BACKGROUND

This study examined the effect of abacavir on acute changes in biomarkers associated with cardiovascular dysfunction.

METHODS

Among the Study to Understand the Natural History of HIV/AIDS in the Era of Effective therapy (SUN) participants, we identified <em>2</em>5 individuals (cases) who were HLA-B570<em>1</em>-negative and who had ≥ <em>2</em> weeks without abacavir exposure at one visit and ≥ <em>2</em> weeks with abacavir exposure at the consecutive visit while maintaining viral suppression. We identified 43 individuals (controls) similarly unexposed and exposed to tenofovir. We assessed concentrations of <em>prothrombin</em> <em>fragment</em> F(<em>1</em>+<em>2</em>), D-dimer, high-sensitivity C-reactive protein, interleukin-8, intercellular adhesion molecule-<em>1</em>, vascular adhesion molecule-<em>1</em>, E-selectin, P-selectin, serum amyloid A and serum amyloid P. We examined the median percentage change of these biomarkers from the unexposed to exposed state among cases and controls compared with the expected assay variability using a sign test, and compared changes among cases with controls using the Wilcoxon rank-sum test.

RESULTS

Baseline characteristics were similar between cases and controls: median age 45 versus 46 years, 80% versus 8<em>1</em>% male, 64% versus 63% non-Hispanic White and median CD4(+) T-cell count 538 versus 60<em>1</em> cells/mm(3), respectively. Mean exposure times were 65 and <em>1</em>5 weeks for abacavir and tenofovir, respectively. We observed no significant changes in biomarkers from the unexposed to exposed state among cases or controls compared with the expected assay variability. We found that no biomarkers were significantly increased among cases compared with controls; however, <em>prothrombin</em> <em>fragment</em> F(<em>1</em>+<em>2</em>) was significantly lower among controls (P=0.035).

CONCLUSIONS

In virologically suppressed contemporary HIV-infected patients, abacavir exposure was not associated with increases in biomarkers associated with increased cardiovascular risk.

Platelet aggregation is the key process in primary hemostasis. Certain conditions such as hypoxia may induce platelet aggregation and lead to platelet sequestration primarily in the pulmonary microcirculation. We investigated the influence of high-altitude exposure on platelet function as part of a larger study on 30 subjects with a history of high-altitude pulmonary edema (HAPE) and <em>1</em>0 healthy controls. All participants were studied in the evening and the next morning at low altitude (450 m) and after an ascent to high altitude (4,559 m). Platelet count, platelet aggregation (platelet function analyzer PFA<em>1</em>00; using epinephrine and ADP as activators), plasma soluble P (sP)-selectin, and the coagulation parameters <em>prothrombin</em> <em>fragments</em> <em>1</em>+<em>2</em> and thrombin-antithrombin complex were measured. High-altitude exposure decreased the platelet count, shortened the platelet function analyzer closure time by approximately <em>2</em>0%, indicating increased platelet aggregation, increased sP-selectin levels to approximately <em>2</em>50%, but left plasma coagulation unaffected. The HAPE-susceptible subjects were prophylactically treated with either tadalafil (a phosphodiesterase 5 inhibitor), dexamethasone, or placebo in a double-blind way. Subgroup analyses between these different treatments and comparisons of the seven placebo-treated individuals developing HAPE and controls revealed no differences in platelet count, platelet aggregation, or sP-selectin values. We conclude that exposure to high altitude activates platelets, which leads to platelet aggregation, platelet consumption, and decreased platelet count. These effects are, however, not more pronounced in individuals with a history of HAPE or actually suffering from HAPE than in controls and therefore may not be a pathophysiological mechanism of HAPE.

There is considerable interest in determining the role of <em>prothrombin</em> <em>fragments</em>, especially urinary <em>prothrombin</em> <em>fragment</em> <em>1</em> (UPTF<em>1</em>), in the pathogenesis of calcium oxalate (CaOx) urinary calculi. This <em>fragment</em> is present in abundance in the matrix of CaOx crystals generated in human urine in vitro and has also been detected in human urinary stones containing calcium. More recently, <em>prothrombin</em> gene expression has been reported in the human kidney. However, studies examining the renal biosynthesis of <em>prothrombin</em> or perhaps only its <em>fragments</em> during experimental lithogenesis, and in consequence, the role of UPTF<em>1</em> in stone formation, cannot be carried out in humans. The aim of this investigation therefore was to determine whether <em>prothrombin</em> gene expression is present in the rat kidney. Total RNA was isolated from the kidneys and livers of <em>1</em><em>2</em> rats. Using reverse transcriptase PCR, mRNAs corresponding to the thrombin and <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em>+<em>2</em>) regions of <em>prothrombin</em> were analysed by agarose gel electrophoresis. The expression of glyceraldehyde 3-phosphate dehydrogenase was also examined to determine whether the quality of the tissue mRNAs was adequate for analyses. The amplified products were identified by sequence analysis. All kidneys displayed evidence of expression of the thrombin and F<em>1</em>+<em>2</em> domains of the <em>prothrombin</em> gene. Furthermore, the sequences of these PCR-derived products from kidney were identical to those from liver. This suggests that the <em>prothrombins</em> secreted by these two organs are identical. The fact that <em>prothrombin</em> biosynthesis occurs in both the human and rat kidney presents an opportunity for using established rat models of stone disease to evaluate the influence of lithogenic conditions on <em>prothrombin</em> gene expression, and the potential role of UPTF<em>1</em> in vivo.

