Despite knowledge of various inherited risk factors associated with venous thromboembolism (VTE), no definite cause can be found in about 50% of patients. The lack of an intermediate phenotype in VTE impedes the discovery of new familial risk factors. We set out to define an intermediate phenotype for VTE by performing global coagulation analyses in unexplained thrombophilic families. Families were selected through a proband with VTE but without one of the known thrombophilic defects and at least one <em>1</em>st or two <em>2</em>nd degree family members with VTE. Clinical data were collected using a standardized questionnaire. Blood samples were collected for overall haemostasis assays (i.e. thrombin generation time [TGT], endogenous thrombin potential [ETP], <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> [F<em>1</em>+<em>2</em>] and activated protein C-sensitivity ratio [APC-sr] and clot lysis time [CLT]). Data were analysed using logistic regression. Coagulation assays were performed in 353 individuals of whom 4<em>1</em> (<em>1</em><em>2</em>%) had a history of VTE; these belonged to <em>1</em>7 thrombophilic families. Of the tested variables only the ETP was associated with VTE (odds ratio [OR] <em>1</em>.03 for each % increase, 95% confidence interval [CI] <em>1</em>.0<em>1</em>-<em>1</em>.05). However, the relatively low number of cases does not firmly exclude the other assays as candidate intermediate phenotypes for venous thrombosis. We found that an increased ETP may serve as an intermediate phenotype for VTE and may be used to discover novel inherited risk factors by genetic linkage analysis.

To evaluate antithrombin III (AT), thrombin (<em>Fragment</em> <em>1</em>+<em>2</em> [F<em>1</em>+<em>2</em>] and thrombin-antithrombin [TAT]) generation markers, as well as other coagulation parameters, such as <em>prothrombin</em> time, partial activated thromboplastin time, thrombin time, fibrinogen, euglobulin lysis time, and platelet count, in postmenopausal women after hormonal therapy.

Forty-five patients who received either 0.6<em>2</em>5 mg/day unopposed oral conjugated equine estrogen (CEE), 0.6<em>2</em>5 mg/day oral CEE plus medroxyprogesterone acetate (MP), or 50 microg/day transdermal <em>1</em>7beta-estradiol plus MP, were included. Tests were performed before (T0) and after 3 (T3), 6 (T6) and <em>1</em><em>2</em> (T<em>1</em><em>2</em>) months of treatment. AT was determined by an amidolytic method, whereas F<em>1</em>+<em>2</em> and TAT complex were measured by ELISA.

There was a significant reduction in the AT level of patients who received oral CEE plus MP at T3. There was no AT reduction in patients taking either oral CEE alone or transdermal <em>1</em>7beta-estradiol plus MP. F<em>1</em>+<em>2</em> increased in all patients, but it reached statistical significance only in patients receiving transdermal <em>1</em>7beta-estradiol MP at T3.

The CEE associated with MP treatment may reduce AT levels, whereas unopposed CEE or transdermal <em>1</em>7beta-estradiol plus MP does not change AT. These changes might not be clinically relevant in the general population; however, hormonal replacement therapy may increase the risk of thrombosis in women with congenital or acquired thrombophilia.

Acute physical exertion may trigger an acute coronary syndrome. Furthermore, acute physical exercise may influence hemostatic markers in healthy individuals. However, the effect of acute exercise on blood fibrinolysis and coagulation in patients with coronary artery disease (CAD) is still not well understood. Nineteen untrained patients with angiographically proven CAD (age, 58 +/- 9 years, <em>1</em><em>2</em> males), and <em>2</em>5 age- and sex-matched controls without CAD (age, 56 +/- 6 years, <em>1</em>6 males) underwent a treadmill exercise test. Global fibrinolytic capacity (GFC) and <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F <em>1</em> + <em>2</em>) levels were measured before exercise, at peak exercise, and <em>2</em> hours after recovery. There were no differences between the groups with respect to left ventricular ejection fraction, history of hypertension, body mass index, and serum lipids. Before exercise, GFC was significantly lower in patients with CAD when compared with controls (<em>1</em>.40 +/- 0.43 versus 3.<em>2</em>8 +/- <em>1</em>.<em>1</em>9 microg/mL, respectively; P < 0.00<em>1</em>). In patients with CAD, F <em>1</em> + <em>2</em> levels were significantly higher than those of controls (<em>1</em>.<em>1</em>5 +/- 0.43 versus 0.79 +/- 0.<em>1</em>0 nmol/L, respectively; P = 0.00<em>2</em>). In both study groups, GFC levels increased significantly at peak exercise and decreased to baseline values <em>2</em> hours after recovery. At peak exercise, F <em>1</em> + <em>2</em> levels significantly increased in both study groups. However, while F <em>1</em> + <em>2</em> levels of controls decreased to baseline values <em>2</em> hours after recovery (0.79 +/- 0.<em>1</em>0 versus 0.80 +/- 0.<em>1</em>0 nmol/L; P>> 0.05), F <em>1</em> + <em>2</em> levels of patients with CAD were still significantly elevated (<em>1</em>.<em>1</em>5 +/- 0.43 versus <em>1</em>.84 +/- 0.06 nmol/L; P = 0.00<em>2</em>). Acute exercise increases coagulation and fibrinolysis both in untrained subjects with and without CAD. However, in patients with CAD, the equilibrium between fibrinolysis and coagulation during peak exercise is disturbed in favor of coagulation after recovery.

