The presence of congenital antithrombin deficiency has been consistently shown to predispose patients to venous thrombosis. We have utilized the <em>prothrombin</em> <em>fragment</em> F<em>1</em>+<em>2</em> radioimmunoassay to quantitate factor Xa activity in the blood of <em>2</em><em>2</em> asymptomatic individuals with this clinical disorder not receiving antithrombotic therapy. The mean level of F<em>1</em>+<em>2</em> was significantly elevated in these patients as compared to normal controls (3.9<em>1</em> vs. <em>1</em>.97 nM, P less than 0.00<em>1</em>). The metabolic behavior of <em>1</em>3<em>1</em> I-F<em>1</em>+<em>2</em> was found to be similar in antithrombin-deficient subjects and normal individuals. The hemostatic system hyperactivity as measured by the F<em>1</em>+<em>2</em> assay could be specifically corrected by raising the plasma antithrombin levels of the above asymptomatic individuals into the normal range. This study provides the first demonstration that the prethrombotic state can be biochemically defined as an imbalance between the production and inhibition of factor Xa enzymatic activity within the human circulation. It is known that antithrombin and alpha <em>1</em>-proteinase inhibitor (PI) are the major inhibitors of factor Xa in human plasma in the absence of heparin. To further evaluate the mechanism by which antithrombin functions as an inhibitor of factor Xa in humans, we studied five patients who exhibited severe congenital deficiencies of alpha <em>1</em>-PI. Our results indicated that the plasma of these subjects showed virtually identical decreases in plasma antifactor Xa activity in the absence of heparin when compared to antithrombin-deficient individuals, but the plasma F<em>1</em>+<em>2</em> levels in the alpha <em>1</em>-PI deficient population were not significantly different than normal. This data suggests that alpha <em>1</em>-PI does not function as a major inhibitor of factor Xa in vivo, and that a tonically active heparin-dependent mechanism exists in humans for accelerating the neutralization of this enzyme by antithrombin.

OBJECTIVE

The effects of ximelagatran, an oral direct thrombin inhibitor (DTI), recombinant hirudin (r-hirudin) and enoxaparin on thrombin generation and platelet activation were studied in humans.

BACKGROUND

Recombinant hirudin (parenteral DTI) and enoxaparin (low molecular weight heparin) have been demonstrated to be clinically effective in acute coronary syndromes. Ximelagatran is currently under investigation for the prevention and treatment of thromboembolism. The shed blood model allows for the study of thrombin generation and platelet activation in humans in vivo.

METHODS

This was an open-label, parallel-group study involving <em>1</em><em>2</em>0 healthy male volunteers randomized to receive one of three oral doses of ximelagatran (<em>1</em>5, 30 or 60 mg), r-hirudin (intravenous) or enoxaparin (subcutaneous) at doses demonstrated to be clinically effective in acute coronary syndromes, or to serve as a control. Thrombin generation (<em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> [F<em>1</em>+<em>2</em>] and thrombin-antithrombin complex [TAT]) and platelet activation (beta-thromboglobulin [beta-TG]) biomarkers were studied using a shed blood model involving blood collection from skin incisions made using standardized bleeding time devices.

RESULTS

Oral ximelagatran, intravenous r-hirudin and subcutaneous enoxaparin rapidly and significantly (p < 0.05) decreased F<em>1</em>+<em>2</em>, TAT and beta-TG levels in shed blood, indicating inhibition of thrombin generation and platelet activation. Statistically significant concentration (melagatran, the active form of ximelagatran)-response relationships for F<em>1</em>+<em>2</em> (p = 0.005), TAT (p = 0.005) and beta-TG (p < 0.00<em>1</em>) levels, with IC(50)s of 0.376 (F<em>1</em>+<em>2</em>), 0.<em>1</em>63 (TAT) and 0.<em>1</em><em>1</em>5 (beta-TG) micromol/l, were detected. Melagatran showed dose-proportional pharmacokinetics with low variability. All drugs were well tolerated.

CONCLUSIONS

Oral administration of the DTI ximelagatran resulted in a rapid inhibition of both thrombin generation and platelet activation in a concentration-dependent manner using a human shed blood model. The inhibition of thrombin generation by 60 mg ximelagatran was comparable to that observed with doses of r-hirudin and enoxaparin demonstrated to be effective for the treatment of acute coronary syndromes.

OBJECTIVE

Decreased matrix metalloproteinase-<em>1</em> (MMP-<em>1</em>) and increased levels of its inhibitor, tissue inhibitor of matrix metalloproteinase-<em>1</em> (TIMP-<em>1</em>), reflect impaired matrix degradation with an increase in fibrosis. A prothrombotic state has been described in atrial fibrillation (AF), increasing the risk of stroke and thromboembolism. Because structural abnormalities and remodeling of atria have been observed in AF, we hypothesized that the prothrombotic state in AF may be related to abnormal indexes of matrix degradation.

METHODS

We studied 48 consecutive patients (30 men; age, 70.5+/-9.0 years) with chronic nonrheumatic AF who were not on anticoagulation. Plasma levels of MMP-<em>1</em>, TIMP-<em>1</em>, and <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>, an index of thrombogenesis) were measured by enzyme-linked immunosorbent assay. M-mode, <em>2</em>-dimensional, and Doppler echocardiographic studies were performed in all patients. Research indexes were compared with data from 3<em>2</em> control subjects in sinus rhythm who were of similar age and sex.

