Thrombocytopenia and altered coagulation characterize all hantavirus infections. To further assess the newly discovered predictive biomarkers of disease severity during acute Puumala virus (PUUV) infection, we studied the associations between them and the variables reflecting coagulation, fibrinolysis and endothelial activation. Nineteen hospital-treated patients with serologically confirmed acute PUUV infection were included. Acutely, plasma levels of pentraxin-3 (PTX3), cell-free DNA (cf-DNA), complement components SC5b-9 and C3 and interleukin-6 (IL-6) were recorded as well as platelet ligands and markers of coagulation and fibrinolysis. High values of plasma PTX3 associated with thrombin formation (<em>prothrombin</em> <em>fragments</em> F<em>1</em>+<em>2</em>; r = 0.46, P = 0.05), consumption of platelet ligand fibrinogen (r = -0.70, P < 0.00<em>1</em>) and natural anticoagulants antithrombin (AT) (r = -0.74, P < 0.00<em>1</em>), protein C (r = -0.77, P < 0.00<em>1</em>) and protein S free antigen (r = -0.8<em>1</em>, P < 0.00<em>1</em>) and a decreased endothelial marker ADAMTS<em>1</em>3 (a disintegrin and metalloproteinase with a thrombospondin type <em>1</em> domain <em>1</em>3) (r = -0.48, P = 0.04). Plasma level of AT associated with C3 (r = 0.76, P < 0.00<em>1</em>), IL-6 (r = -0.56, P = 0.0<em>1</em>) and cf-DNA (r = -0.47, P = 0.04). High cf-DNA coincided with increased <em>prothrombin</em> <em>fragments</em> F<em>1</em>+<em>2</em> (r = 0.47, P = 0.04). Low C3 levels reflecting the activation of complement system through the alternative route predicted loss of all natural anticoagulants (for protein C r = 0.53, P = 0.03 and for protein S free antigen r = 0.64, P = 0.004). Variables depicting altered coagulation follow the new predictive biomarkers of disease severity, especially PTX3, in acute PUUV infection. The findings are consistent with the previous observations of these biomarkers also being predictive for low platelet count and underline the cross-talk of inflammation and coagulation systems in acute PUUV infection.

To explore the coagulation/fibrinolytic balance and its relation with free protein S (f-PS) in subjects with antiphospholipid antibodies (aPLs) outside the setting of autoimmune inflammatory disorders, we carried out a cross-sectional study on <em>1</em>8 thrombotic patients with primary antiphospholipid syndrome and <em>1</em>8 apparently healthy subjects with persistence of idiopathic aPLs. <em>Prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>), thrombin-antithrombin complex (TAT) and D-Dimer (D-D) were taken as markers of thrombin generation and fibrin turnover. Mean F<em>1</em> + <em>2</em> levels were higher in thrombotic (p = 0.006) and non-thrombotic subjects (p = 0.000<em>1</em>) than in controls as were those of D-D (p < 0.000<em>1</em> and p = 0.003 respectively). TAT levels did not differ. Lower mean levels of f-PS were found in thrombotic (p = 0.0006) and non-thrombotic subjects (p = 0.00<em>2</em>) than in controls. Within both groups, mean F<em>1</em> + <em>2</em> levels were higher in subjects who had low f-PS levels compared to those with normal f-PS levels (p = 0.0<em>1</em>). Gender analysed data revealed blunted tPA release (venous occlusion test) in thrombotic females (from <em>1</em>6.80 +/- 0.79 to <em>2</em><em>1</em>.3 +/- 3.9 ng/nl, NS) but not in thrombotic males (from <em>1</em>8.<em>2</em> +/- <em>2</em>.0 to 33.7 +/- 4.9 ng/ml, p=0.0<em>1</em>) nor in asymptomatic subjects of either sex. Also, in both patient groups females had higher mean PAI than males (p < 0.000<em>2</em>) and than control females (p < 0.0<em>2</em>). Low free protein S was found in <em>1</em>00% of non-thrombotic and in 90% of thrombotic patients with defective fibrinolysis. These data are consistent with increased thrombin generation, accelerated fibrin turnover and fibrinolysis abnormalities also in asymptomatic carriers of aPLs and highlight a central role for acquired f-PS deficiency in the thrombotic tendency of the antiphospholipid syndrome.

<em>Prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>) and thrombin-antithrombin complexes (TAT), as well as other coagulation and fibrinolysis parameters, were studied in a series of <em>1</em>3 patients affected by thrombotic thrombocytopenic purpura (TTP) or hemolytic-uremic syndrome (HUS). <em>Fragment</em> F<em>1</em> + <em>2</em> was found to be increased in all patients at diagnosis (patients' range, <em>1</em>.<em>2</em><em>1</em>-<em>1</em>9.03 nmol/l; normal limits, 0.<em>2</em>8-<em>1</em>.08 nmol/l), and remained also higher than normal after treatment with plasma exchange (patients' range, <em>1</em>.5-4.0<em>1</em> nmol/l). Even though the analysis of fibrinolysis markers did not show a definite state of hypo or hyperfibrinolysis in the systemic circulation, enhanced circulating D-dimer levels (0.53-<em>1</em><em>2</em>.6 micrograms/ml, normal levels of 0.03-0.<em>2</em>9 micrograms/ml) indicated that a certain grade of fibrin lysis was present at previously formed thrombi. Plasma PAI-<em>1</em> activities either on admission (9.<em>2</em>-38.<em>2</em> U/ml) and after plasma exchange therapy (<em>2</em>.6-38.6 U/ml) showed a behavior irrespective of t-PA:Ag changes, and post-plasmapheresis values remained high only in patients with fatal neurological outcome. Nevertheless, no correlations between clinical and laboratory data could be established useful for the TTP/HUS prognosis. We conclude that increased thrombin generation occurring in damaged areas is appropriately inhibited by antithrombin III in the systemic circulation, avoiding consumption coagulopathy to develop in uncomplicated patients. In addition, fibrinolysis data suggest that elevated PAI-<em>1</em> may decisively favor the development of microvascular thrombi.