Fibrinolysis and coagulation were studied in <em>1</em>0 neonates undergoing cardiac operations for congenital heart defects. Coagulation was activated during cardiopulmonary bypass as evidenced by highly increased <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> levels compared with preoperative values. <em>Prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> levels remained elevated until postoperative day 3. Unlike coagulation, fibrinolysis was not activated during cardiopulmonary bypass but did show late activation on postoperative day 3, as evidenced by elevated levels of the fibrin degradation product D-dimer. Lack of fibrinolytic activation during bypass and its appearance on postoperative day 3 were partly explained by changes observed in tissue plasminogen activator and its inhibitor. During bypass, levels of tissue plasminogen activator and its inhibitor increased by 3.4-fold and 3.<em>2</em>-fold, respectively. In the postoperative period, levels of plasminogen activator inhibitor normalized rapidly whereas tissue plasminogen activator remained elevated, resulting in late fibrinolytic activation on postoperative day 3. In accordance with elevated <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>, platelet count, antithrombin III, protein C, <em>prothrombin</em>, and factor VII were decreased on postoperative day <em>2</em>, indicating ongoing consumptive coagulopathy. Nine patients had antithrombin III and six had protein C levels below age-specific normal ranges, consistent with an acquired deficiency state. Three had central venous thrombosis by postoperative day 4 or 5. In all three, thrombosis was preceded by antithrombin III deficiency, protein C deficiency, and highly elevated plasminogen activator inhibitor (3.7 to 37 times the mean of the other patients) on postoperative days <em>1</em> to 3. In conclusion, cardiopulmonary bypass in neonates caused rapid and profound alterations in the coagulation and fibrinolytic systems and initiated consumptive coagulopathy lasting until at least postoperative day 3. Thrombophilic abnormalities in antithrombin III, protein C, and fibrinolysis were frequently found and were associated with serious thrombotic complications.

A double blind randomized cross-over multi-center study has been conducted to compare the pharmacokinetic and coagulation activation markers of high-purity factor IX concentrate subjected to both solvent/ detergent (SD) treatment and <em>1</em>5 nm-filtration (FIX-SD-<em>1</em>5) with the licensed product subjected only to solvent-detergent (FIX-SD). This filtration process allows the elimination of small particles, such as non-enveloped viruses (i.e., hepatitis A and parvovirus B<em>1</em>9). Eleven severe hemophilia B patients (FIX coagulant activity (<em>2</em> IU/dl) received one infusion of 60 IU/kg of FIX-SD and one infusion of 60 IU/kg of FIX-SD-<em>1</em>5 at least at <em>1</em>0 days interval. Blood samples were obtained before and at various time up to 7<em>2</em> h after infusion. The decay curves of factor IX (FIX:C and FIX:Ag) were evaluated by a model independent method. Bioequivalence was found between the two concentrates using the Schuirmann test. The mean FIX:C and FIX:Ag recovery of FIX-SD-<em>1</em>5 was <em>1</em>.08 and 0.89 IU/dl/IU/kg respectively with a mean half-life of 33.3 h for FIX:C and <em>2</em>5.6 h for FIX:Ag. Six months after initial enrollment, pharmacokinetic parameters were similar in the 7 patients tested. There was no significant variation of <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> and thrombin-antithrombin complexes measured up to 6 h after infusion, indicating that there was no activation process after administration of FIX. In conclusion, these data demonstrate that the introduction of a <em>1</em>5 nm filtration does not alter the pharmacokinetic profile of a well characterized SD FIX concentrate while providing additional viral safety.

Higher levels of tissue factor (the initiator of blood coagulation) have been found in coronary atherosclerotic plaques of patients with unstable coronary artery disease, but it is not established whether they are associated with a different thrombotic response to in vivo plaque rupture. In 40 patients undergoing directional coronary atherectomy, <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>, a marker of thrombin generation, was measured in intracoronary blood samples obtained proximally and distally to the coronary atherosclerotic plaque before and after the procedure. Before the procedure, plasma <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> levels were significantly increased across the lesion in patients with unstable, but not in those with stable, coronary disease (unstable, median increase, 0.37 nM; range, -0.35-<em>1</em>.<em>1</em>6 nM) (stable, median increase, -0.065 nM; range, -0.58-<em>1</em>.06 nM) (P =.00<em>2</em><em>1</em>). After plaque removal, an increase in <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> across the lesion was observed only in patients with unstable coronary disease (unstable, median increase, 0.<em>2</em>5 nM; range, -<em>1</em>.04-4.9 nM) (stable, 0.0<em>1</em> nM; range, -0.48-3.59 nM) (P =.036)]. There was a correlation between the tissue factor content of the plaque and the increase in thrombin generation across the lesion (rho = 0.33; P =.038). The higher tissue factor content found in plaques obtained from patients with unstable coronary disease was associated with a local increase in thrombin generation, thus suggesting a link with the in vivo thrombogenicity of the plaque.

BACKGROUND

Total hip/knee replacement surgery (THR/TKR respectively) is associated with an increased risk of venous thromboembolism. Dabigatran is recommended as a thromboprophylactic agent post orthopaedic surgery. The aim of this study was to assess the post-operative (Day-<em>1</em> and Day-<em>2</em>) effect of prophylactic Dabigatran on: the thrombin generation (TG) assay; <em>prothrombin</em> <em>fragment</em> <em>1</em>.<em>2</em> (F<em>1</em>.<em>2</em>); thrombin-antithrombin complexes (TAT); D-dimer (D-D); and other coagulation parameters. METHODS AND SAMPLES: Nineteen patients (<em>1</em><em>2</em> THR, 7 TKR) who received <em>1</em><em>1</em>0 mg dabigatran 4 hours post-operatively, then <em>2</em><em>2</em>0 mg the following day, were recruited. Blood was collected: pre-operatively (Pre-); peri-operatively (Peri-); <em>1</em>9 hours after <em>1</em><em>1</em>0 mg dabigatran (Day-<em>1</em>); and <em>1</em>7 hours after <em>2</em><em>2</em>0 mg dabigatran (Day-<em>2</em>). The TG assay was measured using the Calibrated Automated Thrombogram and a low concentration of tissue factor. Other coagulation parameters measured included activated partial thromboplastin time (APTT), thrombin-time (TT), ecarin-clotting time (ECT) and Hemoclot tests.

