OBJECTIVE

To determine whether activation of hemostatic function (thrombosis and fibrinolysis) is associated with cognitive decline in older people.

RESULTS

We studied 5804 people (age, 70-8<em>2</em> years) in the Prospective Study of Pravastatin in the Elderly at Risk (PROSPER). Mean follow-up was 3.<em>2</em> years, including annual measurement of speed of information processing (letter, digit coding, and Stroop), verbal memory (picture-word naming), and basic and instrumental activities of daily living. Raised levels of markers of thrombin generation (d-dimer and <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>) were associated independently with increased rate of cognitive decline (eg, Stroop increased by 4.44 s [SEM, 0.68] in bottom tertile of d-dimer compared to 5.46 [SEM, 0.7<em>1</em>] in highest tertile; P<0.05) and deterioration in activities of daily living. This increased rate of decline was attenuated but not removed when subjects with incident nonfatal stroke were omitted from the analysis. It also persisted when adjustments were made for inflammation (C-reactive protein and IL-6).

CONCLUSIONS

Older patients with increased markers of thrombin generation (d-dimer and <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>) are at increased risk for cognitive decline and deterioration in ability to perform activities of daily living. This is likely attributable to increased risk of cerebral ischemic damage (including covert disease) associated with prothrombotic states.

OBJECTIVE

It is unknown whether a therapeutic combination of aspirin (ASA) and ticlopidine might effectively decrease activation of hemostasis.

BACKGROUND

Percutaneous transluminal coronary angioplasty (PTCA), rotational atherectomy and stent implantation are procedures that fracture or ablate endothelium and plaque, a situation that activates hemostasis.

METHODS

In 85 patients undergoing PTCA for a 77.8 +/- <em>1</em>% stenosis, we measured markers of coagulation and platelet activation (thrombin-antithrombin complexes [TAT], <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> [F<em>1</em> + <em>2</em>] serotonin and the presence of circulating activated platelets reacting with monoclonal antibodies against glycoproteins exposed on platelet membranes). Blood samples were drawn from a peripheral vein and from the coronary ostium before the procedures. Both immediately and <em>1</em>0 min after angioplasty, and <em>1</em>0 min afterward, samples were collected from a probing catheter (0.0<em>1</em>8 in, [0.46 cm]) positioned beyond the stenosis. All patients were being treated with antianginal drugs and ASA, <em>2</em>50 mg/day. Seventy of them had taken ticlopidine, <em>2</em>50 mg, twice daily for < or = <em>1</em> day (< or = <em>2</em>4 h) (n = <em>2</em>8) or for>> or = 3 days >> or = 7<em>2</em> h) (n = 4<em>2</em>). Heparin (<em>1</em>50 U/kg) was administered before angioplasty. Thirty patients underwent PTCA; <em>1</em>5 of them were not treated with ticlopidine and <em>1</em>5 were given ticlopidine >> or = 7<em>2</em> h). Thirty-five patients had stent implantation, <em>2</em>0 rotational atherectomy.

RESULTS

Before and during the procedures, there was greater thrombin generation (expressed by higher TAT and F<em>1</em> + <em>2</em> plasma levels) in patients not taking ticlopidine or taking it for < or = <em>2</em>4 h (p < 0.05). Platelet activation and plasma serotonin levels were also significantly higher in the no ticlopidine or < or = <em>2</em>4-h ticlopidine groups.

CONCLUSIONS

The combined use of ticlopidine, ASA and heparin effectively controls activation of coagulation in patients with stable or unstable angina undergoing coronary dilation.

We investigated whether the anticoagulant effect of idraparinux, a selective long-acting factor Xa inhibitor, could be neutralized by recombinant factor VIIa (rFVIIa) in healthy male volunteers. We performed a randomized, placebo-controlled trial, comparing idraparinux [7.5 mg subcutaneous (s.c.)] followed at 3 h by rFVIIa [90 microg/kg intravenous (i.v.)] (n = 6), or idraparinux (7.5 mg s.c) followed after <em>1</em> week by rFVIIa (90 microg/kg i.v.)(n = 6). rFVIIa, given 3 h after idraparinux, significantly reversed the increased thrombin generation time (TGT), the increased activated partial thromboplastin time (aPTT) and <em>prothrombin</em> time (PT), and the reduced <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>) levels caused by idraparinux, although no clear effect of rFVIIa on the endogenous thrombin potential (ETP) was observed. One week after idraparinux, injection of rFVIIa resulted in a similar relative reduction of the remaining increased aPTT, PT and TGT, with correction to pre-idraparinux values. A clear increase of F<em>1</em>+<em>2</em> was observed, together with a small increase in ETP. We conclude that rFVIIa has significant effects on the idraparinux-inhibited thrombin generation and clotting parameters. These results suggest that rFVIIa may be useful in serious bleeding complications in idraparinux treated patients.

OBJECTIVE

Haemostatic changes may be involved in the pathogenesis and progression of ulcerative colitis. We studied longitudinally inflammatory and haemostatic parameters in patients treated for severe ulcerative colitis.

METHODS

We carried out a descriptive study of longitudinal blood measurements in patients with severe ulcerative colitis from one large regional hospital.

METHODS

Nineteen patients with severe ulcerative colitis were assessed by an endoscopic score and a patient score at baseline. Patients were assessed by patient scores during treatment at scheduled intervals. At each visit, inflammatory and haemostatic parameters were determined.

RESULTS

At baseline, the erythrocyte sedimentation rate, C-reactive protein, leucocyte and granulocyte count, thrombin-antithrombin complexes, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, fibrinogen and degradation products of fibrinogen and fibrin were increased in patients when compared with controls, whereas albumin concentration and factor XIII activity were significantly lower. Antithrombin activity was normal. During treatment, the median patient score diminished significantly from <em>1</em><em>2</em> to 4.5 points after <em>2</em> weeks, decreased further to 4 points after 4 weeks and remained below 4 points throughout the remaining study period. Inflammation parameters returned to within the reference range in two patients after 4 weeks, whereas the coagulation markers <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> and thrombin-antithrombin complexes returned to normal values after 8 weeks and <em>2</em>4 weeks, respectively. In contrast with markers of inflammation, slightly increased concentrations of the degradation products of both fibrinogen and fibrin were found for almost <em>1</em> year, which indicated low-grade activation of coagulation and fibrinolysis.

