OBJECTIVE

To evaluate the efficacy and safety of intravenous infusions of an improved prothrombin complex concentrate (PCC) formulation.

METHODS

Twenty-two adults with haemostatic defects due to severe liver disease (Quick's test 50%), which required rapid haemostasis because of bleeding or before urgent surgery or invasive intervention. Laboratory follow-up, including the response and in-vivo recovery of the substituted coagulation factors II, VII, IX and X and protein C took place before, then <em>1</em>0 min, 30 min and 60 min after PCC substitution. Clinical efficacy (avoidance or cessation of bleeding) was assessed using a scale ranging from 'very good' to 'none'.

RESULTS

Patients received a median PCC dose of <em>2</em>5.7 IU/kg. The response of factor IX and protein C was <em>1</em>.<em>2</em>-<em>1</em>.4 (IU/dl)/(IU/kg), the in-vivo recovery was 49.7-57.4%, and the Quick's test increased from 39% to a maximum of 65%. Levels of activation markers of the coagulation system factor VIIa, <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> and thrombin antithrombin complex (TAT) increased, but without evidence of any thromboembolic events. Clinical efficacy was judged as 'very good' in 76% of patients after the first (n = <em>2</em><em>1</em>) treatment. There were no changes in serological status regarding transmission of HIV, hepatitis A virus, hepatitis B virus and hepatitis C virus. No PCC-related adverse reactions occurred.

CONCLUSIONS

The infusion of pasteurized, nanometre-filtered PCC is an effective, well-tolerated method of correcting prothrombin complex deficiency in patients with severe liver disease with haemorrhage, or before an urgent surgical or invasive diagnostic intervention.

Although coagulopathy is known to be the major contributor to a poor outcome of traumatic brain injury (TBI), the mechanisms that trigger coagulation abnormalities have not been studied in detail. We undertook a prospective observational study at a neurosurgical ICU (NICU) in a university hospital. We examined <em>1</em><em>1</em> patients with severe isolated TBI, at admittance to the hospital and during the next 3 days. We collected cerebrovenous blood samples from a jugular bulb catheter, arterial blood, and cerebrospinal fluid (CSF) samples. We measured concentrations of thrombin-antithrombin complex (TAT), fibrin D-dimer (DD), <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> (F<em>1</em> + <em>2</em>), interleukin-6 (IL-6), and complement complex (C5b-9). All patients had some degree of consumption coagulopathy at the study start and a tendency to thrombocytopenia during the next few days. Levels of DD (3.6 +/- <em>2</em>.7 mg/L), TAT (86 +/- 7<em>2</em> microg/L) and F<em>1</em> + <em>2</em> (5.9 +/- 6.8 nmol/L) were significantly increased shortly after the trauma compared to reference values, with considerable transcranial gradients for TAT (49 microg/L) and F<em>1</em> + <em>2</em> (3.<em>2</em> nmol/L). Compared to controls, IL-6 levels were increased more than a hundredfold in both blood (<em>2</em>83 +/- <em>1</em>9<em>2</em> ng/L) and CSF (4<em>2</em>4 +/- 355 ng/L) samples, with a transcranial gradient at the study start (<em>1</em>07 ng/L). C5b-9 levels were moderately increased in blood samples, <em>2</em>70 +/- <em>1</em><em>1</em>4 microg/L, versus controls, <em>1</em>84 +/- 39 (p < 0.05). We conclude that activation of the coagulation system takes place during the passage of blood through the damaged brain, and is already evident hours after the trauma. IL-6 and activation of the complement system (C5b-9) co-vary with hemostatic parameters in TBI patients.

The present investigation describes a novel approach to prepare a specific antibody against <em>prothrombin</em> activation <em>fragment</em> <em>1</em> + <em>2</em> (F <em>1</em> + <em>2</em>). The antibody discriminates between native <em>prothrombin</em> and F <em>1</em> + <em>2</em> in plasma. A synthetic peptide from the negatively charged region of F <em>1</em> + <em>2</em>, which becomes the carboxy-terminal sequence after cleavage of <em>prothrombin</em> by factor Xa, was used for immunization of rabbits. Obtained antiserum was immunopurified and an enzyme-linked immunosorbent assay (ELISA) was constructed for determination of F <em>1</em> + <em>2</em>. The test system follows the sandwich principle and uses two different antibodies directed against F <em>1</em> + <em>2</em> and <em>prothrombin</em>, respectively. The ELISA was calibrated with purified F <em>1</em> + <em>2</em> added to F <em>1</em> + <em>2</em>-poor plasma. The lower limit of sensitivity of the assay was 0.0<em>2</em> nmol/l. Coefficients of variation of 6.9 to <em>1</em>0.4% (intraassay) and 6.7 to <em>1</em><em>1</em>% (interassay) were found for F <em>1</em> + <em>2</em> concentrations between 0.08 and 4.9 nmol/l. A reference range from 0.3<em>2</em> to <em>1</em>.<em>2</em> nmol/l was calculated from 95 healthy donors (mean value +/- SD: 0.67 +/- 0.<em>1</em>9 nmol/l). In patients with deep vein thrombosis (n = 7) confirmed by phlebography and in patients with pulmonary embolism (n = 8) confirmed by lung scan, F <em>1</em> + <em>2</em> levels were found up to <em>1</em>.5 to 9.5 nmol/l. In plasma samples of patients under oral anticoagulant therapy in the stable state F <em>1</em> + <em>2</em> concentrations were found to be in the range of 0.08 to 0.5 nmol/l.(ABSTRACT TRUNCATED AT <em>2</em>50 WORDS)

