Increased Blood Coagulation Research Articles

Paraneoplastic vasculitis with digital necrosis: a rare presentation of advanced ovarian cancer

We report an interesting case of a rare presentation of ovarian cancer. A 64-year-old woman attended the Accident and Emergency department with a complaint of sudden onset of ‘‘painful, cold and blue fingers’’ (see Fig. 1). On further questioning, she revealed symptoms of myalgia and malaise. Several nail fold infarcts were also noticed. She was found to have a tense distended abdomen; however, no obvious masses were felt. She was referred to a Rheumatologist who carried out a battery of tests for autoimmune vasculitis, which were negative. A pelvic ultrasound revealed moderate ascites and a right sided complex adnexal cyst measuring 4.7 9 5.2 cm. Tumour marker serum CA-125 was raised at over 800 U/ mL and ascitic fluid sent for cytology showed cells suspicious of adenocarcinoma of the ovary. Subsequently, CT scan was carried out which revealed the adnexal mass with ascites and extensive omental as well as peritoneal seedlings. During the course of her admission, her vasculitis became more prominent. She was commenced on a short course of oral prednisolone therapy which resulted in symptomatic relief but did not resolve the vasculitis. The clinical presentation with sudden onset of ‘‘painful, cold and blue fingers’’, negative immunological tests and advanced ovarian malignancy led us to the diagnosis of digital vasculitis affecting the fingers secondary to a paraneoplastic phenomenon. She received six cycles of chemotherapy with carboplatin which resulted in good biochemical and radiological response, with a drop in CA125 to 55 U/mL. The symptoms of digital vasculitis also showed improvement and appeared to resolve completely by the time of third cycle of chemotherapy, further raising the suspicion that the paraneoplastic syndrome is caused by an ovarian tumour. The patient underwent debulking surgery and the vasculitis remained stable during the subsequent course of her treatment without relapse. The first report on the association of digital ischaemia and carcinoma was by O’Connor in 1884. He observed a correlation between breast cancer and gangrenous fingertips [1]. The prevalence of paraneoplastic syndromes associated with ovarian cancer is estimated in the order of only one in 1,000 [2]. With regard to the pathogenesis, induction of vasculitis by antibodies to tumour antigens is proposed as a likely method of paraneoplastic phenomenon [3]. The other pathogenesis could be digital vasospasm caused by sympathetic hyperactivity and digital artery obstruction due to increased blood coagulation associated with malignancy, hyperviscosity and arteritis [4]. Some paraneoplastic syndromes are also idiopathic. While investigating a patient with paraneoplastic vasculitis, apart from the haematological tests, immunological and serological investigations are of the utmost importance. Although the imaging studies required will depend on the individual presentation, the following tests may be of value: chest and sinus radiographs, echocardiograph, upper limb arteriography, ultrasound of abdomen and pelvis, erect abdominal X-ray, abdominal and pelvis CT. Biopsy of the digit or biopsy of the underlying malignancy should be considered [1, 5]. The aim of the treatment is to alleviate symptoms of ischaemia, prevent progression and to treat underlying cause. Although the symptoms of digital ischaemia may improve with medical therapies, definite benefits have been S. Robati K. Razvi K. Madhavan K. Gajjar (&) Department of Obstetrics and Gynaecology, Southend University Hospital NHS Foundation Trust, Westcliff on Sea, Essex, UK e-mail: gajjarkb@yahoo.co.uk

Strong Correlation of High Endogenous Plasma Levels of Tissue Factor Pathway Inhibitor (TFPI) with Poor Blood Clotting and Corrective Action of BAX 513 Under FVIII-Inhibited Conditions

