OBJECTIVE

<em>Prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> measured in spot urine (uF<em>1</em>+<em>2</em>) is an indicator of thrombin generation. We examined whether measured levels of uF<em>1</em>+<em>2</em> can be used to differentiate between patients who do and do not acquire sustained coagulation activation after total hip arthroplasty (THA).

METHODS

We performed two separate studies in patients undergoing THA. Study <em>1</em> was a prospective pilot study aiming to roughly estimate the extent of pre- and postoperative fluctuations in the uF<em>1</em>+<em>2</em> concentration. Study <em>2</em> was a larger prospective cohort study aiming to verify the findings of Study <em>1</em> and to examine the association between the uF<em>1</em>+<em>2</em> concentrations and risk of vascular thrombotic complications (VTC) or death. Finally, we sought to define a cut-off concentration value that could be used to identify patients with a sustained uF<em>1</em>+<em>2</em> elevation after the first postoperative week. The urine samples were analysed by ELISA. In both studies thromboprophylaxis was used for at least 7 days after the operation.

RESULTS

The operative trauma resulted in elevation of the uF<em>1</em>+<em>2</em> level in all patients compared with the preoperative level and levels in the healthy volunteers. Ten out of <em>1</em><em>1</em>3 patients (8.8%) in the second study suffered VTC or death, assumed to be caused by a coagulation problem. Analysis of variance revealed the following statistically significant associations: pre- vs. postoperative log uF<em>1</em>+<em>2</em> levels (P<0.000<em>1</em>), log uF<em>1</em>+<em>2</em> levels comparing patients with and without events (P=0.004); and the individual log uF<em>1</em>+<em>2</em> levels (P<0.000<em>1</em>). A cut-off value of uF<em>1</em>+<em>2</em> concentration between 0.3 and 0.5 nmol/l had a sensitivity and a negative predictive value between<em>1</em>00% and 90%, and specificity between 45% and 63% and overall accuracy between 50% and 65%. This value was obtained by the analysis of a receiver operating characteristic curve with the purpose of identifying patients with sustained coagulation activation on day 5 after operation.

CONCLUSIONS

Our studies suggest that measured levels of uF<em>1</em>+<em>2</em> can be potentially used to assess the individual risk of VTC after THA and to test for non-invasive detection of sustained coagulation activation.

OBJECTIVE

Hemorrhage and thrombosis predisposing to myocardial infarction, multiple organ failure, and thromboembolism account for the majority of the morbidity and mortality associated with repair of ruptured and nonruptured abdominal aortic aneurysms (AAAs). The aim of this study was to examine coagulation and fibrinolysis in patients operated on for ruptured and nonruptured infrarenal AAAs.

METHODS

Ten patients operated on for ruptured and 9 patients operated on for nonruptured AAAs were studied. Tissue plasminogen activator (t-PA) antigen, thrombin-antithrombin (TAT), and D-dimer were measured before induction of anesthesia. Plasminogen activator inhibitor (PAI) activity, t-PA activity, and <em>prothrombin</em> <em>fragment</em> (PF) <em>1</em>+<em>2</em> were measured before induction of anesthesia, immediately before aortic clamp release, and 5 minutes and <em>2</em>4 hours after aortic clamp release.

RESULTS

Preoperatively, ruptured AAA was associated with significantly elevated t-PA antigen (median <em>1</em>5.7 ng/mL, range 9. 0 to <em>2</em><em>2</em>.<em>1</em> ng/mL versus nonrupture: median 6.6 ng/mL, range 4.7 to <em>1</em>6. 4 ng/mL; P <.0<em>1</em>, Mann-Whitney test), increased PAI activity (median 36.5 arbitrary units/mL, range <em>2</em>0.6 to 38.8 arbitrary units/mL versus nonrupture: median 8.<em>2</em> arbitrary units/mL, range 3.<em>2</em> to <em>2</em><em>1</em>.7 arbitrary units/mL; P <.00<em>1</em>), reduced t-PA activity (median 0.<em>1</em><em>2</em> IU/mL, range 0.06 to 0.4 IU/mL versus nonrupture: median 0.49 IU/mL, range 0.<em>1</em>4 to 3.<em>2</em> IU/mL; P <.0<em>1</em>), elevated TAT (median <em>1</em>35.5 microg/L, range 6<em>1</em>.<em>2</em> to <em>2</em>09.4 microg/L versus nonrupture: median <em>2</em><em>1</em>. 6 microg/L, range 6.6 to <em>1</em>80.4 microg/L; P <.0<em>2</em>) and elevated PF <em>1</em>+<em>2</em> (median 9.0 nmol/L, range 5.4 to <em>1</em><em>1</em>.6 nmol/L versus nonrupture: median <em>2</em>.<em>2</em> nmol/L, range 0.7 to 7.<em>1</em> nmol/L, P <.00<em>1</em>). There was no significant difference in preoperative D-dimer levels (median 3460 ng/mL, range <em>1</em><em>2</em>36 to 7860 ng/mL versus nonrupture: median <em>1</em>64<em>2</em> ng/mL, range 7<em>2</em>8 to 5334 ng/mL; P =.07). The differences in PAI activity, t-PA activity, and PF <em>1</em>+<em>2</em> persisted throughout the course of surgery, but there was no significant difference between the groups at <em>2</em>4 hours.

CONCLUSIONS

These novel data demonstrate that ruptured AAA repair is associated with inhibition of systemic fibrinolysis and intense thrombin generation. Similar changes are seen in nonruptured AAA but are of a lesser magnitude. This procoagulant state may contribute to the microvascular and macrovascular thrombosis that leads to myocardial infarction, multiple organ failure, and thromboembolism.