CONCLUSIONS

These results are compatible with a condition of persistent hypercoagulation in patients with ulcerative colitis who are in clinical remission. Persistent hypercoagulation may contribute to the clinical course of ulcerative colitis.

Systemic lupus erythematosus (SLE) is a chronic inflammatory disorder with a high prevalence of cardiovascular disease due to accelerated atherosclerosis, as well as an increased risk of venous thromboembolism. Many of these clinical features have been attributed to the high prevalence of autoantibodies that are directed against phospholipid-bound antigens and that induce prothrombotic effects and disturb endothelial cell function. We conducted a case-control study in a cohort of female patients with SLE and in age-matched and sex-matched normal individuals. Patients had significantly higher levels of plasma inflammatory markers, but their overall coagulation status assessed by <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> and D-dimer plasma levels was not different from controls. Resistance against activated protein C (APC), assessed by a thrombin generation-based as well as an activated partial thromboplastin time-based method, however, was increased in patients. This defect was neither due to factor V Leiden carriership or to the use of oral contraceptives. This acquired form of APC resistance was due to proinflammatory changes associated with lower plasma levels of free protein S. In conclusion, acquired APC resistance may be an important determinant of the risk of thrombosis in patients with SLE, probably due to an active cross-talk between inflammation and coagulation systems.

BACKGROUND

Hemostatic drugs are widely thought to be unnecessary and potentially detrimental in off-pump coronary artery bypass graft surgery (OPCABG), despite well-established use in on-pump surgery. In a randomized, prospective OPCABG trial, we assessed efficacy and safety of aprotinin through a comprehensive assessment of graft patency and hematologic function.

METHODS

Sixty patients were randomly assigned to full-dose aprotinin or placebo. Heparin was titrated to a kaolin-based activated clotting time of greater than 300 seconds. Exclusionary criteria included creatinine greater than <em>2</em> mg/dL, conversion to on-pump CABG, and preoperative GPIIb/IIIa inhibition. Hematologic assessments were obtained preoperatively, at the end of surgery, and on days <em>1</em> and 3: mean platelet volume, thrombin generation (<em>prothrombin</em> <em>fragment</em> <em>1</em>.<em>2</em> assay), and aspirin resistance using a modified thrombelastography, whole blood aggregometry, <em>1</em><em>1</em>-dehydro-thromboxane B<em>2</em> levels, and flow cytometry. Thrombotic events were defined as postoperative myocardial infarction by electrocardiography or elevated troponin I, clinical stroke by examination and head computed tomography, and bypass graft failure by multichannel computed tomography angiography on day 5.

RESULTS

Aprotinin was associated with a significant reduction in intraoperative and postoperative blood loss compared with placebo but had no effect on transfusion rates. Patients treated with aprotinin had significantly fewer thrombotic events (3% versus <em>2</em>3%, p < 0.05, Fisher's exact test) and less postoperative aspirin resistance (<em>2</em>0% versus 46%, respectively, p < 0.05, Fisher's exact test). Postoperative <em>prothrombin</em> <em>fragment</em> <em>1</em>.<em>2</em> level was reduced by aprotinin use.

CONCLUSIONS

Aprotinin reduced perioperative bleeding after OPCABG. Preserved aspirin sensitivity in the aprotinin group may explain the observed reduction in thrombotic events and might be related to the suppression of perioperative and transmyocardial thrombin formation.

BACKGROUND

Inflammatory bowel disease is associated with an increased incidence of thromboembolic complications. The aim of this study was to investigate the role of the soluble CD40 ligand (sCD40L), which displays prothrombotic properties, in patients with ulcerative colitis (UC) and Crohn's disease (CD) in comparison with inflammatory and healthy controls.

METHODS

Plasma levels of sCD40L, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>), thrombin-antithrombin (TAT) complex and soluble P-selectin were measured in <em>1</em>04 inflammatory bowel disease patients (54 ulcerative colitis and 50 Crohn's disease), in <em>1</em>8 cases with other causes of intestinal inflammation and in 80 healthy controls using commercially available enzyme-linked immunosorbent assays. Plasma levels of sCD40L were correlated with disease activity, type, localization and treatment as well as with the measured thrombophilic parameters.

RESULTS

CD patients had significantly higher sCD40L levels than both groups of controls (CD vs HC P < 0.00<em>1</em>; CD vs non-IBD P < 0.05). UC patients had higher but not significantly different sCD40L levels compared with the controls. Both UC and CD patients with active disease had significantly higher sCD40L levels in comparison with patients with inactive disease. Plasma levels of sCD40L were correlated with platelet count (r = 0.<em>2</em>7, P = 0.00<em>1</em>). They also showed a correlation with <em>prothrombin</em> F<em>1</em>+<em>2</em> (r = 0.<em>1</em>6, r = 0.03) and TAT (r = 0.<em>1</em>5, r = 0.04) as well as with P-selectin (r = 0.<em>1</em>9, P = 0.0<em>1</em>).

CONCLUSIONS

The increased sCD40L plasma levels may represent, at least in some degree, a molecular link between inflammatory bowel disease and the procoagualant state.

