Bovine <em>prothrombin</em> <em>fragment</em> <em>1</em> and <em>prothrombin</em> undergo decarboxylation of their gamma-carboxyglutamic acid residues when the lyophilized proteins are heated in vacuo at <em>1</em><em>1</em>0 degrees C for several hours. The fully decarboxylated <em>fragment</em> <em>1</em> product has lost its barium-binding ability as well as the calcium-binding function which causes fluorescence quenching in the presence of <em>2</em> mM Ca<em>2</em>+. There is no sign of secondary structure alteration in solution upon analysis by fluorescence emission and circular dichroic spectroscopy. A family of partially decarboxylated <em>fragment</em> <em>1</em> species generated by heating for shorter periods shows that the initial decrease in calcium-binding ability occurs almost twice as rapidly as the loss of gamma-carboxyglutamic acid. This is consistent with the idea that differential functions can be ascribed to the <em>1</em>0 gamma-carboxyglutamic acid residues in <em>fragment</em> <em>1</em>, including both high- and low-affinity metal ion binding sites. <em>Prothrombin</em> itself also undergoes total decarboxylation without any apparent alteration in secondary structure. However, in this case the latent thrombin activity is progressively diminished during the heating process in terms of both clotting activity and hydrolysis of the amide substrate H-D-Phe-Pip-Arg-pNA. The present results indicate that in vitro decarboxylation of gamma-carboxyglutamic acid in dried proteins is useful for analyzing the detailed calcium-binding proteins of vitamin K dependent coagulation factors.

<em>1</em>. <em>Fragments</em> of breast muscle from a <em>1</em><em>2</em> day old chick embryo have been kept alive in single flasks for an entire year without being transferred. The nutrient materials were supplied by frequent applications of adult fowl serum diluted with Tyrode solution. <em>2</em>. When <em>fragments</em> of fixed tissues are cultivated in serum, cell multiplication and cell death are both reduced to an extremely low level. 3. The presence of a plasma coagulum is not essential to the continued survival and further development of tissues cultivated inserum. 4. The fibrinogen, <em>prothrombin</em>, and fibrin of coagulated plasma are not essential to the development of connective tissue fibers in vitro.

Two pathways are possible during the proteolytic formation of alpha-thrombin (alpha-IIa) from <em>prothrombin</em> (II) or prethrombin <em>1</em> (P<em>1</em>). One of the pathways, with prethrombin <em>2</em> or prethrombin <em>2</em> associated with <em>fragment</em> <em>2</em> (P<em>2</em>F<em>2</em>) as intermediates, has long been known to exist when activation is catalyzed by Factor Xa (Xa) alone. The second pathway, with meizothrombin or meizothrombin (des <em>fragment</em> <em>1</em>) (MzIIa(-F<em>1</em>)) as intermediate, has been shown to exist when Factor Va and phospholipids are present with Xa. Until now, MzIIa(-F<em>1</em>) has not been detected in reactions catalyzed by Xa alone. In this study, we demonstrate that P<em>1</em> activation by Xa alone occurs via both pathways, and we provide rate constants and kinetic equations for calculating the relative contributions of each of the pathways to the formation of alpha-IIa by Xa. Investigation of the initial rates of proteolytic cleavage of P<em>2</em>F<em>2</em> and P<em>1</em> by Xa alone indicated first-order dependence on substrate concentration with no evidence of saturation of Xa with either substrate at concentrations as high as <em>2</em>00 microM. Apparent second-order rate constants (kc/Km) of <em>1</em><em>1</em>3 +/- 9 M-<em>1</em> s-<em>1</em> for the formation of thrombin from P<em>2</em>F<em>2</em> and <em>1</em>,4<em>1</em>0 +/- <em>1</em>9 M-<em>1</em> s-<em>1</em> for the disappearance of P<em>1</em> were determined at pH 7.5, <em>2</em>5 degrees C, <em>1</em>0 mM CaCl<em>2</em>, 0.<em>1</em>5 M ionic strength. A two-step sequential first-order pathway employing these rate constants for thrombin activity production from P<em>1</em> via P<em>2</em>F<em>2</em> could not, however, account for the quantity of thrombin that was produced during the early stages of P<em>1</em> activation. Addition of a parallel first-order reaction to produce thrombin activity from P<em>1</em> independently of P<em>2</em>F<em>2</em>, tentatively identified as the formation of MzIIa(-F<em>1</em>), yielded progress curves in quantitative agreement with the experimental data. kc/Km for the parallel reaction was estimated to be 98 +/- <em>1</em>0 M-<em>1</em> s-<em>1</em>. Independent determination of the second-order rate constant for the cleavage of isolated MzIIa (-F<em>1</em>), <em>1</em>5,000 +/- 4<em>2</em>0 M-<em>1</em> s-<em>1</em>, indicated that MzIIa(-F<em>1</em>) could meet the kinetic requirements for an intermediate in the parallel activation pathway. The transient formation of MzIIa (-F<em>1</em>), as well as the generation of alpha-IIa, was directly demonstrated during activation of P<em>1</em> by active site-affinity labeling of the reaction products with a biotin derivative of D-Phe-Pro-Arg chloromethyl ketone and visualization by semiquantitative Western blotting.(ABSTRACT TRUNCATED AT 400 WORDS)

OBJECTIVE

Bivalirudin has been successfully used as a replacement for heparin during on-pump coronary artery bypass grafting. This study was conducted to assess the effects of the currently suggested protocol for bivalirudin on hemostatic activation during cardiopulmonary bypass with and without cardiotomy suction.

