest that this weight-based regimen may be more effective than standard fixed-dose enoxaparin. Clinical outcome studies are warranted to determine the clinical safety and efficacy of this regimen.

Decreasing warfarin sensitivity during the first three months after heart valve surgery: Implications for dosing

K. Meijer, Y.-K. Kim, S. Schulman — 2010-03-01

Abstract: Introduction: Vitamin K antagonists are prescribed to prevent thromboembolic complications after heart valve surgery. In our experience, patients often show a progressive decrease in sensitivity to warfarin after surgery making it difficult to reach and maintain a therapeutic International Normalized Ratio (INR).We sought to confirm our clinical impression and to gather data for the development of a guide to dosing these patients.Materials and methods: In a large anticoagulation clinic, we retrospectively reviewed 200 patients who were on warfarin (target range of 2.0-3.0) during the first three months after valve surgery. Data on dosing and INR results were collected and time in therapeutic range (TTR) calculated. Controls were patients started on warfarin for atrial fibrillation.Results: A steady increase in warfarin requirements was seen over the three months in patients with mechanical valves, bioprosthetic valves or valve repairs. The mean dose of warfarin increased by 26% while the mean INR decreased from 2.5 to 2.1. In contrast, both the mean dose of warfarin and the INR were stable in controls. TTR in patients after valve surgery was 48.5%, with 40.8% of time spent at an INR below 2.0. A dosing algorithm was modeled from the data in this patient group.Conclusions: Patients steadily become less sensitive to warfarin during the first months after heart valve surgery. This leads to subtherapeutic anticoagulation. A dosing algorithm that takes increasing requirements into account is proposed. This algorithm will need to be validated prospectively.

Hereditary protein C deficiency caused by the Ala267Thr mutation in the protein C gene is associated with symptomatic and asymptomatic venous thrombosis

Lena Tjeldhorn, Per Morten Sandset, Kaia Haugbro, Grethe Skretting — 2010-03-01

Abstract: Introduction: Protein C (PC) is a key anticoagulant that regulates hemostasis, and inherited deficiency of PC is an established risk factor for venous thrombosis (VT). The factor V Leiden mutation causing activated PC (APC) resistance is an additional risk factor for VT. Reduced PC levels in the circulation and/or APC resistance do not necessarily lead to thrombotic disease. In the present study we describe and characterize an ethnic Lebanese family in which individuals with reduced PC levels and APC resistance have various clinical symptoms.Methods: PC activity and antigen levels and APC resistance in the family members were quantified with commercial kits. Sequencing of PC DNA and mRNA was performed with BigDye Terminator Cycle Sequencing kit on the ABI 3730 Genetic Analyzer.Results: PC antigen and anticoagulant activity in the plasma of the proband and family members ranged from 9% to 69% and 3% to 63%, respectively, compared to levels measured in pooled normal plasma. Sequencing analysis of the PC gene of family members revealed that they were either homozygous or heterozygous for the Ala267Thr mutation. In addition, three of them exhibited APC resistance. None of the family members, except the proband, have had a history of VT despite that two of them have two genetic risk factors for thrombosis.

Prophylactic plasma levels of the low molecular weight heparin nadroparin does not affect colon cancer tumor development in mouse liver

2010-03-01

Clinical trials showed life-prolonging effects of low molecular weight heparins (LMWHs) in cancer patients with and without thrombotic complications . An inhibitory effect of anticoagulants on metastasis has been found in animal studies, usually in the mouse model of B16 melanoma tumor development in lung . In this model, anticoagulants have effects only when administered at the time of cancer cell inoculation . The effects are exerted by blocking P- and L- selectins thus inhibiting interactions between cancer cells and endothelium either or not in combination with heparanase inhibition .