The intensity of warfarin therapy for prevention of primary and secondary thromboembolic complications in paediatric patients, is extrapolated from guidelines for adults, which may not be optimal. Therefore, we assessed thrombin regulation ex vivo and in vitro in plasmas from 40 children (<em>1</em> to <em>1</em>8 years old with a median age of <em>1</em>3 years) and <em>2</em>7 adults receiving warfarin with an international normalized ratio of <em>2</em> to 3 (child: <em>2</em>.5 +/- 0.<em>1</em>5; adult: <em>2</em>.4 +/- 0.<em>1</em>4). Ex vivo concentrations of <em>prothrombin</em> <em>fragment</em> <em>1</em>.<em>2</em> were significantly lower in children (0.30 +/- 0.03 nM) compared to adults (0.45 +/- 0.04 nM; p <0.0<em>1</em>). Thrombin generation in defibrinated plasmas (Arvin) was decreased and delayed for children compared to adults when activated by either activated partial thromboplastin time (child = 3<em>2</em> +/- <em>1</em>.7, adult = 45 +/- <em>1</em>.9 microM x s) or <em>prothrombin</em> time (child = 35 +/- 0.7, adult = 46 +/- <em>1</em>.0 microM x s) reagents (p <0.0<em>1</em> for both). Although plasma concentrations of factors (F) II, FVII, FIX, F X, protein C and protein S were similar, more of the thrombin generated was complexed to alpha<em>2</em> macroglobulin (alpha<em>2</em>M) at times close to peak thrombin activity (60 s) in plasma from children (general linear analysis of variance; p <0.03). Thus, increased alpha<em>2</em>M levels may enhance thrombin regulation in paediatric compared to adult patients receiving warfarin, suggesting that clinical trials in children, using less intense warfarin treatment, may be required to determine optimum therapy.

A series of overlapping cDNAs coding for mouse <em>prothrombin</em> (coagulation factor II) have been isolated and the composite DNA sequence has been determined. The complete <em>prothrombin</em> cDNA is <em>1</em>,987 bp in length [excluding the poly(A) tail] and codes for <em>1</em>8 bp of 5' untranslated sequence, an open reading frame coding for 6<em>1</em>8 amino acids, a stop codon, and a 3' untranslated region of <em>1</em><em>1</em><em>2</em> bp followed by a poly(A) tail. The translated amino acid sequence predicts a molecular weight of 66,087, which includes <em>1</em>0 residues of gamma-carboxyglutamic acid. There are five potential N-linked glycosylation sites. Mouse <em>prothrombin</em> is 8<em>1</em>.4% and 77.3% identical to the human and bovine proteins, respectively. Comparison of the cDNA coding for mouse <em>prothrombin</em> to the human and bovine cDNAs indicates 79.9% and 76.5% identity, respectively. Amino acid residues important for the structure and function of human <em>prothrombin</em> are conserved in the mouse and bovine proteins. In the adult mouse and rat, <em>prothrombin</em> is primarily synthesized in the liver, where is constitutes 0.07% of total mRNA as determined by solution hybridization analysis. The genetic locus for mouse <em>prothrombin</em>, Cf-<em>2</em>, has been mapped using an interspecies backcross and DNA <em>fragment</em> differences between the two species. The <em>prothrombin</em> locus lies on mouse chromosome <em>2</em>, <em>1</em>.8 +/- <em>1</em>.3 map units proximal to the catalase locus. The gene order in this region is Cen-Acra-Cf-<em>2</em>-Cas-<em>1</em>-A-Tel. This localization extends the proximal boundary of the known region of homology between mouse chromosome <em>2</em> and human chromosome <em>1</em><em>1</em>p from Cas-<em>1</em> about <em>2</em> map units toward the centromere.

BACKGROUND

Inflammatory and hemostasis-related biomarkers may identify women at risk of stroke.

METHODS

Hormones and Biomarkers Predicting Stroke is a study of ischemic stroke among postmenopausal women participating in the Women's Health Initiative observational study (n = 97<em>2</em> case-control pairs). A Biomarker Risk Score (BRS) was derived from levels of 7 inflammatory and hemostasis-related biomarkers that appeared individually to predict risk of ischemic stroke: C-reactive protein (CRP), interleukin-6, tissue plasminogen activator, D-dimer, white blood cell count, neopterin, and homocysteine. The c index was used to evaluate discrimination.

RESULTS

Of all the individual biomarkers examined, CRP emerged as the only independent single predictor of ischemic stroke (adjusted odds ratio comparing Quartile(4)v Quartile(<em>1</em>) = <em>1</em>.64, 95% confidence interval: <em>1</em>.<em>1</em>5-<em>2</em>.3<em>2</em>, P = .0<em>1</em>) after adjustment for other biomarkers and standard stroke risk factors. The BRS identified a gradient of increasing stroke risk with a greater number of elevated inflammatory/hemostasis biomarkers, and improved the c index significantly compared with standard stroke risk factors (P = .0<em>2</em>). Among the subset of individuals who met current criteria for high-risk levels of CRP (>3.0 mg/L), the BRS defined an approximately <em>2</em>-fold gradient of risk. We found no evidence for a relationship between stroke and levels of E-selectin, fibrinogen, tumor necrosis factor-alpha, vascular cell adhesion molecule-<em>1</em>, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, Factor VIIC, or plasminogen activator inhibitor-<em>1</em> antigen (P>> .<em>1</em>5).

CONCLUSIONS

The findings support the further exploration of multiple biomarker panels to develop approaches for stratifying an individual's risk of stroke.

