During the course of <em>prothrombin</em> activation, as catalyzed by Factor Xa, Factor Va, Ca<em>2</em>+, and negatively-charged phospholipid vesicles, the three proteins distribute between the fluid phase and the vesicle surface. On the vesicle, efficient Factor Xa-catalyzed proteolysis yields thrombin plus <em>Fragment</em> <em>1</em>.<em>2</em>. Further thrombin-catalyzed feedback cleavage of the latter then yields <em>Fragment</em> <em>1</em> plus <em>Fragment</em> <em>2</em>. Prior to this cleavage <em>Fragment</em> <em>1</em>.<em>2</em> might retain thrombin at the site of catalysis since it binds both phospholipid and thrombin through its respective <em>Fragment</em> <em>1</em> and <em>Fragment</em> <em>2</em> domains. In order to study the role of the feedback cleavage, light scattering at right angles was used to deduce the nature of the components associated with the vesicle during <em>prothrombin</em> activation by continuous monitoring of the relative molecular weight of the vesicle-protein complex. When <em>prothrombin</em> (<em>1</em>.4 microM) was added to homogeneously sized phospholipid vesicles of phosphatidylcholine-phosphatidylserine (3:<em>1</em>) at a total phospholipid concentration of <em>2</em>0 microM, the scattering intensity doubled. Upon subsequent addition of Factor Xa and Factor Va (5.0 nM each) the scattering intensity smoothly decreased to a value about <em>1</em>.<em>2</em>5-fold greater than that of the vesicles alone. Analysis of the composition of the reaction mixture at intervals during the course of the reaction by gel electrophoresis and laser densitometry, provided a good correlation between the mass of the vesicle-protein complex measured by light scattering and its mass inferred by composition. In addition, the decrease in mass of the vesicle-protein complex measured by light scattering correlated temporally with cleavage of <em>Fragment</em> <em>1</em>.<em>2</em>. When the reaction was initiated in the presence of the reversible thrombin inhibitor dansylarginine-N-(3-ethyl-<em>1</em>,5-pentanediyl)amide no cleavage of <em>Fragment</em> <em>1</em>.<em>2</em> occurred, as indicated by gel electrophoresis, and no change in the mass of the vesicle-protein complex occurred as indicated by light scattering. The absence of change in scattering intensity in the presence of dansylarginine-N-(3-ethyl-<em>1</em>,5-pentanediyl)amide suggests a <em>1</em>:<em>1</em> replacement of <em>prothrombin</em> at the catalytic surface by components of equivalent mass (<em>Fragment</em> <em>1</em>.<em>2</em> plus thrombin), whereas the decrease in scattering in the absence of dansylarginine-N-(3-ethyl-<em>1</em>,5-pentanediyl)amide suggests replacement of <em>prothrombin</em> by <em>Fragment</em> <em>1</em> only. Together these results indicate that the thrombin-catalyzed cleavage of <em>Fragment</em> <em>1</em>.<em>2</em> promotes release of thrombin from the catalytic surface.

<AbstractText>Thrombin generation, thrombin-antithrombin complex (TAT) levels, and <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>) have shown potential as biomarkers of thromboembolic risk. The aims were to establish reference intervals for these three biomarkers and to assess the levels in patients with localized cancer compared with healthy individuals.</AbstractText><AbstractText>We included <em>1</em><em>2</em>4 healthy individuals (57 females and 67 males; aged <em>2</em><em>1</em>-66 years), 86 patients with low-stage primary lung cancer, and 57 patients with localized head and neck cancer. Thrombin generation was determined by the calibrated automated thrombogram using platelet-poor-plasma reagent containing <em>1</em> pM tissue factor and 4 μM phospholipids. TAT and F<em>1</em>+<em>2</em> were measured using commercial enzyme-linked immunosorbent assays. Reference intervals were calculated as mean ± <em>1</em>.96 × standard deviation (thrombin generation and F<em>1</em>+<em>2</em>) or <em>2</em>.5th to 97.5th percentiles (TAT).</AbstractText><AbstractText>The reference intervals for thrombin generation parameters were: lag time 4.4-9.4 min, peak thrombin 46-<em>2</em>88 nM, time-to-peak thrombin 8-<em>1</em>5 min, and endogenous thrombin potential 554-<em>1</em>95<em>2</em> nM x min. The reference interval for TAT was ≤<em>1</em>3 μg/l, and for F<em>1</em>+<em>2</em> it was 47-3<em>2</em>0 pmol/l. Both low-stage primary lung cancer and head and neck cancer patients had significantly higher TAT (p <0.000<em>1</em>) and F<em>1</em>+<em>2</em> (p < 0.000<em>1</em>) concentrations than healthy individuals. However, this was not reflected in the thrombin generation assay.</AbstractText><AbstractText>Reference intervals for thrombin generation, TAT as well as F<em>1</em>+<em>2</em> were established. Patients with localized cancer had significantly elevated TAT and F<em>1</em>+<em>2</em>. TAT and F<em>1</em>+<em>2</em> may hold potential for identifying hypercoagulation in cancer patients.</AbstractText>

BACKGROUND

Aspirin has been routinely prescribed following transcatheter closure of secundum atrial septal defects (ASDs) but its rationale has not been clinically or biologically evaluated; and despite aspirin, thrombotic complications occur following transcatheter ASD closure. We therefore evaluated the presence, degree and timing of the activation of the coagulation and platelet systems following transcatheter closure of ASDs.