RESULTS

From Pre- to Peri-, ETP/peak-thrombin, F<em>1</em>.<em>2</em>, TAT and D-D increased significantly. From Peri- to Day-<em>1</em> and Day-<em>2</em>: TAT reduced progressively; D-D increased; F<em>1</em>.<em>2</em> did not change significantly; lag-time and time-to-peak prolonged; ETP/Peak-thrombin increased spuriously, due to Dabigatran interfering with the α-<em>2</em> macroglobulin:thrombin complex in the TG assay. APTT, TT, ECT and Hemoclot increased progressively post-operatively; good correlations were seen between these tests.

CONCLUSIONS

The effect of dabigatran on the TG assay, showed a spurious increase in ETP and Peak-thrombin due to its interference with the TG assay. Dabigatran reduced TAT, but not F<em>1</em>.<em>2</em>, suggesting that thrombin was still being generated after surgery, but was blocked by Dabigatran.

Forty healthy female volunteers aged between <em>1</em>9 and 35 years (<em>2</em>7.3 +/- 4.<em>1</em> years) with normal menstrual cycles were included in a double-blind, randomized, placebo-controlled study to investigate the influence on the hemostatic system of an oral contraceptive containing 30 micrograms ethinyl estradiol in combination with <em>2</em>.00 mg dienogest, which is a <em>1</em>9-norprogestin without a <em>1</em>7 alpha-ethinyl group. At baseline and during one treatment cycle, <em>1</em><em>2</em> hemostatic parameters were measured on cycle days 7, <em>1</em>4, and <em>2</em><em>1</em>. The hemostatic parameters were categorized as either procoagulatory, anticoagulatory and profibrinolytic, or antifibrinolytic and indicative of fibrin turnover. Differences between placebo and 30 micrograms ethinyl estradiol and <em>2</em>.00 mg dienogest of plasma levels of hemostatic parameters on cycle days <em>2</em><em>1</em> of the precycle and treatment cycle were chosen as target variables. <em>Prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F <em>1</em> + <em>2</em>) was chosen as the main target variable. Equivalence of F <em>1</em> + <em>2</em> between placebo and active treatment was noted. Among the procoagulatory factors, only factor VII activity was found to be increased over placebo in the active treatment group, but decreased in the placebo group. Protein C activity increased during the treatment with 30 micrograms ethinyl estradiol and <em>2</em>.00 mg dienogest, and was higher than that of the placebo group in which this parameter decreased during the treatment cycle. There was a corresponding increase in fibrinolytic activity being reflected by higher plasminogen levels in the active treatment group in comparison with placebo. An increase was noted for the fibrinolytic parameter D-dimer. Apart from isolated measurements, the parameters remained in their respective normal ranges. The data combine to suggest that 30 micrograms ethinyl estradiol and <em>2</em>.00 mg dienogest has a balanced effect on the hemostatic system stimulating both procoagulatory and fibrinolytic activity.

BACKGROUND

Hypercoagulability is observed in vascular dementia, including Binswanger disease. However, the correlation between hypercoagulability, leukoaraiosis, and dementia remains unclear.

OBJECTIVE

To examine how activation of the coagulation fibrinolysis correlates with leukoaraiosis and dementia.

METHODS

Thrombin-antithrombin complex (TAT), <em>prothrombin</em> <em>fragment</em>(<em>1</em> + <em>2</em>) (F<em>1</em> + <em>2</em>) and cross-linked D-dimer (XDP) were measured consecutively in <em>1</em>8 subjects without dementia and with leukoaraiosis, and in <em>2</em>9 subjects with subcortical vascular dementia and severe leukoaraiosis (Binswanger disease) at either stable or deteriorating stages. They were compared with <em>1</em>9 patients with old lacunar infarctions and <em>2</em>4 patients with other neurological diseases. We also examined the indices of cognitive impairment and brain atrophy. In each group, the ventricular area-cranial space area ratio was measured by an image analyzer.

RESULTS

Patients with Binswanger disease who were exclusively at deteriorating stages showed increased TAT and XDP levels and an increased ventricular area-cranial space area ratio, as compared with the patients with other neurological diseases (P<.00<em>1</em>). The index of cognitive impairment in patients at a deteriorating stage showed a decreasing trend vs that of patients in the stable stage. Among the variables that were significantly associated with a hypercoagulable condition (ie, age, scores on Mini-Mental State Examination or the Hasegawa Dementia Rating Scale, Revised [MMSE/HDRS], white matter lesions, ventricular area-cranial space area ratio, and C-reactive protein), age (odds ratio [OR], <em>2</em>.8<em>2</em>) and MMSE/HDSR scores (OR, 0.43) survived as predictors for coagulation activation, and C-reactive protein survived for fibrinolysis activation (OR, 4.63) in multivariate analysis.

CONCLUSIONS

Hypercoagulability in a subgroup of patients with Binswanger disease and with more severe cognitive impairment and brain atrophy does not support a triggering role for a coagulation-fibrinolysis system, although it may contribute to worsening of neurological deficits.

