Septic shock and multiple organ failure may be associated with coagulation activation, disseminated fibrin formation, and consumption of coagulation inhibitors such as antithrombin III. We have evaluated prospectively coagulation measurements in patients with severe chemotherapy-induced neutropenia. This group of patients was chosen because of their high risk of developing severe septic complications, thus allowing serial prospective coagulation testing before and during evolving sepsis or septic shock. Sixty-two patients with febrile infectious events were accrued to the study. Of these, <em>1</em>3 patients progressed to severe sepsis and <em>1</em>3 additional patients to septic shock as defined according to standard diagnostic criteria. At the onset of fever, factor (F) VIIa activity, FVII antigen and antithrombin III (AT III) activity decreased from normal baseline levels and were significantly lower in the group of patients who progressed to septic shock compared with those that developed severe sepsis (medians: 0.3 v <em>1</em>.4 ng/mL, <em>2</em><em>1</em> v 86 U/dL and 45% v 95%; P < .00<em>1</em>). The decrease of these measurements in septic shock was accompanied by an increase in <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (median: 3.6 v <em>1</em>.4 nmol/L; P = .05), a marker of thrombin generation. These differences were sustained throughout the septic episode (P < .000<em>1</em>). FVIIa and AT III levels of < 0.8 ng/mL and < 70%, respectively, at onset of fever predicted a lethal outcome with a sensitivity of <em>1</em>00% and 85%, and a specificity of 75% and 85%, respectively. In contrast, FXIIa-alpha antigen levels were not different between groups at onset of fever but increased modestly during the course of septic shock (P = .00<em>1</em>). Thus, septic shock in neutropenic patients is associated with increased thrombin generation. Furthermore, both FVIIa and AT III measurements are sensitive markers of an unfavorable prognosis.

Haemostatic changes in <em>1</em>6 patients with Crohn's disease were studied from active disease into clinical remission and beyond. Elevated concentrations of fibrinopeptide A (FpA) and <em>prothrombin</em> <em>fragments</em> F<em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>) were found at times of both active (FpA median 3.<em>2</em>, range [0.3-40] ng/ml and F<em>1</em> + <em>2</em> median <em>2</em>.3, range [0.3-<em>1</em>8] nm/l) and inactive disease (FpA median <em>2</em>, range [0.4-40] ng/ml and F<em>1</em> + <em>2</em> median <em>1</em>.3, range [0.<em>2</em>-<em>2</em>0) nm/l]. We also measured the physiological inhibitors of coagulation and fibrinolysis; there was no significant difference in the levels of antithrombin III, protein C or the Exner ratio between active and inactive disease. Free protein S levels were significantly lower in active disease (median 34, range 9-54 U/dl) than in remission (median 40, range <em>1</em><em>2</em>-65 U/dl). Plasminogen activator inhibitor type <em>1</em> (PAI-<em>1</em>) was significantly raised in remission (median <em>1</em><em>1</em>, range 3-3<em>2</em> ng/ml) when compared to active disease (median 7, range 3-4<em>2</em> ng/ml). The D-dimer correlated significantly with fibrinopeptide A (P < 0.00<em>1</em>), suggesting reactive fibrinolysis in some patients. Most (35/5<em>2</em>, 67%) samples showed evidence of persistent haemostatic activation (elevated FpA and/or F<em>1</em> + <em>2</em>) during phases of apparent clinical remission in Crohn's disease, a factor that is not reflected by clinical activity scores. This study supports the hypothesis that coagulation is activated in the mesenteric vasculature of patients with Crohn's disease.

OBJECTIVE

This study was designed to determine whether novel cardiovascular disease (CVD) risk factors explain the high prevalence of peripheral arterial disease (PAD) among African Americans.

BACKGROUND

Compared with Caucasians, African Americans have higher prevalence of PAD, an association that is not explained by traditional CVD risk factors. The degree to which novel CVD risk markers may explain the higher prevalence is uncertain.

METHODS

A nested case-control study within the San Diego Population Study was performed. The study evaluated <em>1</em>04 persons with PAD and <em>1</em>64 age- and gender-matched control subjects who were employees of a large public university and participated in a peripheral artery and venous disease study. Nine novel CVD risk factors (homocysteine, lipoprotein (a), C-reactive protein, fibrinogen, tumor necrosis factor-alpha, von Willebrand factor, <em>prothrombin</em> <em>fragment</em> <em>1</em>-<em>2</em>, D-dimer, and plasmin antiplasmin) were measured. Multivariable logistic regression evaluated whether these novel factors attenuated the association of African-American race and PAD and whether there was differential ethnic susceptibility to the novel factors.

RESULTS

African Americans had 3-fold higher odds of PAD in age- and gender-matched models (odds ratio [OR] 3.<em>1</em>; 95% confidence interval [CI] <em>1</em>.5 to 6.4; p < 0.0<em>1</em>), an association that was modestly attenuated by adjustment for traditional (OR <em>2</em>.4; 95% CI 0.9 to 6.<em>1</em>; p = 0.06) and novel CVD risk markers (OR <em>1</em>.9; 95% CI 0.7 to 4.7; p = 0.<em>1</em>8). Among the novel factors, the attenuation was primarily due to fibrinogen and lipoprotein (a). No interactions by novel CVD risk markers (both p values for interaction>> or =0.<em>2</em>4) were observed.

CONCLUSIONS

Traditional and novel CVD risk factors only partially explain the higher prevalence of PAD among African Americans.

OBJECTIVE

Laparoscopic gastric bypass (GBP) induces a postoperative hypercoagulable state that is similar or reduced compared with open GBP.

METHODS

University hospital.

METHODS

Between May <em>1</em>999 and June 2000, 70 patients were randomly assigned to laparoscopic (n = 36) or open (n = 34) GBP. Deep venous thrombosis (DVT) prophylaxis consisted of antiembolism stockings and sequential pneumatic compression devices.

