BACKGROUND

NN<em>1</em>73<em>1</em> is a recombinant activated factor VII (rFVIIa) analog with enhanced activity.

OBJECTIVE

This clinical trial aimed to assess the safety and pharmacokinetics of single doses of NN<em>1</em>73<em>1</em> in healthy male subjects.

METHODS

This was a randomized, placebo-controlled dose-escalation trial with four dose tiers (NN<em>1</em>73<em>1</em> 5-30 microg kg(-<em>1</em>)). Eight subjects were randomized to either NN<em>1</em>73<em>1</em> (n = 6) or placebo (n = <em>2</em>) in each tier.

RESULTS

No thromboembolic or serious adverse events were reported and no antibody formation towards NN<em>1</em>73<em>1</em> was detected. NN<em>1</em>73<em>1</em> was demonstrated to be pharmacologically active based on coagulation-related parameters (<em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, activated partial thromboplastin time and <em>prothrombin</em> time). There were five mild/moderate adverse events in three subjects. The FVIIa activity of NN<em>1</em>73<em>1</em> after ascending single-dose administration of NN<em>1</em>73<em>1</em> fits well with a two-compartment model, indicating a bi-exponential decline with a rapid initial distribution of approximately 73% FVIIa activity (half-life = <em>2</em>0 min), followed by a less rapid terminal elimination phase eliminating the remaining <em>2</em>7% (half-life = 3 h). Dose proportionality in healthy male subjects at the dose levels investigated (5-30 microg kg(-<em>1</em>)) was supported by the FVIIa activity data.

CONCLUSIONS

Based on the results of this trial, NN<em>1</em>73<em>1</em> appears safe and well tolerated in healthy subjects at doses up to 30 microg kg(-<em>1</em>). No immunogenic or thromboembolic events were reported. The pharmacokinetic profile of NN<em>1</em>73<em>1</em> as measured by FVIIa activity appears to follow two-compartment pharmacokinetics characterized by an initial rapid distribution phase followed by a less rapid elimination phase.

Increased plasma levels of <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>) found in patients with sickle-cell disease reflect enhanced endogenous thrombin generation. We postulate that hypercoagulability contributes to vaso-occlusion. The intensity of acenocoumarol treatment required to reduce the F<em>1</em> + <em>2</em> level to 50% of pretreatment level was investigated in seven patients with symptomatic sickle-cell anaemia during steady-state disease for a period of <em>2</em> months. All patients had increased levels of F<em>1</em> + <em>2</em> compared with an age-matched control group. Normalization of the F<em>1</em> + <em>2</em> was achieved at a median INR of <em>1</em>.64 (range <em>1</em>.<em>1</em>8-<em>2</em>.<em>2</em>). It is concluded that low-intensity oral anticoagulation normalizes the hypercoagulability in sickle-cell disease.

OBJECTIVE

Low-velocity and nonlaminar flow patterns in the Fontan circulation, as well as abnormal liver function in some patients, may partly account for the coagulation abnormalities seen. We examined (<em>1</em>) coagulation factor abnormalities before and after the Fontan procedure and (<em>2</em>) regional coagulation factor abnormalities in the Fontan circulation.

METHODS

Levels of factors V, VII, VIII, X, antithrombin III, prothrombin fragment F<em>1</em>+<em>2</em>, protein C, and protein S were measured in <em>2</em> groups of patients: In <em>1</em>4 patients undergoing the Fontan procedure, blood was analyzed before the operation and 5 days after the operation (group <em>1</em>). The median age in this group was 3.<em>2</em> years. In <em>1</em>0 patients who had undergone the Fontan procedure, cardiac catheterization was performed and samples were taken from the femoral vein, inferior vena cava, right atrium, and pulmonary artery (group <em>2</em>). The median age in this group was 6.<em>2</em> years and the median follow-up from the Fontan procedure was 4.<em>1</em> years.

RESULTS

In group <em>1</em> a significant increase was noted postoperatively in the concentration of factor VIII (P<.00<em>1</em>), factor X (P<.00<em>1</em>), and prothrombin fraction F<em>1</em>+<em>2</em> (P <.00<em>1</em>). A significant decrease in the levels of antithrombin III (P <.00<em>1</em>), protein C (P<.004), and protein S (P<.0<em>2</em>) was also found. The increase in factors VIII and X persisted at 4 years' follow-up in group <em>2</em> patients. In group <em>2</em>, no significant regional differences were observed between the coagulation factors measured at different sites.

CONCLUSIONS

There is an increased tendency toward coagulation after the Fontan procedure. A prothrombotic state is supported by thrombin generation associated with reduced antithrombin III concentration. This increase in coagulation may contribute to the early and late risks of thromboembolism observed after the Fontan procedure. We did not find any regional differences in coagulation abnormalities in patients late after the Fontan procedure. Therefore, the mechanisms and causes of the coagulation abnormalities remain unclear.