RESULTS

Fourteen consecutive patients (9 females, mean age 4<em>1</em>+/-<em>2</em><em>2</em> years) who underwent successful transcatheter closure of an ASD defect with the Amplatzer septal occluder were prospectively studied. Measurements of the <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>) levels and the percentage of activated platelets (determined by P-selectin expression detected by flow cytometry) were taken at baseline just before the procedure, and at <em>1</em>, 7, 30 and 90 days following device implantation. F<em>1</em>+<em>2</em> levels increased from 0.85+/-0.<em>2</em>9 nmol/l at baseline to a maximal value of <em>1</em>.<em>2</em>0+/-0.5<em>2</em> nmol/l at 7 days, gradually returning to the baseline levels at 90 days (0.79+/-0.54 nmol/l) (p<0.00<em>1</em>). F<em>1</em>+<em>2</em> levels at 7 days were also significantly higher than those obtained in a control group of <em>2</em>0 healthy subjects (p=0.0<em>1</em>6). A greater increase in coagulation activation was observed in cases of residual shunt following ASD closure (r=0.53, p=0.050). No significant variations in the percentage of platelets expressing P-selectin were detected at any time.

CONCLUSIONS

Transcatheter closure of ASDs with the Amplatzer septal occluder was associated with a significant increase in F<em>1</em>+<em>2</em> levels during the first week after device implantation, but there was no detectable effect on platelet system activation. These findings raise the question whether the optimal prophylactic approach following transcatheter ASD closure should be anticoagulant instead of antiplatelet therapy.

To assess the degree of haemostatic system activation, plasma levels of <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>), a direct indicator for thrombin generation in vivo, were measured in 49 patients with thrombotic disease undergoing long-term warfarin therapy (Thrombotest values < or = 40%). In these patients, vitamin K dependent coagulation factors (factors II, VII, IX and X) were decreased together with the anticoagulant proteins C and S, but the mean plasma concentration of F<em>1</em> + <em>2</em> was significantly decreased compared with 48 healthy subjects. In warfarin-treated patients, F<em>1</em> + <em>2</em> was positively correlated with the Thrombotest value, factors II, VII, IX and X. When analysed according to the intensity of anticoagulation, patients with Thrombotest values less than 30% showed a significant decrease in F<em>1</em> + <em>2</em>, but the mean F<em>1</em> + <em>2</em> level was normal in patients with Thrombotests higher than 30%. These findings indicate that long-term oral anticoagulant therapy suppresses thrombin generation approximately in parallel to the decrease in coagulation factors, and levels of F<em>1</em> + <em>2</em> lower than healthy subjects are observed when Thrombotest values are less than 30%.

A monoclonal antibody (mAb 5A5G<em>2</em>) recognized cleaved plasma protein S (PS) but not uncleaved PS. Interestingly, mAb 5A5G<em>2</em> did not recognize thrombin-cleaved recombinant PS. Microsequencing of cleaved plasma PS showed a Q-S-T-N amino-terminal sequence, inferring cleavage after the Arg 60 residue. The mAb epitope was located within the sequence encompassing residues 6<em>1</em> to 73, i.e. the carboxy-terminal part of the thrombin-sensitive region (TSR). We used this mAb to develop an ELISA assay to quantify in vivo cleaved PS. In plasma from <em>1</em>0 normal subjects, about <em>1</em>0% of PS was cleaved (7.<em>1</em>% to <em>1</em>5.4%), with a more than <em>2</em>-fold increase in the corresponding sera. We found increased levels of cleaved PS in 8 patients with disseminated intravascular coagulation (DIC) and decreased levels in <em>2</em><em>2</em> patients on long-term oral anticoagulant therapy, whereas cleaved PS levels were similar in 8 hemophiliacs and the <em>1</em>0 normal subjects. Cleaved PS levels did not correlate with <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> levels released after cleavage by FXa in any of the groups, suggesting that circulating FXa is not the main factor involved in the production of cleaved PS in vivo.

The function of a newly devised bioartificial liver (AMC-BAL) based on viable, freshly isolated porcine hepatocytes has been evaluated in anhepatic pigs. The aim of this study was to assess the contribution of BAL treatment on blood coagulation parameters. Pigs were anesthetized and a total hepatectomy was performed (n = <em>1</em>5). The infrahepatic caval vein and the portal vein were connected to the subdiaphragmatic caval vein using a three-way prosthesis. Animals received standard intensive care (control, n= 5), treatment with an empty BAL (device control, n= 5) or with a cell-loaded BAL (BAL-treatment, n= 5) for a period of <em>2</em>4 h starting <em>2</em>4 h after hepatectomy. Coagulation parameters studied concerned <em>prothrombin</em> time (PT), platelet count, the procoagulant system (factors (F)II, FV, FVII, FVIII and fibrinogen), anticoagulant system (AT III), fibrinolytic system (t-PA, PAI-<em>1</em>) as well as markers of coagulation factor activation (TAT complexes, <em>prothrombin</em> <em>fragment</em> F<em>1</em> + <em>2</em>). FII, FV, FVII, AT III and fibrinogen rapidly decreased after total hepatectomy in pigs in accordance with the anhepatic state of the animals. FVIII levels were not influenced by the hepatectomy. A mild drop in platelet count was seen in all groups. Treatment of anhepatic pigs with the cell-loaded BAL did not restore PT or clotting factor levels. TAT and F<em>1</em> + <em>2</em> complexes, however, were significantly increased in this group. Levels of t-PA and PAI-<em>1</em> were not influenced by cell-loaded BAL treatment. Treatment of anhepatic pigs with the AMC-BAL based on freshly isolated porcine hepatocytes does not result in an improved coagulation state due to extensive consumption of clotting factors. However, increased levels of TAT complexes and <em>prothrombin</em> <em>fragments</em> F<em>1</em> + <em>2</em> during treatment of anhepatic pigs indicate synthesis and direct activation of coagulation factors, leading to thrombin generation. This demonstrates that this bioartificial liver is capable of synthesizing coagulation factors.

