BACKGROUND

In contrast to other extracorporeal treatments no established regime exists for anticoagulation with low-molecular-weight heparin (LMWH) in plasmapheresis therapy. A study was conducted to investigate whether LMWH (dalteparin-Na) is suitable as an effective anticoagulant in plasmapheresis therapy.

METHODS

Eleven patients with autoimmune neurological diseases and the necessity for a plasmapheresis therapy were enrolled. A capillary membrane filter was used. A total of <em>2</em>000 mL of human plasma was isovolumetrically exchanged per plasmapheresis cycle. The anticoagulation was accomplished with a single bolus of LMWH (dalteparin) of 80 to 90 IU per kg of body weight. The system was visually monitored. Anti-factor (F)Xa activity, thrombin-antithrombin III complex (TAT), and <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F <em>1</em>+<em>2</em>) were determined at regular intervals. Samples were taken from the collected plasma pool to determine the loss of LMWH during the plasmapheresis procedure.

RESULTS

All plasmapheresis cycles with LMWH were successful without complications. Approximately 40 percent of the initially administered LMWH bolus was lost by the large porous filter during the plasmapheresis. The anti-FXa values were determined to be 0.5 IU per mL during the entire plasmapheresis. TAT values were elevated (TAT median, <em>1</em>4.3 microg/L). F <em>1</em>+<em>2</em> values measured before the filter cartridge remained within the normal range for the entire plasmapheresis cycle ((<em>1</em>.<em>2</em> nmol/L) and were increasingly elevated after the filter.

CONCLUSIONS

Our initial experiences with LMWH for anticoagulation in plasmapheresis indicate that a body weight adjusted dose of LMWH (dalteparin) is suitable for anticoagulation in plasmapheresis therapy. No complications were observed. The data are encouraging. Further investigations will show if and how the present anticoagulation regime could be further optimized.

Disseminated intravascular coagulation (DIC) constitutes a part of the multiple organ failure (MOF) syndrome seen with such disorders as trauma and sepsis. Early detection of increased coagulation and fibrinolytic activity is important. The dynamic changes in some markers for early detection of the activation of these cascade systems are presented in relation to two patients with brain trauma. The clinical status and the severity of the disease were assessed by an established scoring method (APACHE II). The coagulation activation was noted by the appearance of increased end products of the coagulation cascade, such as soluble fibrin, thrombin-antithrombin complex, and <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>. Fibrinolytic activation and an increased secondary inhibition of fibrinolysis were detected by increased levels of D-dimer and plasminogen activator inhibitor-<em>1</em>. Leukocyte activation was indicated by a rise in elastase. The laboratory results normalized with clinical improvement. These new methods seem to detect DIC earlier than traditional methods and may also be of value for monitoring treatment.

Colistin electrostatically interacts with lipopolysaccharides (LPS). Pre-clinical studies demonstrated beneficial effects of colistin on LPS-induced coagulation and fibrinolysis. The objective of this trial was to investigate the effects of colistin during experimental endotoxaemia. In this randomised, double-blind, placebo-controlled, crossover trial <em>1</em>6 healthy volunteers received a <em>2</em> ng/kg LPS bolus after infusion of <em>2</em>.5 million IU colistin or placebo. Plasma levels of F<em>1</em>+<em>2</em> <em>prothrombin</em> <em>fragments</em>, thrombin-antithrombin complexes (TAT), von Willebrand factor antigen levels (vWF), E-selectin, plasmin-antiplasmin complexes (PAP), tissue-type plasminogen activator (t-PA) antigen and activity, plasminogen activator inhibitor-<em>1</em> (PAI-<em>1</em>) were measured. Infusion of colistin significantly reduced peak concentrations of PAP complexes by 70 %, t-PA antigen levels by 63 % and t-PA activity by 48 %, while PAI-<em>1</em> levels decreased numerically by 63 %. Two hours after the LPS bolus F<em>1</em>+<em>2</em> levels and TAT complexes were slightly reduced in the colistin period, but peak concentrations were similar in both periods. Colistin blunted the LPS induced four-fold increase in soluble E-Selectin levels by ~50 % and the two-fold increase in vWF antigen levels by ~70 %. The LPS-scavenging actions of colistin significantly reduce endothelial activation and fibrinolytic response in the human endotoxaemia model, while the activation of the coagulation system remains largely unaffected.

Women with diabetes mellitus have an increased risk of developing coronary heart disease which may be related at least partially to unfavourable changes in haemostasis. The effect of oestrogen replacement therapy on haemostasis has not been studied systematically in women with non-insulin-dependent diabetes mellitus (NIDDM) and therefore this study was performed for that purpose. Twenty-five postmenopausal women with NIDDM were treated with <em>2</em> mg of <em>1</em>7-beta-oestradiol orally for 3 months in a double-blind, crossover, placebo-controlled trial. During the last <em>1</em>6 days of active treatment, <em>1</em> mg of norethisterone acetate was added for <em>1</em>0 days for endometrial protection. Blood samples were taken at baseline and after 68 days of active or placebo treatment. Treatment with oestradiol was followed by a marked decrease in the activity of plasminogen activator inhibitor, compared with placebo. The activity of tissue plasminogen activator increased significantly. Levels of antithrombin decreased during treatment with oestradiol, whereas no changes were seen in levels of fibrinogen, von Willebrand factor, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, protein S, protein C or resistance to activated protein C. In conclusion, oestrogen replacement therapy in postmenopausal women with NIDDM improved the fibrinolytic activity, while only clinically insignificant alterations in the clotting system were seen. These changes in haemostasis may have a favourable impact on the risk for coronary heart disease in diabetic women.

BACKGROUND

Activation of coagulation and fibrinolysis is common among patients undergoing cardiopulmonary bypass (CPB) surgery. Little is known, however, about the impact of myocardial ischemia and reperfusion on coagulation activation and fibrinolysis in this clinical setting.

METHODS

We determined the levels of coagulation activation and fibrinolysis markers (CAFM) in <em>1</em>9 patients with severe coronary heart disease (CHD) during CPB surgery. FXIIa, tissue factor (TF), FVIIa, tissue plasminogen activator/plasminogen activator inhibitor-<em>1</em> complexes (tPA/PAI-<em>1</em>), <em>prothrombin</em> <em>fragments</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>), D-dimers (DD) and plasmin-plasmin inhibitor complexes (PPI) were measured at baseline, prior to and after cardioplegic myocardial ischemia. Simultaneous blood samples were drawn from the aorta and the coronary sinus to evaluate arteriovenous CAFM plasma level gradients.

RESULTS

Myocardial ischemia induced significant increases in gradients of FXIIa and F<em>1</em>+<em>2</em> levels across the coronary circulation without influencing systemic levels of these markers significantly. Systemic levels of FXIIa, tPA/PAI-<em>1</em>, F<em>1</em>+<em>2</em>, DD and PPI increased significantly during CPB operation. There was a significant linear correlation between FXIIa, FVIIa, F<em>1</em>+<em>2</em>, DD and PPI.

CONCLUSIONS

Myocardial ischemia induces contact activation and thrombin generation rather than release of tPA and might thus contribute to postoperative thromboembolic complications. Surgery itself and CPB cause activation of coagulation and fibrinolysis as already described. A significant association between FXIIa, FVIIa, F<em>1</em>+<em>2</em>, DD and PPI suggests a relationship between contact activation, thrombin generation, fibrin formation and fibrinolysis.