Use of oral contraceptives (OC) that combine a progestogen with synthetic ethinyl estradiol (EE) is associated with increased risk of venous thromboembolism. NOMAC/E<em>2</em> is a new monophasic OC that combines nomegestrol acetate (NOMAC), a highly selective progestogen, with <em>1</em>7β-estradiol (E<em>2</em>). The study objective was to compare the effects on markers of haemostasis of NOMAC/E<em>2</em> (<em>2</em>.5 mg/<em>1</em>.5 mg) versus the second-generation OC, levonorgestrel (LNG)/EE (<em>1</em>00 μg/<em>2</em>0 μg). Healthy women (age <em>1</em>8-38 years) received once-daily treatment for three consecutive <em>2</em>8-day cycles in a double-blind, randomised study: either NOMAC/E<em>2</em> for <em>2</em>4 days with a four-day placebo interval (n=45) or LNG/EE for <em>2</em><em>1</em> days with a seven-day placebo interval (n=45) per cycle. Mean changes from baseline to end-of-treatment in coagulation markers, including <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (primary endpoint), fibrinolysis markers and platelet functions were assessed. Mean <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> levels (primary endpoint) did not increase with NOMAC/E<em>2</em> compared with LNG/EE ( -0.0<em>2</em> vs. +0.08 nM, p<0.0<em>1</em>). Other significant differences between NOMAC/E<em>2</em> and LNG/EE were mean changes in antithrombin (+0.3% vs. -4.4%, p<0.00<em>1</em>), activated protein C resistance - normalised ratio (+0.<em>2</em>0 vs. +0.46, p<0.0<em>1</em>), D-dimer ( -53 vs. +43 ng/ml, p<0.00<em>1</em>), plasminogen (+6% vs. +30%, p<0.000<em>1</em>) and plasminogen activator inhibitor-<em>1</em> ( -3.<em>1</em> vs. -8.0 ng/ml, p<0.00<em>1</em>). There was no effect of either treatment on platelet aggregation. The NOMAC/E<em>2</em> pill regimen has fewer adverse effects on blood biological coagulation and fibrinolysis markers than LNG/EE. This suggests that NOMAC/E<em>2</em> could have a more favourable venous thromboembolism risk profile than LNG/EE; further epidemiological data are required to confirm this.

Hormone replacement therapy (HRT) appears to be cardioprotective in postmenopausal women; however, concerns exist over its thrombogenic effects. To address the effects of combined HRT on coagulation and fibrinolysis, we have measured circulating markers of these processes in a double-blind placebo-controlled trial. Forty-two healthy postmenopausal women aged 50 to 75 years received continuous combined HRT with <em>2</em> mg estradiol+<em>1</em> mg norethisterone or placebo for 6 weeks. Hormone profiles were measured at baseline, and lipid and hemostatic parameters were measured at baseline and after 6 weeks of therapy. Baseline characteristics were similar in the <em>2</em> groups. With change from baseline the main outcome measure, HRT increased the markers of coagulation (<em>prothrombin</em> <em>fragments</em> <em>1</em>+<em>2</em>, 0.<em>2</em>0+/-0.06 versus 0.06+/-0.04 nmol/L, P=0.0005; soluble fibrin, <em>2</em>.3+/-0.4 versus 0.<em>2</em>5+/-0.3 microgram/mL, P=0.0004), reduced plasma fibrinolytic inhibitory activity (plasminogen activator inhibitor-<em>1</em>, -0.67+/-0.<em>1</em>6 versus 0.<em>2</em>4+/-0.<em>2</em><em>1</em> U/mL, P=0.00<em>2</em>), and increased fibrinolysis (D-dimer, <em>2</em>4+/-<em>1</em><em>2</em> versus -6+/-8 ng/mL, P=0.04) compared with placebo. Increases in soluble fibrin and D-dimer were positively correlated (r=0.59, P=0.0<em>2</em>), but changes in plasminogen activator inhibitor-<em>1</em> and D-dimer were unrelated. Although baseline hemostatic and lipid parameters were correlated, there were no associations between changes in hemostatic markers and lipids after treatment. Short-term oral combined continuous HRT (estradiol and norethisterone) increased thrombin and fibrin generation, reduced plasma fibrinolytic inhibitory activity, and increased fibrinolysis. Enhanced fibrinolysis was related to increased fibrin generation but not reduced plasma fibrinolytic inhibitory activity. Coagulation activation may partly explain the increases in venous thrombosis and cardiovascular events reported with the use of combined HRT.

BACKGROUND

Racial differences in coagulation are poorly understood. While some studies suggest a 'prothrombotic' coagulation profile in blacks compared with whites, others report an increased bleeding risk for blacks in various clinical settings. Moreover, preclinical data suggest a link between the Duffy antigen (=DARC, Duffy antigen receptor of chemokines) and coagulation.

OBJECTIVE

Based on our previous research in Duffy antigen negative Africans, we hypothesized that Africans have an attenuated procoagulant response compared with Caucasians in a model of lipopolysaccharide (LPS)-induced, tissue factor (TF)-triggered coagulation activation.

METHODS

Healthy male volunteers (<em>1</em>6 Duffy-negative Africans, <em>1</em>6 Duffy-positive Caucasians) received <em>2</em> ng kg(-<em>1</em>) LPS, and outcome parameters were measured using enzyme immunoassays and real-time polymerase chain reaction (RT-PCR, Taqman).

RESULTS

LPS increased microparticle (MP)-associated TF procoagulant activity (PCA) less in Africans than Caucasians. Africans had reduced in vivo thrombin formation compared with Caucasians: they generated less thrombin-antithrombin (TAT) complexes (<em>1</em>0.4 pg mL(-<em>1</em>) vs. <em>2</em>3.0 pg mL(-<em>1</em>), P<0.000<em>1</em>) and less prothrombin fragments (F<em>1</em>+<em>2</em>) (337 pmol mL(-<em>1</em>) vs. 8<em>1</em>9 pmol mL(-<em>1</em>), P<0.000<em>1</em>). Consistently, Africans also had decreased fibrin formation (D-dimer: 0.3 pg mL(-<em>1</em>) vs. 0.5 pg mL(-<em>1</em>), P=0.0<em>2</em>).

CONCLUSIONS

Duffy-negative subjects of African descent have a markedly reduced procoagulant response in a model of LPS-induced, TF-triggered coagulation activation compared with Duffy-positive healthy Caucasians.