Virally inactivated, high-purity factor XI concentrates are available for treatment of patients with factor XI deficiency. However, preliminary experience indicates that some preparations may be thrombogenic. We evaluated whether a highly purified concentrate produced signs of activation of the coagulation cascade in two patients with severe factor XI deficiency infused before and after surgery. Signs of heightened enzymatic activity of the common pathway of coagulation (elevated plasma levels of <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> and fibrinopeptide A) developed in the early post-infusion period, accompanied by more delayed signs of fibrin formation with secondary hyperfibrinolysis (elevated D-dimer and plasmin-antiplasmin complex). These changes occurred in both patients, but were more severe in the older patient with breast cancer when she underwent surgery, being accompanied by fibrinogen and platelet consumption. There were no concomitant signs of heightened activity of the factor VII-tissue factor mechanism on the factor Xase complex (plasma levels of activated factor VII and of factor IX and X activation peptides did not increase). The observed changes in biochemical markers of coagulation activation indicate that concentrate infusions increased thrombin generation and activity and that such changes were magnified by malignancy and surgery. Because some factor XI concentrates may be thrombogenic, they should be used with caution, especially in patients with other risk factors for thrombosis.

BACKGROUND

Cardiovascular disease with disturbances in the haemostatic system, might lead to thrombotic complications with clinical manifestations like acute myocardial infarction (AMI) and stroke. Activation of the coagulation cascade with subsequent increased thrombin generation, characterizes a prothrombotic phenotype. In the present study we investigated whether prothrombotic markers were associated with risk factors and clinical subgroups in a cohort of patients with angiographically verified coronary artery disease (CAD). The patients were randomized to long-term treatment with the antiplatelet drugs aspirin or clopidogrel, and we further investigated the effect on hypercoagulability of such treatment for <em>1</em> year, of which limited data exists.

METHODS

Venous blood samples were collected in fasting condition between 08:00 and <em>1</em>0:30 am, at baseline when all patients were on aspirin therapy (n = <em>1</em>00<em>1</em>) and in <em>2</em>76 patients after <em>1</em> year follow-up on aspirin or clopidogrel. In vivo thrombin generation was assessed by <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em>+<em>2</em>) and D-dimer, and the endogenous thrombin potentiale (ETP) in the calibrated automated thrombogram (CAT) assay, representing ex vivo thrombin generation. In addition soluble tissue factor (sTF) and free- and total tissue factor pathway inhibitor (TFPI) were measured.

RESULTS

We found age to be significantly associated with F<em>1</em>+<em>2</em> and D-dimer (β = 0.<em>2</em><em>2</em>9 and β =0.4<em>1</em>7 respectively, p <0.00<em>1</em>, both). Otherwise, only weak associations were found. F<em>1</em>+<em>2</em> and D-dimer were higher in women compared to men (p <0.00<em>1</em> and p = 0.033, respectively). Smokers had elevated levels of ETP compared to non-smokers (p = 0.0<em>1</em>4). Additionally, patients on renin-angiotensin system (RAS) inhibition showed significantly higher levels of F<em>1</em>+<em>2</em>, compared to non-users (p = 0.0<em>1</em>3). Both aspirin and clopidogrel reduced levels of ETP after <em>1</em><em>2</em> months intervention (p = 0.003 and p <0.00<em>1</em>, respectively) and the levels of F<em>1</em>+<em>2</em> were significantly more reduced on aspirin compared to clopidogrel (p = 0.0<em>2</em>3).

CONCLUSIONS

In the present population of stable CAD, we could demonstrate a more hypercoagulable profile among women, smokers and patients on RAS medication, assessed by the prothrombotic markers F<em>1</em>+<em>2</em>, D-dimer and ETP. Long-term antiplatelet treatment with aspirin alone seems to attenuate thrombin generation to a greater extent than with clopidogrel alone. The study is registered at http://www.clinicaltrials.gov: NCT00<em>2</em><em>2</em><em>2</em><em>2</em>6<em>1</em>.

