The staphylococcal superantigen-like protein (SSL) family is composed of <em>1</em>4 exoproteins sharing structural similarity with superantigens but no superantigenic activity. Target proteins of four SSLs have been identified to be involved in host immune responses. However, the counterparts of other SSLs have been functionally uncharacterized. In this study, we have identified porcine plasma <em>prothrombin</em> as SSL<em>1</em>0-binding protein by affinity purification using SSL<em>1</em>0-conjugated Sepharose. The resin recovered the prodomain of <em>prothrombin</em> (<em>fragment</em> <em>1</em> + <em>2</em>) as well as factor Xa in pull-down analysis. The equilibrium dissociation constant between SSL<em>1</em>0 and <em>prothrombin</em> was <em>1</em>.36 × <em>1</em>0(-7) M in surface plasmon resonance analysis. On the other hand, the resin failed to recover γ-carboxyglutamic acid (Gla) domain-less coagulation factors and <em>prothrombin</em> from warfarin-treated mice, suggesting that the Gla domain of the coagulation factors is essential for the interaction. SSL<em>1</em>0 prolonged plasma clotting induced by the addition of Ca(<em>2</em>+) and factor Xa. SSL<em>1</em>0 did not affect the protease activity of thrombin but inhibited the generation of thrombin activity in recalcified plasma. S. aureus produces coagulase that non-enzymatically activates <em>prothrombin</em>. SSL<em>1</em>0 attenuated clotting induced by coagulase, but the inhibitory effect was weaker than that on physiological clotting, and SSL<em>1</em>0 did not inhibit protease activity of staphylothrombin, the complex of <em>prothrombin</em> with coagulase. These results indicate that SSL<em>1</em>0 inhibits blood coagulation by interfering with activation of coagulation cascade via binding to the Gla domain of coagulation factor but not by directly inhibiting thrombin activity. This is the first finding that the bacterial protein inhibits blood coagulation via targeting the Gla domain of coagulation factors.

The human protease-activated receptor <em>1</em> (PAR-<em>1</em>) is activated by thrombin at the surface of platelets and endothelial cells, <em>2</em> cells that are implicated in hemostasis and thrombosis. We studied the PAR-<em>1</em> gene in a large case-control study from the Paris Thrombosis Study (PATHROS), and the possible implication of polymorphisms in venous thromboembolism was evaluated. Two polymorphisms were found in the 5' regulatory region. The first is a C to T transition that is <em>1</em>4<em>2</em>6 nucleotides upstream from the translation start site (-<em>1</em>4<em>2</em>6 C/T), and the second is a <em>1</em>3-bp insertion repeating the preceding -506 5'-CGGCCGCGGGAAG-3' sequence (-506 I/D, where I indicates insertion and D indicates deletion), a putative cis-acting element of the Ets family. The third polymorphism is an A to T transversion in the intervening sequence (IVS) that is <em>1</em>4 nucleotides upstream from the exon <em>2</em> start site (IVS-<em>1</em>4 A/T). The distribution of the 3 polymorphisms was otherwise similar in the <em>2</em>50 cases and the <em>1</em><em>2</em><em>1</em>4 controls. A noteworthy sex heterogeneity led us to analyze men and women separately with regard to the -506 I/D polymorphism. We found that allele I was less frequent in male cases than in male controls (0.<em>1</em>54 versus 0.<em>2</em>47, P<0.0<em>1</em>), with an odds ratio at 0.5<em>2</em> (95% CI 0. 3<em>2</em> to 0.8<em>2</em>, P<0.0<em>1</em>). Furthermore, a reduction of <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> levels was observed in homozygous carriers of allele -506 I (P=0.04). Altogether, these data suggested a protective effect in men of -506 I/D polymorphism for venous thromboembolism.

BACKGROUND

Patients with Crohn's disease and ulcerative colitis have an increased risk of thromboembolic events.

METHODS

Data were collected from <em>2</em>4 patients aged 4.5 to <em>2</em>3 years who had inflammatory bowel disease. Platelet count, antithrombin, fibrinogen, <em>prothrombin</em> <em>fragment</em> F<em>1</em>+<em>2</em>, soluble thrombomodulin, tissue plasminogen activator, D-dimer, and plasminogen activator inhibitor-<em>1</em> antigen were investigated. In addition the response to activated protein C, the factor V R506Q mutation, protein C, free protein S antigen, and lipoprotein (a) were analyzed. These data were compared with medical treatment, duration, and disease activity, estimated with the Pediatric Crohn's Disease Activity Index or the Clinical Colitis Activity Index.

RESULTS

Forty-five percent of our patients showed an increase in fibrinogen, <em>2</em>9% in <em>prothrombin</em> <em>fragment</em> F<em>1</em>+<em>2</em>, and <em>2</em>0% in platelet count, plasminogen activator inhibitor- antigen, and soluble thrombomodulin. Thrombomodulin was higher in active disease than in inactive disease and in Crohn's disease than in ulcerative colitis. Fibrinogen was also higher with Crohn's disease and tended to be higher in active disease than in ulcerative colitis and inactive disease. Plasminogen activator inhibitor-<em>1</em> antigen was significantly higher in patients with Crohn's disease than in those with ulcerative colitis and was higher in the patient group treated with steroids.

CONCLUSIONS

As has been shown in adults, young patients with active and inactive inflammatory bowel disease were found to have abnormal coagulation and fibrinolysis. The relevance as a thromboembolic risk factor is discussed.

BACKGROUND

Recombinant FVIIa (rFVIIa) has been shown to improve hemostasis in patients with thrombocytopenia and to prevent or control bleeding episodes in patients with inherited deficiencies of major PLT glycoproteins, but the mechanism of action is not well understood.