In addition to inhibiting platelet (plt) aggregation abciximab, a glycoprotein (GP) IIb/IIIa antagonist, reduces coagulation in blood or platelet-rich plasma. We assessed the effects of abciximab (<em>1</em>0 micro g mL(-<em>1</em>)) on adhesion-dependent procoagulant activity (PCA) of plt upon: (i) collagen to model initial adhesion; or (ii) plasma clot or fibrin to model a preformed thrombus with retained thrombin activity. In a two-stage assay gel-filtered plt (GFP) first adhered on collagen, plasma clot, or fibrin, and plt activation was traced with platelet factor 4 (PF 4) release. Second, PCA was measured on adherent plt (i) by soluble <em>prothrombin</em> <em>fragments</em> (F<em>1</em> + <em>2</em>); and (ii) chromogenically by adding defibrinated plasma and thromboplastin. Abciximab inhibited aggregation upon collagen-adherent plt both in the absence and presence of plasma. In contrast, without plasma abciximab enhanced plt deposition to fibrin surfaces depending on thrombin generation and fibrin polymerization. However, abciximab reduced PCA and generation of F<em>1</em> + <em>2</em> on adherent plt surface-independently by 35%, whereas PF 4 release persisted. Also, a GP Ib inhibitor, mAb SZ<em>2</em>, attenuated PCA by 40% alone, and by 65% together with abciximab, leaving 35% of PCA unaltered. Abciximab decreased generation of new thrombin on both collagen- and clot-adherent plt. However, abciximab did not inhibit alpha-granule release, suggesting distinct pathways for PCA and release reaction. Deposition of isolated plt on clots in the presence of abciximab was dependent on thrombin and polymerizing plt-derived fibrin(ogen). Due to local consumption of natural anticoagulants adjacent to a preformed thrombus the antithrombotic effect of abciximab benefits from additional inhibition of thrombin.

The cross-talk between inflammatory and coagulation cascades has been demonstrated. <em>Prothrombin</em> processing releases the protease domain (thrombin) along with two catalytically inactive kringle-containing derivatives: <em>prothrombin</em> <em>fragments</em> <em>1</em> (F<em>1</em>) and <em>2</em> (F<em>2</em>). It is well established that thrombin is able to trigger an inflammatory response but the possible effects of <em>prothrombin</em> <em>fragments</em> on leukocyte functions are still unknown. In this report, we demonstrate for the first time that both F<em>1</em> and F<em>2</em> <em>prothrombin</em> <em>fragments</em>, interfere with intracellular functional signaling pathways to modulate human neutrophil migration. In addition, we show that thrombin, <em>fragment</em> <em>1</em> and <em>fragment</em> <em>2</em> induce human neutrophil chemotaxis. The effect of <em>fragment</em> <em>2</em>, but not <em>fragment</em> <em>1</em>, was partially inhibited by pertussis toxin, an inhibitor of G(alphai)-signaling. The pre-treatment of cells with <em>fragment</em> <em>2</em> inhibited thrombin-induced chemotaxis, while both <em>fragments</em> impaired neutrophil migration induced by interleukin-8. F<em>1</em> and F<em>2</em> increased the expression and activation of G-protein-coupled receptor kinase-<em>2</em>, which has emerged as a key effector in the desensitization of chemokine receptors. In parallel, <em>prothrombin</em> <em>fragments</em> activated extracellular signal-regulated kinase <em>1</em>/<em>2</em>, stimulating its phosphorylation and nuclear translocation, and induced inhibitor of kappa-B phosphorylation and degradation followed by nuclear factor-kappa B translocation to nucleus. Furthermore, both <em>prothrombin</em> <em>fragments</em> induced interleukin-8 gene expression in human neutrophils. These findings suggest that the interference with neutrophil signaling and function, caused by kringle-containing <em>prothrombin</em> <em>fragments</em> may desensitize these cells to respond to further activation by thrombin and interleukin-8 during inflammatory and coagulation responses.

BACKGROUND

Puumala hantavirus (PUUV) causes haemorrhagic fever with renal syndrome characterized by thrombocytopenia, capillary leakage and acute kidney injury (AKI) with proteinuria and haematuria. Although the typical histologic lesion is acute tubulointerstitial nephritis, the amount of glomerular proteinuria predicts the severity of upcoming AKI. Here, we studied the associations of haematuria and proteinuria with the severity of emerging AKI, thrombocytopenia and markers of coagulation and fibrinolysis in PUUV infection.

METHODS

We examined <em>2</em>05 consecutive patients treated for serologically confirmed acute PUUV infection at Tampere University Hospital during 1997-<em>2</em>014. The patients were divided into three groups according to the combined positive result in urine haemoglobin and albumin dipstick tests: 0-<em>2</em> + (n = 58), 3-4 + (n = 100) and 5-6 + (n = 47).

RESULTS

The medians of maximum creatinine concentrations in the three groups were: 0-<em>2</em> + 100 μmol/L (range 5<em>2</em>-1499), 3-4 + <em>2</em>04 μmol/L (range 65-1071) and 5-6 + 361 μmol/l (range 51-1<em>2</em>85) (p < .001). The number of blood platelets (p = .069), and the levels of fibrinogen, prothrombin fragments F1 + <em>2</em> and d-dimer (p = .60<em>2</em>, p = .113, p = .<em>2</em>89, respectively) were not significantly different between the groups. When the amount of haematuria in the dipstick test was examined separately, no association with thrombocytopenia was detected (p = .307 between groups 0, 1+ and <em>2</em>-3+).

CONCLUSIONS

Combined positive result of haematuria and proteinuria in the dipstick test at hospital admission predicted the severity of upcoming AKI in acute PUUV infection. As haematuria was not associated with the severity of thrombocytopenia, it did not indicate increased bleeding tendency, but was rather a marker of acute kidney injury.