Anti-phospholipid Ab (aPL) are a heterogeneous group of autoantibodies directed against various combinations of phospholipids (PL) and PL-binding proteins. Lupus anticoagulant (LA) Ab, a subset of aPL, exhibit anticoagulant properties in vitro, but are procoagulant in vivo. Most LA Ab are specific for either beta(<em>2</em>)-glycoprotein I (beta(<em>2</em>)GPI) or <em>prothrombin</em> (PT), two PL-binding proteins. We have previously shown that beta(<em>2</em>)GPI and beta(<em>2</em>)GPI-dependent aPL bind specifically to apoptotic, but not viable, thymocytes. In this study, we demonstrate that PT, like beta(<em>2</em>)GPI, binds selectively to the surface of apoptotic, but not viable, Jurkat cells. Furthermore, PT supports the binding of systemic lupus erythematosus-derived polyclonal and murine monoclonal LA Ab to apoptotic cells. Two LA mAb, which differed dramatically in their relative affinities for PT, were studied. Although one mAb (<em>2</em>9J3-6<em>2</em>) had a high affinity for PT alone, the other (<em>2</em>9I4-<em>2</em>4) showed minimal reactivity with PT alone and required PL for elevated binding. Monovalent <em>fragments</em> of <em>2</em>9I4-<em>2</em>4 reacted with PL-bound PT with high affinity, suggesting that this mAb recognizes a PL-dependent epitope. Despite these differences, PT-dependent binding of both mAb to apoptotic cells was 30-fold greater than that to viable cells. Moreover, binding of PT to apoptotic cells was, itself, increased in the presence of bivalent, but not monovalent, forms of either mAb. In summary, our data demonstrate the following: <em>1</em>) specific binding of PT to apoptotic cells, an effect enhanced by PT-dependent LA Ab; <em>2</em>) heterogeneity of PT-dependent LA Ab; and 3) potential pathogenicity of Ab of either low or high affinity for PT.

Plasma F <em>1</em>+<em>2</em> levels, the activation peptide originating from the factor Xa-mediated activation of <em>prothrombin</em>, increase in many clinical conditions associated with hypercoagulability and decrease in patients on oral anticoagulant treatment (OAT). However. the usefulness of F <em>1</em>+<em>2</em> measurement to monitor OAT has not yet been investigated in clinical studies. Before those studies are attempted, the plausibility of its implementation in the laboratory control of OAT should be evaluated. In this respect, a thorough investigation of the pattern of changes of F <em>1</em>+<em>2</em> as a function of increased intensity of anticoagulation expressed as International Normalized Ratio is essential. One hundred and thirty-two patients on long-term warfarin treatment were recruited to cover 8 ranges of anticoagulation from < <em>1</em>.5 to 9.0 INR. F <em>1</em>+<em>2</em> was measured in batch on frozen plasma and INR was determined on fresh plasma. The relationship of F <em>1</em>+<em>2</em> vs. INR showed a hyperbolic pattern with F <em>1</em>+<em>2</em> levels decreasing progressively and significantly as a function of increasing INR up to 3.0. A further decrease in F <em>1</em>+<em>2</em> levels observed at INR up to 4.0 was not statistically significant. At INR greater than 4.0, F <em>1</em>+<em>2</em> reached a plateau, with mean levels not significantly different for patients at increasing INR up to 9.0. Since the risk of bleeding increases at INR greater than 4.5, our results suggest that F <em>1</em>+<em>2</em> is of little value to assess the hemorrhagic risk in patients on OAT.

OBJECTIVE

The patency of small-diameter expanded polytetrafluoroethylene (ePTFE) grafts for vascular reconstruction is impaired by acute thrombotic occlusion. Prosthetic materials are thrombogenic and cause platelet adhesion and activation of the coagulation cascade. Heparin is a potent anticoagulant drug widely used to prevent and treat thrombosis. A new ePTFE graft with long-term bonding of heparin is now commercially available in several European countries, but a basic analysis of its mechanism of action in humans has never been performed. This study was performed to evaluate the thrombogenicity of heparin-bonded ePTFE grafts compared with standard ePTFE in a newly developed human ex vivo model.

METHODS

Nonanticoagulated blood was drawn from antecubital veins of <em>1</em>0 healthy donors with a <em>1</em>9-gauge needle. The proximal end of a 60-cm ePTFE vascular graft with a diameter of 3 mm was connected to the needle while the distal end was connected to a syringe, which was placed in a syringe pump. Every volunteer served as his or her own control by using a heparin-bonded ePTFE graft on one arm and a standard ePTFE graft on the other arm. The perfusions were performed over 6 minutes with a flow rate of <em>2</em>0 mL/min, corresponding to a shear rate of 74/s. Serial samples were taken at the distal end of the graft for determination of <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>, fibrinopeptide A, and P-selectin expression on perfused platelets. Fibrin deposition and platelet deposition were studied by using scanning electronic microscopy.

RESULTS

Fibrinopeptide A production over time was significantly reduced on the heparin-bonded ePTFE grafts compared with standard ePTFE grafts (P < .05). There was no increase in the production of <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> or P selectin over time on either type of graft. Scanning electronic microscopy scanning showed platelet deposition and fibrin formation on standard ePTFE grafts, whereas no platelets or fibrin were observed on heparin-bonded ePTFE grafts.

CONCLUSIONS

Heparin immobilization substantially reduces the thrombogenicity of small-diameter ePTFE in a newly developed human ex vivo model. In this study, we provide evidence that the mechanism of action of the heparin bonding is due not only to anticoagulant but also to antiplatelet effects. Heparin bonding may be an important improvement of ePTFE, resulting in better patency rates for vascular reconstructions.

OBJECTIVE

No studies have yet determined whether antiplatelet or anticoagulant therapy is the more appropriate treatment after transcatheter closure of patent foramen ovale (PFO) in patients with cryptogenic stroke. The objective of this study was to prospectively evaluate the presence, degree, and timing of activation of the platelet and coagulation systems after transcatheter closure of PFO in patients with cryptogenic stroke.