METHODS

Effects of rFVIIa on hemostasis were explored with an in vitro perfusion technique. Blood samples, from healthy donors or from patients with congenital defects of PLT glycoprotein IIb-IIIa (GPIIb-IIIa), were anticoagulated with low-molecular-weight heparin. Experimental thrombocytopenia (<6000 PLTs/microL) was induced by a filtration procedure. rFVIIa was added to blood samples at therapeutic concentrations. A severe GPIIb-IIIa impairment was also induced by exposure of normal blood samples to a specific antibody. Perfusion studies were performed through annular chambers containing damaged vascular segments. The presence of fibrin and PLTs on the perfused subendothelium was morphometrically quantified.

RESULTS

Under conditions of experimental thrombocytopenia, addition of rFVIIa enhanced fibrin formation in a dose-dependent manner (p < 0.05). Improvements in local fibrin generation and partial restoration of PLT interactions were also observed after incubation of blood from patients with Glanzmann's thrombasthenia with rFVIIa at 5 microg per mL (<em>1</em>80 microg/kg). Similar improvements were observed in blood samples incubated with antibodies to GPIIb-IIIa. rFVIIa in whole normal blood also enhanced fibrin formation but PLT deposition was unaffected. Evaluation of <em>prothrombin</em> <em>fragments</em> <em>1</em> and <em>2</em> in the perfusates confirmed that rFVIIa increased thrombin generation in all cases.

CONCLUSIONS

Our data indicate that rFVIIa promotes a procoagulant activity at sites of vascular damage. This mechanism could explain the beneficial hemostatic effect of rFVIIa in patients with thrombocytopenia or with Glanzmann's thrombasthenia.

Factor Xa, as with thrombin, binds to the clot and contributes to the propensity of thrombi to activate the coagulation system. The aim of this work was to compare the extent of <em>prothrombin</em>ase inhibition produced by two factor Xa inhibitors: the antithrombin III-dependent synthetic pentasaccharide (SR 90<em>1</em>07/Org 3<em>1</em>540) and DX-9065A, a direct factor Xa inhibitor. When incubated together with <em>prothrombin</em>, factor Xa, phospholipids, antithrombin III and calcium, clots formed from human plasma exhibited a <em>prothrombin</em>ase activity as measured through <em>fragment</em> <em>1</em>-<em>2</em> (F<em>1</em>+<em>2</em>) generation. Ten washes of the clot were required to achieve complete removal of unbound factor Xa. The absence of F<em>1</em>+<em>2</em> generation brought about by washed clots in buffer when factor V was omitted, or in the presence of annexin V, indicated that they contained bound factor Xa and phospholipids but no factor V/Va. In all tested experimental conditions, clot-bound-factor Xa-induced F<em>1</em>+<em>2</em> generation was inhibited by SR 90<em>1</em>07/AT and DX-9065A with IC50 in the same range of concentrations (0.5 microM). In contrast, the inhibition of <em>prothrombin</em>ase formed with factor Xa, factor Va phospholipids and calcium in buffer was observed at significantly lower concentrations of DX-9065A than of SR 90<em>1</em>07/AT (respective IC50 concentrations: 0.<em>1</em> and 70 microM). In vivo, fibrin accretion onto a preformed thrombus as well as venous thrombosis induced in the jugular vein of rabbits was inhibited by SR 90<em>1</em>07 and DX-9065A in the same range of concentrations therefore showing that inhibition of clot-bound factor Xa is a predominant factor for the antithrombotic activity of both direct and indirect inhibitors for factor Xa.

BACKGROUND

The use of combined hormonal contraceptives with ethinyl estradiol (EE) and a progestin results in alterations in potential biomarkers of venous thromboembolism risk. Evaluation of the impact of delivery route on these changes is difficult due to an interaction between EE and the progestin component.

OBJECTIVE

The aim of the study was to compare the impact of oral and vaginal administration of EE alone on hemostatic variables and estrogen-sensitive liver proteins.

METHODS

This was a single-center, randomized, crossover study with two treatment cycles separated by a washout cycle.

METHODS

The study was conducted in an academic outpatient center.

METHODS

Fourteen healthy postmenopausal women were enrolled; <em>1</em>3 completed the study and were included in the analyses.

METHODS

Participants were randomized to receive EE (<em>1</em>5 microg/d) delivered by oral tablet or vaginal ring for <em>2</em><em>1</em> d in one of two treatment sequences.

METHODS

Changes in plasma concentration or activity of <em>1</em>0 hemostatic variables and six estrogen-sensitive liver proteins between baseline and d <em>2</em><em>1</em> of treatment were the primary outcomes.

RESULTS

<em>Prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> plasma level was unaffected by treatment or delivery route. Angiotensinogen (expressed as plasma level of angiotensin I) increased similarly with oral and vaginal delivery; mean (sd) increases were <em>2</em>757 (<em>1</em>033) and <em>2</em>864 (893) ng /ml, respectively (P = 0.000<em>2</em>). Alterations in other study variables, except total cholesterol, were similar with oral and vaginal administration.

CONCLUSIONS

Our results provide evidence that the customary effects of combined hormonal contraceptives on hemostatic variables and estrogen-sensitive liver proteins are largely related to EE and independent of delivery route during short-term treatment.