<em>Prothrombin</em> (Pro) activation by factor Xa generates the thrombin catalytic site and exosites I and II. The role of <em>fragment</em> <em>1</em> (F<em>1</em>) in the pathway of exosite I expression during Pro activation was characterized in equilibrium binding studies using hirudin(54-65) labeled with 6-(N-(7-nitrobenz-<em>2</em>-oxa-<em>1</em>,3-diazol-4-yl)amino)hexanoate ([NBD]Hir(54-65)(SO3-)) or 5-(carboxy)fluorescein ([5F]Hir(54-65)(SO3-)). [NBD]Hir(54-65)(SO3-) distinguished exosite I environments on Pro, prethrombin <em>1</em> (Pre <em>1</em>), and prethrombin <em>2</em> (Pre <em>2</em>) but bound with the same affinities as [5F]Hir(54-65)(SO3-). Conversion of Pro to Pre <em>1</em> caused a 7-fold increase in affinity for the peptides. Conversely, <em>fragment</em> <em>1</em>.<em>2</em> (F<em>1</em>.<em>2</em>) decreased the affinity of Pre <em>2</em> for [5F]Hir(54-65)(SO3-) by 3-fold. This was correlated with a <em>1</em>6-fold increased affinity of F<em>1</em>.<em>2</em> for Pre <em>2</em> in comparison to thrombin, demonstrating an enhancing effect of F<em>1</em> on F<em>1</em>.<em>2</em> binding. The active intermediate, meizothrombin, demonstrated a 50- to <em>2</em><em>2</em>0-fold increase in exosite affinity. Free thrombin and thrombin.F<em>1</em>.<em>2</em> complex bound [5F]Hir(54-65)(SO3-) with indistinguishable affinity, indicating that the effect of F<em>1</em> on peptide binding was eliminated upon expression of catalytic activity and exosite I. The results demonstrate a new zymogen-specific role for F<em>1</em> in modulating the affinity of ligands for exosite I. This may reflect a direct interaction between the F<em>1</em> and Pre <em>2</em> domains in Pro that is lost upon folding of the zymogen activation domain. The effect of F<em>1</em> on (pro)exosite I and the role of (pro)exosite I in factor Va-dependent substrate recognition suggest that the Pro activation pathway may be regulated by (pro)exosite I interactions with factor Va.

A study was performed to explore the effects of supplemental intake of various marine oils known to be part of the Eskimo diet. Healthy men and women (<em>1</em>34) were randomly selected to consume <em>1</em>5 mL/d of oil from blubber of seal, cod liver, seal/cod liver, blubber of Minke whale, or no oil for ten weeks. Total cholesterol was unchanged in the oil groups, whereas high density lipoprotein cholesterol increased 7% in the seal/cod liver oil (CLO) group (P < 0.05) and <em>1</em><em>1</em>% in the whale oil group (P < 0.005). Triacylglycerol was significantly reduced in the CLO group only. The concentration of <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> was reduced <em>2</em>5% (P < 0.05) after whale oil supplementation. No change in fibrinogen or factor VIIc was detected. Tumor necrosis factor generation in lipopolysaccharide (LPS)-stimulated blood was 30% reduced after whale oil (P < 0.05), but was unaffected by intake of seal or CLO. The LPS-induced tissue factor activity in monocytes was reduced to a significant degree only in the seal/CLO group (34%) and whale oil group (35%) (P < 0.05). The most dramatic change in thromboxane B<em>2</em> in LPS-stimulated blood was seen after whale oil intake with 44% reduction (P < 0.0<em>1</em>). Supplementation of a regular diet with a combination of seal oil and CLO and especially with whale oil seems to have beneficial effects on several products thought to be associated with cardiovascular and thrombotic diseases.

Currently raloxifene, a selective estrogen receptor modulator, is being investigated as a potential alternative for postmenopausal hormone replacement to prevent osteoporosis and cardiovascular disease. We compared the <em>2</em>-year effects of raloxifene on a wide range of cardiovascular risk factors with those of placebo and conjugated equine estrogens (CEEs). Analyses were based on 56 hysterectomized but otherwise healthy postmenopausal women aged 54. 8+/-3.5 (mean+/-SD) years who entered this double-blind study and who were randomly assigned to raloxifene hydrochloride 60 mg/d (n=<em>1</em>5) or <em>1</em>50 mg/d (n=<em>1</em>3), placebo (n=<em>1</em>3), or CEEs 0.6<em>2</em>5 mg/d (n=<em>1</em>5). At baseline and after 6, <em>1</em><em>2</em>, and <em>2</em>4 months of treatment, we assessed serum lipids, blood pressure, glucose metabolism, C-reactive protein, and various hemostatic parameters. Compared with placebo, both raloxifene and CEEs lowered the level of low density lipoprotein cholesterol by 0.53 to 0.79 mmol/L (all P<0.04) and lowered, at <em>2</em>4 months, the level of fibrinogen by 0.7<em>1</em> to 0.86 g/L (all P<0.05). The effects of raloxifene and CEEs did not differ significantly. In contrast to raloxifene, from 6 months on CEEs increased high density lipoprotein cholesterol by 0.<em>2</em>5 to 0.<em>2</em>9 mmol/L and reduced plasminogen activator inhibitor-<em>1</em> antigen by 30.6 to 48.6 ng/mL (all P<0.0<em>2</em> versus both placebo and raloxifene). CEEs transiently increased C-reactive protein by <em>1</em>.0 mg/L at 6 months (P<0.05 versus placebo) and <em>prothrombin</em>-derived <em>fragment</em> F<em>1</em>+<em>2</em> by 0. 79 nmol/L at <em>1</em><em>2</em> months (P<0.00<em>1</em> versus placebo). Finally, from <em>1</em><em>2</em> months on, CEEs increased triglycerides by 0.33 to 0.56 mmol/L (all P<0.05 versus both placebo and raloxifene). Our findings suggest that in healthy postmenopausal women, raloxifene and estrogen monotherapy have similar beneficial effects on low density lipoprotein cholesterol and fibrinogen levels. These treatments differ, however, in their effects on high density lipoprotein cholesterol, triglycerides, and plasminogen activator inhibitor-<em>1</em> and possibly in their effects on <em>prothrombin</em> <em>fragment</em> F<em>1</em>+<em>2</em> and C-reactive protein.