Antiphospholipid antibodies (aPL) have been found to be associated with arterial and venous thrombosis. Percutaneous transluminal coronary angioplasty (PTCA) is an established therapy for ischaemic heart disease (IHD), which is still affected by restenosis at a rate of <em>2</em>0-30%. This study was aimed at investigating the possible role of aPL in restenosis after PTCA. In sixty consecutive IHD patients, aPL (lupus anticoagulant -LA- and anticardiolipin antibodies -aCL) and markers of haemostatic activation were investigated before PTCA, and patients were followed up for restenosis. No infections, autoimmune disease or treatment by drugs that may alter aPL levels occurred in any of the patients. aPL were found in <em>1</em>5/60 patients: aCL in 7/60, LA in 5/60 and aCL and LA in 3/60. No statistically significant difference was found between aPL negative and aPL positive patients in pre PTCA plasma levels of <em>prothrombin</em> activation <em>fragment</em> (F<em>1</em>+<em>2</em>) <em>1</em>.4 nmol/l (0.3-5.7<em>1</em>) vs <em>1</em>.4 nmol/l (0.9-4.0), thrombin-antithrombin complex (TAT) 4.0 microg/l (<em>1</em>.<em>1</em>-34.<em>2</em>) vs 5.<em>2</em> microg/l (<em>2</em>.<em>1</em>-60.0), D-dimer (DD) <em>2</em>5 ng/ml (<em>2</em>-5<em>1</em>5) vs 44 ng/ml (<em>2</em>-<em>1</em>60) or plasminogen activator inhibitor activity (PAI) 4.8 IU/ml (<em>2</em>.5-36.4) vs 4.4 IU/ml (<em>2</em>.5-<em>1</em>3.4). Restenosis was observed in <em>1</em>3/60 patients (7/45-<em>1</em>5% - aPL negative and 6/<em>1</em>5-40% - aPL positive patients) who underwent angiographic tests after PTCA because of recurring angina or positive exercise test. Restenosis occurred after <em>2</em>.<em>2</em> months (0.5-3) in aPL positive patients and after 3.5 months (<em>1</em>-<em>1</em><em>2</em>.8) in aPL negative. These results suggest that <em>1</em>) restenosis with recurrent ischaemia occurs more frequently in aPL positive than in aPL negative patients, <em>2</em>) in aPL positive patients restenosis occurs earlier, and 3) the presence of aPL is not associated with hypercoagulability.

Nafamostat mesilate (FUT) is a synthetic serine protease inhibitor with a short half-life that is used during hemodialysis (HD) in patients with a high risk of bleeding because it does not prolong the systemic coagulation time. To evaluate whether or not FUT is able to effectively prevent clot formation in the extracorporeal circuit without increasing systemic bleeding, HD using FUT was carried out for 33 sessions in <em>1</em><em>2</em> patients with a high risk of bleeding. FUT was continuously infused during HD at <em>2</em>0-40 mg/h to maintain a <em>2</em>-fold prolongation of activated partial thromboplastin time (APTT) at the dialyzer outlet on the venous side of the circuit. No APTT prolongation was observed on the arterial side of the circuit before FUT infusion, and none of the patients showed increased bleeding during or after HD. However, clots formed in the arterial chamber (30.3%), the dialyzer (36.6%), and the venous chamber (<em>1</em>5.<em>1</em>%). In <em>2</em> of the <em>1</em><em>2</em> patients, HD was discontinued due to clot formation despite sufficient prolongation of APTT. The mean levels of the thrombin-antithrombin III complex and <em>prothrombin</em> activation <em>fragment</em> <em>1</em> + <em>2</em> in the circuit gradually increased on both the arterial and venous sides during HD using FUT, and protein C activity decreased. No significant changes in these parameters occurred during heparin HD in the same patients after the bleeding episode had resolved. Despite sufficient prolongation of APTT in the circuit, FUT was less effective in suppressing thrombin generation when compared to heparin.(ABSTRACT TRUNCATED AT <em>2</em>50 WORDS)

BACKGROUND

Heparin inhibits prothrombotic tissue factor (TF) and releases its inhibitor, tissue factor pathway inhibitor (TFPI), from the endothelium, but repeated administration of heparin depletes vascular stores of TFPI. We studied the anticoagulant effects of unfractionated heparin (UFH) vs low-molecular-weight enoxaparin-used for thrice-weekly maintenance haemodialysis (HD)-on plasma levels of total TF and TFPI and on those of an activated coagulation marker <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (PF <em>1</em>+<em>2</em>).

METHODS

Twenty-five patients dialysed using a single injection of enoxaparin (at a mean dose of 0.68 mg/kg) were randomly assigned to either receive UFH administered as a mean bolus of 4<em>2</em>.<em>1</em> IU/kg and continuous infusion of 57.8 IU/kg (n=<em>1</em><em>2</em>) or to be maintained on enoxaparin (n=<em>1</em>3), and were followed prospectively for <em>1</em><em>2</em> weeks. Plasma immunoreactive TF, TFPI and PF <em>1</em>+<em>2</em> were measured at the start and after <em>1</em>0 and <em>1</em>80 min of HD, and compared with values in <em>1</em>5 healthy controls.

RESULTS

Pre-dialysis TF, TFPI and PF <em>1</em>+<em>2</em> were higher than normal (all P<0.000<em>1</em>). TF and PF <em>1</em>+<em>2</em> did not change, while TFPI levels, compared with baseline, increased at each interval in enoxaparin-anticoagulated HD patients (all P<0.000<em>1</em>). TFPI increments correlated inversely with pre-dialysis TFPI (both P<0.0007). In patients switched to UFH, TF levels remained unchanged compared with pre-randomization values, TFPI increased at each interval of HD sessions (all P<0.035) and PF <em>1</em>+<em>2</em> increased pre-dialysis (P=0.0<em>1</em>5). The over-dialysis effects of UFH resembled those of enoxaparin. In contrast, baseline TFPI and its <em>1</em>0-min rise correlated inversely with the UFH loading dose (both P<0.040). Pre-dialysis PF <em>1</em>+<em>2</em> was inversely associated with TFPI increments (both P<0.034), and directly with pre-dialysis TFPI (P=0.0<em>1</em>8) and the UFH loading dose (P=0.045).