The purified factor IX concentrates Nanotiv (Kabi Pharmacia), Immunine (Immuno), Factor IX VHP (Bio-transfusion), Alphanine (Alpha) and Mononine (Armour) have been studied in vitro and compared with the <em>prothrombin</em> complex concentrates (PCCs) Preconativ (Kabi Pharmacia) and Prothromplex TIM4 (Immuno). The measured values for factor IX coagulant activity (IX:C) were in good agreement with the manufacturer's label values. In contrast to the PCCs, most of the purified concentrates were virtually devoid of other vitamin K-dependent coagulation factors, the inhibitors protein C and S as well as fibrinogen, fibronectin and immunoglobulins. Indicators of thrombin generation, namely <em>prothrombin</em> <em>fragments</em> <em>1</em> and <em>2</em> (F <em>1</em> + <em>2</em>) and thrombin-antithrombin complex (TAT), were present in varying amounts in all preparations. The specific activity in the purified concentrates exceeded that in the PCCs by a factor of 50-<em>1</em>00. Some differences in purity were found between the purified concentrates. In vivo, Nanotiv was compared with Preconativ and Immunine with Prothromplex TIM4 in crossover studies in patients with severe hemophilia B, and Mononine was tested in a single drug study. Most of the preparations yielded postinfusion increases in TAT, but not in F <em>1</em> + <em>2</em>. Pharmacokinetic variables were analyzed with non-linear curve-fitting combined with model-independent methods. In retrospective comparisons, there were no apparent differences between Nanotiv, Preconativ and Mononine, whereas in vivo recovery seemed lower and the apparent clearance higher for Immunine and Prothromplex. Purified factor IX concentrates were successfully used as cover for surgery or in immune tolerance induction.

Bothrojaracin (BJC) is a <em>2</em>7-kD snake venom protein from Bothrops jararaca that has been characterized as a potent thrombin inhibitor. BJC binds to exosites I and II, with a dissociation constant of 0.7 nM, and influences but does not block the proteinase catalytic site. BJC also binds <em>prothrombin</em> through an interaction that has not been characterized. In the present work we characterize the interaction of BJC with <em>prothrombin</em> quantitatively for the first time, and identify the BJC binding site on human <em>prothrombin</em>. Gel filtration chromatography demonstrated calcium-independent, <em>1</em>:<em>1</em> complex formation between fluorescein-labeled BJC ([5F]BJC) and <em>prothrombin</em>, whereas no interactions were observed with activation <em>fragments</em> <em>1</em> or <em>2</em> of <em>prothrombin</em>. Isothermal titration calorimetry showed that binding of BJC to <em>prothrombin</em> is endothermic, with a dissociation constant of 76 +/- 3<em>2</em> nM. The exosite I-specific ligand, hirudin(54-65) (Hir(54-65) (SO(3)(-)), displaced competitively [5F]BJC from <em>prothrombin</em>. Titration of the fluorescent hirudin(54-65) derivative, [5F]Hir(54-65)(SO(3)(-)), with human <em>prothrombin</em> showed a dissociation constant of 7.0 +/- 0.<em>2</em> microM, indicating a approximately <em>1</em>00-fold lower binding affinity than that exhibited by BJC. Both ligands, however, displayed a similar, approximately <em>1</em>00-fold increase in affinity for exosite I when <em>prothrombin</em> was activated to thrombin. BJC efficiently displaced [5F]Hir(54-65)(SO(3)(-)) from complexes formed with thrombin or <em>prothrombin</em> with dissociation constants of 0.7 +/- 0.9 nM and <em>1</em><em>1</em> +/- 80 nM, respectively, indicating that BJC and Hir(54-65)(SO(3)(-)) compete for the same exosite on these molecules. The results indicate that BJC is a potent and specific probe of the partially exposed anion-binding exosite (proexosite I) of human <em>prothrombin</em>.

BACKGROUND: In spite of using heparin-coated extracorporeal circuits, cardiopulmonary bypass (CPB) is still associated with an extensive thrombin generation, which is only partially suppressed by the use of high dosages of heparin. Recent studies have focused on the origins of this thrombotic stimulus and the possible role of retransfused suctioned blood from the thoracic cavities on the activation of the extrinsic coagulation pathway. The present study was designed to find during CPB an association between retransfusion of suctioned blood from the pericardium and pleural space, containing activated factor VIIa and systemic thrombin generation. METHODS: Blood samples taken from <em>1</em><em>2</em> consenting patients who had elective cardiac surgery were assayed for plasma factor VIIa, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>), and thrombin-antithrombin (TAT) concentrations. Blood aspirated from the pericardium and pleural space was collected separately, assayed for F<em>1</em>+<em>2</em>, TAT, and factor VIIa and retransfused to the patient after the aorta occlusion. RESULTS: After systemic heparinization and during CPB thrombin generation was minimal, as indicated by the lower than base line plasma levels of F<em>1</em>+<em>2</em>, and TAT after correction for hemodilution. In contrast, blood aspirated from the thoracic cavities had significantly higher levels of factor VIIa, F<em>1</em>+<em>2</em>, and TAT compared to the simultaneous samples from the blood circulation (P < 0.05). Furthermore, after retransfusion of the suctioned blood (range, <em>2</em>00-<em>1</em>600 mL) circulating levels of F<em>1</em>+<em>2</em>, and TAT rose significantly from <em>1</em>.6 to <em>2</em>.9 nmol/L (P = 0.00<em>2</em>) and from 5.<em>1</em> to 37.5 μg/L (P = 0.0<em>1</em>), respectively. The increase in both F<em>1</em>+<em>2</em>, and TAT levels correlated significantly with the amount of retransfused suctioned blood (r = 0.68, P = 0.0<em>2</em><em>1</em> and r = 0.90, P = 0.00<em>1</em>, respectively). However, the circulating factor VIIa levels did not correlate with TAT and F<em>1</em>+<em>2</em> levels. CONCLUSIONS: These data suggest that blood aspirated from the thoracic cavities during CPB is highly thrombogenic. Retransfusion of this blood may, therefore, promote further systemic thrombin generation during CPB.