Whether hormone replacement therapy (HRT) is beneficial for coronary heart disease (CHD) is controversial. We hypothesized that continuous combined transdermal HRT may have benefits on CHD risk markers without the potential adverse effects seen with certain other HRT regimens. Sixty apparently healthy postmenopausal women, aged 40-65 years, entered a prospective, double-blind, randomized, placebo-controlled clinical trial; 55 women completed the 6-month study. Women received either transdermal oestradiol <em>1</em>7beta 0.05 mg and norethisterone acetate 0.<em>1</em><em>2</em>5 mg daily, or identical placebo. Circulating markers of vascular function and remodelling, forearm blood flow, lipids and lipoproteins, glucose and insulin, and haemostatic safety parameters were measured at baseline and after treatment. Compared with placebo after 6 months, HRT administration resulted in decreased E-selectin (P < 0.0<em>1</em>), and angiotensin-converting-enzyme (ACE; P = 0.05). Cholesterol (P < 0.05), low-density lipoproteins (LDL; P < 0.05), high-density lipoprotein3 (HDL3; P < 0.05) and apolipoproteins AII (P < 0.05) and B (P < 0.05), and fasting insulin (P < 0.05) also decreased in the HRT group. Factor VII coagulation activity decreased (P < 0.0<em>1</em>) and plasminogen activator inhibitor-<em>1</em> and fibrin D-dimer increased (P < 0.05) in the HRT group, whilst <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (P < 0.05) decreased, more so in the placebo group. There were no changes in matrix metalloproteinase (MMP)-<em>2</em>, or in LDL particle size. This transdermal HRT had beneficial effects on vascular function and CHD risk markers.

BACKGROUND

Anti-phospholipid antibodies (aPLs) were associated with an ongoing prothrombotic state in patients with systemic lupus erythematosus (SLE). Because aPLs are able to shift endothelial function toward procoagulant activity in vitro, we investigated the relationship among aPLs, ongoing prothrombotic state, and endothelial perturbation in SLE patients.

RESULTS

We measured aPLs, anti-EC antibodies, circulating levels of <em>prothrombin</em> <em>fragment</em> F<em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>), tumor necrosis factor-alpha (TNF-alpha), tissue-type plasminogen activator (TPA), and von Willebrand factor (vWF) in 43 SLE patients and <em>2</em>5 healthy subjects. Patients positive for aPLs (n = <em>2</em>3) had a higher prevalence of anti-EC antibodies (P = .0<em>2</em>) and higher levels of F<em>1</em> + <em>2</em> (P = .003) than aPL(-) patients. Endothelial perturbation, defined by elevated plasma levels of both TPA and vWF, was significantly associated with aPL positivity (P = .00<em>1</em>). F<em>1</em> + <em>2</em>>> <em>1</em> nmol/L (mean +/- <em>2</em> SD of controls) was detected in all but one patient in whom aPL positivity and endothelial perturbation coexisted and in no aPL(+) patient without endothelial perturbation (P = .0039). F<em>1</em> + <em>2</em> was significantly correlated with vWF (rho = .6, P = .004) and TPA (<em>1</em><em>1</em>4 = .70, P = .0006) only in aPL(+) patients. Endothelial perturbation was closely associated with high values of TNF-alpha (P = .000<em>1</em>), anti-phospholipid (P = .00<em>1</em>), and anti-EC antibodies (P = .0<em>1</em><em>2</em>). In 3<em>1</em> patients without a clinical history of thrombosis followed up for 3 years, aPL(+) patients with endothelial perturbation showed higher F<em>1</em> + <em>2</em> and TNF-alpha values than aPL(+) patients without endothelial dysfunction.

CONCLUSIONS

This study shows that in SLE patients, aPL positivity is associated with an ongoing prothrombotic state only in the presence of endothelial perturbation. Our findings also suggest that aPLs and TNF-alpha might cooperate in inducing endothelial perturbation.

OBJECTIVE

Solvent/detergent-treated plasma (SDP) contains markedly lower protein S (PS) and plasmin inhibitor (PI) activity than standard fresh-frozen plasma (FFP). It has also been reported that SDP contains no alpha(<em>1</em>)-antitrypsin. Despite the lack of clinical data, it is suspected that SDP may be less effective than FFP in the treatment of complex coagulopathies. We therefore conducted a prospective trial to study the impact of SDP and FFP on haemostasis and fibrinolysis in complex coagulopathy after open-heart surgery.

METHODS

Patients received either 600 ml of SDP (n = 36) or 600 ml of FFP (n = 3<em>1</em>) at an infusion rate of 30 ml/min. The following parameters were measured before treatment and 60 min after termination of plasma infusion: prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen, factor VIII, antithrombin, protein C (PC), free PS and PS activity, prothrombin fragments F<em>1</em>+2 (F<em>1</em>+2), D-dimers (DD), fibrinogen degradation products (FDP), plasmin-plasmin inhibitor complexes (PPI), plasminogen, PI and alpha(<em>1</em>)-antitrypsin.

RESULTS

The rise in fibrinogen, factor VIII, antithrombin, PC, free PS, alpha(<em>1</em>)-antitrypsin and plasminogen, and the decrease in PT and APTT, did not significantly differ between the two study arms. However, PS activity did not increase after SDP infusion but did show a significant elevation after infusion with FFP. PI declined significantly after SDP and remained uninfluenced by FFP. Neither SDP nor FFP had any significant influence on F<em>1</em>+2, DD or FDP. However, a significant decrease in PPI levels caused by both types of plasma indicated a reduction in hyperfibrinolysis. Clinical haemostasis evaluation revealed no significant difference between the two treatment regimens. No adverse reactions were observed.

CONCLUSIONS

With the exception of PS and PI, SDP and FFP improved haemostasis and fibrinolysis to a similar degree. The clinical significance of these findings has to be determined in patients with severe acquired PS and PI deficiency requiring plasma transfusions.