RESULTS

Patients with AF had lower levels of MMP-<em>1</em> (P=0.0<em>1</em><em>1</em>) but increased levels of TIMP-<em>1</em> (P=0.033) and F<em>1</em>+<em>2</em> (P<0.00<em>1</em>) and a higher ratio of TIMP-<em>1</em> to MMP-<em>1</em> (P=0.009) compared with control subjects. After adjustment for sex, age, hypertension, and diabetes, TIMP-<em>1</em> levels and the ratio of TIMP-<em>1</em> to MMP-<em>1</em> correlated with F<em>1</em>+<em>2</em> levels (r=0.<em>2</em>4, P=0.038; and r=0.<em>2</em>6, P=0.0<em>2</em>3, respectively). In multivariate analysis, there was no independent relationship between MMP-<em>1</em>, TIMP-<em>1</em>, or ratio of TIMP-<em>1</em> to MMP-<em>1</em> and the presence of AF.

CONCLUSIONS

Patients with AF have evidence of impaired matrix degradation, but this was not independently associated with the presence of AF on multivariate analysis. However, an independent relationship was found between the MMP/TIMP system and prothrombotic state (assessed by F<em>1</em>+<em>2</em> levels).

A sequence variation in the 3'-untranslated region of the <em>prothrombin</em> (PT) gene (<em>2</em>0<em>2</em><em>1</em>0 G->>A) was recently claimed to be associated with elevated plasma <em>prothrombin</em> levels and an increased risk for venous and arterial thrombosis. We examined the prevalence of the <em>2</em>0<em>2</em><em>1</em>0 A allele in the <em>prothrombin</em> gene in 400 healthy controls and in <em>2</em>63 patients with proven premature atherosclerotic disease. In addition, we measured <em>prothrombin</em>, <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>, thrombin-antithrombin (TAT) complex and D-dimer levels in plasma from carrier and non-carrier patients. The frequency of the variant allele was <em>1</em>% in the control subjects and <em>2</em>.7% in the patient group, yielding a relative risk (RR) for the <em>2</em>0<em>2</em><em>1</em>0 A allele of <em>2</em>.7 (95% CI 0.8-9.4). All heterozygotes in the patient group were found to have had a myocardial infarction (MI), yielding a RR for MI of 4.<em>2</em> (95% CI <em>1</em>.<em>2</em>-<em>1</em>4.6). Plasma <em>prothrombin</em> levels in carriers (<em>1</em><em>2</em>6+/-<em>1</em>0) were higher than in non-carriers (<em>1</em>03+/-<em>1</em>, P=0.0<em>2</em>). The levels of TAT complexes (<em>1</em>6+/-9 v 6+/-<em>1</em> microg/ml, P=0.0<em>2</em>) as well as of <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (<em>1</em>.5+/-0.3 v <em>1</em>.0+/-0.<em>1</em> nmol/l, P=0.0<em>2</em>) were also elevated in carriers of the mutation. Our findings suggest that the <em>2</em>0<em>2</em><em>1</em>0 G->>A mutation in the <em>prothrombin</em> gene is a genetic risk factor for MI. In addition, our data provide evidence for an association of the mutation with excessive thrombin generation, which may contribute to the understanding of its role in venous and arterial disease.

Protease nexin-<em>2</em>/amyloid beta-protein precursor (PN-<em>2</em>/A beta PP) is a Kunitz-type protease inhibitor which has been shown to be a tight-binding inhibitor of coagulation factors XIa and IXa. Here we show that PN-<em>2</em>/A beta PP and its KPI domain also inhibited isolated factor Xa with a Ki of <em>1</em>0(-8) M. On a solid phase binding assay, PN-<em>2</em>/A beta PP formed a complex with factor Xa. Incubation of molar excess factor Xa to PN-<em>2</em>/A beta PP produced a single cleavage within PN-<em>2</em>/A beta PP's heparin binding domain liberating a 8.<em>2</em>-kDa amino-terminal peptide. PN-<em>2</em>/A beta PP and its KPI domain equally inhibited factor Xa in the <em>prothrombin</em>ase complex with a Ki of <em>1</em>.9 x <em>1</em>0(-8) M and <em>1</em>.3 x <em>1</em>0(-8) M, respectively. A beta PP695 which does not contain the KPI domain was a substrate of factor Xa but did not inhibit it, indicating the PN-<em>2</em>/A beta PP inhibition of factor Xa was not substrate inhibition. All of the factor Xa inhibition in the <em>prothrombin</em>ase complex by PN-<em>2</em>/A beta PP and its KPI domain on the chromogenic assay was accounted for by inhibition of release of <em>prothrombin</em> <em>fragment</em> F<em>1</em>+<em>2</em> as determined on immunochemical assay. In the <em>prothrombin</em>ase complex, PN-<em>2</em>/A beta PP inhibited factor Xa with a kassoc = <em>1</em>.8 +/- 0.7 x <em>1</em>0(6) M-<em>1</em> min-<em>1</em> similar to antithrombin III and heparin inhibition (kassoc of 3.0 +/- 0.<em>2</em> x <em>1</em>0(6) M-<em>1</em> min-<em>1</em>). These studies indicated that PN-<em>2</em>/A beta PP in the assembled <em>prothrombin</em>ase complex inhibited factor Xa comparable to antithrombin III in the presence of heparin. PN-<em>2</em>/A beta PP's factor Xa inhibitory activity along with its known inhibition of factors XIa and IXa suggest that this protease inhibitor and related proteins could be regulators of hemostatic reactions on membranes of cells in the intravascular compartment.