OBJECTIVE

To evaluate the effect of IVF-ET on the hemostatic system.

METHODS

Prospective clinical study.

METHODS

Apparently healthy age-matched women of the hospital staff at various stage of the menstrual cycle.

METHODS

Twenty-five women involved in a IVF-ET program at the Department of Obstetrics and Gynecology, Montpellier University Hospital.

METHODS

Twenty-six hemostasis parameters evaluated repeatedly in patients undergoing IVF-ET.

METHODS

Blood cell-dependent hemostasis parameters and plasmatic coagulation factors, determined at pituitary desensitization, maximal E<em>2</em> level, and P plateau.

RESULTS

Activation of the hemostatic system is evidenced at the P plateau, when D-dimers and <em>fragments</em> <em>1</em> + <em>2</em> of the <em>prothrombin</em> levels rose dramatically. At E<em>2</em> peak, no significant modification of hemostasis markers was noted.

CONCLUSIONS

The present results indicate that ovarian hyperstimulation may induce hemostasis activation at the P plateau. The role of supraphysiologic sex hormone levels on the hemostatic system requires further investigation.

Previous studies from this laboratory have been directed toward elucidation of the roles of individual gamma-carboxyglutamic acid (Gla) residues in Gla domain-related Ca(<em>2</em>+)-directed properties of human protein C (PC) and activated protein C (APC). On the basis of results using recombinant variants of PC containing highly conservative (Asp) mutations of individual Gla residues, it was previously proposed that Gla6, Gla<em>1</em>4, and Gla<em>1</em>9 may not be essential for properties associated with the Ca(<em>2</em>+)-dependent conformation of the Gla domain of these proteins. In this study, we have demonstrated that radical mutations to Val of Gla residues <em>1</em>4 and <em>1</em>9 resulted in 94% and 8<em>2</em>%, respectively, of the Gla domain-related, Ca(<em>2</em>+)- and phospholipid- (PL-) dependent anticoagulant (APTT) activity of wild-type recombinant (wtr) APC, while [Gla6->>Val]r-APC showed a complete loss of this same activity. The more conservative mutant [Gla6->>Gln]r-APC possessed 4% of the APTT activity of wtr-APC, whereas [Gla6->>Asp]r-APC was nearly fully active. As with wtr-PC, both [Gla6->>Val]r-PC and [Gla6->>Gln]r-PC displayed Ca(<em>2</em>+)-dependent intrinsic fluorescence quenching, suggesting that they adopted a Ca(<em>2</em>+)-induced conformation. However, Ca<em>2</em>+ titration data suggested that these conformations were not identical to that undergone by wtr-PC. In addition, the Ca(<em>2</em>+)-mediated binding parameters of [Gla6->>Val]r-PC and [Gla6->>Gln]r-PC to acidic PL vesicles were found to be defective. These data were interpreted at the molecular level using a model for the Gla domain of PC based on the X-ray crystal structure of the Ca<em>2</em>+/bovine <em>prothrombin</em> <em>fragment</em> <em>1</em> complex.(ABSTRACT TRUNCATED AT <em>2</em>50 WORDS)

OBJECTIVE

Tissue factor pathway inhibitor (TFPI) is bound to vascular endothelium (presumably to heparan sulfate) and circulates in complex with plasma lipoproteins. It directly binds and inhibits factor Xa. The purpose of the study is to investigate whether plasma TFPI activity is altered in IDDM and nephropathy and to evaluate the possible determinants of the alteration.

METHODS

We assessed plasma concentration of TFPI (total, truncated, and domain 3 TFPI) and plasma activity of factor Xa inhibition in nondiabetic control subjects (n = <em>2</em><em>2</em>) and in IDDM patients with normoalbuminuria (urinary albumin excretion rate [UAE] < 30 mg/<em>2</em>4h, n = <em>1</em>7), incipient nephropathy (UAE 30-300 mg/<em>2</em>4 h, n = <em>1</em>7), clinical nephropathy (UAE>> 300 mg/<em>2</em>4h, n = <em>2</em>5).

RESULTS

Total, truncated, and domain 3 TFPI concentrations were increased in IDDm patients compared with those in control subjects and were more pronounced in IDDM patients with nephropathy. Plasma activity of factor Xa inhibition measured by HEPTEST (Haemachem, St. Louis, MO) assay was increased in IDDM patients, especially in those with nephropathy. TFPI-dependent factor Xa inhibition, obtained as the difference in clotting time with and without adding activity-neutralizing anti-TFPI antibody to samples, was increased in IDDm patients with nephropathy. This was, however, not sufficient to inhibit the biological activity of factor Xa as demonstrated by increased levels of <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>. LDL cholesterol and HbA<em>1</em>c were independently correlated to plasma TFPI.

CONCLUSIONS

Inhibition of factor Xa activity is increased in IDDM patients with nephropathy, mainly because of increased plasma TFPI activity. The increased plasma TFPI activity in these patients may be associated with and regulated by LDL in plasma and metabolic control. The anticoagulant activity of TFPI may attenuate the hypercoagulable state in diabetes but does not seem to be able to normalize hemostasis.

BACKGROUND

In patients with suspected heparin-induced thrombocytopenia (HIT) who need renal replacement therapy, a nonheparin anticoagulant has to be chosen to prevent thrombosis in the extracorporeal circuit. Danaparoid, a low-molecular-weight heparinoid consisting of heparan sulphate, dermatan sulphate, and chondroitin sulphate, is recommended for systemic anticoagulation in patients with HIT. However, there are few data on the use of danaparoid in patients with acute renal failure, especially in patients dependent on renal replacement therapy such as continuous venovenous hemofiltration (CVVH). In the present study, we analyzed the pharmacokinetics and pharmacodynamics of danaparoid during CVVH in patients with suspected HIT.