Cell saving systems are commonly used during cardiac operations to improve hemoglobin levels and to reduce blood product requirements. We analyzed the effects of residual pump blood salvage through a cell saver on coagulation and fibrinolysis activation and on postoperative hemoglobin levels. Thirty-four elective coronary artery bypass graft (CABG) patients were randomized. In <em>1</em>7 patients, residual cardiopulmonary bypass (CPB) circuit blood was transfused after the cell saving procedure (cell salvage group). In the other <em>1</em>7 patients, residual CPB circuit blood was discarded (control group). Activation of the coagulative, fibrinolytic and inflammatory systems was evaluated pre-operatively (Pre), <em>2</em> hours after the termination of CPB (T0) and <em>2</em>4 hours postoperatively (T<em>1</em>), measuring <em>prothrombin</em> <em>fragment</em> <em>1</em>.<em>2</em> (PF <em>1</em>.<em>2</em>), plasmin-anti-plasmin (PAP), plasminogen activator inhibitor-<em>1</em> (PAI-<em>1</em>) and interleukin-6 (IL-6). The cell salvage group of patients had a significant improvement in hemoglobin levels after processed blood infusion (<em>2</em>.7 ± <em>1</em>.7 g/dL vs <em>1</em>.<em>2</em> ± <em>1</em>.<em>1</em> g/dL; p=0.003). PF<em>1</em>.<em>2</em> levels were significantly higher after infusion (T0: <em>1</em><em>1</em>75 ± 770 pmol/L vs 730 ± <em>2</em>37 pmol/L; p=0.037; T<em>1</em>: 33<em>1</em> ± <em>2</em>35 pmol/L vs <em>1</em>74 ± <em>1</em>34 pmol/L; p=0.0<em>2</em>6). Also, PAP levels were higher in the cell salvage group, although not significantly (T0: <em>2</em>53 ± <em>2</em>5<em>1</em> ng/mL vs <em>1</em>68 ± 96 ng/mL; p: NS; T<em>1</em>: 95 ± 60 ng/mL vs 53 ± 3<em>2</em> ng/mL; p: NS). No differences were found for PAI-<em>1</em>, IL-6, heparin levels or for red blood cell (RBC) transfusions. The cell salvage group of patients had increased chest tube drainage (749 ± 3<em>2</em>0 vs 59<em>2</em> ± <em>2</em>64; p: NS) and fresh frozen plasma transfusion rate (5 (<em>2</em>9%) pts vs 0 pts; p<0.04). Pump blood salvage with a cell saving system improved postoperative hemoglobin levels, but induced a strong thrombin generation, fibrinolysis activation and lower fibrinolysis inhibition. These conditions could generate a consumption coagulopathy.

BACKGROUND

Oxidative stress and thrombosis have been reported to be increased in diabetic patients and involved in the pathogenesis of cardiovascular complications. It has been demonstrated in diabetic patients that consumption of a meal is accompanied by the generation of an oxidative stress and of a hypercoagulable state. It is well recognized that red wine shows antithrombotic activity and that its ingestion increases plasma antioxidant capacity in man. In this study the possibility that red wine consumption may reduce the oxidative stress and thrombosis produced postprandially in diabetic patients has been evaluated.

METHODS

Twenty type <em>2</em> diabetic patients were studied during fasting consumption of 300 mL of red wine, or during a meal accompanied, or not, by red wine ingestion.

RESULTS

Plasma glucose, insulin, triglycerides, total plasma radical-trapping capacity, activated factor VII and <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em> were measured in basal state and at 60, <em>1</em><em>2</em>0 and <em>1</em>80 min after the start of each experiment. Low-density lipoprotein (LDL) oxidation was also evaluated at baseline and after <em>1</em><em>2</em>0 min Plasma glucose, insulin, triglycerides and LDL oxidation significantly increased, while the total plasma radical-trapping parameter significantly decreased during the meal test. Consumption of red wine in the fasting state significantly increased total plasma radical-trapping parameter activity, while wine ingestion with a meal counterbalanced the decrease of total plasma radical-trapping parameter and the increase of LDL oxidation. Meal consumption induced an increase in plasma <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em> and activated factor VII in diabetic patients. Wine ingestion with the meal significantly reduced the production of both <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em> and activated factor VII. Fasting consumption of red wine alone did not show effects on coagulation or LDL oxidation.

CONCLUSIONS

This finding confirms that in the absorptive phase free radicals are produced in diabetic patients, which reduce serum antioxidant defences, increase LDL oxidation and activate the coagulation system. Red wine consumption during a meal significantly preserves plasma antioxidant defences and reduces both LDL oxidation and thrombotic activation. The consumption of a moderate amount of red wine during meals may have a beneficial effect in the prevention of cardiovascular disease in diabetic patients.

OBJECTIVE

Oral estrogens were an effective treatment for prostate cancer but were abandoned because of an increased risk of cardiovascular toxicity and particularly thromboembolism. We have recently shown that transdermal estradiol produces an effective tumor response and negligible cardiovascular toxicity. Here we report the influence of transdermal estradiol therapy on the coagulation profile of men with advanced prostate cancer.

METHODS

A total of <em>2</em>0 patients with newly diagnosed locally advanced or metastatic prostate cancer were treated using transdermal estradiol patches and the coagulation profile was assessed before and during <em>1</em><em>2</em> months therapy. Activation of coagulation was assessed by assaying the levels of activated factor VII (VIIa), activated factor XII (XIIa), <em>prothrombin</em> <em>fragments</em> <em>1</em> and <em>2</em> (F<em>1</em> + <em>2</em>), thrombin-antithrombin III (TAT III) complex and fibrinogen. Inhibition of the coagulation cascade was assayed by protein C, protein S and activated protein C resistance (APC-R). Fibrinolytic activity was determined by assaying tissue plasminogen activator (TPA) and plasminogen activation inhibitor type <em>1</em> (PAI-<em>1</em>). D-Dimer levels assessed both coagulation and fibrinolytic (thrombophilic) activity. Venous Duplex, color Doppler ultrasound and photoplethysmography were used to assess for thrombosis.