BACKGROUND

Growing evidence indicates that ambient air pollution is associated with exacerbation of chronic diseases like chronic pulmonary disease. A prospective panel study was conducted to investigate short-term changes of blood markers of inflammation and coagulation in response to daily changes in air pollution in Erfurt, Germany. <em>1</em><em>2</em> clinical visits were scheduled and blood parameters were measured in 38 male patients with chronic pulmonary disease during winter <em>2</em>00<em>1</em>/<em>2</em>00<em>2</em>. Additive mixed models with random patient intercept were applied, adjusting for trend, weekday, and meteorological parameters. Hourly data on ultrafine particles (UFP, 0.0<em>1</em>-0.<em>1</em> mum), accumulation mode particles (ACP, 0.<em>1</em>-<em>1</em>.0 mum), PM<em>1</em>0 (particulate matter (<em>1</em>0 mum in diameter), elemental (EC) and organic carbon (OC), gaseous pollutants (nitrogen monoxide [NO], nitrogen dioxide [NO<em>2</em>], carbon monoxide [CO], and sulphur dioxide [SO<em>2</em>]) were collected at a central monitoring site and meteorological data were received from an official network. For each person and visit the individual <em>2</em>4-hour average of pollutants immediately preceding the blood withdrawal (lag 0) up to day 5 (lag<em>1</em>-4) and 5-day running means were calculated.

RESULTS

Increased levels of fibrinogen were observed for an increase in one interquartile range of UFP, PM<em>1</em>0, EC, OC, CO, and NO revealing the strongest effect for lag 3. E-selectin increased in association with ACP and PM<em>1</em>0 with a delay of one day. The ACP effect was also seen with the 5-day-mean. The pattern found for D-dimer was inconsistent. <em>Prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> decreased with lag 4 consistently for all particulate pollutants. Von Willebrand factor antigen (vWF) showed a consistent decrease in association with almost all air pollutants with all lags except for lag 0. No associations were found for C-reactive protein, soluble intercellular adhesion molecule <em>1</em>, serum amyloid A and factor VII.

CONCLUSIONS

These results suggest that elevated concentrations of air pollution are associated with changes in some blood markers of inflammation and coagulation in patients with chronic pulmonary disease. The clinical implications of these findings need further investigation.

The amino acid sequence of the nonthrombin half of human <em>prothrombin</em> is presented. <em>Prothrombin</em> <em>fragment</em> <em>1</em> has <em>1</em>55 amino acid residues as compared with <em>1</em>56 for the bovine equivalent. Ten gamma-carboxyglutamic acid residues are at the same location in each species. Human <em>prothrombin</em> <em>fragment</em> <em>2</em> contains <em>1</em><em>1</em>8 amino acid residues, as does the similar bovine <em>fragment</em>. Comparing bovine and human <em>prothrombin</em> <em>fragment</em> <em>1</em> we found <em>1</em>3<em>1</em> residues to be identical (84%). In <em>prothrombin</em> <em>fragment</em> <em>2</em>, 84 residues were identical (7<em>1</em>%). Assuming a time span of 90 million years since the radiation of several orders of placental mammals, <em>prothrombin</em> <em>fragments</em> <em>1</em> and <em>2</em> incorporated one substitution site per <em>1</em>00 amino acid sites every <em>1</em><em>1</em>.<em>2</em> and 6.3 million years, respectively. Internal homology is acribed to partial gene duplication, with the most likely crossover point located between residues 60-6<em>1</em> and residues <em>1</em>65-<em>1</em>66.

OBJECTIVE

To determine the relationship between <em>prothrombin</em> and crystal matrix protein (CMP). CMP is the predominant protein found in the organic matrix of calcium oxalate (CaOx) crystals generated from human urine and is a 3<em>1</em> kDa glycoprotein, whose N-terminal amino acid sequence shares homology with human <em>prothrombin</em>.

METHODS

CaOx crystallization was induced in ultrafiltered (UF) human urine containing either plasma or serum derived from the same healthy donor, by the addition of sodium oxalate. The crystals were demineralized and the resulting protein extracts analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, using antibodies raised against human <em>prothrombin</em> and the C-terminus of <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>).

RESULTS

Prothrombin was detected in extracts of crystals precipitated from the UF urine in the presence of plasma, while CMP was completely absent. Crystals precipitated from UF urine supplemented with serum contained relatively large amounts of F<em>1</em> + <em>2</em> and a protein with the same electrophoretic mobility as CMP. Analysis of a standard preparation of F<em>1</em> + <em>2</em> which also contained <em>prothrombin</em> <em>fragment</em> <em>1</em> (F<em>1</em>) as a minor contaminant, showed a protein with electrophoretic and staining properties comparable to CMP.

CONCLUSIONS

CMP is a urinary form of F<em>1</em>, a degradation product of <em>prothrombin</em> possessing the domain rich in gamma-carboxyglutamic acid, which may have undergone some molecular modification either before or after its release into the urine.

The effects of a fish-enriched diet or dietary supplements consisting of either fish oil or a docosahexaenoic acid-rich oil (DHA-oil) on platelet aggregation and hemostatic factors were studied in healthy male students. After an experimental period of <em>1</em>5 weeks, the levels of tissue factor pathway inhibitor, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> and fibrinogen as well as factor VII activity were not changed. Factor X activity was slightly decreased by the fish diet (P < 0.05). Collagen but not ADP-induced maximum platelet aggregation decreased in the fish diet and the fish oil groups (P < 0.05 in both). In the DHA-oil group there was a slight, statistically insignificant, increase of platelet aggregation which correlated significantly with the decrease of plasma triglycerides. Platelet aggregation measured 4 h after a standardized fat meal was lower than in the fasting state and this decrease correlated with the increase of plasma triglycerides. These results show that a fish diet and fish oil, but not DHA-oil, inhibit in vitro platelet aggregation and that hemostatic factors are not affected by moderate n-3 fatty acid supplementation.