Flow-based measurements of von Willebrand factor (VWF) function: Binding to collagen and platelet adhesion under physiological shear rate

2010-03-01

Abstract: Introduction: VWF circulates in plasma as a series of heterogeneous multimers, mediating platelet tethering, translocation and finally adhesion to areas of injured endothelium under physiological high arterial blood flow. VWF-platelet binding requires conformational changes in VWF, which are induced by immobilization and shear. Because of unavailability of a simple flow-based measurement system, VWF activity assays are generally performed under static conditions. We describe an easily reproducible in vitro flow-chamber model using commercially available flow devices to examine VWF-collagen binding and VWF-mediated platelet adhesion under physiological flow conditions.Methods: The collagen surface of the flow-chamber was analyzed by atomic force microscopy. Collagen-bound VWF was characterized by multimer analysis and multi labelling immunofluorescence detection of exposed GPIb binding domains. Platelet adhesion was captured by time-lapse microscopy.Results: The described flow-chamber system facilitates multimer analysis of collagen-bound VWF, whereas all VWF multimers bound to collagen under physiological low to high shear rates. Multi labelling immunofluorescence detection exhibited exposed GPIb binding domains co-localized with VWF molecules. VWF-dependent platelet adhesion using time-lapse microscopy showed values comparable to experiments done with whole blood, and platelet adhesion was dependent on the VWF concentration.Conclusions: The established flow-chamber model represents an easy-to-set-up and customized tool for the characterization of VWF-binding to collagen as well as the determination of VWF-dependent platelet adhesion under defined flow conditions in real-time.

The effect of the long term aspirin administration on the progress of atherosclerosis in apoE-/- LDLR-/- double knockout mouse

Y. Yamamoto, T. Yamashita, F. Kitagawa, K. Sakamoto, J.C. Giddings, J. Yamamoto — 2010-03-01

Abstract: We have investigated the effects of differential aspirin doses on atherogenesis. Aspirin was given to homozygous, apoE-/- and LDLR-/- double deficient mice for 12weeks. The development of arteriosclerosis was determined morphologically by image analysis and endothelial cell function was assessed by measurement of peripheral nitric oxide (NO).Methods: ApoE-/- LDLR-/-double knockout mice were bred and maintained with a high fat diet containing aspirin (4 and 40mg/kg B.W. /day) for twelve weeks. The development of arteriosclerosis was monitored by estimating the total area of atherosclerotic lesions in the entire aorta. Acetylcholine-induced NO release was measured in vivo using electrochemical sensors. The expression of eNOS on the endothelial surface was determined by immuno-staining. Plasma prostaglandin F1α (PGF1α), serum thromboxian B2 (TXB2) and total cholesterol were measured using enzymatic assay. Bleeding time was measured by tail cut method.Results: Arteriosclerosis in the 4mg/kg/day aspirin group was decreased significantly compared with the placebo group, but not in the 40mg/kg/day aspirin group. Acetylcholine-induced NO release was significantly depressed in the 40mg/kg/day aspirin group. Immunochemical analysis with anti-eNOS antibody supported these findings. In the 4mg/kg/day aspirin group, the severe suppression of PGI2 production was not confirmed in spite of decreasing TXB2 production, but not in the 40mg/kg/day aspirin group.Conclusion: Our results suggest that endothelial dysfunction with low dose aspirin improved, reduced progression of atherosclerosis in apoE-/- and LDLR-/- double deficient mice and provides a pathophysiological basis for the beneficial effects of aspirin in atherosclerosis, and low doses appeared to be more efficient than high doses.

Presence of Integrin alpha(IIb)beta3 in early gestation human trophoblasts: Possible involvement of fibrin as a matrix ligand

Ayelet Snir, Benjamin Brenner, Baram. Paz, Naomi Lanir — 2010-03-01

Abstract: Introduction: Various integrins are expressed on trophoblast membrane and participate in placenta anchoring, cell adhesion , migration and vascular development. Although integrin αIIbβ3 is known to be associated mainly with megakaryocytes and platelets, here we show for the first time its presence in human trophoblasts and in ex-vivo placental villi.Methods: Trophoblasts were isolated and cultured from early term placentas (9-13weeks of gestation). The presence of αIIbβ3 integrin was demonstrated in the cells by means of immunofluorescence , flow cytometry and Western blot analysis and in villous placentas by immunohistochemistry. Interaction of trophoblasts with fibronectin and fibrin was evaluated by adhesion assays.Results: Expression of αIIb protein in trophoblasts decreased with time in culture and was more prominent when cells were cultured on fibrin as compared to fibronectin. Presence of αIIb on villous trophoblasts was documented in placenta sections of 8 and 15weeks of gestation but not in term placentas.Trophoblasts adhesion to fibrin was reduced by 45% in the presence of blocking antibodies for αIIb, but only by 10% to fibronectin, suggesting fibrin as a ligand for trophoblast αIIbβ3.Conclusions: Our finding demonstrate the transient presence and participation of αIIbβ3 in the orchestrated adhesion molecules of trophblasts and villi. The increased adhesion and expression of αIIb in trophoblasts on fibrin suggest its involvement as a ligand in the extracellular milieu of the early fetal placenta.