In acute myocardial infarction (AMI), monocyte procoagulant activity is increased and may contribute to the risk for recurrence and other thrombotic events. This study sought to investigate the role tissue factor (TF) and tissue factor pathway inhibitor-<em>1</em> (TFPI-<em>1</em>) in the regulation of monocyte procoagulant activity in AMI. Serial venous blood samples were obtained from 40 patients with AMI undergoing revascularization by stent placement. Twenty patients with elective stenting for stable angina served as control subjects. TF proteolytic activity was measured with spectrozyme factor Xa (FXa), TF and TFPI-<em>1</em> surface expression on monocytes by flow cytometry, RNA expression in whole blood by reverse transcription-polymerase chain reaction, and concentrations of plasma <em>prothrombin</em> <em>fragments</em> F(<em>1</em> + <em>2</em>) by immunoassay. Forty-eight hours after AMI, an increase was found in TF RNA, followed by an increase in TF surface expression by <em>2</em>4% +/- 4% and in plasma concentration of F(<em>1</em> + <em>2</em>) by <em>1</em>03% +/- <em>1</em>7% (P <.05). These changes could not be attributed to the intervention because they did not occur in the control group. TFPI-<em>1</em> RNA and binding to the monocyte surface remained unchanged. FXa generation by monocytes of patients with AMI increased 53.6% +/- 9% in the presence of polyclonal antibodies to TFPI-<em>1</em>, indicating that cell-associated TFPI-<em>1</em> inhibits monocyte TF activity. The increased monocyte procoagulant activity in AMI was caused by an up-regulation of TF that was partially inhibited by surface-bound TFPI-<em>1</em>. Anticoagulant therapy by direct inhibition of TF activity may, thus, be particularly effective in AMI. (Blood. <em>2</em>00<em>1</em>;97:37<em>2</em><em>1</em>-37<em>2</em>6)

It has been previously demonstrated that hyperglycaemia activates haemostasis; diabetes mellitus is considered a thrombosis-prone state. Acarbose, by inhibiting dietary carbohydrate absorption, reduces post-meal hyperglycaemia. In this study we evaluated the effect of post-meal hyperglycaemia on two markers of coagulation activation: <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em> and D-dimer. Seventeen non-insulin-dependent diabetic patients maintained on diet therapy alone were randomly assigned to receive- with a cross-over study design-acarbose (<em>1</em>00 mg orally) or placebo before a standard meal. Blood samples for measurement of plasma glucose, insulin, <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em> and D-dimer were drawn at 0, 60, <em>1</em><em>2</em>0 and <em>2</em>40 min. After both placebo and acarbose, hyperglycaemia and hyperinsulinaemia which followed a standard meal were accompanied by a significant increase of plasma concentration of <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em> and D-dimer in comparison to their baseline values. Acarbose administration significantly reduced the rise of glucose, insulin, <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em> and D-dimer from 0 to <em>2</em>40 min in comparison to placebo. We conclude that post-meal hyperglycaemia, at the level reached by many diabetic patients on diet therapy alone, induces a coagulation activation. Acarbose, by decreasing post-meal hyperglycaemia, may be useful in reducing meal-induced activation of haemostasis in diabetic patients.

OBJECTIVE

Adhesion of urinary crystals to renal tubular cells could be a critical event that triggers a cascade of responses ending in kidney stone formation. We clarified the role of urinary macromolecules during calcium oxalate monohydrate (COM) crystal adhesion to cells.

METHODS

To assess COM crystal binding to cells in the presence of whole urine and fractions thereof we used monolayer cultures of distal nephron derived Madin-Darby canine kidney, type I cells as a model system.

RESULTS

COM crystal adhesion to cells was decreased in the presence of whole urine compared with an ultrafiltrate prepared by passing urine through a <em>1</em>0 kDa cutoff membrane. Supplementing the ultrafiltrate with urinary concentrate containing proteins greater than <em>1</em>0 kDa returned crystal adhesion to low levels, similar to whole urine. Macromolecules in whole urine acted to decrease binding to cells by coating crystals and 4 proteins previously implicated in the pathogenesis of nephrolithiasis were detected on coated crystals (bikunin, osteopontin, <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em> and Tamm-Horsfall glycoprotein). Crystals precipitated and grown in whole urine also bound less avidly to cells than crystals precipitated in artificial urine.

CONCLUSIONS

This study confirms that macromolecules present in whole urine can coat crystals and, thereby, block their adhesion to renal tubular cells. Preventing crystal retention in the kidney could be an important mechanism whereby these macromolecules protect against kidney stones.

Venous thromboembolism (VTE) occurs frequently in patients with cancer and contributes to elevated morbidity and mortality. Risk factors for the occurrence of VTE events in patients with cancer have been investigated in numerous clinical studies. For now more than <em>1</em>0 years, the Vienna Cancer and Thrombosis Study (CATS) has focused on the identification of parameters predictive of future VTE occurrence. CATS has contributed to new findings, which may help identify patients at high risk of developing VTE, by means of biomarkers (such as D-dimer, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>, soluble P-selectin, platelet count, coagulation factor VIII activity, thrombin generation potential, etc.). The association of tissue factor bearing microparticles and the mean platelet volume with the risk of VTE was also elaborately investigated in the framework of CATS. More recently CATS has researched clinical and clinicopathologic parameters which contribute to identification of patients at risk of VTE. The type of cancer is one of the most important risk factor for VTE occurrence. Also the stage of cancer and the histological grade of a tumor have been found to be associated with the occurrence of cancer-related VTE. In further investigations, venous diseases including a history of previous VTE, a history of superficial thrombophlebitis and the presence of varicose veins, have been associated with the risk of VTE in CATS.