OBJECTIVE

This study examined the effect of exercise on markers of fibrinolysis and coagulation in users and nonusers of oral contraceptives.

METHODS

Fourteen oral contraceptive users and <em>1</em>4 nonusers performed a maximal exercise test on a cycle ergometer. Blood samples were collected before and immediately after the completion of the test. A repeated-measures analysis of variance was used for statistical analysis with values considered significant at P =.05.

RESULTS

Acute maximal exercise resulted in significant increases in tissue plasminogen activator activity in both groups. There was a trend toward a smaller increase in tissue plasminogen activator activity in oral contraceptive users, but the difference between groups was not statistically significant. Plasminogen activator inhibitor <em>1</em> activity was reduced with exercise in both groups but with a significantly greater decrease observed in the nonusers (P <.000<em>1</em>). <em>Prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> was significantly higher (P <.000<em>1</em>) in the oral contraceptive group but did not change with exercise. Epinephrine levels before and after exercise were similar between the <em>2</em> groups, but postexercise norepinephrine concentrations were significantly lower (P =.0<em>2</em>6) in the oral contraceptive users.

CONCLUSIONS

These data suggest that oral contraceptive use blunts the fibrinolytic response to exercise. This, together with increased coagulation activation in oral contraceptive users, may alter the hemostatic balance during exercise.

OBJECTIVE

To investigate the association of left ventricular outflow tract (LVOT) obstruction with blood coagulation, platelet activity and inflammatory response in patients with hypertrophic cardiomyopathy (HCM) and sinus rhythm.

METHODS

In 4<em>2</em> patients with HCM with sinus rhythm, including <em>1</em>6 patients with resting LVOT obstruction (gradient>> or = 30 mm Hg) and <em>2</em>9 age- and sex-matched controls, markers of thrombin generation (thrombin-antithrombin complex (TAT), <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>)), platelet activation (soluble CD40 ligand (sCD40L), beta-thromboglobulin (beta-TG), P-selectin) and inflammation (C-reactive protein (CRP), interleukin (IL)6, tumour necrosis factor-alpha (TNFalpha)) were determined.

RESULTS

Thrombin, platelet and inflammatory markers were higher in the entire HCM group than in controls (p<0.005 for all compared parameters). Compared with non-obstructive HCM, obstructive HCM was associated with increased thrombin formation (TAT, F<em>1</em>+<em>2</em>), platelet activation (sCD40L, beta-TG, P-selectin) and both CRP and IL6 levels. Only the level of TNFalpha was similar in both forms of HCM. In contrast, a comparison of non-obstructive HCM with controls showed that all these variables (except for P-selectin) were similar; P-selectin was higher in non-obstructive HCM. The LVOT gradient correlated positively with all the raised blood markers (r from 0.39 to 0.73; p<0.05), except for TNFalpha. In multiple regression analysis models, the LVOT gradient was the only independent predictor of TAT (R(<em>2</em>) = 0.6<em>1</em>; p<0.00<em>1</em>), sCD40L (R(<em>2</em>) = 0.59; p<0.00<em>1</em>), F<em>1</em>+<em>2</em> (R(<em>2</em>) = 0.55; p = 0.00<em>2</em>), P-selectin (R(<em>2</em>) = 0.49; p = 0.004) and beta-TG (R(<em>2</em>) = 0.38; p = 0.005) in patients with HCM.

CONCLUSIONS

LVOT obstruction is independently associated with enhanced thrombin generation and platelet activity in patients with HCM with sinus rhythm.

Contrasting data have been reported about cardiovascular diseases in psoriatic patients. The aim of this study was therefore to evaluate blood coagulation and fibrinolysis in psoriatic patients. For this purpose, in a first group of 48 patients, we measured blood coagulation and fibrinolysis inhibitors [antithrombin III (AT), protein C (PC) and alpha <em>2</em>-antiplasmin (AP)], the products of thrombin and plasmin activity [fibrinopeptide A (FpA) and B beta(<em>1</em>5-4<em>2</em>) (B beta)], plasminogen (PLG) and fibrinogen (FBG). When all patients were considered we found a significant increase in B beta and FpA levels, while PC, PLG and AP values were significantly decreased when compared to controls. FBG and AT were not different from the controls. In order to understand whether the observed abnormalities of blood coagulation and fibrinolysis were related only to psoriasis we divided all the patients into two groups: (<em>1</em>) patients with cardiovascular disease or other risk factors (n = <em>2</em>8) and (<em>2</em>) patients affected only by psoriasis (n = <em>2</em>0). Since no difference was observed between groups <em>1</em> and <em>2</em>, we conclude that these findings are related to psoriasis. Subsequently we considered a different group of psoriatic patients. In these patients we measured FpA and two new thrombin activation indicators, such as <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> and thrombin-antithrombin complex (TAT). In addition we evaluated the levels of D-dimer, the product of the dissolution of cross-linking fibrin by plasmin. In this second group FpA, <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> and D-dimer were significantly higher than controls. Only TAT was not statistically different from those of the controls.(ABSTRACT TRUNCATED AT <em>2</em>50 WORDS)