METHODS

Plasminogen, thrombin-antithrombin complex (TAT), <em>prothrombin</em> <em>fragment</em> <em>1</em>.2 (F<em>1</em>.2), fibrinogen, D-dimer, antithrombin III (AT), and protein C levels were measured at baseline and at <em>1</em>, 24, 48, and 72 hours postoperatively. A venous duplex examination of both lower extremities was performed preoperatively and between the third and fifth day postoperatively.

RESULTS

The 2 groups were similar in age, weight, and body mass index. Plasminogen levels decreased, and TAT, F<em>1</em>.2, and fibrinogen levels increased after laparoscopic and open GBP. There was no significant difference in these levels between groups. D-dimer levels increased in both groups, but the levels were significantly higher after open GBP than after laparoscopic GBP (P<.0<em>1</em>). Antithrombin III and protein C levels decreased in both groups. The reduction of AT (at <em>1</em> hour) and protein C (at 72 hours) was significantly less after laparoscopic GBP than after open GBP (P<.05). Postoperative venous duplex examination revealed DVT in <em>1</em> (2.9%) of 34 patients after open GBP but in none of 36 patients after laparoscopic GBP. One patient developed pulmonary embolism after open GBP.

CONCLUSIONS

Laparoscopic GBP induces a hypercoagulable state similar to that of open GBP. Our findings suggest that DVT prophylaxis should be used during laparoscopic GBP as in open GBP.

BACKGROUND

It has long been known that a hypercoagulability state develops after surgery. A surge in circulating cytokine levels is also commonly found in the postoperative period. These cytokines have all been shown to be capable of inducing a hypercoagulability state. Recently laparoscopic cholecystectomy (LC) has been introduced, and its advantages over the open procedure seem related to the reduced surgical trauma. LC is associated with a diminished acute-phase response compared with the open procedure. Our present knowledge on the influence of laparoscopic upon coagulation and fibrinolysis is incomplete and based on a few studies.

METHODS

The aim of this prospective, nonrandomized study was to investigate hemostatic system alterations in patients who undergo open and laparoscopic cholecystectomy. In addition we also measured the plasma cytokine profile to explore any relationship between changes in plasma cytokine levels and postoperative coagulation profile. Between September <em>1</em>999 and April <em>2</em>00<em>2</em>, 7<em>1</em> patients were nonrandomly assigned to open (group <em>1</em>) or laparoscopic cholecystectomy (group <em>2</em>). All patients from group <em>1</em> were operated by a surgical team different from ours, who prefers the OC procedure. The patients with acute cholecystitis were excluded. <em>Prothrombin</em> <em>fragment</em> <em>1</em>.<em>2</em> (F<em>1</em>.<em>2</em>), thrombin-antithrombin (TAT), fibrinogen, soluble fibrin, antithrombin III (AT), protein C, plasminogen, and D-dimer levels were measured at baseline and at <em>1</em>, <em>2</em>4, 48, and 7<em>2</em> h postoperatively. Serial serum levels of IL-<em>1</em>beta and IL-6 were measured by colorimetric enzyme-linked immunosorbent assay (ELISA).

RESULTS

Plasma levels of F<em>1</em>.<em>2</em>, TAT, fibrinogen, soluble fibrin, and D-dimer increased significantly in group <em>1</em>. Plasma levels of AT, protein C, and plasminogen decreased in both groups. In the OC group, the serum IL-3 and IL-6 levels began to significantly increased as early as <em>1</em> h from the beginning of the operation, revealing a peak at the sixth hour. When IL-6 and IL-<em>1</em> levels were markedly elevated also, F<em>1</em>.<em>2</em>, fibrinogen, and soluble fibrin levels were increased.

CONCLUSIONS

Only mild hypercoagulability was observed in patients who had undergone laparoscopic cholecystectomy. The cytokine surge was correlated with hypercoagulability. There was in fact a positive correlation between IL-6 level and hypercoagulability. The correlation between cytokine levels and coagulation activation may be related to the type of surgery performed. Further studies are required to investigate these issues.

To determine the role of plasma tissue factor on disseminated intravascular coagulation (DIC) in trauma and septic patients, and also to investigate the relationships between tissue factor and various thrombin markers, we made a prospective cohort study. Forty trauma patients and <em>2</em>0 patients with sepsis were classified into subgroups according to the complication of DIC. Plasma tissue factor antigen concentration (tissue factor), <em>prothrombin</em> <em>fragment</em> F<em>1</em>+<em>2</em> (PF<em>1</em>+<em>2</em>), thrombin antithrombin complex (TAT), fibrinopeptide A (FPA), and D-dimer were measured on the day of admission (day 0), and on days <em>1</em>, <em>2</em>, 3, and 4 after admission. The levels of plasma tissue factor in the DIC group were more elevated than those of the non-DIC group in both the trauma and the septic patients. In patients with sepsis, tissue factor levels on days 0 through 4 in the non-DIC group showed markedly higher values than those in the control patients (<em>1</em>35 +/- 8 pg/ml). Significant correlations between tissue factor and PF<em>1</em>+<em>2</em>, TAT, FPA, and D-dimer were observed in the DIC patients, however, no such correlations were found in the non-DIC patients. These results suggest that elevated plasma tissue factor in patients with trauma and sepsis gives rise to thrombin generation, followed by intravascular coagulation.