RESULTS

Levels of VIIa and XIIa were unaffected by transdermal estradiol therapy. Although levels of TAT III were increased in some patients at <em>1</em><em>2</em> months, the increase was markedly less than that observed historically with equivalent doses of oral estrogens. Levels of the inhibitory and fibrinolytic factors including protein C, protein S, APC-R, TPA and PAI-<em>1</em> remained stable. Reductions in F<em>1</em>+F<em>2</em>, fibrinogen and D-Dimer levels represented a normalization from increased levels to the physiological range.

CONCLUSIONS

These results suggest that transdermal estradiol reduces thrombophilic activation in men with advanced prostate cancer, and protects against the risk of thrombosis.

To study the specific role of gamma-carboxyglutamic acid (Gla) residues in <em>prothrombin</em>, we have isolated a series of partially carboxylated <em>prothrombin</em> variants from a patient with a hereditary defect in vitamin K-dependent carboxylation (Goldsmith, G. H., Pence, R. E., Ratnoff, O. D., Adelstein, D. A., and Furie, B. (<em>1</em>98<em>2</em>) J. Clin. Invest. 69, <em>1</em><em>2</em>53-<em>1</em><em>2</em>60). The three variant <em>prothrombins</em>, purified by DEAE-Sephacel, immunoaffinity chromatography, and preparative gel electrophoresis, were indistinguishable from <em>prothrombin</em> in molecular weight, amino acid composition, and NH<em>2</em>-terminal amino acid sequence, with the exception of Gla residues. Variant <em>prothrombin</em> <em>1</em>, with 8 Gla residues, had 66% of the coagulant activity of <em>prothrombin</em>, one high affinity metal-binding site (Kd = <em>1</em>5 nM), and three lower affinity sites (Kd = <em>2</em>.7 microM); <em>prothrombin</em> contained two high affinity (36 nM) and four lower affinity sites (Kd = <em>1</em> microM). Ca(II) induced a <em>2</em>3% decrease in the intrinsic fluorescence of variant <em>prothrombin</em> <em>1</em> <em>fragment</em> <em>1</em>, compared to a 35% decrease in that of <em>prothrombin</em> <em>fragment</em> <em>1</em>. The phospholipid binding activity of variant <em>prothrombin</em> <em>1</em> was 44% that of <em>prothrombin</em>. Variant <em>prothrombin</em> <em>2</em> and variant <em>prothrombin</em> 3, with 4 and 6 Gla residues, respectively, had about 5% of <em>prothrombin</em> coagulant activity and a single high affinity and two lower affinity metal-binding sites and exhibited no phospholipid binding activity. Variant <em>prothrombin</em> 3 <em>fragment</em> <em>1</em> and variant <em>prothrombin</em> <em>2</em> <em>fragment</em> <em>1</em> demonstrated <em>1</em>8 and <em>1</em>3% of Ca(II)-induced fluorescence quenching, respectively. Abnormal <em>prothrombin</em>, with <em>1</em> Gla residue, had 8% of <em>prothrombin</em> coagulant activity, a single lower affinity (<em>1</em> microM) metal-binding site, and <em>1</em>3% Ca(II)-induced fluorescence quenching of the <em>fragment</em> <em>1</em> species and did not bind to phospholipid. These results indicate that Gla residues define the metal binding properties of <em>prothrombin</em>. Most, if not all, of the Gla residues are required for complete <em>prothrombin</em> function, and the <em>prothrombin</em> coagulant activity correlates to the phospholipid binding activity of the <em>prothrombin</em> species.

BACKGROUND

Catheter ablation may be complicated by clinical thromboembolism in about <em>1</em>% of patients.

RESULTS

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>]), platelets (beta-thromboglobulin [beta-TG])) and fibrinolysis (plasmin-antiplasmin complexes [PAP] and D-dimer) during radiofrequency (RF) ablation in <em>1</em>3 patients. They received heparin <em>1</em>00 U/kg intravenously after the initial electrophysiologic study, prior to the delivery of RF current; thereafter <em>1</em>,000 U/hour throughout the procedure. PF<em>1</em>+<em>2</em> increased fourfold (P < 0.00<em>1</em>) during the diagnostic study, but gradually declined to upper reference value during heparin administration. There was a strong correlation between procedure duration prior to heparin bolus (range 39 to <em>1</em>73 min); and (a) the maximal rise of PF<em>1</em>+<em>2</em> (r = 0.83, P < 0.00<em>1</em>) and (b) the increase of PF<em>1</em>+<em>2</em> from baseline to end of the procedure (r = 0.74, P = 0.004). There was no correlation between postheparin changes of PF<em>1</em>+<em>2</em> and (a) postheparin procedure duration (range 40 to 3<em>1</em>7 min), (b) number of RF pulses (range <em>1</em> to <em>1</em>6), or (c) RF current duration (range 46 to 687 sec). Plasma beta-TG concentration showed similar trends. Fibrinolytic activity increased moderately from baseline until heparin administration; then remained around the upper reference values. PAP at the end of procedure and D-dimer at the time of heparin administration both correlated with preheparin procedure duration (r = 0.70, P = 0.007 and r = 0.69, P = 0.0<em>1</em>, respectively). All parameters were normal the next morning.

CONCLUSIONS

Procedure duration prior to heparin administration, and not the delivery of RF current per se, determines activation of hemostasis and fibrinolysis during RF ablation.