During factor Xa-catalyzed <em>prothrombin</em> activation, several other reaction products accumulate as a result of proteolysis of <em>prothrombin</em> and its activation products by thrombin and meizothrombin. Gel electrophoretic analysis and N-terminal sequencing of reaction products showed that in the absence of Ca<em>2</em>+ ions thrombin cleaved the following peptide bonds: Arg5<em>1</em>-Thr5<em>2</em>/Arg54-Asp55 in the <em>fragment</em> <em>1</em> (F<em>1</em>) domain (k = 0.4 x <em>1</em>0(4) M-<em>1</em> s-<em>1</em>), Arg<em>1</em>55-Ser<em>1</em>56 in <em>prothrombin</em> (k = <em>2</em> x <em>1</em>0(4) M-<em>1</em> s-<em>1</em>), and Arg<em>2</em>84-Thr<em>2</em>85 in prethrombin <em>1</em> (k = 0.0<em>2</em> x <em>1</em>0(4) M-<em>1</em> s-<em>1</em>). In the presence of <em>2</em>.5 mM CaCl<em>2</em>, cleavage in <em>fragment</em> <em>1</em> (Arg5<em>1</em>-Thr5<em>2</em>/Arg54-Asp55) was not detectable, whereas cleavage at Arg<em>1</em>55-Ser<em>1</em>56 (i.e., removal of F<em>1</em>) was inhibited <em>2</em>5-fold. Cleavage at Arg<em>2</em>84-Thr<em>2</em>85 (formation of prethrombin <em>2</em> des-<em>1</em>-<em>1</em>3) was not affected by the presence of Ca<em>2</em>+ ions. Meizothrombin rapidly converted itself into meizothrombin des-F<em>1</em>. The half-life (t<em>1</em>/<em>2</em> = approximately 30 s) of this reaction was independent of the meizothrombin concentration (0.<em>1</em>-<em>1</em> microM meizothrombin), which is indicative for intramolecular autocatalysis (k = 0.0<em>2</em> s-<em>1</em> in the presence of <em>2</em>.5 mM Ca<em>2</em>+ ions). Since the rapid removal of <em>fragment</em> <em>1</em> precludes investigations of the cleavage at Arg<em>2</em>84-Thr<em>2</em>85 in intact meizothrombin, we analyzed the cleavage of this peptide bond in R<em>1</em>55A-meizothrombin, a recombinant product that is resistant to autocatalytic removal of the <em>fragment</em> <em>1</em> domain. In the absence of phospholipids, R<em>1</em>55A-meizothrombin converted itself into thrombin des-<em>1</em>-<em>1</em>3 by a combination of intramolecular (k = 0.8 x <em>1</em>0(-4) s-<em>1</em>) and intermolecular autocatalysis (k = 0.<em>2</em> x <em>1</em>0(3) M-<em>1</em> s-<em>1</em>). Intramolecular autocatalytic conversion of R<em>1</em>55A-meizothrombin into thrombin was not affected by the presence of phospholipids (k = 0.8 x <em>1</em>0(-4) s-<em>1</em>), whereas intermolecular autocatalysis was accelerated <em>2</em>5-fold (k = 5.6 x <em>1</em>0(3) M-<em>1</em> s-<em>1</em>) by phospholipid vesicles. Since factor Xa/Va-catalyzed conversion of meizothrombin into thrombin occurs with k = 5.5 x <em>1</em>0(8) M-<em>1</em> s-<em>1</em>, we conclude that in reaction systems containing purified proteins autocatalysis of meizothrombin hardly contributes to thrombin formation during factor Xa-catalyzed <em>prothrombin</em> activation.

BACKGROUND

Elevated coagulative molecular markers could reflect the prothrombotic state in the cardiovascular system of patients with non-valvular atrial fibrillation (NVAF). A prospective, cooperative study was conducted to determine whether levels of coagulative markers alone or in combination with clinical risk factors could predict subsequent thromboembolic events in patients with NVAF.

METHODS

Coagulative markers of <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, D-dimer, platelet factor 4, and beta-thromboglobulin were determined at the enrollment in the prospective study.

RESULTS

Of 509 patients with NVAF (mean age, 66.6 +/- <em>1</em>0.3 years), <em>2</em>63 patients were treated with warfarin (mean international normalized ratio, <em>1</em>.86), and <em>1</em>63 patients, with antiplatelet drugs. During an average follow-up period of <em>2</em>.0 years, 3<em>1</em> thromboembolic events occurred. Event-free survival was significantly better in patients with D-dimer level < <em>1</em>50 ng/ml than in those with D-dimer level>or==<em>1</em>50 ng/ml. Other coagulative markers, however, did not predict thromboembolic events. Age >>or==75 years), cardiomyopathies, and prior stroke or transient ischemic attack were independent, clinical risk factors for thromboembolism. Thromboembolic risk in patients without the clinical risk factors was quite low (0.7%/year) when D-dimer was < <em>1</em>50 ng/ml, but not low (3.8%/year) when D-dimer was>>or==<em>1</em>50 ng/ml. It was >5%/year in patients with the risk factors regardless of D-dimer levels. This was also true when analyses were confined to patients treated with warfarin.

CONCLUSIONS

D-dimer level in combination with clinical risk factors could effectively predict subsequent thromboembolic events in patients with NVAF even when treated with warfarin.