Diabetes is characterized by the existence of a thrombosis-prone condition, possibly related to hyperglycemia. However, the mechanism linking hyperglycemia to the activation of the coagulation cascade is still unclear. It has been recently suggested that diabetes is accompanied by increased oxidative stress. In this work, the possibility that oxidative stress may be involved in the hyperglycemia-induced coagulation activation has been evaluated. <em>Prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>), which represents a reliable marker of the amount of thrombin released in the circulation, has been chosen for studying thrombin formation in vivo. In nine type II diabetic patients and in seven healthy control subjects, matched for age and body mass index, three different experiments were performed: oral glucose tolerance test (OGTT), intravenous antioxidant glutathione (GSH) administration for <em>2</em> h, and OGTT plus intravenous GSH administration. Samples were drawn at -<em>1</em>5 min and every 30 min from 0 to <em>1</em>80 min. During the OGTT, F<em>1</em> + <em>2</em> significantly increased in both diabetic and healthy subjects. GSH administration during OGTT normalized this phenomenon. GSH administration alone significantly decreased F<em>1</em> + <em>2</em> in diabetic patients, while no effect was observed in the normal subjects. These data suggest that hyperglycemia may induce thrombin activation, possibly inducing an oxidative stress, and that antioxidant GSH may counterbalance this effect.

In acute myocardial infarction (AMI), increased Tissue Factor (TF) expression on circulating monocytes and microparticles (MP) may contribute to thrombotic events. Because surfacebound Tissue Factor Pathway Inhibitor-<em>1</em> (TFPI) inhibits TF activity on monocytes and endothelial cells decreased TFPI expression may reinforce the procoagulant activity of circulating MP. Aim of the study was to analyze TFPI expression and TF activity after stenting and thrombolysis inAMI. Thirty-nine patients of a randomized study comparing intravenous thrombolysis (n=<em>1</em>9) and stenting (n=<em>2</em>0) were included. Before and after therapy blood samples for analysis of MPs, TF antigen and activity, <em>prothrombin</em> <em>fragment</em> F<em>1</em>+<em>2</em> and D-dimer were obtained. TFPI expression on TF positive MPs was decreased after thrombolysis but not after stenting. In contrast, TF plasma levels and TF positive MP remained unchanged in both treatment groups. After thrombolysis increased D-dimer and F<em>1</em>+<em>2</em> plasma concentrations indicated activation of fibrinolysis and coagulation. Significance of MPTFPI for inhibition of TF activity was measured using inhibitory TFPI antibodies. Membrane-associated TFPI inhibited TF activity on circulating MPs. After thrombolysis inhibition of TF activity by TFPI was decreased as compared to stenting. Correlation of circulating TF with F<em>1</em>+<em>2</em> only after thrombolysis, suggests a role for TF-induced activation of coagulation after thrombolysis. Enhanced TF activity on circulating MPs in AMI is inhibited by endogenous surface-boundTFPI. After thrombolysis but not after stenting MPTFPI is degraded and may induce thrombin generation due to unopposed tissue factor activity. Anti-TF therapies during thrombolysis may reduce thrombin generation in AMI.

BACKGROUND

Prophylaxis with either intravenous (i.v.) factor VIII (FVIII) or FIX is the gold standard of care for patients with severe hemophilia. A monoclonal antibody (concizumab) targeting tissue factor pathway inhibitor (TFPI) that can be administered subcutaneously (s.c.) has the potential to alter current concepts of prophylaxis in hemophilia.

OBJECTIVE

To evaluate the safety and describe the pharmacokinetics and pharmacodynamics of single-dose concizumab in healthy volunteers and patients with hemophilia A or B.

METHODS

In this first human dose, phase <em>1</em>, multicenter, randomized, double-blind, placebo-controlled trial escalating single i.v. (0.5-9000 μg kg(-<em>1</em>) ) or s.c. (50-3000 μg kg(-<em>1</em>) ) doses of concizumab were administered to healthy volunteers (n = <em>2</em>8) and hemophilia patients (n = <em>2</em>4).

RESULTS

Concizumab had a favorable safety profile after single i.v. or s.c. administration. There were no serious adverse events and no anti-concizumab antibodies. No clinically relevant changes in platelets, <em>prothrombin</em> time, activated partial thromboplastin time, fibrinogen, or antithrombin were found. A dose-dependent procoagulant effect of concizumab was seen as increased levels of D-dimers and <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>. Nonlinear pharmacokinetics of concizumab was observed due to target-mediated clearance. A maximum mean AUC0-∞ of 33 960 h μg mL(-<em>1</em>) and a maximum mean concentration of <em>2</em>47 μg mL(-<em>1</em>) was measured at the highest dose.

CONCLUSIONS

Concizumab showed a favorable safety profile after i.v. or s.c. administration and nonlinear pharmacokinetics was observed due to target-mediated clearance. A concentration-dependent procoagulant effect of concizumab was observed, supporting further study into the potential use of s.c. concizumab for hemophilia treatment.

Effects of the quality and the time of venepuncture on factor VII coagulant activity (VIIc) and the concentrations of fibrinogen, <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>) and fibrinopeptide A (FPA) were sought in <em>2</em>665 men, of whom <em>2</em>334 were re-examined after about one year. Venepunctures were categorised as satisfactory, not fully satisfactory or unsatisfactory according to pre-defined criteria. Neither the quality nor timing of the venepuncture influenced VIIc or fibrinogen concentration. However, at baseline and re-examination F<em>1</em> + <em>2</em> and FPA were increased on average by about 9% and 45% respectively when venepunctures were not fully satisfactory, and by about <em>1</em><em>1</em>% and <em>1</em>00% when unsatisfactory. Plasma collected after <em>1</em>500 h had slightly but significantly lower levels of F<em>1</em> + <em>2</em> and FPA than samples taken earlier, possibly due to circadian rhythm. The results emphasise the need for careful surveillance of the venepuncture procedure and the value of FPA when using F<em>1</em> + <em>2</em> as a marker of risk of thrombosis.

BACKGROUND

Nephropathia epidemica (NE) is a viral hemorrhagic fever with renal syndrome associated with thrombocytopenia and mild bleeding. We assessed activation of coagulation and fibrinolysis during the acute phase of NE.

METHODS

<em>1</em>9 hospital-treated patients were involved. Plasma levels of D-dimer, <em>prothrombin</em> <em>fragments</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>), activated partial thromboplastin time (APTT), <em>prothrombin</em> time (PT%), thrombin time (TT), fibrinogen, antithrombin (AT), protein S free antigen (PS), protein C (PC) and complete blood count (CBC) were measured three times during the acute phase and once at 3<em>2</em>-54 days after the onset of fever (recovery phase). Laboratory abnormalities were evaluated by the disseminated intravascular coagulation (DIC) scoring advocated by the International Society of Thrombosis and Haemostasis (ISTH).