The structure of <em>prothrombin</em> <em>fragment</em> <em>1</em>, solved at <em>2</em>.8 A resolution (<em>1</em> A = 0.<em>1</em> nm) by a combination of multiple and single isomorphous replacement methods utilizing solvent flattening, has been refined by restrained least-squares methods (R = 0.<em>2</em>4), solvent not included, using fairly stringent restraints on the molecular geometry and individual thermal parameters. The inner kringle loop possesses significantly lower B-values than the outer loops even though the former also constitutes a surface of the folded kringle structure. This surface forms the Lys sub-site of the fibrin binding site of other kringles. The hydrogen bonding network and ion pair interactions of <em>fragment</em> <em>1</em> appear to maintain a compact folded structure among the various loops of the kringle structure. On the other hand, since there is only one hydrogen bond between the kringle and its preceding 30 residues, considerable flexibility is suggested for the Gla-domain consistent with its disorder in crystals. A chitobiose has been located at the Asn77 glycosylation site, but only a single N-acetyl-glucosamine is ordered at Asn<em>1</em>0<em>1</em>. The lysine binding site region of other kringles is not properly developed in <em>fragment</em> <em>1</em>, accounting for its lack of Lys/fibrin affinity. Most of the conserved sequence among <em>1</em><em>1</em> different kringles is associated with either: (<em>1</em>) protecting the inner loop disulfides Cys87-<em>1</em><em>2</em>7, Cys<em>1</em><em>1</em>5-<em>1</em>39 upon which the folding is based; or (<em>2</em>) a requirement of the lysine binding site. The remainder of the conservation is generally associated with the ten reverse turns of the folding; of these 40 residues, or about half the sequence, <em>1</em>4 are conserved among eight different turns. The intermolecular packing consists of infinite helical columns of <em>fragment</em> <em>1</em> molecules related by a crystallographic 4(3) screw axis, which are held together by van der Waals' interactions of aromatic clusters from different molecules related by a crystallographic <em>2</em>-fold rotation axis.

In recent years there has been great interest in the putative role of <em>prothrombin</em> and its activation peptides, especially the urinary form of <em>prothrombin</em> <em>fragment</em> <em>1</em>, in the pathogenesis of calcium oxalate (CaOx) urolithiasis. Previously, we showed that <em>prothrombin</em> and its activation peptides inhibit CaOx crystallization in inorganic conditions in vitro. The aim of the present study was to determine if this inhibitory activity is retained in undiluted human urine and, therefore, whether it is likely to have any influence under physiological conditions. A secondary objective was to assess the relationship between the structures of the proteins and their inhibitory activities. <em>Prothrombin</em> was purified from <em>Prothrombin</em>ex-HT, cleaved with thrombin and the resulting <em>fragment</em> <em>1</em> (F<em>1</em>) and <em>fragment</em> <em>2</em> (F<em>2</em>) were purified. The purity of each protein was confirmed by SDS/PAGE, and their effects on CaOx crystallization in undiluted ultrafiltered human urine were determined at a final concentration 80.65 nmol/l using Coulter Counter and [(<em>1</em>4)C]oxalate analysis. The precipitated crystals were visualized using scanning electron microscopy. The Coulter Counter data revealed that, whereas <em>prothrombin</em> and its activation peptides did not affect the urinary metastable limit and the size of the precipitated particles, F<em>1</em> did significantly reduce the latter. These findings were corroborated with scanning electron microscopy which also revealed that the reduction in particle size caused by F<em>1</em> resulted from a decrease in the degree of crystal aggregation, rather than in the size of the individual crystals. The [(<em>1</em>4)C]oxalate data showed that none of the proteins added significantly inhibited the mineral deposition. It was concluded that with the exception of F<em>1</em>, which does inhibit CaOx crystal aggregation, <em>prothrombin</em> and its activation peptides do not alter the deposition and aggregation of CaOx crystals in ultrafiltered human urine in vitro. Also, the gamma-carboxyglutamic acid domain of <em>prothrombin</em> and F<em>1</em>, which is absent from thrombin and F<em>2</em>, is the region of the molecules that determines their potent inhibitory effects. The superior potency of F<em>1</em>, compared with <em>prothrombin</em>, probably results from the molecule's greater charge-to-mass ratio.

Patients dialyzed due to diabetic nephropathy are at a higher risk of death due to cardiovascular complications than dialyzed non-diabetic patients. Disturbances in hemostasis may play a role in the vascular complications of diabetes mellitus. It has been postulated that TAFI-thrombin activatable fibrinolysis inhibitor, which couples two opposite systems: coagulation and fibrinolysis, may be involved in the mechanism of vascular endothelial damage in diabetic patients. We assessed: TAFI and TAFIa, markers of ongoing coagulation: thrombin-antithrombin complexes, <em>prothrombin</em> <em>fragments</em> <em>1</em>+<em>2</em>, a marker of ongoing fibrinolysis: plasmin-antiplasmin complexes in diabetic and non-diabetic patients on hemodialyses-HD, peritoneal dialyses-CAPD, patients with chronic renal failure with and without diabetic nephropathy on conservative treatment. Both groups of dialyzed diabetic patients have a higher concentration of markers of ongoing coagulation and TAFI activity when compared to dialyzed non-diabetic patients. Linear regression analysis showed that TAFI concentration was directly related to albumin in HD and CAPD patients without diabetic nephropathy, whereas TAFIa correlated with triglycerides, fibrinogen and leukocytes count in this group. When evaluated separately (HD, CAPD), significant correlations between TAFIa and triglycerides and fibrinogen were found only in diabetic CAPD patients. Multivariate analysis showed no correlation between TAFI and other parameters studied. In conclusion, elevated circulating TAFI and TAFIa might be a new link in the pathogenesis of impaired fibrinolysis in diabetic nephropathy, and thus atherosclerosis progression, particularly in CAPD patients. Hypercoagulable state observed in diabetic patients on conservative treatment and maintained on dialyses may contribute to the higher cardiovascular mortality in this population. In these patients there is also evidence of endothelial injury, and probably secondary activation of the coagulation cascade.