The effect of homocysteine-lowering treatment on thrombin generation was investigated in <em>1</em>7 subjects with hyperhomocysteinemia (aged <em>2</em><em>2</em>-60 years), <em>1</em><em>1</em> of whom had symptomatic atherosclerotic vascular disease. All subjects had fasting total homocysteine levels above <em>1</em>6 micromol/L. The formation of thrombin was assessed by measuring thrombin-antithrombin III complexes and <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> in peripheral venous blood and in the bleeding time blood collected at 30-second intervals from skin incisions on a forearm. All the tests were performed before and after an 8-week treatment with folic acid p.o. 5 mg/day, vitamin B6 p.o. 300 mg/day, and vitamin B<em>1</em><em>2</em> i.m. <em>1</em>000 microg given on a weekly basis. Following the 8-week therapy, the median plasma homocysteine concentration became significantly reduced from <em>2</em>0 to <em>1</em>0 micromol/L, while plasma levels of fibrinogen, <em>prothrombin</em>, and antithrombin III as well as activity of protein C, S, and factor VII showed no changes. Vitamin treatment was associated with a significant fall in thrombin-antithrombin III complexes and <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> concentrations in peripheral venous blood. Bleeding time became prolonged by about 60 seconds. At sites of hemostatic plug formation, plasma concentrations of both thrombin markers significantly decreased. Compared with pretreatment values, significantly less thrombin was produced during the first 3 minutes of bleeding after homocysteine-lowering therapy. In subjects with hyperhomocysteinemia a reduction of plasma fasting homocysteine concentration by folic acid and vitamins B<em>1</em><em>2</em> and B6 administration is associated with attenuation of thrombin generation both in peripheral blood and at sites of hemostatic plug formation.

OBJECTIVE

To determine the degree of clotting activation that occurs with the usual anticoagulation regimen with systemic heparinization.

METHODS

To allow a standardized comparison of the patients, this study focused on the first 48 hours of extracorporeal membrane oxygenation (ECMO) in term newborn infants. The ECMO perfusion circuit consisted of a roller pump, silicone membrane lungs, and silicone rubber tubing. Coagulation was controlled routinely by measuring <em>prothrombin</em> time, fibrinogen, antithrombin III, and reptilase time. Platelet counts, activated clotting time, and heparin concentration were controlled regularly. The following specific activation markers of the clotting system were measured: <em>prothrombin</em> activation <em>fragment</em> <em>1</em> + <em>2</em>(F<em>1</em>+<em>2</em>), thrombin-antithrombin III complexes, and D-dimer. Measurements were done before the start of ECMO, after 5 minutes, and at hours <em>1</em>, <em>2</em>, 3, 4, 6, <em>1</em><em>2</em>, <em>2</em>4 and 48.

RESULTS

All seven term infants had excessively high levels of clotting activation markers within the first <em>2</em> hours of ECMO: F<em>1</em>+<em>2</em>, <em>1</em><em>1</em>.6(+/- O.9) nmol/L (mean +/- SEM); thrombin-antithrombin, 9<em>2</em>0(+/- <em>2</em>.<em>2</em>) microg/L; D-dimer, <em>1</em>5.5<em>2</em><em>2</em>(+/- 3.689) ng/L. During the next 46 hours of ECMO, F<em>1</em>+<em>2</em> and thrombin-antithrombin III complexes decreased from those high values, whereas D-dimer did not. The increase of activation markers was accompanied by low fibrinogen, low platelet counts. and prolongation of reptilase time.

CONCLUSIONS

These findings fit the pattern of consumptive coagulopathy during neonatal ECMO, especially in the first <em>2</em>4 hours.

<em>Prothrombin</em> is a vitamin K-dependent plasma protein composed of several functional domains, which is proteolytically activated into thrombin by factor Xa in the presence of factor Va, Ca<em>2</em>+, and phospholipids. During the activation, <em>prothrombin</em> is cleaved into three <em>fragments</em>: <em>fragment</em> <em>1</em>, containing a domain rich in gamma-carboxyglutamic acid residues and kringle <em>1</em> domain; <em>fragment</em> <em>2</em>, containing the kringle <em>2</em> domain; and a protease catalytic domain, thrombin. Here we studied the interaction site for factor Xa in human <em>prothrombin</em> during the activation. The isolated <em>fragment</em> <em>2</em> inhibited the activation of <em>prothrombin</em> by either <em>prothrombin</em>ase complex or factor Xa alone in a dose-dependent manner, whereas <em>fragment</em> <em>1</em> and diisopropylphosphate (DIP)-thrombin did not. Factor Xa directly bound to <em>fragment</em> <em>2</em> immobilized to microwell plates with a Kd of 9.0 x <em>1</em>0(-8) M, but not to <em>fragment</em> <em>1</em> or DIP-thrombin. Factor Xa also bound to immobilized <em>prothrombin</em> and prethrombin <em>1</em> with Kds of <em>2</em>.0 x <em>1</em>0(-7) and <em>1</em>.5 x <em>1</em>0(-7) M, respectively, suggesting that factor Xa interacts with the kringle <em>2</em> domain in these molecules. The binding of factor Xa to immobilized <em>fragment</em> <em>2</em> was Ca(<em>2</em>+)-dependent with an optimal concentration at 6 mM. In the presence of Ca<em>2</em>+, the interaction was enhanced by phospholipids in a concentration-dependent manner. To localize the factor Xa-binding site in the kringle <em>2</em> domain, <em>fragment</em> <em>2</em> was digested with lysyl endopeptidase and then trypsin after reduction and S-carboxymethylation. The resulting peptides were immobilized to microwell plates and assayed for factor Xa binding ability. The amino acid sequence of the peptide positive in the assay was determined to be residues His<em>2</em>05 to Arg<em>2</em><em>2</em>0. Factor Xa bound to a synthetic peptide corresponding to the residues His<em>2</em>05 to Arg<em>2</em><em>2</em>0 immobilized to microwell plates. The peptide inhibited factor Xa-catalyzed activation of <em>prothrombin</em>, but a peptide with the reversed sequence of His<em>2</em>05 to Arg<em>2</em><em>2</em>0 did not. These findings indicate that factor Xa interacts with at least a linear sequence, His<em>2</em>05 to Arg<em>2</em><em>2</em>0, in the kringle <em>2</em> domain of <em>prothrombin</em> during its activation into thrombin.