PAR1 antagonist mediated antithrombotic activity in extracorporeal arterio-venous shunt in the rat

2010-03-01

The activation of platelets by thrombin is mediated at least in part by cleavage of protease-activated receptors (PARs) . Four distinct PARs have been cloned to date, with PAR1, PAR3 and PAR4 being activated by thrombin, whereas PAR2 is sensitive to trypsin . Human platelets express PAR1 and PAR4 and activation of either is sufficient to trigger platelet aggregation and secretion . Studies with selective neutralizing antibodies have indicated that PAR1 mediates activation of human platelets at low thrombin concentrations and that, in the absence of PAR1 function, PAR4 can mediate platelet activation but only at high thrombin concentrations . Curiously, PAR1 appears not to play a role in rat platelets. PAR1-activating peptides do not activate rat platelet aggregation . In contrast to this, PAR3 is not expressed by human or guinea-pig platelets, but by mouse and rat platelets . Recently, Leger et al. elegantly showed that in human platelets PAR1 and PAR4 form a stable heterodimer that enables thrombin to act as a bivalent functional agonist. Collectively, all these studies reveal the complexity of the PAR-mediated platelet activation in different animal species. Thus, the aim of the present study was to investigate the putative in vivo role of PAR1 in a thrombosis model conducted in anesthetized rats. We have investigated the effects of selective PAR4 antagonist, YD-3 but also relatively selective PAR1 antagonists ER-121958 and SCH-203099 and conventional antithrombotic agents on an extracorporeal arteriovenous shunt model in the rat.

Impaired secretion of carboxyl-terminal truncated factor VII due to an F7 nonsense mutation associated with FVII deficiency

2010-03-01

Abstract: Introduction: Factor VII (FVII) is a vitamin K-dependent glycoprotein secreted into the blood circulation from hepatic cells. We investigated the molecular basis of the congenital FVII deficiency found in a Japanese patient.Materials and Methods: We analyzed the F7 gene of the patient, who was diagnosed with a FVII deficiency at pregnancy. We expressed a carboxyl-terminal truncated FVII (Arg462X FVII) corresponding to the identified mutation in CHO-K1 cells. To study roles of the carboxyl-terminus in the secretion of FVII, we also expressed a series of recombinant FVIIs deleted of limited numbers of carboxyl-terminal amino acids (462Arg-466Pro).Results: We identified a nonsense mutation (c.1384C>T: p.Arg462X) in F7, leading to a lack of five amino acids in the carboxyl-terminus. In expression experiments, Arg462X FVII was undetectable not only by Western blotting, but also by ELISA. A Western blot analysis of the truncated FVIIs revealed that all mutants were expressed in the cells the same as the wild type, but were secreted into the culture medium in lesser amounts than the wild type depending on the length of the deletion, which was confirmed by ELISA. Arg462X FVII did not colocalize with the Golgi on immunofluorescence staining, suggesting that it might be retained in the ER and degraded in the cell.Conclusion: The carboxyl-terminal amino acids of FVII play an important role in its secretion, and the p.Arg462X mutation was likely to have caused the FVII deficiency in this patient.

