BACKGROUND

Tissue injury after trauma and surgery may induce alterations in blood coagulation and fibrinolysis, and the hypercoagulable state observed after surgery can be associated with the risk of postoperative thromboembolic complications. Recently, videolaparoscopic (VLPS) cholecystectomy has been introduced, and its advantages over the open procedure seem related to the reduced surgical trauma. The aim of this study was to investigate hemostatic system alterations in patients who undergo open and VLPS cholecystectomy.

METHODS

Fibrinogen, <em>prothrombin</em> <em>fragment</em> F<em>1</em>+<em>2</em>, D-dimer, and plasminogen activator inhibitor type-<em>1</em> (PAI-<em>1</em>) activity was determined in <em>1</em>0 patients who underwent open (group A) and <em>1</em>0 patients who underwent VLPS cholecystectomy (group B), respectively. Blood samples were obtained the day of surgery in the morning (B<em>1</em>), after anesthesia (A<em>1</em>), <em>1</em> hour after the start of surgery (S<em>1</em>), then 30 min (E.05) and <em>2</em>4 h (E.<em>2</em>4) after the surgery.

RESULTS

No significant differences were observed in baseline values between groups A and B for the parameters investigated. At <em>2</em>4 h after surgery, fibrinogen increased significantly (p < 0.05) in group A and also was significantly higher than in group B (p < 0.05). In group A, a marked increase in F<em>1</em>+<em>2</em> levels (p < 0.0<em>1</em>) was observed in all the samples, with the maximum values on the first day after surgery (3.7 nmol/l; <em>1</em>.<em>2</em>-6.0 nmol/l), whereas in group B, a slight but significant increase in F<em>1</em>+<em>2</em> levels (<em>2</em>.<em>1</em> nmol/l; <em>1</em>.<em>1</em>-3.9 nmol/l; p < 0.0<em>1</em>) was observed only 30 min after the end of surgery. In both groups A and B, D-dimer markedly increased after surgery, without statistical significant differences between the two groups at any time. The PAI-<em>1</em> activity plasma levels remained in the normal range during and after surgery in both groups.

CONCLUSIONS

These results indicate that VLPS surgery induces an activation of the clotting system that, although of low degree and short duration, can lead to a transient prothrombotic state.

Ximelagatran, an oral direct thrombin inhibitor, whose active form is melagatran, was studied using a model of thrombin generation in humans. Healthy male volunteers (<em>1</em>8 per group) received ximelagatran (60 mg p.o.), dalteparin (<em>1</em><em>2</em>0 IU/kg s.c.) or a control (water p.o.). Shed blood, collected after incision of the forearm with standardised bleeding time devices at pre-dose, and at <em>2</em>, 4 and <em>1</em>0 h post-dosing, was analysed for markers of thrombin generation. Statistically significant reductions (p < 0.05) in levels of <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>) and thrombin-antithrombin complex (TAT) in shed blood were detected at <em>2</em> and 4 h post-dosing in both the ximelagatran and dalteparin groups. Shed blood F<em>1</em>+<em>2</em> and TAT levels had returned to pre-dose levels at <em>1</em>0 h post-dosing. Using a shed blood model, we demonstrate that the reversible thrombin inhibitor melagatran and, therefore, oral administration of ximelagatran, inhibits thrombin generation in humans after acute activation of coagulation.

Diarrhoea-associated haemolytic uraemic syndrome (D+ HUS) is usually caused by verotoxin producing Eschericia coli. We hypothesized that verotoxin binding to glomerular endothelial cells causes localised endothelial cell activation and thus activation of coagulation and reduction of fibrinolytic potential. We also proposed that treatment with fresh frozen plasma or dialysis would not affect these changes. Markers of activation of coagulation and fibrinolysis were measured in 30 children with acute D+ HUS serially, in healthy children and in children on dialysis. In acute D+ HUS, levels of thrombin-antithrombin III complex and <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> were significantly increased (p <0.00<em>1</em>). The source of thrombin generation was unclear. Factor XIIa levels were increased in patients and controls with renal failure. Factor VIIa levels were not significantly raised in children with acute D+ HUS. D-dimers were increased, but fibrinolytic potential as measured by fibrin plate was reduced. Levels of plasminogen activator inhibitor antigen and activity and tissue plasminogen activator antigen were increased. Neither peritoneal dialysis nor administration of blood products, the most common treatments, altered parameters of coagulation or fibrinolysis.

To examine whether exercise-induced thrombin formation is accompanied by increased in vivo plasmin formation, we measured molecular markers and neoantigens of the hemostatic system in <em>1</em>0 male subjects (mean <em>2</em>9 yr. range <em>1</em>9-38) before, immediately after, and <em>2</em>, 8, and <em>2</em><em>1</em> h after a triathlon lasting <em>1</em><em>2</em>8-<em>1</em>63 min. Thrombin-antithrombin (TAT) complexes, fibrinopeptide A (FPA), and tissue plasminogen activator (t-PA) antigen were maximally increased immediately after exercise and decreased thereafter rapidly. <em>Prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (PTF<em>1</em> + <em>2</em>), fibrin degradation products (FbDP) and plasmin-antiplasmin (PAP) complexes rose to a similar extent 0 and <em>2</em> h after exercise decreased thereafter. The maximal levels of PTF<em>1</em> + <em>2</em>, TAT, FPA, and FbDP were <em>1</em>.5-, <em>2</em>.<em>1</em>-, <em>1</em>.8-, and <em>1</em>.9-fold above baseline, respectively. This investigation shows that strenuous prolonged exercise leads to a moderate activation of blood coagulation resulting in thrombin and fibrin formation which is accompanied by a greatly enhanced plasmin generation. It is concluded that the hemostatic of healthy individuals is well kept in balance when stimulated by prolonged strenuous exercise.