METHODS

Ten patients scheduled for coronary artery bypass grafting were enrolled. Bivalirudin was given with a bolus of 50 mg in the priming solution and <em>1</em>.0 mg/kg for the patient, followed by an infusion of <em>2</em>.5 mg . kg(-<em>1</em>) . h(-<em>1</em>) until <em>1</em>5 minutes before the conclusion of cardiopulmonary bypass. Cardiopulmonary bypass was performed with a closed system in 5 patients with and in 5 patients without the use of cardiotomy suction. Blood samples were obtained before and after cardiopulmonary bypass. D-dimers, fibrinopeptide A, <em>prothrombin</em> <em>1</em> and <em>2</em> <em>fragments</em>, thrombin-antithrombin, and factor XIIa were determined.

RESULTS

Values for factor XIIa remained almost unchanged in both groups, indicating a minor effect of contact activation. In patients without cardiotomy suction, post-cardiopulmonary bypass values for D-dimers, fibrinopeptide A, <em>prothrombin</em> <em>1</em> and <em>2</em> <em>fragments</em>, and thrombin-antithrombin were not significantly increased compared with pre-cardiopulmonary bypass values. In patients with cardiotomy suction, values obtained for these parameters had significantly increased compared with pre-cardiopulmonary bypass values and the values obtained in the group without cardiotomy suction after cardiopulmonary bypass.

CONCLUSIONS

With this protocol, hemostatic activation during cardiopulmonary bypass was almost completely attenuated when cardiotomy suction was avoided. Cardiotomy suction results in considerable activation of the coagulation system and should therefore be restricted and replaced by cell saving whenever possible.

RF catheter ablation is complicated by thromboembolism in about <em>1</em>% of patients. Limited knowledge exists concerning when and how to use anticoagulation or antithrombotic treatment. We studied the activation of coagulation (<em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> [PF<em>1</em> + <em>2</em>] and D-dimer), platelets (beta-thromboglobulin [beta-TG]) and fibrinolysis (plasmin-antiplasmin complexes [PAP]) during RF ablation of accessory pathways in 30 patients. They were randomized to receive heparin (<em>1</em>00 IU/kg, intravenously) (<em>1</em>) immediately after introduction of the femoral venous sheaths (group I) or (<em>2</em>) after the initial electrophysiological study, prior to the delivery of RF current (groups II and III). Group II additionally received saline irrigation of all femoral sheaths. After the initial bolus, <em>1</em>,000 IU of heparin was supplied hourly in all groups. Within groups II and III, median plasma values of PF<em>1</em> + <em>2</em> and beta-TG more than tripled (P < or = 0.007) during the diagnostic study and gradually declined during heparin administration despite RF current delivery. Median D-dimer tripled (P = 0.005) and PAP doubled (NS) before heparin administration; then both remained around the upper reference values. In the early heparin group, however, PF<em>1</em> + <em>2</em>, D-dimer, and PAP did not rise at all, and beta-TG showed only a slight increase towards the end of the procedure. The differences between group I versus groups II and III were statistically significant prior to the first RF current delivery (PF<em>1</em> + <em>2</em>, D-dimer, and beta-TG) and by the end of the procedure (PF<em>1</em> + <em>2</em>, D-dimer, and PAP). In conclusion, "late" heparin administration allows hemostatic activation during the initial catheterization and diagnostic study. By administering intravenous heparin immediately after introduction of the venous sheaths, hemostatic activation is significantly decreased. Saline irrigation of the venous sheaths added nothing to late heparin administration.

BACKGROUND

Renal transplant recipients are at increased risk of venous thromboembolic events, which is in part caused by their treatment with maintenance immunosuppressive drugs. Because we observed an increased incidence of venous thromboembolic events in renal transplant recipients treated with the mTOR inhibitor (mTORi) everolimus, we aimed to identify prothrombotic mechanisms of this immunosuppressive drug.

METHODS

In a single center study, nested in a multi-center randomized controlled trial, we measured parameters of coagulation, anti-coagulation and fibrinolysis in renal transplant recipients, receiving the mTORi everolimus (n=<em>1</em>6, mTOR group) and compared them to a similar patient group, receiving a calcineurin inhibitor and/or mycophenolate sodium (n=<em>2</em>0, non-mTOR group). All patients were at least 6 months following transplantation with a stable transplant function.