CONCLUSIONS

Depletion of heparin-releasable stores of TFPI is an untoward effect of repeated anticoagulation during maintenance HD therapy. The traditional UFH regimen is more prothrombotic than single enoxaparin injections, with high loading doses of UFH being involved in TFPI exhaustion and subsequent hypercoagulability.

BACKGROUND

Low-molecular-weight heparins (LMWHs) are an alternative to unfractionated heparin (UFH) for anticoagulation during hemodialysis (HD). We performed a prospective randomized crossover study of the effect of enoxaparin, nadroparin, and dalteparin on some hemostatic factors, including tissue factor pathway inhibitor (TFPI), in patients with maintenance HD.

METHODS

Plasma levels (immunoassays) of total TFPI, platelet-derived growth factor-AB (PDGF-AB), and <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (PF <em>1</em> + <em>2</em>) were evaluated pre-HD, after <em>1</em>0 (T<em>1</em>0) and <em>1</em>80 (T<em>1</em>80) minutes of HD in <em>2</em><em>1</em> patients, who completed a 3-period (for <em>2</em> months each) crossover study in 6 groups (Latin-square design).

RESULTS

The baseline TFPI, PDGF-AB, and PF <em>1</em> + <em>2</em> levels were comparable under all LMWH treatments. Tissue factor pathway inhibitor levels, compared with the baseline, significantly increased (all P < <em>1</em>0(-4)), whereas PDGF-AB levels remained stable at each interval during enoxaparin, nadroparin, and dalteparin anticoagulated HD. Interestingly, TFPI increment at T<em>1</em>0 was the highest, dose-dependent, and accompanied by PF <em>1</em> + <em>2</em> decrease under enoxaparin administration.

CONCLUSIONS

The switch from enoxaparin to nadroparin and dalteparin used as anticoagulants had no long-term effect on the baseline total TFPI and PF <em>1</em> + <em>2</em> levels in chronically HD patients. Only short-term, overdialytic differences were noticed, indicating a single bolus of enoxaparin (0.75 mg/kg) as the most potent stimulus for endothelial TFPI.

Molecular imaging of inflammatory mediators in atria may contribute to thrombotic risk assessment of atrial fibrillation (AF). We investigated the feasibility of ultrasound molecular imaging (UMI) targeted to P-selectin to assess thrombotic risk in AF. Rat AF models were established with rapid atrial pacing. Microbubbles targeted to P-selectin were injected into the rats, followed by left atrial (LA) UMI examination. Furthermore, P-selectin, platelets (PLTs), fibrin and tissue factor (TF) of LA were detected by histopathology and scanning electron microscopy. Plasma levels of P-selectin, thrombin-antithrombin complex (TAT) and <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>) were measured by enzyme-linked immunosorbent assay. The data showed that P-selectin in LA was correlated with PLT, fibrin and TF (r = 0.735, p < 0.05; r = 0.8<em>2</em>7, p < 0.05; r = 0.785, p < 0.05, respectively). The plasma level of P-selectin was correlated with the expression of TAT and F<em>1</em> + <em>2</em> (r = 0.866, p < 0.05; r = 0.9<em>1</em>6, p < 0.05, respectively). The contrast video intensity of adhered microbubbles targeted to P-selectin was correlated with the levels of P-selectin, PLT and fibrin in LA (r = 0.768, p < 0.05; r = 0.798, p < 0.05; r = 0.745, p < 0.05, respectively). In conclusion, P-selectin may serve as a biomarker for thrombotic risk in AF and can be quantified by UMI to assess thrombotic risk.

BACKGROUND

Precise evaluation of the haemocompatibility of prototype membranes, flow configurations and anticoagulant regimens is an essential step in the development of dialysis systems minimizing blood activation. An ex vivo model in humans currently employed in our laboratory has recently been adapted to allow the parallel evaluation of two minimodule dialysers with blood from a single donor, thus eliminating differences due to donor variability in the comparison of test and control dialysis modules.

METHODS

The ex vivo flow system is designed to reproduce the haemodynamic conditions of clinical dialysis on a <em>1</em>/50 scale. A blood line from the forearm vein of the volunteer donor is divided at a Y-shaped junction, two roller pumps assure equivalent blood flow (5 ml/min) in the branches leading to two minimodule dialysers and heparin (0.<em>1</em> IU/ml final concentration) is injected into each branch immediately after the Y junction. Samples for analysis of blood activation markers are collected at the exits of the two minimodules over a test period of <em>2</em>7 min. In the present series of tests, a new polyacrylonitrile membrane (PAN) was evaluated relative to standard commercial polysulphone (PS), acrylonitrile copolymer (AN 69) and cuprophan (CUP) membranes.

RESULTS

A steady minimal level of anticoagulation corresponding to a slightly less than two-fold prolongation of APTT (activated partial thromboplastin time) was maintained throughout testing in both branches of the ex vivo flow system. Time curves for the accumulation of activation markers (thrombin-antithrombin III complexes, <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>, platelet beta-thromboglobulin, and complement <em>fragment</em> C3a) showed all four types of minimodule dialyser to induce comparable low levels of activation of coagulation parameters and platelets, together with similar mild activation of complement for AN 69, PAN, and PS dialysers as compared to stronger activation for CUP modules. Overall results thus confirmed the acceptable haemocompatibility of the prototype polyacrylonitrile (PAN) membrane.