METHODS

Twenty-four consecutive patients (mean age, 44+/-<em>1</em>0 years; <em>1</em><em>1</em> men) with previous cryptogenic stroke who had undergone successful transcatheter closure of PFO were included in the study. <em>Prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>) and thrombin-antithrombin III (TAT) were used as markers of coagulation activation, and soluble P-selectin and soluble CD40 ligand were used as markers of platelet activation. Measurements of all hemostatic markers were taken at baseline just before the procedure and at 7, 30, and 90 days after device implantation.

RESULTS

F<em>1</em>+<em>2</em> and TAT levels increased from 0.4<em>1</em>+/-0.<em>1</em>6 nmol/L and <em>2</em>.34+/-<em>1</em>.8<em>1</em> ng/mL, respectively, at baseline to a maximal value of 0.6<em>1</em>+/-0.<em>1</em>6 nmol/L and 4.34+/-<em>1</em>.83 ng/mL, respectively, at 7 days, gradually returning to baseline levels at 90 days (P<0.00<em>1</em> for both markers). F<em>1</em>+<em>2</em> and TAT levels at 7 days after PFO closure were higher than those obtained in a group of <em>2</em>5 healthy controls (P<0.00<em>1</em> for both markers). Levels of soluble P-selectin and soluble CD40 ligand did not change at any time after PFO closure.

CONCLUSIONS

Transcatheter closure of PFO is associated with significant activation of the coagulation system, with no increase in platelet activation markers. These findings raise the question of whether optimal antithrombotic treatment after PFO closure should be short-term anticoagulant rather than antiplatelet therapy.

The diagnosis of thromboembolic diseases is still difficult to establish before the occurrence of the pathological event, although it is now known that they are the result of a progressive alteration of the cardiovascular system. Introduction of new diagnostic tools for the evaluation of the thromboresistance capacity of the body or for the measurement of molecular markers allows the testing of the body defenses against thrombosis which is becoming a routine clinical diagnosis. Antithrombin III (AT III), protein C, protein S, and parameters of fibrinolysis have been recognized to be very important anticoagulant proteins and regulators of thrombin formation and thrombus extension. Furthermore, a normal factor V is necessary for the normal function of the protein C pathway. The presence of a factor V mutation leads to the activated protein C resistance syndrome. However, the major incidence of thrombotic events concerns the overall population. It has been epidemiologically related to the existence of risk factors producing blood activation, which progressively saturates the body's thromboresistance. This period is clinically silent for a long time. The new molecular markers recently introduced can show the existence of a preclinical state of blood activation at the plasma level (fibrinopeptide A, thrombin-antithrombin complexes, modified antithrombin III, <em>fragments</em> <em>1</em> + <em>2</em> of <em>prothrombin</em>, D-dimer) or at the platelet level (B-thromboglobulin, platelet factor 4, and thrombospondin), and promising developments concern the endothelial level (soluble thrombomodulin). The most universally used blood activation test is the D-dimer assay. This analyte has become very popular in past years for its high sensitivity, its long half-life, and its easy detection directly on citrated plasma. Its negative predictive value (in deep venous thrombosis or pulmonary embolism) as well as its use for monitoring of thrombotic risk in the post-operative period have been well documented clinically. New investigations are initiated to find analytes reflecting endothelial damage, an early platelet activation, or the involvement of blood cells (mainly monocytes and neutrophils) in abnormal processes. It also becomes possible to evaluate directly pathological causes inducing blood activation, such as the presence of antiphospholipid antibodies or other autoimmune antibodies.

BACKGROUND

Recombinant human C<em>1</em>-inhibitor (rhC<em>1</em>INH; Ruconest®) has been developed for treatment of acute angioedema attacks in patients with hereditary angioedema (HAE) due to heterozygous deficiency of C<em>1</em>INH. Previous reports suggest that administration of plasma-derived C<em>1</em>INH products may be associated with an increased risk for thromboembolic complications.

OBJECTIVE

Our aim is to evaluate the effects of rhC<em>1</em>INH on coagulation and fibrinolysis in symptomatic HAE patients.

METHODS

Levels of various coagulation and fibrinolytic parameters were determined in pre- and post-exposure plasma samples from HAE patients included in a randomized clinical trial. Patients were treated with either saline, or 50 or <em>1</em>00 U/kg rhC<em>1</em>INH for an acute angioedema attack.

RESULTS

Prior to rhC<em>1</em>INH treatment, the majority of patients had low to normal activated partial thromboplastin times (aPTT) and increased levels of <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, thrombin-antithrombin complexes, D-dimers and plasmin-antiplasmin complexes, all of which indicate activation of both coagulation and fibrinolysis. Infusion of rhC<em>1</em>INH at doses up to <em>1</em>00 U/kg did not affect these parameters except for a dose-dependent prolongation of aPTT, confirming that rhC<em>1</em>INH is an inhibitor of the contact system, and that F<em>1</em>+<em>2</em> levels decreased.

CONCLUSIONS

Coagulation and fibrinolytic systems are activated in HAE patients suffering from an acute angioedema attack. Treatment with rhC<em>1</em>INH at 50 or <em>1</em>00 U/kg had no effect on parameters reflecting activation of these systems except for a significant effect on aPTT, which likely reflects a pharmacodynamic effect of rhC<em>1</em>INH, and a reduction on plasma levels of the <em>prothrombin</em> activation <em>fragment</em> F<em>1</em>+<em>2</em>. We conclude that these results argue against a prothrombotic effect of treatment with this rhC<em>1</em>INH product in HAE patients.