Abstract 1201▪▪This icon denotes a clinically relevant abstractTFPI is the major physiological inhibitor of the extrinsic pathway of blood coagulation. Thus, blocking TFPI activity is a possible approach to treat bleeding disorders such as hemophilia A and B. BAX 513, a fucoidan derived from the brown seaweed L. japonica, and other non-anticoagulant sulfated polysaccharides (NASPs) improve coagulation in hemophilic blood and plasma (Liu et al. Thromb Haemost 2006;95:68). This procoagulant activity of NASPs can at least partially be attributed to the inhibition of TFPI. For this study, we predicted that endogenous TFPI levels are associated with decreased hemostatic capacity under hemophilic conditions. We correlated clotting parameters measured by global hemostatic assays with the total and full-length (FL) TFPI levels in human plasma samples in the absence or presence of BAX 513.Tissue factor-triggered coagulation of normal and FVIII-inhibited plasma or blood from 30 healthy subjects was monitored by calibrated automated thrombograph (CAT) assay and rotation thromboelastometry (ROTEM). The experiments were performed with or without factor VIII blockade with a goat anti-human Factor VIII antibody to simulate acquired hemophilia. The total and FL-TFPI plasma levels were quantified by ELISA. We observed longer clotting and clot formation times by ROTEM and decreased thrombin generation in the presence of high TFPI levels in normal plasma (p<0.05). These correlations became substantially stronger when FVIII was inhibited (reaching up to r2= 0.64; p<0.001). In the presence of BAX 513 (0.1–1.2 μg/mL), most plasma samples showed a pro-coagulant response with an at least 50% increase in thrombin peak and a decrease in time to peak. The BAX 513-mediated increase in thrombin generation correlated strongly with the individual FL-TFPI plasma level. The effect was even more apparent when FVIII was inhibited, where the extrinsic pathway is the only driver of thrombin formation.These data indicate that high endogenous TFPI levels critically compromise hemostasis in acquired factor VIII deficiency and that TFPI is a threshold factor for the intrinsic amplification of thrombin formation. As low TFPI levels are associated with increased blood clotting, TFPI inhibition is a rationale treatment approach in patients with hemophilia. Importantly, our findings validate the inhibition of TFPI activity as a mode of action for BAX 513 and related pro-coagulant compounds. Disclosures:Dockal:Baxter Innovations GmbH: Employment. Gorczyca:Medical University of Vienna: Employment. Knappe:Baxter Innovations GmbH: Employment. Palige:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment.

Vitamin K in fish nutrition

Vitamin K belongs to the lipid soluble vitamins, and occurs naturally as phylloquinone (vitamin K1) and menaquinone (vitamin K2). In addition, there is a synthetic provitamin, menadione (vitamin K3), primarily used as a vitamin K source in animal feed. Menadione is unstable during feed processing and storage and the dietary content may reach critically low levels. Recent publications also question the availability of menadione in feed for salmonids. Vitamin K plays vital roles in blood coagulation and bone mineralization in fish, but the suggested minimum requirement varies considerably depending on the vitamin K source used. Vitamin K deficiency is characterized by mortality, anaemia, increased blood clotting time and histopathological changes in liver and gills. However, one should assess both inherent and supplemented forms of vitamin K in feeds for exact determinations, as relevant novel feed ingredients of plant origin may be sufficient to meet the requirement for vitamin K. The current review gives an overview of the biochemical role of vitamin K, and discusses vitamin K requirement in fish in light of updated literature, with special emphasis on salmonids.

Is HIV-1 infection associated with endothelial dysfunction in a population of African ancestry in South Africa?

The chronic infection status suffered by HIV-infected individuals promotes chronic arterial inflammation and injury, which leads to dysfunction of the endothelium, atherosclerosis and thrombosis. Although HIV-1 subtype C is prevalent in South Africa and accounts for almost a third of the infections worldwide, this subtype differs genetically from HIV-1 subtype B on which the majority of studies have been done. The objective of this study was to assess whether newly identified, never-treated, HIV-1-infected South African participants showed signs of endothelial dysfunction, accelerated atherosclerosis and increased blood coagulation. We compared 300 newly diagnosed (never antiretroviraltreated) HIV-infected participants to 300 age-, gender-, body mass index- and locality-matched uninfected controls. Levels of high-density lipoprotein cholesterol (HDL-C), triglycerides, interleukin-6 (IL-6), C-reactive protein (CRP), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), fibrinogen and plasminogen activator inhibitor-1 (PAI-1), and carotid radialis pulse wave velocity (cr-PWV) were determined. The HIV-infected participants showed lower HDL-C and higher IL-6, CRP, ICAM-1 and VCAM-1 levels compared to the uninfected controls. No differences in fibrinogen and PAI-1 levels were detected. A continuous positive trend of increasing age with cr-PWV was detected in the HIV-infected group. Our findings suggest inflammatory injury of the endothelium, pointing to endothelial dysfunction of never-treated HIV-1-infected South Africans of African ancestry. Although no indication of a prothrombotic state could be detected, there was an indication of accelerated vascular aging and probable early atherosclerosis in the older HIV-infected participants.