BACKGROUND

Colon cancer (CC) is frequently complicated by thromboembolic episodes. Thrombin plays a role in angiogenesis and among others induces the synthesis of vascular endothelial growth factor (VEGF) and its receptors (VEGFR-<em>1</em> and VEGFR-<em>2</em>). The aim of this study was to assess the expression of <em>prothrombin</em> <em>fragment</em> F<em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>), a byproduct in thrombin generation (indicating the presence of thrombin), in relation to the presence of VEGFR-<em>2</em>-bound VEGF (VEGF:VEGFR-<em>2</em>), as an indicator of VEGFR-<em>2</em> activation in human CC tissue.

METHODS

Immunohistochemical ABC and double staining studies were performed using antibodies against F<em>1</em>+<em>2</em> and VEGF:VEGFR-<em>2</em> in 59 specimens obtained from CC patients.

RESULTS

Medium and high expression of both F<em>1</em>+<em>2</em> and VEGF:VEGF<em>2</em> in association with CC cells and endothelial cells was demonstrated. Moreover, coexpression of F<em>1</em>+<em>2</em> and VEGF:VEGFR-<em>2</em> was observed in the cells.

CONCLUSIONS

The results may suggest a possible functional interaction between thrombin and VEGF-R<em>2</em> stimulation in human CC in vivo.

BACKGROUND

The risk of thrombosis is clearly increased in the postpartum period. Mice with a targeted deletion of the transmembrane domain of tissue factor (TF) develop serious activation of blood coagulation and widespread thrombosis after delivery.

OBJECTIVE

We hypothesized that TF, abundantly present in placental tissue, is released during delivery, resulting in the activation of blood coagulation. We measured sensitive markers for TF-dependent activation of coagulation before and after induction of labor in two groups: a vaginal delivery (VAG) group and a cesarean section (CS) group.

RESULTS

One hour after delivery, soluble TF (sTF) significantly increased in both groups [VAG group (mean +/- SD) <em>2</em><em>2</em>6 +/- 4<em>2</em> to 380 +/- 4<em>2</em> pg mL(-<em>1</em>) and CS group <em>1</em>93 +/- <em>1</em>7 to 355 +/- 44 pg mL(-<em>1</em>)]. The day after delivery, sTF was somewhat less increased. Both groups also showed an increase in factor VIIa, indicating activation of the TF pathway of coagulation. Indeed, after delivery, TF-dependent coagulation, as measured by the TF clotting time assay, was significantly enhanced. Increased plasma levels of <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> and thrombin-antithrombin complexes demonstrated thrombin generation following delivery. TF pathway-dependent activation of coagulation upon delivery was not blocked by TF pathway inhibitor and was not dependent on the mode of delivery.

CONCLUSIONS

The postdelivery increase in TF-dependent activation of coagulation is likely to be a natural mechanism to prevent excessive blood loss during and after delivery, and may also indicate a novel mechanism by which puerperal women have an increased risk of venous thromboembolism.

The aim of this study was to compare fibrinolysis in normal pregnancy and pre-eclampsia using individual markers of thrombosis and fibrinolysis with the contribution of a new parameter, global fibrinolytic capacity. Coagulation was determined with thrombin-antithrombin complex and <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F <em>1</em>+<em>2</em>) and fibrinolysis markers. Tissue plasminogen activator, plasminogen activator inhibitor-<em>1</em> and global fibrinolytic capacity were determined in <em>1</em>4 normal pregnancies and <em>2</em>9 women with pre-eclampsia. global fibrinolytic capacity was also determined in <em>1</em>4 age-matched healthy women. The Mann-Whitney U test and Pearson correlation test were used for statistical analysis. Thrombin-antithrombin complex, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> levels, and global fibrinolytic capacity levels in pre-eclamptic women were significantly higher than in women with normal pregnancies (P < 0.05). Tissue plasminogen activator, plasminogen activator inhibitor-<em>1</em> levels were also significantly higher in the pre-eclampsia group (P < 0.00<em>1</em> and P < 0.05 respectively). No significant correlation was found between global fibrinolytic capacity and thrombin-antithrombin complex, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> levels, tissue plasminogen activator or plasminogen activator inhibitor-<em>1</em> activity. Our results suggest that both thrombin formation and fibrinolysis are increased in pre-eclampsia compared with normal pregnancy. The increased global fibrinolytic capacity indicates that fibrinolysis remains preserved in pre-eclampsia. We suggest that global fibrinolytic capacity may be a useful parameter for accurately measuring in-vivo fibrinolysis globally, instead of with single parameters which may overlook the complex interactions between coagulation and fibrinolytic systems.

BACKGROUND

Both Type <em>2</em> diabetes and cardiovascular disease have been associated with enhanced coagulation and suppressed fibrinolysis.

OBJECTIVE

To investigate a possible relationship between selected hemostatic variables and abnormal glucose regulation (AGR) in patients with acute ST-elevation myocardial infarction (STEMI) without known diabetes and to study changes in selected hemostatic variables from baseline to follow-up in STEMI patients with or without AGR.

METHODS

Plasminogen activator inhibitor-<em>1</em> (PAI-<em>1</em>) activity, tissue plasminogen activator (t-PA) antigen, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F(<em>1</em>+<em>2</em>)) and von Willebrand factor (vWF) were measured in fasting blood samples from <em>1</em>99 STEMI patients <em>1</em>6.5 h (median time) after admission and 3 months later. All patients were classified into normal glucose regulation (NGR) or AGR based on an oral glucose tolerance test at follow-up, according to the WHO criteria.

RESULTS

High PAI-<em>1</em> activity (≥ 75th percentile) measured in-hospital was associated with AGR (n = 49) with an adjusted odds ratio of <em>2</em>.<em>2</em> (95% confidence interval, <em>1</em>.<em>1</em>, 4.4). In addition, high levels of t-PA antigen (≥ 75th percentile) were associated with AGR (adjusted odds ratio, 3.5; 95% confidence inteval, <em>1</em>.5, 8.<em>2</em>), but only in men. Changes in the levels of F(<em>1</em>+<em>2</em>) were significantly more pronounced in patients with AGR compared with NGR (adjusted P = 0.04).

CONCLUSIONS

Elevated levels of PAI-<em>1</em> activity and t-PA antigen measured in-hospital in STEMI patients were associated with AGR classified at 3-month follow-up. Additionally, changes in the levels of F(<em>1</em>+<em>2</em>) were more pronounced in patients with AGR compared with NGR. The data suggest an enhanced prothrombotic state after an acute STEMI in patients with AGR without known diabetes.

Systemic inflammation and activation of haemostasis are common in patients with lung cancer. Both conditions support tumour growth and metastasis. Therefore, inflammatory and haemostatic biomarkers might be useful for prediction of survival and therapy response. Patients with unresectable/metastatic lung cancer initiating <em>1</em>st-line chemotherapy (<i>n</i> = <em>2</em>77, 83% non-small cell lung cancer) were followed in a prospective observational cohort study. A comprehensive panel of haemostatic biomarkers (D-dimer, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, soluble P-selectin, fibrinogen, coagulation factor VIII, peak thrombin generation), blood count parameters (haemoglobin, leucocytes, thrombocytes) and inflammatory markers (neutrophil-lymphocyte ratio, lymphocyte-monocyte ratio, platelet-lymphocyte ratio, C-reactive protein) were measured at baseline. We assessed the association of biomarkers with mortality, progression-free-survival (PFS) and disease-control-rate (DCR). A biomarker-based prognostic model was derived. Selected inflammatory and haemostatic biomarkers were strong and independent predictors of mortality and therapy response. The strongest predictors (D-dimer, LMR, CRP) were incorporated in a unified biomarker-based prognostic model (<em>1</em>-year overall-survival (OS) by risk-quartiles: 79%, 69%, 5<em>1</em>%, <em>2</em>4%; <em>2</em>-year-OS: 53%, 36%, <em>2</em>3%, 8%; log-rank <i>p</i> < 0.00<em>1</em>). The biomarker-based model further predicted shorter PFS and lower DCR. In conclusion, inflammatory and haemostatic biomarkers predict poor prognosis and treatment-response in patients with advanced lung cancer. A biomarker-based prognostic score efficiently predicts mortality and disease progression beyond clinical characteristics.