Anabolic-androgenic steroid abuse has recently been linked with acute vascular events in athletes. To date, the relationship between steroid abuse and the potential for cardiovascular disease has been considered almost exclusively in terms of lipid metabolism. However, recent reports of thrombosis in androgen abusing athletes with no evidence of atherosclerosis suggests the hypothesis that thrombosis risk in such athletes could be mediated through androgen induced abnormalities of coagulation. To determine if anabolic-androgenic steroid abuse in weight lifters is associated with an activation of the hemostatic system we studied forty-nine weight lifters recruited through advertisements. History of androgen use or abstinence was confirmed via urine assays. Plasma was assayed for clotting and fibrinolytic activity by measuring thrombin/antithrombin complexes (TAT), <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>1</em> (F<em>1</em> + <em>2</em>), and D-dimers (D-di); markers of the endothelial based fibrinolytic components were assayed by measuring tissue plasminogen activator antigen (t-PA Ag) and its inhibitor (PAI-<em>1</em>); finally, the activity of antithrombin III, protein C, and protein S were measured. Abnormally high concentrations of TAT complexes were noted in <em>1</em>6% of our confirmed steroid using weight lifters compared to 6% of our confirmed nonusers (P = .0<em>1</em>). Steroid users also demonstrated abnormally high concentrations of F<em>1</em> + <em>2</em> and D-dimers when compared to nonusers (44 vs. <em>2</em>4%, P < .00<em>1</em>, and 9 vs. 0%, respectively). Non-steroid users were more likely to have elevated levels of t-PA Ag and PAI-<em>1</em> than our steroid using weight lifters (both P < .00<em>1</em>). The activities of antithrombin III and protein S were more likely to be higher in users compared to nonusers (<em>2</em><em>2</em> vs. 6%, P = .005; <em>1</em>9 vs. 0%, respectively). Some anabolic-androgenic steroid using weight lifters have an accelerated activation of their hemostatic system as evidence by increased generation of both thrombin and plasmin. These changes could reflect a thrombotic diatheses that may contribute to vascular occlusion reported in young athletes using these drugs. The predictive value of these coagulation abnormalities in terms of risk of thrombosis to individual steroid using weight lifters or the population as a whole remains to be studied.

OBJECTIVE

Clinical trials show that interleukin (IL)-6 represents a predictive marker in human sepsis. Furthermore, IL-6 has been proposed as a candidate mediator for endotoxin (lipopolysaccharide)-induced coagulation activation: In a primate model, an (alphaIL-6 antibody (alphaIL-6 Ab) almost abolished lipopolysaccharide-induced coagulation activation. Therefore, we wished to determine if an alphaIL-6 Ab (B-E8) may also attenuate lipopolysaccharide-induced activation of coagulation in humans.

METHODS

The study was a randomized, double blind, placebo-controlled parallel group trial (n = <em>1</em><em>2</em> per group).

METHODS

University medical center.

METHODS

Healthy volunteers.

METHODS

Healthy volunteers were randomized to receive either 80 mg of a monoclonal anti-IL-6 Ab (B-E8) or placebo intravenously before bolus infusion of <em>2</em> ng/kg lipopolysaccharide.

RESULTS

B-E8 effectively decreased IL-6 bioactivity as measured by a Bg-bioassay in vitro and concentrations of C reactive protein. However, B-E8 did not decrease lipopolysaccharide-induced tissue factor-messenger RNA transcription or plasma concentrations of downstream coagulation variables (<em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>, thrombin-antithrombin III complexes, and D-dimer concentrations). Similarly, tumor necrosis factor-alpha concentrations, fibrinolytic activity (plasmin-antiplasmin complexes), endothelial activation (soluble E-selectin), and IL-<em>1</em>0 were unaffected.

CONCLUSIONS

IL-6 does not appear to mediate early-phase lipopolysaccharide-induced coagulation activation in humans.

Familial Mediterranean fever (FMF) patients in clinical remission are reported to have increased baseline inflammation. Normal function of the natural anticoagulant pathways is particularly needed in diminishing inflammatory responses. In the presence of subclinical inflammation, natural anticoagulant response may be exaggerated. We aimed to observe the anticoagulant-procoagulant status in attack-free FMF patients. Twenty-seven FMF patients diagnosed in accordance with Tel-Hashomer criteria, and <em>2</em>6 healthy controls were included. All patients were attack-free under regular colchicine treatment. Amyloidosis, autoimmunity, accompanying liver and renal disease, and vasculitis were excluded. Predisposing factors for thrombosis were not present. Acute phase reactants (APRs), anticardiolipin antibody positivity, <em>prothrombin</em> time (PT), activated <em>prothrombin</em> time, thrombin time (TT) and d-dimer, protein C activity, activated protein C resistance, free protein S, antithrombin, lupus anticoagulant, human <em>prothrombin</em> <em>fragment</em> F <em>1</em> + <em>2</em>, and human thrombin/antithrombin III complex were analyzed for all subjects. APRs were comparable with controls. Autoimmune markers were negative in all. Anti-streptolysin titers were significantly different than the control group. PT, TT, protein C activity, and F <em>1</em> + <em>2</em> levels were significantly different from those of healthy controls. Shortened PT and TT, decreased protein C activity vs increased levels of F <em>1</em> + <em>2</em> suggested a hypercoagulable state in our patients. The hypercoagulable state detected in FMF patients suggests that screening with abnormal coagulation tests may be beneficial for tracing the future consequences of subclinical inflammation in these patients. Studies covering larger groups of patients are needed to verify the currently observed hypercoagulable status in FMF.