Effects of high-dose megestrol acetate on blood coagulation and fibrinolysis were investigated in patients with gynecological (n = <em>1</em>3) and breast (n = <em>1</em>0) cancer. Patients received either <em>1</em>60 mg or 3<em>2</em>0 mg/day megestrol acetate orally. Blood sampling was performed prior to and after months <em>1</em>, 3 and 6 of treatment. Pretreatment values of global clotting times, fibrinogen, factor VII, thrombin-antithrombin III complex, anticoagulation, fibrinolysis and antifibrinolysis were found to be within the reference range. Elevated plasma levels were demonstrated for <em>prothrombin</em> <em>fragments</em> <em>1</em> and <em>2</em>, fibrin degradation products and the plasmin-antiplasmin complex. We demonstrated a significant <em>2</em>0-30% reduction of factor VII until the 3rd month of therapy. No further effects were seen within the remaining 3 months of treatment. For the other analyzed parameters of hemostasis, no significant influence of high-dose progestin treatment was found. Furthermore, we observed no clinically relevant differences between the two dosages. Our results do not provide any evidence that there is a thrombogenic effect of high-dosage megestrol acetate with <em>1</em>60 mg or 3<em>2</em>0 mg per day amongst patients with advanced gynecological malignancies. The observed incidence of thrombosis might be the consequence of other risk factors such as tumor-induced hypercoagulability, simultaneous chemotherapy or other individual thrombosis risk factors.

BACKGROUND

Cardiopulmonary bypass and coronary artery bypass grafting (CABG) result in significant thrombin generation and activation of fibrinolysis. Thrombin contributes to myocardial ischemia-reperfusion injury in animal studies, but the role of thrombin in myocardial damage after CABG is unknown.

OBJECTIVE

We measured thrombin generation and fibrin turnover during reperfusion after CABG to evaluate their associations with postoperative hemodynamic changes and myocardial damage.

METHODS

One hundred patients undergoing primary, elective, on-pump CABG were prospectively enrolled. Plasma <em>prothrombin</em> <em>fragment</em> F(<em>1</em>+<em>2</em>) and D-dimer were measured preoperatively and at seven time points thereafter. Mass of the Mb fraction of creatine kinase (Ck-Mbm) and troponin T (TnT) were measured on the first postoperative day.

RESULTS

Reperfusion induced an escalation of thrombin generation and fibrin turnover despite full heparinization. F(<em>1</em>+<em>2</em>) during early reperfusion associated with postoperative pulmonary vascular resistance index. F(<em>1</em>+<em>2</em>) at 6 h after protamine administration correlated with Ck-Mbm (r = 0.40, P < 0.00<em>1</em>) and TnT (r = 0.44, P < 0.00<em>1</em>) at <em>1</em>8 h postoperatively. Patients with evidence of myocardial damage (highest quintiles of plasma Ck-Mbm and TnT) had significantly higher F(<em>1</em>+<em>2</em>) during reperfusion than others (P < 0.00<em>2</em>). Logistic regression models identified F(<em>1</em>+<em>2</em>) during reperfusion to independently associate with postoperative myocardial damage (odds ratios <em>2</em>.5-4.4, 95% confidence intervals <em>1</em>.04-<em>1</em>5.7).

CONCLUSIONS

Reperfusion caused a burst in thrombin generation and fibrin turnover despite generous heparinization. Thrombin generation during reperfusion after CABG associated with pulmonary vascular resistance and postoperative myocardial damage.

Platelet dysfunction after cardiopulmonary bypass (CPB) can contribute to excessive post-operative bleeding. Most trials of the protective effect of aprotinin in this setting have involved hypothermic CPB, which is more deleterious for platelets than normothermic CPB. Here we investigated the effect of aprotinin on platelet function during normothermic CPB in pediatric patients. Twenty patients (9 newborns [(<em>1</em> month old] and <em>1</em><em>1</em> infants [<36 month old]), weighting less than <em>1</em>5 kg and undergoing normothermic CPB (35-36 degrees C) were randomly assigned to two equal groups, one of which received high-dose aprotinin. Platelet function was assessed by flow cytometry just before CPB and 5 minutes after heparin neutralization. F<em>1</em> + <em>2</em> <em>fragments</em> were measured by ELISA before and 5 minutes after CPB. Platelet activation marker expression (CD6<em>2</em>P and activated alphaIIbbeta3) induced by ADP or TRAP was lower after CPB than before CPB, suggesting a deleterious effect of normothermic CPB on platelet function. <em>Prothrombin</em> <em>fragment</em> F<em>1</em> + <em>2</em> levels increased after CPB. Aprotinin administration did not influence the level of <em>prothrombin</em> <em>fragments</em> or platelet marker expression measured in basal condition. However, after CPB, the capacity for platelet activation was higher in the aprotinin group, as shown by measuring CD6<em>2</em>P expression after TRAP activation (p = 0.05). This study suggests that pediatric normothermic CPB causes platelet dysfunction, and that high-dose aprotinin has a protective effect.

OBJECTIVE

The aim of the experiments shown here, is to demonstrate exemplarily that thrombin can be a survival factor for malignant cells.

METHODS

Activation of the coagulation system has been examined in patients with acute myeloid leukemia (AML) and non-Hodgkin lymphoma (NHL) before and after chemotherapy as well as in malignant effusions of heavily pretreated patients with solid tumors. Thrombin receptor expression (PAR-I) has been examined on HL-60 cells; the effect ofthrombin on the proliferation of the cells and inhibition of apoptosis induction by idarubicin has been shown.

RESULTS

Using fibrinopeptide A as an indirect parameter for thrombin activation, we found elevated levels in patients with AML and NHL before and a significant <em>2</em>-fold increase after chemotherapy (p < 0.0<em>2</em> for the AML group; p < 0.0006 for the NHL group). Apparently, this does not only affect patients with hematological diseases, but also with solid tumors. In order to find out if the tumor cells directly activate thrombin, we examined malignant effusions of patients with different solid tumors. Comparing <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> in ascites and pleural effusions with the patients' serum levels, we found it significantly increased in all cases (mean of <em>1</em>.96 +/- 0.5 nmol/l in the serum vs. <em>1</em><em>2</em>.<em>1</em> +/- 3.6 nmol/l in effusions; p < 0.00<em>1</em>). The majority of patients presented elevated serum levels. Additionally, we incubated HL-60 cells (human promyelocytic leukemia) with thrombin prior to treatment with idarubicin. Expression of thrombin receptor (PAR-<em>1</em>) could be verified by FACS-analysis using a monoclonal antibody. HL-60 cells responded with increased proliferation to thrombin exposure with concentrations between 0.3 and 3 U/ml. This effect could be abolished by the addition of hirudin, demonstrating thrombin specificity. In these concentrations, thrombin was able to abrogate the induction of apoptosis by idarubicin completely (p < 0.005).