We have demonstrated the presence of a saturable, reversible, and Ca(<em>2</em>+)-dependent binding site for <em>1</em><em>2</em>5I-labeled factor X ([<em>1</em><em>2</em>5I]factor X) on human platelets (<em>1</em>6000 +/- <em>2</em>000 sites per platelet, Kd = 3<em>2</em>0 +/- 40 nM, n = <em>1</em><em>2</em>) activated with either thrombin or the thrombin receptor agonist peptide, SFLLRN-amide, but not with ADP. Bound [<em>1</em><em>2</em>5I]factor X could be completely removed by the addition of a Ca<em>2</em>+ chelator or an excess of unlabeled factor X. Antibodies that inhibit binding of factor X to the MAC-<em>1</em> integrin receptor of monocytes and those directed against human factor V, failed to disrupt [<em>1</em><em>2</em>5I]factor X binding to platelets. <em>Prothrombin</em>, but neither factor VII, factor IX, protein C, nor protein S, was an effective competitor of [<em>1</em><em>2</em>5I]factor X binding with a K<em>1</em> approximately Kd. [<em>1</em><em>2</em>5I]<em>Prothrombin</em> also binds to activated (but not unactivated) platelets in a saturable, reversible, and Ca(<em>2</em>+)-dependent manner (<em>2</em>0500 +/- <em>1</em>500 sites, Kd = 470 +/- <em>1</em><em>1</em>0 nM, n = 3). Annexin V potently inhibited the binding of both [<em>1</em><em>2</em>5I]factor X and [<em>1</em><em>2</em>5I]<em>prothrombin</em> (IC50 approximately 3 nM). Factor X, <em>prothrombin</em>, and <em>prothrombin</em> <em>fragment</em> <em>1</em> (residues <em>1</em>-<em>1</em>55) were equipotent inhibitors of [<em>1</em><em>2</em>5I]<em>prothrombin</em> and [<em>1</em><em>2</em>5I]factor X binding, whereas Gla-domain-less factor X was unable to compete with [<em>1</em><em>2</em>5I]factor X for platelet binding sites. Thus, it is the Gla-domains of factor X and <em>prothrombin</em> that appear to contain the regions necessary for platelet binding. The results of studies utilizing artificial phospholipid surfaces have led to the hypothesis that the substrates (FX and <em>prothrombin</em>) for the intrinsic pathway FXase and <em>prothrombin</em>ase complexes are bound to the phospholipid surface. The factor X/<em>prothrombin</em> binding site we have described on the surface of activated platelets permits the utilization of surface-bound substrates by these complexes when they are assembled on a physiologic surface.

Thromboembolism frequently complicates gastric cancer. This study examined the solid phase interaction between gastric cancer and coagulation proteins in situ that may explain coagulation activation and that may contribute to tumor progression and angiogenesis in this tumor type. Immunohistochemical techniques were applied to tissues from 37 cases of adenocarcinoma of the stomach obtained at surgical resection. Fibrinogen was present throughout the tumor stroma. Fibrin and its D-dimer cross-link sites occurred at the host-tumor interface. Subunit "a" of factor (F) XIII and F VII, IX, X, and XII were observed on cancer cells. <em>Prothrombin</em> and <em>prothrombin</em> <em>fragment</em> F<em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>) were demonstrated in the tumor stroma on cancer cells and on small blood vessels. Tissue factor (TF) was present on cancer cells and tumor-associated macrophages. Protein C was observed on cancer cells and small blood vessels, whereas protein S was present only in the vascular bed. There was no staining for tissue factor pathway inhibitor (TFPI). High-molecular-weight (HMW) urokinase plasminogen activator (u-PA) antigen was not detected, but weak and inconsistent staining for low-molecular-weight (LMW) u-PA was demonstrated on cancer cells. Weak staining for tissue plasminogen activator (t-PA) occurred on cancer cells and in the tumor stroma. In contrast, plasminogen activator inhibitor-<em>1</em> (PAI-<em>1</em>) expression was strong in the tumor stroma, along with PAI-<em>2</em> and PAI-3. The endothelium of small stromal blood vessels, particularly near the host-tumor interface, demonstrated von Willebrand factor antigen (vWF Ag). Vascular endothelial growth factor (VEGF) was present on cancer cells and stromal macrophages. These results demonstrate tumor cell-associated TF-dependent extravascular coagulation activation in situ in gastric cancer that does not appear to be counterbalanced by TFPI or sufficient fibrinolytic activity. Colocalization of VEGF with hemostatic proteins suggests that they may cooperate in the pathogenesis of gastric cancer.

OBJECTIVE

Activated protein C (APC) resistance not related to the factor V Leiden mutation is a risk factor for venous thrombosis. Oral estrogen replacement therapy (ERT) has been reported to induce APC resistance. Little is known about the effect of transdermal estrogen.

RESULTS

We enrolled <em>1</em>96 postmenopausal women who were randomly allocated to receive either <em>1</em> mg <em>1</em>7beta-estradiol orally (n=63) or 50 microg <em>1</em>7beta-estradiol transdermally per day (n=68), both associated with <em>1</em>00 mg progesterone daily or placebo (n=65) for 6 months. An activated partial thromboplastin time (APTT)-based test and the effect of APC on thrombin potential (ETP) were used. Oral ERT induced an ETP-based APC resistance compared with both placebo (P=0.006) and transdermal ERT (P<0.00<em>1</em>), but there was no significant effect of transdermal ERT compared with placebo (P=0.<em>1</em>9<em>1</em>). There was no significant effect of ERT on the APTT-based APC sensitivity ratio. <em>Prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> plasma levels were significantly higher after 6 months of treatment in women allocated to oral ERT compared with those on placebo and transdermal ERT and were positively and significantly correlated with changes in ETP-based APC sensitivity ratio.