RESULTS

The use of an mTORi was associated with significantly higher levels of von Willebrand factor, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, thrombin-activatable fibrinolysis inhibitor and plasminogen activator inhibitor-<em>1</em> as compared to a non-mTORi based immunosuppressive regimen.

CONCLUSIONS

Treatment with an mTORi leads to increased endothelial activation, thrombin formation and impaired fibrinolysis in renal transplant recipients. This suggests an increased risk of thrombotic events in renal transplant recipients treated with mTOR inhibitors. A prospective study to establish the precise risk of thrombotic events in these patients is urgently needed.

Disturbances of coagulation and fibrinolysis in type <em>2</em> diabetes mellitus (DM<em>2</em>) contribute to increased rates of macrovascular complications such as myocardial infarction and ischemic stroke. The aim of the study was to investigate the relationship among plasminogen activator inhibitor <em>1</em> (PAI-<em>1</em>), thrombin-activable fibrinolysis inhibitor (TAFI), tissue plasminogen activator (t-PA), <em>prothrombin</em> <em>fragments</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>), glycemic control, hypertension, sex and body mass index (BMI) in DM<em>2</em> patients with normoalbuminuria and microalbuminuria. Forty-two normoalbuminuric (NAU), 4<em>2</em> microalbuminuric (MAU) patients with DM<em>2</em> and 4<em>2</em> blood donors as control group were enrolled. TAFI, PAI-<em>1</em>, t-PA and F<em>1</em>+<em>2</em> were assessed by enzyme-linked immunosorbent assay (ELISA) in all patients. TAFI was significantly increased in the MAU group, PAI-<em>1</em> and F<em>1</em>+<em>2</em> were increased in both groups and t-PA was not elevated in either group compared to controls. We found positive correlations in the NAU: TAFI and fibrinogen (r=0.65, P=0.0<em>2</em>), PAI-<em>1</em> and triglycerides (r=0.67, P=0.0<em>1</em>), in the MAU: TAFI and F<em>1</em>+<em>2</em> (r=0.48, P=0.0<em>2</em>), TAFI and systolic blood pressure (r=0.53, P=0.0<em>1</em>), PAI-<em>1</em> and BMI (r=0.43, P<0.05). We found decreased fibrinolysis in DM<em>2</em> patients presented with increased PAI-<em>1</em> in both NAU and MAU. Hypofibrinolysis in MAU is further accented by the elevation of TAFI. TAFI-mediated inhibition of fibrinolysis in DM<em>2</em> is regulated independently from PAI-<em>1</em>. Patient[Combining Acute Accent]s sex does not affect diabetes-related changes in hemostasis and fibrinolysis.

OBJECTIVE

To assess the effects of oral E<em>2</em> replacement therapy combined with nomegestrol acetate, a <em>1</em>9-norprogesterone derivative, on cardiovascular risk factors.

METHODS

A double-blind randomized prospective study comparing the effect of a placebo and two oral E<em>2</em>-nomegestrol acetate combinations (<em>1</em> mg-<em>2</em>.5 mg and <em>1</em>.5 mg-3.75 mg) over a three-cycle trial.

METHODS

Department of Internal Medicine and Nutrition, Hotel-Dieu, Paris, France.

METHODS

Fifty-seven nonhysterectomized women with natural menopause.

METHODS

Blood pressure, renin substrate, glucose, total cholesterol, high-density and low-density lipoprotein cholesterol, triglycerides, apoproteins A<em>1</em> and B, lipoprotein(a), antithrombin III, fibrinogen, plasminogen, <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>, protein C, and total and free protein S.

RESULTS

Both treatments significantly reduced menopausal complaints, total cholesterol, low-density lipoprotein cholesterol and lipoprotein(a). Treatment with the <em>1</em>.5 mg-3.75 mg combination resulted in a significant increase in apolipoprotein A<em>1</em>. No significant change were observed in other parameters.

CONCLUSIONS

Sequentially combined with oral E<em>2</em> in hormone replacement therapy, nomegestrol acetate had favorable effects on plasma lipids and lipoproteins. This nonandrogenic progestin decreased lipoprotein(a) levels as observed previously with medroxyprogesterone acetate combined with conjugated equine estrogens.

Simvastatin, a widely used 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor, effectively reduced cardiac death and ischemic events in patients with coronary heart disease (CHD) and diabetes mellitus (DM). The mechanism of cardiovascular benefits of statins in DM remains unclear. We examined how simvastatin influences the levels of several in vivo markers for coagulation and fibrinolysis in <em>2</em>6 Type <em>2</em> DM patients. The diabetic patients received <em>2</em>0 mg/day of simvastatin up to <em>1</em><em>2</em> months. The levels of total cholesterol (TC), low density lipoprotein-cholesterol (LDL-c) and triglycerides in peripheral circulation of patients were significantly reduced after>> or =6 weeks of simvastatin treatment. Levels of <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>), factor VII, plasminogen activator inhibitor-<em>1</em> (PAI-<em>1</em>) and tissue factor pathway inhibitor (TFPI) antigens, but not tissue plasminogen activator (tPA) antigen, in the pre-simvastatin plasmas of the diabetic patients were significantly higher than the levels found in plasmas of healthy subjects. Significant reductions in F<em>1</em>+<em>2</em> and PAI-<em>1</em> levels were evident>> or =6 weeks after the diabetic patients received simvastatin. Levels of total tPA, TFPI, FVII, hemoglobin A<em>1</em>c, fasting blood glucose, and insulin in the diabetic patients' plasma were not significantly altered by simvastatin treatment. Positive correlations were found between PAI-<em>1</em> versus TC, PAI-<em>1</em> versus LDL-c, and FVII versus triglycerides in the plasmas of simvastatin-treated patients. The results suggest that simvastatin reduces in vivo <em>prothrombin</em>ase activity and PAI-<em>1</em> levels in type <em>2</em> DM patients. These actions may contribute to the protective properties of simvastatin against ischemic events in diabetic patients.