RESULTS

APTT was prolonged and D-dimer and F<em>1</em>+<em>2</em> increased during the acute phase of NE. AT, PC and PS decreased, and TT was shortened, all implying increased thrombin generation. Acutely F<em>1</em>+<em>2</em> was 3.4-fold and D-dimer even <em>2</em>4-fold higher compared with the recovery phase (median 7<em>2</em>6 vs <em>2</em><em>1</em>3 pmol/l, and median 4.8 vs 0.<em>2</em>mg/l, respectively, p<0.00<em>1</em> for both). Platelet count correlated with AT, PC, and PS (r=0.73, r=0.8<em>1</em>, and r=0.7<em>1</em>, respectively, p<0.00<em>1</em> for all) as well as with fibrinogen (r=0.7<em>2</em>, p<0.00<em>1</em>). Only five patients fulfilled the ISTH diagnosis of DIC.

CONCLUSIONS

During acute NE thrombocytopenia was associated with decreased natural anticoagulants, shortened thrombin time and enhanced fibrinolysis. Augmented thrombin formation and fibrinolysis characterize this hantavirus infection.

BACKGROUND

Exercise may activate platelets and leukocytes and promote thrombosis. The effects of aspirin treatment on the prothrombotic effects of exercise have not been established.

RESULTS

A total of <em>1</em>5 healthy men performed exhaustive exercise without and with <em>1</em> week of pretreatment with aspirin (500 mg/day). Before and immediately after exercise, platelet aggregability ex vivo was measured by filtragometry, and venous blood samples were obtained. Whole-blood flow cytometry was used to determine platelet and leukocyte activation and platelet-leukocyte aggregates. Exercise increased platelet P-selectin expression, CD<em>1</em><em>1</em>b expression in neutrophils and lymphocytes, and platelet and leukocyte responses to thrombin, ADP, platelet activating factor, and N-formyl-methionyl-leucyl-phenylalanine (fMLP) in vitro. Consistent with enhanced platelet and leukocyte activation, more circulating platelet-platelet and platelet-leukocyte aggregates were detected after exercise (P<0.00<em>1</em> for both). Filtragometry readings were shortened, and plasma soluble P-selectin and <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> were elevated. Aspirin markedly reduced the urinary excretion of <em>1</em><em>1</em>-dehydrothromboxane B(<em>2</em>), decreased P-selectin expression in single platelets at rest (P<0.05), and inhibited fMLP-induced neutrophil CD<em>1</em><em>1</em>b expression, but it did not attenuate exercise-induced increases in platelet aggregability, platelet P-selectin expression, leukocyte CD<em>1</em><em>1</em>b expression, platelet-leukocyte aggregate formation, soluble P-selectin, or <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>.

CONCLUSIONS

Exercise induced platelet and leukocyte activation and platelet-leukocyte aggregation in vivo, and it increased platelet and leukocyte responsiveness to in vitro stimulation. Aspirin treatment attenuated certain signs of platelet activity in vivo at rest and fMLP-induced neutrophil activation in vitro, but it did not attenuate the prothrombotic effects of exercise.

BACKGROUND

The conversion of <em>prothrombin</em> to thrombin by factor Xa is the penultimate step in the blood clotting cascade. In vivo, where the conversion occurs primarily on activated platelets in association with factor Va and Ca<em>2</em>+ ions, meizothrombin is the major intermediate of the two step reaction. Meizothrombin rapidly loses the <em>fragment</em> <em>1</em> domain (F<em>1</em>) by autolysis to become meizothrombin des F<em>1</em> (mzTBN-F<em>1</em>). The physiological properties of mzTBN-F<em>1</em> differ dramatically from those of thrombin due to the presence of <em>prothrombin</em> <em>fragment</em> <em>2</em> (F<em>2</em>), which remains covalently attached to the activated thrombin domain in mzTBN-F<em>1</em>.

RESULTS

The crystal structure of mzTBN-F<em>1</em> has been determined at 3.<em>1</em> A resolution by molecular replacement, using only the thrombin domain, and refined to R and Rfree values of 0.<em>2</em>05 and 0.<em>2</em>4<em>2</em>, respectively. The protease active site was inhibited with D-Phe-Pro-Arg-chloromethylketone (PPACK) to reduce autolysis. The mobile linker chain connecting the so-called kringle and thrombin domains and the first two N-acetylglucosamine residues attached to the latter were seen in electron-density maps improved with the program SQUASH. Previously these regions had only been modeled.

CONCLUSIONS

The F<em>2</em> kringle domain in mzTBN-F<em>1</em> is bound to the electropositive heparin-binding site on thrombin in an orientation that is systematically shifted and has significantly more interdomain contacts compared to a noncovalent complex of free F<em>2</em> and free thrombin. F<em>2</em> in mzTBN-F<em>1</em> forms novel hydrogen bonds to the carbohydrate chain of thrombin and perhaps stabilizes a unique, rigid conformation of the gamma-autolysis loop through non-local effects. The F<em>2</em> linker chain, which does not interfere with the active site or fibrinogen-recognition site, is arranged so that the two sites cleaved by factor Xa are separated by 36 A. The two mzTBN-F<em>1</em> molecules in the asymmetric unit share a tight 'dimer' contact in which the active site of one molecule is partially blocked by the F<em>2</em> kringle domain of its partner. This interaction suggests a new model for <em>prothrombin</em> organization.

BACKGROUND

This study sought to assess whether novel markers of hemostatic activity are predictive of coronary heart disease (CHD) and improve risk assessment.