BACKGROUND

Enhanced thrombin activity has been associated with acute and long-term complications following balloon angioplasty (percutaneous transluminal coronary angioplasty (PTCA). We evaluated, in a <em>2</em>-to-<em>1</em> randomized, double-blind trial, the effects of recombinant hirudin, CGP 39 393, relative to unfractionated sodium heparin on periprocedural events, bleeding, early angiographic outcome, and coagulation in <em>1</em><em>1</em>3 patients with stable angina undergoing PTCA.

RESULTS

Prior to PTCA, <em>2</em>0 mg CGP 39 393 was administered as a bolus, followed by continuous infusion at a rate of 0.<em>1</em>6 mg.kg-<em>1</em> x h-<em>1</em>, or <em>1</em>0,000 IU sodium heparin was administered as a bolus and continued at a rate of <em>1</em><em>2</em> IU.kg-<em>1</em> x h-<em>1</em> for <em>2</em>4 hours. Infusion was adjusted to activated partial thromboplastin time (APTT) levels. ST segment was monitored for <em>2</em>4 hours, and angiograms were analyzed with quantitative technique (QCA). In 74 CGP 39 393- and 39 heparin-treated patients, <em>1</em>3<em>2</em> lesions were dilated. Myocardial infarction and/or emergency coronary bypass surgery occurred in <em>1</em> (<em>1</em>.4%) CGP 39 393 patient compared with 4 (<em>1</em>0.3%) heparin patients (relative risk, 7.6; 95% confidence interval, 0.9, 65.6). At <em>2</em>4 hours, complete perfusion was present in 9<em>1</em>% heparin and <em>1</em>00% CGP 39 393 patients. Significant ST segment displacement was found in <em>1</em><em>1</em>% of heparin versus 4% of CGP 39 393 subjects. Bleeding occurred only at the puncture site in 4 CGP 39 393-treated patients. QCA did not reveal significant differences between the groups. APTT values were more often in the target range and more stable in CGP 39 393 patients. Levels of thrombin-antithrombin III complexes, prothrombin fragment F<em>1</em>+<em>2</em>, and fibrinopeptide A indicated that CGP 39 393 was an effective inhibitor of thrombin activity.

CONCLUSIONS

CGP 39 393 can safely be administered to patients undergoing elective PTCA for stable anginal symptoms and may have a more favorable anticoagulant profile than heparin.

OBJECTIVE

Recently emerging evidence suggests that oxidative stress (SOX) may participate in atherogenesis. The aim of the present study was to establish whether enhanced SOX, involving endothelial injury, activation of coagulation, and inflammatory reaction, could be implicated in atherosclerotic diseases in haemodialysis (HD) patients.

METHODS

Markers of SOX, endothelial injury, coagulation, and cytokines, were measured in the plasma of HD patients with and without cardiovascular disease (CVD), and of healthy controls by ELISA methods. Remodeling of the carotid arteries was assessed by measuring the intima-media thickness (IMT) as a surrogate of atherosclerotic disease in all groups.

RESULTS

Markers of SOX, endothelial injury, and extrinsic coagulation pathway activation and IMT values were significantly elevated in HD patients, especially in those with CVD when compared with the control group. The von Willebrand factor antigen (vWF:Ag) levels were more increased in the patients with CVD than in those without. Furthermore, the plasma levels of tumour necrosis factor alpha, monocyte chemo-attractant protein <em>1</em>, and macrophage inflammatory protein <em>1</em> beta were significantly higher only in the HD group with CVD when compared with the controls. The IMT was strongly and directly correlated with Cu/Zn superoxide dismutase. Both IMT and Cu/Zn superoxide dismutase were positively correlated with age, thrombomodulin, vWF:Ag, tissue factor, tissue factor pathway inhibitor, <em>prothrombin</em> <em>fragment</em> F<em>1</em> + <em>2</em>, monocte chemo-attractant protein <em>1</em>, macrophage inflammatory protein <em>1</em> beta, and tumour necrosis factor alpha levels. Multivariate analysis identified vWF:Ag as the only independent variable significantly associated with an increased IMT.

CONCLUSIONS

The present study suggests that enhanced SOX, involved pro-atherogenic cytokine and chemokines levels, endothelial injury, and coagulation activation may constitute a pathway for accelerated atherosclerosis in HD patients. The significant, independent association between IMT and vWF:Ag should be assessed in future studies to determine whether vWF:Ag elevation is causative or a by-product of the increased IMT.

BACKGROUND

Patients with chronic kidney disease exhibit features of a hypercoagulable state and have endothelial dysfunction, which may contribute to their increased cardiovascular risk. We examined the relationship between coagulation activation and vascular function in patients with chronic kidney disease.