The endothelial protein C receptor (EPCR) facilitates protein C activation and plays a protective role in the response to Escherichia coli-mediated sepsis in primates. Previously, a soluble form of EPCR (sEPCR) in human plasma was characterized, and several studies indicated that generation of sEPCR is regulated by inflammatory mediators, including thrombin-mediated up-regulation of surface metalloproteolytic activity in vitro. This study addressed the question of whether plasma sEPCR levels reflect changes in thrombin generation in patients undergoing anticoagulant treatment. The sEPCR levels in patients treated with coumarin-type oral anticoagulants were significantly lower than those in healthy asymptomatic adult volunteers (<em>1</em>05.3 +/- 70.8 ng/mL [n = 55] versus <em>1</em>65.8 +/- <em>1</em><em>1</em>5.8 ng/mL [n = <em>2</em>00]; P <.000<em>1</em>). A similar decline in plasma sEPCR levels was found in patients treated with unfractionated heparin. In healthy volunteers, sEPCR levels declined to about <em>1</em>00 ng/mL within 3 days after initiation of an 8-day period of warfarin administration and increased within <em>2</em> days after its cessation. Plasma sEPCR levels returned to pretreatment values within <em>1</em> week, and the changes in plasma sEPCR levels mirrored changes in values for international normalized ratios. A similar decline in sEPCR levels with time was observed in 7 patients beginning treatment with warfarin for a thrombotic disorder. <em>Prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> levels also decreased in volunteers and patients given warfarin. These results show that plasma sEPCR levels decline in response to treatment with anticoagulants whose mechanism of action is known to decrease in vivo thrombin production.

BACKGROUND

Tamoxifen citrate is being evaluated for primary prevention of breast cancer, but this drug with estrogen-like properties may cause changes in the hemostatic system that would increase the risk of thrombosis.

METHODS

Women who had undergone hysterectomy were consecutively enrolled in the placebo-controlled, randomized, double-blind Breast Carcinoma Chemoprevention Tamoxifen Study, which was designed to evaluate the efficacy of oral tamoxifen citrate (<em>2</em>0 mg/d). Our substudy of hemostasis and lipid measurements included the first 68 consecutive women assigned to tamoxifen (n = 3<em>1</em>) or placebo (n = 37). Blood specimens were obtained before treatment and after <em>1</em>,<em>2</em>,4, and 6 months of treatment. Measurements included blood cell counts, lipid levels, coagulation activation markers, clotting factors, and anticoagulant and fibrinolysis proteins.

RESULTS

Hematocrit and hemoglobin and platelet levels fell slightly but significantly in women treated with tamoxifen. No between-treatment differences were observed in any of the clotting factors. Naturally occurring anticoagulant proteins such as antithrombin and protein C fell slightly in women treated with tamoxifen. However, no significant changes were observed in any of the markers of activated coagulation or fibrinolysis (fibrinopeptide A, <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>, thrombin-antithrombin complex, D-dimer). Total and low-density lipoprotein cholesterol levels fell significantly in women treated with tamoxifen.

CONCLUSIONS

Tamoxifen induced a modest decrease in anticoagulant proteins, but without biochemical signs of activation of coagulation and fibrinolysis. Tamoxifen improved the lipid profile and induced changes in blood cell counts, which should determine an improvement in blood rheologic factors. These preliminary findings seem to justify continuation of the double-blind study in healthy women, but only direct comparison of thromboembolic complications in the <em>2</em> treatment groups will establish whether tamoxifen carries a risk of thrombosis.

OBJECTIVE

Patients with myeloproliferative disease (MPD) have an increased risk of thrombosis. We studied markers of platelet and coagulation activation in a large cohort of patients with MPD (n = <em>1</em><em>1</em>8) and related this to Janus Kinase <em>2</em> (JAK<em>2</em>) V6<em>1</em>7 F mutation status, a marker of clonality, and the presence of antiphospholipid antibodies (APA), all of which have been associated with thrombosis in MPD.

METHODS

D-dimer, thrombin-antithrombin complexes (TAT), <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em> (F(<em>1</em>+<em>2</em>)), soluble E-selectin (sE-selectin), and soluble P-selectin (sP-selectin) levels were compared between patients and hypertensive controls (n = <em>1</em><em>2</em>7). Assays for lupus anticoagulant (LA), anticardiolipin antibodies (ACA), antibeta<em>2</em> glycoprotein <em>1</em> antibodies (anti-beta(<em>2</em>)GP<em>1</em>), and anti<em>prothrombin</em> antibodies (alpha-Pro) were also performed. The JAK<em>2</em> V6<em>1</em>7F mutation status was determined in the cohort using amplification refractory mutation system (ARMS) polymerase chain reaction. Disease clonality was determined in 54 patients using the HUMARA assay.

RESULTS

sP-selectin was significantly increased in patients with MPD (P <or= 0.00<em>1</em>). sP-selectin levels were significantly elevated in JAK<em>2</em> V6<em>1</em>7F-positive patients compared to wild-type (P = 0.006), or controls (P < 0.00<em>1</em>). There was no correlation between proportion clonality and any activation marker. We found no significant difference in the incidence of APA between patients and controls (<em>1</em><em>1</em>% vs. <em>1</em>4%, P = 0.46), and no significant association between APA status and any activation marker.

CONCLUSIONS

The JAK<em>2</em> V6<em>1</em>7F mutation is associated with platelet activation, as measured by elevated sP-selectin levels, in MPD. In contrast to previous reports, we found no excess of APA in patients with MPD.