Assessment of oral toxicity and safety of 9-cis-UAB30, a potential chemopreventive agent, in rat and dog studies

9-cis-UAB30 is a potential chemopreventative agent that has been shown to be effective on many different types of tumors. The safety and toxicity of 9-cis-UAB30 had not been previously established. These studies were conducted to evaluate the potential toxicity and pharmacokinetics in a rodent and a nonrodent species for the purpose of investigational new drug submission. Oral gavage administration of 9-cis-UAB30 at the doses 0, 3, 15, and 100 mg/kg/day to CD® rats for 28 days showed a dose-dependent (although not dose-proportional) increase in plasma drug levels in week 4. The liver was the target organ for toxicity of 9-cis-UAB30. Hepatomegaly along with increases in serum aspartate-aminotransferase and alkaline-phosphatase levels were seen in rats. Moderate hypoalbuminemia and hyperglobulinemia resulted in a decreased albumin/globulin ratio. Histopathology revealed hepatocellular change consistent with hepatic glycogen deposition. Toxicity studies in dogs did not show treatment-related toxicity at doses as high as 100 mg/kg/day (highest dose tested) administered by capsules for 28 days. No effects on the central nervous system (functional observational battery in rats) or cardiovascular function (safety pharmacology study in telemeterized dogs) were seen. The no observed adverse effect level (NOAEL) in the rat studies was 3 mg/kg/day; however, the adverse effects seen in rats receiving 15 mg/kg/day (the least observed adverse effect level) was a slight, but statistically significant, elevation in fibrinogen and decrease in prothrombin time, which may be a sign of some tendency for increased blood coagulation. The NOAEL in the dog study was at least 100 mg/kg/day.

Aerosol exposure to Zaire ebolavirus in three nonhuman primate species: differences in disease course and clinical pathology

There is little known concerning the disease caused by Zaire ebolavirus (ZEBOV) when inhaled, the likely route of exposure in a biological attack. Cynomolgus macaques, rhesus macaques, and African green monkeys were exposed to aerosolized ZEBOV to determine which species might be the most relevant model of the human disease. A petechial rash was noted on cynomolgus and rhesus macaques after fever onset but not on African green monkeys. Fever duration was shortest in rhesus macaques (62.7 ± 16.3 h) and longest in cynomolgus macaques (82.7 ± 22.3 h) and African green monkeys (88.4 ± 16.7 h). Virus was first detectable in the blood 3 days after challenge; the level of viremia was comparable among all three species. Hematological changes were noted in all three species, including decreases in lymphocyte and platelet counts. Increased blood coagulation times were most pronounced in African green monkeys. Clinical signs and time to death in all three species were comparable to what has been reported previously for each species after parenteral inoculation with ZEBOV. These data will be useful in selection of an animal model for efficacy studies.

Comparison of changes in blood coagulation, fibrinolysis and endothelial damage in patients undergoing laparoscopic cholecystectomy with different durations of carbon dioxide pneumoperitoneum

Objective To compare the changes in blood coagulation, fibrinolysis and endothelial damage in patients undergoing laparoscopic cholecystectomy with different durations of carbon dioxide pneumoperitoneum. Methods Sixty-four ASA Ⅰ orⅡpatients, aged 23-60 yr, weighing 45-82 kg, scheduled for elective laparoscopic cholecystectomy, were randomly divided into 3 groups according to the duration of pneumoperitoneum: duration of pneumoperitoneum ≤30 min group (group Ⅰ, n=21), 30 min ＜ duration of pneumoperitoneum ＜ 60 min (group Ⅱ, n=23) and duration of pneumoperitoneum≥ 60 min (group Ⅲ , n=20).The intra-abdominal pressure was maintained at 12-14 mm Hg. Venous blood samples were taken before surgery (baseline, T0 ),at the end of surgery(T1), and at 1, 2 and 3 d after surgery (T2-4) for determination of prothrombin time, activated partial thromboplastin time, concentrations of prothrombin fragment 1+2(F1+2), fibrinogen (Fib), tissue plasminogen activator and plasminogen activator inhibitor type-1 (PAI-1), and activities of antithrombin Ⅲ(AT-Ⅲ)and von Willebrand factor(vWF).Results Compared with groupⅠ , the vWF activity and PAI-1 concentration at T2 , concentrations of Fib, F1+2, PAI-1 and activity of vWF at T3 and concentrations of Fib and F1+2 at T4 were significantly increased, while the AT-IE activity at T3 was significantly decreased in group Ⅲ(P＜0.05) .Conclusion When the duration of pneumoperitoneum is short, no obvious changes in the blood coagulation, fibrinolysis and endothelial damage are observed postoperatively in patients undergoing laparoscopic cholecystectomy, and along with the prolongation of the duration of pneumoperitoneum, increased blood coagulation, reduced fibrinolysisand aggravated endothelial damage are observed postoperatively. Key words: Pneumoperitoneum,artifical; Laparoscopy; Blood coagulation tests; Fibrinolysis; Tunica intima