Markers of haemostasis and angiogenesis in placentae from gestational vascular complications: Impairment of mechanisms involved in maintaining intervillous blood flow

2010-03-01

Abstract: Introduction: Preeclampsia (PE) and fetal growth restriction (FGR) are multifactorial diseases, whose pathogenesis is largely unknown. A significant relationship between haemostasis and angiogenesis in placentae from uneventful pregnancies was previously shown.Materials and Methods: RNA expression of haemostasis (TF, TFPI, TFPI-2, PAI-2, Anx V, TM) and angiogenesis (Ang-1, Ang-2, PlGF, VEGF) markers in placentae from PE (n=12), PE+FGR (n=17) and FGR (n=20) in respect of placentae from uncomplicated pregnancies (n=21) were investigated.Results: Placentae from complicated pregnancies showed a significant lower expression (p≤0.05 Mann-Whitney U test) of TF, TFPI, TFPI-2, Anx V, PAI-2 than those from in uncomplicated ones. VEGF and PlGF were not different in the considered groups; Ang-1 and Ang-2 were significantly higher (p≤0.05 Mann-Whitney U test) in the PE group.Correlations between factors involved in haemostasis and those involved in angiogenesis, observed in placentae from uneventful pregnancies are lacking in those from complicated ones.Conclusions: Haemostasis factors are reduced in placentae from complicated pregnancies. The relationship between haemostasis and angiogenesis observed in uncomplicated pregnancies is impaired in PE and FGR.

Prothrombin complex concentrate (Beriplex P/N) for control of bleeding after kidney trauma in a rabbit dilutional coagulopathy model

Ingo Pragst, Franz Kaspereit, Bärbel Dörr, Gerhard Dickneite — 2010-03-01

Abstract: Introduction: Fluid resuscitation after trauma often results in dilutional coagulopathy that may hinder control of bleeding and, once initial hemostasis has been secured, heighten risk of perioperative bleeding when further surgery is required. Since multiple coagulation factor deficiencies typically accompany fluid resuscitation, prothrombin complex concentrate (PCC) containing factors II, VII, IX and X may potentially offer greater hemostatic efficacy than coagulation factor monotherapy.Materials and methods: Anesthetized normothermic rabbits were hemodiluted 50-60% by phased blood withdrawal and infusion of hydroxyethyl starch and erythrocytes. The animals were randomly assigned to receive saline placebo, 25IU·kg-1 PCC (Beriplex P/N) or 180μg·kg-1 activated recombinant factor VII (rFVIIa; NovoSeven). Immediately thereafter, bleeding was precipitated by a standardized kidney incision.Results: PCC accelerated hemostasis compared both with saline and rFVIIa (p=0.002 for both comparisons). The median times to hemostasis in the PCC, saline and rFVIIa groups were 12, 19 and 28min, respectively. PCC reduced blood loss by a median of 43mL with a 95% confidence interval (CI) of 8.0-67.5mL vs. saline and 82mL (CI, 35.0-110.0mL) vs. rFVIIa. PCC augmented peak thrombin generation by a median of 104.1 nM (CI, 78.3-142.3 nM) compared with saline and 105.8 nM (CI, 70.7-139.5 nM ) relative to rFVIIa. At the respective 180μg·kg-1 and 25IU·kg-1 doses tested, rFVIIa displayed thrombogenicity in the Wessler stasis model, while PCC did not.Conclusions: In an animal model of dilutional coagulopathy and kidney trauma, PCC accelerated hemostasis and diminished blood loss compared with rFVIIa monotherapy.

Increased thrombin generation in inflammatory bowel diseases

2010-03-01

Abstract: Background: Inflammatory bowel diseases (IBD) are characterized by an increased thrombotic risk of uncertain etiology. Endogenous thrombin potential (ETP), a parameter of the thrombin generation curve, represents a new tool in the evaluation of thrombotic and bleeding disorders.Aims: To study ETP in IBD patients and to correlate the results with clinical and biochemical features.Methods: Seventy-four IBD patients (37 ulcerative colitis and 37 Crohn's disease) and 74 sex- and age-matched healthy individuals. ETP was measured upon activation of coagulation with small amounts of tissue factor and phospholipids in the presence or absence of thrombomodulin; results were expressed as nM thrombin·minutes.Results: Mean±SD ETP values were significantly higher in patients (1,499±454) than controls (1,261±385) (p<0.001) only when the test was performed in the presence of thrombomodulin. ETP evaluated as ratio (with/without thrombomodulin), taken as an index of hypercoagulability, was significantly higher in patients (0.69±0.14) than controls (0.62±0.18) (p<0.006). Patients with increased C-reactive protein (CRP) had significantly higher mean ETP (1,721±458) than those with normal CRP (1,357±394) or controls (1,261±385) (p<0.001). Patients who at the time of blood sampling were classified as having a clinically active disease had ETP higher than those who were quiescent (1,655±451 versus 1,388±427, p<0.001) or controls (1,261±385, p<0.001).Conclusions: ETP measured in the presence of thrombomodulin or as ratio (with/without thrombomodulin) is increased in IBD patients, mainly in those with increased CRP or active disease. It may be considered as a candidate test for prospective studies aimed at assessing the risk of thrombosis in IBD patients.