Membrane binding by <em>prothrombin</em>, mediated by its N-terminal <em>fragment</em> <em>1</em> (F<em>1</em>) domain, plays an essential role in its proteolytic activation by <em>prothrombin</em>ase. Thrombin is produced in two cleavage reactions. One at Arg(3<em>2</em>0) yields the proteinase meizothrombin that retains membrane binding properties. The second, at Arg(<em>2</em>7<em>1</em>), yields thrombin and severs covalent linkage with the N-terminal <em>fragment</em> <em>1</em>.<em>2</em> (F<em>1</em><em>2</em>) region. Covalent linkage with the membrane binding domain is also lost when prethrombin <em>2</em> (P<em>2</em>) and F<em>1</em><em>2</em> are produced following initial cleavage at Arg(<em>2</em>7<em>1</em>). We show that at the physiological concentration of <em>prothrombin</em>, thrombin formation results in rapid release of the proteinase into solution. Product release from the surface can be explained by the weak interaction between the proteinase and F<em>1</em><em>2</em> domains. In contrast, the zymogen intermediate P<em>2</em>, formed following cleavage at Arg(<em>2</em>7<em>1</em>), accumulates on the surface because of a approximately <em>2</em>0-fold higher affinity for F<em>1</em><em>2</em>. By kinetic studies, we show that this enhanced binding adequately explains the ability of unexpectedly low concentrations of F<em>1</em><em>2</em> to greatly enhance the conversion of P<em>2</em> to thrombin. Thus, the utilization of all three possible substrate species by <em>prothrombin</em>ase is regulated by their ability to bind membranes regardless of whether covalent linkage to the F<em>1</em><em>2</em> region is maintained. The product, thrombin, interacts with sufficiently poor affinity with F<em>1</em><em>2</em> so that it is rapidly released from its site of production to participate in its numerous hemostatic functions.

Cardiovascular risk factors were compared between <em>1</em><em>2</em>6 people with non-insulin-dependent diabetes mellitus (NIDDM) and 530 non-diabetics (controls), in a random sample of people (Chinese, Malays, and Asian Indians) aged 40-69 years from the general population of Singapore. Data were adjusted for age and ethnicity. For both genders, people with NIDDM had higher mean body mass indices, waist-hip ratios and abdominal diameters. They also had a higher prevalence of hypertension, higher mean levels of fasting serum triglyceride, slightly lower mean levels of serum high-density-lipoprotein cholesterol, and higher mean levels of plasma plasminogen activator inhibitor-<em>1</em> and tissue plasminogen activator (antigen). These factors are components of syndrome X (metabolic syndrome) and increase the risk of atherosclerosis and thrombosis. In contrast, there were no important differences for cigarette smoking, serum total and low-density-lipoprotein cholesterol, serum apolipoproteins A<em>1</em> and B, plasma factor VIIc and plasma <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>. Females with NIDDM, but not males, had a higher mean serum fibrinogen level than non-diabetics, which could explain why NIDDM has a greater cardiovascular effect in females than males. Serum lipoprotein(a) concentrations were lower in people with NIDDM. Mean levels of serum ferritin, a pro-oxidant, were higher in people with NIDDM than controls, but there were no important differences for plasma vitamins A, C and E, and serum selenium, which are anti-oxidants.

Pediatric patients with acute lymphoblastic leukemia (ALL) are at an increased risk of thromboembolic events. Potential responsible mechanisms include the disease process itself, treatment with chemotherapeutic agents (particularly L-Asparaginase [ASP]), or a combination of the disease and treatment. We studied thrombin regulation in <em>2</em>6 consecutive children with ALL and <em>1</em>4 healthy age-matched controls by: (<em>1</em>) plasma concentrations of <em>prothrombin</em>; (<em>2</em>) plasma inhibition of <em>1</em><em>2</em>5I-alpha-thrombin; and (3) four biochemical markers of in vivo thrombin activation (thrombin complexed to its inhibitor antithrombin III [ATIII; TAT], <em>prothrombin</em> <em>fragment</em> <em>1</em>.<em>2</em> (F<em>1</em>.<em>2</em>), activated protein C complexed to the inhibitors alpha <em>1</em> antitrypsin [APCAT]), and protein C inhibitor (APC-PCI). Measurements were made at presentation before treatment, after treatment with ASP alone, and during combination chemotherapy with and without ASP. At presentation, the capacity to generate thrombin (reflected by plasma <em>prothrombin</em> concentrations) and the capacity to inhibit thrombin (<em>1</em><em>2</em>5I-alpha-thrombin--inhibitor complex formation) were similar in children with ALL compared with that for healthy children. After ASP alone or as part of combination chemotherapy, <em>prothrombin</em> levels were preserved, whereas plasma inhibition of <em>1</em><em>2</em>5I-alpha-thrombin decreased significantly because of a decrease in plasma concentrations of inhibitors, most importantly ATIII. After combination chemotherapy without ASP, plasma concentrations of ATIII and the capacity to inhibit <em>1</em><em>2</em>5I-alpha-thrombin returned to normal values, whereas <em>prothrombin</em> levels increased above control values. Thrombin generation in vivo also differed from healthy controls. At presentation, plasma concentrations of three of four markers of in vivo thrombin activity (TAT, F<em>1</em>.<em>2</em>, APCAT, but not APC-PCI) were increased in children with ALL. Neither ASP alone nor combination chemotherapy with or without ASP significantly altered values of these three markers. In summary, although the in vitro capacity to generate thrombin was preserved, the in vitro capacity to inhibit <em>1</em><em>2</em>5I-alpha-thrombin decreased after ASP therapy. Evidence for increased endogenous thrombin generation was documented in children with ALL at presentation and throughout treatment. We speculate that poor regulation of this thrombin may contribute to thrombotic complications in children with ALL.

BACKGROUND

Increased hypercoagulability has been reported with low doses of direct thrombin inhibitors but not with direct factor Xa inhibitors.