BACKGROUND

Melioidosis, which is caused by infection with the Gram-negative bacterium Burkholderia pseudomallei, is an important cause of sepsis in South-East Asia with a mortality of up to 40%. Knowledge of the involvement of coagulation and fibrinolysis in the pathogenesis of melioidosis is highly limited.

OBJECTIVE

To define the involvement of the coagulation and fibrinolytic systems in patients with severe melioidosis.

METHODS

Parameters of coagulation and fibrinolysis were measured in 34 patients with culture proven septic melioidosis and 3<em>2</em> healthy controls.

RESULTS

Patients demonstrated strong activation of the coagulation system, as reflected by high plasma levels of soluble tissue factor, the <em>prothrombin</em> <em>fragment</em> F(<em>1</em>+<em>2</em>) and thrombin-antithrombin complexes (TATc), and consumption of coagulation factors resulting in a prolonged <em>prothrombin</em> time and activated partial thromboplastin time. Concurrently, anticoagulant pathways were downregulated in patients: protein C, protein S, and antithrombin levels were all decreased when compared to controls. Patients also demonstrated evidence of activation and inhibition of fibrinolysis, as reflected by elevated concentrations of tissue-type plasminogen activator (tPA), plasminogen activator inhibitor type <em>1</em>, plasmin-alpha<em>2</em>-antiplasmin complexes (PAPc) and D-dimer. High TATc/PAPc ratios in patients pointed to a predominance of the prothrombotic pathway in melioidosis. Furthermore, soluble thrombomodulin levels were increased. The extent of coagulation activation correlated with mortality; patients who went on to die had higher TATc, F(<em>1</em>+<em>2</em>), tPA and PAPc and lower protein C and antithrombin levels on admission than patients who survived.

CONCLUSIONS

The coagulation system is strongly activated during melioidosis. A high degree of activation of the coagulation system is an indicator of poor outcome in patients with melioidosis.

OBJECTIVE

The purpose of this study was to determine the mechanistic basis for thrombin generation and increased prothrombotic potential after the abrupt cessation of intravenous (i.v.) unfractionated heparin among patients with acute coronary syndromes.

BACKGROUND

A "rebound" increase in prothrombotic potential has been observed biochemically and clinically after the abrupt cessation of unfractionated heparin (UFH) among patients with acute coronary syndromes. Although the mechanism is unknown, tissue factor and the extrinsic coagulation cascade, both operative in atherosclerotic vascular disease and arterial thrombosis, are thought to be centrally involved.

METHODS

In a single-center, pilot study, 30 patients with either unstable angina or non-ST segment elevation myocardial infarction who had received a continuous i.v. infusion of UFH for 48 h were randomly assigned to: <em>1</em>) abrupt cessation, <em>2</em>) i.v. weaning over <em>1</em><em>2</em> h or 3) subcutaneous weaning over <em>1</em><em>2</em> h.

RESULTS

Thrombin generation (prothrombin fragment <em>1</em>.<em>2</em>) was evident within <em>1</em> h of UFH cessation, increased progressively (by nearly two-fold) at <em>2</em>4 h (p = 0.00<em>2</em>) and correlated inversely with tissue factor pathway inhibitor concentration (r = -0.6<em>1</em>). Thrombin generation was greatest among patients randomized to abrupt cessation (<em>1</em>.6-fold increase at <em>2</em>4 h) and least in those with i.v. weaning.

CONCLUSIONS

Thrombin generation after the abrupt cessation of UFH may represent a drug-induced impairment of physiologic vascular thromboresistance in response to locally generated tissue factor. A dosing strategy of abbreviated i.v. weaning attenuates but does not prevent heparin rebound among patients with acute coronary syndromes.

OBJECTIVE

This prospective study investigated the behavior of thrombin generation and activity during thrombolysis and concomitant heparin administration.

BACKGROUND

It has been shown that during thrombolytic therapy there is an increase in thrombin generation and activity. Increased thrombin activity is suppressed by concomitant intravenous heparin, but it is unknown whether thrombin generation is also affected.

METHODS

Thrombin generation was assessed by measuring <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> and thrombin-antithrombin complex plasma levels and thrombin activity by measuring fibrinopeptide A plasma levels. Serial blood samples were obtained before and at 90 min and <em>2</em>4 and 48 h after the administration of streptokinase (<em>1</em>5 patients), recombinant tissue-type plasminogen activator (<em>1</em>5 patients) or anistreplase (<em>1</em>3 patients). An intravenous bolus of heparin (5,000 IU) was administered before the start of thrombolytic therapy, followed by an infusion of <em>1</em>,000 U/h to maintain an activated partial thromboplastin time>> <em>1</em>.5 times baseline.

RESULTS

During thrombolytic and concomitant heparin therapy, there was an increase in the plasma levels of <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (baseline <em>1</em>.08 vs. <em>2</em>.73 nmol/liter, p < 0.00<em>1</em>) and thrombin-antithrombin complex (baseline 6.5 vs. <em>1</em>7.<em>1</em> micrograms/ml, p < 0.0<em>1</em>) at 90 min, whereas no change was observed in fibrinopeptide A at 90 min (baseline <em>2</em>.8 vs. 3.0 nmol/liter, p = NS).

CONCLUSIONS

During thrombolytic therapy with both fibrin-specific and non-fibrin-specific drugs, there is an increase in thrombin generation despite concomitant administration of intravenous heparin.

BACKGROUND

Maximal exercise may be a trigger for cardiovascular events. The aim of the study was to investigate changes in blood coagulation and fibrinolysis following maximal short-term exercises with different durations up to 90 s.