To determine in vivo effects of interleukin (IL)-<em>1</em><em>2</em> on host inflammatory mediator systems, 4 healthy chimpanzees received recombinant human IL-<em>1</em><em>2</em> (<em>1</em> microg/kg) by intravenous injection. IL-<em>1</em><em>2</em> induced increases in plasma concentrations of IL-<em>1</em>5, IL-<em>1</em>8, and interferon-gamma (IFN-gamma), plus a marked antiinflammatory cytokine response (IL-<em>1</em>0, soluble tumor necrosis factor [TNF] receptors, IL-<em>1</em> receptor antagonist) and secretion of alpha-chemokines (IL-8, IFN-gamma-inducible protein <em>1</em>0) and beta-chemokines (monocyte chemoattractant protein-<em>1</em>, macrophage inflammatory protein-<em>1</em>beta). In addition, IL-<em>1</em><em>2</em> elicited neutrophilic leukocytosis, neutrophil degranulation (elastase-alpha<em>1</em>-antitrypsin complexes), coagulation activation (F<em>1</em> + <em>2</em> <em>prothrombin</em> <em>fragment</em>, thrombin-antithrombin III complexes), and fibrinolytic activation (tissue-type plasminogen activator, plasmin-alpha<em>2</em>-antiplasmin complexes). IL-<em>1</em><em>2</em>-induced activation of multiple host mediator systems was found only after 8-<em>2</em>4 h, remained detectable until the end of the 48-h observation period, and occurred in the absence of detectable TNF and IL-<em>1</em>beta. These data may contribute to understanding the role of IL-<em>1</em><em>2</em> in the pathogenesis of sepsis syndrome and the toxicity found after repeated injections of IL-<em>1</em><em>2</em>.

Edoxaban, an oral direct factor Xa (FXa) inhibitor, is in phase III clinical development for stroke prevention in atrial fibrillation and treatment of venous thromboembolism. The shed blood model allows for study of activated coagulation at a site of standardised tissue injury due to local release of tissue factor. The objective of this study was to evaluate the effect of three doses of edoxaban on markers of coagulation in shed and venous blood versus placebo and a standard prophylactic dose of fondaparinux. A total of <em>1</em>00 healthy male subjects were randomised to receive single doses of one of five treatments: subcutaneously administered fondaparinux <em>2</em>.5 mg; orally administered edoxaban 30, 60, or <em>1</em><em>2</em>0 mg; or placebo. The primary objective was measurement of blood coagulation markers <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>) and thrombin-antithrombin (TAT) complex, and platelet activation marker β-thromboglobulin (β-TG), in venous and shed blood. Secondary objectives included pharmacokinetics, shed blood volume, and safety of edoxaban. Single doses of edoxaban caused rapid and significant decreases of F<em>1</em>+<em>2</em>, TAT, and β-TG in the shed blood model, indicating inhibition of thrombin generation and platelet activation. Inhibition was significantly less for fondaparinux versus edoxaban. Baseline-corrected F<em>1</em>+<em>2</em>, TAT, and β-TG values demonstrated sustained inhibition up to <em>2</em>4 hours for shed blood in the edoxaban groups but no significant inhibition in venous blood. Overall, edoxaban treatments were well tolerated. In conclusion, single oral doses of edoxaban 30, 60, or <em>1</em><em>2</em>0 mg caused rapid and sustained inhibition of coagulation up to <em>2</em>4 hours in the shed blood model.

OBJECTIVE

Antiphospholipid antibodies (APA) and other coagulation abnormalities have been associated with an increased risk of venous, arterial, and placental thrombosis and recurrent pregnancy loss (RPL). Factor V Leiden (a point mutation [1691G->>A] in the factor V gene), the prothrombin 20210G->>A mutation, and homozygosity for a common polymorphism in the methylene tetrahydrofolate reductase (MTHFR) gene (677C->>T) have been associated with arterial and venous thrombosis and arterial occlusive disease. We explored an association between these markers of thrombophilic states and RPL.

METHODS

Prospective case-control evaluation.

METHODS

University-associated private practice.

METHODS

Fifty nonpregnant women with three or more pregnancy losses and 50 healthy, nonpregnant controls.

METHODS

None.

METHODS

Anticardiolipin and antiphosphatidylserine antibodies were detected in serum by ELISA. Polymerase chain reaction was performed to identify the factor V Leiden (1691G->>A) mutation, the thermobile MTHFR (677C->>T) mutation, and the prothrombin 20210G->>A mutation.

RESULTS

The following were identified by restriction fragment-linked polymorphism analyses: 1 (2%) factor V Leiden heterozygosity; 1 (2%) prothrombin 20210G->>A heterozygosity; and 4 (8%) thermolabile MTHFR homozygosity. None of these mutation frequencies in women with RPL were statistically significantly different from controls.

CONCLUSIONS

These data suggest that factor V Leiden, thermolabile MTHFR (677C->>T), and prothrombin 20210G->>A are not found at an increased frequency in women with a history of early RPL.

BACKGROUND

Thrombelastograph analysis (TEG) is used to evaluate blood coagulation. Ideally, whole blood is immediately processed. If impossible, blood may be citrated and assessed after recalcification. No data describe the effect of such treatment and storage on TEG parameters.

METHODS

Three studies were performed in 90 surgical patients. In 30 patients, blood was citrated (<em>1</em>:<em>1</em>0, 0. <em>1</em><em>2</em>9 M) and recalcified (<em>2</em>0 microl <em>2</em> M CaCl<em>2</em> to 340 microl citrated blood), and TEG was performed with native blood and after recalcification after 0, <em>1</em>5, and 30 min of citrate storage. In another 30 patients, TEG was performed with citrated blood recalcified immediately and after <em>1</em>-7<em>2</em> h storage. In a third study, thrombin-antithrombin complex, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, and beta-thromboglobulin were measured (using enzyme-linked immunoabsorbant assay tests) at corresponding time points. Data were compared using repeated-measures analysis of variance and post hocpaired t tests.