BACKGROUND

In addition to acquired and inherited risk factors, the growth of venous thrombus under static conditions and endothelial injury play important roles in the development of deep venous thrombosis (DVT), for which risk factors include increased plasma levels of coagulation factor XI (FXI). The aim of this study is to understand the role of FXI in venous thrombus formation under conditions of endothelial denudation and/or blood stasis.

METHODS

The contribution of FXI to venous thrombus formation was investigated in a rabbit model and a flow chamber system. Thrombi were induced in the rabbit jugular veins by (<em>1</em>) endothelial denudation, (<em>2</em>) vessel ligation (blood stasis) or (3) by combined endothelial denudation and vessel ligation. Blood samples were perfused on immobilized type III collagen at a wall shear rate of 70/s and then the surface area covered by platelets and fibrin was morphometrically evaluated. <em>Prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>) generation was also measured before and after perfusion.

RESULTS

All thrombi induced in rabbit jugular veins were composed of platelets, fibrin and erythrocytes. Anti-FXI antibody significantly reduced ex vivo plasma thrombin generation initiated by ellagic acid but not by tissue factor, and in vivo thrombus formation under endothelial denudation and/or vessel ligation. The antibody significantly reduced surface areas covered by platelets and fibrin, as well as F<em>1</em>+<em>2</em> generation at a wall shear rate of 70/s in flow chambers.

CONCLUSIONS

These results suggest that FXI contributes to venous thrombus growth under conditions of endothelial denudation and/or blood stasis, and that thrombin generation by FXI interaction promotes further platelet aggregation and fibrin formation at low shear rates.

Fibrin D-Dimer (D-Di), <em>prothrombin</em> activation <em>fragment</em> (F <em>1</em>+<em>2</em>) and thrombin-antithrombin III complexes (TAT) were measured using ELISA procedures in the plasma of patients with an acute deep venous thrombosis (DVT), at presentation and on days <em>2</em>, 6 and <em>1</em>0 after initiation of heparin treatment. Patients were randomly allocated into two treatment groups: 44 patients received adapted doses of continuous intravenous unfractionated heparin (UH) whereas 47 received <em>1</em> mg/kg every twelve hours of a low molecular weight heparin (enoxaparin) subcutaneously. A phlebography and a perfusion lung scan were performed before inclusion and on day <em>1</em>0. Failure of therapy (n = 9) was defined by venogram worsening or confirmed pulmonary embolism. Improvement (n = 44) or stationary state (n = 38) were defined by venogram evolution in the absence of new leg scan defects. At presentation, D-Di, F <em>1</em>+<em>2</em> and TAT were above cut-off values in 97, 66 and 89% of patients respectively. D-Di levels correlated with the extent of venous thrombosis whereas TAT and F <em>1</em>+<em>2</em> did not. Mean levels of D-Di decreased sharply during the first days of treatment but were still abnormal on day <em>1</em>0. A secondary increase of D-Di on days 6 or <em>1</em>0 by more than 3 micrograms/ml occurred in 4 of the 9 patients who developed a thromboembolic recurrence but in none of the 7<em>2</em> patients who had a more favorable outcome. F <em>1</em>+<em>2</em> and TAT time-courses were not related to clinical evolution. In the Enoxaparin group, there was no relationship between antifactor Xa activities and any biological markers.(ABSTRACT TRUNCATED AT <em>2</em>50 WORDS)

We studied the effects of stimulated skin mast cells on bleeding time and thrombin generation which was measured using <em>prothrombin</em> <em>fragment</em> F <em>1</em>+<em>2</em> (F <em>1</em>+<em>2</em>) and thrombin-antithrombin-III-complex (TAT). In <em>1</em>0 patients with urticaria pigmentosa (chronic cutaneous mast cell accumulation) the mean bleeding time was significantly prolonged in wounds made on urticaria pigmentosa lesions vs. normal skin (460 +/- 34 vs. 34<em>2</em> +/- <em>2</em>7 s, p = 0.005). In <em>1</em>0 atopic subjects skin incisions were made on prick-tested sites 30, 60, <em>1</em><em>2</em>0 and <em>2</em>40 min after administration of an allergen (acute mast cell stimulation), histamine or vehicle. The mean bleeding time was significantly prolonged at all time points, being maximal at <em>1</em><em>2</em>0 min (60% prolonged) in wounds made on allergen-stimulated skin areas (p < 0.0<em>1</em>) compared with histamine or vehicle sites. Administration of allergen or histamine lowered the TAT concentration in the bleeding-time blood. Furthermore, TAT and F <em>1</em>+<em>2</em> levels in the bleeding-time blood were lower at 60, <em>1</em><em>2</em>0 and <em>2</em>40 min after allergen or histamine application in comparison with samples collected at 30 min. We conclude that skin mast cells can regulate primary hemostasis by prolonging bleeding time and by inhibiting thrombin generation.

BACKGROUND

The pathophysiological mechanism of chronic urticaria is still poorly understood and its aetiology is considered to have an autoreactive basis. Autologous serum skin tests (ASSTs) and autologous plasma skin tests (APSTs) comprise the simplest ways for diagnosing autoreactive urticaria (with autoantibodies, histamine-releasing factor and coagulation factors, especially thrombin) in a clinical setting. However, there are still some questions about the specificity of these tests.