CONCLUSIONS

Here we give evidence for the role of thrombin as a resistance factor for tumor cells towards chemotherapy. In the light of the fact that thrombin is regularly activated in cancer patients, these findings indicate that thrombin is a clinically relevant cellular resistance factor. A number of pre-clinical and clinical studies imply that inhibition of the coagulation system, e.g. by low-molecular weight heparins or warfarin, increases the effect of chemotherapy.

Human plasminogen kringle 4, which crystallizes in the orthorhombic system a = 3<em>2</em>.<em>1</em>5(<em>2</em>), b = 49.0<em>1</em>(<em>2</em>), c = 49.04(3) A, space group P<em>2</em>(<em>1</em>)<em>2</em>(<em>1</em>)<em>2</em>(<em>1</em>), four molecules per unit cell, protein volume fraction 0.6<em>2</em>, has been determined at <em>1</em>.9 A resolution. The structure was solved by rotation-translation methods using the structure of bovine <em>prothrombin</em> kringle <em>1</em> as a model and it has been refined at <em>1</em>.9 A resolution to an R-value of 0.<em>1</em>4<em>2</em>. The root mean square (rms) deviation between the main-chain atoms of the two kringles is about 0.5 A while that between 3<em>1</em> conserved side chains is a surprisingly large <em>1</em>.<em>2</em> A. The structure of the lysine binding subsite of fibrin binding of kringle 4 is approximated well by <em>prothrombin</em> kringle <em>1</em> but with some notable exceptions. The latter transform the site from a non-binding kringle to one which recognizes lysine and other omega-amino-carboxylic acids. The binding site of the observed kringle 4 structure is also compared with one that was modelled from the structure of kringle <em>1</em> of <em>prothrombin</em> <em>fragment</em> <em>1</em> and NMR observations. Arginine residues of the binding site of a neighbouring molecule make ion pairs with aspartic acid residues in the binding site of another molecule in the kringle 4 structure.

<em>Prothrombin</em> denaturation was examined in the presence of Na<em>2</em>EDTA, 5mM CaCl<em>2</em>, and CaCl<em>2</em> plus membranes containing <em>1</em>-palmitoyl-<em>2</em>-oleoyl-3-sn-phosphatidylcholine (POPC) in combination with either bovine brain phosphatidylserine (PS) or <em>1</em>,<em>2</em>-dioleoyl-phosphatidylglycerol (DOPG). Heating denaturation of <em>prothrombin</em> produced thermograms showing two peaks, a minor one at approximately 59 degrees C previously reported to correspond to denaturation of the <em>fragment</em> <em>1</em> region (Ploplis, V. A., D. K. Strickland, and F. J. Castellino <em>1</em>98<em>1</em>. Biochemistry. <em>2</em>0:<em>1</em>5-<em>2</em><em>1</em>), and a main one at approximately 57-58 degrees C, reportedly due to denaturation of the rest of the molecule (prethrombin <em>1</em>). The main peak was insensitive to the presence of 5mM Ca<em>2</em>+ whereas the minor peak was shifted to higher temperature (Tm approximately 65 degrees C) by Ca<em>2</em>+. Sufficient concentrations of POPC/bovPS (75/<em>2</em>5) large unilamellar vesicles to guarantee binding of 95% of <em>prothrombin</em> resulted in an enthalpy loss in the main endotherm and a comparable enthalpy gain in the minor endotherm accompanying an upward shift in peak temperature (Tm approximately 73 degrees C). Peak deconvolution analysis on the <em>prothrombin</em> denaturation profile and comparison with isolated <em>prothrombin</em> <em>fragment</em> <em>1</em> denaturation endotherms suggested that the change caused by POPC/PS vesicles reflected a shift of a portion of the enthalpy of the prethrombin <em>1</em> domain to higher temperature (Tm approximately 77 degrees C). The enthalpy associated with this high-temperature endotherm increased in proportion to the surface concentration of PS. By contrast, POPC/DOPG (50/50) membranes shifted the prethrombin <em>1</em> peak by 4 degrees C to a lower temperature and the <em>fragment</em> <em>1</em> peak by 5 degrees C to a higher temperature. The data lead to a hypothesis that the <em>fragment</em> <em>1</em> and prethrombin <em>1</em> domains of <em>prothrombin</em> do not denature quite independently and that binding of <em>prothrombin</em> to acidic-lipid membranes disrupts the interaction between these domains. It is further hypothesized that PS containing membranes exert the additional specific effect of decoupling the denaturation of two subdomains of the prethrombin <em>1</em> domain of <em>prothrombin</em>.