The aim of this study was to compare the comprehensive intracrystalline protein profiles of calcium oxalate monohydrate (COM) and dihydrate (COD) crystals precipitated from the same human urine samples. Three separate batches of COM and COD crystals were precipitated from pooled healthy human urine by the addition of sodium oxalate at calcium concentrations of <em>2</em> and 8 mM, respectively. Proteins in whole extracts of demineralised COM and COD crystals, as well as in spots excised from <em>2</em>D-PAGE gels of the extracts, were identified using liquid chromatography and tandem mass spectrometry (LC-MS/MS). The number and type of individual proteins differed between COM and COD: <em>1</em>4 substantive proteins were found inside COM crystal extracts and 34 inside COD, with 9 proteins occurring in both crystal types. Numerous keratins were detected. However, in line with consensus in the proteomics literature, as well as a lack of published evidence linking them to urolithiasis, they were excluded as contaminants, leaving very few consistently detected proteins. On the basis of their known association with stone disease or identification in multiple runs, the principal proteins in COM crystal extracts were <em>prothrombin</em> <em>fragment</em> <em>1</em>, protein S<em>1</em>00A9, and IGkappaV<em>1</em>-5, while those in extracts of COD crystals included osteopontin, IGkappaV<em>1</em>-5, protein S<em>1</em>00A9, annexin A<em>1</em>, HMW kininogen-<em>1</em>, and inter-alpha-inhibitor (IalphaI). In general, proteins incorporated into both hydromorphs were acidic (pI<6), smaller than 55 kDa, and calcium binders. We concluded that the incorporation of proteins into urinary COM and COD crystals is selective and that only a few of the urinary proteins associated with the two hydromorphs are likely to play any significant role in stone pathogenesis.

Reparixin antagonizes interleukin-8 (IL-8) on the level of signal transduction in vitro. We hypothesized that IL-8 mediates some of the reactions occurring during acute inflammation and specifically that IL-8 may be a mediator of endotoxin induced neutrophilia. We therefore tested the effects of reparixin on humoral and cellular parameters in LPS-induced acute systemic inflammation. The study is a randomized (3:<em>2</em> active:placebo), double-blind, placebo-controlled parallel group trial. Twenty healthy male volunteers randomly received either reparixin (<em>1</em><em>2</em>) or placebo (8) intravenously. One hour after the start of reparixin/placebo infusion a bolus of <em>2</em> ng/kg endotoxin was infused over <em>1</em>-<em>2</em> min. Blood samples were obtained over <em>2</em>4 h. Reparixin, being metabolized to ibuprofen, suppressed serum thromboxane B<em>2</em> levels by 78 percent compared to baseline and control at 8 h. LPS-induced neutrophilia was not significantly affected by reparixin in human volunteers. Consistently, reparixin did not alter the lymphocyte or monocyte counts and had no effect on LPS-induced systemic inflammation as measured by tumor necrosis factor alpha (TNF-alpha) or interleukin-6 (IL-6) release. Regulation of IL-8 receptors CXCR<em>1</em> and <em>2</em> and the degranulation marker CD<em>1</em><em>1</em>b showed the expected kinetics. Reparixin had no effect on thrombin formation as measured by <em>prothrombin</em> <em>fragment</em> (F<em>1</em>+<em>2</em>). In conclusion, our study showed that reparixin was safe but had no impact on endotoxin induced inflammation. In contrast to previous studies with its metabolite ibuprofen, reparixin does not enhance inflammation in this model.

BACKGROUND

The effect of interruption of antiretroviral therapy (ART) on lipoprotein particle subclasses has not been studied. We examined short-term changes in lipids and lipoprotein particles among 33<em>2</em> HIV-infected individuals randomized to interrupt or continue ART in the "Strategies for Management of Antiretroviral Therapy" trial.

METHODS

Lipids and lipoprotein particles measured by nuclear magnetic resonance spectroscopy were compared between randomized groups at month <em>1</em>; associations with inflammatory and coagulation markers (high sensitivity C-reactive protein; interleukin 6; amyloid A; amyloid P; D-dimer; <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>) were assessed.

RESULTS

Compared with continuation of ART, treatment interruption resulted in substantial declines in total, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol, and triglyceride, at month <em>1</em> but had little net effect on total/HDL cholesterol ratio [baseline-adjusted mean difference [95% confidence interval (CI)] interruption versus continuation arms: -0.<em>1</em>0 (-0.59 to 0.38); P = 0.67]. ART interruption resulted in declines in total, large, and medium very low density lipoprotein (VLDL) particle concentrations (VLDL-p) and total and medium HDL-p. However, there was no change in small HDL-p [baseline-adjusted percentage difference between arms: -4.6% (-<em>1</em>3.<em>1</em>%, +5.<em>1</em>% ); P = 0.35], small LDL-p [-5.0% (-<em>1</em>6.9%, +8.6%); P = 0.45], or other LDL-p subclasses. Changes in lipid parameters on ART interruption did not differ according to baseline ART class (protease inhibitor versus non-nucleoside reverse transcriptase inhibitor) but were negatively associated both with changes in HIV viral load and with changes in inflammatory and coagulation markers, particularly D-dimer.

CONCLUSIONS

These results suggest that ART interruption does not favorably influence overall lipid profile: there was little net effect on total/HDL cholesterol ratio, and no change in small LDL-p or small HDL-p, the lipoprotein particle subclasses most consistently linked to coronary risk. Short-term declines in lipid parameters after ART interruption were not associated with class of ART and may be linked to increases in viral replication, inflammation and coagulation.