OBJECTIVE

To evaluate the role of autoreactivity in the pathogenesis of chronic urticaria by means of measuring plasma <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em>, which are used as markers of thrombin, and to compare the APST with the ASST.

METHODS

Forty-two patients (<em>1</em>9 men and <em>2</em>3 women; mean age 35·7 years, range <em>2</em>8-76) and 35 healthy volunteers (<em>1</em>9 men and <em>1</em>6 women; mean age 30·3 years, range <em>2</em>0-80) were included in the study. APST, negative (ASST, sodium citrate, saline) and positive (histamine) control tests were performed in the patient and control groups. The levels of plasma <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em> were also assessed.

RESULTS

When the APST was evaluated without negative controls, it was positive in 67% of patients. However, the APST was positive in 0% when it was evaluated with negative controls. Levels of <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em> were found to be elevated in patients with chronic idiopathic urticaria.

CONCLUSIONS

We suggest that as negative control tests were not performed along with the APST in previous studies, the APST showed a high rate of positivity. Thus, the use of APST for evaluating autoreactivity in clinical practice is not superior to the use of ASST and further studies should be conducted.

OBJECTIVE

The aim of this study was to compare effects of the transdermal contraceptive patch, a desogestrel/ethinyl estradiol (EE)-containing, monophasic combination oral contraceptive (COC) and a levonorgestrel/EE-containing, triphasic COC on hemostasis variables.

METHODS

This was a randomized, open-label study of <em>1</em>04 young women who received six cycles of treatment. Blood was collected at baseline and on treatment; changes by Day <em>2</em>0/Cycle 6 in baseline hemostasis markers [<em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F <em>1</em>+<em>2</em>), plasmin-plasmin inhibitor complex (PAP) and fibrin degradation products (D-dimer)] were assessed.

RESULTS

All contraceptives induced similar increases in F <em>1</em>+<em>2</em> and D-dimer. Patch-induced PAP increases were less than with the monophasic and similar to the triphasic COC. Decreases in protein S and increases in sex hormone-binding globulin were greater with the patch than with either COC. Patch-induced increases in activated protein C resistance were greater than with the triphasic and similar to the monophasic COC.

CONCLUSIONS

These contraceptives appeared to accelerate baseline procoagulation processes to a similar extent and to change coagulation potency variables differently.

Interleukin-6 levels, but not <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>, correlates with a point-based score for stroke risk in atrial fibrillation, even after oral anticogulation.

BACKGROUND

A hypercoagulable state is often associated with an acute stroke in cerebrovascular disease (CVD). However, in Binswanger disease (BD), no information is available on the coagulation-fibrinolysis pathway except for the presence of high plasma fibrinogen levels.

OBJECTIVE

To determine the association of BD and coagulation-fibrinolysis pathway activation.

METHODS

We examined the levels of fibrinogen, thrombin-antithrombin complex, <em>prothrombin</em> <em>fragment</em>(<em>1</em>+<em>2</em>), and cross-linked D-dimer in <em>1</em>7 patients with BD, <em>2</em>4 neurologic patients without CVD, and <em>2</em>6 patients with lacunar infarction in either the acute or chronic stage.

RESULTS

As compared with the non-CVD and lacunar infarction groups, the patients with BD had significantly elevated levels of thrombin-antithrombin complex (P<.00<em>1</em>), <em>prothrombin</em> <em>fragment</em>(<em>1</em>+<em>2</em>) (P<.05), and cross-linked D-dimer (P<.0<em>1</em>). There was also a significant increase in fibrinogen levels compared with the non-CVD group (P<.05). In the BD group, 8 patients in stable condition (ie, those without obvious neurologic deficits in the past 3 months) showed normal levels or a mild increase in their fibrinogen, thrombin-antithrombin complex, <em>prothrombin</em> <em>fragment</em>(<em>1</em>+<em>2</em>), or cross-linked D-dimer levels. In contrast, 9 patients with BD with a subacute aggravation of their focal or subcortical cerebral functions (deteriorating group) showed a significant increase in their thrombin-antithrombin complex levels compared with the stable patients (P<.0<em>1</em>). Similarly, the fibrinogen, <em>prothrombin</em> <em>fragment</em>(<em>1</em>+<em>2</em>), and cross-linked D-dimer levels were elevated in the deteriorating patients, but this trend did not reach statistical significance.

CONCLUSIONS

These results indicate that the coagulation-fibrinolysis pathway is activated in patients with BD with a subacute aggravation. Coagulation activation may result in the formation of microthrombi and microcirculatory disturbances in the brains of these patients, and thus promote further biological and neurologic insults.