Keywords: chemotherapy; haemostatic biomarker; lung cancer; prognostic model; systemic inflammation.

BACKGROUND

The heparin protocols used during cardiopulmonary bypass (CPB) in children undergoing surgical repair for congenital heart disease are extrapolated from adult data. Studies are needed that assess the optimal heparin dosing in these children, whose heparin clearance is increased compared with that in adults.

METHODS

We assessed the effects of two commonly used doses of heparin in the prime solution at the start of CPB operation on plasma heparin levels, on thrombin production (thrombin-antithrombin III complexes, <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>, D-dimer, and antithrombin III), and on the risk of hemorrhage. Before CPB, 48 children with congenital heart disease received heparin intravenously in a loading dose of 300 U/kg, followed by either <em>1</em> U/mL of heparin in the prime (low-dose group: <em>2</em><em>2</em> patients-acyanotic, 9; cyanotic, <em>1</em>3) or 3 U/mL of heparin in the prime (group: high-dose, <em>2</em>6 patients-acyanotic, <em>1</em>5; cyanotic, <em>1</em><em>1</em>).

RESULTS

In all patients, CPB resulted in the generation of thrombin. The duration of CPB was a significant covariate factor for heparin levels (p = 0.00<em>2</em>), thrombin production (p < 0.00<em>1</em>), and postoperative blood loss (p < 0.00<em>1</em>). In the patients in the high-dose group, the total heparin dose and the plasma heparin levels were higher (p = 0.0005 and 0.005, respectively) and the D-dimer levels tended to be lower (p = 0.06). The postoperative blood loss was higher in the cyanotic patients (p = 0.0<em>2</em>; both high-dose and low-dose groups), with <em>2</em> cyanotic patients (<em>1</em> in low-dose group, <em>1</em> in high-dose group) requiring reoperation, one of whom subsequently died. The increased heparin dose had no significant effect on the rate or volume of postoperative blood loss.

CONCLUSIONS

Increasing the heparin dose in the prime solution from <em>1</em> to 3 U/mL increased the plasma heparin levels and showed a trend toward reducing the postoperative laboratory values indicative of fibrinolysis. Thrombin generation during CPB and the incidence of postoperative hemorrhage were not significantly altered. Larger randomized trials are needed to determine the optimal heparin-dosing regimen in patients with congenital heart disease.

To date there have been no standard methods for assessing the thrombogenicity of central venous catheters. A procedure for testing the thrombogenicity of intravenous lines such as the silver-impregnated catheter by continuous blood flow in vitro was therefore developed. For this test, fresh blood was drawn from healthy human donors and anti-coagulated with sodium citrate (<em>1</em>:9). All material tested (catheter tubes with and without silver manufactured in the same way, polyethylene tubes and tubes with potentially thrombogenic material) were perfused through their lumen with anticoagulated blood for up to 3<em>1</em> hours. Blood samples were collected at different times from the test system at sites before and after the perfusion of the test catheters. The hemoglobin concentration, erythrocyte, leukocyte and thrombocyte counts and markers for thrombin activation (thrombin-antithrombin III-complex, F<em>1</em> + <em>2</em>)-<em>prothrombin</em> <em>fragments</em>) and for hyperfibrinolysis (d-dimers) were determined. No thrombin activation or signs of hyperfibrinolysis were detected in any material tested. Polyethylene tubes were found to cause hemolysis, as shown by a decrease in hemoglobin content from <em>1</em>5 g% to 4.5 g%. Tecothane tubes with and without silver did not induce hemolysis.

OBJECTIVE

: In this study, we measured the activity of coagulation and fibrinolysis and clarified the presence of certain differences between off-pump coronary artery bypass grafting (OPCAB) cases and awake off-pump coronary artery bypass grafting (AOCAB) cases to evaluate whether AOCAB is actually safe from the viewpoint of coagulability.

METHODS

: 8 underwent OPCAB and 6 underwent AOCAB. The following factors inducing coagulation and fibrinolysis were measured for upto 5 days after the operation: platelet counts, <em>prothrombin</em> time, activated partial thromboplastin time, fibrinogen, fibrin degeneration products, d-dimer, thrombin-antithrombin III complex (TAT), α<em>2</em>-plasmin inhibitor-plasmin complex, <em>prothrombin</em> <em>fragment</em> <em>1</em>, <em>2</em> (F<em>1</em>+<em>2</em>), thrombomodulin, β-thromboglobulin (β-TG), and platelet factor-4.

RESULTS

: At 5 days after the operation, fibrin degeneration products, d-dimer, α<em>2</em>-plasmin inhibitor-plasmin complex, and F<em>1</em>+<em>2</em> levels of the OPCAB group were significantly higher compared with their baseline values and those of the AOCAB group. At 5 days after the operation, thrombin-antithrombin III complex levels of the OPCAB group were significantly higher than those of the AOCAB group. Fibrinogen levels of the OPCAB group were significantly higher than their baseline values at 3 days after the operation (POD3) and 5 days after the operation (POD5).

CONCLUSIONS

: In this study, the hypercoagulable state at POD5 was suggested in the patients in the OPCAB group, but not in those in the AOCAB group. Further study is necessary to confirm these results, and future studies would evaluate the potential benefit of AOCAB procedure from the viewpoint of perioperative coagulability.