Analytical Microsystems for Biomedical and Environmental Applications

Two types of analytical microsystems for the detection of species of interest in biomedical diagnosis and in environmental monitoring are specifically described in this paper. We describe a novel device that will measure whole blood concentration of D-dimer, a recognized biomarker of increased blood clotting activity and that will then offer opportunity to use the test in the point of care setting. The device combines innovation in antibody bio-engineering for high specificity immunoassay-based diagnostics and nano/ micro engineered impedimetric analysis electrodes incorporating a biocompatible polymer substrate with development of a disposable microfluidic manifold, enabling diagnostics at the point-of-first-contact. The feasibility of a generic microsytem integrating a microfluidic system of concentration and a module of electrochemical detection is demonstrated for the four metals of the European directive (DCE 2000/60/EC) for the quality of water resource: cadmium, mercury, lead and nickel.

P53 Impairs Endothelial Function by Transcriptionally Repressing Kruppel-Like Factor 2

To evaluate if p53 decreases Kruppel-Like Factor 2 (KLF2) expression and determine whether p53-mediated suppression of KLF2 plays a role in p53-induced endothelial dysfunction. Endothelial KLF2 mediates endothelium-dependent vascular homeostasis by differentially regulating endothelial genes, leading to an anti-inflammatory and antithrombotic endothelial surface with normal vasodilatory function. In contrast, the tumor suppressor p53 leads to inflammatory gene expression and impairs endothelium-dependent vasodilatation, thus promoting endothelial dysfunction. The effect of p53 on KLF2 expression was determined. p53 inhibited KLF2 transcription in a histone deacetylase-dependent and a histone acetyltransferase-independent fashion. KLF2 expression was suppressed by p53 via a conserved p53-binding repressor sequence in its promoter. p53 bound to, and stimulated, deacetylation of Histone H3 at the KLF2 promoter. The effect of p53 on endothelial KLF2 target genes was examined. Downregulation of p53 increased expression of endothelial NO synthase and thrombomodulin and inhibited expression of plasminogen activator inhibitor 1. Conversely, overexpression of p53 suppressed endothelial NO synthase and thrombomodulin expression and stimulated plasminogen activator inhibitor 1 and endothelin-1 expression. Knockdown of KLF2 abolished the p53-induced decrease in thrombomodulin and increase in endothelin-1. Both, overexpression of p53 and knockdown of KLF2 in endothelial cells increased blood coagulation on an endothelial cell monolayer. The p53-induced increase in coagulation was rescued by forced expression of KLF2. p53 also impaired endothelium-dependent vasodilatation and decreased bioavailable vascular NO, both of which were rescued by forced KLF2 expression. These findings illustrate a novel p53-dependent mechanism for the regulation of endothelial KLF2 expression. In addition, they show that downregulation of KLF2, in part, mediates a p53-stimulated dysfunctional endothelium.

