We have reported that <em>prothrombin</em> (<em>1</em> microm) is able to replace high molecular weight kininogen (45 nm) as a cofactor for the specific binding of factor XI to the platelet (Baglia, F. A., and Walsh, P. N. (<em>1</em>998) Biochemistry 37, <em>2</em><em>2</em>7<em>1</em>-<em>2</em><em>2</em>8<em>1</em>). We have also determined that <em>prothrombin</em> <em>fragment</em> <em>2</em> binds to the Apple <em>1</em> domain of factor XI at or near the site where high molecular weight kininogen binds. A region of 3<em>1</em> amino acids derived from high molecular weight kininogen (HK3<em>1</em>-mer) can also bind to factor XI (Tait, J. F., and Fujikawa, K. (<em>1</em>987) J. Biol. Chem. <em>2</em>6<em>2</em>, <em>1</em><em>1</em>65<em>1</em>-<em>1</em><em>1</em>656). We therefore investigated the role of <em>prothrombin</em> <em>fragment</em> <em>2</em> and HK3<em>1</em>-mer as cofactors in the binding of factor XI to activated platelets. Our experiments demonstrated that <em>prothrombin</em> <em>fragment</em> <em>2</em> (<em>1</em> microm) or the HK3<em>1</em>-mer (8 microm) are able to replace high molecular weight kininogen (45 nm) or <em>prothrombin</em> (<em>1</em> microm) as cofactors for the binding of factor XI to the platelet. To localize the platelet binding site on factor XI, we used mutant full-length recombinant factor XI molecules in which the platelet binding site in the Apple 3 domain was altered by alanine scanning mutagenesis. The recombinant factor XI with alanine substitutions at positions Ser(<em>2</em>48), Arg(<em>2</em>50), Lys(<em>2</em>55), Leu(<em>2</em>57), Phe(<em>2</em>60), or Gln(<em>2</em>63) were defective in their ability to bind to activated platelets. Thus, the interaction of factor XI with platelets is mediated by the amino acid residues Ser(<em>2</em>48), Arg(<em>2</em>50), Lys(<em>2</em>55), Leu(<em>2</em>57), Phe(<em>2</em>60), and Gln(<em>2</em>63) within the Apple 3 domain.

Total hip/knee replacement surgeries are associated with an increased risk of venous thromboembolism and post-operative thromboprophylaxis has become standard treatment. This study aimed to: (i) assess the impact of hip/knee replacement surgery on ex vivo thrombin generation (TG), <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>), thrombin-antithrombin complexes (TAT) and D-dimer; (ii) compare the anticoagulant effects of dalteparin and rivaroxaban on TG <em>2</em>4 h after surgery. Haemostatic variables were assessed in plasma samples of 5<em>1</em> patients taken pre-operatively, peri-operatively, and <em>2</em>4 h post-operatively. Prophylaxis, once a day, with dalteparin or rivaroxaban, starting 6–8 h post-operatively, was administered in <em>2</em>5 (<em>1</em>4 knee/<em>1</em><em>1</em> hip) and <em>2</em>6 patients (<em>1</em>3 knee/<em>1</em>3 hip) respectively. TG, F<em>1</em> + <em>2</em>, TAT and D-dimer increased during surgery. Dalteparin patients showed a variable TG response <em>2</em>4 h after surgery: conversely, the effect of rivaroxaban on TG was consistent across individuals. Good correlation was seen between rivaroxaban levels and TG-lag-time (rs = 0·46, P = 0·0<em>1</em>); TG-time-to-Peak (rs = 0·53, P = 0·005); TG-peak-thrombin (rs = −0·59, P = 0·00<em>1</em>); and TG-velocity-index-rate (rs = −0·6<em>1</em>, P = 0·0009). Patients who received rivaroxaban showed a greater decrease of TG, F<em>1</em> + <em>2</em> and TAT (but not D-dimer) than those on dalteparin. TG increases during hip/knee replacement surgery. Rivaroxaban inhibits TG more than dalteparin at <em>2</em>4 h after surgery.

Prolonged physical exercise is associated with multiple changes in blood hemostasis. Eccentric muscle activation induces microtrauma of skeletal muscles, inducing an inflammatory response. Since there is a link between inflammation and coagulation we speculated that downhill running strongly activates the coagulation system. Thirteen volunteers participated in the Tyrolean Speed Marathon (4<em>2</em>,<em>1</em>95 m downhill race, 795 m vertical distance). Venous blood was collected 3 days (T<em>1</em>) and 3 h (T<em>2</em>) before the run, within 30 min after finishing (T3) and <em>1</em> day thereafter (T4). We measured the following key parameters: creatine kinase, myoglobin, thrombin-antithrombin complex, <em>prothrombin</em> <em>fragment</em> F<em>1</em> + <em>2</em>, D-dimer, plasmin-alpha(<em>2</em>)-antiplasmin complexes, tissue-type plasminogen activator antigen, plasminogen-activator-inhibitor-<em>1</em> antigen and thrombelastography with ROTEM [intrinsic pathway (InTEM) clotting time, clot formation time, maximum clot firmness, alpha angle]. Thrombin generation was evaluated by the Thrombin Dynamic Test and the Technothrombin TGA test. Creatine kinase and myoglobin were elevated at T3 and further increased at T4. Thrombin-antithrombin complex, <em>prothrombin</em> <em>fragment</em> F<em>1</em> + <em>2</em>, D-dimer, plasmin-alpha(<em>2</em>)-antiplasmin complexes, tissue-type plasminogen activator antigen and plasminogen-activator-inhibitor-<em>1</em> antigen were significantly increased at T3. ROTEM analysis exhibited a shortening of InTEM clotting time and clot formation time after the marathon, and an increase in InTEM maximum clot firmness and alpha angle. Changes in TGA were indicative for thrombin generation after the marathon. We demonstrated that a downhill marathon induces an activation of coagulation, as measured by specific parameters for coagulation, ROTEM and thrombin generation assays. These changes were paralleled by an activation of fibrinolysis indicating a preserved hemostatic balance.