METHODS

Based on a mathematical model, a dosing scheme for danaparoid was designed, aiming at anti-Xa levels of 0.5 to 0.7 U/mL, with a maximum of <em>1</em>.0 U/mL. This dosing scheme was prospectively tested in the first CVVH run of a cohort of five patients with suspected HIT. CVVH with a blood flow rate of <em>1</em>50 mL/minute and a substitution rate of <em>2</em>,000 mL/hour was performed with a cellulose triacetate membrane. Danaparoid was administered as a continuous infusion of <em>1</em>00 anti-Xa-U/hour after a loading dose of 3,500 anti-Xa-U. Serial measurements of anti-Xa activity and <em>prothrombin</em> <em>fragment</em> F<em>1</em>+<em>2</em> were performed at baseline, at t = 5, <em>1</em>5, and 30 minutes, and at t = <em>1</em>, <em>2</em>, 4, 8, <em>1</em>6, and <em>2</em>4 hours after the danaparoid loading dose.

RESULTS

The median anti-Xa activity reached a maximum of <em>1</em>.0<em>2</em> (0.66 to <em>1</em>.3<em>1</em>) anti-Xa-U/mL after <em>1</em>5 minutes and gradually declined to 0.40 (0.<em>1</em>5 to 0.58) anti-Xa-U/mL over the span of <em>2</em>4 hours. Target anti-Xa levels were reached from <em>2</em> to <em>1</em><em>2</em> hours after the loading dose. Median <em>prothrombin</em> <em>fragment</em> F<em>1</em>+<em>2</em> gradually decreased from 43<em>2</em> (<em>2</em>00 to 768) to <em>2</em>6<em>2</em> (<em>2</em>48 to 3<em>1</em>7) pmol/L after <em>2</em>4 hours. No bleeding or thromboembolic events occurred throughout the described treatment period.

CONCLUSIONS

Danaparoid administered by a continuous infusion of <em>1</em>00 anti-Xa-U/hour after a loading dose of 3,500 anti-Xa-U elicited target anti-Xa levels from <em>2</em> to <em>1</em><em>2</em> hours after the loading dose, without bleeding or thromboembolic events during the described CVVH treatment in patients with suspected HIT.

To evaluate the suitability of two anticoagulants (heparin vs. argatroban) as adjunctive drugs during and after elective conventional percutaneous transluminal coronary angioplasty, we compared the changes in inflammatory, hemostatic, and endothelium-derived markers in groups of patients with stable angina treated with the two drugs during percutaneous transluminal coronary angioplasty. Twenty-seven patients were randomly allocated to either group <em>1</em> (<em>1</em>5 patients who received an empiric dose of heparin and aspirin as anticoagulant), or group <em>2</em> (<em>1</em><em>2</em> patients who received an alternative regimen of argatroban and aspirin). Both drugs were administered as a bolus followed by continuous infusion for 96 hours during and after percutaneous transluminal coronary angioplasty. There were no differences in the inflammatory response induced by percutaneous transluminal coronary angioplasty in both groups, but the fibrinogen concentration significantly decreased during percutaneous transluminal coronary angioplasty in group <em>2</em>. Decreased platelet counts and increased mean platelet volume were observed during percutaneous transluminal coronary angioplasty in both groups. The levels of <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> and thrombin antithrombin III complex increased markedly during percutaneous transluminal coronary angioplasty. Group <em>2</em> showed a more rapid return to the baseline levels of these two markers than group <em>1</em>. Secondary fibrinolysis was evidenced by a steep increase of D-dimer after percutaneous transluminal coronary angioplasty in both groups. In contrast to the significant decrease in antithrombin activities during percutaneous transluminal coronary angioplasty in group <em>1</em>, no marked change in these markers was found in group <em>2</em>. Although the levels of von Willebrand factor and plasminogen activator inhibitor-<em>1</em> showed essentially the same changes in both groups during and after percutaneous transluminal coronary angioplasty, more markedly increased levels of tissue type plasminogen activator and type plasminogen activator-plasminogen activator inhibitor-<em>1</em> complex after percutaneous transluminal coronary angioplasty were found in group <em>1</em> than in group <em>2</em>. While neither drug had any effect on the percutaneous transluminal coronary angioplasty-induced inflammatory response, argatroban may more effectively inhibit the generated thrombin and prevent antithrombin consumption during and after percutaneous transluminal coronary angioplasty.

BACKGROUND

Thromboembolism occurs in 0.4% to <em>2</em>% of the subjects undergoing radiofrequency ablation (RFA), but its mechanisms remain unclear. Our aim was to evaluate several parameters of the hemostatic system in relation to the electrophysiologic procedure.

METHODS

Thirty consecutive patients were enrolled in the study. Fifteen underwent electrophysiologic study and <em>1</em>5 underwent radiofrequency ablation. Before the ablation procedure, all subjects were given an intravenous heparin bolus (<em>2</em>500 IU). Blood samples were drawn immediately before, at the end of, and <em>2</em>4 hours after the procedures. Spontaneous platelet aggregation in whole blood and in platelet-rich plasma, markers of clotting activation (<em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> and the thrombin-antithrombin complex) and the fibrinolytic system (plasminogen activator inhibitor and D-dimer) levels were evaluated.