METHODS

We measured parameters of the tissue factor pathway of blood coagulation (tissue factor, factor VIIc and factor X); natural inhibitors (tissue factor pathway inhibitor, protein C, free and total protein S, antithrombin III) and markers of coagulation activation (thrombin-antithrombin complexes, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>) in 66 stage 4&5 chronic kidney disease patients and 36 healthy controls. Their relationship with markers of vascular function (flow mediated dilatation, soluble E-selectin and thrombomodulin) and a mediator of inflammation (interleukin-6) was determined.

RESULTS

Up-regulation of the tissue factor pathway (increased tissue factor and factor VIIc), increased <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> and significant reductions in antithrombin III and the ratio of free protein S: total protein S were found in patients compared to healthy controls. Increased tissue factor antigen was significantly and independently correlated with creatinine and interleukin-6 (P<0.00<em>1</em>). Factor X and antithrombin III were both reduced in chronic kidney disease and correlated (r=0.58; P<0.00<em>1</em>). Changes in coagulation and anti-coagulation were independent of all measures of endothelial function.

CONCLUSIONS

Significant activation of the TF pathway of coagulation and depletion or reduction of some natural anticoagulants in chronic kidney disease was correlated with the degree of renal dysfunction, but not correlated with the abnormalities of vascular function. These data are consistent with a hypercoagulable state in chronic kidney disease that may be independent of endothelial based regulation but associated with an inflammatory state.

Staphylocoagulase (SC) secreted by Staphylococcus aureus is a potent non-proteolytic activator of the blood coagulation zymogen <em>prothrombin</em> and the prototype of a newly established zymogen activator and adhesion protein (ZAAP) family. The conformationally activated SC.<em>prothrombin</em> complex specifically cleaves fibrinogen to fibrin, which propagates the growth of bacteria-fibrin-platelet vegetations in acute bacterial endocarditis. Our recent <em>2</em>.<em>2</em> A X-ray crystal structures of an active SC <em>fragment</em> [SC(<em>1</em>-3<em>2</em>5)] bound to the <em>prothrombin</em> zymogen catalytic domain, prethrombin <em>2</em>, demonstrated that SC(<em>1</em>-3<em>2</em>5) represents a new type of non-proteolytic activator with a unique fold. The observed insertion of the SC(<em>1</em>-3<em>2</em>5) N-terminus into the 'Ile <em>1</em>6' cleft of prethrombin <em>2</em>, which triggers the activating conformational change, provided the first unambiguous structural evidence for the 'molecular sexuality' mechanism of non-proteolytic zymogen activation. Based on the SC(<em>1</em>-3<em>2</em>5) fold, a new family of bifunctional zymogen activator and adhesion proteins was identified that possess N-terminal domains homologous to SC(<em>1</em>-3<em>2</em>5) and C-terminal domains that mediate adhesion to plasma or extracellular matrix proteins. Further investigation of the ZAAP family may lead to new insights into the mechanisms of bacterial factors that hijack zymogens of the human blood coagulation and fibrinolytic systems to promote and disseminate endocarditis and other infectious diseases.

Alterations in blood coagulation may explain the poorer neurological outcome with diabetes mellitus and hyperglycemia after acute ischemic stroke. We studied the relationships between diabetes mellitus, hyperglycemia, whole blood tissue factor procoagulant activity (TF-PCA) and plasma factorVIIa (FVIIa) in ten patients with type <em>2</em> diabetes mellitus and <em>1</em><em>1</em> non-diabetic patients at baseline and 6, <em>1</em><em>2</em>, <em>2</em>4, and 48 hours (h) after presentation for acute stroke. In addition, we examined plasma <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> (F<em>1</em>.<em>2</em>) and thrombin-antithrombin complexes (TAT) as markers of thrombin generation. Stroke severity, assessed by National Institute of Health Stroke Scale (NIHSS), was similar at baseline (p=0.<em>2</em>6) but worse in diabetic (8.<em>2</em>0+/-4.3) than nondiabetic patients (<em>2</em>.67+/-<em>2</em>.<em>1</em>, p=0.0<em>2</em>3) at 48 h. At presentation, diabetic patients had higher FVIIa (p=0.004) and lower TF-PCA (p=0.0<em>2</em>7) than non-diabetic patients but both were higher than in normal control subjects. FVIIa levels remained higher in diabetic patients at 6, <em>1</em><em>2</em> and <em>2</em>4 h after stroke. In diabetic patients, FVIIa (r=0.40, p=0.0<em>2</em>) and TF-PCA (r=0.50, p=0.0<em>2</em>) correlated with blood glucose; and, FVIIa correlated with plasma F<em>1</em>.<em>2</em> (r=0.34, p=0.00<em>2</em>) and TAT levels (r=0.6<em>2</em>, p<0.000<em>1</em>). In non-diabetic patients, TF-PCA, but not FVIIa, correlated with F<em>1</em>.<em>2</em> (r=0.40<em>2</em>, p=0.0<em>1</em>0) and TAT (r=0.39, p=0.0<em>1</em><em>1</em>). Combining both groups, NIHSS scores were positively related to FVIIa levels (r=0.50, p=0.0<em>2</em><em>1</em>) and inversely related to TF-PCA levels (r=-0.498, p=0.0<em>2</em>). Acute ischemic stroke patients with diabetes and hyperglycemia have a more intense procoagulant state compared with nondiabetic patients. This is related to glucose levels and provides a potential mechanism for the observed worse prognosis in such patients after acute stroke.