<em>Prothrombin</em> deficiency is an autosomal recessive disorder associated with a moderately severe bleeding tendency. In this study, <em>1</em>3 patients with <em>prothrombin</em> deficiency were screened for the presence of alterations in the <em>prothrombin</em> gene, and nine novel candidate mutations were identified. Of <em>1</em><em>1</em> patients with hypo<em>prothrombin</em>emia, ten are homozygous for five mutations and one patient is a compound heterozygote. The two patients with dys<em>prothrombin</em>emia are homozygous for two mutations. Eight of nine mutations are missense ones associated with single amino acid substitutions in the propeptide (Arg-<em>1</em>Gln, Arg-<em>2</em>Trp), the kringle-<em>1</em> (Asp<em>1</em><em>1</em>8Try) and kringle-<em>2</em> (Arg<em>2</em><em>2</em>0Cys) domains and the catalytic serine protease domain (Gly330Ser, Ser354Arg. Arg38<em>2</em>His and Arg538Cys). The ninth mutation is an in-frame deletion of 3 bp that results in the omission of one amino acid (del Lys 30<em>1</em>/30<em>2</em>). The combination of these missense mutations with crystal structures for alpha-thrombin and the <em>prothrombin</em> <em>fragments</em> <em>1</em> and <em>2</em> resulted in new insight into the function of alpha-thrombin. The hypo<em>prothrombin</em>emia mutations were inferred to affect either the cleavage of the propeptide from the Gla domain, the stability of the kringle-<em>1</em> and -<em>2</em> domains, or the close association of the A and B chains of the serine protease domain. The dys<em>prothrombin</em>emia mutations were inferred to directly affect catalytic function through their location at the active site crevice or exosite <em>1</em> within the serine protease domain.

Lonomia obliqua venom causes a severe consumptive coagulopathy, which can lead to a hemorrhagic syndrome. The crude bristles extract displays a procoagulant activity due to a Factor X and to a <em>prothrombin</em> activating activity. Here, we describe a 69 kDa <em>prothrombin</em> activator serine protease purified from L. obliqua caterpillar bristle extract using gel filtration (Sephadex G 75) and HPLC (C(4) column). The purified protein was able to activate <em>prothrombin</em> in a dose-dependent manner, and calcium ions increased this activity. The <em>prothrombin</em>-derived fluorogenic peptide (Abz-YQTFFNPRTGSQ-EDDnp) had its main cleavage site at the Arg-Thr bond. The kinetic parameters obtained for this substrate were Kmapp of 4.5 microM, kcat of 5.3<em>2</em> s(-<em>1</em>), and a kcat/Kmapp of <em>1</em>.<em>2</em> x <em>1</em>0(6) M(-<em>1</em>) s(-<em>1</em>). The <em>prothrombin</em> <em>fragments</em> generated by the purified enzyme corresponded to the molecular masses of prethrombin <em>2</em>, <em>fragment</em> <em>1</em>, <em>fragment</em> <em>2</em>, and thrombin as seen in SDS-PAGE. The thrombin generated was able to clot purified fibrinogen. The partial amino acid sequence of the purified protein, named Lopap (L. obliqua <em>prothrombin</em> activator protease), showed no similarity to any known <em>prothrombin</em> activator.

In patients with acute deep-vein thrombosis (DVT), apixaban, a direct oral factor Xa inhibitor, showed efficacy and safety similar to low-molecular-weight heparin followed by vitamin K antagonist (LMWH/VKA). We evaluated biomarkers of coagulation activity in relation to treatment dose, duration and clinical outcome. Patients (N = 5<em>2</em>0) with symptomatic DVT were randomised to receive apixaban (5 mg bid, <em>1</em>0 mg bid or <em>2</em>0 mg qd) or LMWH/VKA for <em>1</em><em>2</em> weeks. Plasma D-dimer, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>) and thrombin-antithrombin complex (TAT) levels were measured at baseline, and weeks 3 and <em>1</em><em>2</em> after treatment. Median plasma levels of D-dimer, F<em>1</em>+<em>2</em> and TAT were elevated at baseline. At weeks 3 and <em>1</em><em>2</em>, biomarker levels were normalised in most patients in all treatment groups, consistent with the low rate of venous thromboembolism (VTE) observed. Median reduction in D-dimer was similar in all treatment groups; percentage of patients with D-dimer above upper limit of normal decreased from 95% to <em>2</em>4-40% at week <em>1</em><em>2</em>. F<em>1</em>+<em>2</em> decline was greater with LMWH/VKA than apixaban. F<em>1</em>+<em>2</em> in the apixaban groups changed to a similar extent (>84% of patients had F<em>1</em>+<em>2</em> within reference range at week <em>1</em><em>2</em>). Magnitude of TAT reduction was not quantifiable. In conclusion, levels of coagulation biomarkers decreased over <em>1</em><em>2</em> weeks of treatment with apixaban or LMWH/VKA in most patients with acute VTE. Baseline D-dimer and F<em>1</em>+<em>2</em> were higher in patients with recurrent symptomatic VTE than in those without. Plasma levels of coagulation biomarkers did not appear to correlate with total bleeding events.

<em>Prothrombin</em> <em>fragment</em> <em>2</em> (the second kringle) has been co-crystallized with PPACK (D-Phe-Pro-Arg)-thrombin and the structure of the non-covalent complex has been determined and refined (R = 0.<em>1</em>6) at 3.<em>2</em> A resolution using X-ray crystallographic methods. The kringles interact with thrombin at a site that has previously been proposed to be the heparin binding region. The latter is a highly electropositive surface near the C-terminal helix of thrombin abundant in arginine and lysine residues. These form salt bridges with acidic side chains of kringle <em>2</em>. Somewhat unexpectedly, the negative groups of the kringle correspond to an enlarged anionic center of the lysine binding site of lysine binding kringles such as plasminogen K<em>1</em> and K4 and TPA K<em>2</em>. The anionic motif is DGDEE in <em>prothrombin</em> kringle <em>2</em>. The corresponding cationic center of the lysine binding site region has an unfavorable Arg7<em>1</em>Phe substitution but Lys35 is conserved. However, the folding of <em>fragment</em> <em>2</em> is different from that of <em>prothrombin</em> kringle <em>1</em> and other kringles: the second outer loop possesses a distorted two-turn helix and the hairpin beta-turn of the second inner loop pivots at V64 and D70 by 60 degrees. The Lys35 is located on a turn of the helix, which causes it to project into solvent space in the <em>fragment</em> <em>2</em>-thrombin complex, thereby devastating the cationic center of the lysine binding site.(ABSTRACT TRUNCATED AT <em>2</em>50 WORDS)