CONCLUSIONS

Among current methods for evaluation of the biocompatibility of haemodialysis systems, ex vivo flow models in humans avoid problems arising from species differences and may be designed to closely reproduce the conditions of clinical dialysis. A parallel configuration eliminates artefacts due to individual variations in donor response. This not only facilitates the direct comparison of test and control membranes under close to identical experimental conditions, but also provides a model particularly well adapted to studies of the effects of different anticoagulation regimens, flow configurations, and dialysates, or alternative methods of sterilization, rinsing, and priming of the dialysers.

Chronic Fatigue and/or Fibromyalgia have long been diseases without definition. An explanatory model of coagulation activation has been demonstrated through use of the ISAC panel of five tests, including, Fibrinogen, <em>Prothrombin</em> <em>Fragment</em> <em>1</em>+<em>2</em>, Thrombin/ AntiThrombin Complexes, Soluble Fibrin Monomer, and Platelet Activation by flow cytometry. These tests show low level coagulation activation from immunoglobulins (Igs) as demonstrated by Anti-B<em>2</em>GPI antibodies, which allows classification of these diseases as a type of antiphospholipid antibody syndrome. The ISAC panel allows testing for diagnosis as well as monitoring for anticoagulation protocols in these patients.

We describe a patient with congenital afibrinogenemia who showed elevated <em>prothrombin</em> activation <em>fragments</em> (F<em>1</em> + <em>2</em>) indicating increased thrombin formation. This finding was unexpected since it has hitherto been thought that patients with congenital hypo- or afibrinogenemia have no evidence of increased utilization or accelerated consumption of coagulation factors. No other possible reasons for the elevation of F <em>1</em> + <em>2</em> were found. Upon fibrinogen substitution F<em>1</em> + <em>2</em> decreased and were again increasing when fibrinogen concentration in plasma fell to very low levels. These findings raise the question of whether increased thrombin formation should be understood as a compensatory mechanism in congenital afibrinogenemia.

OBJECTIVE

Periodontitis is associated with cardiovascular diseases (CVD). In our previous studies a prothrombotic state has been observed in periodontitis, which contributes to the risk of CVD. The aim of this study was to investigate whether serum IgG levels against Aggregatibacter actinomycetemcomitans (Aa) and Porphyromonas gingivalis (Pg) in periodontitis were associated with a prothrombotic state.

METHODS

Patients with moderate (n = 38) and severe periodontitis (n = 30) and controls (n = <em>2</em>4) were recruited. We explored correlations between serum anti-Aa and anti-Pg IgG and plasma levels of markers of prothrombotic state (von Willebrand Factor [vWF], <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> [F<em>1</em>+<em>2</em>], plasminogen activator inhibitor-<em>1</em> [PAI-<em>1</em>] and D-dimer). Multivariate analyses were performed considering several major potential contributing factors.

RESULTS

Periodontitis patients showed higher anti-Aa IgG (p = 0.0<em>1</em>5) than controls but not for Pg (p = 0.3<em>2</em>0). In periodontitis patients, body mass index and anti-Aa IgG showed a positive correlation with vWF (β = 0.<em>2</em>97, p = 0.0<em>1</em>0 and β = 0.<em>2</em>48, p = 0.033 respectively).

CONCLUSIONS

In periodontitis, infection with Aa together with other well accepted risk factors for CVD, may play a role in increasing the risk for prothrombotic state.

BACKGROUND

Hemorrhage causes significant morbidity and mortality in people aged <65 years. A lyophilized platelet-derived hemostatic agent (Thrombosomes) demonstrated hemostatic efficacy in animal models. We report the results of the first safety trial of autologous Thrombosomes given to normal subjects.

METHODS

Ten subjects received autologous Thrombosomes prepared from their apheresis platelets, and five control subjects received a buffer solution. There were five cohorts, with three subjects per cohort (two in the Thrombosomes group and one in the control group). Doses escalated from <em>1</em>/<em>1</em>,000 to <em>1</em>/<em>1</em>0 of a proposed efficacious dose. Cohorts 4 and 5 received the highest dose, but in Cohort 5, one-half the dose was infused <em>2</em> hours apart. Cohorts <em>1</em> through 3 were monitored for 4<em>2</em> days, Cohorts 4 and 5 were monitored for 60 days using hematology, coagulation, and chemistry assays and antibody testing.

RESULTS

There were no serious adverse events (AEs) and no subject withdrawals. There were eight treatment-related AEs (TRAEs) in 5 of <em>1</em>5 subjects (33%) (four in the Thrombosomes group and one in the control group). Of four subjects receiving the highest doses, three had TRAEs. One had elevated D-dimer, <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>, and white blood cell count (subject had concurrent upper respiratory tract infection); one had T-wave inversions in precordial leads V<em>2</em> and V3 without elevated troponin or symptoms; and one had a platelet autoantibody without change in platelet count. All subjects' TRAEs resolved by Day <em>2</em><em>1</em>.

CONCLUSIONS

There were no serious AEs in this small study. Thrombosomes were considered safe at the doses assessed. Future, larger trials will be needed to further assess safety and efficacy.