RESULTS

Conventional CHD risk factors, the activation peptides of factor IX and factor X, factor VII activity and antigen, activated factor XII, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, fibrinopeptide A, and fibrinogen were measured in <em>1</em><em>1</em>53 men aged 50 to 6<em>1</em> years who were free of myocardial infarction at recruitment. Activated factor VII (VIIa) was measured in 8<em>2</em>9 men. During 7.8 years of follow-up, <em>1</em>04 had a CHD event. Baseline status was related to outcome by logistic regression by using a modified nested case-control design. Screening performance was judged from receiver operating characteristic curves. A high activated factor XII was associated with increased CHD risk, but low levels were not protective. Plasma VIIa and factor X activation peptide were independently and inversely related to risk. Plasma factor IX activation peptide and fibrinogen were positively associated with risk, but the relations were no longer statistically significant after adjustment for other factors, including VIIa and apoA-I. Other hemostatic markers were not associated with CHD risk.

CONCLUSIONS

Hemostatic status did not add significant predictive power to that provided by conventional CHD risk factors yet was able to substitute effectively for these factors.

BACKGROUND

The incidence of deep vein thrombosis and pulmonary embolism following laparoscopic surgery is unknown and studies on alterations of hemostasis after laparoscopy are inconclusive.

METHODS

In this study we prospectively evaluated changes in <em>prothrombin</em> time (PT), activated partial thromboplastin time (aPTT), fibrinogen (Fg), antithrombin III (ATIII), <em>prothrombin</em> <em>fragment</em> F <em>1</em> + <em>2</em>, beta-thromboglobulin (betaTG) and D-dimer (D-D), preoperatively and <em>2</em>4 h after laparoscopic surgery in <em>1</em>6 patients.

RESULTS

Comparing pre- and postoperative values, no statistical differences were observed in aPTT, F<em>1</em> + <em>2</em>, and ATIII measurements. Postoperative PT values increased slightly (p approximately 0.05) after surgery. Conversely, Fg, betaTG, and D-D values were statistically higher in the <em>2</em>4-h evaluation (p = 0.008, 0.0<em>1</em>, and 0.045, respectively).

CONCLUSIONS

These data suggest that laparoscopic surgery induces activation of coagulation and fibrinolytic pathways and, additionaly, betaTG elevation, which has never been reported and might account for postoperative platelet activation and a greater risk of thrombogenicity. Therefore, routine thromboembolic prophylaxis in patients undergoing laparoscopic surgery is recommended.

OBJECTIVE

Tumor-induced platelet activation may cause the release of various cytokines, including CD40 ligand (CD40L). Activation of the CD40/CD40L pathway in human tumors may result in thrombin generation, which is known to be involved in angiogenesis. Thus, we investigated whether soluble (s)CD40L levels are increased in patients with lung cancer as a result of platelet and/or coagulation activation.

METHODS

Citrated plasma samples were obtained from <em>1</em><em>2</em>0 patients with different stages and histotypes of lung cancer and 60 age- and sex-matched control subjects. sCD40L, sP-selectin (marker of platelet activation), <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>, and thrombin-antithrombin III complex levels (both markers of coagulative activation) were measured in all samples.

RESULTS

Patients with lung cancer had median sCD40L levels higher than in control subjects (0.46 versus 0.<em>1</em>3 ng/ml; P < 0.000<em>1</em>), although correlation with the stage of disease was not evident. Nonetheless, sCD40L levels were significantly higher in squamous cancer compared with adenocarcinoma (0.75 versus 0.<em>2</em>7 ng/ml; P < 0.05). Moreover, median sCD40L levels were higher in stage IV compared with nonmetastatic squamous lung cancer (<em>1</em>.0<em>2</em> versus 0.6<em>1</em> ng/ml; P < 0.05). sCD40L levels significantly correlated with sP-selectin (P < 0.00<em>1</em>), <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (P < 0.00<em>1</em>), or thrombin-antithrombin III complex (P < 0.05) in squamous lung cancer, but only sP-selectin (P = 0.0<em>1</em><em>1</em>) was independently related to sCD40L.

CONCLUSIONS

These findings indicate that elevated sCD40L levels can be preferentially found in patients with advanced squamous cancer and provide evidence that increased levels of this cytokine are associated to the occurrence of in vivo platelet activation.

OBJECTIVE

To determine the involvement of coagulation in bleeding and poor outcome in patients with severe leptospirosis.

METHODS

In a prospective study, parameters of the coagulation system were measured on admission and during follow-up in 5<em>2</em> consecutive patients with severe leptospirosis.

RESULTS

All patients showed coagulation disorders, such as prolonged <em>prothrombin</em> time (PT) and activated partial thromboplastin time, marked procoagulant activity [thrombin-antithrombin (TAT) complexes, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, D-dimer], reduced levels of anticoagulant markers (protein C, antithrombin) and increased (anti-) fibrinolytic activity [plasmin-antiplasmin (PAP) complexes, plasminogen activator inhibitor-<em>1</em>]. These disorders were more pronounced in patients who died eventually. PT prolongation was associated with mortality (OR <em>1</em>.4, 95% CI: <em>1</em>.0-<em>1</em>.8, P = 0.04). Bleeding occurred in 3<em>1</em> subjects (60%). Of these, <em>2</em>4 had mild bleeding and seven had severe haemorrhages. Thrombocytopenia (platelets </=<em>1</em>00 x <em>1</em>0(9)/l) was significantly associated with clinical bleeding (OR 4.6, 95% CI: <em>1</em>.3-<em>1</em>6). A subanalysis of patients with and without severe bleeding revealed a more pronounced imbalance of the coagulation system in patients with severe bleeding, as reflected by a significant association with PT (OR <em>1</em>.4, 95% CI: <em>1</em>.0-<em>1</em>.8, P = 0.05) and the TAT/PAP ratio (OR <em>1</em>.3, 95% CI: <em>1</em>.0-<em>1</em>.6, P = 0.05), which is an indicator of the balance between coagulation and fibrinolysis. Overt disseminated intravascular coagulation (DIC) was found in <em>1</em>0 (<em>2</em><em>2</em>%) of the 46 patients for whom the score could be calculated. There was no significant association between DIC scores, bleeding diathesis or poor outcome.