We compared peritoneal dialysis effluents from <em>1</em>8 CAPD patients who had not suffered from peritonitis during the last 6 months (group <em>1</em>) with the effluents from five patients with acute peritonitis (group <em>2</em>), measuring activation markers of coagulation and fibrinolysis. These markers included <em>prothrombin</em> <em>fragment</em> F<em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>), thrombin-antithrombin III complex (TAT), fibrin monomer (FM), and fibrin degradation products (FbDP). In the dialysate of group <em>1</em> we found remarkably high levels of F<em>1</em> + <em>2</em>, TAT and FM concomitant with a high concentration of FbDP, indicating a high rate of intraperitoneal fibrin turnover. The balance between peritoneal generation and degradation of fibrin was disturbed in untreated patients of group <em>2</em>, who had significantly higher levels of coagulation markers and a higher ratio between FM and FbDP. Seven days after treatment with intraperitoneal administration of antibiotics and heparin, F<em>1</em> + <em>2</em>, TAT, FM and FbDP decreased significantly. To evaluate the role of mesothelial cells (MC) in the high peritoneal fibrin turnover we investigated the expression of tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), plasminogen activator inhibitor type-<em>1</em> (PAI-<em>1</em>), and tissue factor in cultured human peritoneal MC under basal conditions and after exposure to tumour necrosis factor alpha (TNF alpha), interleukin-<em>1</em> alpha (IL-<em>1</em> alpha), or bacterial lipopolysaccharide (LPS). The exposure of MC to TNF alpha or to a lesser extent IL-<em>1</em> alpha or LPS reduced their fibrinolytic activity by decreasing t-PA production and increasing PAI-<em>1</em> synthesis.(ABSTRACT TRUNCATED AT <em>2</em>50 WORDS)

OBJECTIVE

To investigate the clinical and laboratory features of neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) and to characterize the molecular basis and prognosis of this disease.

METHODS

Twenty-six patients with NICCD were collected because of idiopathic intrahepatic cholestasis and jaundice. The diagnosis was made by routine laboratory data collection, tandem mass spectrometry (MS-MS) and gas chromatography mass spectrometry (GC-MS) analyses. SLC<em>2</em>5A13 gene mutation was analyzed by using polymerase chain reaction (PCR), direct DNA sequencing and restriction <em>fragment</em> length polymorphism analyses. The patients were followed up for nearly <em>2</em> years.

RESULTS

The NICCD patients showed low birth weight and the average onset of jaundice was <em>2</em>9 days. Laboratory data showed liver dysfunction, hyperbilirubinemia, hypoproteinemia, high levels of alpha-fetoprotein, prolonged prothrombin time, hypoglycemia and hyperammonemia. MS-MS analysis of the blood samples revealed specific elevation of citrulline, methionine, threonine, tyrosine and elevation of free carnitine, short-chain and long-chain acylcarnitines. GC-MS analysis of the urine samples showed elevated 4-hydroxyl phenyllactic acid and 4-hydroxyl phenylpyruvic acid. Twelve different mutations were identified, including 4 novel mutations, i.e., G386V, R467X, K453R and 119<em>2</em>-1193delT. Forty-four mutated alleles were identified in the 5<em>2</em> alleles (84.6% ). Among them, 851del4, 1638ins<em>2</em>3 and IVS6+5G>A mutations were the most frequent mutations, accounting for 40.9%, <em>2</em>0.5% and 11.4% of the total alleles examined respectively. Five of the <em>2</em>6 patients have not been recovered, including 4 died and 1 accepted liver transplantation. No obvious relationship was found between the genotype and phenotype in NICCD.

CONCLUSIONS

The 851del4, 1638ins<em>2</em>3 and IVS6+5G>A mutations are the hot-spot mutations in Chinese NICCD patients. Some NICCD patients have poor prognosis.

The prevalence of hypertension and cardiovascular disease increases dramatically after menopause in women, implicating estrogen as having a protective role in the cardiovascular system. However, recent large clinical trials have failed to show cardiovascular benefit, and have even demonstrated possible harmful effects, of opposed and unopposed estrogen in postmenopausal women. While these findings have led to a revision of guidelines such that they discourage the use of estrogen for primary or secondary prevention of heart disease in postmenopausal women, many investigators have attributed the negative results in clinical trials to several flaws in study design, including the older age of study participants and the initiation of estrogen late after menopause.Because almost all clinical trials use oral estrogen as the primary form of hormone supplementation, another question that has arisen is the importance of the route of estrogen administration with regards to the cardiovascular outcomes. During oral estrogen administration, the concentration of estradiol in the liver sinusoids is four to five times higher than that in the systemic circulation. This supraphysiologic concentration of estrogen in the liver can modulate the expression of many hepatic-derived proteins, which are not observed in premenopausal women. In contrast, transdermal estrogen delivers the hormone directly into the systemic circulation and, thus, avoids the first-pass hepatic effect.Although oral estrogen exerts a more favorable influence than transdermal estrogen on traditional cardiovascular risk factors such as high- and low-density lipoprotein-cholesterol levels, recent studies have indicated that oral estrogen adversely influences many emerging risk factors in ways that are not seen with transdermal estrogen. Oral estrogen significantly increases levels of acute-phase proteins such as C-reactive protein and serum amyloid A; procoagulant factors such as <em>prothrombin</em> <em>fragments</em> <em>1</em>+<em>2</em>; and several key enzymes involved in plaque disruption, while transdermal estrogen does not have these adverse effects.Whether the advantages of transdermal estrogen with regards to these risk factors will translate into improved clinical outcomes remains to be determined. Two ongoing clinical trials, KEEPS (Kronos Early Estrogen Prevention Study) and ELITE (Early versus Late Intervention Trial with Estradiol) are likely to provide invaluable information regarding the role of oral versus transdermal estrogen in younger postmenopausal women.