BACKGROUND

Data are lacking on the impact that the intensity of serial donor plasmapheresis has on the quality of source plasma. A study was conducted to examine the quality of source plasma produced by intensive plasmapheresis and slow deep-freezing and to compare it to source plasma manufactured by moderate plasmapheresis and rapid freezing.

METHODS

Seventy-five plasma samples from intensive plasmapheresis programs (Group <em>1</em>) and 75 plasma units from moderate plasmapheresis programs (Group <em>2</em>) were examined. The plasma had been deep-frozen either slowly at -30 degrees C in walk-in freezers (Group <em>1</em>) or rapidly within <em>1</em> hour to a core temperature below -30 degrees C (Group <em>2</em>). Determinations were made of the plasma levels of citrate; total protein; albumin; IgG; fibrinogen; factors II, V, VII, VIII, and IX; vWF; antithrombin; protein C; D-dimers; and <em>prothrombin</em> <em>fragments</em> <em>1</em>+ <em>2</em>.

RESULTS

Plasma units of Group <em>2</em> contained substantially greater levels of citrate, IgG, FVIII, and FV than samples of Group <em>1</em> (p<0.000<em>1</em>). Plasma levels of total protein, albumin, and fibrinogen also were higher in Group <em>2</em> (p<0.000<em>1</em>, p = 0.007, and p = 0.006, respectively). Neither plasmapheresis intensity nor freezing procedure had any influence on the levels of factors II, VII, and IX, antithrombin, or protein C. There was no evidence of substantial coagulation activation in the plasma units of either group. However, higher FVIII clotting activity/chromogenic substrate activity ratios in rapidly frozen plasmas and a significant correlation between these ratios and <em>prothrombin</em> fragment <em>1</em>+ <em>2</em> levels suggest that rapid freezing yields both more native FVIII and greater partial activation of FVIII.

CONCLUSIONS

Source plasma collected from donors undergoing intensified plasmapheresis contains markedly lower levels of IgG than plasma units produced by moderate serial plasmapheresis. The combination of intensified plasmapheresis and slower freezing of source plasma results in substantially lower levels of FV and FVIII than does moderate plasmapheresis with rapid freezing. Prospective studies should establish the optimum conditions required for the safe and economic production of source plasma for fractionation.

BACKGROUND

The available evidence suggests strongly that intravascular thrombosis is mediated predominantly by tissue-factor and its activation of factor X, which in the presence of factor Va, calcium, and phospholipid (<em>prothrombin</em>ase complex) effectively converts <em>prothrombin</em> to thrombin. In vitro experiments have shown that low molecular weight heparins (LMWHs) have greater anti-Xa activity than unfractionated heparin; however, it remains unclear as to whether their antithrombotic effects in vivo are determined by a similar mechanism. We determined the ability of plasma obtained from patients with either unstable angina or non-ST segment elevation myocardial infarction (MI) receiving the LMWH enoxaparin (anti Xa:IIa ratio 3:<em>1</em>) to inhibit tissue factor-mediated thrombin generation and to inactivate platelet <em>prothrombin</em>ase.

METHODS

Platelet rich plasma was prepared by suspending washed donor platelets in the plasma of 7 patients participating in the TIMI <em>1</em><em>1</em>A study. Samples were obtained before, <em>1</em> hour after a 30-mg IV bolus of enoxaparin and 6 hours after the third subcutaneous injection (<em>1</em>. 0-<em>1</em>.25 mg/kg given subcutaneously every <em>1</em>2 hrs). Tissue factor (0.<em>1</em> ng/ml) and <em>1</em>0 mM CaCl(2) were added to initiate extrinsic coagulation. At timed intervals <em>prothrombin</em> activation fragment <em>1</em>.2 (F<em>1</em>.2) levels (thrombin generation) were measured using an ELISA technique. Inactivation of reformed platelet <em>prothrombin</em>ase by samples obtained at the same time points was also determined.

RESULTS

Patient plasma obtained <em>1</em> hr after treatment initiation and 6 hours after the third subcutaneous injection inhibited tissue factor mediated <em>prothrombin</em>ase assembly by 3<em>1</em>% and <em>1</em><em>1</em>%, respectively and platelet <em>prothrombin</em>ase activity by 27% and 22%, respectively.