OBJECTIVE

To compare the effects of rivaroxaban with those of melagatran and dabigatran on thrombin generation (TG) and tissue factor-induced hypercoagulability and to explore the possible involvement of the thrombin-thrombomodulin/activated protein C system.

METHODS

In normal human plasma and in protein C-deficient plasma, TG was investigated in vitro in the presence and absence of recombinant human soluble thrombomodulin (rhs-TM). TG was determined by calibrated automated thrombography and an ELISA for <em>prothrombin</em> <em>fragments</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em> ). In an in vivo rat model, hypercoagulability was induced by tissue factor; levels of thrombin-antithrombin (TAT) and fibrinogen and the platelet count were determined.

RESULTS

Rivaroxaban inhibited TG in a concentration-dependent manner. In the absence of rhs-TM, melagatran and dabigatran also inhibited TG concentration dependently. However, in the presence of rhs-TM, lower concentrations of melagatran (<em>1</em><em>1</em>9-474 nmol L(-<em>1</em>) ) and dabigatran (68-545 nmol L(-<em>1</em>) ) enhanced endogenous thrombin potential, peak TG, and F<em>1</em>+<em>2</em> formation in normal plasma but not in protein C-deficient plasma. In vivo, rivaroxaban dose-dependently inhibited TAT generation, whereas melagatran showed a paradoxical effect, with an increase in TAT and a small decrease in fibrinogen and platelet count at lower doses.

CONCLUSIONS

Low concentrations of the direct thrombin inhibitors melagatran and dabigatran enhanced TG and hypercoagulability, possibly via inhibition of the protein C system. In contrast, rivaroxaban reduced TG and hypercoagulability under all conditions studied, suggesting that it does not suppress this negative-feedback system.

BACKGROUND

Chest pain is a frequent symptom in the emergency department and often presents a diagnostic challenge. Because coronary thrombosis is a hallmark of acute ischemic syndromes, the substrates of the coagulation and fibrinolysis cascades may be markers of coronary ischemia. The objective of this study was to determine the diagnostic value of several hemostatic markers in patients presenting to the emergency department (ED) with chest pain syndromes.

METHODS

Two hundred fifty-seven consecutive patients with acute chest pain were studied in this prospective study conducted in an urban ED. D-Dimer levels were measured at admission to the ED in all patients. We also measured thrombin-antithrombin complexes, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, activated factor VII, and fibrinogen. We used regression analysis to estimate the likelihood of myocardial infarction and the diagnostic value of D-dimer.

RESULTS

D-Dimer and fibrinogen levels were significantly higher in patients with acute ischemic events (myocardial infarction and unstable angina) than in nonischemic patients (P <.0<em>1</em> and P =.0<em>2</em>, respectively). The other hemostatic markers were not significantly elevated in patients with ischemic events. D-Dimer level >500 microg/L had an independent diagnostic value for myocardial infarction and increased the diagnostic sensitivity of the electrocardiogram and history from 73% to 9<em>2</em>%.

CONCLUSIONS

D-Dimer, an expression of ongoing thrombus formation and lysis, is a marker of substantial incremental value for the early diagnosis of acute coronary syndromes presenting with chest pain. It adds independent information to the traditional assessment for myocardial infarction. D-Dimer can be incorporated into clinical decision models in the ED.

The complement system and the Toll-like (TLR) co-receptor CD<em>1</em>4 play important roles in innate immunity and sepsis. Tissue factor (TF) is a key initiating component in intravascular coagulation in sepsis, and long pentraxin 3 (PTX3) enhances the lipopolysaccharide (LPS)-induced transcription of TF. The aim of this study was to study the mechanism by which complement and CD<em>1</em>4 affects LPS- and Escherichia coli (E. coli)-induced coagulation in human blood. Fresh whole blood was anti-coagulated with lepirudin, and incubated with ultra-purified LPS (<em>1</em>00 ng/ml) or with E. coli (<em>1</em> × <em>1</em>0(7) /ml). Inhibitors and controls included the C3 blocking peptide compstatin, an anti-CD<em>1</em>4 F(ab')<em>2</em> antibody and a control F(ab')<em>2</em> . TF mRNA was measured using quantitative polymerase chain reaction (qPCR) and monocyte TF surface expression by flow cytometry. TF functional activity in plasma microparticles was measured using an amidolytic assay. <em>Prothrombin</em> <em>fragment</em> F <em>1</em>+<em>2</em> (PTF<em>1</em>.<em>2</em>) and PTX3 were measured by enzyme-linked immunosorbent assay (ELISA). The effect of TF was examined using an anti-TF blocking antibody. E. coli increased plasma PTF<em>1</em>.<em>2</em> and PTX3 levels markedly. This increase was reduced by 84->99% with compstatin, 55-97% with anti-CD<em>1</em>4 and>> 99% with combined inhibition (P < 0·05 for all). The combined inhibition was significantly (P < 0·05) more efficient than compstatin and anti-CD<em>1</em>4 alone. The LPS- and E. coli-induced TF mRNA levels, monocyte TF surface expression and TF functional activity were reduced by>> 99% (P < 0·05) with combined C3 and CD<em>1</em>4 inhibition. LPS- and E. coli-induced PTF<em>1</em>.<em>2</em> was reduced by 76-8<em>1</em>% (P < 0·05) with anti-TF antibody. LPS and E. coli activated the coagulation system by a complement- and CD<em>1</em>4-dependent up-regulation of TF, leading subsequently to <em>prothrombin</em> activation.