Prothrombinase formation at the site of microvascular injury and aspirin resistance: The effect of simvastatin

2010-03-01

The production of thrombin is determined by the formation of the prothrombinase complex, which assembles predominantly on activated platelets. Aspirin that suppresses the synthesis of thromboxane A2 (TXA2) has been shown to reduce thrombin formation at the site of injury . We have reported that impaired platelet sensitivity to aspirin, called aspirin resistance, is associated with faster thrombin generation following injury .

Efficacy and safety of enoxaparin in Japanese patients undergoing curative abdominal or pelvic cancer surgery: Results from a multicenter, randomized, open-label study

Masato Sakon, Takao Kobayashi, Toru Shimazui — 2010-03-01

Abstract: Background: Enoxaparin sodium (enoxaparin) is used worldwide for the prevention of venous thromboembolism (VTE). Registration trials of enoxaparin have been conducted primarily in Caucasian populations, and its preventive use in Japanese patients has yet to be established. To address this, we evaluated the efficacy and safety of postoperative enoxaparin in Japanese patients undergoing surgery for abdominal cancer.Methods: This multicenter, open-label study randomized 151 Japanese patients undergoing curative surgery for abdominal cancer to enoxaparin 20mg twice daily for 14days, started 24–36hours after surgery (n=113) or intermittent pneumatic compression (IPC) as a reference (n=38). IPC was performed at least once in both groups between randomization and surgery. The primary efficacy endpoint was the incidence of VTE in the modified intention-to-treat (mITT) population. The primary safety outcome was the incidence of any bleeding during treatment and follow-up.Results: Incidence of VTE was 1.2% (95% CI, 0.03–6.53%) (1/83 patients) in the enoxaparin group and 19.4% (95% CI, 7.45–37.47%) (6/31 patients) in the IPC group. In the safety population, 10/109 patients in the enoxaparin group (9.2%; 95% CI, 4.49–16.23%) and 3/38 patients in the IPC group (7.9%; 95% CI, 1.66–21.38%) experienced a bleeding event. There were no cases of fatal bleeding or bleeding into any critical organ.Conclusions: These favorable efficacy and safety data support the use of enoxaparin (20mg twice daily for 14days started 24–36hours after surgery) in Japanese patients undergoing abdominal or pelvic cancer surgery.

Expression of protein C (PC), protein S (PS) and thrombomodulin (TM) in human colorectal cancer

Ewa Sierko, Marek Z. Wojtukiewicz, Roman Zawadzki, Lech Zimnoch, Walter Kisiel — 2010-03-01

Abstract: Introduction: Colorectal cancer (CRC) is often complicated by thromboembolic episodes. It has been recognized that blood coagulation proteins play a role in cancer progression. An important inhibitory mechanism is provided by the protein C (PC) system consisting of PC, protein S (PS) and thrombomodulin (TM). Recently, novel biological activities have been ascribed to the PC system that do not relate to their hemostatic functions, eg. in angiogenesis, apoptosis and inflammation.Objectives: The purpose of the study was to elucidate the solid phase interactions between CRC tissue and components of the PC system that may contribute to tumor progression.Material and methods: CRC tissues were obtained at surgical resection during treatment of 66 patients. Immunohistochemical studies were performed using polyclonal antibodies against PC, PS and TM. A semiquantitative analysis of the protein expression was also performed.Results: Weak expression of PC was observed in cancer cells of two-thirds of the specimens examined, while in 3/66 cases there was no staining for PC in cancer cells. One fourth of CRCs exhibited strong expression of PC. The presence of PS was demonstrated in 64/66 cases of CRC. However, its expression was irregular in terms of intensity of staining and percentage of cancer cells exhibiting protein expression. Weak expression of TM was demonstrated in two thirds of the cases examined, while a strong TM staining was revealed in one third of colon cancers.Conclusion: Heterogeneous expression of the PC system components in CRC tissue may point to their biological activity modulating tumor growth.