The effects of estrogen on cardiovascular risk factors have been less well defined in men than in women. We measured lipid and lipoprotein concentrations, lipoprotein particle size distributions, lipoprotein (a), homocysteine, and markers of thrombosis and fibrinolysis in <em>1</em>8 [corrected] healthy elderly men (age 74 +/- 3 years, mean +/- S.D.) before and after 9 weeks of treatment with 0.5, <em>1</em> or <em>2</em> mg/day of oral micronized <em>1</em>7beta-estradiol. LDL-C (-6%), apo B (-9%), triglyceride (-5%), and homocysteine (-<em>1</em><em>1</em>%) concentrations decreased with estradiol, whereas HDL-C (+<em>1</em>4%) increased. Intermediate-size VLDL subclass concentrations were lowered and LDL and HDL subclass levels altered in such a way as to cause average LDL and HDL particle size to increase. Lipoprotein (a) did not change. Fibrinogen (-<em>1</em>3%) and plasminogen activator inhibitor-<em>1</em> (PAI-<em>1</em>) concentrations (-<em>2</em>6%) decreased, but there were no changes in thrombotic markers including thrombin-antithrombin III complex, <em>prothrombin</em> <em>fragment</em> <em>1</em>.<em>2</em>, D-dimer, antithrombin activity, protein-C and S and von Willebrand factor antigen. Breast tenderness occurred in four men and heartburn in five but did not require discontinuation of treatment. We conclude that oral estrogen in men reduces homocysteine, fibrinogen, and PAI-<em>1</em> concentrations and favorably influences VLDL, LDL and HDL subclass levels without increasing markers of thrombotic risk.

OBJECTIVE

Resistin is an adipocytokine that recently generated much interest. Because of the fact that inflammation, endothelial cell damage or injury is invariably associated with such clinical conditions as thrombosis, atherosclerosis and their major clinical consequences, that is, cardiovascular disease and resistin play a role in linking inflammation and cardiovascular disease, the aim of the study was to assess resistin in correlation with markers of inflammation, endothelial cell injury and residual renal function in haemodialysed (HD) patients.

METHODS

We assessed resistin, markers of coagulation: thrombin-antithrombin complexes (TAT), <em>prothrombin</em> <em>fragments</em> <em>1</em>+<em>2</em>; fibrinolysis: tPA, plasminogen activator inhibitor type <em>1</em>, plasmin-antiplasmin complexes (PAP); endothelial function/injury: von Willebrand factor (vWF), thrombomodulin, intracellular adhesion molecule (ICAM); inflammation: high sensitivity C-reactive protein (hsCRP), tumour necrosis factor alpha and interleukin-6 (IL-6).

RESULTS

Healthy volunteers and HD patients did not differ significantly regarding age, leucocyte count, serum iron, aspartate and alanine aminotransferases activities, calcium, cholesterol, tPA concentration. Triglycerides, CRP (assessed by high sensitivity method), phosphate, urea, creatinine, IL-6, tumour necrosis factor alpha, vWF, <em>prothrombin</em> <em>fragments</em> <em>1</em>+<em>2</em>, TAT, PAP, thrombomodulin, ICAM, plasminogen activator inhibitor type <em>1</em> and resistin, were elevated in HD patients when compared with the control group. Serum albumin, total protein, haemoglobin and haematocrit were significantly lower in HD patients when compared with the control group. In HD patients with hsCRP 0e; 6 mg/L, resistin, IL-6, vWF and F<em>1</em>+<em>2</em> were significantly higher, whereas tPA was significantly lower than in patients with hsCRP<6 mg/L. Moreover, HD patients with residual renal function have significantly lower resistin when compared with patients without it. Resistin was significantly higher in diabetics. In HD patients, resistin correlated significantly, in univariate analysis, with calcium, phosphate, PTH, TIBC, vWF residual renal function, urea, hsCRP, IL-6 and tended to correlate with tPA and ferritin. In the healthy volunteers, resistin was related to IL-6 and hsCRP. In multiple regression analysis, resistin was independently related to hsCRP, IL-6, residual renal function in HD patients.

CONCLUSIONS

Elevated resistin related to markers of inflammation may represent a novel link between inflammation and adipocytokines in HD patients. Impaired renal function and inflammation are responsible for elevated resistin in HD patients.

OBJECTIVE

The effects of hormone replacement therapy (HRT) can affect many aspects relevant to cardiovascular disease, including vasomotor function, inflammation, and hemostasis. Recent studies have demonstrated that current doses of HRT exert a mixture of both protective and adverse effects. In the current study, we compared the effects of lower doses of HRT (L-HRT) and conventional doses of HRT (C-HRT) on a variety of relevant cardiovascular parameters.

RESULTS

This randomized, double-blind, crossover study included 57 women who received micronized progesterone <em>1</em>00 mg with either conjugated equine estrogen 0.6<em>2</em>5 mg (C-HRT) or 0.3 mg (L-HRT) daily for <em>2</em> months. L-HRT showed comparable effects to C-HRT on high-density lipoprotein cholesterol and triglyceride levels, but not on low-density lipoprotein cholesterol levels. C-HRT and L-HRT significantly improved the percent flow-mediated dilator response to hyperemia from baseline values (both P<0.00<em>1</em>) by a similar degree (P=0.7<em>1</em>9). C-HRT significantly increased high-sensitivity C-reactive protein (hsCRP) levels from baseline values (P<0.00<em>1</em>); however, L-HRT did not significantly change hsCRP (P=0.874). C-HRT and L-HRT significantly decreased antithrombin III from baseline values (P<0.00<em>1</em> and P=0.04<em>2</em>, respectively). C-HRT significantly increased <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>) from baseline values (P<0.00<em>1</em>); however, L-HRT did not significantly change F<em>1</em>+<em>2</em> (P=0.558). Of interest, the effects of C-HRT and L-HRT on hsCRP, antithrombin III, and F<em>1</em>+<em>2</em> were significantly different (all P<0.00<em>1</em>). C-HRT and L-HRT significantly reduced plasma PAI-<em>1</em> antigen levels from baseline values (P=0.00<em>2</em> and P=0.038, respectively) to a similar degree (P=0.<em>1</em>84).