METHODS

A total of <em>1</em>5 healthy nonsmokers underwent three isokinetic maximal tests on an SRM cycle ergometry system with durations of <em>1</em>5, 45, and 90 s. Blood samples were taken after a 30-min rest, immediately before and after exercise, <em>1</em>5 min, and <em>1</em> h after completion of exercise. For the investigation of blood coagulation, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>), thrombin-antithrombin III complex (TAT), intrinsic and extrinsic total (TTPin+ex), and endogenous thrombin potential (ETPin+ex) were measured. For testing fibrinolysis, determinations of plasmin-alpha(<em>2</em>)-antiplasmin complex (PAP), tissue-type plasminogen activator (tPA)-antigen, plasminogen activator inhibitor (PAI)-<em>1</em>-antigen and D-dimer were used.

RESULTS

Immediately after the exercise tests, only F<em>1</em>+<em>2</em> (<em>1</em>5- and 90-s test) and TTPin (45 and 90 s) showed a moderate increase (p<0.05), while TAT and ETP was unchanged. In contrast, a clear increase in PAP and tPA-antigen already after <em>1</em>5 s maximal exercise in relation to the exercise duration time could be investigated. These effects were not totally reversed to baseline <em>1</em>5 min after exercise; D-dimer and PAI-<em>1</em>-antigen still remained unchanged after these types of exercise.

CONCLUSIONS

Maximal short-term exercise does not lead to a relevant activation of blood coagulation in healthy young subjects, it is only slightly altered within the normal range. In contrast, fibrinolysis is clearly activated, and the increase is directly dependent on exercise duration. Additionally, it could be shown for the first time that fibrinolysis is already activated after <em>1</em>5 s maximal exercise duration.

The kringle <em>2</em> domain of <em>prothrombin</em> has been shown to interact with factor Va during the activation of <em>prothrombin</em> by the <em>prothrombin</em>ase complex composed of factor Xa, factor Va, negatively charged phospholipids and Ca<em>2</em>+ ions. However, contradictory results have been reported about the role of the kringle <em>1</em> domain of <em>prothrombin</em> during the assembly of the <em>prothrombin</em>ase complex. In an attempt to clarify the role of the kringle <em>1</em> domain of <em>prothrombin</em>, its effect on the activation of <em>prothrombin</em> by the <em>prothrombin</em>ase complex and its direct binding to human factor Va were assessed. Comparative evaluation with the effects caused by other <em>prothrombin</em> structural components [a <em>fragment</em> <em>1</em> (gamma-carboxyglutamic acid and kringle <em>1</em> domains), a kringle <em>2</em> domain and a catalytic protease domain] was also performed. In the presence of factor Va, each kringle <em>1</em> and kringle <em>2</em> <em>fragment</em> significantly inhibited the factor Xa-catalysed <em>prothrombin</em> activation in the absence of phospholipids. However, in the absence of both factor Va and phospholipids, kringle <em>2</em> <em>fragment</em>, but not kringle <em>1</em> <em>fragment</em>, inhibited <em>prothrombin</em> activation. Evaluation of the molecular interaction of the kringle domains with factor Va in assays with solid-phase phospholipid vesicles showed that each kringle <em>1</em> and kringle <em>2</em> <em>fragment</em> inhibited the <em>prothrombin</em>ase complex activity. Assessment of the direct binding of <em>prothrombin</em> and each kringle domain of <em>prothrombin</em> with factor Va by fluorescence polarization showed that <em>prothrombin</em>, kringle <em>1</em> and kringle <em>2</em> <em>fragments</em> bind directly to factor Va with dissociation constants of <em>1</em>.9+/-0.<em>1</em>, <em>2</em>.3+/-0.<em>1</em> and <em>2</em>.0+/-0.4 microM (means+/-S.D.) respectively. These findings suggest that both kringle <em>1</em> and <em>2</em> domains of <em>prothrombin</em> interact with factor Va during the assembly of the <em>prothrombin</em>ase complex.

Novel monoclonal antibodies that specifically recognize gamma-carboxyglutamyl (Gla) residues in proteins and peptides have been produced. As demonstrated by Western blot and time-resolved immunofluorescence assays the antibodies are pan-specific for most or all of the Gla-containing proteins tested (factors VII, IX, and X, <em>prothrombin</em>, protein C, protein S, growth arrest-specific protein 6, bone Gla protein, conantokin G from a cone snail, and factor Xa-like proteins from snake venom). Only the Gla-containing light chain of the two-chain proteins was bound. Decarboxylation destroyed the epitope(s) on <em>prothrombin</em> <em>fragment</em> <em>1</em>, and Ca(<em>2</em>+) strongly inhibited binding to <em>prothrombin</em>. In Western blot, immunofluorescence, and surface plasmon resonance assays the antibodies bound peptides conjugated to bovine serum albumin that contained either a single Gla or a tandem pair of Gla residues. Binding was maintained when the sequence surrounding the Gla residue(s) was altered. Replacement of Gla with glutamic acid resulted in a complete loss of the epitope. The utility of the antibodies was demonstrated in immunochemical methods for detecting Gla-containing proteins and in the immunopurification of a factor Xa-like protein from tiger snake venom. The amino acid sequences of the Gla domain and portions of the heavy chain of the snake protein were determined.