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs), together with extrinsic coagulation pathway activation and increased oxidative stress (SOX) have all been implicated as important factors in atherosclerosis and vascular remodelling. The aim of the present study was to investigate whether the MMPs/TIMPs system is associated with activation of the extrinsic coagulation pathway in conditions of increased SOX in hemodialysis (HD) patients. In HD patients, with and without cardiovascular disease (CVD), and in controls, we compared pre-dialysis levels of MMP-<em>2</em>, MMP-9,TIMP-<em>1</em>,TIMP-<em>2</em>; the markers of extrinsic coagulation pathway-tissue factor (TF) and its inhibitor (TFPI), <em>prothrombin</em> <em>fragment</em> F<em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>); a marker of SOX-Cu/Zn superoxide dismutase (Cu/Zn SOD) and a surrogate of atherosclerotic disease-intima media thickness (IMT). Hemodialysis patients, particularly those with CVD, showed a significant increase in values of MMP-<em>2</em>/TIMPs, markers of the extrinsic coagulation pathway, Cu/Zn SOD and IMT as compared to controls. The markers of coagulation activation positively correlated with the MMP-<em>2</em>/TIMPs system, whereas they did not correlate with MMP-9. In addition, both MMP-<em>2</em>/TIMPs as well as extrinsic coagulation parameters were related to the prevalence of increased SOX, IMT and CVD. Multiple stepwise regression analysis showed that low TIMP-<em>2</em> followed by increased Cu/Zn SOD and MMP-<em>2</em> levels independently and significantly predicted elevated IMT on maintenance HD patients. In conclusion, our data suggest that the MMP-<em>2</em>/TIMPs system and an activated extrinsic coagulation pathway could cooperate in conditions of elevated SOX and could influence arterial remodeling resulting in the presence of CVD in haemodialysis patients.

Chemical modification of bovine <em>prothrombin</em> <em>fragment</em> <em>1</em> according to the procedure of D. J. Welsch and G. L. Nelsestuen (<em>1</em>988) [Biochemistry <em>2</em>7, 4946-495<em>2</em> and ealier papers] provided a series of <em>fragment</em> <em>1</em> derivatives in which various nitrogen-containing side chains were N-acetylated and/or N-<em>2</em>,4,6-trinitrophenylated. In addition the des-[Ala-<em>1</em>,Asn-<em>2</em>]- and des-[Ala-<em>1</em>,Asn-<em>2</em>,Lys-3]-<em>fragment</em> <em>1</em> derivatives were prepared by limited enzymatic hydrolysis of <em>fragment</em> <em>1</em> using cathepsin C and plasmin, respectively. Quantitative studies on the Ca(II) binding of these proteins have been accomplished using 45Ca(II) equilibrium dialysis. Binding of these <em>fragment</em> <em>1</em> derivatives to phosphatidylserine/phosphatidylcholine (PS/PC) vesicles (<em>2</em>5:75) in the presence of Ca(II) ions has been studied using the light-scattering technique. Acylation of the 5 lysine residues of <em>fragment</em> <em>1</em> by the action of acetic anhydride (500-fold molar excess) in the presence of 75 mM Ca(II), pH 8.0, results in loss of positive cooperativity in Ca(II) binding (Scatchard plot) and an increase in the number of Ca(II) ions bound. The Ca(II)-dependent PS/PC binding of the acylated protein is reduced. Removal of <em>2</em> and 3 residues from the amino terminus likewise leads to loss of positive cooperativity in Ca(II) binding and reduced binding affinity to PS/PC vesicles. The important role of the amino-terminal <em>1</em>-<em>1</em>0 sequence is discussed. We conclude that positive cooperativity in Ca(II) binding is not a prerequisite for the Ca(II)-dependent binding of bovine <em>prothrombin</em> <em>fragment</em> <em>1</em> to PS/PC vesicles.

OBJECTIVE

The present study is aimed at gaining insight into coagulation and fibrinolysis in the peritoneal cavity of patients on continuous ambulatory peritoneal dialysis (CAPD). For this purpose we measured coagulation- and fibrinolysis-related antigens in plasma and dialysate, comparing patients with and without peritonitis.

METHODS

Markers of activated coagulation and fibrinolysis in plasma and dialysate of CAPD patients were determined at different time points (0 hr, <em>2</em> hr, 4 hr) after infusion of the dialysis solution in the peritoneal cavity. <em>Prothrombin</em> <em>fragment</em> (F<em>1</em> + <em>2</em>), thrombin-antithrombin III complex (TAT), and fibrin monomer (FM) were chosen as parameters of activated coagulation. Fibrin degradation products (FbDP), D-dimer (DD), tissue-type plasminogen activator (t-PA), and plasminogen activator inhibitor type <em>1</em> (PAI-<em>1</em>) were measured as parameters for ongoing fibrinolysis. Beta <em>2</em>-microglobulin, albumin, and IgG were used as marker proteins for the diffusion of proteins of intravascular origin into the peritoneal cavity.

METHODS

Eleven clinically stable CAPD patients, who had not suffered from peritonitis during the last six months, and 5 CAPD patients with an acute episode of bacterial peritonitis were studied.

RESULTS

In the dialysate of stable CAPD patients (n = <em>1</em><em>1</em>) the concentration of activation markers of coagulation and fibrinolysis increased continuously with dwell time. After four hours we found remarkably high levels of the coagulation markers F<em>1</em> + <em>2</em> (0.4 +/- 0.<em>1</em> nmol/L), TAT (6.5 +/- <em>1</em>.0 ng/mL), and FM (<em>2</em>4.5 +/- 7.<em>1</em> micrograms/mL), and the fibrinolysis markers DD (85<em>1</em> +/- <em>2</em>6 ng/mL), FbDP (<em>1</em>.0 +/- 0.3 microgram/mL), t-PA (3.3 +/- 0.8 ng/mL), and PAI-<em>1</em> (<em>2</em>.6 +/- <em>1</em>.<em>2</em> ng/mL). The dialysate-to-plasma (D/P) ratios of all of these antigens were significantly higher compared to the D/P ratios of proteins with similar molecular weight, which are not produced intraperitoneally (beta <em>2</em>-microglobulin, albumin, and IgG). These findings point to a local, thrombin-induced intraperitoneal fibrin generation during regular CAPD. Compared with clinically stable CAPD patients, the patients with bacterial peritonitis (n = 5) had significantly higher levels of F<em>1</em> + <em>2</em> (5.3 +/- <em>1</em>.6 nmol/L), TAT (57.8 +/- <em>1</em>0.7 ng/mL), FM (97<em>2</em> +/- 3.<em>2</em> micrograms/L), FbDP (<em>1</em>6.4 +/- <em>2</em>.9 micrograms/L), and PAI-<em>1</em> (7.3 +/- <em>2</em>.4 ng/mL) in the dialysate (4-hr dwell time), and a <em>2</em>.4-times higher ratio between FM and FbDP. These results can be interpreted as an intraperitoneal imbalance between coagulation and fibrinolysis during peritonitis.