CONCLUSIONS

Our data show that oral, unlike transdermal, estrogen induces APC resistance and activates blood coagulation. These results emphasize the importance of the route of estrogen administration.

Several parameters of primary hemostasis and markers of activation of coagulation and fibrinolysis were measured in 48 patients with severe (creatinine clearance < <em>2</em>0 ml/min) chronic renal failure (CRF) without dialysis and disease or drugs affecting hemostasis. Bleeding time (BT) was prolonged in <em>2</em>5/48 patients, and was correlated with age of patients, severity of renal failure, hematocrit, impairment in platelet aggregation-secretion and decrease in platelet ATP content. Defects in von Willebrand factor played no role in the prolongation of the BT. Multivariate analysis showed that only platelet dysfunction and severity of renal disease were independent predictors of the BT in uremia. The platelet functional disorder was significantly correlated with a reduction in platelet ATP and ADP. High levels of plasma thrombin-antithrombin complexes (TAT), <em>prothrombin</em> <em>fragment</em> F<em>1</em> + <em>2</em>, fibrinogen and factor VIIc were observed in patients with CRF, as described in prethrombotic states. Plasmin-antiplasmin complexes (PAP), fibrinogen and fibrin degradation products (FgDP, FnDP) were significantly increased, and the activity of plasminogen activator inhibitor (PAI-<em>1</em>) was slightly reduced, denoting an activation of fibrinolysis. A negative correlation was found between platelet levels of ATP and ADP with plasma TAT, F<em>1</em> + <em>2</em> and PAP. Furthermore, plasma PAI-<em>1</em> activity was negatively correlated with the BT and was lower in patients with prolonged BT as compared with controls and patients with normal BT. These links between primary hemostasis and activation of coagulation and fibrinolysis suggest that increased intravascular generation of thrombin and/or plasmin is an important mediator of the defects in primary hemostasis, prolongation of the BT and, probably, bleeding in CRF.

BACKGROUND

Smoking is associated with endothelial dysfunction. Cytokines released by injured endothelium promote vascular interactions with leukocytes and platelets. We investigated whether (a) cigarette smoking is linked to increased cytokine production, which may mediate platelet activation and thrombin generation in chronic coronary artery disease (CAD), and (b) aspirin treatment inhibits smoking-related changes on cytokines, platelets, and thrombin.

RESULTS

Plasma macrophage-colony-stimulating factor (M-CSF) and C-reactive protein (CRP) were measured in <em>1</em>00 patients with chronic CAD, 60 of whom were chronic smokers. <em>Prothrombin</em> <em>fragments</em> <em>1</em>+<em>2</em> and urinary <em>1</em><em>1</em>-dehydro-thromboxane B<em>2</em> (TXB<em>2</em>) were additionally measured in 60 of <em>1</em>00 patients (30 of whom were smokers) and in <em>2</em>4 healthy controls. Smokers (n = <em>2</em>0) matched for age, myocardial ischemia, and other risk factors with <em>2</em>0 nonsmokers entered a double-blind crossover trial of aspirin (300 mg/d for 3 weeks) versus placebo. Blood and urine measurements were repeated after each treatment. Compared with nonsmokers, smokers had 3-fold median M-CSF (<em>1</em>499 vs 476 pg/mL), <em>2</em>-fold CRP (<em>1</em>.5 vs 0.8 mg/L), and higher <em>1</em><em>1</em>-dehydro-TXB <em>2</em> (3.6 vs <em>2</em>.<em>1</em> ng/mg creatinine, P < .0<em>1</em> for all comparisons). After aspirin treatment, M-CSF, CRP, <em>1</em><em>1</em>-dehydro-TXB <em>2</em> , and <em>prothrombin</em> <em>fragments</em> <em>1</em>+<em>2</em> remained higher in smokers compared with nonsmokers despite a significant reduction of these markers by aspirin (P < .05). M-CSF remained related to <em>1</em><em>1</em>-dehydro-TXB <em>2</em> excretion during both treatment phases (P < .0<em>1</em>) suggesting that cytokine-mediated thromboxane A <em>2</em> production was not altered by aspirin.

CONCLUSIONS

Smoking is associated with increased M-CSF, CRP, and platelet activity. Although aspirin treatment reduces the proinflammatory and procoagulant markers in smokers, it does not abolish the proinflammatory effects of smoking in patients with chronic CAD.

BACKGROUND

Renalase is an enzyme released by the kidneys, which breaks down catecholamines in the blood and thus may regulate blood pressure. In kidney transplant recipients, endothelial dysfunction is often present.

OBJECTIVE

The aim of the study was to assess associations between renalase, blood pressure, and kidney function in kidney allograft recipients.

METHODS

We studied 6<em>2</em> kidney allograft recipients. Complete blood count, urea and creatinine levels, serum lipids, and fasting glucose were measured by standard laboratory methods. We also assessed markers of coagulation: <em>prothrombin</em> <em>fragments</em> <em>1</em>+<em>2</em>; fibrinolysis: tissue plasminogen activator (tPA), plasminogen activator inhibitor, plasmin-antiplasmin complexes; endothelial function/injury: von Willebrand factor (vWF), thrombomodulin, intercellular adhesion molecule, vascular cell adhesion molecule (VCAM); and inflammation: high‑sensitivity C‑reactive protein and interleukin 6. Renalase levels were assessed using a commercially available kit.

RESULTS

Mean serum renalase levels in kidney allograft recipients correlated with age, time after transplantation, soluble CD44 (sCD44), VCAM, serum creatinine, estimated glomerular filtration rate (eGFR; measured by CKD-EPI, MDRD, and Cockcroft‑Gault formulas), serum phosphate, urea, sCD<em>1</em>46, vWF, and thrombomodulin and tended to correlate with tPA. In patients with eGFR above 60 ml/min, renalase was lower than in those with lower eGFR. In hypertensive allograft recipients, renalase was significantly higher than in normotensives. A multiple regression analysis showed that renalase was predicted in 58% by serum creatinine.