深部静脈血栓症を合併し,血液凝固・線溶マーカーの異常高値が長期間持続した維持血液透析患者の一例

A 70-year-old woman on maintenance hemodialysis for 3 years was admitted to our hospital because of deep vein thrombosis (DVT) in the right femoral vein. Seven days before admission, she suddenly noticed severe pain in her right inguinal region while she was walking on the street. A wide range of stenosis from the iliac to the distal femoral region was detected by both CT scanning and venography. Her hematocrit reading was 30% and her serum erythropoietin level was 10.5 MU/ml, which was within the normal range, on the day of admission. The results of routine coagulation tests, including prothrombin time, activated partial thrombin time and plasma fibrinogen values, were normal. Plasma anti-thrombin III and plasminogen were also normal. In contrast, beta-thromboglobulin, platelet factor IV, fibrinogen degradation product, D-dimer, thrombin-antithrombin III complex (TAT) and fibrinopeptide A were abnormally elevated. In the venous occlusion test which was performed in the forearm of the opposite side of the arterio-venous shunt, plasma tissue type plas-minogen activator values showed little response to occlusion indicating that the vessel endothelium may have been partially damaged. These data suggest that the DVT had been induced by imbalance of increased blood coagulation and decreased fibrinolytic activity. Damaged vascular wall may also have contributed to the production of DVT. Furthermore, it is surprising that the patient had elevated values of D-dimer and TAT for many years without recurrence of the DVT. Spontaneous DVT in an apparently healthy individual on maintenance hemodialysis seems to be rare, compared with arterial thrombosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis of 2-(3-spiro-cyclohexyl-3,4-dihydroisoquinolyl-1)acetic acid derivatives and their effects on blood clotting

Cyclocondensation of the esters and amides of cyanoacetic acid with 1-spiro-cyclohexylcarbinols yielded derivatives of 2-(3-spiro-cyclohexyl-3,4-dihydroisoquinolyl-1)acetic acid containing and not containing methylenedioxo groups at positions 6,7. Compounds containing this group were found to increase blood clotting; those without it, conversely, decreased clotting. The most active compounds had effects comparable to those of ethamsylate and heparin.

Synthesis of 1-aroylmethyl-and 1-aryloxymethyl-3,4-dihydroisoquinolines and their effects on blood clotting

Acylation of a number of tertiary enamines with aroylchlorides was used to synthesize 1-aroylmethyl-3,4-dihydroisoquinolines. Reaction of 1-chloromethyl-3,3-dimethyl-3,4-dihydroisoquinoline with phenols with interphase catalysis was also used to make 1-aryloxymethyl-3,4-dihydroisoquinoline derivatives. 1-Aroylmethylisoquinolines characteristically had a hemostatic effect, while 1-aryloxymethyl-3,4-dihydroisoquinolines had an anticoagulant effect. The most active compound increased blood clotting by 17.7%.

Synthesis and blood coagulation properties of drotaverine derivatives

Quaternary ammonium salts have been obtained via the reaction of drotaverine base and its tetrahydroisoquinoline derivative with alkyl iodides. Boiling of drotaverine with HBr leads to the cleavage of ethoxy groups with the formation of hydroxy groups. It is established that all the synthesized compounds increase blood coagulation. For the most active compounds, the effect reaches 26%.

Effects of KAATSU training on haemostasis in healthy subjects

The KAATSU training is a novel method for muscle training performed under the reduction of muscle blood flow by a specially-designed belt (KAATSU belt). Under the conditions of restricted muscle blood flow, even short-term, low-intensity exercise such as walk training can induce muscle strength, and increased muscle mass (Takarada et al. 2000a; b; c; Takarada et al., 2002a,b; Abe et al. 2006). In addition, KAATSU femoral blood flow restriction induces lower-body venous pooling and reduces venous return (Takano et al., 2006), resulting in stimulating cardiovascular effects of orthostasis in 1G like lower body negative pressure (LBNP) (Iida et al., 2007). Since the most effective countermeasure regimen to prevent cardiovascular deconditioning in space flight would be a gravitation-like stress combined with exercises, the use of KAATSU in combination with low-intensity resistance exercise would provide a significant countermeasure like LBNP for space flight countering both cardiovascular and musculoskeletal decline. Thus, the KAATSU training may be a promising training in astronauts to prevent cardiovascular deconditioning and muscle atrophy in space flights (Iida et al., 2006) as well as athletes and healthy persons as described previously (Nakajima et al., 2006). Haemostasis is achieved through a delicate equilibrium between the coagulation and fibrinolytic cascades (Wu and Thiagarajan, 1996). Exercises have been shown to affect activation of both the coagulation and fibrinolytic cascades (Davis et al., 1976; Andrew et al., 1986; Boman et al., 1994; Weiss et al., 1998; El-Sayed et al., 2000; Ribeiro et al., 2006). Regular exercise preferentially activates fibrinolysis, which is generally associated with favorable alterations in risk from cardiovascular morbidity, while strenuous exercise may increase blood coagulation, and promote thrombus formation (Hilberg et al., 2002), resulting in exertion-related ischemic events (Giri et al., 1999). Thus, it is likely that effects of exercise on the haemostatis are Effects of KAATSU training on haemostasis in healthy subjects