Previously we defined binding sites for high molecular weight kininogen (HK) and thrombin in the Apple <em>1</em> (A<em>1</em>) domain of factor XI (FXI). Since <em>prothrombin</em> (and Ca(<em>2</em>+)) can bind FXI and can substitute for HK (and Zn(<em>2</em>+)) as a cofactor for FXI binding to platelets, we have attempted to identify a <em>prothrombin</em>-binding site in FXI. The recombinant A<em>1</em> domain (rA<em>1</em>, Glu(<em>1</em>)-Ser(90)) inhibited the saturable, specific and reversible binding of <em>prothrombin</em> to FXI, whereas neither the rA<em>2</em> domain (Ser(90)-Ala(<em>1</em>8<em>1</em>)), rA3 domain (Ala(<em>1</em>8<em>1</em>)-Val(<em>2</em>7<em>1</em>)), nor rA4 domain (Phe(<em>2</em>7<em>2</em>)-Glu(36<em>1</em>)) inhibited <em>prothrombin</em> binding to FXI. Kinetic binding studies using surface plasmon resonance showed binding of FXI (K(d) approximately 7<em>1</em> nm) and the rA<em>1</em> domain (K(d) approximately <em>2</em>39 nm) but not rA<em>2</em>, rA3, or rA4 to immobilized <em>prothrombin</em>. Reciprocal binding studies revealed that synthetic peptides (encompassing residues Ala(45)-Ser(86)) containing both HK- and thrombin-binding sites, inhibit (<em>1</em><em>2</em>5)I-rA<em>1</em> (Glu(<em>1</em>)-Ser(90)) binding to <em>prothrombin</em>, (<em>1</em><em>2</em>5)I-<em>prothrombin</em> binding to FXI, and (<em>1</em><em>2</em>5)I-<em>prothrombin</em> <em>fragment</em> <em>2</em> (Ser(<em>1</em>56)-Arg(<em>2</em>7<em>1</em>)) binding to FXI. However, homologous prekallikrein-derived peptides (encompassing Pro(45)-Gly(86)) did not inhibit FXI rA<em>1</em> binding to <em>prothrombin</em>. The peptides Ala(45)-Arg(54), Phe(56)-Val(7<em>1</em>), and Asp(7<em>2</em>)-Ser(86), derived from sequences of the A<em>1</em> domain of FXI, acted synergistically to inhibit (<em>1</em><em>2</em>5)I-rA<em>1</em> binding to <em>prothrombin</em>. Mutant rA<em>1</em> peptides (V64A and I77A), which did not inhibit FXI binding to HK, retained full capacity to inhibit rA<em>1</em> domain binding to <em>prothrombin</em>, and mutant rA<em>1</em> peptides Ala(45)-Ala(54) (D5<em>1</em>A) and Val(59)-Arg(70) (E66A), which did not inhibit FXI binding to thrombin, retained full capacity to inhibit rA<em>1</em> domain binding to <em>prothrombin</em>. Thus, these experiments demonstrate that a <em>prothrombin</em> binding site exists in the A<em>1</em> domain of FXI spanning residues Ala(45)-Ser(86) that is contiguous with but separate and distinct from the HK- and thrombin-binding sites and that this interaction occurs through the kringle II domain of <em>prothrombin</em>.

OBJECTIVE

There exists a current lack of information about the impact of different inline filters, used for the leucoreduction of whole blood (WB), on the levels of clotting factors and markers of coagulation, complement and cell activation in plasma. Only a few small comparisons of different types of WB inline filters have been published to date.

METHODS

This study compared two plasma types of <em>2</em>00 units each. Both study groups were derived from WB, inline-filtered and held for <em>2</em> h at <em>2</em>0 degrees between donation and filtration. Then, <em>2</em>00 units (Group A) were filtered using a positively charged polyester filter (Baxter RZ<em>2</em>000) and the other <em>2</em>00 units (Group B) were filtered using an uncharged polyester filter (Fresenius). After filtration, both groups were analysed for fibrinogen, factors V and VIII:C (FV and FVIII:C, respectively), immunoglobulin G (IgG), residual leucocytes and platelets, and markers of coagulation, complement and cell activation. Predonation plasma samples from CPDA<em>1</em>-anticoagulated blood were obtained from <em>1</em>00 different individuals and served as controls.

RESULTS

WB inline filtration did not influence fibrinogen, FV, FVIII:C or IgG levels. Neither filter induced thrombin or fibrin formation. The charged filter caused substantial complement activation and neutrophil elastase and platelet factor 4 release. In contrast, the plasma filtered through the uncharged filter showed markedly lower levels of C3a-desArg, C5a, neutrophil elastase and platelet factor 4, and moderately reduced levels of <em>prothrombin</em> <em>fragments</em> <em>1</em>+<em>2</em> and D-dimer, compared with controls.

CONCLUSIONS

Filter type has a significant impact on the quality of plasma derived from WB filtered through inline filtration systems. Some filters produce substantial coagulation and complement activation and cell release, while others appear to reduce the plasma levels of activation markers. The clinical significance of these findings remains to be determined.

Hypercoagulability can be defined as a condition of procoagulant imbalance due to heightened enzymatic activation of coagulation zymogens, but with no laboratory evidence of fibrin deposition nor clinical signs of thrombosis. The imbalance can be detected by measuring the plasma levels of <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>), fibrinopeptide A (FPA) and thrombin-antithrombin III (TAT) complexes. The aims of this study were to establish the frequency of existence and biochemical pattern of hypercoagulability in patients with cancer and autoimmune disorders, clinical conditions associated with an increased risk of thrombosis, and to ascertain the most sensitive method for its diagnosis. In approximately one-fourth of the patients hypercoagulability was identified by finding high levels of FPA F<em>1</em> + <em>2</em> or TAT unaccompanied by signs of fibrin deposition (expressed by normal levels of D-dimer). In a smaller proportion of patients (approximately <em>1</em>0%), the concomitant presence of high levels of D-dimer indicated that the activation of the coagulation cascade had gone beyond the stage of heightened enzymatic activity to the point of cross-linked fibrin deposition. Of the markers used to detect hypercoagulability. FPA seems to be the most sensitive, being significantly increased in all clinical conditions studied.

<em>Prothrombin</em> is conformationally activated by von Willebrand factor-binding protein (vWbp) from Staphylococcus aureus through insertion of the NH(<em>2</em>)-terminal residues of vWbp into the <em>prothrombin</em> catalytic domain. The rate of <em>prothrombin</em> activation by vWbp(<em>1</em>-<em>2</em>63) is controlled by a hysteretic kinetic mechanism initiated by substrate binding. The present study evaluates activation of <em>prothrombin</em> by full-length vWbp(<em>1</em>-474) through activity progress curve analysis. Additional interactions from the COOH-terminal half of vWbp(<em>1</em>-474) strengthened the initial binding of vWbp to <em>prothrombin</em>, resulting in higher activity and an ∼<em>1</em>00-fold enhancement in affinity. The affinities of vWbp(<em>1</em>-<em>2</em>63) or vWbp(<em>1</em>-474) were compared by equilibrium binding to the <em>prothrombin</em> derivatives prethrombin <em>1</em>, prethrombin <em>2</em>, thrombin, meizothrombin, and meizothrombin(des-<em>fragment</em> <em>1</em>) and their corresponding active site-blocked analogs. Loss of <em>fragment</em> <em>1</em> in prethrombin <em>1</em> enhanced affinity for both vWbp(<em>1</em>-<em>2</em>63) and vWbp(<em>1</em>-474), with a 30-45% increase in Gibbs free energy, implicating a regulatory role for <em>fragment</em> <em>1</em> in the activation mechanism. Active site labeling of all <em>prothrombin</em> derivatives with D-Phe-Pro-Arg-chloromethyl ketone, analogous to irreversible binding of a substrate, decreased their K(D) values for vWbp into the subnanomolar range, reflecting the dependence of the activating conformational change on substrate binding. The results suggest a role for <em>prothrombin</em> domains in the pathophysiological activation of <em>prothrombin</em> by vWbp, and may reveal a function for autocatalysis of the vWbp·<em>prothrombin</em> complexes during initiation of blood coagulation.

BACKGROUND

The role of blood coagulation in the pathogenesis of aortic stenosis (AS) is unknown. Recently, tissue factor (TF) expression in stenotic aortic valves has been reported in animal model.

OBJECTIVE

The aim of the study was to investigate TF expression in valve leaflets obtained from AS patients and to determine its associations with circulating coagulation markers and echocardiographic variables.

METHODS

We studied <em>2</em>0 patients (<em>1</em>0 men, <em>1</em>0 women) with dominant AS (age 6<em>2</em>.9 +/-9.6, years, mean gradient 43.6<em>2</em> +/-<em>1</em>4.6<em>2</em> mmHg), and <em>2</em>0 well-matched patients with dominant aortic insufficiency (AI) undergoing elective aortic valve replacement. Immunofluorescence was measured on decalcified leaflets using antibodies against human TF and macrophages. <em>Prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>) and circulating TF were determined in plasma prior to surgery.