In a prospective study, the role of various hemostatic factors known to be associated with thrombotic risk was investigated in 7<em>1</em> patients with peripheral arterial occlusive disease (PAOD, stages II through IV, Fontaine; aged 68 +/- <em>1</em>3 years). Laboratory investigations were done before; <em>1</em>, <em>2</em>4, and 48 hours after; and 3 and 6 months after percutaneous transluminal angioplasty (PTA). Thirty of 7<em>1</em> (4<em>2</em>.3%) patients developed restenosis >> 50% reduction of the lumen diameter) at the site of PTA within 6 months, verified by color-coded duplex sonography. Significantly increased levels of thrombin-antithrombin III complexes (P < .0<em>1</em>), <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em> (P < .0<em>1</em>), and D-dimers (P < .0<em>1</em>) were found <em>1</em> hour, as well as <em>2</em>4 to 48 hours, after PTA. Fibrinogen (P < .0<em>1</em>) and von Willebrand factor (P < .0<em>1</em>) were significantly higher 48 hours after PTA. Restenotic patients as a whole had higher plasma fibrinogen (3.46 +/- <em>1</em>.<em>1</em><em>2</em> versus <em>2</em>.95 +/- 0.6<em>2</em> g/L, P < .0<em>1</em>) and C-reactive protein (<em>2</em>5.4 +/- 46.7 versus 7.9 +/- 6.9 mg/L, P < .05) at baseline, as well as higher fibrinogen (P < .05) and <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em> (P < .0<em>1</em>) during months 3 to 6 after PTA. There was a nonsignificant tendency for higher values of von Willebrand factor (<em>2</em>06 +/- 98% versus <em>1</em>84 +/- <em>1</em>00%, P = .<em>2</em>) at baseline in patients with restenosis, whereas tissue plasminogen activator, plasminogen activator inhibitor, coagulation screening tests, blood cell counts, and serum lipids showed no significant difference between the two groups. The relative risk for developing restenosis within 6 months while having high fibrinogen >> <em>2</em>.8 g/L) or C-reactive protein at baseline was <em>2</em>.80 (95% CI: <em>1</em>.30-6.0<em>2</em>, P < .0<em>1</em>) and <em>1</em>.96 (95% CI: <em>1</em>.07-3.58, P < .05), respectively. Patients with critical limb ischemia (stage III/IV, Fontaine) had significantly higher fibrinogen and von Willebrand factor at repeated points of time, as well as significantly higher C-reactive protein and lower creatinine clearance at entry. In the logistic regression risk factor analysis, baseline plasma fibrinogen, C-reactive protein concentration, and the severity of the arterial disease were significantly predictive of restenosis. Our results indicate that high procoagulant factors and persistent thrombin generation of the hemostatic system might promote restenosis, particularly in patients with extended atherosclerosis. This finding suggests that new treatment strategies should be taken under consideration for patients with PAOD and PTA.

Previous work has shown that bovine <em>prothrombin</em> <em>fragment</em> <em>1</em> binds to substrate-supported planar membranes composed of phosphatidylcholine (PC) and phosphatidylserine (PS) in a Ca(<em>2</em>+)-specific manner. The apparent equilibrium dissociation constant is <em>1</em>-<em>1</em>5 microM, and the average membrane residency time is approximately 0.<em>2</em>5 s-<em>1</em>. In the present work, fluorescence pattern photobleaching recovery with evanescent interference patterns (TIR-FPPR) has been used to measure the translational diffusion coefficients of the weakly bound <em>fragment</em> <em>1</em>. The results show that the translational diffusion coefficients on fluid-like PS/PC planar membranes are on the order of <em>1</em>0(-9) cm<em>2</em>/s and are reduced when the <em>fragment</em> <em>1</em> surface density is increased. Control measurements were carried out for <em>fragment</em> <em>1</em> on solid-like PS/PC planar membranes. The dissociation kinetics were similar to those on fluid-like membranes, but protein translational mobility was not detected. TIR-FPPR was also used to measure the diffusion coefficient of the fluorescent lipid NBD-PC in fluid-like PS/PC planar membranes. In these measurements, the diffusion coefficient was approximately <em>1</em>0(-8) cm<em>2</em>/s, which is consistent with that measured by conventional fluorescence pattern photobleaching recovery. This work represents the first measurement of a translational diffusion coefficient for a protein weakly bound to a membrane surface.

OBJECTIVE

To correlate the concentration of plasma coagulation markers at baseline and during follow-up in patients with solid tumors and venous thromboembolic disease with the risk of recurrence and death.

METHODS

Patients (N = <em>2</em><em>2</em>3) with first episode of venous thromboembolic disease received oral anticoagulation with warfarin for a target international normalized ratio of <em>2</em> to 3. Plasma coagulation markers were measured before instituting warfarin and at 3 monthly intervals, thereafter.

RESULTS

The median duration of oral anticoagulation was 6.7 months (range <em>2</em> weeks to 11 months). Major bleeding episodes occurred in 18 patients (8%), and minor hemorrhagic events occurred in 15 (6.7%) patients. Patients with advanced malignancy (P = 0.03<em>2</em>), history of surgery (P = 0.057), and those with poor performance status (P = 0.001) were more likely to encounter major bleeding episodes. Recurrence of venous thromboembolic disease was diagnosed in 31 patients (14%). At univariate analysis, advanced stage of cancer (P = 0.03), performance status>> 1 (P = 0.001), treatment with chemotherapy (P = 0.01), the presence of metastatic liver disease (P = 0.03), higher d-dimer (P = 0.001), and thrombin antithrombin complex levels (P = 0.01) were features predictive of recurrent venous thromboembolic disease. At multivariate analysis, poor performance status (P = 0.01) and d-dimer levels (P = 0.001) were predictors of recurrent venous thromboembolic disease. Persistent activation of coagulation as indicated by an upward trend in d-dimer (P = 0.001) and antithrombin (P = 0.001) was observed in patients who developed recurrent thrombosis. Similar upward trends in d-dimer (P = 0.001), antithrombin (P = 0.001), and prothrombin fragment F1 + <em>2</em> (P = 0.001) was observed in the 76 patients who died during the study period and in the patients who received chemotherapy.

CONCLUSIONS

Successful oral anticoagulation with warfarin in patients with cancer and venous thromboembolic disease is more likely to be achieved in patients with early stage tumors and good performance status. The persistence of activation of hemostasis as shown by plasma coagulation markers is a strong predictor of recurrence and poor outcome.