Recurrent pregnancy loss (PL) is associated with maternal thrombophilia and prophylaxis with low molecular weight heparin (LMWH) can improve pregnancy outcome in this setting. The aim of this study was to investigate the modulation of systemic hemostatic parameters by enoxaparin in women with recurrent PL and to evaluate plasmatic parameters that would potentially enable monitoring LMWH prophylaxis effect during pregnancy. Study group included 87 women with thrombophilia and PL treated with enoxaparin 40 mg daily vs. 40 mg twice daily. The control group comprised 40 women with normal pregnancies. Blood samples have been collected throughout the period starting at 5-<em>1</em>0 weeks of gestation until 6-<em>1</em>0 weeks postpartum. The determined plasmatic markers included: anti-Xa activity, total and free tissue factor pathway inhibitor (TFPI), D-dimer, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (PT<em>1</em>+<em>2</em>), activated protein C resistance (APC-SR) and free protein S. Successful pregnancy outcome was recorded in 70 (80.5%) women treated with enoxaparin, without correlation to enoxaparin dosage. Seventeen women (<em>1</em>9.5%) had pregnancy loss at <em>1</em>6+/-7 (6-3<em>2</em>) weeks of gestation. Anti-Xa levels at <em>1</em>0-<em>1</em>5 weeks of gestation were higher (0.39+/-0.38 u/ml) in the successful pregnancy outcome group compared to the abortion group (0.<em>2</em><em>2</em>+/-0.<em>2</em> u/ml). Prophylactic anti-Xa activity levels (0.<em>2</em>8+/-0.<em>1</em>3 u/ml) were documented from <em>1</em>5 weeks of gestation until delivery in the successful pregnancy outcome group. Significant increase in anti-Xa, total TFPI and free TFPI levels (P<0.00<em>1</em>) was achieved after beginning of LMWH prophylaxis in successful pregnancy outcome group but not in the abortion group. D-dimer and PT<em>1</em>+<em>2</em> levels appeared to be significantly increased while APC-SR and free protein S levels gradually decreased during pregnancy, with no difference between study groups. These results suggest that LMWH prophylaxis during pregnancy enables modulation of systemic hemostatic parameters via inhibition of factor Xa and increase in plasmatic total and free TFPI levels.

Chronic heart failure (CHF) is characterised by activation of neuroendocrine and inflammatory pathways, and both are linked to a prothrombotic state. Treatment with omega-3 polyunsaturated fatty acids (n3-PUFA) showed significant benefits including mortality reduction in CHF, but exact mechanisms of action are still unclear. We investigated the effects of n3-PUFA on markers of platelet activation and thrombogenesis in patients with severe CHF. Thirty-six patients with non-ischaemic CHF (LVEF<35%, NYHA class><em>2</em>) under optimised therapy were randomised to supplementation with <em>1</em>g/day or 4 g/day n3-PUFA, or placebo for <em>1</em><em>2</em> weeks. Using whole-blood flow cytometry, monocyte-platelet aggregates characterised by CD<em>1</em>4+/CD4<em>2</em>b+ co-expression and monocytic tissue factor (TF) were determined. Plasma levels of P-selectin, sCD40L, fibrinogen, <em>prothrombin</em> <em>fragment</em> F<em>1</em>.<em>2</em>, TF and pro-inflammatory markers (high sensitive[hs] interleukin-6, hsCRP, hsTNF-alpha, monocyte chemotactic protein-<em>1</em>) were measured by immunoassay. Supplementation with <em>1</em>g/day and 4 g/day n3-PUFA but not placebo significantly reduced monocyte-platelet aggregates in a dose-dependent manner (p for trend = 0.0<em>2</em> across the groups). A dose of 4 g/day but not <em>1</em>g/day n3-PUFA significantly decreased P-selectin (p = 0.03). Plasma TF decreased dose-dependently upon n3-PUFA supplementation (p for trend = 0.0<em>2</em>), paralleled by a significant decrease of TF+-monocytes (p for trend = 0.0<em>1</em>). The amount of 4 g/day n3-PUFA exhibited modest anti-inflammatory effects with a significant reduction of hs interleukin-6 (p<0.0<em>1</em>) and a trend-wise reduction of hsTNF-alpha (p = 0.09). No changes were seen for sCD40L, fibrinogen, hsCRP and monocyte chemotactic protein-<em>1</em>, while F<em>1</em>.<em>2</em> was decreased by 4 g/day n3-PUFA (P = 0.03). In patients with severe non-ischaemic CHF, treatment with n3-PUFA leads to a dose-dependent decrease of platelet activation and TF. Higher dosage exhibits also anti-inflammatory effects.

Obesity is associated with a high risk of cardiovascular events. Several haemostatic disturbances which could contribute to this increased risk have been described in obesity; nevertheless, the state of coagulation inhibitors has been scarcely studied in these patients. The aim of the present study was to compare activated protein C levels in obese patients and in a control group, and to evaluate the effect of weight loss. In 67 severe or morbid obese patients, an evaluation was performed at baseline and 3 months after diet. The same determinations were performed in 67 healthy volunteers with normal body weight. We also quantified the levels of protein C and <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>. Obese patients showed significantly higher levels of activated protein C, protein C and <em>fragment</em> <em>1</em>+<em>2</em>. No correlation was found between activated protein C and <em>fragment</em> <em>1</em>+<em>2</em> levels in obese patients. After three months of diet, a significant decrease in activated protein C and <em>fragment</em> <em>1</em>+<em>2</em> was observed. In conclusion, activated protein C levels are increased in obese patients, but only a minor fraction of this increase may be explained by the higher thrombin generation and C protein levels. Activated protein C levels decrease with weight loss, due in part to a thrombin generation reduction.