CONCLUSIONS

We conclude that enoxaparin in plasma concentrations achieved routinely in clinical practice is able to: (<em>1</em>) inhibit tissue factor mediated extrinsic coagulation by preventing platelet surface <em>prothrombin</em>ase assembly, and (2) inactivate platelet <em>prothrombin</em>ase activity and resulting thrombin generation. These observations suggest that a LMWH's anti-Xa activity (and anti-Xa:IIa profile) is important in determining its overall antithrombotic potential. Clinical trials comparing agents with differing anti-Xa:IIa properties will be required, however, to provide proof of concept.

Oligodeoxynucleotide phosphorothioates (PS-oligos) are being studied as novel therapeutic agents based on their ability to inhibit gene expression. Preclinical studies produced unanticipated complement and coagulation effects in monkeys receiving high-dose PS-oligo. In the present in vitro studies, PS-oligo inhibited normal human blood clotting as well as subsequent assays for <em>prothrombin</em> <em>fragment</em> PF(<em>1</em>+<em>2</em>) and hemolytic complement. PS-oligo treatment of normal donor plasma produced concentration-dependent prolongations of clotting times, with the activated partial thromboplastin time more sensitive than <em>prothrombin</em> time or thrombin clotting time. PS-oligo treatment of normal donor serum similarly reduced hemolytic complement activity in a concentration-dependent manner. Reduced hemolysis correlated with increased levels of complement <em>fragment</em> C4d. The anti-heparin drug protamine sulfate inhibited in vitro effects of PS-oligo in both complement and coagulation assays, suggesting that charged residues in internucleotide linkages of PS-oligo mediated the observed activities. Therefore, oligonucleotides with varying internucleotide linkages, nucleotide sequence, or secondary structure were compared. Both complement and coagulation effects appeared to be independent of nucleotide sequence but were strongly related to the nature of internucleotide linkages. Several of these modified oligonucleotides have been shown previously to retain potent antisense activity and thus may represent viable alternatives for antisense therapeutics.

Beta-thalassemia/hemoglobin (Hb) E is a hereditary hemolytic anemia with varying degrees of severity. Severely affected patients are treated with blood transfusion and/or splenectomy in order to maintain an optimum level of hemoglobin for normal growth and physical activities. As thrombosis has been observed among splenectomized patients, we have investigated alterations in coagulation and fibrinolysis in beta-thalassemia/Hb E patients. Plasma levels of <em>prothrombin</em>, fibrinogen, factors V, VII, VIII, IX and XI, protein C, protein S, thrombin activatable fibrinolysis inhibitor (TAFI) and <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> were determined in 6<em>1</em> patients (<em>2</em><em>1</em> non-severe non-splenectomized, <em>1</em>8 severe non-splenectomized, <em>2</em><em>2</em> severe splenectomized) and <em>2</em>8 healthy individuals. Serum levels of D-dimer, ferritin, aspartate transaminase and alanine transaminase were also measured. All severe patients received regular blood transfusion. <em>Prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> and D-dimer were significantly elevated in splenectomized patients compared to the healthy control subjects, whereas levels of proteins C, protein S, TAFI, fibrinogen, and factors V and VIII in the splenectomized groups were statistically lower than those in control group. There are no statistical differences for the other parameters measured between patients and controls. Coagulation tests showed only significantly reduction in TAFI and factor V and VIII levels in severe splenectomized group in comparison with severe non-splenectomized patients. These results demonstrate the existence of a low grade consumptive coagulopathy among blood-transfused splenectomized patients with severe clinical manifestations, indicating that these patients may have a higher risk for thrombosis than comparable patients with intact spleen.

Atrial fibrillation (AF) is a well-defined risk factor for ischemic stroke. Patients with lone AF represent a subgroup of AF patients with the lowest lifelong stroke risk. Nonvalvular atrial fibrillation (NVAF) confers a hypercoagulable state resulting in an increased risk of thromboembolism. This study was performed to determine the contributory role of alteration in the hemostatic markers of thrombin generation and fibrinolysis in patients with lone AF during acute ischemic stroke episode. We studied thrombin-antithrombin complexes (TAT), <em>prothrombin</em> <em>fragments</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>), tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor type-<em>1</em> (PAI-<em>1</em>) concentrations in patients with acute middle cerebral artery ischemic stroke due to atherosclerotic large artery disease (n=50), lone AF (n=<em>2</em>4) and cardioembolism (n=<em>2</em><em>1</em>). The values were compared with those of age-matched control subjects with lone AF and sinus rhythm (n=<em>2</em><em>1</em> and <em>1</em>5, respectively). The mean F<em>1</em>+<em>2</em> concentration was higher in the control subjects with lone AF in comparison with those without AF (p=0.0<em>1</em>4). Patients with stroke due to possible cardioembolism, from lone AF or other causes, had higher TAT (and marginally higher F<em>1</em>+<em>2</em>) concentrations than those with atherosclerotic stroke (p<0.00<em>1</em>). tPA concentrations were not different among groups (p=0.89). PAI-I levels were marginally higher in stroke patients with lone AF and atherothrombotic large artery disease compared to the controls without AF (p=0.05). These results suggest that in the acute period of ischemic stroke secondary to lone AF, enhancement of the coagulatory activity occurs as a result of increased thrombin generation, similar to other possible sources of cardioembolism. Observed hemostatic alterations in acute ischemic stroke associated with lone AF may indicate some therapeutic and prognostic implications.