RESULTS

AS valves were characterized by an increased (all, p <0.00<em>1</em>) percentage of TF-positive (<em>2</em>4.6%) and macrophage-containing (<em>2</em>7.3%) areas detected mainly on the aortic side of the leaflets, compared with AI valves (6.3% and 7.4%, respectively). Patients with AS had elevated F<em>1</em>+<em>2</em> (<em>2</em>6<em>2</em>.<em>1</em> +/-<em>2</em>7.8 pmol/l, p <0.00<em>1</em>) and plasma TF (median <em>1</em>3<em>1</em>.8, interquartile range [9<em>1</em>.4<em>2</em>-3<em>1</em>0.56] pg/ml, p = 0.0<em>1</em>8) compared with AI subjects (<em>1</em>36.<em>1</em> +/-<em>1</em><em>1</em>.9 pmol/l, 65.38 [49.5<em>1</em>-87.8<em>1</em>] pg/ml, respectively). Percentage of TF-positive areas correlated with plasma TF (r = 0.68, p <0.000<em>1</em>), but not with F<em>1</em>+<em>2</em>. Maximum transvalvular gradient >75 mmHg, but not the aortic valve area, showed associations with percentage of TF-positive areas (r = 0.88, p = 0.0039).

CONCLUSIONS

This study is the first full-length report demonstrating the presence of TF associated with macrophage infiltration in human aortic valve leaflets in AS patients.

OBJECTIVE

To investigate the pathophysiology underlying raised lactate levels after cardiac surgery with cardiopulmonary bypass (CPB).

METHODS

Prospective observational study.

METHODS

Medical and surgical intensive care unit of a tertiary hospital.

METHODS

A total of 40 patients undergoing first-time coronary artery bypass grafting with CPB.

METHODS

The prothrombotic response to cardiac surgery with CPB was assessed by measuring plasma levels of <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> and plasminogen activator inhibitor (PAI) activity. The hemodynamic responses to cardiac surgery with CPB were also measured using standard techniques.

RESULTS

After cardiac surgery, <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> levels increased 6-fold and PAI activity increase <em>2</em>- to 3-fold (p <.000<em>1</em>). Lactate levels were not associated with <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> and PAI activity levels after CPB. Lactate levels were associated with baseline PAI activity (p =.006), a history of hypertension (p =.0<em>2</em>), raised baseline lactate levels (p =.0<em>2</em>), an early increase in body temperature after CPB (p =.05), a late increase in oxygen consumption after CPB (p =.03), and a raised white cell count after CPB (p =.06). Lactate levels were inversely associated with the maximum activated clotting time level reached during CPB (p =.0<em>2</em>). Multivariate linear regression demonstrated lactate levels were independently associated with baseline PAI activity.

CONCLUSIONS

We found cardiac surgery with CPB was associated with a marked prothrombotic response. Lactate levels were associated with elevated baseline PAI activity and evidence of an amplified inflammatory response to cardiac surgery with CPB. Our findings implicate aspects of the inflammatory response, including microvascular thrombosis, in the development of raised lactate levels after cardiac surgery with CPB.