RESULTS

TEG parameters were different in recalcified citrated blood compared with native blood (P < 0.05) and changed significantly during 30-min (P < 0.0<em>2</em>5) and 7<em>2</em>-h (P < 0.00<em>1</em>) citrate storage. TEG parameters measured between <em>1</em> and 8 h of citrate storage were stable. Thrombin-antithrombin complex and <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> values were not elevated in native blood. After 30 min of citrate storage a gradual thrombin activation was observed, as evidenced by increasing thrombin-antithrombin complex and <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> values (P < 0.05). beta-Thromboglobulin level was increased after <em>2</em> and 8 h of citrate storage (P < 0.0<em>1</em>).

CONCLUSIONS

Analysis of native blood yields the most reliable TEG results. Should immediate TEG processing not be possible, citrated blood may be used if recalcified after <em>1</em>-8 h.

Steroid hormones may profoundly influence hemostasis; for example, as discussed for hormone replacement therapy, pregnancy and, being less obvious, for the ovarian cycle. We investigated primary hemostasis parameters using a platelet function analyzer (PFA-<em>1</em>00) during the follicular and luteal phases in <em>1</em>8 healthy young women without oral contraceptives. During the follicular phase, the mean closure time (CT) was <em>1</em>64.7 +/- 56.7 s, and it decreased to <em>1</em>30.<em>2</em> +/- 30.6 s in collagen/epinephrine cartridges in the luteal phase (P = 0.0095). No significant difference could be found for CT values in collagen/adenosine diphosphate cartridges during the follicular phase as compared with the luteal phase (97.<em>2</em> +/- <em>2</em>4.<em>2</em> s versus 89.6 +/- <em>1</em>8.4 s, P = 0.<em>1</em>74). Negative correlations between the CT values in collagen/epinephrine cartridges and von Willebrand factor from both phases of the cycle were found (follicular phase: r = -0.53; luteal phase: r = -0.54). Fibrinogen and fibrinogen degradation products were significantly increased in the luteal phase (<em>2</em>.49 +/- 0.6<em>2</em> g/l versus <em>2</em>.05 +/- 0.59 g/l and 0.<em>1</em><em>2</em> +/- 0<em>1</em>4 versus 0.04 +/- 0.04, P = 0.0<em>2</em> for both parameters) as compared with the follicular phase. No significant differences could be detected for plasminogen, plasmin-antiplasmin complex, <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> and D-dimer between the groups. This study indicates that platelet function is periodically altered during the ovarian cycle due to the influence of progesterone and estrogen on von Willebrand factor concentrations.

OBJECTIVE

Closed circuit extracorporeal circulation (CCECC) has been developed to reduce deleterious effects of standard cardiopulmonary bypass (CPB). This study compares the effects of CCECC (CORx system), CPB, and off-pump coronary artery bypass grafting (OPCAB) on red blood cell damage, coagulation activation, fibrinolysis and cytokine expression.

METHODS

Thirty patients underwent coronary artery bypass grafting (CABG). Twenty of them were randomized into two groups: CCECC (n = <em>1</em>0), CPB (n = <em>1</em>0). While not randomized, OPCAB (n = <em>1</em>0) served as a separate reference group. CCECC and CPB patients received cardioplegic arrest. Interleukin 6 (IL-6), free hemoglobin (fHb), von Willebrand factor activity (vWf), thrombin-antithrombin-III-complex (TATc), <em>prothrombin</em> <em>fragment</em> <em>1</em>.<em>2</em> (F <em>1</em>+<em>2</em>) and plasmin-antiplasmin complex (PAPc) were assessed preoperatively, perioperatively and <em>2</em>4 h postoperatively.

RESULTS

CCECC showed significantly lower red blood cell damage than CPB (fHb: CCECC, 7.<em>1</em>+/- 5.7 micromol/l; CPB, <em>1</em>6.8+/-<em>1</em><em>1</em>.4 micromol/l; P = 0.0<em>2</em>5; OPCAB, 3.4+/-<em>1</em>.<em>1</em> micromol/l). Perioperatively, CCECC exhibited significantly lower activation of coagulation and fibrinolysis than CPB, but did not differ from OPCAB (vWf: CCECC, <em>1</em>33+/-5<em>2</em>%; CPB, <em>2</em>4<em>1</em>+/-<em>1</em><em>2</em>8%; P = 0.05<em>2</em>; OPCAB, <em>1</em>53+/-58%; TATc: CCECC, 4.7+/-0.9 ng/ml; CPB, 3<em>1</em>.<em>1</em>+/-<em>1</em>5.8 ng/ml; P < 0.00<em>1</em>; OPCAB, <em>2</em>.4+/-0.6 ng/ml; PAPc: CCECC, <em>2</em><em>1</em>4+/-30 ng/ml; CPB, 897+/-367 ng/ml; P < 0.00<em>1</em>; OPCAB, <em>2</em>53+/-98 ng/ml). In contrast, fibrinolysis markers and IL-6 were markedly increased in CCECC postoperatively (PAPc: CCECC, 458+/-98 ng/ml; CPB, <em>1</em>59+/-<em>1</em><em>2</em>8 ng/ml; P < 0.00<em>1</em>; OPCAB, <em>2</em>6<em>2</em>+/-<em>1</em>74 ng/ml; IL-6: CCECC, <em>1</em><em>2</em>3.4+/-49.8 pg/dl; CPB, <em>1</em>8.8+/-<em>1</em>3.<em>1</em> pg/dl; P < 0.00<em>1</em>; OPCAB, 3<em>1</em>.6+/-<em>2</em>6.<em>2</em> pg/dl).

CONCLUSIONS

CCECC for CABG is associated with a significant reduction of red blood cell damage and activation of coagulation cascades similar to OPCAB when compared with conventional CPB while a delayed fibrinolytic and inflammatory activity was observed. These findings require further investigation to verify the promising concept of CCECC.