Platelet activation and endogenous thrombin potential in pre-eclampsia

2010-03-01

Abstract: Introduction: Platelets and the coagulation system may be involved in the pathogenesis of pre-eclampsia. We investigated whether platelet and coagulation activation markers, are elevated in pre-eclampsia.Materials/methods: Case-control study in which activated platelets, platelet-monocyte/ neutrophil aggregates, platelet microparticles (measured by flow cytometry) and four markers of thrombin generation capacity (endogenous thrombin potential (ETP), peak height, lag time and time to peak) using the Calibrated Automated Thrombogram system were assessed in pregnant women of similar gestational age with (n=46) and without (n=46) pre-eclampsia, and in healthy non-pregnant women (n=42).Results: The percentage of, CD62P+ platelets (p=0.013), CD62P+ platelet microparticles (p=0.029) and platelet-monocyte aggregates (p=0.019) were significantly higher in women with pre-eclampsia than the pregnant controls. Both groups of pregnant women had significantly higher ETP and peak height (p <0.001) than the healthy non pregnant group and the women with pre-eclampsia had significantly higher ETP and peak height (p<0.001) than the normotensive pregnant controls.Conclusion: In the most comprehensive laboratory analysis to date, we found evidence of both platelet and coagulation activation in women with pre-eclampsia.

Bolus tenecteplase for right ventricle dysfunction in hemodynamically stable patients with pulmonary embolism

2010-03-01

Abstract: Introduction: The clinical benefit of thrombolytic treatment over heparin in patients with pulmonary embolism without hemodynamic compromise remains controversial. In these patients bolus tenecteplase has the potential to provide an effective and safe thrombolysis.Methods: We evaluated the effect of tenecteplase on right ventricle dysfunction (RVD) assessed by echocardiography in hemodynamically stable patients with PE in a multicenter, randomized, double-blind, placebo-controlled study. RVD was defined as right/left ventricle end-diastolic dimension ratio >1 in the apical 4-chamber view. Patients were randomized to receive weight-adjusted single-bolus tenecteplase or placebo. All patients received unfractionated heparin. Reduction of RVD at 24hours was the primary efficacy end-point and was evaluated by an independent committee unaware of treatment allocation.Results: Overall, 58 patients were randomized. Echocardiograms were adequate for efficacy analysis in 51 patients, 23 randomized to tenecteplase and 28 to placebo. The reduction of right to left ventricle end-diastolic dimension ratio at 24hours was 0.31±0.08 in patients randomized to tenecteplase as compared to 0.10±0.07 in patients randomized to placebo (p=0.04). One patient randomized to tenecteplase suffered a clinical event (recurrent pulmonary embolism) in comparison to three patients randomized to placebo (1 recurrent pulmonary embolism; 1 clinical deterioration and 1 non pulmonary embolism-related death). Two non fatal major bleedings occurred with tenecteplase (1 intracranial) and one with placebo.Conclusion: In hemodynamically stable patients with PE, treatment with single bolus tenecteplase is feasible at the same dosages used for acute myocardial infarction and is associated with reduction of RVD at 24hours. Whether this benefit is associated with an improved clinical outcome without excessive bleeding is currently explored in a large clinical trial.