Chloroprene is used almost exclusively in the manufacture of neoprene (polychloroprene). Chloroprene was chosen for study because it is a high-volume production chemical with limited information on its carcinogenic potential and because it is the <em>2</em>-chloro analogue of <em>1</em>,3-butadiene, a potent, multi-species, multi-organ carcinogen. Male and female F344/N rats and B6C3F<em>1</em> mice were exposed to chloroprene (greater than 96% pure) by inhalation for <em>1</em>6 days, <em>1</em>3 weeks, or <em>2</em> years. Genetic toxicology studies were conducted in Salmonella typhimurium, Drosophila melanogaster, and B6C3F<em>1</em> mice (bone marrow cells and peripheral blood erythrocytes). <em>1</em>6-Day Study in Rats: Groups of <em>1</em>0 male and <em>1</em>0 female F344/N rats were exposed to 0, 3<em>2</em>, 80, <em>2</em>00, or 500 ppm chloroprene by inhalation, 6 hours per day, 5 days per week, for <em>1</em>6 days. Three 500 ppm males died on day <em>2</em> or 3 of the study. Mean body weight gains of <em>2</em>00 ppm males and females and 500 ppm females were significantly less than those of the chamber control groups. On the first day of exposure, rats exposed to 500 ppm were hypoactive and unsteady and had rapid shallow breathing. These effects were also observed to some degree in animals exposed to <em>2</em>00 ppm. After the second day of exposure, the effects in these groups worsened, and hemorrhage from the nose was observed. A normocytic, normochromic, responsive anemia; thrombocytopenia; and increases in serum activities of alanine aminotransferase, glutamate dehydrogenase, and sorbitol dehydrogenase occurred on day 4 in <em>2</em>00 ppm females and 500 ppm males. Kidney weights of 80 and 500 ppm females were significantly greater than those of the chamber control group, as were the liver weights of <em>2</em>00 and 500 ppm females. The incidences of minimal to mild olfactory epithelial degeneration of the nose in all exposed groups of males and females were significantly greater than those in the chamber control groups. The incidence of squamous metaplasia of the respiratory epithelium was significantly increased in 500 ppm males. The incidences of centrilobular to random hepatocellular necrosis in 500 ppm males and <em>2</em>00 ppm females were significantly greater than those in the chamber control groups. <em>1</em>6-Day Study in Mice: Groups of <em>1</em>0 male and <em>1</em>0 female B6C3F<em>1</em> mice were exposed to 0, <em>1</em><em>2</em>, 3<em>2</em>, 80, or <em>2</em>00 ppm chloroprene by inhalation, 6 hours per day, 5 days per week, for <em>1</em>6 days. All males and females exposed to <em>2</em>00 ppm died on day <em>2</em> or day 3 of the study. Mean body weight gains of males exposed to 3<em>2</em> or 80 ppm were significantly less than that of the chamber control group. Mice exposed to <em>2</em>00 ppm exhibited narcosis during exposure and were hypoactive with reduced body tone after the first day of exposure. In general, hematology and clinical chemistry parameters measured for exposed males and females were similar to those of the chamber control groups. Thymus weights of 80 ppm males and females were significantly less than those of the chamber control groups. Liver weights of 80 ppm females were significantly greater than those of the chamber control groups. Increased incidences of multifocal random hepatocellular necrosis occurred in males and females exposed to <em>2</em>00 ppm. Hypertrophy of the myocardium, foci of hemorrhage, and mucosal erosion were observed in three males and three females exposed to <em>2</em>00 ppm. Squamous epithelial hyperplasia of the forestomach was observed in two males and two females exposed to 80 ppm. Thymic necrosis, characterized by karyorrhexis of thymic lymphocytes, was observed in all males and females in the <em>2</em>00 ppm groups. <em>1</em>3-Week Study in Rats: Groups of <em>1</em>0 male and <em>1</em>0 female F344/N rats were exposed to chloroprene at concentrations of 0, 5, <em>1</em><em>2</em>, 3<em>2</em>, 80, or <em>2</em>00 ppm by inhalation, 6 hours per day, 5 days per week, for <em>1</em>3 weeks. One male exposed to <em>2</em>00 ppm died during the study. The final mean body weights and body weight gains of all exposed groups of males and females were similar to those of the chamber control groups. Clinical findings in <em>2</em>00 ppm males included red or clear discharge around the nose and eye region. At week <em>1</em>3, a norm a normocytic, normochromic, and non-responsive anemia occurred in <em>2</em>00 ppm males and females. A thrombocytopenia occurred in <em>2</em>00 ppm males and females on day <em>2</em> and in 80 and <em>2</em>00 ppm females on day <em>2</em><em>2</em>. However, at week <em>1</em>3, platelet counts rebounded and were minimally increased in <em>2</em>00 ppm males and females. On day <em>2</em>, a minimal to mild increase in activated partial thromboplastin time and <em>prothrombin</em> time occurred in <em>2</em>00 ppm males and females. The <em>2</em>00 ppm males and females also had increased activities of serum alanine aminotransferase, glutamate dehydrogenase, and sorbitol dehydrogenase on day <em>2</em><em>2</em>; these increases were transient, and by week <em>1</em>3 the serum activities of these enzymes were similar to those of the chamber controls. An alkaline phosphatase enzymeuria occurred in <em>2</em>00 ppm females on day <em>2</em><em>2</em>; at week <em>1</em>3, an alkaline phosphatase enzymeuria oc-curred in 3<em>2</em>, 80, and <em>2</em>00 ppm males and <em>2</em>00 ppm females. At week <em>1</em>3, a proteinuria occurred in <em>2</em>00 ppm males. Liver nonprotein sulfhydryl concentrations in male rats immediately following <em>1</em> day or <em>1</em><em>2</em> weeks of exposure to <em>2</em>00 ppm and in females exposed to <em>2</em>00 ppm for <em>1</em><em>2</em> weeks were significantly less than those of the chamber control groups. Kidney weights of <em>2</em>00 ppm males and females and 80 ppm females were significantly greater than those of the chamber control groups. Sperm motility of <em>2</em>00 ppm males was significantly less than that of the controls. In neurobehavioral assessments, horizontal activity was increased in male rats exposed to 3<em>2</em> ppm or greater and total activity was increased in 3<em>2</em> and <em>2</em>00 ppm males. Increased incidences of minimal to mild olfactory epithelial degeneration and respiratory metaplasia occurred in males and females exposed to 80 or <em>2</em>00 ppm. The incidence of olfactory epithelial degeneration in 3<em>2</em> ppm females was also significantly greater than that in the chamber control group. The incidence of hepatocellular necrosis in <em>2</em>00 ppm females was significantly greater than that in the chamber control group. Scattered chronic inflammation also occurred in the liver of male and female rats in the <em>2</em>00 ppm groups; the incidence in <em>2</em>00 ppm females was significantly greater than that in the chamber control group. The incidences of hemosiderin pigmentation were significantly increased in males and females exposed to <em>2</em>00 ppm. <em>1</em>3-Week Study in Mice: Groups of <em>1</em>0 male and <em>1</em>0 female B6C3F<em>1</em> mice were exposed to chloroprene at concentrations of 0, 5, <em>1</em><em>2</em>, 3<em>2</em>, or 80 ppm by inhalation, 6 hours per day, 5 days per week, for <em>1</em>3 weeks. All male and female mice survived to the end of the study. The final mean body weight and body weight gain of males exposed to 80 ppm were significantly less than those of the chamber control group. Hematocrit concentrations of females exposed to 3<em>2</em> or 80 ppm and erythrocyte counts of 80 ppm females were significantly less than those of the chamber control group. Platelet counts of 3<em>2</em> and 80 ppm females were also greater than that of the chamber control group. Increased incidences of squamous epithelial hyperplasia of the forestomach occurred in males and females exposed to 80 ppm. <em>2</em>-Year Study in Rats: Groups of 50 male and 50 female F344/N rats were exposed to chloroprene at concentrations of 0, <em>1</em><em>2</em>.8, 3<em>2</em>, or 80 ppm by inhalation, 6 hours per day, 5 days per week, for <em>2</em> years. Survival, Body Weights, and Clinical Findings: Survival of males exposed to 3<em>2</em> or 80 ppm was significantly less than that of the chamber control group. Mean body weights of males exposed to 80 ppm were less than those of the chamber controls after week 93. Masses of the torso were observed during the study in exposed female groups, and these clinical findings correlated with mammary gland fibroadenomas observed at necropsy. Pathology Findings: The incidences of squamous cell papilloma and squamous cell papilloma or squamous cell carcinoma (combined) of the oral cavity in male rats exposed to 3<em>2</em> ppm and male and female rats exposed to 80 ppm were significantly greater than those in the chamber controls and exceeded the historical control ranges. The incidences of thyroid gland follicular cell adenoma or carcinoma (combined) in males exposed to 3<em>2</em> or 80 ppm were significantly greater than that in the chamber control group and exceeded the historical control range. Although the incidences of follicular cell adenoma and follicular cell adenoma or carcinoma (combined) in 80 ppm females were not significantly greater than those in the chamber controls, they did exceed the historical control range for these neoplasms. The incidences of alveolar epithelial hyperplasia of the lung were significantly greater in all exposed groups of males and females than in the chamber control groups. The incidences of alveolar/bronchiolar carcinoma and alveolar/bronchiolar adenoma or carcinoma (combined) in 80 ppm males were slightly greater than those in the chamber control group. Although these neoplasm incidences were not significant, they exceeded the historical control range. The incidence of alveolar/bronchiolar adenoma, although not significant, was greater in 80 ppm females than in the chamber control group. The incidences of multiple fibroadenoma of the mammary gland in all exposed groups of females were greater than that in the chamber control group. The incidences of fibroadenoma (including multiple fibroadenoma) in 3<em>2</em> and 80 ppm females were significantly greater than that in the chamber controls. The incidences of fibroadenoma in the chamber control group and in all exposed groups of females exceeded the historical control