CONCLUSIONS

The coagulation system was strongly activated in patients with leptospirosis. This was more pronounced in the deceased and in patients with severe bleeding than in than the survivors and in those without severe bleeding.

OBJECTIVE

To establish the physiologic changes in the coagulation and fibrinolytic systems during normal pregnancy and puerperium.

METHODS

One hundred and seventeen normal pregnant women were investigated in a longitudinal study involving five measurements: blood samples were collected at <em>1</em>0, <em>2</em>0, 30, 36 weeks and on the second day puerperium and were assayed for <em>prothrombin</em> time (PT expressed in INR), activated partial thromboplastin time (PTT), fibrinogen (FBG), antithrombin III activity (AT III), protein C activity (PC), protein S activity (PS), <em>prothrombin</em> <em>fragments</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>), type <em>1</em> plasminogen activator inhibitor activity (PAI) and tissue-plasminogen activator antigen (t-PA). Student t-test, One Way Analysis of Variance (ANOVA) and Bonferroni test were used for statistical analysis. P<0.05 (two tails) was assumed to indicate a significant difference.

RESULTS

Fibrinogen concentrations were always increased with respect to controls (P<0.00<em>1</em>), while protein S was always decreased, with values averaging 60% of those of controls from the <em>1</em>0th week of pregnancy onwards (P<0.00<em>1</em>). Variance analysis showed a statistically significant increase with gestational age for procoagulant factors (INR: P<0.00<em>1</em>; FBG: P<0.00<em>1</em>), a reduction for anticoagulants (PC: P<0.000<em>1</em>; PS: P<0.000<em>1</em>), and a rise for F<em>1</em>+<em>2</em> (P<0.000<em>1</em>). With regard to fibrinolysis, there was an increase both for t-PA (P<0.000<em>1</em>) and PAI-<em>1</em> (P<0.000<em>1</em>) during pregnancy. The t-PA values were always comprised in the normal range. PAI-<em>1</em> were increased with respect to control values starting from 3<em>1</em>st week. The most significant variations in the procoagulants (expressed by PT and FBG) were recorded up to the <em>2</em>0th week (P<0.00<em>1</em>); from the 30th week onwards, they remained stable until after the delivery. The same was true for protein S levels (P<0.00<em>1</em>), except that the difference between the <em>1</em>0th and the <em>2</em>0th weeks was not statistically significant. The level of F<em>1</em>+<em>2</em> gradually increased throughout pregnancy (P<0.00<em>1</em>), and then fell in the puerperium (P<0.00<em>1</em>).

CONCLUSIONS

The parameters showing the greatest variation during pregnancy were PT, FBG, PS, F<em>1</em>+<em>2</em> and PAI-<em>1</em>. The existence of a hypercoagulable state in pregnancy was suggested by the increased levels of F<em>1</em>+<em>2</em>.

Investigations to date have demonstrated that ligand binding to exosites <em>1</em> or <em>2</em> on thrombin produces conformational changes at the active site. In this study, we directly compared the effect of ligand binding to exosites <em>1</em> and <em>2</em> on the structure and function of the active site of thrombin and investigated functional linkage between the two exosites. Binding studies were performed in solution with fluorescein-Phe-Pro-Arg-CH<em>2</em>Cl (FPR)-thrombin. Hirudin-(54-65) and sF<em>2</em>, a synthetic peptide corresponding to residues 63-<em>1</em><em>1</em>6 of <em>prothrombin</em> <em>fragment</em> <em>2</em>, were used as ligands for exosites <em>1</em> and <em>2</em> of thrombin, respectively. The two ligands produce diametric changes in the fluorescence of fluorescein-FPR-thrombin and also have opposing effects on the rate of thrombin hydrolysis of a number of chromogenic substrates. These results indicate that sF<em>2</em> and hirudin-(54-65) differentially affect the conformation of the active site. Experiments then were performed to investigate whether both ligands can bind to thrombin simultaneously. When thrombin-bound fluorescein-sF<em>2</em> is titrated with hirudin-(54-65), complete displacement of fluorescein-sF<em>2</em> is observed. Likewise, when thrombin-bound fluorescein-hirudin-(54-65) is titrated with sF<em>2</em>, complete displacement occurs. Additional support for reciprocal binding was obtained in fluorescence experiments where both probes were labeled and in experiments monitoring ligand binding to agarose-immobilized thrombin. This mutually exclusive binding of either ligand can be explained by reciprocal, allosteric modulation of ligand affinity between the two exosites. Thus, not only do the two exosites differentially influence the active site, they also affect the binding properties of the opposing exosite.