The behavior of hemostatic system activation during protracted physical exercise is well known, but the duration of its modification is not yet defined. In order to evaluate the time of hemostatic system activation after prolonged strenuous endurance physical exercise (typical marathon race: 4<em>2</em>.<em>1</em>95 km, v=<em>1</em>5.35 km/h; mean length of time run <em>2</em>.45+/-0.<em>1</em>5 hours) <em>1</em><em>2</em> well-trained long-distance male runners (mean age: 35+/-7, range <em>2</em>5-47 years) were investigated. Blood samples were drawn in the morning on the day before the performance, immediately after the race, and <em>2</em>4 hours and 48 hours after the end of run. With respect of baseline, immediately after the race, a significant decrease of fibrinogen (-<em>2</em>5%) and significant increases of <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (+633%) and thrombin-antithrombin complex (+848%) were observed. A significant acceleration of euglobulin lysis time (-4<em>1</em>%), and rises of plasma levels of tissue plasminogen activator antigen (+36<em>1</em>%), plasminogen activator inhibitor type <em>1</em> antigen (+<em>2</em>35%), d-dimer (+<em>2</em><em>1</em>5%), and plasma fibrinogen degradation products (+<em>1</em><em>2</em>00%) were also found. Only a slight, yet not significant, decrease in plasminogen activator inhibitor type <em>1</em> activity was observed. One day after the end of marathon different parameters were still unchanged. Forty-eight hours after the competition all parameters investigated returned to baseline values. These results indicate a persistence of clotting as well as fibrinolysis activation up to <em>2</em>4 hours after the end of the race.

For rehabilitation training it is recommended that the intensity of exercise should be clearly below the individual anaerobic threshold (IAT). We investigated blood coagulation, particularly endogenous thrombin potential (ETP) and fibrinolysis following a standardized treadmill (TR) ergometer test at 90% IAT for 60-<em>1</em><em>2</em>0 min. Sixteen healthy male non-smokers underwent the TR test. Blood samples were taken after a 30-min rest, immediately after exercise, and <em>2</em> h after exercise completion. Extrinsic and intrinsic total (TTP(ex+in)) and endogenous (ETP(ex+in)) thrombin potential, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>), thrombin-antithrombin complex (TAT), plasmin-alpha<em>2</em>-antiplasmin complex (PAP), D-dimer, tissue plasminogen activator antigen and activity (tPA-AG and tPA-ACT) and plasminogen activator inhibitor type <em>1</em> antigen and activity (PAI-<em>1</em>-AG and PAI-<em>1</em>-ACT) were measured. Immediately after TR, F<em>1</em>+<em>2</em>, TAT and TTP(ex+in) were increased ( P<0.05) while ETP(ex+in) remained unchanged. In contrast, PAP, D-dimer, tPA-AG, tPA-ACT ( P<0.05) were distinctly enhanced while PAI-<em>1</em>-ACT was decreased ( P<0.05) immediately after exercise. The changes in tPA-AG, tPA-ACT, and PAI-<em>1</em>-ACT were reversed to nearly baseline while the enhancement in PAP and D-dimer was prolonged by more than <em>2</em> h after exercise. Long-duration exercise between 60 and <em>1</em><em>2</em>0 min controlled by IAT (90%) on a TR ergometer only implicates a small increase in thrombin generation markers and total (free and alpha(<em>2</em>)-macroglubulin-bound thrombin), but not in endogenous (free) thrombin potential alone. In contrast, fibrinolysis is distinctly increased after this type of exercise. Endurance exercise with an intensity below 90% IAT and a duration below <em>2</em> h generates a more favourable condition for fibrinolysis than for blood coagulation in healthy young subjects. Data are given as mean (SD).

OBJECTIVE

To evaluate thrombogenicity of prothrombin complex concentrates (PCCs) in critically ill patients.

METHODS

Prospective clinical study.

METHODS

Medical intensive care unit at a university hospital.

METHODS

16 consecutive patients suffering from acquired deficiencies of coagulation factors and with either overt bleeding from any site or a planned invasive procedure.

METHODS

2000 factor IX units of PCCs intravenously.

RESULTS

Prothrombin time (PT), activated partial prothrombin time, fibrinogen, platelet count, plasma levels of coagulation factors II, V, VII, VIII, IX, X, antithrombin, protein C, thrombin-antithrombin complex (TAT), prothrombin fragment F(1+2), and the fibrin degradation product D-dimer were measured prior to and 1, 3, and 24 h after administration of PCCs. PT as well as coagulation factors II, VII, IX, and X, TAT, and F(1+2) showed a significant increase after administration of PCCs. All other parameters remained unchanged.

CONCLUSIONS

Administration of PCCs induces thrombin generation. No evidence for induction of disseminated intravascular coagulation in biochemical terms could be found. When rapid correction of acquired coagulation factor disturbances is warranted, the use of PCCs seems reasonable, but the elevated risk of intravascular thrombus formation should be kept in mind.

OBJECTIVE

Periodontitis is a chronic infectious disease and has been associated with cardiovascular diseases (CVD). We investigated whether plasma levels of markers of a prothrombotic state were elevated in patients with periodontitis in comparison with healthy controls.

METHODS

Untreated patients with moderate (n=53) and severe periodontitis (n=38) and healthy controls (n=39) were recruited. Levels of von Willebrand factor (vWF), <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>), plasminogen activator inhibitor-<em>1</em> (PAI-<em>1</em>) activity and D-dimer were measured as markers of a prothrombotic state.

RESULTS

The erythrocyte sedimentation rate (ESR), plasma C-reactive protein (CRP) and leucocyte counts (WBC) were significantly higher in patients with periodontitis. No statistically significant difference was found among the three groups for vWF (p=0.<em>2</em>64), F<em>1</em>+<em>2</em> (p=0.<em>2</em>95) and D-dimer (p=0.57<em>2</em>). However, PAI-<em>1</em> was clearly elevated in the severe periodontitis group (p=0.00<em>1</em>), even after adjusting for potential confounding factors (p(adj)=0.004). Moreover, more patients than controls were having vWF and PAI-<em>1</em> levels above the respective population medians.