OBJECTIVE

This study was designed to investigate whether or not combination aspirin-clopidogrel therapy would reduce markers of thrombogenesis and platelet activation in atrial fibrillation (AF), in a manner similar to warfarin.

BACKGROUND

Dose-adjusted warfarin is beneficial as thromboprophylaxis in AF, but potentially serious side effects and regular monitoring leave room for alternative therapies. METHODS; We randomized 70 patients with nonvalvular AF who were not on any antithrombotic therapy to either dose-adjusted warfarin (international normalized ratio <em>2</em> to 3) (Group I) or combination therapy with aspirin 75 mg and clopidogrel 75 mg (Group II). Plasma indices of thrombogenesis (fibrin D-dimer, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em>) and platelet activation (beta-thromboglobulin [TG] and soluble P-selectin) were quantified, along with platelet aggregation responses to standard agonists, at baseline (pretreatment) and at six weeks posttreatment. RESULTS; Pretreatment levels of fibrin D-dimer (p = 0.00<em>1</em>), beta-TG (p = 0.0<em>1</em>) and soluble P-selectin (p = 0.03) were raised in patients with AF, whereas plasma <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> levels and platelet aggregation were not significantly different compared with controls. Dose-adjusted warfarin reduced plasma levels of fibrin D-dimer, <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> and beta-thromboglobulin levels at six weeks (all p < 0.00<em>1</em>), enhanced plasma levels of soluble P-selectin (p < 0.00<em>1</em>) and had no significant effect on platelet aggregation. Aspirin-clopidogrel combination therapy made no difference to the plasma markers of thrombogenesis or platelet activation (all p = NS), but the platelet aggregation responses to adenosine diphosphate (p < 0.00<em>1</em>) and epinephrine (p = 0.0<em>2</em>) were decreased.

CONCLUSIONS

Aspirin-clopidogrel combination therapy failed to reduce plasma indices of thrombogenesis and platelet activation in AF, although some aspects of ex vivo platelet aggregation were altered. Anticoagulation with warfarin may be superior to combination aspirin-clopidogrel therapy as thromboprophylaxis in AF.

BACKGROUND

Tissue injury increases blood levels of extracellular histones and nucleic acids, and these may influence hemostasis, promote inflammation and damage the endothelium. Trauma-induced coagulopathy (TIC) may result from an endogenous response to the injury that involves the neurohumoral, inflammatory and hemostatic systems.

OBJECTIVE

To study the contribution of extracellular nucleic constituents to TIC, inflammation and endothelial damage.

METHODS

Prospective observational study.

METHODS

We investigated histone-complexed DNA fragments (hcDNA) along with biomarkers of coagulopathy, inflammation and endothelial damage in plasma from 80 trauma patients admitted directly to the Trauma Centre from the scene of the accident. Blood was sampled a median of 68 min (IQR 48-88) post injury. Trauma patients with hcDNA levels>>median or ≤median were compared.

RESULTS

Trauma patients with high plasma hcDNA had higher Injury Severity Score (ISS) and level of sympathoadrenal activation (higher adrenaline and noradrenaline) and a higher proportion of prolonged activated partial thromboplastin time (APTT) and higher D-dimer, tissue-type plasminogen activator (tPA), Annexin V and soluble CD40 ligand (sCD40L) concurrent with lower plasminogen activator inhibitor (PAI)-1) and prothrombin fragment (PF) 1 + 2 (all P < 0.05), all indicative of impaired thrombin generation, hyperfibrinolysis and platelet activation. Furthermore, patients with high hcDNA had enhanced inflammation and endothelial damage evidenced by higher plasma levels of terminal complement complex (sC5b-9), IL-6, syndecan-1, thrombomodulin and tissue factor pathway inhibitor (all P < 0.05).

CONCLUSIONS

Excessive release of extracellular histones and nucleic acids seems to contribute to the hypocoagulability, inflammation and endothelial damage observed early after trauma.

Deep venous thrombosis and pulmonary embolism represent two expressions of a similar clinical pathological process traditionally referred to as venous thromboembolism. Several population studies evidence venous thromboembolism as a leading healthcare problem worldwide, highlighting the need for early and reliable diagnosis to enable appropriate triage of affected patients and to optimize outcome. There is still debate, however, on which thrombotic markers to use, as well as their most suitable position within diagnostic algorithms. This article aims to review the pathophysiology and clinical usefulness of past, present and future markers of thrombosis, including soluble fibrin monomers, fibrin/fibrinogen degradation products, thrombin-antithrombin complex, plasmin-antiplasmin complex, fibrinopeptide A and B, <em>prothrombin</em> <em>fragments</em> <em>1</em> + <em>2</em>, thrombus precursor protein, D-dimer, activated protein C-protein C inhibitor complex, myeloperoxidase, thrombin generation assays and proteomic analysis. Several lines of evidence now attest that the global diagnostic performances of some D-dimer assays largely outperform those of any other coagulation or fibrinolytic marker proposed thus far, and a "negative" D-dimer measured with rapid enzyme linked fluorescent immunoassay is now considered the biochemical gold standard for ruling out an acute episode of venous thromboembolism in a patient with a low pretest probability for venous thromboembolism, so that additional testing can be safely omitted. However, to further improve clinical outcomes, the diagnostic efficiency of combining D-dimer testing with other markers covering different pathophysiological aspects of thrombosis such as continuous and progressive thrombin generation (e.g., activated protein C-protein C inhibitor complex) or neutrophil activation (i.e., myeloperoxidase) merits further investigation. Proteomic analysis, which would help to characterize the structure and function of each protein and the complexities of protein-protein interactions in physiological and pathological hemostasis, also holds promise for identifying novel markers and developing effective diagnostic protocols in the future.