range. The severity of nephropathy in exposed groups of male and female rats was slightly greater than in the chamber controls. Renal tubule adenoma and hyperplasia were observed in males and females. Additional kidney sections from male and female control and exposed rats were examined to provide a clearer indication of the potential effects of chloroprene on the kidney. The combined single- and step-section incidences of renal tubule hyperplasia in 3<em>2</em> and 80 ppm males and 80 ppm females and the incidences of adenoma and adenoma or carcinoma (combined) in all exposed males were significantly greater than those in the chamber controls. A slight increase in the incidence of transitional epithelium carcinoma of the urinary bladder was observed in 80 ppm females. In addition, one 3<em>2</em> ppm male had a transitional epithelium carcinoma and one 80 ppm male had a transitional cell papilloma. These findings are noteworthy because no urinary bladder neoplasms have been observed in chamber control male or female F344/N rats. In the nose, the incidences of atrophy, basal cell hyperplasia, metaplasia, and necrosis of the olfactory epithelium in 3<em>2</em> and 80 ppm males and females and of atrophy and necrosis in <em>1</em><em>2</em>.8 ppm males were significantly greater than those in the chamber control groups. The incidences of chronic inflammation were significantly increased in males exposed to <em>1</em><em>2</em>.8 or 3<em>2</em> ppm and in males and females exposed to 80 ppm. The incidences of fibrosis and adenomatous hyperplasia in 80 ppm males and females were significantly greater than those in the chamber controls. Generally, lesions in the nasal cavity were mild to moderate in severity. <em>2</em>-Year Study in Mice: Groups of 50 male and 50 female B6C3F<em>1</em> mice were exposed to chloroprene at concentrations of 0, <em>1</em><em>2</em>.8, 3<em>2</em>, or 80 ppm by inhalation, 6 hours per day, 5 days per week, for <em>2</em> years. Survival, Body Weights, and Clinical Findings: Survival of males exposed to 3<em>2</em> or 80 ppm and of all exposed female groups was significantly less than that of the chamber controls. The mean body weights of 80 ppm females were significantly less than those of the chamber control group after week 75. Clinical findings included masses of the head, which correlated with harderian gland adenoma and/or carcinoma in 3<em>2</em> ppm males and 80 ppm males and fe-males. Dorsal and lateral torso masses of female mice correlated with mammary gland neoplasms in 3<em>2</em> and 80 ppm females and subcutaneous sarcomas in <em>1</em><em>2</em>.8, 3<em>2</em>, and 80 ppm females. Pathology Findings: The incidences of alveolar/bronchiolar neoplasms in the lungs of all groups of exposed males and females were significantly greater than those in the chamber control groups and generally exceeded the historical control ranges. The incidences of multiple alveolar/bronchiolar adenoma and alveolar/bronchiolar carcinoma were increased in all exposed groups of males and females. The incidences of bronchiolar hyperplasia in all exposed groups of males and females were significantly greater than those in the chamber control groups. Male mice had a pattern of nonneoplastic liver lesions along with silver-staining helical organisms within the liver consistent with infection with Helicobacter hepaticus. An organism compatible with H. hepaticus was confirmed with a polymerase chain reaction-restriction <em>fragment</em> length polymorphism (PCR-RFLP)-based assay. In NTP studies with H. hepaticus-associated hepatitis, increased incidences of hemangiosarcoma have been seen in the livers of male mice. Therefore, hemangiosarcomas of the liver were excluded from the analyses of circulatory (endothelial) neoplasms in males in this study. Even with this exclusion, the combined occurrence of hemangioma or hemangiosarcoma at other sites was significantly increased at all chloroprene exposure concentrations in males and in 3<em>2</em> ppm females. Incidences of neoplasms at other sites in this study of chloroprene were not considered to have been significantly impacted by the infection with H. hepaticus or its associated hepatitis. The incidences of harderian gland adenoma and harderian gland adenoma or carcinoma (combined) in males exposed to 3<em>2</em> or 80 ppm and females exposed to 80 ppm were significantly greater than those in the chamber controls. The incidences of harderian gland adenoma or carcinoma (combined) in 3<em>2</em> ppm males and 80 ppm males and females exceeded the historical control ranges. The incidences of mammary gland carcinoma and adenoacanthoma or carcinoma (combined) in 80 ppm females were significantly greater than those in the chamber control group. The incidences of mammary gland carcinoma and of adenoacanthoma in 3<em>2</em> and 80 ppm females exceeded the historical control ranges. Multiple mammary gland carcinomas occurred in exposed females. The incidences of hepatocellular carcinoma in all exposed female groups and hepatocellular adenoma or carcinoma (combined) in 3<em>2</em> and 80 ppm females were significantly greater than those in the chamber controls; in the 80 ppm group, the incidence exceeded the historical control ranges for carcinoma and adenoma or carcinoma (combined). The incidence of eosinophilic foci in 80 ppm females was also significantly greater than that in the chamber controls. The incidences of sarcoma of the skin were significantly greater in all exposed groups of females than in the chamber controls. The incidences of sarcoma of the mesentery were also increased in all exposed groups of females. The incidence of squamous cell papilloma in 80 ppm females was greater than that in the chamber controls; the difference was not significant, but the incidence exceeded the historical control range. Males also showed a positive trend in the incidence of squamous cell papilloma of the forestomach. In males and females exposed to 80 ppm, the incidences of hyperplasia of the forestomach epithelium were significantly greater than those in the chamber controls. Carcinomas of the Zymbal's gland were seen in three 80 ppm females, and two carcinomas metastasized to the lung. Zymbal's gland carcinomas have not been reported in control female mice in the NTP historical database. The incidence of renal tubule adenoma in 80 ppm males was greater than that in the chamber controls. Though this difference was not significant, the incidence of this rare neoplasm exceeded the historical control range. The incidences of renal tubule hyperplasia in males exposed to 3<em>2</em> or 80 ppm were significantly greater than that in the chamber controls. Additional sections of kidney were examined from control and exposed males to verify these findings. The combined single- and step-section incidence of renal tubule adenoma in 80 ppm males and the combined incidences of renal tubule hyperplasia in all groups of exposed male mice were greater than those in the chamber controls. The incidences of olfactory epithelial atrophy, adenomatous hyperplasia, and metaplasia in 80 ppm males and females were significantly greater than those in the chamber controls. The incidences of hematopoietic proliferation of the spleen in 3<em>2</em> and 80 ppm males and in all groups of exposed females were significantly greater than those in the chamber controls. Genetic Toxicology: Chloroprene was not mutagenic in any of the tests performed by the NTP. No induction of mutations was noted in any of four strains of S. typhimurium in the presence or the absence of S9 metabolic activation enzymes, and no induction of sex-linked recessive lethal mutations was observed in germ cells of male D. melanogaster treated with chloroprene via feeding or injection. In male mice exposed to chloroprene by inhalation for <em>1</em><em>2</em> days over a <em>1</em>6-day period, no induction of chromosomal aberrations, sister chromatid exchanges, or micronucleated erythrocytes in bone marrow or peripheral blood occurred. Results of a second micronucleus assay in male and female mice after <em>1</em>3 weeks of exposure to chloroprene via inhalation were also negative. Conclusion: Under the conditions of these <em>2</em>-year inhalation studies, there was clear evidence of carcinogenic activity of chloroprene in male F344/N rats based on increased incidences of neoplasms of the oral cavity; increased incidences of neoplasms of the thyroid gland, lung, and kidney were also attributed to chloroprene exposure. There was clear evidence of carcinogenic activity of chloroprene in female F344/N rats based on increased incidences of neoplasms of the oral cavity; increased incidences of neoplasms of the thyroid gland, mammary gland, and kidney were also attributed to exposure to chloroprene. Low incidences of urinary bladder neoplasms in male and female rats and lung neoplasms in female rats may also have been related to exposure to chloroprene. There was clear evidence of carcinogenic activity of chloroprene in male B6C3F<em>1</em> mice based on increased incidences of neoplasms of the lung, circulatory system (hemangiomas and hemangiosarcomas), and harderian gland; increased incidences of neoplasms of the forestomach and kidney were also attributed to exposure to chloroprene. There was clear evidence of carcinogenic activity of chloroprene in female B6C3F<em>1</em> mice based on increased incidences of neoplasms of the lung, circulatory system (hemangiomas and hemangiosarcomas), harderian gland, mammary gland, liver, skin, and mesentery; increased incidences of neoplasms of the forestomach and Zymbal's gland were also attributed to exposure to chloroprene. Exposure of male and female rats to chloroprene was associated with increased incidences of alveolar epithelial hyperplasia in the lung; nephropathy; and several nonneoplastic effects in the nose including olfactory epithelial atrophy, fibrosis, adenomatous hyperplasia, basal cell hyperplasia, chronic inflammation, respiratory metaplasia, and necrosis. Exposure of male and female mice to chloroprene was associated with increased incidences of bronchiolar hyperplasia and histiocytic cell infiltration in the lung; epithelial hyperplasia in the forestomach; renal tubule hyperplasia (males only); several effects in the nose including olfactory epithelial atrophy, respiratory metaplasia, and adenomatous hyperplasia; and hematopoietic cell proliferation in the spleen. Synonyms: Chlorobutadiene, <em>2</em>-chlorobuta-<em>1</em>,3-diene, <em>2</em>-chloro-<em>1</em>,3-butadiene, -chloroprene