CONCLUSIONS

Our study demonstrates a high intraperitoneal fibrin formation, not only during peritonitis but also in clinically stable CAPD patients. The remarkably high levels of coagulation (F<em>1</em> + <em>2</em>, TAT, FM) and fibrinolysis (FbDP, DD, t-PA, PAI-<em>1</em>) related antigens in the dialysate of patients without peritonitis cannot be explained by transport from plasma into the peritoneal cavity and may reflect a high rate of intraperitoneal fibrin turnover. The balance between peritoneal generation and degradation of fibrin is obviously disturbed in CAPD patients with peritonitis, who had significantly higher levels of coagulation markers in the dialysate and a higher ratio between FM and FbDP.

Several prospective studies have demonstrated that high plasma fibrinogen levels are associated with an increased risk of ischemic heart disease. Since in most patients an increased thrombin generation has been reported, we investigated whether the control of thrombin generation could affect plasma fibrinogen levels. Forty male outpatients (<em>2</em>0 asymptomatic with previous myocardial infarction and <em>2</em>0 with stable effort angina) were enrolled in a randomized medium-term (6 months) cross-over study. Clottable fibrinogen, according to Clauss, <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>, thrombin-antithrombin complex, and fibrinopeptide A were evaluated in relation to treatment with low-dose heparin. After a <em>1</em>5-day wash-out period, during which patients had been treated only with nitrates if needed, patients were allocated to two sequential periods of treatment with standard heparin (<em>1</em><em>2</em>,500 U, subcutaneously daily) plus antianginal treatment or antianginal treatment alone, separated by a second <em>1</em>5-day wash-out period. At the end of the treatment period with low-dose heparin significant decreases in the plasma fibrinogen (<em>2</em>.5 +/- 0.6 g/l vs. 3.3 +/- 0.5 g/l, P < 0.00<em>1</em>), <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (<em>1</em>.4 +/- 0.5 nmol/l vs. <em>1</em>.9 +/- 0.7 nmol/l, P < 0.00<em>1</em>), thrombinantithrombin (4.5 +/- <em>2</em>.4 ng/ml vs. 9.7 +/- 3.6 ng/ml, P < 0.00<em>1</em>), and fibrinopeptide A (<em>2</em>.<em>1</em> +/- <em>1</em>.<em>1</em> ng/ml vs. 3.5 +/- <em>2</em>.<em>1</em> ng/ml, P < 0.00<em>1</em>) were observed compared with the period without heparin. The present results indicate that low-dose heparin can effectively control the increased abnormal thrombin generation and elevated fibrinogen levels in patients with ischemic heart disease, possibly decreasing the risk of cardiovascular death.

Plasma and red blood cell quality are affected both by citrate concentration and the levels of extracellular leukocyte and platelet derived substances, accumulated during storage of blood. The effect of leukocyte filtration on the storage stability of whole blood was therefore studied in blood collected in standard CPD and 0.5CPD (CPD with half strength citrate concentration). A total of 5<em>2</em> units, <em>1</em><em>2</em> of them with reduced citrate concentration, were leukocyte-filtered with Pall( whole blood filter (WBF<em>1</em> or 3). No differences in leukocyte or platelet reduction were observed with the two citrate concentrations. However, with 0.5CPD a significantly longer filtration time and increased complement activation was observed. The effect of pre-storage leukocyte filtration on the plasma quality of whole blood was therefore only studied with standard CPDA<em>1</em> anticoagulant solution (normal strength citrate concentration). Leukocyte filtration did not affect the von Willebrand factor concentration, while a small reduction (7%, p=0.04) in factor VIII (FVIII) concentration was observed. During storage, however, FVIII decreased more slowly in the filtered than in the unfiltered product, and, from day two, the FVIII content was significantly higher in the filtered product (46% versus 30% at <em>2</em>8 days, p<0.00<em>1</em>). Factor V (FV) demonstrated a <em>1</em>6% reduction (p<0.00<em>1</em>) upon filtration, followed by an additional 8% in the next <em>2</em>4 h and only a 4% reduction the next <em>2</em>7 days, while unfiltered products demonstrated a continuous reduction to <em>2</em>6% at <em>2</em>8 days. While the beta-thromboglobulin (beta-TG) concentration significantly increased (from 836 to <em>2</em>483 IU/ml, p<0.00<em>1</em>) during leukocyte filtration, no further increase was observed during storage. In contrast, unfiltered products demonstrated an increase to 576<em>2</em> IU/ml (p<0.00<em>1</em>) at <em>1</em>4 days, followed by a slight, not significant, reduction. This indicates platelet activation during filtration and explains a parallel reduction in FV. Filtration induced no increase in <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, while a slight increase was observed in some unfiltered products after <em>2</em>8 days of storage.Pre-storage leukocyte depletion thus improves the coagulation factor content of plasma in stored whole blood.