CONCLUSIONS

Renalase, which is highly elevated in kidney transplant recipients, is dependent primarily on kidney function, which deteriorates with age and time after transplantation. Further studies are needed to establish the putative role of renalase in the pathogenesis of hypertension after transplantation and its possible use in novel targeted therapies.

OBJECTIVE

Symptomatic intracerebral hemorrhage (ICH) is a major complication of thrombolysis in patients with acute ischemic stroke. We analyzed whether baseline hemostatic markers could predict symptomatic ICH (SICH).

METHODS

In a multicenter study of patients treated with intravenous tissue plasminogen activator (t-PA) within 3 hours of stroke onset, we analyzed the following variables: demographic data, vascular risk factors, blood glucose at admission, time from the onset of symptoms to t-PA infusion, blood pressure, neurological deficit measured by the National Institutes of Health Stroke Scale (NIHSS) score, early signs of ischemia on the baseline computed tomography (CT) scan, and protocol deviations. In blood samples, the following markers of coagulation/fibrinolysis were measured before treatment: fibrinogen, <em>prothrombin</em> <em>fragments</em> <em>1</em>+<em>2</em>, Factor XIII, Factor VII, alpha<em>2</em> antiplasmin, plasminogen activator inhibitor-<em>1</em> (PAI-<em>1</em>), and thrombin-activatable fibrinolysis inhibitor. ICH was classified according to the European Cooperative Acute Stroke Study (ECASS) II criteria. SICH was defined as a parenchymal hematoma-<em>1</em> (PH<em>1</em>) or PH<em>2</em> type, associated with an increase in>> or =4 points on the NIHSS score appearing within 36 hours after infusion.

RESULTS

We studied <em>1</em><em>1</em>4 patients. Mean age was 68.4+/-<em>1</em><em>2</em>.7 years, and 6<em>1</em>% were men. The median baseline NIHSS score was <em>1</em>4. Mean time to treatment was <em>1</em>53+/-33 minutes. Eight patients had SICH (7%), and <em>1</em>8 patients (<em>1</em>5.7%) had asymptomatic ICH. None of the baseline markers of coagulation/fibrinolysis were associated with SICH. In the multivariate analysis, only NIHSS on admission was an independent risk factor for SICH.

CONCLUSIONS

None of the hemostatic markers analyzed in our study predicted symptomatic cerebral hemorrhage in patients with ischemic stroke treated with t-PA.

OBJECTIVE

To relate factor XIII levels and other prothrombotic markers to inflammatory bowel disease and investigate the frequency of valine34leucine and its effect on factor XIII cross-linking activity in patients with inflammatory bowel disease.

METHODS

Fifty patients with active inflammatory bowel disease but no venous thromboembolism (3<em>2</em> with ulcerative colitis, <em>1</em>8 with Crohn's disease), 50 patients with inactive inflammatory bowel disease but no venous thromboembolism (3<em>2</em> with ulcerative colitis, <em>1</em>8 with Crohn's disease), two age- and gender-matched healthy control groups of <em>1</em>00 subjects each were recruited. To further explore the relationship between valine34leucine and inflammatory bowel disease, <em>2</em><em>1</em> patients with the disease (<em>1</em>3 with ulcerative colitis and eight with Crohn's disease) and venous thromoembolism (male to female ratio = 7 : <em>1</em>4, median age 59.5 years (range, <em>1</em>9-80 years)) were recruited. Two hundred and fifteen control subjects (M : F = <em>1</em><em>2</em><em>1</em> : 94, median age 6<em>2</em> years (<em>2</em>8-74 years)), with venous thromboembolism (<em>1</em><em>1</em>9 with deep venous thrombosis, and 96 with pulmonary embolism) were drawn from the same geographical area as the patients.

METHODS

Factor XIII A, B-subunit antigen and A<em>2</em>B<em>2</em> tetramer levels were measured using an in-house sandwich enzyme-linked immunoassay method.

RESULTS

Factor XIII A<em>2</em>B<em>2</em> tetramer and the A-subunit were significantly decreased in patients with active inflammatory bowel disease compared with controls (59% vs 95%, P < 0.000<em>1</em> and 75% vs <em>1</em>0<em>2</em>%, P < 0.000<em>1</em>, respectively), but not between the inactive inflammatory bowel disease group and controls. The D-dimer and <em>prothrombin</em> <em>1</em>+<em>2</em> <em>fragment</em> levels in patients with active inflammatory bowel disease were raised compared with controls (<em>1</em>78 (<em>1</em>5<em>2</em>) vs <em>1</em>09 (84), P = 0.0007 and 8<em>2</em> (43) vs 55 (<em>2</em>8), P = 0.000<em>1</em>, respectively). The factor XIII B-subunit and factor XIII cross-linking activity were not significantly different between patients with active or inactive inflammatory bowel disease and controls. There was no significant difference in genotype distribution in inflammatory bowel disease patients with or without venous thromboembolism and respective control subjects. Levels of tissue plasminogen activator antigen were significantly increased in patients with active inflammatory bowel disease when compared to inactive inflammatory bowel disease and controls (8.9 (3.7) vs 6.7 (3.4) vs 6.9 (3.4), P < 0.00<em>1</em>).

CONCLUSIONS

Active inflammatory bowel disease is associated with activation of coagulation. Factor XIII A and A<em>2</em>B<em>2</em> tetramer levels were markedly decreased in active inflammatory bowel disease. Variations in the level of factor XIII in patients with inflammatory bowel disease could be multifactorial and in part may result from the increased formation of microthrombi and accelerated turnover of the factor XIII. We found no evidence of association of factor XIII valine34leucine polymorphism and inflammatory bowel disease.