OBJECTIVE

To evaluate effects of postmenopausal hormone replacement therapy (HRT) on von Willebrand factor, factor (F)VIII, factor (F)VII, fibrinogen, antithrombin (AT) III, <em>prothrombin</em> <em>fragments</em> <em>1</em> and <em>2</em>, protein C, total and free protein S, plasminogen activator inhibitor-<em>1</em> (PAI-<em>1</em>), tissue plasminogen activator (tPA) and resistance to activated protein C.

METHODS

Part <em>1</em>: double blind randomized trial for 3 months. Part <em>2</em>: open study for 9 months.

METHODS

Department of Endocrinology, University Hospital, Malmö, Sweden.

METHODS

Fifty-one postmenopausal women with a history of amenorrhoea of at least 6 months and body mass index>> or = <em>2</em>4 kg m-<em>2</em> participated in part <em>1</em> and 46 participated in part <em>2</em>.

METHODS

Randomization for placebo (n=<em>2</em>4) or HRT (n=<em>2</em>7). HRT was given as <em>2</em> mg oestradiol valerate for the first 3 months, with the addition of <em>1</em>0 mg medroxyprogesterone for <em>1</em>0 days every third month thereafter.

METHODS

At baseline and after 3 and <em>1</em><em>2</em> months.

RESULTS

During 0-3 months in the HRT group, FVII increased (P < 0.0<em>1</em>), whereas fibrinogen, AT III and total protein S all decreased (P < 0.00<em>1</em> for all). Changes in variables were expressed as Delta-values. After 3 months Delta-values differed between groups for fibrinogen (P < 0.05), AT III (P < 0.00<em>1</em>), total protein S (P < 0.00<em>1</em>), and PAI-<em>1</em> (P < 0.00<em>1</em>). During 0-<em>1</em><em>2</em> months, fibrinogen, total protein S, tPA (P < 0.0<em>1</em> for all) and AT III (P < 0.05) decreased. In the control group, all variables were unchanged during the study, except for increases (P < 0.05) in total protein S after 3 and <em>1</em><em>2</em> months, and a decrease (P < 0.0<em>1</em>) in FVIII after <em>1</em><em>2</em> months. After <em>1</em><em>2</em> months Delta-values differed for fibrinogen (P < 0.05), AT III (P < 0.05) and total protein S (P < 0.00<em>1</em>).

CONCLUSIONS

Unopposed oestrogen substitution was associated with both potentially beneficial effects, such as decreases in fibrinogen, and potentially thrombogenic effects such as decreasing AT III and protein S and increasing FVII. During prolonged follow-up and addition of progesterone, differences between groups concerning FVII were attenuated. These data suggest that effects of HRT upon coagulation are most pronounced early after institution of unopposed treatment.

The effects of inhibition of tumor necrosis factor (TNF) on cell and protease activation were evaluated in <em>1</em>8 normal volunteers given endotoxin (4 ng/kg, i.v.) after an infusion of low (<em>1</em>0 mg/m<em>2</em> i.v., n = 6) or high dose (60 mg/m<em>2</em> i.v., n = 6) recombinant human dimeric TNF receptor protein (TNFR:Fc) or its vehicle (placebo n = 6). Activation of the coagulation system occurred by <em>2</em> h in the TNFR:Fc vehicle-placebo group manifested by decreased prekallikrein functional levels and increased levels of <em>prothrombin</em> F<em>1</em>+<em>2</em> <em>fragments</em> (p < 0.000<em>1</em>). High or low dose TNFR:Fc delayed the fall in prekallikrein functional levels by <em>1</em> h and 4 h, respectively (p < 0.000<em>2</em>), but did not inhibit the increase in circulating levels of <em>prothrombin</em> F<em>1</em>+<em>2</em> <em>fragments</em>. In contrast, endothelium activation, characterized by increased levels of tissue plasminogen activator, plasminogen activator inhibitor-<em>1</em>, and von Willebrand Factor antigen was blunted by both low and high dose TNFR:Fc (p < 0.00<em>1</em>). While the endotoxin-associated decrease in platelet number was not altered, platelet-derived beta-thromboglobulin peak levels were blunted and delayed by TNFR:Fc (p < 0.0<em>2</em>). Increased levels of neutrophil elastase were attenuated by low and high dose TNFR:Fc (p < 0.00<em>1</em>). These results suggest that although TNF is functionally linked to the activation of endothelium, neutrophils, coagulation, and fibrinolysis, alternative pathways are present in vivo that result in activation of the kallikrein-kinin system after endotoxin-induced TNF release. These alternative pathways may limit some of the anti-inflammatory effects of TNFR:Fc.