OBJECTIVE

To evaluate whether cisplatin-induced stroke is mediated by vascular toxicity with release of prothrombotic endothelial and platelet-derived microparticles (MPs).

METHODS

Endothelial (CD3<em>1</em>(+)CD4<em>1</em>(-)), platelets (CD3<em>1</em>(+)CD4<em>1</em>(+)) and prothrombotic (Annexin V(+)) circulating MPs were quantified by flow cytometry in <em>1</em>8 patients with cancer, before and 3 days after administration of cisplatin, and compared with <em>1</em>8 healthy controls. Thrombin-antithrombin complex and <em>prothrombin</em> <em>fragments</em> (F(<em>1</em>+<em>2</em>)) were measured as markers of the activation of the coagulation.

RESULTS

In patients with cancer, baseline levels of circulating prothrombotic, endothelial and platelet-derived MPs were similar to healthy controls and decreased significantly after administration of cisplatin. High-baseline MPs levels were observed in 5 patients who received cisplatin for a second or third cycle. A high-baseline activation of the coagulation was observed in all patients without further increase after cisplatin infusion.

CONCLUSIONS

Cisplatin treatment is immediately followed by a decrease in circulating levels of endothelial and platelet-derived MPs. However, a transient increase in MPs is observed at the second and third infusion, and this may contribute to the cisplatin-induced stroke.

The molecular basis of the substrate and inhibitor specificity of factor Xa, the serine proteinase of the <em>prothrombin</em>ase complex, was investigated by constructing two mutants of human antithrombin (HAT) in which the reactive site loop of the serpin from the P4-P4' site was replaced with the corresponding residues of the two factor Xa cleavage sites in <em>prothrombin</em> (HAT/Proth-<em>1</em> and HAT/Proth-<em>2</em>). These mutants together with prethrombin-<em>2</em>, the smallest zymogen form of thrombin containing only the second factor Xa cleavage site, were expressed in mammalian cells, purified to homogeneity and characterized in kinetic reactions with factor Xa in both the absence and presence of cofactors; factor Va, high affinity heparin and pentasaccharide <em>fragment</em> of heparin. HAT/Proth-<em>1</em> inactivated factor Xa approximately 3-4-fold better than HAT/Proth-<em>2</em> in either the absence or presence of heparin cofactors. In the absence of a cofactor, factor Xa reacted with the HAT/Proth-<em>2</em> and prethrombin-<em>2</em> with similar second-order rate constants (approximately <em>2</em>-3x<em>1</em>0(<em>2</em>) M(-<em>1</em>)s(-<em>1</em>)). Pentasaccharide catalyzed the inactivation rate of factor Xa by the HAT mutants 300-500-fold. A similar <em>1</em>0(4)-<em>1</em>0(5)-fold enhancement in the reactivity of factor Xa with prethrombin-<em>2</em> and the HAT mutants was observed in the presence of the cofactors Va and heparin, respectively. Factor Va did not influence the reactivity of factor Xa with either one of the HAT mutants. These results suggest that (<em>1</em>) in the absence of a cofactor, the P4-P4' residues of HAT and prethrombin-<em>2</em> primarily determine the specificity reactions with factor Xa, (<em>2</em>) factor Va binding to factor Xa is not associated with allosteric changes in the catalytic pocket of enzyme that would involve interactions with the P4-P4' binding sites, and (3) similar to allosteric activation of HAT by heparin, a role for factor Va in the <em>prothrombin</em>ase complex may involve rearrangement of the residues surrounding the scissile bond of the substrate to facilitate its optimal docking into the catalytic pocket of factor Xa.

OBJECTIVE

This study investigated levels of coagulation and fibrinolysis factors in cerebrospinal fluid (CSF) from adults with bacterial meningitis in relation to development of brain infarction.

METHODS

CSF was collected from 9<em>2</em> adults with community-acquired bacterial meningitis, who participated in the prospective Dutch Meningitis Cohort Study; 8 patients with viral meningitis and 9 healthy control subjects. Levels of proteins involved in the coagulation cascade were determined by means of immunoassays.

RESULTS

Bacterial meningitis was accompanied by local activation of coagulation, as shown by significantly higher CSF soluble tissue factor (P<0.00<em>1</em>) and <em>prothrombin</em> <em>fragment</em> F<em>1</em>+<em>2</em> concentrations (P<0.00<em>1</em>) as compared to viral meningitis patients and controls. This was accompanied by a significantly higher D-dimer formation (P<0.00<em>1</em>). In addition, in bacterial meningitis fibrinolysis was attenuated, since CSF plasminogen activator inhibitor (PAI)-<em>1</em> levels were significantly higher as compared to the controls (P=0.0<em>2</em>). In patients with bacterial meningitis who developed brain infarction, CSF PAI-<em>1</em> levels were higher than in those without infarction (P=0.04).

CONCLUSIONS

Activation of coagulation and attenuation of fibrinolysis in the CSF are important features of bacterial meningitis; the net effect on fibrin turnover may contribute to the development of brain infarction.

BACKGROUND

C-reactive protein (CRP) plays a major role in the immune system and is an independent risk marker of cardiovascular disease. However, CRP's role in atherogenesis as innocent bystander, causative, or even protective agent, remains unresolved. The (+)<em>1</em>444C/T alteration in the CRP gene has been reported to determine basal CRP concentrations. We hypothesized that this alteration may also be associated with the degree of inflammatory response and coagulation activation in a well-standardized model of systemic inflammation.