We evaluated the serum/plasma levels of cytokines [interleukin (IL)-6, vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-beta<em>2</em>] and markers of coagulation, fibrinolysis, endothelial and platelet activation during the first 4 wk of treatment with the thalidomide analogue Actimid (CC-4047) in <em>1</em>5 patients with relapsed/refractory myeloma. There was evidence of activation of endothelium (soluble vascular cell adhesion molecule, sVCAM), coagulation (<em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>, PF<em>1</em> + <em>2</em>) and fibrinolysis (D-dimers) but no evidence of platelet activation or endothelial cell damage in myeloma patients. These parameters were not affected by the use of CC-4047. Three of four patients with baseline D-dimers levels >500 microg/L subsequently developed deep vein thrombosis (DVT). The hypothesis that D-dimer level >500 microg/L may predict for those patients most at risk of thromboembolism with multiple myeloma undergoing treatment is worthy of further study.

Alterations of coagulation and fibrinolytic systems might contribute to the increased cardiovascular and cerebrovascular mortality observed in patients with both chronic growth hormone (GH) excess (acromegaly) and deficiency (GHD). However, contrasting results have been so far reported. To assess the importance of GH in modulating haemostatic system, several haemostatic variables in patients with GHD and acromegaly were measured. Twenty-four adult patients with GHD (8 childhood- and <em>1</em>6 adult-onset; age: 4<em>1</em>+/-<em>1</em><em>2</em> years, insulin like growth factor-I, IGF-I: 6.7+/-4 nmol/L), <em>1</em>0 non-diabetic acromegalic patients (age: 39+/-<em>1</em>5 years; IGF-I: <em>1</em>09+/-37 nmol/L) and 64 healthy volunteers age- and sex-matched with cases were studied. The plasma levels of tissue-type plasminogen activator antigen (t-PA), <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>) and thrombin-antithrombin complex (TAT) were measured by ELISA. Plasminogen activator inhibitor type I (PAI-<em>1</em>) was measured by an immunoactivity assay and fibrinogen by von Clauss method. GH levels were measured by IFMA and IGF-I by RIA. GHD patients had higher PAI-<em>1</em> (<em>1</em><em>2</em>.7+/-<em>1</em>6.7 vs 4.8+/-5.3 U/ml, p<0.0<em>1</em>), fibrinogen (363+/-<em>1</em>04 vs <em>2</em>9<em>1</em>+/-7<em>1</em> mg/dL, p< 0.05) and TAT levels (6.8+/-9 vs 3.6+/-<em>2</em>.8 ng/ml, p<0.05) than controls. Taking the 95th pecentile of the normal distribution in the control group as the cut-off point for normal plasma levels of the haemostatic variables, high PAI levels were found in <em>2</em>5% of patients with GHD (P<0.0<em>1</em>), while high fibrinogen and TAT levels were observed in <em>2</em><em>1</em>% (P<0.05). The alterations were mostly present in patients with adult-onset GHD, with the exception of hyperfibrinogenaemia which was equally present in adult- and childhood-onset patients. Acromegalic patients had higher mean fibrinogen levels than controls (398+/-<em>1</em><em>1</em><em>1</em> vs <em>2</em>9<em>1</em>+/-7<em>1</em> mg/dL, p< 0.05), 40% having hyperfibrinogenaemia (P<0.0<em>1</em>, vs controls). They also had t-PA levels lower than controls and GHD. No correlations between hormonal and haemostatic variables were found. Body mass index and waist to hip ratio correlated positively with PAI-<em>1</em> levels in GHD patients only. In conclusion, this study shows that several abnormalities of coagulation variables (increased PAI-<em>1</em>. fibrinogen and TAT levels) are present in patients with GHD, while only hyperfibrinogenaemia is found in patients with acromegaly. These changes do not appear to be directly related to IGF-I levels or to the degree of GH deficiency/excess. However, these abnormalities may be an additional trigger for the development of coronary heart disease and thromboembolic complications mostly in patients with GHD.

Venous thromboembolism (VTE; deep venous thrombosis and pulmonary embolism) is associated with a poor prognosis in most malignancies and is a major cause of death among cancer patients. Universal anticoagulation for primary thromboprophylaxis in the outpatient setting is precluded by potential bleeding complications, especially without sufficient evidence that all patients would benefit from such prophylaxis. Therefore, appropriately targeting cancer patients for thromboprophylaxis is key to reducing morbidity and perhaps mortality. Predictive biomarkers could aid in identifying patients at high risk for VTE. Possible biomarkers for VTE include C-reactive protein, platelet and leukocyte counts, D-dimer and <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, procoagulant factor VIII, tissue factor, and soluble P-selectin. Evidence is emerging to support the use of risk assessment models in selecting appropriate candidates for primary thromboprophylaxis in the cancer setting. Further studies are needed to optimize these models and determine utility in reducing morbidity and mortality from cancer-associated thromboembolism.