Patients on rivaroxaban requiring percutaneous coronary intervention (PCI) represent a clinical conundrum. We aimed to investigate whether rivaroxaban, with or without an additional bolus of unfractionated heparin (UFH), effectively inhibits coagulation activation during PCI. Stable patients (n=<em>1</em>08) undergoing elective PCI and on stable dual antiplatelet therapy were randomised (<em>2</em>:<em>2</em>:<em>2</em>:<em>1</em>) to a short treatment course of rivaroxaban <em>1</em>0 mg (n=30), rivaroxaban <em>2</em>0 mg (n=3<em>2</em>), rivaroxaban <em>1</em>0 mg plus UFH (n=30) or standard peri-procedural UFH (n=<em>1</em>6). Blood samples for markers of thrombin generation and coagulation activation were drawn prior to and at 0, 0.5, <em>2</em>, 6-8 and 48 hours (h) after start of PCI. In patients treated with rivaroxaban (<em>1</em>0 or <em>2</em>0 mg) and patients treated with rivaroxaban plus heparin, the levels of <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> at <em>2</em> h post-PCI were 0.<em>1</em>6 [0.<em>1</em>] nmol/l (median) [interquartile range, IQR] and 0.<em>1</em>7 [0.<em>2</em>] nmol/l, respectively. Thrombin-antithrombin complex values at <em>2</em> h post-PCI were 3.90 [6.8]µg/l and 3.90 [<em>1</em>0.<em>1</em>] µg/l, respectively, remaining below the upper reference limit (URL) after PCI and stenting. This was comparable to the control group of UFH treatment alone. However, median values for thrombin-antithrombin complex passed above the URL with increasing tendency, starting at <em>2</em> h post-PCI in the UFH-alone arm but not in rivaroxaban-treated patients. In this exploratory trial, rivaroxaban effectively suppressed coagulation activation after elective PCI and stenting.

Deep vein thrombosis (DVT) is a frequent event in patients with spinal cord injury, even with prophylactic anticoagulant therapy. Lower limb paralysis is a known major risk factor for venous thrombosis, supposedly due to the venostasis in relation with total immobility. The main goal of this study was to evaluate the endothelial response to anoxia to determine whether recovery of fibrinolytic potential occurs in patients subjected to forced bedrest because of a spinal cord injury and whether this recovery is related to the incidence and/or evolution of DVT. We evaluated vascular endothelium reactivity in the lower limbs no longer submitted to the hydrostatic pressure of the erected position in <em>1</em>5 patients with paraplegia or tetraplegia and in <em>1</em>0 normal volunteers after venous occlusion produced by the application of <em>1</em>0 cm Hg pressure to the lower limb for <em>1</em>5 min comparatively to the upper limb used as reference. Among the <em>1</em>5 patients, <em>1</em>0 whose spinal cord injury had occurred <em>1</em> to 6 months earlier were still receiving prophylactic anticoagulant therapy, whereas the five other patients were not receiving prophylactic anticoagulants because the injury dated back 6 months or more. After venostasis, tissue plasminogen activator (tPA) increased significantly in both patients and controls in the upper limb (tPA levels twofold and threefold respectively in controls and patients) but showed no significant changes in the lower limb; prolonged immobility did not allow recovery in the lower limbs of a level of fibrinolytic responsiveness identical to that in the upper limbs. The plasminogen activator inhibitor (PAI<em>1</em>) remained unchanged after anoxia, although wide interindividual variations were seen. Natural coagulation inhibitors and circulating blood stigmates of hypercoagulability were measured. None of the patients had abnormally low levels of coagulation inhibitors (ie, antithrombin III, protein C and protein S levels were normal). Seventy-five per cent of patients (prophylactically anticoagulated or not) had very high levels of fibrin degradation products (D. Dimer levels sevenfold to eightfold those of the controls), but all patients had normal levels of thrombin-antithrombin complexes and <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em>. The permanence of the thrombotic process characterized by an increase in D. Dimer levels without recovery of fibrinolytic potential suggests a proposal for the patients an indefinite antithrombotic treatment at curative doses.

The optimal intensity of oral anticoagulant therapy for the prevention of thromboembolism in patients with antiphospholipid antibodies (APLA) and systemic lupus erythematosus is controversial. Retrospective studies have suggested that patients with APLA are resistant to oral anticoagulant therapy, with a targeted International Normalization Ratio (INR) of <em>2</em>.0 to 3.0, and that a higher intensity of anticoagulation (INR: <em>2</em>.6 to 4.5) is required to prevent recurrent thromboembolism. To investigate if patients with APLA are resistant to the anticoagulant effect of low intensities of warfarin therapy, we performed a randomized trial in which <em>2</em><em>1</em> patients with APLA and systemic lupus erythematosus were allocated to receive one of three intensities of warfarin (INR: <em>1</em>.<em>1</em> to <em>1</em>.4, <em>1</em>.5 to <em>1</em>.9 or <em>2</em>.0 to <em>2</em>.5) or placebo for four months. The main outcome was the effect of each intensity of warfarin therapy on <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> level (F<em>1</em>+<em>2</em>), that was used as a marker of coagulation activation. When F<em>1</em>+<em>2</em> levels in patients allocated to the three warfarin intensities were compared to F<em>1</em>+<em>2</em> levels in the placebo group, there was a statistically significant decrease (p<0.05) in the patient group receiving warfarin with a targeted INR of <em>2</em>.0 to <em>2</em>.5 at two, three and four months, and in the patient group with a targeted of INR <em>1</em>.5 to <em>1</em>.9 at three months. We conclude that in patients with APLA and systemic lupus erythematosus, warfarin therapy, with a targeted INR of <em>2</em>.0 to <em>2</em>.5, is effective in suppressing coagulation activation, and therefore, might be effective in preventing thromboembolism.