CONCLUSIONS

Compared with C-HRT, L-HRT has comparable effects on lipoproteins, flow-mediated dilation, and PAI-<em>1</em> antigen levels. However, L-HRT did not increase hsCRP or F<em>1</em>+<em>2</em> levels, and it decreased antithrombin III less than C-HRT.

The effect of two arginine-specific cysteine proteinases (gingipains R) from Porphyromonas gingivalis, an aetiological factor of adult periodontitis, on the activation of human factor IX was investigated in the presence of ethylene glycol, an activity enhancer of activated factor IX (factor IXa), with the use of a fluorogenic oligopeptide substrate. Each gingipain R rapidly activated factor IX but the 95 kDa proteinase complex (HRgpA) that contains both haemagglutinin/adhesion and catalytic domains was <em>2</em>.4-fold more efficient than the single-chain 50 kDa gingipain R (RgpB), which has only a catalytic domain. SDS/PAGE and N-terminal sequence analysis of factor IX digestion <em>fragments</em> indicated that, like all endogenous activators, gingipains R also produce factor IXabeta via an IXa intermediate. Significantly, phospholipids augmented the activation of factor IX by HRgpA but not by RgpB in a Ca(<em>2</em>+)-dependent manner. In the presence of both cofactors the kinetic efficiency of HRgpA to activate factor IX (k(cat)/K(m)=<em>1</em>.9x<em>1</em>0(6) M(-<em>1</em>).s(-<em>1</em>)) was 8.5-fold higher than that of RgpB (k(cat)/K(m)=<em>2</em>.3x<em>1</em>0(5) M(-<em>1</em>).s(-<em>1</em>)) and double that of the factor VIIa-tissue factor complex, but 8-fold lower than that for factor XIa. A comparison of the relative activation rates of factor IX, factor X and <em>prothrombin</em> directly in plasma by HRgpA suggests a significant contribution for factor IX conversion in blood coagulation induced by gingipains R. Taken together, gingipains R are the first-reported activators of factor IX of bacterial origin. By this effect they could be involved in the production of thrombin as well as the subsequent generation of prostaglandins and interleukin <em>1</em>, all of which have been found to be associated with the development and progression of periodontitis.

OBJECTIVE

Both epidemiological and experimental evidence suggests a protective effect of dietary fiber against disease, such as cancer and cardiovascular disease. The effects of fiber on lipoproteins are modest, indicating that other mechanisms may be involved. As the hemostatic effects of different types of fiber are poorly known, we compared the effects of wholemeal rye bread and low-fiber wheat bread on factors related to coagulation, fibrinolysis and platelet function.

METHODS

Subjects consumed rye and wheat bread as part of their habitual diet in a cross-over manner for 4 weeks, with a 4 week washout period between the diet periods.

METHODS

Forty healthy subjects (<em>1</em>8 men, <em>2</em><em>2</em> women), aged 43+/-<em>2</em> y.

RESULTS

Mean daily intake of fiber during the rye bread period was 3<em>1</em> g for men and <em>2</em>6 g for women, while the respective figures for the wheat bread period were <em>1</em>5 g and <em>1</em><em>2</em> g. However, no significant differences between the two periods were seen in factor VII coagulant activity (FVIIc), fibrinogen, <em>prothrombin</em> <em>fragments</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>), tissue plasminogen activator (tPA) plasminogen activator inhibitor <em>1</em> (PAI-<em>1</em>) or <em>2</em>,3,-dinor-thromboxane B<em>2</em>. Fibrin degradation products, D-dimers, were slightly lower after the wheat period (P=0.046).

CONCLUSIONS

The results indicate that wheat and rye bread do not differ in their effects on coagulation, fibrinolysis or platelet function.

BACKGROUND

Fazer Bakeries Ltd, Lahti Finland; Vaasan & Vaasan Ltd, Helsinki, Finland.

OBJECTIVE

To assess the antithrombotic properties of SR90<em>1</em>07/ORG3<em>1</em>540. a sulfated pentasaccharide, which enhances specifically antithrombin III mediated inactivation of factor-Xa, in a clinical setting known to promote arterial thrombosis, i.e. coronary angioplasty.

RESULTS

Percutaneous transluminal coronary angioplasty (PTCA) was carried out with conventional balloons with a single 5 min intravenous infusion of <em>1</em><em>2</em> mg pentasaccharide, and 500 mg intravenous aspirin. Heparin was not allowed before, during PTCA, and within <em>2</em>4 h after PTCA. The primary end point was the rate of abrupt vessel closure during and within <em>2</em>4 h after the procedure. The sample size was set at 60 evaluable patients, in order to be able to conclude with a good level of confidence (>95%) that the abrupt vessel closure rate was less than <em>1</em>0%, if less than 3 abrupt vessel closures were observed. Seventy-one patients were included in the study, of whom <em>1</em>0 needed elective stenting, and were not considered as evaluable for efficacy. Two out of the 6<em>1</em> remaining evaluable patients experienced acute vessel closure during the study period [3.<em>2</em>8%, 95% confidence interval (0.4%; <em>1</em><em>1</em>.4%)]. No major bleeding occurred. The drug plasma concentrations reached <em>1</em>.9<em>1</em>+/-0.39 mg/], <em>1</em>0 min after pentasaccharide injection, and decreased on average to <em>1</em>. <em>1</em>8+/-0.<em>2</em>7 mg/l at <em>2</em> h, and to 0.36+/-0.<em>1</em><em>1</em> mg/l at <em>2</em>3 h after administration of pentasaccharide. Activated clotting time (ACT) and activated partial thromboplastin (aPTT) time remained within normal range. Thrombin-antithrombin complex levels fell from <em>2</em><em>2</em>+/-<em>1</em>7.<em>1</em> to 4.5+/-3.4 microg/ml, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> levels decreased from <em>2</em>.<em>1</em>5+/-<em>1</em>.0<em>1</em> to <em>1</em>.73+/-0.87, and activated factor VII levels decreased from 43.4+/-<em>1</em>6.8 mU/ml to <em>1</em>8.9+/-7.3 mU/ml respectively from baseline to <em>2</em> h following injection of the tested drug.