In the current study, we investigated molecular markers of coagulation activity, ie, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>), thrombin-antithrombin (TAT) complex, soluble fibrin (SF), and D-dimer, and their relation to death, myocardial infarction, and refractory angina during and after anticoagulant treatment in unstable coronary artery disease. Patients with unstable coronary artery disease (N=3<em>2</em>0) were randomized to a 7<em>2</em>-hour infusion with either inogatran, a low-molecular-mass direct thrombin inhibitor, or unfractionated heparin. During the 30-day follow-up, a 40% lower event rate was seen in patients with high compared with low baseline levels of TAT or SF. High baseline levels of coagulation activity were correlated with a larger decrease during treatment. Patients with decreased compared with raised F<em>1</em>+<em>2</em> or TAT levels after 6 hours of treatment had a 50% lower event rate at 30 days (F<em>1</em>+<em>2</em>, P=0.04; TAT, P=0.0<em>2</em>). At the cessation of antithrombin treatment, there was a clustering of cardiac events that tended to be related to a rise in the levels of TAT and the other markers. During long-term follow-up (median, <em>2</em>9 months), there was a relation between higher baseline levels of D-dimer (P=0.003) and increased mortality. High baseline levels of molecular markers of coagulation activity might identify patients with a thrombotic condition (as the major cause of instability) who are good responders to anticoagulant therapy, with a larger decrease in coagulation activity during treatment and a decreased risk of ischemic events. However, this early benefit is lost during long-term follow-up when high baseline levels of coagulation activity are associated with a raised risk of early reactivation and increased mortality.

The aim of the study was to evaluate the effect of two antithrombotic therapies on platelet function and on coagulation in patients with nonvalvular atrial fibrillation (NVAF). Twenty patients with NVAF were treated with aspirin (300 mg/day) and clopidogrel (75 mg/day) for <em>2</em> weeks immediately followed by oral anticoagulation (target international normalized ratio <em>2</em>.0-3.0). Parameters of platelet function and coagulation were evaluated before antithrombotic therapy, at the end of aspirin plus clopidogrel and during subsequent anticoagulation treatment. Aspirin plus clopidogrel significantly inhibited platelet aggregation, fibrinogen receptor activation and release of P-selectin and prolonged in vitro bleeding time (p < 0.0<em>1</em>). Coagulation parameters (platelet-dependent thrombin generation, antithrombin III, thrombin-antithrombin III complex, <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>) were not significantly affected. During the subsequent oral anticoagulation phase platelet function was not substantially reduced; however, coagulation parameters were significantly inhibited (p < 0.00<em>1</em>). The results indicate that combined antiplatelet therapy is superior to aspirin monotherapy in inhibiting platelet function but does not seem to substantially modulate coagulation cascade in patients with NVAF.

BACKGROUND

The study was conducted to investigate the effect of a combined oral contraceptive (COC) containing 30 mcg ethinylestradiol and <em>2</em> mg dienogest with two different regimens on various hemostasis variables.

METHODS

Hemostatic parameters were measured in 59 women treated with a monophasic COC containing 30 mcg ethinylestradiol and <em>2</em> mg dienogest (EE/DNG) either conventionally (<em>1</em>3 cycles with <em>2</em><em>1</em> days of treatment+7 days without hormones) or with an extended-cycle regimen (4 extended cycles with 84 days of continuous administration of EE/DNG, followed by a hormone-free interval of 7 days). Blood samples were taken on Days <em>2</em><em>1</em>-<em>2</em>6 of the preceding control cycle and on Days <em>1</em>9-<em>2</em><em>1</em> of the 3rd and <em>1</em>3th conventional cycle or on Days 8<em>2</em>-84 of the first and fourth extended cycle.

RESULTS

After 3 and <em>1</em><em>2</em> months, significant increases in fibrinogen (<em>2</em>0%), factor VII antigen (50-60%), factor VII activity (45%), activated factor VII (30-45%) and factor VIII activity (<em>1</em>0-<em>2</em>0%) occurred in both treatment regimens. In both groups, there was a small but significant decrease in the level and activity of antithrombin, a <em>2</em>0-<em>2</em>5% decrease in total and free protein S and a <em>1</em>5-<em>2</em>0% rise in the level and activity of protein C, but no significant change of the thrombin-antithrombin complex. A significant over-time rise by about <em>2</em>5% of <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> occurred only in the extended-cycle group, but this effect did not differ significantly from that observed during conventional treatment. Plasminogen was elevated by 50% in both groups, while tissue-plasminogen activator (t-PA) activity rose by <em>1</em>5% in the conventional group and by <em>2</em>5-30% in the extended-cycle group. In both groups, t-PA antigen was reduced by about 30% and plasminogen activator inhibitor-<em>1</em> by 40-60%. The levels of the plasmin-antiplasmin complex rose by 30-40% and those of D-dimers by <em>2</em>0-55%. The <em>prothrombin</em> time was slightly increased and the activated partial thromboplastin time was slightly decreased.

CONCLUSIONS

In general, these results were in agreement with those observed during treatment with other COCs. The study demonstrated that during conventional and extended-cycle treatment with EE/DNG, a steady-state in the effects on hemostasis variables was reached within 3 months, and that the effects observed after 3 and <em>1</em><em>2</em> months of treatment did not substantially differ between conventional and extended-cycle regimen.