Controlling clots with Clk1

Platelets are critical blood cells involved in clotting. An early step in clot construction requires tissue factor (TF), but whether platelets make TF was unknown. New work by Schwertz et al. (page 2433) reveals that these nuclei-free cells doproduce TF by cytoplasmic splicing and further identify the kinase Clk1 as a splicing activator. After injury to a blood vessel, platelets are the first cells on the scene to plug the hole. Platelets get activated by attachment to exposed collagen (amongst other things), and promote a TF-dependent coagulation cascade on their cell membranes. This ultimately results in large-scale fibrin production and the assembly of a tough fibrin meshwork over the platelet plug. TF is released from the injured tissue, but whether platelets themselves express TF is a debated question. Schwertz et al. found that activated human platelets contained TF mRNA. Resting platelets instead contained unspliced TF pre-mRNA. Activation, the team showed, induced splicing and subsequent translation of TF in as little as 5 min. Cytoplasmic splicing was described for the first time by this group a year ago. The precise mechanism is unknown, but splicing factor SF2/ASF was identified in platelet cytoplasms. In nucleated cells, Clk1 activates SF2/ASF by phosphorylation. Interrupting this modification, the team now shows, prevents processing of TF pre-mRNA. A number of conditions lead to increased blood clotting. In recent unpublished work, the team found that platelets isolated from septic patients, in whom thrombosis occurs, are more prone to clotting and contain increased levels of spliced TF mRNA. Clk1-controlled splicing might thus be a good target for anticoagulation treatments.

Is APC activation of endothelial cell PAR1 important in severe sepsis?: No

Is APC activation of endothelial cell PAR1 important in severe sepsis?: No

Kruppel-Like Factor 2 (KLF2) Regulates Endothelial Thrombotic Function

The vascular endothelium maintains blood fluidity by inhibiting blood coagulation, inhibiting platelet aggregation, and promoting fibrinolysis. Endothelial cells lose these nonthrombogenic properties on exposure to proinflammatory stimuli. We recently identified the Kruppel-like factor KLF2 as a novel regulator of endothelial proinflammatory activation. Here it is found that KLF2 differentially regulates key factors involved in maintaining an antithrombotic endothelial surface. Overexpression of KLF2 strongly induced thrombomodulin (TM) and endothelial nitric oxide synthase (eNOS) expression and reduced plasminogen activator inhibitor-1 (PAI-1) expression. Furthermore, overexpression of KLF2 inhibited the cytokine-mediated induction of tissue factor (TF). In contrast, siRNA mediated knockdown of KLF2 reduced antithrombotic gene expression while inducing the expression of pro-coagulant factors. The functional importance of KLF2 was verified by in vitro clotting assays. By comparison to control infected cells, KLF2 overexpression increased blood clotting time as well as flow rates under basal and inflammatory conditions. In contrast, siRNA-mediated knockdown of KLF2 reduced blood clotting time and flow rates. These observations identify KLF2 as a novel transcriptional regulator of endothelial thrombotic function. The full text of this article is available online at http://circres.ahajournals.org.

Increased Blood Clotting, Microvascular Density, and Inflammation in Eotaxin-Secreting Tumors Implanted into Mice

An important theme that is emerging in cancer research is the interaction between tumor cells and the host stroma. Because many types of human cancer are infiltrated by eosinophils that are believed to mediate an anti-tumor cytotoxic effect, we developed and studied a transfected B16 murine melanoma cell line that secretes high levels (510 pg/ml/100,000 cells/day) of eotaxin, a chemokine that recruits and activates primarily eosinophils. Here we report that there was increased inflammation (eosinophils, mast cells, mononuclear cells), blood clotting, and microvascular density within the tumors produced by subcutaneous implants of eotaxin-secreting tumor cells in 10 C57BL/6 compared to tumors produced by wild-type tumor cells. The extensive blood clotting in the eotaxin-transfected tumors was associated with significantly decreased blood flow to the tumors as measured by magnetic resonance imaging [(mean maximum signal enhancement of eotaxin-secreting tumors, 147 +/- 57 (n = 7) compared to 202 +/- 36 signal enhancement units (n = 8) for the wild-type melanoma cells; P = 0.04 by two-tailed, unpaired t-test]. Surprisingly, there was no significant difference between the growth rates or mean masses of the eotaxin-secreting tumors (750 +/- 280 mg, n = 10) and the wild-type tumors (780 +/- 290, n = 10) after 20 days of growth in vivo, despite the significantly slower growth rate in vitro of the eotaxin-secreting tumor cells. We conclude that eotaxin and the resultant tumor-infiltrating inflammatory cells are not likely to mediate a significant anti-tumor effect in vivo. Instead, elevated eotaxin is associated with increased inflammation, microvascular density, and blood clotting. Thus, eotaxin and eosinophils may play a more complex role in modulating the growth of tumors than the simple, anti-tumor cytotoxic effect that has been previously proposed.