The activation of protein C by thrombin is thought to occur at the endothelial cell surface in the presence of an essential membrane glycoprotein cofactor, thrombomodulin. In the present study it is demonstrated that, in the presence of hirudin, the most potent known inhibitor of thrombin, human protein C can be activated by human factor Xa (<em>2</em>0 nM), but by a thrombomodulin-independent mechanism requiring only the presence of Ca<em>2</em>+ and phospholipid vesicles bearing a high proportion of negative charges (30-75% phosphatidylserine, depending on the conditions). At an optimal concentration of phosphatidylserine/phosphatidylcholine (<em>1</em>:<em>1</em>, w/w) of 75 microM, the apparent Km was <em>1</em> microM with a kcat. of <em>1</em> min-<em>1</em>. At <em>2</em>5 microM-phospholipid the Km was unchanged and the kcat. was 0.67 min-<em>1</em>. At either lipid concentration, increasing the density of negative charges by the adjunction of sulphated polysaccharides, like pentosan polysulphate or standard heparin at optimal concentrations of <em>2</em>-5 micrograms/ml and 5-<em>1</em>0 micrograms/ml respectively, resulted in a 4-fold increase of the kcat. without affecting the Km. Sulphated polysaccharides alone were poor promoters of protein C activation by factor Xa. In any case the presence of Ca<em>2</em>+ was essential, the dependence being sigmoidal with Hill coefficients ranging from <em>1</em>.4 to <em>2</em>.0. No significant activation of 4-carboxyglutamic acid-domainless protein C, a chymotrypic derivative lacking the phospholipid-binding domain, could be detected in the presence of phospholipids and Ca<em>2</em>+, with or without pentosan polysulphate. In a large molar excess, other phospholipid-binding entities like <em>prothrombin</em> <em>fragments</em> F<em>1</em> or F<em>1</em>+<em>2</em> could inhibit protein C activation by factor Xa, but pentosan polysulphate exerted a clear protective effect. Factor Xa irreversibly inhibited at its active centre, but not di-isopropyl phosphoro-thrombin, behaved as an inhibitor but in a more complex manner than simple Michaelis-Menten kinetics. Among several derivatives of pentosan polysulphate or of heparin which were tested, those having the higher degree of sulphation and/or molecular mass were the most efficient in enhancing the rate of activation of protein C by factor Xa in the presence of phospholipids. These results suggest that human factor Xa, at physiological concentrations, could activate human protein C in the presence of anionic phospholipids and that this activation could be potentiated by therapeutic concentrations of sulphated polysaccharides.

Men have an increased cardiovascular risk as compared to women, which is largely ascribed to the cardioprotective effects of female sex steroids. We hypothesised that this may be reflected by differences in the activation status of the coagulation system. Hence the aim of this study was to compare plasma levels of activated factor VII (FVIIa) in men and women, and to study the influence of the menstrual cycle on FVIIa levels. In a prospective study we investigated <em>2</em>0 healthy young women and <em>2</em>0 men. Men had significantly higher levels of activated factor VII (60 mU/ml, CI: 5<em>2</em> to 67) than women during all phases of the menstrual cycle. In women FVIIa was higher during the follicular phase (4<em>1</em> mU/ml, CI: 33 to 50) than during midcycle (34 mU/ml, CI: <em>2</em>4 to 45; p = 0.0<em>2</em><em>2</em> vs. follicular phase) and during the luteal phase (33 mU/ml, CI: <em>2</em>4 to 4<em>2</em>; p = 0.006 vs. follicular phase). <em>Prothrombin</em> <em>fragment</em> (F<em>1</em> + <em>2</em>) levels decreased from 0.86 nmol/l (CI: 0.5<em>1</em>-<em>1</em>.<em>2</em><em>1</em>) by -<em>2</em>3% (-39% to -8%; p = 0.0<em>1</em><em>1</em>) during midcycle and by -<em>2</em>5% (CI: -5<em>1</em>% to <em>1</em>%; p = 0.0<em>2</em>3) during the luteal phase. These data support the contention that plasma levels of FVIIa, a key enzyme of the coagulation cascade, may be down-regulated by endogenously produced female sex hormones during the menstrual cycle. This may at least partially explain the marked gender differences found in FVIIa.

Blood coagulation (fibrinogen, thrombin-antithrombin III complexes, TAT, and <em>prothrombin</em> <em>fragment</em> F<em>1</em> + <em>2</em>) and fibrinolytic parameters [fibrin split-product D-dimer, tissue plasminogen activator (t-PA) activity, plasminogen activator inhibitor-<em>1</em> activity (PAI-<em>1</em>), and plasmin-antiplasmin-complexes (PAP)] were evaluated in <em>1</em>6 women on low estrogen (EE) oral contraceptive (OC) therapy. Blood samples were taken before and between days <em>1</em>8 and <em>2</em><em>2</em> of the first, third, and sixth treatment cycle. Fibrinogen levels were found significantly elevated during OC treatment compared with pretreatment values, while TAT and also F<em>1</em> + <em>2</em> levels remained unchanged. Treatment-induced activation of fibrinolysis was documented by elevated D-dimer [pretreatment (pt): <em>1</em>7<em>2</em> ng/ml (range: 65-640 ng/ml), cycle 6 (c.6): 35<em>1</em> ng/ml (range: 93-960 ng/ml), p < 0.05)] and PAP [(pt: 46.6 ng/ml (<em>1</em>3-<em>2</em><em>2</em>0 ng/ml), c.6: 66.4 ng/ml (<em>2</em><em>1</em>-<em>2</em>00 ng/ml), p < 0.05] plasma levels. Among the fibrinolytic components a decrease in PAI-<em>1</em> [pt: <em>1</em>0.8 ng/ml (<em>2</em>-56 ng/ml), c.6: 5.3 ng/ml (<em>2</em>.<em>2</em>-<em>1</em>4.4 ng/ml), p < 0.05] and an increase in t-PA activity [pt: 0.<em>2</em>3 U/ml (0.<em>1</em>7-0.45 U/ml), c.6: 0.33 U/ml (0.<em>2</em>-0.9 U/ml), p < 0.05] were detected. Experiments with cultured human endothelial cells (EC) showed that EE influenced neither EC hemostatic regulatory activities (tissue factor, thrombomodulin) nor the secretion of the fibrinolytic components t-PA and PAI-<em>1</em>.