BACKGROUND

Exsanguination associated with acute traumatic coagulopathy is a leading cause of death following injury. While platelets occupy a pivotal role in clot formation, clinical research has been scant because of complexities resulting from the need for rapid handling and complex testing of platelet functions. While the thrombin pathway has been proposed as a mediator of platelet dysfunction in trauma, it has not been systematically investigated. The purpose of this study was to evaluate the thrombin pathway in platelet dysfunction.

METHODS

Forty trauma patients and <em>2</em>0 noninjured controls were enrolled in the study at a Level I trauma center. Platelet aggregation was tested by light transmission aggregometry with two agonists, adenosine diphosphate (ADP) and thrombin receptor agonist peptide (TRAP). Mean fluorescence intensity and percent positivity of CD6<em>2</em> on ADP-activated platelets were evaluated using flow cytometry. Enzyme-linked immunosorbent assays were performed to evaluate the concentrations of D-dimer, thrombin-antithrombin complex (TAT), and <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (PF <em>1</em> + <em>2</em>) in each sample.

RESULTS

Compared with healthy controls, trauma patients had significantly decreased ADP- and TRAP-mediated platelet aggregation and ADP-mediated CD6<em>2</em> expression. In trauma patients, TRAP-mediated aggregation was inversely proportional to head Abbreviated Injury Scale (AIS) score. Glasgow Coma Scale (GCS) score was directly proportional to TRAP- and ADP-mediated aggregation. When compared with controls, significant differences of D-dimer, TAT, and PF <em>1</em> + <em>2</em> were found. Measures of shock, including admission blood pressure, pulse, base deficit, and lactate level, did not correlate with platelet dysfunction.

CONCLUSIONS

Trauma patients have significantly lower levels of platelet activation and aggregation compared with healthy controls. Severity of head injury was significantly correlated with platelet dysfunction in a stepwise fashion. Trauma patients also have significantly increased levels of D-dimer, TAT, and PF <em>1</em> + <em>2</em> when compared with healthy controls. Our data suggest that the thrombin receptor pathway plays an important role in platelet dysfunction in trauma.

METHODS

Prognostic and epidemiologic study, level III.

OBJECTIVE

Hypoglycemia produces thrombosis activation, but little attention has been paid to the effects of hyperglycemia following recovery from hypoglycemia on thrombosis activation.

RESULTS

In both twenty-two healthy subjects and twenty-one matched persons with type <em>1</em> diabetes, recovery from a <em>2</em>-h induced hypoglycemia was obtained by reaching normo-glycemia or hyperglycemia for another <em>2</em> h. After this, normal glycemia was maintained for the following 6 h. Hyperglycemia after hypoglycemia was also repeated with the concomitant infusion of vitamin C. In both controls and people with diabetes, the recovery with normo-glycemia was accompanied by a significant improvement of Von Willebrand factor (vWF), <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>), thrombin-antithrombin III-complexes (TAT), P-selectin, plasminogen activator inhibitor-<em>1</em> (PAI-<em>1</em>), nitrotyrosine and 8-iso-prostaglandin F<em>2</em>α (8-iso-PGF<em>2</em>α) (p < 0.0<em>1</em> vs hypoglycemia for all the parameters), all directly affected by hypoglycemia itself (p < 0.0<em>1</em> vs baseline for all the parameters). On the contrary, the recovery with hyperglycemia after hypoglycemia worsens all these parameters (p < 0.0<em>1</em> vs normoglycemia for all the parameters), an effect persisting even after the additional 6 h of normo-glycemia. The effect of hyperglycemia following hypoglycemia was partially counterbalanced when vitamin C was infused (p < 0.0<em>1</em> vs hyperglycemia alone for all the parameters), suggesting that hyperglycemia following hypoglycemia may activate thrombosis through the oxidative stress production.

CONCLUSIONS

This study shows that, in type <em>1</em> diabetes as well as in controls, the way in which recovery from hypoglycemia takes place could play an important role in favoring the activation of thrombosis and oxidative stress, widely recognized cardiovascular risk factors.

OBJECTIVE

We sought to study the association of the silent cerebral infarct (SCI), a predisposing condition of stroke, with hyperinsulinemia and hemostatic abnormalities in older hypertensive subjects.

BACKGROUND

Hypertension is a powerful risk factor for stroke. However, the role of other risk factors for stroke in hypertensive subjects remains incompletely understood.

METHODS

We performed brain magnetic resonance imaging and measured cardiovascular risk factors, by administering the 75-g oral glucose tolerance test and measuring plasma insulin and hemostatic variables, in <em>1</em><em>2</em>3 asymptomatic hypertensive subjects (mean age 69 years).

RESULTS

At least one SCI was detected in 80 subjects (65%), and multiple SCIs were found in 48 subjects (39%). The presence of SCIs was associated with older age, higher levels of <em>2</em>4-h systolic blood pressure, <em>2</em>-h insulin, thrombin-generation markers (<em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> and thrombin-antithrombin complexes), plasminogen activator inhibitor-<em>1</em> (PAI-<em>1</em>), D-dimer and von Willebrand factor (vWF), but not with plasmin-alpha<em>2</em>-plasmin complex (PIC) levels. The <em>2</em>-h insulin area under the curve (AUC) was positively correlated with PAI-<em>1</em> and vWF levels (p < 0.0<em>1</em>), and the PAI-<em>1</em> level was negatively correlated with the PIC level (p < 0.0<em>2</em>). Multiple logistic regression analysis revealed that age and the <em>2</em>-h insulin AUC were significantly associated with SCIs, particularly those located in the subcortical white matter, and hemostatic abnormalities were significantly associated with the presence of multiple SCIs, particularly those located in the basal ganglia.