RESULTS

At the end of the procedure, spontaneous platelet aggregation in whole blood, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, thrombin-antithrombin complex, and D-dimer levels increased significantly in all patients. The hemostatic changes were more marked after RFA than after electrophysiology. Spontaneous aggregation in whole blood, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, and thrombin-antithrombin complex levels at <em>2</em>4 hours after the procedure were similar to those observed before the procedure in both groups; D -dimer levels were still elevated with respect to preprocedure levels, with a trend toward higher levels in patients undergoing RFA rather than electrophysiology. A significantly more marked activation of coagulation (<em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, P <.005) was found in patients in whom the mean duration of energy application was higher than <em>2</em>3.5 seconds.

CONCLUSIONS

Our data suggest that antithrombotic prevention with a prolonged administration of heparin and/or the association of antiplatelet agents should be considered in patients undergoing RFA.

Mouse and human <em>prothrombin</em> (ProT) active site specifically labeled with D-Phe-Pro-Arg-CH(<em>2</em>)Cl (FPR-ProT) inhibited tissue factor-initiated thrombin generation in platelet-rich and platelet-poor mouse and human plasmas. FPR-prethrombin <em>1</em> (Pre <em>1</em>), <em>fragment</em> <em>1</em> (F<em>1</em>), <em>fragment</em> <em>1</em>.<em>2</em> (F<em>1</em>.<em>2</em>), and FPR-thrombin produced no significant inhibition, demonstrating the requirement for all three ProT domains. Kinetics of inhibition of ProT activation by the inactive ProT(S<em>1</em>95A) mutant were compatible with competitive inhibition as an alternate nonproductive substrate, although FPR-ProT deviated from this mechanism, implicating a more complex process. FPR-ProT exhibited ∼<em>1</em>0-fold more potent anticoagulant activity compared with ProT(S<em>1</em>95A) as a result of conformational changes in the ProT catalytic domain that induce a more proteinase-like conformation upon FPR labeling. Unlike ProT and ProT(S<em>1</em>95A), the pathway of FPR-ProT cleavage by <em>prothrombin</em>ase was redirected from meizothrombin toward formation of the FPR-prethrombin <em>2</em> (Pre <em>2</em>)·F<em>1</em>.<em>2</em> inhibitory intermediate. Localization of ProT labeled with Alexa Fluor® 660 tethered through FPR-CH(<em>2</em>)Cl ([AF660]FPR-ProT) during laser-induced thrombus formation in vivo in murine arterioles was examined in real time wide-field and confocal fluorescence microscopy. [AF660]FPR-ProT bound rapidly to the vessel wall at the site of injury, preceding platelet accumulation, and subsequently to the thrombus proximal, but not distal, to the vessel wall. [AF660]FPR-ProT inhibited thrombus growth, whereas [AF660]FPR-Pre <em>1</em>, lacking the F<em>1</em> membrane-binding domain did not bind or inhibit. Labeled F<em>1</em>.<em>2</em> localized similarly to [AF660]FPR-ProT, indicating binding to phosphatidylserine-rich membranes, but did not inhibit thrombosis. The studies provide new insight into the mechanism of ProT activation in vivo and in vitro, and the properties of a unique exosite-directed <em>prothrombin</em>ase inhibitor.

The factor V (Arg506->>Gln) mutation confers an increased risk of deep vein thrombosis, whereas its role in saphenous vein graft closure after coronary artery bypass grafting (CABG) remains unclear. This study examined the anticoagulant response to activated protein C (APC ratio) in relation to the surgical trauma and the significance of the factor V Leiden mutation in determining postoperative thrombin generation and fibrin formation and the risk of early vein graft occlusion. A total of <em>1</em>08 men undergoing elective CABG for exertional angina pectoris (mean age 6<em>1</em>.<em>1</em> +/- 8.7 years) were examined. The patency of saphenous vein grafts was studied at routine reangiography three months after CABG. Of <em>1</em>00 patients who underwent reangiography, <em>2</em>3 had one or more occluded vein grafts at reangiography. Heterozygosity for the factor V (Arg506->>Gln) mutation tended to be associated with early saphenous vein graft occlusion (5/<em>1</em><em>1</em> carriers vs. <em>1</em>8/89 non-carriers with graft occlusion, chi<em>2</em> = 3.5<em>2</em>, p = 0.06), whereas pre- and postoperative APC ratios did not. Pre- and postoperative determinations of <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, thrombin-antithrombin complexes and soluble fibrin levels did not differ between patients with and without the mutation. Early saphenous vein graft occlusion after CABG could tentatively be added to deep vein thrombosis as a vascular complication that can be attributed to the factor V (Arg506->>Gln) mutation.

Inherited resistance to activated protein C (APC-resistance), caused by a point mutation in the factor V gene leading to replacement of Arg(R)506 with a Gln (Q), and inherited protein S deficiency are associated with functional impairment of the protein C anticoagulant system, yielding lifelong hypercoagulability and increased risk of thrombosis. APC-resistance is often an additional genetic risk factor in thrombosis-prone protein S deficient families. The plasma concentration of <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>), which is a marker of hypercoagulable states, was measured in <em>2</em>05 members of 34 thrombosis-prone families harbouring the Arg506 to Gln mutation (APC-resistance) and/or inherited protein S deficiency. The plasma concentration of F<em>1</em> + <em>2</em> was significantly higher both in 38 individuals carrying the FV:Q506 mutation in heterozygous state (<em>1</em>.7 +/- 0.7 nM; mean +/- SD) and in 48 protein S deficient cases (<em>1</em>.9 +/- 0.9 nm), than in <em>1</em>00 unaffected relatives (<em>1</em>.3 +/- 0.5 nM). Warfarin therapy decreased the F<em>1</em> + <em>2</em> levels, even in those four patients who had combined defects (0.5 +/- 0.3 nM). Our results agree with the hypothesis that individuals with APC-resistance or protein S deficiency have an imbalance between pro- and anti-coagulant forces leading to increased thrombin generation and a hypercoagulable state.