We investigated the effects of statin treatment on platelet-derived microparticles (PMPs) and thrombin generation in atherothrombotic disease. Nineteen patients with peripheral arterial occlusive disease were randomised to eight weeks of treatment with atorvastatin or placebo in a cross-over fashion. Expression of GPIIIa (CD6<em>1</em>), P-selectin (CD6<em>2</em>P), tissue factor (TF, CD<em>1</em>4<em>2</em>) and phosphatidylserine (PS; annexin-V or lactadherin binding) was assessed on PMPs. Thrombin generation in vivo was assessed by measurement of <em>prothrombin</em> <em>fragment</em> <em>1</em>+<em>2</em> in plasma (F<em>1</em>+<em>2</em>) and ex vivo by using the calibrated automated thrombogram (CAT). During atorvastatin treatment, expression of TF, P-selectin and GPIIIa was significantly reduced vs. placebo (p<0.00<em>1</em> for all). No effect on annexin-V or lactadherin binding was seen. Thrombin generation was significantly reduced during atorvastatin as assessed by both the CAT assay (p<0.00<em>1</em>) and by measurements of F<em>1</em>+<em>2</em> (p<0.0<em>1</em>). Subsequent in vitro experiments showed that when TF on microparticles (MPs) was blocked by antibodies, the initiation of thrombin generation was slightly but significantly delayed. Blocking PS on MPs using annexin-V or lactadherin resulted in almost complete inhibition of thrombin generation. In conclusion, atorvastatin reduces thrombin generation and expression of TF, GPIIIa and P-selectin on PMPs in patients with peripheral vascular disease. Microparticle-bound TF slightly enhances initiation of thrombin generation whereas negatively charged surfaces provided by MPs or lipoproteins could reinforce thrombin generation. Statins may inhibit initiation of thrombin generation partly through a microparticle dependent mechanism but the main effect is probably through reduction of lipoprotein levels.

The zymogen <em>prothrombin</em> is composed of <em>fragment</em> <em>1</em> containing a Gla domain and kringle-<em>1</em>, <em>fragment</em> <em>2</em> containing kringle-<em>2</em>, and a protease domain containing A and B chains. The <em>prothrombin</em>ase complex assembled on the surface of platelets converts <em>prothrombin</em> to thrombin by cleaving at Arg-<em>2</em>7<em>1</em> and Arg-3<em>2</em>0. The three-dimensional architecture of <em>prothrombin</em> and the molecular basis of its activation remain elusive. Here we report the first x-ray crystal structure of <em>prothrombin</em> as a Gla-domainless construct carrying an Ala replacement of the catalytic Ser-5<em>2</em>5. <em>Prothrombin</em> features a conformation 80 Å long, with <em>fragment</em> <em>1</em> positioned at a 36° angle relative to the main axis of <em>fragment</em> <em>2</em> coaxial to the protease domain. High flexibility of the linker connecting the two kringles suggests multiple arrangements for kringle-<em>1</em> relative to the rest of the <em>prothrombin</em> molecule. Luminescence resonance energy transfer measurements detect two distinct conformations of <em>prothrombin</em> in solution, in a 3:<em>2</em> ratio, with the distance between the two kringles either fully extended (54 ± <em>2</em> Å) or partially collapsed (≤34 Å) as seen in the crystal structure. A molecular mechanism of <em>prothrombin</em> activation emerges from the structure. Of the two sites of cleavage, Arg-<em>2</em>7<em>1</em> is located in a disordered region connecting kringle-<em>2</em> to the A chain, but Arg-3<em>2</em>0 is well defined within the activation domain and is not accessible to proteolysis in solution. Burial of Arg-3<em>2</em>0 prevents <em>prothrombin</em> autoactivation and directs <em>prothrombin</em>ase to cleave at Arg-<em>2</em>7<em>1</em> first. Reversal of the local electrostatic potential then redirects <em>prothrombin</em>ase toward Arg-3<em>2</em>0, leading to thrombin generation via the prethrombin-<em>2</em> intermediate.

P38 mitogen-activated protein kinase (MAPK) is an important component of intracellular signaling cascades that initiate various inflammatory cellular responses. To determine the role of p38 MAPK in the procoagulant response to lipopolysaccharide (LPS), <em>2</em>4 healthy subjects were exposed to an intravenous dose of LPS (4 ng/kg), preceded 3 hours earlier by orally administered 600 or 50 mg BIRB 796 BS (a specific p38 MAPK inhibitor), or placebo. The 600-mg dose of BIRB 796 BS strongly inhibited LPS-induced coagulation activation, as measured by plasma concentrations of the <em>prothrombin</em> <em>fragment</em> F<em>1</em> + <em>2</em>. BIRB 796 BS also dose dependently attenuated the activation and subsequent inhibition of the fibrinolytic system (plasma tissue-type plasminogen activator, plasmin-alpha<em>2</em>-antiplasmin complexes, and plasminogen activator inhibitor type <em>1</em>) and endothelial cell activation (plasma soluble E-selectin and von Willebrand factor). Activation of p38 MAPK plays an important role in the procoagulant and endothelial cell response after in vivo exposure to LPS.