Contrary to low-fat meals, high-fat meals are known to cause postprandial factor VII (FVII) activation, but the mechanism is unknown. To study the postprandial FVII activation in detail, <em>1</em>8 young men consumed in randomized order high-fat or low-fat test meals. Fasting and non-fasting blood samples were collected. The high-fat test was associated with an increase in plasma triglyceride and kallikrein concentrations and postprandial FVII activation (p<0.00<em>1</em>). Plasma kallikrein was strongly associated with triglycerides in fasting and non-fasting samples (r<em>2</em>=0.74-0.87, p<0.000<em>1</em>), suggesting that triglyceride-rich lipoproteins may activate prokallikrein. Neither plasma triglycerides nor kallikrein and activated FVII were statistically associated. This may suggest that additional factors are involved in the postprandial FVII activation. No clear evidence for a role of tissue factor expression by monocytes, factor XII or insulin in postprandial FVII activation was observed. Tissue factor pathway inhibitor and <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, a marker of thrombin generation, were not affected postprandially after either the high-fat or the low-fat meals. Our findings indicate that triglyceride-rich lipoproteins activate prokallikrein postprandially, which might form an important initial event in FVII activation after consumption of high-fat meals.

Recombinant human <em>prothrombin</em> (r-<em>prothrombin</em>) and recombinant mutant <em>prothrombin</em> with active site Asp4<em>1</em>9 substituted by Asn (D4<em>1</em>9N-<em>prothrombin</em>) were expressed in recombinant CHO cells, isolated and purified from the fermentation supernatant. The r-<em>Prothrombin</em> and D4<em>1</em>9N-<em>prothrombin</em> were digested by both Echis carinatus venom and Oxyuranus scutellatus venom. Prior to, during and after activation, generation of thrombin activity and the proteolytic degradation of the <em>prothrombin</em> polypeptide chain were analysed. Owing to the recombinant preparation and inactivity of D4<em>1</em>9N-<em>prothrombin</em> and its activation products, the proteolytic action of E.carinatus and O.scutellatus venoms could be studied without addition of thrombin inhibitor, without interference from autocatalytic digestion of <em>prothrombin</em> and in the absence of any other blood coagulation protease. The comparison between the activation of r-<em>prothrombin</em> and D4<em>1</em>9N-<em>prothrombin</em> by snake venoms permitted differentiation between proteolytic activation and autocatalytic conversion of <em>prothrombin</em>. Incubation of D4<em>1</em>9N-<em>prothrombin</em> with E.carinatus venom resulted in the generation of stable D4<em>1</em>9N-meizothrombin by hydrolysis of the peptide bond Arg3<em>2</em>0-Ile3<em>2</em><em>1</em>. By contrast, O.scutellatus venom exhibited activity towards peptide bonds Arg3<em>2</em>0-Ile3<em>2</em><em>1</em> and Arg<em>2</em>7<em>1</em>-Thr<em>2</em>7<em>2</em> and lower activity towards peptide bond Arg<em>1</em>55-Ser<em>1</em>56, thus converting D4<em>1</em>9-<em>prothrombin</em> into D4<em>1</em>9N-thrombin and also liberating <em>Fragment</em>-<em>1</em>, <em>Fragment</em>-<em>2</em> and <em>Fragment</em>-<em>1</em>/<em>2</em> activation peptide. Activation of r-<em>prothrombin</em> by E.carinatus and O.scutellatus venoms demonstrated the autocatalytic potential of <em>prothrombin</em>-derived molecules and indicated that meizothrombin hydrolysed the cleavage between <em>Fragment</em>-<em>2</em> and thrombin A-chain in the meizothrombin molecule, but not in <em>prothrombin</em>, preferentially at position Arg<em>2</em>84-Thr<em>2</em>85. By contrast, both meizothrombin and thrombin exhibited no detectable activity towards peptide bond Arg3<em>2</em>0-Ile3<em>2</em><em>1</em> between thrombin A- and B-chain, although this site exhibits the optimum sequence for thrombin cleavage.

Oral combined hormone replacement therapy (HRT) with oestradiol and norethisterone increases plasma levels of <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>), indicating an increase in thrombin generation, but the mechanisms underlying this increase are uncertain. The aim of this randomized, placebo-controlled study was to determine whether an increase in factor VII, a factor that combines with tissue factor to activate the extrinsic pathway, or a decrease in tissue factor pathway inhibitor (TFPI), an inhibitor of extrinsic pathway activation, may contribute to increases in thrombin generation occurring with HRT. Healthy postmenopausal women aged 50-75 years received placebo (n=<em>1</em>9) or oral combined HRT (n=<em>1</em>8) and had blood collected for measurement of factor VII coagulation activity (VIIc), activated factor VII (VIIa) and TFPI at baseline and at 6 weeks. Baseline characteristics were similar in the two groups, including age, body mass index and cholesterol levels. As reported previously, HRT increased the F<em>1</em>+<em>2</em> concentration by <em>2</em>0%. Placebo had no effect on VIIc, VIIa or TFPI, but 6 weeks of combined HRT decreased VIIc [from <em>1</em>.<em>1</em><em>1</em>+/-0.06 (mean+/-S.E.M.) to <em>1</em>.03+/-0.06 i.u./ml; P<0.03], VIIa [from 43.9; <em>1</em>0.8-<em>1</em>98.3 (median; range) to 35.0; 6.3-66.8 m-units/ml; P<0.03] and TFPI [from 8<em>1</em>.3+/-6.5 to 60.4+/-5.5 ng/ml; P<0.000<em>1</em>]. The decrease in TPFI with HRT was not correlated with the elevation in F<em>1</em>+<em>2</em> levels. In conclusion, the increase in thrombin generation seen with HRT is not due to an effect on factor VII; in addition, while a contribution from the decrease in TFPI is possible, increased thrombin generation is not directly related to the decrease in TFPI.

BACKGROUND

Both systemic inflammatory reaction and regional myocardial ischemia/reperfusion injury may elicit hypercoagulability after off-pump coronary artery bypass grafting (OPCAB). We investigated the influence of ulinastatin, which suppresses the activity of polymorphonuclear leukocyte elastase and production of pro-inflammatory cytokines, on coagulation in patients with elevated high-sensitivity C-reactive protein (hsCRP) undergoing OPCAB.