METHODS

We administered <em>2</em> ng/kg endotoxin [Escherichia coli bacterial lipopolysaccharide (LPS)] intravenously to stimulate inflammation in 9<em>1</em> healthy young Caucasian male paid volunteers (age range, <em>1</em>9-40 years). Participants were confined to bed rest and fasted for 8.5 h after LPS infusion. We collected blood samples before LPS infusion and at 0, <em>2</em>, 6, and <em>2</em>4 h after LPS infusion to measure inflammation markers [interleukin 6 (IL6), tumor necrosis factor-alpha (TNFalpha)], temperature, and coagulation markers (<em>prothrombin</em> <em>fragment</em> F(<em>1</em>+<em>2</em>), D-dimer). We analyzed the CRP 3' untranslated variant with a mutagenic separated PCR assay.

RESULTS

Basal concentrations of high-sensitivity CRP were approximately 40% lower in (+)<em>1</em>444CC alteration carriers than in T homozygous (TT) allele carriers (P = 0.04). In contrast, basal IL6 concentrations were <em>2</em>-fold higher in wild-type C homozygous (CC) than in TT individuals (P = 0.0<em>1</em>). In response to the LPS challenge, CC individuals had 4-fold higher peak TNFalpha concentrations (P <0.0<em>1</em>),>><em>2</em>.5-fold higher peak IL6 concentrations (P <0.0<em>1</em>), and increased temperature (P <0.0<em>1</em>). Twenty-four hours after LPS challenge, <em>prothrombin</em> <em>fragment</em> F(<em>1</em>+<em>2</em>) concentrations were 75% higher and D-dimer concentrations 50% higher in CC than in TT individuals (P <0.05).

CONCLUSIONS

Genetic factors regulating CRP concentrations also modulate the individual response to endotoxin-stimulated inflammation.

The esterolytic activity of bovine alpha-thrombin on the synthetic substrate N-alpha-p-tosyl-L-arninine methyl ester (TosArgOMe) is stimulated when the <em>prothrombin</em> activation <em>fragment</em>, <em>prothrombin</em> <em>fragment</em> <em>2</em>, is added as previously reported by this laboratory (Heldebrant, C. M., and Mann, K. G. (<em>1</em>973), J. Biol. Chem. <em>2</em>48, 364<em>2</em>). A similar stimulation of beta-thrombin is observed upon addition of <em>prothrombin</em> <em>fragment</em> <em>2</em>. The binding constant of <em>prothrombin</em> <em>fragment</em> <em>2</em> to alpha-thrombin has been determined by the method of Gutfreund ((<em>1</em>97<em>2</em>), Enzymes, Physical Principles, Wiley, New York, N.Y., pp 67-7<em>1</em>). The dissociation constant is 7.7 X <em>1</em>0(-<em>1</em>0)M, and there is one molecule of <em>prothrombin</em> <em>fragment</em> <em>2</em> bound per molecule of alpha-thrombin. Prethrombin-<em>2</em> competes for <em>prothrombin</em> <em>fragment</em> <em>2</em>, so the enhancement of the esterolytic activity of alpha-thrombin by <em>prothrombin</em> <em>fragment</em> <em>2</em> was used as a probe to determine the dissociation constant for the binding of <em>prothrombin</em> <em>fragment</em> <em>2</em> to prethrombin <em>2</em>. The dissociation constant for this association is <em>1</em>.3 X <em>1</em>0(-<em>1</em>0)M. The kinetic parameters for the reaction of alpha-thrombin on TosArgOMe were determined in the absence and presence of <em>prothrombin</em> <em>fragment</em> <em>2</em> and are as follows: (a) in the absence of <em>prothrombin</em> <em>fragment</em> <em>2</em>, Km(app) = <em>1</em>.9<em>2</em> X <em>1</em>0(-4)M, and k3(app) = 35.8 mol of TosArgOMe/mol of alpha-thrombin s(-<em>1</em>); (b) in the presence of <em>prothrombin</em> <em>fragment</em> <em>2</em>,Km(app = <em>1</em>.76 X <em>1</em>0(-4)M, and k3(app) = 60.5 mol of TosArgOMe/mol of alpha-thrombin s(-<em>1</em>). Thus, the stimulatory effect of bovine <em>prothrombin</em> <em>fragment</em> <em>2</em> on bovine alpha-thrombin is reflected in k3(app) and not in Km(app). In contrast to the stimulatory effect of <em>prothrombin</em> <em>fragment</em> <em>2</em> on the thrombin-catalyzed hydrolysis of TosArgOMe, it inhibits the activity of alpha-thrombin toward N-alpha-benzoyl-L-arginine ethyl ester and N-alpha-benzoyl-L-arginine p-nitroanilide. The inhibition of activity toward these substrates by <em>prothrombin</em> <em>fragment</em> <em>2</em> is also reflected in k3(app). Activity toward the nonspecific substrate p-nitrophenyl butyrate was completely inhibited by the addition of <em>prothrombin</em> <em>fragment</em> <em>2</em>. <em>Prothrombin</em> <em>fragment</em> <em>2</em> has no effect on the inhibition of alpha-thrombin activity by the active-site serine inhibitors diisopropyl phosphofluoridate, phenylmethanesulfonyl fluoride, or p-nitrophenyl guanidinobenzoate. Inhibition by the active-site-histidine-modifying inhibitor, N-alpha-p-tosyl-L-arginine chloromethyl ketone, was enhanced by the addition of <em>prothrombin</em> <em>fragment</em> <em>2</em>. Soybean trypsin inhibitor reduces the stimulation by <em>prothrombin</em> <em>fragment</em> <em>2</em>, but only at high molar ratios. <em>Prothrombin</em> <em>fragment</em> <em>2</em> has no effect on the clotting activity of alpha-thrombin, nor inhibition of this activity by heparin, hirudin, or diisopropyl phosphafluoridate. Bovine <em>prothrombin</em> <em>fragment</em> <em>2</em> enhances the esterolytic activity of both human and bovine alpha-thrombin, but human <em>prothrombin</em> <em>fragment</em> <em>2</em> does not enhance the esterolytic activity of either human or bovine alpha-thrombin.