Activation of <em>prothrombin</em> by factor X(a) requires proteolysis of two bonds and is commonly assumed to occur via by two parallel, sequential pathways. Hydrolysis of Arg(3<em>2</em><em>2</em>)-Ile(3<em>2</em>3) produces meizothrombin (MzII(a)) as an intermediate, while hydrolysis of Arg(<em>2</em>73)-Thr(<em>2</em>74) produces prethrombin <em>2</em>-<em>fragment</em> <em>1</em>.<em>2</em> (Pre<em>2</em>-F<em>1</em>.<em>2</em>). Activation by human factor X(a) of human <em>prothrombin</em> was examined in the absence of factor V(a) and in the absence and presence of bovine phosphatidylserine (PS)/palmitoyloleoylphosphatidylcholine (<em>2</em>5:75) membranes. Four sets of data were collected: fluorescence of an active site probe (DAPA) was sensitive to thrombin, MzII(a), and Pre<em>2</em>-F<em>1</em>.<em>2</em>; a synthetic substrate (S-<em>2</em><em>2</em>38) detected thrombin or MzII(a) active site formation; and SDS-PAGE detected both intermediates and thrombin. The fluorescence data provided an internal check on the active site and SDS-PAGE measurements. Kinetic constants for conversion of intermediates to thrombin were measured directly in the absence of membranes. Both MzII(a) and Pre<em>2</em>-F<em>1</em>.<em>2</em> were consumed rapidly in the presence of membranes, so kinetic constants for these reactions had to be estimated as adjustable parameters by fitting three data sets (thrombin and MzII(a) active site formation and Pre<em>2</em> appearance) simultaneously to the parallel-sequential model. In the absence of membranes, this model successfully described the data and yielded a rate constant, 44 M(-<em>1</em>) s(-<em>1</em>), for the rate of MzII(a) formation. By contrast, the parallel-sequential model could not describe <em>prothrombin</em> activation in the presence of optimal concentrations of PS-containing membranes without assuming that a pathway existed for converting <em>prothrombin</em> directly to thrombin without release from the membrane-enzyme complex. The data suggest that PS membranes (<em>1</em>) regulate factor X(a), (<em>2</em>) alter the substrate specificity of factor X(a) to favor the meizothrombin intermediate, and (3) "channel" intermediate (MzII(a) or Pre<em>2</em>-F<em>1</em>.<em>2</em>) back to the active site of factor X(a) for rapid conversion to thrombin.

Several studies have shown that thrombosis and inflammation play an important role in the pathogenesis of Ischaemic Heart Disease (IHD). In particular, Tissue Factor (TF) is responsible for the thrombogenicity of the atherosclerotic plaque and plays a key role in triggering thrombin generation. The aim of this study was to evaluate the TF/Tissue Factor Pathway Inhibitor (TFPI) system in patients with IHD. We have studied 55 patients with IHD and not on heparin [<em>1</em>8 with unstable angina (UA), <em>2</em>4 with effort angina (EA) and <em>1</em>3 with previous myocardial infarction (MI)] and 48 sex- and age-matched healthy volunteers, by measuring plasma levels of TF, TFPI, <em>Prothrombin</em> <em>Fragment</em> <em>1</em>-<em>2</em> (F<em>1</em>+<em>2</em>), and Thrombin Antithrombin Complexes (TAT). TF plasma levels in IHD patients (median <em>2</em><em>1</em>5.4 pg/ml; range 7<em>2</em>.6 to 834.3 pg/ml) were significantly (p<0.00<em>1</em>) higher than those found in control subjects (median <em>1</em>4<em>2</em>.5 pg/ml; range <em>2</em>8.0-<em>2</em>55.3 pg/ml). Similarly, TFPI plasma levels in IHD patients were significantly higher (median <em>1</em><em>2</em>9.0 ng/ml; range 30.3-3<em>1</em>6.8 ng/ml; p<0.00<em>1</em>) than those found in control subjects (median 60.4 ng/ml; range <em>2</em>0.8-<em>1</em>5<em>1</em>.3 ng/ml). UA patients showed higher amounts of TF and TFPI plasma levels (TF median <em>2</em>55.6 pg/ml; range <em>1</em>48.8-834.3 pg/ml; TFPI median <em>1</em>37.7 ng/ml; range 38.3-3<em>1</em>6.8 ng/ml) than patients with EA (TF median <em>1</em>8<em>2</em>.0 pg/ml; range 7<em>2</em>.6-380.0 pg/ml; TFPI median <em>1</em><em>1</em>5.<em>2</em> ng/ml; range 47.0-<em>1</em>96.8 ng/ml) and MI (TF median <em>2</em><em>1</em>3.9 pg/ml; range <em>1</em><em>2</em>5.0 to 34<em>1</em>.9 pg/ml; TFPI median <em>1</em>30.5 ng/ml; range 94.0-<em>2</em>07.8 ng/ml). Similar levels of TF and TFPI were found in patients with mono- or bivasal coronary lesions. A positive correlation was observed between TF and TFPI plasma levels (r = 0.57, p<0.00<em>1</em>). Excess thrombin formation in patients with IHD was documented by TAT (median 5.<em>2</em> microg/l; range <em>1</em>.7-<em>2</em><em>1</em>.0 microg/l) and F<em>1</em>+<em>2</em> levels (median <em>1</em>.4 nmol/l; range 0.6 to 6.<em>2</em> nmol/l) both significantly higher (p<0.00<em>1</em>) than those found in control subjects (TAT median <em>2</em>.3 microg/l; range <em>1</em>.4-4.<em>2</em> microg/l; F<em>1</em>+<em>2</em> median 0.7 nmol/l; range 0.3-<em>1</em>.3 nmol/l). As in other conditions associated with cell-mediated clotting activation (cancer and DIC), also in IHD high levels of circulating TF are present. Endothelial cells and monocytes are the possible common source of TF and TFPI. The blood clotting activation observed in these patients may be related to elevated TF circulating levels not sufficiently inhibited by the elevated TFPI plasma levels present.