CONCLUSIONS

In older asymptomatic hypertensive subjects, hyperinsulinemia appears to be associated with lacunar-type SCIs, particularly those located in the subcortical white matter, and hemostatic abnormalities show an association with the presence of multiple SCIs, particularly those located in the basal ganglia.

BACKGROUND

Prothrombotic abnormalities within the coagulation system, the presence of microvascular thrombi in intestinal mucosa, and the increased risk of thromboembolic complications in patients with Inflammatory bowel disease, suggest that a hypercoagulable state may be an important contributing factor in disease pathogenesis. The activation of the coagulation system in a cohort of ulcerative colitis patients was investigated.

METHODS

Markers of coagulation activation in blood (thrombin-antithrombin complex, TAT; <em>prothrombin</em> <em>fragments</em> <em>1</em> and <em>2</em>, F<em>1</em>+<em>2</em>; and D-dimers) and markers of inflammation (erythrocyte sedimentation rate, ESR; C-reactive protein, CRP; and fibrinogen) were measured in 38 patients with active and <em>1</em>3 patients with long-standing quiescent ulcerative colitis. Disease activity was assessed by clinical, endoscopic, and histological criteria. The markers of coagulation activation were also measured in <em>2</em>8 healthy volunteers.

RESULTS

There were no differences in TAT, F<em>1</em>+<em>2</em>, and D-dimer plasma levels between active and inactive ulcerative colitis. D-dimer and F<em>1</em>+<em>2</em> levels were significantly higher in the active ulcerative colitis patients than in the healthy controls. Plasma levels of TAT, F<em>1</em>+<em>2</em>, and D-dimers did not differ between inactive ulcerative colitis patients and healthy controls. However, both active and inactive ulcerative colitis patients had significantly higher proportions of elevated (above-normal) values of coagulation markers than the healthy controls. Correlation analyses revealed strong correlation between ESR, fibrinogen, and D-dimers, which also correlated with the severity and extent of ulcerative colitis.

CONCLUSIONS

A chronic low-grade activation of coagulation exists in ulcerative colitis, regardless of disease activity, and it might be implicated in disease pathogenesis.

Chronic spontaneous urticaria (CSU) is a mast cell-driven disease that is defined as the recurrence of weals, angioedema or both for>> 6 weeks due to known or unknown causes. As of yet, disease diagnosis is purely clinical. Objective tools are needed to monitor the activity of CSU and the efficacy of treatment. Recently, several reports have suggested that blood parameters may be considered as potential disease-related biomarkers. Here, we reviewed available literature on blood biomarkers for CSU diagnosis, activity monitoring, duration, patient subgroup allocation or response to treatment. We performed a PubMed, Google Scholar and Web of Science search and identified and analysed <em>1</em>5<em>1</em> reports published prior to January <em>2</em>0<em>1</em>6. We found strong evidence for significant differences between patients with CSU and healthy controls in blood levels or values of D-dimer, C-reactive protein (CRP), matrix metalloproteinase-9 (MMP-9), mean platelet volume (MPV), factor VIIa, <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>), tumour necrosis factor, dehydroepiandrosterone sulphate and vitamin D. Also, there is strong evidence for a significant association between CSU activity and blood levels or values of D-dimer, F<em>1</em> + <em>2</em>, CRP, IL-6 and MPV. Strong evidence for reduced basophil count and high levels of IgG anti-FcεRI in the subgroup of CSU patients with positive autologous serum skin test was shown. In contrast, the evidence for all reported blood biomarkers for differentiating CSU from other diseases, or a role in prognosis, is weak, inconsistent or non-existent. Taken together, we identified <em>1</em>0 biomarkers that are supported by strong evidence for distinguishing patients with CSU from healthy controls, or for measuring CSU activity. There is a need for further research to identify biomarkers that predict outcome or treatment response in CSU.

Long-distance travel in a cramped position by aircraft or by bus and car has been suggested to be associated with an increased risk for thromboembolic events. Recently, we demonstrated moderate activation of coagulation after a long-haul flight. At present the single contributing factors (i.e. hypoxia and low humidity on board an aircraft and prolonged sitting in an aircraft, car or bus inducing venous stasis) have not yet been investigated. Therefore we measured markers of coagulation and fibrinolysis as well as functional parameters of coagulation using activated thrombelastography in <em>1</em>9 healthy volunteers before, during and after a real <em>1</em>0-h bus journey. In addition, changes in leg volume were measured. Thrombelastography revealed moderate activation of coagulation in all travelers, which was accompanied by a significant increase in <em>prothrombin</em> <em>fragment</em> F<em>1</em> + <em>2</em>. Thrombin-antithrombin III complexes and D-dimer remained unchanged, and tissue-type plasminogen activator and plasminogen-activator inhibitor <em>1</em> decreased after travel. After the travel we found a significant increase in leg volume that was exclusively distributed in the calf. We conclude that beside long-haul flights also long-distance bus travel induces a certain activation of the coagulation system. Thus, it is questionable whether hypoxia is the crucial risk factor for thromboembolic events after long-haul flights.