Inflammation and coagulation occur concomitantly in sepsis. Thrombin activates platelet that leads to P-selectin translocation, which upregulate tissue factor (TF) generation. Tissue factor pathway inhibitor (TFPI) is an anticoagulant that modulates coagulation induced by TF. The term non-overt disseminated intravascular coagulation (DIC) refers to a state of affairs prevalent before the occurrence of overt DIC. It was suggested that an initiation of treatment in non-overt DIC has better outcome than overt DIC. This study investigated the role of TFPI level, P-selectin, and thrombin activation markers in non-overt and overt DIC induced by sepsis and its relationship to outcome and organ dysfunction as measured by the Sequential Organ Failure Assessment (SOFA) score. It included <em>1</em>76 patients with sepsis. They were admitted to the pediatric intensive care unit (ICU).They included <em>1</em>44 cases of non-overt DIC and 3<em>2</em> cases of overt DIC. There was a significant difference in hemostatic markers, platelet count, partial thromboplastin time (PTT), P-selectin, thrombin activation markers, TFPI, and DIC score between overt and non-overt DIC in both groups. It was noticed that P-selectin was positively correlated with DIC score, fibrinogen consumption, fibrinolysis (D-dimer), thrombin activation markers, and TFPI. Tissue factor pathway inhibitor was significantly correlated with fibrinolysis, DIC score, and <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>. Sequential Organ Failure Assessment score was correlated with DIC score and other hemostatic markers in patients with overt DIC. To improve the outcome of patients with DIC, there is a need to establish more diagnostic criteria for non-overt-DIC. Plasma levels of TFPI and P-selectin may be helpful in this respect.

Acute exercise and exercise training may influence putative endothelial progenitor cell (EPC) number and colony forming units (CFU-ECs), although the mechanisms remain unclear. This study examined the effects of in vitro thrombin supplementation and acute exercise on CFU-EC gene expression, associated with cellular proliferation and differentiation. The effect of habitual physical activity was evaluated through analysis of EPCs from chronically high- and low-active men. Participants were healthy high- and low-active men (n=<em>2</em>3), aged 55-80 yr. Circulating CD34+/VEGFR<em>2</em>+ number, CFU-ECs, plasma <em>prothrombin</em> <em>fragment</em> (F<em>1</em>+<em>2</em>), and thrombin-antithrombin III were measured at rest and after 30 min of exercise. Gene expression of cyclin A<em>2</em>, cyclin D<em>1</em>, p<em>2</em>7, VE-cadherin, and VEGFR<em>2</em> was assessed in postexercise CFU-ECs and resting CFU-ECs treated with 0, <em>1</em>, 5, or <em>1</em>0 U/ml of thrombin. Outcomes were compared between high- and low-active participants. F<em>1</em>+<em>2</em> and thrombin-antithrombin III, but not CD34+/VEGFR<em>2</em>+ number and CFU-ECs, increased with exercise. Exercise-induced changes in F<em>1</em>+<em>2</em> correlated with changes in CD34+/VEGFR<em>2</em>+ number in both groups. Thrombin treatments and acute exercise increased cyclin A<em>2</em> and cyclin D<em>1</em> expression and decreased p<em>2</em>7 expression. One unit per milliliter thrombin increased VEGFR<em>2</em> and VE-cadherin expression, whereas 5 U/ml, <em>1</em>0 U/ml, and acute exercise did not elicit any changes. An exercise training effect was observed with greater decreases in p<em>2</em>7 expression with 5 and <em>1</em>0 U/ml thrombin and greater increases in VEGFR<em>2</em> and VE-cadherin expression with <em>1</em> U/ml thrombin in high-active men. Exercise-induced changes in putative EPC gene expression are associated with thrombin production and may be modulated by long-term exercise training.

BACKGROUND

Age-related changes in blood coagulation and fibrinolytic factors are associated with an increase in risk of thrombotic events. The purpose of this study was to assess the effects of age, regular aerobic exercise and detraining on blood coagulation and fibrinolytic factors in men.

METHODS

Initially, 4<em>1</em> sedentary and 4<em>2</em> physically active men (<em>2</em>0-64 years) were analyzed for plasma levels of coagulation and fibrinolytic factors. Twelve sedentary men were then subjected to <em>1</em>6-week aerobic exercise training and subsequent <em>2</em>-week detraining. Their blood samples taken at rest were assayed for activity levels of <em>prothrombin</em>, coagulation factor (F) V, VII, VIII, IX, X, XI and XIII, antithrombin III, protein C and plasminogen, and for antigen levels of fibrinogen, <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>), FIX, protein C, tissue-type plasminogen activator (tPA), plasminogen activator inhibitor <em>1</em> (PAI-<em>1</em>) and tPA/PAI-<em>1</em> complex.

RESULTS

Plasma levels of most coagulation factors, particularly for fibrinogen and FIX antigens as well as FXIII activity significantly increased with aging in sedentary men, while that tendency disappeared in physically active men. By the exercise training, plasma antigen and/or activity levels of most blood coagulation factors except for <em>prothrombin</em> and FIX decreased. These training-effects, however, disappeared after detraining, and in some cases even rebounded to higher levels than those of pre-training. Plasma antigen levels of tPA, PAI-<em>1</em> and tPA/PAI-<em>1</em> complex decreased with the training and remained low even after detraining.

CONCLUSIONS

Regular aerobic exercises give complex effects on expression of hemostatic factors, overall favoring the hemostatic balance to less thrombotic, partly cancelling out the age effects.