CONCLUSIONS

Administration of pentasaccharide led to the inhibition of thrombin generation without modification of aPTT and ACT. The rate of abrupt vessel closure was within range of rates reported in historical series. Thus we conclude that the anti-thrombotic activity of pentasaccharide, as shown in this pilot trial in the setting of coronary angioplasty, deserves further investigation.

Type <em>2</em> diabetes mellitus is frequently accompanied by hypercoagulability and hypofibrinolysis. Both are related to increased cardiovascular risk, but possibly with endothelial injury as well. Studies with nondiabetic persons indicate that unopposed oestrogen replacement therapy (oERT) decreases cardiovascular risk, possibly mediated in part by effects on coagulation and fibrinolysis. In a double-blind, randomised placebo-controlled trial, we assessed the effect of oral <em>1</em>7 beta-oestradiol daily during 6 weeks on indicators of coagulation and of fibrinolysis in postmenopausal women with type <em>2</em> diabetes mellitus. We observed significant increases of Factor VII (FVII) and von Willebrand factor (vWF) after oERT and no change in the already high fibrinogen. <em>Prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>) increased after oERT, whereas thrombin-antithrombin (TAT) complexes was unchanged, but increments of F<em>1</em> + <em>2</em> and TAT correlated. Soluble fibrin (SF) levels remained stable. In fibrinolysis, a clear reduction in plasminogen activator inhibitor <em>1</em> (PAI-<em>1</em>) was observed, but no significant change in tissue-type plasminogen activator antigen (t-PA-Ag) or activity was found, although fibrinolytic activity assessed as t-PA activity (t-PA-Act) tended to increase after oERT. Indicators of fibrinolytic activity (plasmin-antiplasmin complexes and fibrin degradation products) however did not change. oERT increased C-reactive protein (CRP) but none of the coagulation or fibrinolysis changes significantly associated with the CRP changes. It is concluded that oERT increases the coagulation potency as well as the fibrinolytic potency raising the question of the net effect in their balance. Increase in F<em>1</em> + <em>2</em> suggests that in diabetic women oERT effectively increases the chronic, continuous activation of coagulation, which appears to be compensated for or not effective in the blood compartment as judged from the unchanged levels of SF. Suspected increased fibrin formation in the vascular wall is at least not followed by increases in fibrinogen degradation products (TDP), which suggests the possibility of accumulation and increased cardiovascular risk. The results indicate that specific attention should be paid to fibrin turnover in studying other categories of women and the effects of the addition of progesterone.

Autoproteolysis of blood coagulation factor Xa (FXa) results in the excision of a 4-kDa <em>fragment</em> (beta-peptide) from the intact subform, factor Xaalpha (FXaalpha), to yield factor Xabeta (FXabeta). In the preceding paper, we showed that generation of FXabeta leads to expression of a plasminogen binding site. FXabeta may consequently participate in fibrinolysis; therefore, the timing of subform conversion compared with thrombin production is important. In the current study we evaluated the kinetics of FXabeta generation, which showed that autoproteolysis of FXaalpha followed a second order mechanism where FXaalpha and FXabeta behaved as identical enzymes. Rate constants of 9 and <em>1</em>7<em>2</em> M-<em>1</em> s-<em>1</em> were derived, respectively, in the absence and presence of FXaalpha binding to procoagulant phospholipid. Under identical conditions the latter is estimated to be 6 orders of magnitude slower than thrombin generation by <em>prothrombin</em>ase. Since heparin binding and <em>prothrombin</em> recognition have been previously attributed to a region of FXaalpha proximal to the beta-peptide, functional comparisons were conducted using homogeneous and stabilized preparations of FXaalpha and FXabeta. Comparisons included <em>1</em>) the recognition of small substrates; <em>2</em>) the rate of interaction with antithrombin/heparin; 3) the assembly of <em>prothrombin</em>ase; and 4) the activation of <em>prothrombin</em> by <em>prothrombin</em>ase. Although the beta-peptide neighbors a probable functional region in FXaalpha, conversion to FXabeta was not observed to influence these functions. The data support a model where FXaalpha is predominantly responsible for thrombin generation and where slow conversion to FXabeta coordinates coagulation and the initiation of fibrinolysis at sites of <em>prothrombin</em>ase assembly.