Measurements to test for a proposed Ca<em>2</em>+-independent interaction of <em>prothrombin</em> with membranes containing acidic phospholipids are described. Fluorescein-labeled bovine <em>prothrombin</em> and its amino- and carboxy-terminal peptides, <em>prothrombin</em> <em>fragment</em> <em>1</em> and prethrombin <em>1</em>, were added at various concentrations in the presence or absence of Ca<em>2</em>+ to the aqueous space bathing substrate-supported planar membranes composed of <em>1</em>-palmitoyl-<em>2</em>-oleoyl-3-sn-phosphatidylcholine (POPC), POPC/bovine brain phosphatidylserine (bovPS) (70:30 mol/mol), or POPC/<em>1</em>,<em>2</em>-dioleoyl-3-sn-phosphatidylglycerol (DOPG) (70:30 mol/mol). Total internal reflection fluorescence microscopy (TIRFM) at the membrane-solution interface showed a significant enhancement by acidic lipids of <em>prothrombin</em> and <em>prothrombin</em> <em>fragment</em> <em>1</em> binding in the presence of 5 mM Ca<em>2</em>+, with apparent dissociation constants of 0.4 and <em>1</em> microM, respectively. TIRFM measurements indicated that bovPS and DOPG also significantly enhanced the binding of fluorescein-labeled <em>prothrombin</em> to the planar membranes in the absence of Ca<em>2</em>+, with apparent dissociation constants (<em>1</em>3-30 microM) at least an order of magnitude larger than the Ca(<em>2</em>+)-dependent constant for <em>prothrombin</em> binding. Association of prethrombin <em>1</em> but not <em>prothrombin</em> <em>fragment</em> <em>1</em> with membranes in the absence of Ca<em>2</em>+ was enhanced by the presence of bovPS in the membranes, which suggests that the Ca(<em>2</em>+)-independent binding site(s) is (are) in the prethrombin <em>1</em> but not the <em>fragment</em> <em>1</em> portion of <em>prothrombin</em>.

The effects of two doses of aspirin (75 and 500 mg/day during <em>1</em> week) on thrombin generation was investigated in healthy volunteers. Thrombin generation in whole blood was monitored by repeated measurements of <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>) in plasma prepared from untreated whole blood left to clot at 37 degrees C. Experiments with a platelet inhibiting agent (iloprost, a prostacyclinanalogue) and platelet-activating compounds (collagen and a thromboxane analogue), indicated that the formation of thrombin in this system is partly dependent on platelet function. High dose aspirin (500 mg daily) attenuated thrombin generation, whereas low-dose treatment (75 mg daily) failed to attenuate thrombin formation significantly. Collagen-induced platelet aggregation in whole blood, used to monitor antiplatelet effects of aspirin, showed profound inhibition of platelet aggregation already at 75 mg of aspirin; 500 mg did not inhibit platelet aggregation further. Our results show that aspirin suppresses thrombin formation in whole blood in a dose-dependent fashion and that the "antithrombin" effects of aspirin require higher doses than the antiaggregating effects. The mechanism(s) behind the "antithrombin" effects of aspirin is at present unclear but may involve thromboxane-independent mechanisms, such as acetylation of platelet membrane receptors or coagulation factors.

OBJECTIVE

Although the route of estrogen administration is known to be an important determinant of the thrombotic risk among postmenopausal women using hormone therapy, recent data have shown that norpregnane derivatives but not micronized progesterone increase venous thromboembolism risk among transdermal estrogens users. However, the differential effects of progesterone and norpregnanes on hemostasis have not yet been investigated.

METHODS

We set up a cross-sectional study among healthy postmenopausal women aged 45 to 70 years. The impact of activated protein C (APC) on endogenous thrombin potential was investigated in the plasma samples of <em>1</em>08 women who did not use any hormone therapy (n = 40) or who were treated with transdermal estrogens combined with micronized progesterone (n = 30) or norpregnane derivatives (n = 38).

RESULTS

After exclusion of women with factor V Leiden and/or G<em>2</em>0<em>2</em><em>1</em>0A <em>prothrombin</em> gene mutations, there was no significant change in APC sensitivity among women who used transdermal estrogens combined with micronized progesterone compared with nonusers. Women using transdermal estrogens combined with norpregnanes were less sensitive to APC than were nonusers (P = 0.003) or users of transdermal estrogens combined with micronized progesterone (P = 0.004). In addition, <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> concentration was higher in users of transdermal estrogens plus norpregnanes than in nonusers (P = 0.004). Other hemostatic parameters did not vary significantly across the different subgroups.