Platelet activation, commonly found in lung cancer patients, may cause the release of angiogenic factors, such as vascular endothelial growth factor (VEGF-A). The present study was designed to investigate whether plasma VEGF-A levels were associated to different stages of non-small cell lung cancer (NSCLC). Moreover, sP-selectin, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>), thrombin-antithrombin III complex (TATc) and D-dimer levels were measured to test the hypothesis of an involvement of platelet and coagulation activation in tumor angiogenesis. VEGF-A, sP-selectin, F<em>1</em>+<em>2</em>, TATc and D-dimer levels were elevated in 65 patients with NSCLC, particularly in metastatic patients. sP-selectin (p <0.003) and F<em>1</em>+<em>2</em> (p <0.005) levels were independently associated to VEGF-A. In addition, patients with positive levels of both sP-selectin and F<em>1</em>+<em>2</em> had the highest levels of VEGF-A. In conclusion, our findings support the hypothesis that thrombin generation might induce platelet activation and VEGF-A release in NSCLC.

BACKGROUND

The aim of this study was to assess whether the quality of FFP produced from whole blood stored at 4 degrees C overnight is adequate for its intended purpose.

METHODS

Fresh-frozen plasma (FFP) separated from whole blood (n = 60) leukodepleted (LD) after storage at 4 degrees C overnight (<em>1</em>8-<em>2</em>4 hr from donation, Day <em>1</em> FFP) was compared with that LD within 8 hours of donation (Day 0 FFP, the current standard method).

RESULTS

In more than 95 percent of Day <em>1</em> FFP units, levels of factor (F) II, FV, FVII, FVIII, F IX, FX, FXI, and FXII were greater than 0.50 U per mL except for von Willebrand factor (VWF) antigen and FVIII, where 9<em>2</em> and 87 percent of units, respectively, contained greater than 0.50 IU per mL. Compared with historical data on FFP stored for 8 hours, fibrinogen, FV, FVIII, and FXI were reduced by <em>1</em><em>2</em>, <em>1</em>5, <em>2</em>3, and 7 percent, respectively, but other factors were not significantly reduced. Levels of VWF-cleaving protease activity were not different between FFP prepared from paired units of blood (n = 3) held for 8 or <em>2</em>4 hours, but were below the reference range in an additional <em>2</em> of 6 units held for <em>2</em>4 hours. The activities of protein S, protein C, antithrombin III, and alpha(<em>2</em>)-antiplasmin were reduced by less than <em>1</em>0 percent in Day <em>1</em> FFP (n = <em>2</em>0), but with final levels above the lower limit of the normal range in greater than 95 percent of units. Activated FXII antigen was not significantly raised in plasma stored for <em>1</em>8 to <em>2</em>4 hours, but levels of <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> were slightly increased (0.88 ng/mL, <em>1</em>8-<em>2</em>4 hr; 0.65 ng/mL, < 8 hr).

CONCLUSIONS

These data suggest that there is good retention of relevant coagulation factor activity in plasma produced from whole blood stored at 4 degrees C for <em>1</em>8 to <em>2</em>4 hours and that this would be an acceptable product for most patients requiring FFP.

The pathogenesis of antiphospholipid antibody (aPL) related thrombosis is multifactorial and includes, amongst others, enhanced coagulation activation measured as <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>), elevated plasma levels of von Willebrand factor (vWF), plasminogen activator inhibitor (PAI) and endothelin-<em>1</em> (ET-<em>1</em>) as well as heightened thromboxane generation and lipid peroxidation. To evaluate the antioxidant susceptibility of some of the above pathways, probucol (500 mg/d orally, a cholesterol lowering agent bearing antioxidant properties) was administered for a three week period to <em>1</em>4 subjects with aPL and to seven healthy controls. At baseline aPL participants showed higher plasma levels of vWF (P = 0.006), ET-<em>1</em> (P = 0.000<em>2</em>) and enhanced urinary excretion of <em>1</em><em>1</em>-dehydro-thromboxane-B<em>2</em> (TXB<em>2</em>) (P = 0.0004), F<em>2</em>-isoprostanes (marker of lipid peroxidation) (P = 0.0<em>2</em>) and albumin (P = 0.04) than controls. In the aPL group baseline IgG anticardiolipin (aCL) titre positively related with urinary TXB<em>2</em> (r<em>2</em> = 0.43, P = 0.0<em>1</em>) and inversely with urinary NOx (r<em>2</em> = -0.6, P = 0.005) whereas urinary NOx and TXB<em>2</em> were negatively correlated (r<em>2</em> = -0.4<em>2</em>, P = 0.0<em>1</em>). After the treatment period significant decreases from baseline values were noted for PAI (P = 0.0<em>1</em>), ET-<em>1</em> (P = 0.006), TXB<em>2</em> (P = 0.0<em>2</em>), F<em>2</em>-isoprostanes (P = 0.0<em>1</em>) and albuminuria (P = 0.0<em>1</em>) in aPL participants but not in controls. These pilot data support oxidative sensitive mechanisms and a potential role for antioxidant treatment in the pathogenesis of aPL induced vasculopathy.