Chronic inflammation represents a key pathogeneric event in the progression of lung disease in cystic fibrosis (CF). To identify novel mechanisms of the inflammatory reaction in CF and analyze its relation with coagulative activation, we carried-out a cross-sectional study to evaluate circulating levels of the inflammatory mediators soluble (s) CD40L, C-reactive protein (CRP), interleukin (IL)-<em>1</em>beta, the coagulation markers activated factor VII (FVIIa) and <em>prothrombin</em> <em>fragment</em> (F) <em>1</em>+<em>2</em>, as well as urinary <em>1</em><em>1</em>-dehydro-thromboxane (TX)B<em>2</em>, an index of in vivo platelet activation, in 34 CF patients and 34 matched healthy subjects. We observed that CF patients displayed significantly increased circulating levels of sCD40L compared to controls [<em>2</em>.8 (0.4-<em>1</em>5.6) vs <em>1</em>.<em>1</em> (0.<em>2</em>-<em>2</em>.7) ng mL(-<em>1</em>), P = 0.0003]. sCD40L levels inversely correlated with forced expiratory volume at <em>1</em> second (FEV<em>1</em>) (rho = -0.788, P = 0.000<em>1</em>), whereas it directly correlated with CRP and IL-<em>1</em>beta levels (rho = 0.6<em>2</em><em>1</em>, P = 0.0004; and rho = 0.745, P = 0.000<em>1</em>, respectively), which were also elevated in CF patients. CF patients had also enhanced levels of FVIIa and F<em>1</em>+<em>2</em> compared to controls [39.<em>2</em> (<em>2</em><em>2</em>.6-69.8) vs <em>2</em><em>2</em>.3 (<em>1</em>6.<em>2</em>-3<em>2</em>.4) mU mL(-<em>1</em>), P = 0.000<em>1</em>; 0.60 (0.30-<em>1</em>.80) vs 0.<em>1</em>7 (0.<em>1</em>0-0.40) nmol L(-<em>1</em>), P = 0.000<em>1</em>, respectively]. A direct correlation was observed between sCD40L and both plasma FVIIa (rho = 0.69<em>1</em>, P = 0.000<em>1</em>) and F<em>1</em>+<em>2</em> (rho = 0.545, P = 0.00<em>1</em>7) as well as between sCD40L and urinary <em>1</em><em>1</em>-dehydro-TXB<em>2</em> (rho = 0.433, P = 0.0<em>1</em><em>2</em>9). Our findings suggest that in CF patients, sCD40L could represent a biochemical link between the inflammatory state, and endothelial damage and coagulative activation, leading to progressive impairment of pulmonary function.

Sepsis is commonly associated with disturbances of the hemostatic balance. Most of the pathophysiological changes in sepsis are caused by endotoxin acting directly through endothelial injury or indirectly through release of cytokines with procoagulant effects. The relation between cytokines and hemostatic parameters was assessed in 3<em>2</em> patients with sepsis. <em>Prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>+<em>2</em>), thrombin-antithrombin III complexes (TAT), tissue type plasminogen activator (t-PA) functional and antigen, plasminogen activator inhibitor-<em>1</em> (PAI-<em>1</em>), plasminalpha<em>2</em>-antiplasmin complexes (PAP), D-Dimer, thrombomodulin (TM) and von Willebrand factor (vWF) were measured in patients and in 30 healthy subjects. The levels of cytokines TNF-alpha and interleukin-6 (IL-6) also were determined. A significant increase of F<em>1</em>+<em>2</em>, TAT, PAI-<em>1</em>, PAP, and D-Dimer was observed in septic patients as compared with controls (p<0.000<em>1</em>), whereas t-PA activity was significantly reduced (p<0.0<em>1</em>). The markers of endothelial cell activation TM, vWF, and t-PA antigen also were elevated significantly as compared with the control group (p<0.0<em>1</em>). Finally, we found a marked increase of TNF-alpha and IL-6 (p<0.000<em>1</em>). Whereas the increase of cytokine levels could be partially responsible for the hemostatic activation, it did not correlate with markers of endothelial activation in patients with sepsis.

OBJECTIVE

Hormonal treatment of endometriosis is often continued for long periods and has the potential to affect many essential metabolic processes. The current study aimed to determine the effects and safety of high-dose dienogest as a medical endometriosis therapy.

METHODS

The effects and safety of high-dose dienogest, <em>2</em>0-30 mg/day for <em>2</em>4 weeks, were examined in <em>2</em><em>1</em> women aged <em>1</em>8-5<em>2</em> years with laparoscopically and histologically proven endometriosis stage I-IV (according to revised American Society of Reproductive Medicine criteria). At baseline and week <em>2</em>4, sera were obtained and stored at -<em>2</em>0°C prior to analysis.

RESULTS

The study showed no clinically significant effect of high-dose dienogest on thyroid or adrenal function, electrolyte balance or haematopoiesis. High-dose dienogest therapy also had no appreciable effects on glucose and lipid metabolism, liver enzymes or haemostasis. For instance, although dienogest mediated small increases in the haemostatic variables <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>, antithrombin III and protein C, final levels (at week <em>2</em>4) remained within normal reference ranges for these parameters. The exception was the HDL-3 cholesterol concentration at week <em>2</em>4 (0.97 mmol/l), which increased beyond the normal range of 0.<em>2</em>8-0.64 mmol/l.

CONCLUSIONS

This investigation yielded a unique dataset on the safety of high-dose dienogest in endometriosis stage I-IV. High-dose dienogest (<em>2</em>0-30 mg/day) had little influence upon all the parameters measured. It is therefore likely that lower doses of dienogest would have similarly neutral safety effects: an important consideration in the use of dienogest for the treatment of endometriosis.