METHODS

Fifty patients whose preoperative hsCRP>> 3.0 mg/L were randomly allocated into the ulinastatin (600,000 U) or control group. Serum concentrations of thrombin-antithrombin complex (TAT) and <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>) were measured preoperatively, immediately after surgery, and at <em>2</em>4 h after surgery, respectively. Secondary endpoints included platelet factor (PF)-4, amount of blood loss, and transfusion requirement.

RESULTS

All baseline values of TAT, F<em>1</em>+<em>2</em>, and PF-4 were higher than the normal range in both groups. F<em>1</em>+<em>2</em> was elevated in both groups at immediate, and at <em>2</em>4 h after surgery as compared to baseline value, without any significant intergroup differences. Remaining coagulation parameters, transfusion requirement and blood loss during operation and postoperative <em>2</em>4 h were not different between the two groups.

CONCLUSIONS

Intraoperative administration of ulinastatin did not convey beneficial influence in terms of coagulation and blood loss in high-risk patients with elevated hsCRP undergoing multivessel OPCAB, who already exhibited hypercoagulability before surgery.

Urinary form of <em>prothrombin</em> (PT) <em>fragment</em> <em>1</em> is the most abundant protein in calcium oxalate crystals generated in human urine. The protein has also been detected in human calcium-containing stones. In its purified form, the protein inhibits calcium oxalate crystal growth and, more importantly, aggregation in aqueous inorganic solutions and undiluted human urine. Recently, PT gene expression has been reported in human kidneys. However, access to human renal tissue for studies is limited, and it is not possible to easily manipulate PT biosynthesis in human subjects. The aim of this investigation, therefore, was to determine whether PT gene expression is present in rat kidneys. Samples of total RNA were isolated from the kidneys and livers (positive controls) of <em>1</em><em>2</em> male hooded Wistar rats. Using reverse transcription-PCR, mRNA corresponding to the thrombin and F<em>1</em>+<em>2</em> regions of PT was analyzed by agarose gel electrophoresis. The expression of the "housekeeping" gene glyceraldehyde-3-phosphate dehydrogenase was also examined, to determine the availability of amplifiable substrate in each specimen. The amplified products were also sequenced, to determine their identities. All rat kidneys displayed evidence of expression of the thrombin and F<em>1</em>+<em>2</em> domains of the PT gene. This similarity between human and rat kidneys allows the possibility of using established rat models of stone disease to evaluate therapeutic strategies to reduce stone formation.

Two dangerous obstetric complications, amniotic fluid embolism and preterm prelabor rupture of membranes (PROM), can be caused by amniotic fluid (AF) constituents. Disseminated intravascular coagulation (DIC) is related to the former complication, whereas local thrombin/plasmin-dependent collagenolysis in the decidua and fetal membranes is associated with the latter. In AF, most proteins of the coagulation and fibrinolysis system, known as plasma constituents, have been identified based on the activity and/or presence of antigen. The AF levels of most of these proteins are low (< <em>2</em> to 5% of the respective maternal plasma levels). However, there are some exceptions: tissue factor (TF), urokinase plasminogen activator (uPA) and its receptor (uPAR), as well as plasminogen activator inhibitors. The AF level of fetal fibrinogen is trace, which is a particular exception. The key enzymes of coagulation and fibrinolysis, thrombin and plasmin, are generated in AF. Thrombin generation is four- to fivefold higher than in maternal plasma as measured by the concentration of the <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em> (F <em>1</em> + <em>2</em>) and thrombin-antithrombin complexes, whereas plasmin generation is relatively low as measured by the plasmin-α-<em>2</em>-antiplasmin complexes. Phosphatidylserine, a phospholipid, and thrombin-activatable fibrinolysis inhibitor (TAFI) are novel components of AF. Phosphatidylserine contributes to DIC in AF embolism; TAFI is considered a link between coagulation and fibrinolysis. uPA and uPAR are the factors contributing to PROM via plasmin-dependent proteolysis. Intriguing is the assumption that TF and its inhibitor can be risk factors for PROM through thrombin-dependent activation of matrix prometalloproteinases in the decidua and fetal membranes. It is unknown whether the amniotic pool of hemostatic components is involved in pre-eclampsia pathogenesis.

Plasma was subjected to methylene blue (MB) photochemical virus inactivation using the Maco Pharma Maco-Tronic system which allows three units to be illuminated together, thus reducing processing time. The plasma bag system used incorporates an integral membrane plasma filter and a dry MB pill which dissolves in the plasma to give a <em>1</em>-microM concentration. There is computer-controlled processing and datalogging. In an assessment of <em>1</em>0 pools of Group A plasma, the losses of coagulation factors, following MB/light treatment, were <em>2</em>3% fibrinogen, <em>1</em>0% FV, <em>2</em>6% FVIII, <em>1</em><em>1</em>% FIX and <em>1</em>3% FXI. Group O, Group B and Group AB plasmas were not tested. Von Willebrand factor (vWf) multimers showed no substantial change when treated with MB, and no losses were seen for antithrombin III (ATIII), protein C and vWf:Ag. Measurements of C3a, C5a, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> and FXIIa indicated that there was no activation as a result of filtration.

BACKGROUND

Non-dipper hypertensive patients are at increased risk for cardiovascular disease. Coagulation and fibrinolysis activation factors are considered as risk factors for cardiovascular disease. The aim of this study was to examine the relationship between the haemostatic and platelet activation markers and the non-dipping pattern in treated hypertensive patients.

METHODS

Seventy-one treated hypertensive patients (53 with essential and <em>1</em>8 with secondary hypertension, due to chronic kidney disease-stage 4), aged 33 to 8<em>1</em> years (30 men), were classified as dippers and non-dippers, according to the presence or absence, respectively, of a decline of nocturnal average systolic blood pressure (BP) by more than <em>1</em>0% of the diurnal BP (non-dipping pattern) on <em>2</em>4-hour ambulatory BP monitoring. Plasma levels of factors VIII and IX, fibrinogen, <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>, thrombin-antithrombin complex, protein C, plasmin-alpha-<em>2</em> antiplasmin complex, D-dimer and platelet factor 4 were measured in all patients.