Homocysteine decreases extracellular nucleotide hydrolysis in rat platelets

2010-03-01

Abstract: Hyperhomocysteinemia is an independent risk factor for atherothrombotic disease. Platelets play an important role in cardiovascular disease and release pro-aggregates mediators when activated, such as ADP, a physiological agonist involved in normal hemostasis and thrombosis. NTPDases and 5'-nucleotidase are ecto-enzymes that hydrolyze ATP, ADP and AMP to adenosine playing an important role on blood flow and thrombogenesis by regulating ADP catabolism. The aim of the present study was evaluate extracellular adenine nucleotide hydrolysis of rat platelets exposed to homocysteine in vitro and in vivo. In vitro homocysteine (Hcy) in the concentration range of 20 to 500μM caused a significant decrease on ATP (around 30%) and ADP (around 45%) hydrolysis, respectively, while AMP hydrolysis was not altered. Hcy was not able to inhibit the hydrolysis of ATP and ADP catalyzed by purified apyrase at the same concentrations tested in vitro on platelets, suggesting an indirect effect. The inhibitory effect of Hcy on platelets was prevented by antioxidants agents in vitro and in vivo. Furthermore homocysteine treatment increased platelet aggregation induced by ADP. Based on the results presented herein, we propose that inhibition of extracellular ATP and ADP hydrolysis caused by homocysteine was probably due oxidative stress, since antioxidants prevented such effects. These findings may contribute to an increase platelet response to ADP and consequence development of thrombotic risk attributed to hyperhomocysteinemia.

The novel P2Y12 antagonist AZD6140 rapidly and reversibly reduces platelet activation in diabetic rats

2010-03-01

Abstract: Introduction: Excessive platelet activation fundamentally contributes to cardiovascular events and mortality in patients with diabetes mellitus. Functional resistance has been described for current antiplatelet therapies in broad populations that include patients with diabetes. We investigated acute and chronic effects of AZD6140, a reversible oral rapid-onset P2Y12 antagonist, on platelet reactivity in diabetic rats.Materials and Methods: Diabetes was induced by streptozotocin injection in male Wistar rats. After 4weeks, AZD6140 was administered (5mg/kg by gavage) and achieved sufficient plasma levels within 30minutes. Platelet reactivity was determined by ADP-induced P-selectin expression, aggregation and adhesion on fibrinogen coated membranes under arterial flow conditions.Results: At 0.5hour, AZD6140 strongly reduced ADP-induced P-selectin surface expression, inhibited ADP-induced platelet aggregation, and significantly reduced platelet adhesion to fibrinogen under arterial flow conditions. Chronic treatment with AZD6140 (10mg/kg bid for 2weeks, based on data obtained in the acute study) starting at day 14 reduced P-selectin surface expression on circulating platelets, indicating lower in vivo platelet activation. Platelet reactivity was improved 12hours after the last dose, while basal platelet activity remained reduced. AZD6140 was rapidly absorbed in diabetic rats and inhibited platelet reactivity. Chronic treatment lowered in vivo platelet activation of circulating platelets.Conclusion: AZD6140 inhibits platelet reactivity in diabetic rats rapidly and reversibly. Markers of tonic platelet activation, which were increased in diabetic rats, were lowered to levels comparable to non-diabetic rats following chronic treatment with AZD6140.

‘Pergularain e I’ – a plant cysteine protease with thrombin-like activity from Pergularia extensa latex

2010-03-01

Abstract: Pergularain e I, a cysteine protease with thrombin-like activity, was purified by ion exchange chromatography from the latex of Pergularia extensa. Its homogeneity was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), native PAGE and reverse-phase high-performance liquid chromatography (RP-HPLC). The molecular mass of pergularain e I by matrix-assisted laser desorption ionization–time of flight (MALDI–TOF) was found to be 23.356kDa and the N-terminal sequence is L–P–H–D–V–E. Pergularain e I is a glycoprotein containing ∼20% of carbohydrate. Pergularain e I constituted 6.7% of the total protein with a specific activity of 9.5 units/mg/min with a 2.11-fold increased purity. Proteolytic activity of the pergularain e I was completely inhibited by iodoacetic acid (IAA). Pergularain e I exhibited procoagulant activity with citrated plasma and fibrinogen similar to thrombin. Pergularain e I increases the absorbance of fibrinogen solution in concentration-dependant and time-dependant manner. At 10μg concentration, an absorbance of 0.48 was reached within 10min of incubation time. Similar absorbance was observed when 0.2 NIH units of thrombin were used. Thrombin-like activity of pergularain e I is because of the selective hydrolysis of Aα and Bβ chains of fibrinogen and γ-chain was observed to be insusceptible to hydrolysis. Molecular masses of the two peptide fragments released from fibrinogen due to the hydrolysis by pergularain e I at 5-min incubation time were found to be 1537.21 and 1553.29 and were in close agreement with the molecular masses of 16 amino acid sequence of fibrinopeptide A and 14 amino acid sequence of fibrinopeptide B, respectively. Prolonged fibrinogen–pergularain e I incubation releases additional peptides and their sequence comparison of molecular masses of the released peptides suggested that pergularain e I hydrolyzes specifically after arginine residues.