CONCLUSIONS

Transdermal estrogens combined with norpregnanes may induce APC resistance and activate blood coagulation. These results provide a biological support to epidemiological data regarding the potential thrombogenic effects of norpregnanes. However, these findings need to be confirmed in a randomized trial.

BACKGROUND

End-stage renal disease (ESRD) patients, not uncommonly, might exhibit thrombotic complications, as well as they may present with a bleeding diathesis. Changes in vessel wall and/or blood flow in native arteriovenous fistula (AVF) might also augment these disarrangements, as vascular endothelium is predominantly involved in the regulation of haemostatic pathways.

OBJECTIVE

This study was designed to evaluate the state of coagulation and fibrinolysis and the role of AVF on haemostatic defects, in ESRD patients on maintenance haemodialysis.

METHODS

Plasma samples for <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, thrombin-antithrombin III complex, plasmin-alpha<em>2</em> antiplasmin complex, tissue type plasminogen activator antigen, urokinase type plasminogen activator antigen, u-PA activity, plasminogen activity, alpha<em>2</em>-antiplasmin and alpha<em>2</em>-macroglobulin assays were obtained from AVF and contralateral large veins of ESRD patients and from peripheral veins of the control group.

RESULTS

Our results indicate a predominant thrombotic state as evidenced by activated coagulation markers and enhanced fibrinolysis in systemic circulation of ESRD patients. However the most novel finding is the probable contribution of AVF on haemostatic activation, as proven by the statistically different and positively correlated concentrations of both coagulation, fibrinolysis, and fibrinolysis inhibitors in AVF when compared to the levels in peripheral venous circulation.

CONCLUSIONS

In addition to systemic derangements of haemostasis in ESRD patients, AVF individually might have a substantial role in the modulation of coagulation and fibrinolytic cascade.

The <em>2</em>0<em>2</em><em>1</em>0A <em>prothrombin</em> mutation has recently been associated with an increased risk of venous thrombosis, but the mechanism of the increased thrombotic risk in affected persons has not been elucidated. We report on a thrombophilic family in which the proband presented with cerebral vein thrombosis and homozygosity for the <em>2</em>0<em>2</em><em>1</em>0A <em>prothrombin</em> mutation as her only identifiable risk factor for venous thrombosis. Extended genotyping of family members revealed seven other affected, but asymptomatic, first-degree relatives (one A/A homozygote and six G/A heterozygotes). Plasma levels of <em>prothrombin</em>, <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em> and thrombin-antithrombin complexes were highest in A/A homozygotes, intermediate in G/A heterozygotes and lowest in those with the G/G homozygous normal genotype, while D-dimer levels were elevated only in A/A homozygotes. Our results suggest that the <em>2</em>0<em>2</em><em>1</em>0A <em>prothrombin</em> mutation is associated with activation of coagulation and increased thrombin generation, not only in patients with a past history of thrombosis but also in otherwise healthy asymptomatic persons. In a similar fashion to the homozygous factor V Leiden mutation, patients with the homozygous <em>2</em>0<em>2</em><em>1</em>0A <em>prothrombin</em> mutation could be at highest risk of thrombosis, as suggested by our patient who presented with unusual thrombosis.

The mechanisms by which blood levels of <em>prothrombin</em> (PT) are regulated in the vitamin K-sufficient state are unknown. We have studied PT synthesis by Reuber H-35 rat hepatoma cells exposed to vitamin K and [3H]leucine in serum-free cultures. Administration to the culture system of exogenous bovine PT and rat PT was characterized by increases in endogenous PT synthesis and secretion of <em>2</em>- and 3-fold, respectively. This induction required endogenous proteolytic degradation of PT. Studies conducted with bovine PT <em>fragment</em> <em>1</em> (residues <em>1</em>-<em>1</em>56) demonstrated up to 5-fold increases in PT synthesis. This induction was dose dependent and saturable. Addition of bovine PT chymotryptic <em>fragments</em> to the cells indicated that the NH<em>2</em>-terminal peptide of <em>prothrombin</em> (residues <em>1</em>-4<em>2</em>) contained the requisite structural elements for the induction. Peptide-bound gamma-carboxyglutamate residues were required for the observed stimulation of PT synthesis. These results suggest that PT synthesis might be regulated physiologically by the products formed during its normal turnover and consumption during blood coagulation.