BACKGROUND

Serum markers of inflammation and platelet activation are related to cardiovascular risk. Cardiovascular risk reduction is a major treatment goal in patients with peripheral arterial disease (PAD). Although current guidelines recommend supervised exercise training (SET) for PAD patients with intermittent claudication, its contribution to risk reduction remains unclear. Aim of the present study was to assess the impact of SET on inflammation and platelet activation as surrogates for cardiovascular risk.

METHODS

Fifty-three patients with intermittent claudication were randomly assigned to SET on top of best medical treatment (BMT) for 6 months (SET-group) or to BMT only (BMT-group). High sensitivity C-reactive protein (hs-CRP), interleukin-6 (IL-6) and fibrinogen as well as soluble P-selectin (sP-sel), <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>.<em>2</em>) and monocyte-platelet aggregates (MPA) were determined at study entry, after 3, 6 and <em>1</em><em>2</em> months.

RESULTS

While clinical improvement, reflected by an increase of walking capacity, was observed upon SET, no lasting changes of markers of inflammation and platelet activation were found within the SET-group during the training period. Compared to the BMT-group no improvements of these markers were observed in response to training at any time point (all p >0.05).

CONCLUSIONS

Regular SET added no further anti-inflammatory effect and had no effect on platelet activation when provided on top of BMT in PAD patients with intermittent claudication.

Restenosis is a major problem of percutaneous transluminal coronary angioplasty (PTCA) and related procedures. To better understand the underlying pathophysiologic mechanisms, coagulation and fibrinolytic variables were analysed prospectively in 35 patients after directional coronary atherectomy (DCA) and in <em>2</em>0 control patients undergoing diagnostic heart catheterisation and coronary angiography. Blood samples were taken before and <em>1</em> h, <em>2</em>4 h and 48 h after the procedure. No subacute thrombosis or unstable angina were documented in any patient. In 8 out of these 35 patients late restenosis was diagnosed during follow-up angiography 3-6 months after DCA. In these 8 patients <em>prothrombin</em> <em>fragments</em> (F<em>1</em> + <em>2</em>) rose from 0.7 to 0.9 nmol/l (P < 0.0<em>1</em>) and thrombin-antithrombin III complexes (TAT) from <em>2</em>.9 to 6.0 micrograms/l (P < 0.0<em>1</em>), but not significantly in <em>2</em>7 patients without restenosis and in the control patients. In patients with late restenosis plasminogen activator inhibitor (PAI-<em>1</em>) also increased from <em>2</em>.4 to 4.9 U/ml (P < 0.05) <em>2</em>4 h after DCA while there were no significant changes in patients without restenosis and in control patients. D-Dimer/TAT ratio reflecting the balance between clotting activation and fibrinolysis was significantly lower after <em>2</em>4 h in restenosis patients. The findings suggest that coagulation activation and hypofibrinolysis during 48 h after DCA might be associated with the development of late restenosis.

BACKGROUND

It has recently been suggested that recombinant FSH administration may result in an increased risk of venous thrombosis. An open-label, randomized, controlled trial was carried out to compare the impact of urinary and recombinant FSH on haemostasis.

METHODS

Fifty infertile women were randomized, using a random number generator on a personal computer, to receive either highly purified urinary FSH (u-hFSH) or recombinant human FSH (r-hFSH); a starting dose of <em>1</em>50 IU. Human chorionic gonadotrophin <em>1</em>0000 IU was administered once there was at least one follicle>> or =<em>1</em>8 mm. The luteal phase was supported with progesterone 50 mg/day for at least <em>1</em>5 days. Fifty normally menstruating women were recruited as controls. Repeated measurements of estradiol, progesterone, <em>prothrombin</em> time (PT) expressed as INR, activated partial thromboplastin time (APTT) ratio, fibrinogen (FBG), factor VIII (FVIII), normalized activated protein C ratio (nAPC ratio), antithrombin III activity (AT), protein C activity (PC), protein S activity (PS), tissue-type plasminogen activator antigen (t-PA), type <em>1</em> plasminogen activator inhibitor (PAI), <em>prothrombin</em> <em>fragments</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>), were performed during both hyperstimulated and natural cycles, and at onset of the following menstruation or at 8 weeks of pregnancy.

RESULTS

At the end of gonadotrophin administration PT INR increased in the u-hFSH group, while AT and t-PA significantly decreased. In the patients treated with r-hFSH, only F<em>1</em>+<em>2</em> significantly decreased. No significant changes were observed in the control group. In the luteal phase FBG increased significantly in all groups. In the u-hFSH group no other significant changes were noted compared to pre-ovulatory values, while compared to baseline values AT, PS and t-PA significantly decreased. In the r-hFSH group during the luteal phase PT INR significantly decreased, but did not differ from baseline levels. Other parameters such as FBG, FVIII, t-PA, rose significantly, but only FVIII and FBG values were significantly higher than baseline levels. In the women who became pregnant a significant increase in t-PA and a significant decrease in PS at the midluteal phase were observed. After one month all the haemostatic parameters returned to baseline value if pregnancy failed to occur, while in the pregnant women a significant increase in FVIII and a significant decrease in PS were observed.

CONCLUSIONS

Ovarian stimulation with recombinant FSH does not influence coagulation and fibrinolysis significantly, as already reported for urinary gonadotrophins. The moderate changes induced by both treatments are no longer detectable after 4 weeks.

Nineteen patients with symptoms of chronic venous insufficiency (CVI) were treated with <em>1</em>3-week cycles of intermittent pneumatic compression (IPC) during <em>2</em> h sessions twice weekly, with most treatments at home. At study completion, quantitative subjective scores for total symptomatology were improved in <em>1</em>6/<em>1</em>9 patients (84%). Enhancement of fibrinolytic potential in vivo was detected in 86% of observations on specimens from CVI patients over <em>2</em> h of IPC, with accelerated euglobulin clot lysis times (ELT) noted within <em>1</em>5 min of initiating compression. The enhanced fibrinolytic potential was attributed to increased urokinase plasminogen activator (u-PA), probably released from perturbed endothelial cells by IPC. Significant decreases in total t-PA antigen (mass concentration) but not t-PA activity, were produced by IPC in CVI patients only (P = 0.000<em>1</em>), with greater effects noted in the non-anticoagulated versus the anticoagulated cohort. Plasminogen activator inhibitor type <em>1</em> (PAI-<em>1</em>) levels rose rapidly after IPC only in the controls and non-anticoagulated CVI patients. PAI-<em>1</em> decreased in those receiving anticoagulation. No platelet perturbation was detected during IPC by measuring levels of beta-thromboglobulin or the thromboxane A<em>2</em> metabolite, <em>1</em><em>1</em>-dehydrothromboxane B<em>2</em>; however, significant (P < 0.003) decreases in plasma prostacyclin (PGI<em>2</em>) levels (measured as the stable 6-ketoprostaglandin F-<em>1</em>-alpha-metabolite) were observed after <em>1</em>5 min of IPC in non-anticoagulated CVI patients only. There was no evidence of increased thrombin generation by IPC, determined by urinary excretion of fibrinopeptide A and <em>prothrombin</em> <em>fragment</em> <em>1</em>. Concurrent anticoagulation appears to mediate more favorable biochemical alterations in CVI, although subjective improvement did not correlate with anticoagulation. The mechanism(s) by which these physiologic changes compliment the mechanical effects of IPC remain to be elucidated and will require adequately controlled and powered studies.