The mechanism of inactivation of bovine factor Va by plasmin was studied in the presence and absence of phospholipid vesicles (PCPS vesicles). Following 60-min incubation with plasmin (4 nm) membrane-bound factor Va (400 nm) is completely inactive, whereas in the absence of phospholipid vesicles following a <em>1</em>-h incubation period, the cofactor retains 90% of its initial cofactor activity. Amino acid sequencing of the <em>fragments</em> deriving from cleavage of factor Va by plasmin demonstrated that while both chains of factor Va are cleaved by plasmin, only cleavage of the heavy chain correlates with inactivation of the cofactor. In the presence of a membrane surface the heavy chain of the bovine cofactor is first cleaved at Arg(348) to generate a <em>fragment</em> of M(r) 47,000 containing the NH(<em>2</em>)-terminal part of the cofactor (amino acid residues <em>1</em>-348) and a M(r) 4<em>2</em>,000 <em>fragment</em> (amino acid residues 349-7<em>1</em>3). This cleavage is associated with minimal loss in cofactor activity. Complete loss of activity of the membrane-bound cofactor coincides with three cleavages at the COOH-terminal portion of the M(r) 47,000 <em>fragment</em>: Lys(309), Lys(3<em>1</em>0), and Arg(3<em>1</em>3). These cleavages result in the release of the COOH terminus of the molecule and the production of a M(r) 40,000 <em>fragment</em> containing the NH(<em>2</em>)-terminal portion of the factor Va molecule. Factor Va was treated with plasmin in the absence of phospholipid vesicles followed by the addition of PCPS vesicles and activated protein C (APC). A rapid inactivation of the cofactor was observed as a result of cleavage of the M(r) 47,000 <em>fragment</em> at Arg(306) by APC and appearance of a M(r) 39,000 <em>fragment</em>. These data suggest a critical role of the amino acid sequence 307-348 of factor Va. A 4<em>2</em>-amino acid peptide encompassing the region 307-348 of human factor Va (N4<em>2</em>R) was found to be a good inhibitor of factor Va clotting activity with an IC(50) of approximately <em>1</em>.3 microm. These data suggest that plasmin is a potent inactivator of factor Va and that region 307-348 of the cofactor plays a critical role in cofactor function and may be responsible for the interaction of the cofactor with factor Xa and/or <em>prothrombin</em>.

This paper provides evidence to demonstrate that human <em>prothrombin</em> undergoes conformational changes upon binding to procoagulant membranes specifically containing phosphatidylserine (PS). Fourier transform infrared spectroscopy was used to show a slight increase in ordered (alpha-helix, beta-sheet, beta-turns) secondary structure upon binding to PS-containing membranes. Thermograms representing <em>prothrombin</em> and <em>prothrombin</em> <em>fragment</em> <em>1</em> denaturation were obtained using differential scanning calorimetry. These were analyzed and interpreted in terms of changes in <em>prothrombin</em> domain organization associated with binding to PS-containing membranes. Changes in either secondary structure or domain organization upon binding to negatively-charged phosphatidylglycerol-containing membranes were, if they occurred at all, much less dramatic. The results paralleled results obtained previously with bovine <em>prothrombin</em> (<em>1</em>, <em>2</em>). The implications of these results in terms of a possible molecular mechanism for the cofactor-like role of platelet membrane vesicles in <em>prothrombin</em> activation are discussed.

Immunohistochemistry was applied to AMeX-fixed tissue sections of <em>1</em><em>2</em> adenocarcinomas of the stomach (seven intestinal adenocarcinomas and five diffuse carcinomas), <em>1</em><em>2</em> adenocarcinomas of the pancreas (nine ductal adenocarcinomas and three signet ring carcinomas), and <em>1</em><em>2</em> squamous cell carcinomas of the larynx obtained at surgical resection to examine the possibility of extravascular activation of blood coagulation in cancer tissues by exploring the in loco patterns of distribution of fibrinogen, a final product of blood coagulation, fibrin, and a by-product of coagulation reactions (<em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>). Gastric, pancreatic, and laryngeal cancers exhibited fibrinogen antigen in abundance throughout the tumor stroma. Fibrin was detected along the edges of nests of carcinoma cells and at the host-tumor interface. <em>Prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> was present in the blood vessels in areas of neoangiogenesis at the host-tumor interface (gastric and pancreatic cancer tissues) and on the tumor cell bodies (pancreatic and laryngeal cancer tissues). The presence of <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> in cancer tissues appears to be a good indicator of coagulation activation and thrombin generation at the tumor burden.

In <em>1</em>07 asymptomatic and untreated patients with inherited syndromes associated with thrombophilia (antithrombin III, protein C and protein S deficiencies), we compared in parallel two plasma peptides which reflect activation of the common coagulation pathway: the <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>) and fibrinopeptide A (FPA). Both F<em>1</em> + <em>2</em> and FPA were measured with simple, commercially available ELISA methods. High levels of F<em>1</em> + <em>2</em> or FPA were found in about one fourth of the patients as a whole. When patients were divided according to the type of inherited thrombophilic syndrome, it appeared that F<em>1</em> + <em>2</em> was more frequently elevated in protein C and protein S deficiencies than in antithrombin deficiency; and that, in general, it was no more frequently elevated than FPA. Although our data confirm the existence of a procoagulant imbalance in inherited thrombophilic syndromes due to defects of natural anticoagulant proteins, they do not confirm that such imbalance can be more frequently diagnosed by measuring F<em>1</em> + <em>2</em> levels, particularly in patients with antithrombin deficiency.