Release of tissue-type plasminogen activator (t-PA) in the splanchnic circulation of the anaesthetised pig during high sympathetic tone

Jan-Arne Björkman, Arnfinn Ilebekk, Christina Jern — 2010-03-01

Abstract: Background: There is a substantial local release of tissue-type plasminogen activator (t-PA) in the splanchnic vascular bed, and this release is increased at high sympathetic tone. Coronary t-PA release is also significant, and this release increases during cardiac nerve stimulation and during reperfusion after 10min of local myocardial ischemia. However, by repeated cycles of myocardial ischemia/reperfusion coronary t-PA release progressively declines.Objectives: Accordingly, we hypothesised that splanchnic t-PA release might decrease after an initial peak during maintained and long-lasting sympathetic stimulation.Methods: In 6 anaesthetised pigs sympathetic tone was augmented by bleeding (20–25mL/kg over 30minutes). During the subsequent 2hours period portal vein (draining the splanchnic vasular bed) - and arterial blood samples were drawn every 20min and portal vein blood flow was recorded continuously in order to estimate t-PA release in the splanchnic vascular bed.Results: Relatively stable haemodynamic conditions were obtained after bleeding with mean arterial blood pressure at 50 to 65mmHg and a portal vein flow at about the 50% of baseline value. Net splanchnic t-PA release rose to a peak 40min after bleeding, but subsequently declined towards baseline values. Arterial t-PA activity rose after the bleeding period and to a peak value at end of the observation period.Conclusions: Net splanchnic t-PA release increased only transiently during the period with increased sympathetic stimulation, whereas the arterial t-PA level remained elevated. During a strong and longlasting sympathetic stimulation the lack of a continuously augmented splanchnic t-PA release might increase the risk for intravenous splanchnic thrombosis.

A multicolor flow cytometric assay for measurement of platelet-derived microparticles

2010-03-01

Abstract: Introduction: Flow cytometry (FCM) is the most commonly used method for detection of platelet-derived microparticles (PDMPs), but it is poorly standardized and mainly used for “bedside” analyses in fresh samples. If PDMPs could be analyzed in previously frozen samples it would increase the usefulness of the method. However, cell membrane fragments from contaminating cells created during freezing/thawing may cause artifacts and disturb measurements.Materials and Methods: PDMPs were labeled with monoclonal antibodies directed against CD42a and CD62P, or CD42a and CD142. The PDMP gate was determined using forward scatter (FSC) and CD42a expression. The mean fluorescence intensities (MFIs) of CD62P or CD142 positive particles were translated into MESF -values (Molecules of Equivalent Soluble Fluorochrome) using a standard curve. FITC-labeled phalloidin (which binds to intracellular actin) was used to detect destroyed cells/cell fragments.Results: Phalloidin-positive particles were significantly more common in supernatants of frozen/thawed platelet rich and platelet poor plasma samples compared with supernatants of platelet free plasma. High-speed centrifugation was then used to obtain PDMP samples with low contamination of cell fragments. Electron microscopy showed that these samples contained numerous round stained particles with cellular membranes of a size of 100–700nm. Reproducibility experiments using plasma samples from healthy individuals showed that the coefficients of variation (CVs) of MESF values of CD62P and CD142 (both intra- and interassay) were <10%, and the variation between two cytometers in two different laboratories was <5%. We also found that PDMP expression of CD142 (i.e. tissue factor [TF]) and CD62P (i.e P-selectin) was around two times higher in samples from type 1-diabetes patients compared with those from healthy controls (p<0.001).Conclusions: The use of MESF values to quantify PDMP expression of P-selectin and TF yields reproducible data and enables comparison of data between laboratories. If high-speed centrifugation is performed, contamination of cell fragments is low in frozen/thawed samples.