The sequence of coagulant reactions in vivo following vascular injury is poorly characterized. Using quantitative immunoassays, the time courses were evaluated for activation of <em>prothrombin</em>, factor (F)V, FXIII, fibrinogen (Fbg) cleavage, and FVa inactivation in bleeding-time blood collected at 30-second intervals from <em>1</em><em>2</em> healthy subjects both before and after aspirin ingestion. <em>Prothrombin</em> decreased at a maximum rate of <em>1</em>4.<em>2</em> +/- 0.6 nM per second to <em>1</em>0% of initial values at the end of bleeding. Significant amounts of alpha-thrombin B chain appeared rapidly at 90 seconds of bleeding and increased at a maximum rate of 0.<em>2</em><em>2</em>4 +/- 0.03 nM per second to a peak value of 38 nM. Kinetics of prethrombin <em>2</em> generation was almost identical. <em>Prothrombin</em>ase concentration reached a peak value of <em>2</em><em>2</em> pM at <em>1</em>50 seconds and then decreased to 9 pM at the end of bleeding. <em>Prothrombin</em> <em>fragment</em> <em>1</em>.<em>2</em> (F<em>1</em>.<em>2</em>) was produced explosively (0.673 +/- 0.05 nM per second), whereas thrombin-antithrombin III (TAT) complexes were generated at a much slower rate (0.<em>1</em><em>1</em> +/- 0.008 nM per second; P =.00<em>2</em>). FVa light chain was detectable 30 seconds later than the heavy chain (<em>1</em>50 seconds) and was produced at a slightly slower rate (0.0<em>2</em>7 +/- 0.00<em>1</em> nM per second) when compared with the heavy chain (0.03<em>2</em> +/- 0.00<em>2</em> nM per second; P =.04<em>1</em>). The 30 000 <em>fragment</em> (residues 307-506) of FVa heavy chain produced by activated protein C appeared as early as at 90 seconds and increased with time. Fbg was removed from the blood shed with a high rate of 0.047 +/- 0.0<em>2</em> microM/s and became undetectable at approximately <em>1</em>80 seconds of bleeding. The velocity of FXIII activation correlated with thrombin B-chain formation. A 7-day aspirin administration (75 mg/d) resulted in significant reductions in maximum rates of (<em>1</em>) <em>prothrombin</em> removal (by <em>2</em>9%; P =.008); generation of alpha-thrombin B-chain (by <em>2</em>7.<em>2</em>%; P =.0<em>2</em><em>2</em>), and prethrombin <em>2</em> (by <em>2</em>6%; P =.0<em>1</em>4); formation of F<em>1</em>.<em>2</em> (by 3<em>1</em>.4%; P =.009) and TAT (by 30.3%; P = 0.0<em>1</em>3); (<em>2</em>) release of FVa heavy chain (by <em>2</em>5%; P =.003) and FVa light chain (by <em>2</em>9.6%; P =.007); (3) Fbg depletion from solution (by 30.5%; P =.00<em>2</em>); and (4) FXIII activation (by <em>2</em>8.6%; P =.003). Total amounts of the proteins studied, collected at every interval, also significantly decreased following aspirin ingestion. These results indicate that low-dose aspirin impairs thrombin generation and reactions catalyzed by this enzyme at the site of the injury.

BACKGROUND

A prothrombotic or hypercoagulable state has been described in AF, which could increase the risk of thromboembolism. As inflammation has been related to thrombogenesis and endothelial activation, we hypothesised that the prothrombotic state in AF (as assessed by an index of thrombogenesis, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> [F<em>1</em>+<em>2</em>]) and endothelial activation (soluble E-selectin (sEsel)) could be related to an index of inflammation (interleukin-6 (IL-6)).

METHODS

We studied <em>1</em>9<em>1</em> consecutive patients (98 male; mean age 7<em>2</em>.3+/-9.<em>2</em> years) with chronic non-rheumatic AF who were not on anticoagulant therapy. Plasma IL-6, sEsel and F<em>1</em>+<em>2</em> were measured by ELISA. Research indices were compared to 74 controls in sinus rhythm matched for age and sex. In 43 patients with AF, the effects of introducing anticoagulation (INR <em>2</em>.0-3.0) were also studied.

RESULTS

Patients with AF had elevated levels of F<em>1</em>+<em>2</em> (p<0.00<em>1</em>) and IL-6 (p=0.045), but not sEsel. There was no significant correlation between F<em>1</em>+<em>2</em> and IL-6. In multivariate analysis, only F<em>1</em>+<em>2</em> levels were independently associated with the presence of AF (p=0.00<em>1</em>). After oral anticoagulation, plasma levels of F<em>1</em>+<em>2</em> and sEsel were significantly decreased (both p<0.0<em>1</em>).

CONCLUSIONS

High levels of IL-6 in AF suggest an inflammatory state, which appears to be more related to clinical variables of the patients, rather than to the presence of AF per se. There was no association of inflammation with endothelial activation (sEsel) or the presence of abnormal thrombogenesis (high F<em>1</em>+<em>2</em> levels) in AF. Moreover, no changes in IL-6 levels were found despite the reduction of the other markers by oral anticoagulant therapy.

OBJECTIVE

Hemostasis and thrombosis may be important contributors to cognitive decline and dementia. Certain blood markers may assist in diagnosis or management.

OBJECTIVE

To collate evidence for the association of circulating hemostatic variables and dementia or cognitive impairment.

METHODS

A systematic review of studies describing blood markers of hemostatic function and cognition/dementia. Abstracts were reviewed by two independent assessors and studies selected based on pre-specified criteria. We described methodological quality and performed meta-analyzes where data allowed.

RESULTS

From 7<em>1</em>03 titles, 485 abstracts and included <em>2</em><em>1</em> studies (n = 3<em>2</em>,773) were assessed. In two longitudinal studies, the incident of vascular dementia risk was greater for higher D-dimer [hazard ratio (HR): <em>1</em>.50, 95% confidence interval (CI): <em>1</em>.<em>1</em>5-<em>1</em>.96]. For case-control data, we calculated standardized mean differences (SMD) and 95% CI. Higher levels of: factor (F)VII (SMD: 0.93; 95% CI: 0.60-<em>1</em>.<em>2</em>6), fibrinogen (SMD: <em>1</em>.53; 95% CI: <em>1</em>.<em>1</em>7-<em>1</em>.87), <em>prothrombin</em> <em>fragment</em> <em>1</em> and <em>2</em> (SMD: 0.64; 95% CI: 0.3<em>2</em>-0.96), plasminogen activator inhibitor (SMD: 0.68; 95% CI: 0.<em>2</em>6-<em>1</em>.<em>1</em>0), D-dimer (SMD: <em>2</em>.00; 95% CI: <em>1</em>.59-<em>2</em>.40) and von Willebrand factor (VWF) (SMD: <em>1</em>.68; 95% CI: <em>1</em>.30-<em>2</em>.06) showed modest but significant associations with vascular dementia. For patients with any dementia diagnosis, associations were with higher D-dimer (SMD: 0.36; 95% CI: 0.<em>1</em>5-0.56) and VWF (SMD: 0.3<em>1</em>; 95% CI: 0.<em>1</em><em>1</em>-0.5<em>1</em>). For specific cognitive domains, significant (P < 0.00<em>1</em>) positive correlations were fibrinogen and speed of processing (0.76; 95% CI: 0.67-0.84), verbal memory (0.69; 95% CI: 0.59-0.79) and non-verbal reasoning (0.57; 95% CI: 0.49-0.65).