Protein S (PS) is a vitamin K-dependent plasma protein and serves as a cofactor for the anticoagulant activities of activated protein C (APC). We investigated the effects of different PS concentrations on <em>prothrombin</em> activation and thrombin generation in cord and adult plasma containing APC and different amounts of alpha <em>2</em>-macroglobulin (a<em>2</em>-M). <em>Prothrombin</em> activation was assessed by monitoring the time-course of <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>) generation. Thrombin generation curves were determined by means of a subsampling technique using the chromogenic substrate S-<em>2</em><em>2</em>38. We demonstrate a dose-dependent inhibition of the anticoagulant action of PS by a<em>2</em>-M: suppression of F<em>1</em>+<em>2</em> and thrombin generation due to addition of PS was stronger in plasma containing low amounts of a<em>2</em>-M than in plasma with elevated a<em>2</em>-M levels. Since no complex formation between a<em>2</em>-M and PS was observed by means of SDS-PAGE, we attribute decreased anticoagulant action of PS at high a<em>2</em>-M levels to enhanced complex formation between APC and a<em>2</em>-M. Thereby, APC is subtracted from its cofactor PS, resulting in suppressed formation of the anticoagulant APC/PS complex. Thus, our data suggest that a<em>2</em>-M, besides its well-known anticoagulant effects, also acts as a procoagulant by suppressing the formation of the anticoagulant APC/PS complex. Our findings have implications particularly on thrombin generation and inhibition in cord plasma, since a<em>2</em>-M levels in newborns are elevated over adult values and the antithrombotic APC/PS pathway is up-regulated at birth. Therefore, elevated levels of a<em>2</em>-M might restrict the up-regulation of the APC/PS pathway.

The binding of Ca<em>2</em>+ to <em>prothrombin</em> and the intermediates of <em>prothrombin</em> activation was investigated by equilibrium dialysis using 45Ca<em>2</em>+ as the ligand. Scatchard plots of these data indicate that <em>prothrombin</em> (Mr = 70,000) has <em>1</em>0 to <em>1</em><em>1</em> Ca<em>2</em>+ binding sites which can be differentiated in terms of their binding affinity. Six of these Ca<em>2</em>+ binding sites have log Kassoc = 3.5 and all are found intact in the NH<em>2</em>-terminal segment (activation intermediate 3, Mr = <em>2</em>3,000) of the <em>prothrombin</em> molecule. Four or five additional weaker binding sites for Cz<em>2</em>+ with log Kassoc = <em>2</em>.7 present in <em>prothrombin</em> are found intact in the remaining COOH-segment (activation intermediate <em>1</em>, Mr = 5<em>1</em>,000) of the <em>prothrombin</em> molecule. Upon further activation the Ca<em>2</em>+ binding sites residing in intermediate <em>1</em> are found intact in activation intermediate 4 (which constitutes the NH<em>2</em>-terminal segment of the intermediate <em>1</em> molecule). The remaining COOH-terminal portion (activation intermediate <em>2</em>, Mr = 4<em>1</em>,000) of the intermediate <em>1</em> molecule has no affinity for Ca<em>2</em>+. The activation of <em>prothrombin</em> and activation intermediates <em>1</em> and <em>2</em> was studied using these activators: Factor Xa alone, Factor Xa-Ca+, AND Factor Xa-Ca<em>2</em>+-phospholipid. The rate of thrombin production from <em>prothrombin</em> was progressively increased as Ca<em>2</em>+ and phospholipid were added to the system, whereas no significant increase in the rates of activation of intermediate <em>1</em> and <em>2</em> was observed. When Factor V was added to the Factor Xa-Ca<em>2</em>+-phospholipid system, the rate of activation of intermediate <em>1</em> was greatly enhanced. In the absence of Ca<em>2</em>+, Factor V had no effect on the rate of thrombin formation from intermediate <em>1</em>. Factor V had no stimulatory effects on the rate of intermediate <em>2</em> activation. However, in the presence of an equimolar amount of intermediate 4, Factor V accelerated the conversion of intermediate <em>2</em> to thrombin. These studies indicate that the Ca<em>2</em>+ binding sites of the <em>prothrombin</em> molecule are contained in the "pro" <em>fragment</em> (intermediates 3 and 4) of the <em>prothrombin</em> molecule. Intermediate <em>1</em> and intermediate <em>2</em>, both of which lack the strong Ca<em>2</em>+ binding sites of <em>prothrombin</em>, are poor substrates for the Factor Xa-Ca<em>2</em>+-phospholipid complex activation when compared to <em>prothrombin</em>. The addition of Factor V to the catalyst results in acceleration of the activation rate of intermediate <em>1</em> and an equimolar mixture if intermediates <em>2</em> and 4. These results lead us to conclude that the strong Ca<em>2</em>+ binding sites are the sites of phospholipid binding (intermediate 3), whereas the seak binding sites are the sites of Factor V binding (intermediate 4).

The optimal management of asymptomatic overanticoagulated patients remains unknown. We measured international normalized ratio (INR), activated partial thromboplastin time (APTT) and <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>) over 7 d in <em>2</em>4 asymptomatic or mildly haemorrhagic patients on warfarin with prolonged INR of>> 7.0 who were randomized to receive 0.5 mg, <em>1</em> mg or <em>2</em> mg intravenous vitamin K. Of six severely overanticoagulated patients (INR>> 9.5 with APTT ratio>> <em>2</em>), five failed to achieve an INR < or = 4.0 on day <em>1</em>, irrespective of vitamin K dose given. In the remaining <em>1</em>8 cases, an optimal response (INR <em>2</em>-4 at day <em>1</em>) was observed in 67% of those receiving 0.5 mg vitamin K, but only in 33% of those receiving <em>1</em> or <em>2</em> mg, the majority of whom developed an INR < <em>2</em>.0. Our results support an optimal dose of 0.5 mg i.v. vitamin K for most overanticoagulated patients, with possibly a repeat dose in the small group of severely overanticoagulated patients.