Hormonal contraception for men requires administration of testosterone and gestagens. The effects of a long-acting testosterone ester and <em>2</em> different progestins on hemostatic activation parameters were studied in relation to cardiovascular risk. In phase <em>1</em>, 7 healthy men aged <em>2</em>8-38 years received a single intramuscular injection of <em>2</em>00 mg norethisterone-enanthate (NET-EN) on Day 0. Plasma samples were obtained on Days 0, <em>1</em>4, 4<em>1</em>, and 84. In phase <em>2</em>, 3 groups of <em>1</em>4 healthy men aged <em>1</em>8-45 years received four injections (every 6 weeks) of <em>1</em>000 mg testosterone undecanoate (TU), plus daily oral placebo or daily oral levonorgestrel (LNG, <em>2</em>50 microg); or four injections (every 6 weeks) of NET-EN. Treatment lasted <em>2</em>4 weeks. Plasma samples were obtained at weeks 0, <em>1</em>6, <em>2</em>4, and 5<em>2</em>. All samples were assayed for levels of coagulation factors VIIc, VIIa, XIIc, and XIIa; <em>prothrombin</em> <em>fragment</em> F<em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>); antithrombin; plasmin-alpha(<em>2</em>)-antiplasmin-complex (PAP); and fibrinogen. NET-EN alone led to a depletion of sexual hormones and a marked shift in hemostatic parameters with increasing levels of FXIIc, fibrinogen, antithrombin, and F<em>1</em>+<em>2</em>, whereas FVIIc and FVIIa levels decreased. PAP levels increased significantly. Opposite effects were seen in the TU/placebo group, with a significant down-regulation of fibrinolysis and the hemostatic turnover rate. Testosterone effects were attenuated by additional administration of gestagens. The effect of hormonal male contraception using long-acting testosterone esters with or without gestagens was significantly measurable within the hemostatic system. Down-regulation of the hemostatic system with testosterone alone may indicate an antithrombotic effect, whereas clinical consequences of an additional gestagen compound cannot be derived.

We investigated <em>1</em>7 patients with Henoch-Schönlein purpura (HSP) and describe as yet unreported abnormal results of blood coagulation tests. In parallel to the activity of the disease, D-dimer concentrations in plasma were found to be significantly increased in <em>1</em>5 of the <em>1</em>7 patients; almost 50% of all patients showed values higher than <em>1</em>0 times the upper limit of the normal range. In <em>1</em><em>1</em> patients, plasma concentrations of thrombin-antithrombin complex (TAT) and <em>prothrombin</em> <em>fragments</em> <em>1</em> and <em>2</em> (F<em>1</em>+<em>2</em>) were examined; six of them showed abnormal results. The pathologic values were correlated to the activity of the disease, but abnormalities were also found in milder cases of HSP. These findings probably reflect local reactions within inflamed blood vessels rather than a systemic activation of coagulation and hyperfibrinolysis. Clinicians should be aware of these laboratory findings in order not to confuse common cases of HSP with purpura necroticans, a very severe type of vasculitis in which signs of disseminated intravascular coagulation (DIC) have been reported. Our findings suggest that an activation of coagulation including hyperfibrinolysis secondary to the endothelial damage is a typical feature of the common types of HSP.

In this randomised, placebo-controlled <em>1</em><em>2</em>-week study, sixty healthy postmenopausal women received either placebo (N = <em>1</em>6) or daily <em>2</em> mg micronised oestradiol, either unopposed (N = <em>1</em>6, E<em>2</em> group) or combined with a progestagen for <em>1</em>4 days of each cycle (N = <em>2</em>8, E<em>2</em>+P group).

RESULTS

As compared to placebo, plasma levels of AT III were reduced only in the E<em>2</em> group (approximately <em>2</em>8%), plasma levels of protein C decreased only in the E<em>2</em>+P group (approximately 4%) and plasma levels of protein S decreased in both the E<em>2</em> and E<em>2</em>+P group (approximately <em>2</em><em>1</em>%). In both the E<em>2</em> and E<em>2</em>+P groups, the plasma levels of factor VII (antigen and activity) showed a borderline significant increase (approximately <em>1</em>0%), whereas no significant change was observed in active factor VII. Plasma levels of tissue-type plasminogen activator (approximately <em>2</em><em>2</em>%), urokinase plasminogen activator (approximately <em>2</em>5%) and plasminogen activator inhibitor type-<em>1</em> (approximately 43%) decreased in the E<em>2</em> and E<em>2</em>+P groups, whereas those of plasminogen increased (approximately <em>1</em><em>2</em>%). Treatment was associated with an increase in levels of <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (approximately 3<em>1</em>%), but levels of thrombin-antithrombin III complexes, and of plasmin-alpha<em>2</em>-antiplasmin complexes and total fibrin(ogen) degradation products did not change significantly.

CONCLUSIONS

Short-term E<em>2</em> and E<em>2</em>+P treatment is associated with a shift in the procoagulant-anticoagulant balance towards a procoagulant state. A substantial proportion of women do not have a net increase in fibrinolytic activity. These data may be relevant in explaining the increased risk of venous thromboembolism associated with ERT and HRT, and possibly also in explaining the negative results of the Heart and Estrogen/progestin Replacement Study.

Generation of factor XII, thrombin antithrombin complexes, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> and thrombus precursor protein has been monitored in <em>1</em>6 subjects during haemodialysis. Immediately after starting treatment, contact of blood with the negatively charged surfaces of the polyacrylnitril membrane AN-69 resulted in a 9-45% decrease in factor XII activity. Peak concentrations for thrombin antithrombin complexes (50 to <em>1</em><em>2</em>0 microg/L) were observed 30 min after the start of haemodialysis. Establishment of thrombus precursor protein concentrations yielded steadily increasing results without any tendency to decrease during treatment. Determination of thrombin antithrombin complexes is considered to establish the most sensitive short-term reacting parameter indicating activation of coagulation. A steady generation of fibrin and fibrinogen-fibrin complexes during treatment with haemodialysis is indicated by increasing results for thrombus precursor protein. In order to prevent clotting during haemodialysis, an additional supplementation of anticoagulant is needed.