Endothelial injury in vivo induced by Rickettsia Conorii, the etiologic agent of Mediterranean Spotted Fever (MSF) has been recently demonstrated. We sought to determine whether platelet and/or coagulative activation in vivo can be demonstrated in the acute phase of MSF, through measurements of a major metabolite of thromboxane (TX) in the urine (<em>1</em><em>1</em>-dehydro-TXB<em>2</em>) and of plasma <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>, whose levels reflect activation of <em>prothrombin</em> to thrombin. Moreover, we measured plasma endothelin-<em>1</em> as marker of endothelial dysfunction. Our results provide biochemical evidence for the occurrence of TXA<em>2</em>-dependent platelet activation and thrombin generation in vivo, together with endothelial dysfunction. These phenomena could account for clinical manifestations of MSF, such as vasculitis and focal microthrombus formation. These results could also provide a rationale for testing the efficacy of aspirin or heparin in reducing the prothrombotic status of Rickettsiae diseases.

BACKGROUND

Cancer is one of the most common acquired causes of venous thromboembolism.

OBJECTIVE

To evaluate haemostasis disorders in patients with non-metastatic gastric cancer.

METHODS

We studied <em>1</em><em>1</em> patients with non-metastatic gastric cancer (9 males and <em>2</em> females, median age 54 years) and <em>2</em>0 healthy subjects (<em>1</em>5 males and 5 females, median age 48 years) control. We measured <em>prothrombin</em> time, activated partial thromboplastin time, coagulation time, clot lysis time, fibrinogen, clotting factors (II, VII, VIII, IX, X), C protein, S protein, AT III, activated protein C resistance, <em>prothrombin</em> <em>1</em>+<em>2</em> <em>fragment</em>, tissue plasminogen activator and D-Dimer in all subjects.

RESULTS

Fibrinogen plasma levels were significantly higher in patients with non-metastatic gastric cancer than in control group (505+/-<em>2</em>4 mg/dl vs 336+/-30 mg/dl, p<0.00<em>1</em>). We also found a significant increase in <em>prothrombin</em> <em>1</em>+<em>2</em> <em>fragment</em> plasma concentration compared with controls (3.8+/-0.6 nM vs 0.83+/-0.09 nM, p<0.00<em>1</em>). Plasma D-dimer levels were <em>2</em>0-fold higher in patients with non-metastatic gastric cancer compared with controls (9.57+/-0.4 ng/dl vs 0.4+/-0.05 ng/dl, p<0.00<em>1</em>). Also tissue plasminogen activator was significantly higher in gastric cancer patients than in controls (<em>2</em>0.8+/-<em>2</em>.3<em>2</em> ng/ml vs 9.<em>1</em>+/-<em>1</em>.37 ng/ml, p<0.0<em>1</em>). Finally clot lysis time was significantly accelerated in gastric cancer patients compared with control subjects (8<em>1</em>+/-37 min vs <em>2</em>33+/-74 min, p<0.0<em>1</em>).

CONCLUSIONS

Patients with non-metastatic gastric cancer are at risk for thrombotic events due to the combined increase in fibrinogen plasma levels and thrombin formation.

Activation of <em>prothrombin</em> (Pro) by factor Xa to form thrombin occurs by proteolysis of Arg<em>2</em>7<em>1</em>-Thr<em>2</em>7<em>2</em> and Arg3<em>2</em>0-Ile3<em>2</em><em>1</em>, resulting in expression of regulatory exosites I and II. Cleavage of Pro by thrombin liberates <em>fragment</em> <em>1</em> and generates the zymogen analog, prethrombin <em>1</em> (Pre <em>1</em>). The properties of exosite I on Pre <em>1</em> and its factor Xa activation intermediates were characterized in spectroscopic and equilibrium binding studies using the fluorescein-labeled probe, hirudin(54-65) ([5F]Hir(54-65)-(SO3-)). Prethrombin <em>2</em> (Pre <em>2</em>), formed by factor Xa cleavage of Pre <em>1</em> at Arg<em>2</em>7<em>1</em>-Thr<em>2</em>7<em>2</em>, had the same affinity for hirudin(54-65) peptides as Pre <em>1</em> in the absence or presence of near-saturating <em>fragment</em> <em>2</em> (F<em>2</em>). Pre <em>2</em> and thrombin also had indistinguishable affinities for F<em>2</em>. By contrast, cleavage of Pre <em>1</em> at Arg3<em>2</em>0-Ile3<em>2</em><em>1</em>, to form active meizothrombin des-<em>fragment</em> <em>1</em> MzT(-F<em>1</em>), showed a <em>1</em><em>1</em>- to <em>2</em>0-fold increase in affinity for hirudin(54-65), indistinguishable from the <em>1</em>3- to <em>2</em>0-fold increase seen for conversion of Pre <em>2</em> to thrombin. Thus, factor Xa cleavage of Pre <em>1</em> at Arg<em>2</em>7<em>1</em>-Thr<em>2</em>7<em>2</em> does not effect exosite I expression, whereas cleavage at Arg3<em>2</em>0-Ile3<em>2</em><em>1</em> results in concomitant activation of the catalytic site and exosite I. Furthermore, expression of exosite I on the Pre <em>1</em> activation intermediates is not modulated by F<em>2</em>, and exosite II is not activated conformationally. The differential expression of exosite I affinity on the Pre <em>1</em> activation intermediates and the previously demonstrated role of (pro)exosite I in factor Va-dependent substrate recognition suggest that changes in exosite I expression may regulate the rate and direction of the Pre <em>1</em> activation pathway.