RESULTS

Thirty-seven patients were classified as dippers and 34 as non-dippers. The percentages of patients with essential and with secondary hypertension were similar in the dippers and in the non-dippers groups (both P = 0.754). Multivariate analysis of variance showed statistically significant differences in all measured variables between dippers and non-dippers (P = 0.043). Plasma levels of factors VIII and IX, fibrinogen, <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>, protein C, plasmin-alpha-<em>2</em>-antiplasmin complex, and D-dimers were significantly higher in non-dippers when compared to dippers (P < 0.05 for all). In contrast, there were no significant differences in plasma levels of thrombin-antithrombin complex (P = 0.955) and platelet factor 4 (P = 0.43<em>1</em>) between the two groups.

CONCLUSIONS

This study provides evidence that non-dipper treated hypertensive patients exhibit alterations in haemostasis, which may affect their cardiovascular risk.

Molecular makers such as thrombin-antithrombin complex (TAT), <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>), soluble fibrin (SF), and D-dimer, are useful markers in the diagnosis and assessment of various thrombotic conditions. These markers are measured in plasma after blood sampling. Difficult blood sampling is known to falsely elevate plasma TAT levels. However, it is not known exactly why this occurs. In the present study, we examined how levels of molecular markers of haemostatic and fibrinolytic activation change under various sampling conditions using vacuum tube samples from healthy volunteers. When blood was sampled continuously by taking <em>1</em>0 consecutive vacuum tube samples following application of a tourniquet, blood sampling resulted in an accurate assessment of these molecular makers. When blood was sampled continuously by taking vacuum tube samples every one minute over a total of 9 minutes to investigate possible changes in the levels of the molecular markers over time, plasma levels of TAT, SF, and F<em>1</em>+<em>2</em> gradually increased with time. Plasma levels of TAT, F<em>1</em>+<em>2</em>, and SF increased beyond the normal range over the course of nine minutes. When blood was sampled using three alternative methods, which varied in terms of the duration of needle puncture (sampling B), duration of tourniquet use (sampling C), or both (sampling A), plasma TAT and SF levels were significantly increased with all three methods, compared to control samples. Plasma F<em>1</em>+<em>2</em> levels were significantly increased with sampling methods A and B, compared to control samples, but not with sampling method C. On the other hand, plasma D-dimer levels were not significantly altered by any of the sampling methods. In conclusion, the results suggest that molecular markers of haemostatic and fibrinolytic activation, except for D-dimer, may be affected by sampling method, particularly the duration of needle puncturing. Therefore, care needs to be taken when using TAT, F<em>1</em>+<em>2</em>, and SF levels to diagnose and estimate activation of the coagulation system.

BACKGROUND

The aim of this study was to investigate the effect of gamma irradiation with 30 Gy on the coagulation system in leukoreduced fresh-frozen plasma (FFP).

METHODS

In 74 FFP units that had been stored for 35<em>2</em> +/- <em>1</em>03 days below -30 degrees C, the following variables were determined in parallel in an irradiated and not irradiated half: <em>prothrombin</em> time (PT); activated partial thromboplastin time (APTT); thrombin time; antithrombin III; protein C; protein S; von Willebrand factor antigen; ristocetin cofactor; plasminogen-alpha(<em>2</em>)-antiplasmin; the coagulation factors fibrinogen, factor (F)II, FV, FVII, VIII, F IX, FX, FXI, FXII, FXIII, and activated factor XII (FXIIa); D-dimer; fibrin monomer; thrombin-antithrombin complex; <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em>+<em>2</em>); plasmin-alpha(<em>2</em>)-antiplasmin complexes (PAPs); and platelet factor 4. The FVII activity ratio was assayed to quantify activation of FVII.

RESULTS

Irradiation with 30 Gy resulted in a reduction of APTT (35.0 +/- 4.<em>1</em> sec vs. 34.4 +/- 4.<em>1</em> sec; p = 0.00000006) and PT (89.8 +/- 8.<em>2</em>% vs. 90.7 +/- 8.0%; p = 0.00<em>2</em>) and a significant increase of the activities of the coagulation factors FII, FV, FVII, F IX, FX, and FXII. FVIII activity decreased from <em>1</em><em>1</em>8 +/- 3<em>1</em> to <em>1</em><em>1</em>6 +/- 3<em>2</em> percent (p = 0.0<em>2</em>). Activation of the coagulation system was shown by an increase in the FVII activity ratio (<em>1</em>.<em>1</em>9 +/- 0.<em>2</em>9 vs. <em>1</em>.3<em>1</em> +/- 0.34; p = 0.000000<em>1</em>), FXIIa (0.8<em>1</em> +/- 0.50 ng/mL vs. 0.90 +/- 0.5<em>1</em> ng/mL; p = 0.006), and F<em>1</em>+<em>2</em> (<em>1</em>.<em>1</em>9 +/- 0.<em>2</em>0 nmol/L vs. <em>1</em>.<em>2</em>4 +/- 0.<em>2</em>0 nmol/L; p = 0.000005) after irradiation with 30 Gy, whereas an increase of PAP (<em>1</em>6.<em>2</em> +/- <em>1</em><em>1</em>.5 ng/mL vs. <em>2</em>0.<em>2</em> +/- <em>1</em><em>2</em>.0 ng/mL; p = 0.0004) demonstrated activation of the fibrinolytic system. No negative influence of irradiation with 30 Gy on inhibitors of coagulation was observed.

CONCLUSIONS

Gamma irradiation of leukoreduced FFPs with 30 Gy results in a significant but very weak activation of the coagulation and fibrinolytic system in FFPs.