Increased blood coagulation and platelet activation in patients with infective endocarditis and embolic events.

Inflammation-induced procoagulant changes and alterations in platelet activity appear to play an important role in thromboembolic complications of infective endocarditis (IE). The aim of this study was to investigate systemic coagulation activity, fibrinolytic capacity, and platelet activation in patients with IE with and without embolic events by measuring the plasma levels of prothrombin fragment 1+2 (PF1+2), thrombin-antithrombin III complex (TAT), plasminogen activator inhibitor-1 (PAI-1), beta-thromboglobulin (beta-TG), and platelet factor 4 (PF4), respectively. The study included 76 consecutive patients (female = 55, male = 21, mean age 26 years, range 8-64 years) with definite IE according to the Duke criteria; of these, 13 (17.1%) had embolic events. Plasma concentrations of PF1+2 (3.2 +/- 1.3 vs. 1.7 +/- 0.7 and 1.4 +/- 0.7 nmol/l, p < 0.001, respectively) and TAT (7.3 +/- 1.5 vs. 2.9 +/- 1.2 and 2.2 +/- 1.1 ng/ml, p < 0.001, respectively) were elevated in patients with embolic events compared with patients without embolic events and control subjects. Similarly, patients with embolic events had increased plasma levels of beta-TG (63.3 +/- 10.9 vs. 33.1 +/- 11.6 and 19.1 +/- 10.6 ng/ml, p < 0.001, respectively) and PF4 (106.0 +/- 28.7 vs. 50.3 +/- 16.7 and 43.0 +/- 15.8 ng/ml, p < 0.001, respectively) compared with those without embolic events and the control group. Embolic patients also had higher PAI-1 levels than nonembolic patients and healthy subjects (14.4 +/- 6.4 vs. 8.6 +/- 5.9 and 5.4 +/- 4.3 ng/ml, p = 0.002, respectively). Patients with IE and with subsequent thromboembolism have increased systemic coagulation activation, enhanced platelet activity/damage, and impaired fibrinolysis. The resulting imbalance produces a sustained hypercoagulable state, which contributes to the increased risk of thromboembolic events in this particular group.

Mast cell inhibitor cromolyn increases blood clotting and hypoxia in murine breast cancer.

Human breast cancer is extensively infiltrated by mast cells that contain powerful anticoagulants such as heparin, tryptase and chymase. To determine if human breast cancer is associated with mast cell activation, we measured the levels of mast cell tryptase (an indicator of mast cell activation) in the blood of 20 women with varying stages of breast cancer. The mean level of tryptase in women with breast cancer (10.3 +/- 4.2 microg/L) was significantly higher than in 50 normal healthy women without breast cancer (3.0 +/- 2.5 microg/L, p < 0.05 by two-tailed t-test). To explore the role of mast cells in breast cancer in more detail, we then carried out experiments that were aimed at determining if an inhibitor of mast cell function, sodium cromolyn, could increase blood clotting and hypoxia within subcutaneous implants of the 4T1 mammary adenocarcinoma cell line in mice. We treated tumor-bearing mice with 5 consecutive daily doses of sodium cromolyn (10 mg/kg, i.p.). An average of 30% of the periphery of the tumors from the 5 drug-treated mice contained large lakes of clotted blood that were not evident in any of the tumors from the control (untreated) mice. By computerized image analysis of tumors immunostained for a hypoxia marker (pimonidazole), the tumors from the treated mice had significantly more hypoxia (35 +/-12 % hypoxic regions, n = 5) than the tumors from untreated (control) mice (16 +/- 7%, n = 5). We conclude that sodium cromolyn enhanced peri-tumoral blood clotting and intratumoral hypoxia. These results suggest that mast cells may play an important role in regulating blood clotting and hypoxia within breast cancer.