CONCLUSIONS

The present results suggest a modest association between hemostasis and vascular dementia including increased levels of thrombin generation markers (D-dimer and <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>) and endothelial dysfunction (VWF and plasminogen activator inhibitor). Associations are weaker for specific cognitive tests and when all dementias are combined.

Warfarin, like all the 4-hydroxycoumarin compounds, has an asymmetric carbon atom. The clinically available warfarin preparations consist of a racemic mixture of equal amounts of <em>2</em> distinct S and R isomers, the former being 4-times more potent as anticoagulant and more susceptible to drug interaction. Warfarin is highly water soluble and rapidly absorbed from the stomach and the upper gastrointestinal tract; its plasma concentrations peak 60 to 90 minutes after oral administration. Warfarin binds to the enzyme vitamin K <em>2</em>,3-epoxide reductase in liver microsomes, stopping the cycle of vitamin K and reducing gamma-carboxylation of the precursors of vitamin D-dependent pro- and anticoagulant factors. A variable fraction of the binding with the target enzyme, albeit small, can be reversed by competitive displacers, such as dithiol-reducing agent activity. Differences in dithiol-reducing activity have been suggested as a contributing factor to the wide interindividual differences in sensitivity to oral anticoagulants. The anticoagulant effect is caused by a small fraction of the drug, since most (97 to 99%) is protein bound (mainly to albumin) and ineffective. Drugs that can displace the albumin binding will increase the action of warfarin, even though this effect is counteracted by a more rapid elimination of the drug. The elimination half-life of warfarin varies greatly among individuals, ranging from 35 to 45 hours; the S isomer has, however, an average half-life shorter than the R isomer. The plasma levels of vitamin K-dependent proteins are determined by a dynamic equilibrium between their synthesis and half-life times. The delay before warfarin takes effect reflects the half-life of the clotting proteins; the levels of factor VII and protein C (with shorter half-lives) are reduced earlier, reaching steady inhibited levels in about <em>1</em> day, whereas factor II takes more than <em>1</em>0 days. Oral anticoagulant therapy (OAT) with warfarin or other coumarin derivatives is increasingly administered to patients for primary or secondary prevention of various arterial or venous thromboembolic diseases. If in some clinical conditions OAT is given indefinitely, in others--such as venous thromboembolism or after tissue heart valve replacement--anticoagulants are usually given only for the high risk period of thrombotic complication. A recent large prospective study performed by the Italian Federation of Anticoagulation Clinics showed that about 30% of the patients who began OAT for various clinical indications stopped treatment at different times, confirming that withdrawal from OAT is an occurrence that affects a large number of patients. The expression 'rebound phenomenon' was adopted to indicate a hypercoagulant condition occurring after warfarin withdrawal. A possible more frequent recurrence of thromboembolism after cessation of anticoagulation became a matter of controversy and many clinical studies, mostly observational and noncontrolled, reported on the issue with inconsistent results. Most authoritative commentators agreed that rebound phenomenon, though possible, was not clinically relevant and did not differ in frequency and intensity according to mode of withdrawal. Scientific interest in the topic waned until more sensitive methods for investigating blood hypercoagulability became available. In recent years, many studies (reviewed in the text) have investigated the levels of different markers of hypercoagulability [fibrinopeptide A, activated factor VII, <em>prothrombin</em> <em>fragments</em> F<em>1</em>+<em>2</em>, thrombin-antithrombin complexes, D-dimers (DD)], consistently finding an increase in their values after cessation of anticoagulation. Changes in the levels of markers of activated blood coagulation were prospectively investigated by our group in 3<em>2</em> patients with venous thromboembolism who were randomly withdrawn abruptly or gradually from warfarin treatment. Our results indicate that interruption of anticoagulant treatment frequently elicits low grade acti

Vitamin K antagonists are most commonly used in long-term thrombosis prophylaxis and the use in patients with cardiovascular disease seems to be increasing. By interfering with the normal hemostatic mechanism, an increased risk of bleeding will arise and administration of human plasma or <em>prothrombin</em> complex concentrates may be necessary. It can be difficult to normalize hemostasis using plasma and <em>prothrombin</em> complex concentrates, because these may be associated with thromboembolic side-effects. The level of factor VII, one of the vitamin-K-dependent coagulation factors, decreases during oral anticoagulant therapy and the administration of recombinant factor VIIa normalizes the prolonged <em>prothrombin</em> time in warfarin-treated rats. After administration of acenocoumarol (International Normalized Ratio>> <em>2</em>), decreased levels of factor X and factor IX (<em>1</em>9-46%), protein C (<em>2</em>-<em>2</em>0%) and factor VII (4-<em>1</em>7%) were found in <em>2</em>8 healthy volunteers. After one dose of recombinant factor VIIa (5, <em>1</em>0, <em>2</em>0, 40, 80, <em>1</em><em>2</em>0, <em>1</em>60, <em>2</em>40, or 3<em>2</em>0 microg/kg) the International Normalized Ratio and <em>prothrombin</em> time normalized, which may imply an effect on bleeding in individuals receiving oral anticoagulant therapy. The lowest dose (5 microg/kg) normalized the International Normalized Ratio for <em>1</em><em>2</em> h and doses>> <em>1</em><em>2</em>0 microg/kg normalized it for <em>2</em>4 h. <em>Fragment</em> <em>1</em>+<em>2</em> stayed within its normal range in all dose groups, indicating that no systemic coagulation occurred.