During cardiopulmonary bypass (CPB) mechanical stress and the contact of blood with artificial surfaces lead to the activation of pro- and anticoagulant systems and the complement cascade, and to changes in cellular components. This phenomenon causes the "postperfusion-syndrome", with leukocytosis, increased capillary permeability, accumulation of interstitial fluid, and organ dysfunction. In this study, we focused on the influence of the extracorporeal circulation, sternotomy, and heparin administration on the activation of coagulation and fibrinolysis. In <em>1</em>5 patients we investigated coagulation parameters before, during and post CPB, i.e., fibrinogen, antithrombin (AT) III, thrombin-antithrombin complex (TAT), <em>prothrombin</em> <em>fragments</em> F<em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>), factor (F) XIIa, tissue factor (TF), and parameters of the fibrinolytic system, i.e., plasmin-antiplasmin-complex (PAP), D-dimer, tissue-plasminogen-activator (tPA), urokinase-type plasminogen activator (uPA), and plasminogen-activator inhibitor type <em>1</em> (PAI <em>1</em>). The results demonstrate distinct alterations in the above mentioned parameters. Despite administration of a high dose of heparin (activated clotting time [ACT]>> 450s) combined with a low dose of aprotinin, activation of the coagulation and fibrinolytic pathways was observed. We found this activation was mainly caused by CPB and not by sternotomy. The activation of coagulation was due to foreign surface contact (F XII >> F XIIa) as well as to an effect of tissue factor release in the late phase of CPB. The enhanced fibrinolytic activity during CPB was, at least in part, caused by tPA and was followed by PAI <em>1</em> release.

We investigated the anticoagulant effects of argatroban, a direct thrombin inhibitor, versus heparin in extracorporeal membrane oxygenation (ECMO) circuits. Three sham circuits were prepared according to our hospital's standard practice and run for six hours simultaneously. Two circuits were anticoagulated with argatroban (one with heparin in the wet prime and one without). One circuit had heparin in the initial prime and was then anticoagulated with heparin. We measured thrombin generation (<em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, D-dimer and thrombin-antithrombin complexes), activated clotting times (ACTs) and partial thromboplastin times (aPTTs), and monitored thrombus formation using thromboelastography. ACTs were>><em>1</em>000 s in each circuit throughout assessment. No clot initiation was detected by thromboelastography. Thrombin generation was decreased in circuits anticoagulated with argatroban versus heparin, despite aPTTs being less prolonged. These results suggest that argatroban may be more efficacious than heparin for anticoagulation in ECMO. Additional studies are warranted to further evaluate argatroban in this setting.

Previously, our group showed a prothrombotic state in asymptomatic patients with chronic Chagas disease. The current paper studies the inflammatory status and endothelial function in these patients.

METHODS

In 40 patients and 40 healthy volunteers, we evaluated prothrombotic state, blood parasitemia (molecular biology: polymerized chain reaction [PCR]-amplification), tissue factor pathway inhibitor antibodies (aTFPI), interleukin 6 (IL-6), and vascular cell adhesion molecule-<em>1</em> (VCAM-<em>1</em>). Endothelial function was determined by reactive hyperemia (pulse plethysmography).

RESULTS

In patients, <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>, d-dimer, PAI-<em>1</em>, and fibrinogen were higher. Amplification of <em>1</em><em>2</em><em>1</em>/<em>1</em><em>2</em><em>2</em> primers (Trypanosoma cruzi) was positive in 45% of the patients. Patients presented higher values of aTFPI- immunoglobulin G (IgG; P < .05), aTFPI-IgM (P < .00<em>1</em>), IL-6 (P = .004), and VCAM-<em>1</em> (P = .0000<em>1</em>). In both groups, endothelial function was preserved.

CONCLUSIONS

We found that asymptomatic patients with chronic Chagas disease presented a prothrombotic/inflammatory status. The fact that endothelial function is still preserved suggests that prothrombosis and inflammation are primarily implicated in the beginning of cardiovascular damage.

We studied 33 women during normal uneventful pregnancies and with no history of previous adverse pregnancy events for markers of activated coagulation and thrombin activity including <em>prothrombin</em> <em>fragment</em> <em>1</em>.<em>2</em>(PF<em>1</em>.<em>2</em>), thrombin- antithrombin (TAT), and soluble fibrin polymer (SFP). In addition, we measured potential thrombin generation through the addition of thromboplastin to patient plasma in the presence of a thrombin-specific chromogenic substrate determined serially over a period of time--Endogenous Thrombin Potential assay (ETP). This assay was performed with plasma treated and untreated with activated protein C (APC). The fibrinolytic system was assessed by measurement of thrombin activatable fibrinolysis inhibitor (TAFI). These findings were correlated with the levels of pro-inflammatory cytokines, interleukine-<em>1</em> beta and tumor necrosis factor-alpha. Our data supports previous reports that indicate that resistance to activated protein C and coagulation activation markers are commonly increased in the later <em>2</em>/3rds of pregnancy. There are no differences in thrombin generation potential, as determined by the ETP assay without the addition of APC, in the three trimesters. However, the thrombin reserve (TR), the ETP result without APC divided by the ETP result with the addition of APC, is increased above the reference range in the <em>2</em>nd and 3rd trimesters. Patients with increased TR and resistance to APC had increased levels of TNF-alpha. Increased proinflammatory cytokines are reportedly associated with changes in the APC system with a decrease in the ability to generate APC. A sub-group of pregnancies with APC resistance had increased levels of TNF-alpha and may be important in the risk for adverse pregnancy outcomes.