Previously, we reported that the rabbit <em>prothrombin</em> <em>fragment</em> <em>2</em> (kringle <em>2</em> domain) has an anti-endothelial cell proliferative effect (Lee et al., J. Biol. Chem., in press). In this report, we show that not only rabbit <em>prothrombin</em> <em>fragment</em> <em>2</em> but also human <em>prothrombin</em> <em>fragment</em> <em>1</em> and <em>2</em> have an inhibitory effect on bFGF-stimulated BCE cell growth. Human <em>prothrombin</em> <em>fragment</em> <em>1</em> and <em>2</em> obtained as proteolytic <em>fragments</em> of human <em>prothrombin</em> display potent inhibitory effects on bovine capillary endothelial cells with a half-maximal concentration (ED50) of approximately <em>1</em>00 nM and <em>1</em><em>2</em>0 nM, respectively. As rabbit <em>prothrombin</em> <em>fragment</em> <em>2</em>, the human <em>prothrombin</em> <em>fragment</em> <em>1</em> and <em>2</em> also inhibit angiogenesis in the chorioallantoic membrane (CAM) of chick embryos.

OBJECTIVE

To investigate changes and establish reference values in coagulation, anticoagulation, fibrinolysis, anti-fibrinolysis and hemodynamics during normal pregnancy.

METHODS

A total of 58 women with singleton pregnancies were recruited. Blood and ultrasound examinations were performed in the <em>1</em>0th-<em>1</em>4th, <em>2</em>0th-<em>2</em>4th, and 30th-34th weeks of pregnancy. The same examinations were performed in 50 non-pregnant women who were selected as the control group.

RESULTS

Levels of fibrinogen, thrombin time, fibronectin, <em>prothrombin</em> activated <em>fragments</em> <em>1</em>+<em>2</em> and thrombomodulin were higher in early pregnancy than those in the control group (P < 0.05). Fibrinogen, <em>prothrombin</em> time, activated partial thromboplastin time, thrombin time, thromboxane B<em>2</em>, <em>prothrombin</em> activated <em>fragments</em> <em>1</em>+<em>2</em>, thrombomodulin, D-dimer, and plasminogen activator inhibitor-<em>2</em> were statistically different between the mid pregnancy and the control group (P < 0.05). Meanwhile, fibrinogen, <em>prothrombin</em> time, activated partial thromboplastin time, thrombin time, fibronectin, thromboxane B<em>2</em>, <em>prothrombin</em> activated <em>fragments</em> <em>1</em>+<em>2</em>, thrombomodulin, and plasminogen activator inhibitor-<em>2</em> were obviously elevated in late pregnancy as compared with the control group (P < 0.05). Moreover, fibrinogen, thromboxane B<em>2</em>, <em>prothrombin</em> activated <em>fragment</em> <em>1</em>+<em>2</em>, D-dimer plasminogen, and activator inhibitor-<em>2</em> gradually increased during pregnancy with some fluctuation. <em>Prothrombin</em> time, activated partial thromboplastin time, thrombin time, international normalized ratio, and thrombomodulin as well as systolic/diastolic ratio, pulsatility index, and resistance index in uterine arteries showed a tendency to decrease in pregnant women.

CONCLUSIONS

Coagulation, anti-coagulation, fibrinolytic and anti-fibrinolytic activities are enhanced and balanced at a higher level during pregnancy. In addition, uterine artery and umbilical artery hemodynamics become more baby friendly (i.e., high flow and low resistance).

OBJECTIVE

Plasma markers of coagulation and fibrinolysis have proved sensitive in the initial diagnosis of acute deep venous thrombosis (DVT). The purpose of this study was to examine the evolution and utility of measuring D-dimer and <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F <em>1</em>+<em>2</em>) levels after an acute DVT.

METHODS

Subjects with DVT confirmed by ultrasonography had quantitative plasma D-dimer and F <em>1</em>+<em>2</em> levels determined before anticoagulation. Ultrasound scan and coagulation studies were repeated at 3, 7, and <em>1</em>4 days; <em>1</em> month; and every 3 months for <em>1</em> year.

RESULTS

Sixty-one patients with a median initial thrombus score of 3 (interquartile range, <em>2</em>-7) were followed up for <em>2</em>66 days (interquartile range, 9<em>1</em>.5-364 days). Initial D-dimer levels were elevated in 9<em>2</em>.7% of patients and were associated with thrombus extent (P =.003), whereas F <em>1</em>+<em>2</em> levels were increased in 94.5% of patients and were lower in patients with isolated calf vein thrombosis (P =.00<em>1</em>). Initial D-dimer (P =.00<em>2</em>) and F <em>1</em>+<em>2</em> levels (P =.009) were significantly higher in the <em>2</em>6 (43%) patients with recurrent thrombosis during follow-up. Initial D-dimer levels of <em>2</em>000 ng/mL or greater were predictive of recurrent events after both proximal and isolated calf vein thrombosis. Although interval increases in these markers had little value in detecting recurrent thrombotic events, D-dimer levels of <em>1</em>000 ng/mL or greater and 500 ng/mL or greater had respective sensitivities of 89.3% and <em>1</em>00% in detecting early and late recurrences. Corresponding specificities were 35.6% and 53.9%.

CONCLUSIONS

Initial D-dimer levels are determined by total thrombus load and remain elevated long after an acute DVT. F <em>1</em>+<em>2</em> levels are less sensitive to thrombus score and return to baseline more quickly. Initial levels of these markers may have some utility in predicting the risk of ultrasound scan-documented recurrences, whereas increased D-dimer levels are a sensitive but nonspecific marker of these events.