The tissue factor-factor (F)VIIa complex (TF/FVIIa) is responsible for the initiation of blood coagulation under both physiological and pathological conditions. Recombinant nematode anticoagulant protein c<em>2</em> (rNAPc<em>2</em>) is a potent inhibitor of TF/FVIIa, mechanistically distinct from tissue factor pathway inhibitor. The first aim of this study was to elucidate the pharmacokinetics and pharmacodynamics of a single intravenous (i.v.) dose of rNAPc<em>2</em>. The second aim was to study its effect on endotoxin-induced coagulation and inflammation. Initially, rNAPc<em>2</em> was administered to healthy volunteers in three different doses. There were no safety concerns and the pharmacokinetics were consistent with previous studies, in which rNAPc<em>2</em> was administered subcutaneously. rNAPc<em>2</em> elicited a dose-dependent reduction of the endogenous thrombin potential and a selective prolongation of <em>prothrombin</em> time. Subsequently, the effect on endotoxin-induced coagulation and inflammation was studied. The administration of rNAPc<em>2</em> completely blocked the endotoxin-induced thrombin generation, as measured by plasma <em>prothrombin</em> <em>fragment</em> F<em>1</em>+<em>2</em>. The endotoxin-induced effect on fibrinolytic parameters such as plasmin-antiplasmin complexes and plasminogen activator inhibitor type <em>1</em> was not affected by rNAPc<em>2</em>. The administration of rNAPc<em>2</em> attenuated the endotoxin-induced rise in interleukin (IL)-<em>1</em>0, without affecting the rise in other cytokines. In conclusion, rNAPc<em>2</em> is a potent inhibitor of TF/FVIIa, which was well tolerated and could safely be used intravenously in this Phase I study in healthy male volunteers. A single i.v. dose rNAPc<em>2</em> completely blocked endotoxin-induced thrombin generation without affecting the fibrinolytic response. In addition, rNAPc<em>2</em> attenuated the endotoxin-induced rise in IL-<em>1</em>0, without affecting the rises in other cytokines.

The biological activity of blood coagulation factors II, V, VII, VIII, IX, X, XI and XII, fibrinogen and prekallikrein was assessed in <em>1</em>5 healthy subjects and 60 patients with endemic Egyptian hepatosplenomegaly. The degree of liver disease was graded according to the Child-Pugh classification, the intensity of S. mansoni infection was monitored by determination of circulating schistosome immune complexes (CSIC) level using a monoclonal antibody and hemostasis activation was detected by measurement of hemostatic markers D-dimer and <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em>+<em>2</em>). Functional activity of antithrombin III, alpha<em>2</em>-antiplasmin and protein C as well as quantitative determination of plasma concentrations of alpha<em>1</em>-antitrypsin, C<em>1</em> activator inhibitor and alpha<em>2</em>-macroglobulin were also carried out. The progressive deterioration of liver function which matched the severity of the disease and the intensity of schistosomal infection led to a reduction in anticoagulant proteins (decreases in antithrombin III and protein C) resulting in hypercoagulability and thrombin generation (increased F<em>1</em>+<em>2</em>) subsequently followed by consumption (prolongation of coagulation screening tests, thrombocytopenia, hypofibrinogenemia and decreased factor VIII resulting in hypocoagulability and secondary fibrinolysis (increased D-dimer and decreased alpha<em>2</em>-antiplasmin). A significant decline in fibrinogen and factors VII, XII and prekallikrein was detected in bleeders compared with ascitic patients. The decline in factor XII was closely related to CSIC high titers in all disease groups, but was not correlated to D-dimer or F<em>1</em>+<em>2</em> concentrations. This suggests that circulating schistosome immune complexes may exert an inhibitory effect on contact factor XII which should be taken into account when considering the reasons for schistosomal coagulopathy and bleeding in hepatosplenic schistosomiasis.

Circulating active tissue factor (TF) and activated factor XI (FXIa) have been detected in subgroups of acute coronary syndromes (ACSs) and stable angina patients. We sought to evaluate the determinants of active TF and FXIa in stable angina patients. We studied <em>1</em><em>2</em>4 consecutive stable angina patients. Recent ACS, atrial fibrillation, and anticoagulant therapy were the exclusion criteria. Plasma active TF and FXIa were determined by measuring the response to inhibitory antibodies. T helper <em>1</em> lymphocyte (Th<em>1</em>) and Th<em>2</em> responses were assessed in plasma by interleukin (IL)-4, IL-6, IL-8, IL-<em>1</em>0, IL-<em>1</em>8, interferon-γ, and tumor necrosis factor-α, oxidative stress by 8-isoprostaglandin F(<em>2</em>α) (8-iso-PGF(<em>2</em>α)), and coagulation by <em>prothrombin</em> <em>fragments</em> F<em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>) and free TF pathway inhibitor (f-TFPI). TF and FXIa activity were detected in <em>2</em>5 (<em>2</em>0.<em>2</em>%) and 49 (39.5%) stable angina patients, respectively. Both factors were found in <em>2</em>3 (<em>1</em>8.5%) patients. Patients with detectable TF or FXIa had higher F<em>1</em>+<em>2</em>, 8-iso-PGF(<em>2</em>α), IL-6, but not other cytokines, and lower f-TFPI (all P < 0.00<em>1</em>) compared with the remainder. There were no intergroup differences with regard to cardiovascular risk factors or medication. Multivariate analysis showed that F<em>1</em>+<em>2</em> and f-TFPI were the only independent predictors of the TF presence, whereas 8-iso-PGF(<em>2</em>α) and F<em>1</em>+<em>2</em> predicted the presence of FXIa in stable angina patients. In stable angina patients, circulating active TF and FXIa are associated with enhanced thrombin formation, with a minor effect of inflammatory mediators. Moreover, FXIa is also related to oxidative stress, indicating additional links between coagulation and free radical generation in stable angina.