In this open label, randomized study we compared the influence of a dose-reduced oral contraceptive containing <em>2</em>0 microg ethinyl estradiol (EE) and <em>1</em>00 microg levonorgestrel (<em>2</em>0 EE) with a reference preparation containing 30 microg EE and <em>1</em>50 microg levonorgestrel (30 EE) on hemostatic, lipids, and carbohydrate metabolism variables. Data from 48 volunteers were obtained. The direction of the change (increase or decrease) in most of the hemostatic variables were similar in both treatment groups. In particular, <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> increased during treatment, reaching a median percent change of 40% in the <em>2</em>0 EE group and of <em>1</em>7% in the 30 EE group after one year. D-Dimer fibrin split products remained virtually unchanged, with no change at Cycle <em>1</em>3. The median HDL<em>2</em> cholesterol levels decreased by <em>2</em>6% in the <em>2</em>0 EE group and by 39.8% in 30 EE group (p = 0.0045 for group difference) after one year. The median one year change for LDL cholesterol was 3.<em>2</em>3% in the <em>2</em>0 EE group, compared to <em>2</em>5% in the 30 EE group, for VLDL <em>1</em><em>1</em>.<em>1</em>% compared to 38.8%, respectively, and for total triglycerides <em>1</em>0.0% compared to 37.5%, respectively. The median absolute change for the area under the curve (AUC)(0-3h) for glucose at treatment Cycle <em>1</em>3 was 4<em>1</em>.<em>2</em>5 mmol/L x min in the <em>2</em>0 EE group and 73.50 mmol/L x min in the 30 EE group. The AUC(0-3h) insulin at treatment Cycle <em>1</em>3 decreased in the <em>2</em>0 EE group by <em>1</em>635.0 pmolL x min and increased in the 30 EE group by <em>1</em><em>1</em>797.5 pmolL x min (p = 0.049<em>1</em> for group difference). Both study treatments were safe and well tolerated by the volunteers. In conclusion, the balanced one-third dose reduction in this new oral contraceptive evoked similar effects on the hemostatic variables, but favorable results for the lipid and carbohydrate profiles.

OBJECTIVE

In chronic renal failure in dialyzed patients vascular damage is frequently observed and it is probable that disturbances in fibrinolytic activity and endothelial dysfunction may play a role in vascular complications such as stroke or ischemic heart disease. There have been a few data concerning hemostasis in chronic renal failure. Since hemostatic disturbances in nephrotic syndrome mimick those observed in patients maintained on chronic ambulatory peritoneal dialyses (CAPD), the aim of the study was to assess adhesion molecules (P-selectin, E-selectin, ICAM, VCAM and markers of endothelial cell injury), von Willebrand factor, thrombomodulin, and TFPI (tissue factor pathway inhibitor) in CAPD patients as well as in subjects with chronic renal failure (CRF) treated conservatively.

METHODS

The studies were performed on <em>2</em>3 CAPD patients, <em>2</em>4 patients with nephrotic syndrome and <em>2</em>4 sex- and age-matched healthy volunteers. TFPI total, full length, truncated, von Willebrand factor, trombomodulin, P-selectin, E-selectin, ICAM, VCAM and vascular endothelial growth factor (VEGF) and its receptor sFlt3 were assayed using commercially available kits. We evaluated also thrombin activity (thrombin-antithrombin complexes (TAT), <em>prothrombin</em> <em>fragments</em> <em>1</em> and <em>2</em>) and the degree of plasmin generation.

RESULTS

In CAPD and CRF patients, concentrations of the adhesion molecules P-selectin, E-selectin, ICAM and VCAM were significantly higher when compared to the control group. Concentrations of total, free and truncated TFPI were significantly higher in CAPD and CRF patients when compared to the healthy volunteers. Concentrations of 'classical' markers of endothelial cell injury, von Willebrand factor and thrombomodulin, were significantly higher in CAPD and CRF patients when compared to the control group. In CAPD patients, VCAM and thrombomodulin were significantly elevated when compared to the CRF patients.

CONCLUSIONS

The elevated levels of adhesion molecules in CAPD patients probably reflect inadequate clearance as well as enhanced synthesis/release. They may also indicate endothelial cell injury as well as elevated levels of von Willebrand factor and trombomodulin and increased ICAM and VCAM in CAPD patients. Our studies indicate that in renal failure patients, particularly on CAPD, there is evidence of endothelial cell injury and a high degree of hypercoagulation relative to healthy subjects. It may lead to fibrin deposition in the vascular wall, thrombus formation, and development and progression of atherosclerosis with its complications.

There is a current controversy over the hypothesis that a number of thromboembolic events could be related to hypercoagulable state in patients with chronic Chagas disease. This study was designed to determine whether a prothrombotic state existed in chronic Trypanosoma cruzi-infected patients and, if so, to describe its evolution after treatment with Benznidazole. Twenty-five patients with chronic Chagas disease and <em>1</em>8 controls were evaluated. The markers used were <em>prothrombin</em> time, activated partial thromboplastin time, fibrinogen, antithrombin, plasminogen, protein C, total protein S, free protein S, factor VIII, D-dimer, activated factor VIIa, tissue-type plasminogen activator inhibitor-<em>1</em>, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F₁+₂), plasmin-antiplasmin complexes, soluble P-selectin and endogenous thrombin potential (ETP). Despite statistically significant differences between cases and controls in several markers, only ETP (which quantifies the ability of plasma to generate thrombin when activated through tissue factor addition) (p<0.000<em>1</em>) and F₁+₂ (a marker of thrombin generation in vivo) (p<0.000<em>1</em>) showed values outside the normal levels in patients compared with controls. Similar results were obtained in these markers six months after treatment in the cohort of cases (p<0.0008 and p<0.004, respectively). These results may be relevant in clinical practice. Though current treatment for Chagas disease is still controversial, if it were considered as a thromboembolic risk factor the antiparasitic treatment strategy could be reinforced. The results also support further research on haemostasis parameters as candidates for early surrogate biomarkers of cure or progression of Chagas disease.