CONCLUSIONS

In periodontitis, elevated levels of PAI-<em>1</em> activity are observed compared with healthy controls. This may increase the potential for impaired fibrinolysis, a condition that results in a prothrombotic state. We suggest that this state, if left untreated, may contribute to an increased risk for CVD.

A prospective cohort study was performed in 50 patients with dengue haemorrhagic fever (DHF) to determine the potential role of the contact activation system and factor XI activation (intrinsic pathway) in the coagulation disorders in DHF. To establish whether TAFI (thrombin-activatable fibrinolysis inhibitor) was involved in the severity of the coagulation disorders, the TAFI antigen and activity levels were also determined. Markers of contact activation (kallikrein--C<em>1</em>-inhibitor complexes), the intrinsic pathway of coagulation (factor XIa--C<em>1</em>-inhibitor complexes) and TAFI were measured and correlated to thrombin generation markers (thrombin--anti-thrombin complexes (TAT), <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>)) and a marker for fibrinolysis [plasmin--alpha <em>2</em>--anti-plasmin complexes (PAP)]. Activation of the intrinsic pathway of coagulation was clearly demonstrated by elevated levels of factor XIa--C<em>1</em>-inhibitor complexes, without evidence of contact activation, reflected by undetectable kallikrein--C<em>1</em>-inhibitor complexes. Both TAFI antigen and activity levels were decreased in all patients, which may contribute to the severity of bleeding complications in DHF because of the impaired capacity of the coagulation system to protect the fibrin clot from fibrinolysis. These findings in a human viral infection model are in accordance with earlier findings in bacterial sepsis.

OBJECTIVE

The objective of this study was to determine the cause of purpura fulminans, disseminated intravascular coagulation, or thrombosis in seven children with varicella. All children were found to have a lupus anticoagulant and acquired protein S deficiency. Thrombosis in five children was associated with presumed or documented infection with streptococcus.

METHODS

Coagulation tests included determinations of the activated partial thromboplastin time, the <em>prothrombin</em> time, the dilute Russell viper venom time, the <em>prothrombin</em> F <em>1</em> + <em>2</em> <em>fragment</em>, the C4b-binding protein (C4b), total and free protein S antigen, and clotting activities of factors II, V, VII, and X and of protein C and protein S. Autoantibodies to phospholipids, cardiolipin, and protein S were determined in enzyme-linked immunosorbent assays.

RESULTS

All children had a lupus anticoagulant and acquired protein S deficiency. Thrombosis in five children was associated with presumed or documented infection with streptococcus. All children transiently expressed free protein S deficiency, elevated levels of IgG, IgM, or both binding to protein S, the lupus anticoagulant, and increased concentration of the F <em>1</em>+<em>2</em> <em>fragment</em>. Four children also had antiphospholipid or anticardiolipin antibodies. In one child a purified IgG fraction cross-reacted with both protein S and a specific varicella antigen.

CONCLUSIONS

A subset of children with varicella infection, some of whom are coinfected with streptococcus, are prone to development of a lupus anticoagulant and an autoantibody to protein S, which results in acquired free protein S deficiency. Such children are at risk of having life-threatening thrombotic events.

Incorporation of factor (F) Va into <em>prothrombin</em>ase directs <em>prothrombin</em> activation by FXa through the meizothrombin pathway, characterized by initial cleavage at Arg(3<em>2</em>0). We have shown that a pentapeptide with the sequence DYDYQ specifically inhibits this pathway. It has been also established that Hir(54-65)(SO(3)(-)) is a specific inhibitor of <em>prothrombin</em>ase. To understand the role of FVa within <em>prothrombin</em>ase at the molecular level, we have studied thrombin formation by <em>prothrombin</em>ase in the presence of various <em>prothrombin</em>-derived <em>fragments</em> alone or in combination. Activation of prethrombin <em>1</em> is slow with cleavages at Arg(3<em>2</em>0) and Arg(<em>2</em>7<em>1</em>) occurring with similar rates. Addition of purified <em>fragment</em> <em>1</em> to prethrombin <em>1</em> accelerates both the rate of cleavage at Arg(3<em>2</em>0) and thrombin formation. Both reactions were inhibited by Hir(54-65)(SO(3)(-)) while DYDYQ had no significant inhibitory effect on prethrombin <em>1</em> cleavage in the absence or presence of <em>fragment</em> <em>1</em>. Similarly, activation of prethrombin <em>2</em> by <em>prothrombin</em>ase, is inhibited by Hir(54-65)(SO(3)(-)), but is not affected by DYDYQ. Addition of purified <em>fragment</em> <em>1</em>*<em>2</em> to prethrombin <em>2</em> accelerates the rate of cleavage at Arg(3<em>2</em>0) by <em>prothrombin</em>ase. This addition also results in a significant inhibition of thrombin formation by DYDYQ and is concurrent with the elimination of the inhibitory effect of Hir(54-65)(SO(3)(-)) on the same reaction. Finally, a membrane-bound ternary complex composed of prethrombin <em>2</em>/<em>fragment</em> <em>1</em>*<em>2</em>/Hir(54-65)(SO(3)(-)) is inhibited by DYDYQ. Altogether, the data demonstrate that membrane-bound <em>fragment</em> <em>1</em> is required to promote optimum Fva cofactor activity which in turn is translated by efficient initial cleavage of <em>prothrombin</em> by <em>prothrombin</em>ase at Arg(3<em>2</em>0).