OBJECTIVE

The prevalence of thromboembolism might be higher than previously recognized in patients with atrial flutter (AFL) based on findings of transesophageal echocardiography (TEE). To evaluate the potential prothrombotic state in patients with AFL, TEE findings and hemostatic markers were compared among patient groups with AFL, normal sinus rhythm (NSR) and chronic nonvalvular atrial fibrillation (AF).

METHODS

Cross-sectional study at a university hospital.

METHODS

In <em>2</em>8 patients (mean age, 63 years) with AFL, 58 patients (mean age, 66 years) with AF, and <em>2</em>7 patients (mean age, 6<em>1</em> years) with NSR who underwent TEE, plasma levels of markers for platelet activity (platelet factor 4 and beta-thromboglobulin [beta-TG]), thrombotic status (thrombin-antithrombin III complex and <em>prothrombin</em> <em>fragments</em> <em>1</em> and <em>2</em>) and fibrinolytic status (d-dimer and plasmin-alpha(<em>2</em>)-plasmin inhibitor complex) were determined.

RESULTS

Left atrial appendage (LAA) blood flow velocity in patients with AFL was higher (p < 0.05) than that in patients with AF, but was lower (p < 0.05) than that in patients with NSR (AF, <em>2</em>5 +/- <em>2</em>; AFL, 44 +/- 4; NSR, 60 +/- 4 cm/s). Dense left atrial spontaneous echo contrast (SEC) was found in 4 patients (<em>1</em>4%) with AFL and <em>1</em>6 patients (<em>2</em>8%) with AF. There was no significant difference in plasma levels of hemostatic markers between the AFL group and the NSR group. AFL patients with impaired LAA function (LAA flow < 30cm/s, dense SEC, or both), however, showed higher level of d-dimer and beta-TG than those without impaired LAA function (d-dimer, <em>1</em>.9 +/- 0.6 microg/mL vs 0.4 +/- 0.<em>1</em> microg/mL; beta-TG, 73 +/- <em>1</em>7 ng/mL vs 33 +/- 5 ng/mL, p < 0.05).

CONCLUSIONS

Patients with AFL as a whole are not in the prothrombotic state as compared with those with AF. However, patients with AFL and impaired LAA function are at potentially high risk for thromboembolism and might require anticoagulation.

OBJECTIVE

Cirrhosis is commonly associated with haemostatic dysfunction. The similarities of laboratory tests of disseminated intravascular coagulation (DIC) to those found in cirrhosis has led to the belief that DIC is a feature of the haemostatic failure of cirrhosis.

METHODS

The aim of this study was to determine whether DIC is part of the coagulopathy of cirrhosis by applying quantitative tests for <em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>, antithrombin III, thrombin-antithrombin complex, and specific fribrinogen degradation products levels (XDP), as well as the thrombelastograph for detecting the Clot Lysis Index.

RESULTS

Fifty-two stable cirrhotic patients (33 men, <em>1</em>9 women; mean age, 58.8 yr; range, <em>2</em>4-7<em>2</em> yr) with differing etiologies were studied. On tests of thrombin generation: thrombin-antithrombin complexes, fibrin(ogen) degradation products, and <em>prothrombin</em> <em>fragment</em>s <em>1</em> + <em>2</em> were not found to be significantly different from an age- and gender-matched control group (p = 0.<em>1</em>8, 0.3, and 0.67, respectively), whereas albumin, Factor V, fibrinogen, antithrombin III, and alpha<em>2</em>-antiplasmin were all significantly low (p = 0.0004, 0.00<em>2</em>, 0.06, 0.004, and 0.004, respectively), reflecting reduced synthetic function and correlation in ascitic and non-ascitic patients. There was no correlation between impaired synthesis (antithrombin III and alpha<em>2</em>-antiplasmin) and indices of DIC (<em>prothrombin</em> <em>fragment</em> <em>1</em> + <em>2</em>, thrombin-antithrombin complexes, and XDP) (p = not significant). The percentage of patients with high <em>prothrombin</em> <em>fragment</em>s <em>1</em> + <em>2</em> and thrombin antithrombin levels in each Child grade group was similar. Thrombin time was significantly elevated in the cirrhotic group (a manifestation of low fibrinogen levels). The Clot Lysis Index as measured by thrombelastography was significantly abnormal, indicating mild hyperfibrinolysis.

CONCLUSIONS

We conclude that DIC is not part of the coagulopathy in stable liver cirrhosis without recent complications.

OBJECTIVE

The progression of diabetic angiopathy is, in most cases, unpredictable. The aim of this study was to investigate early events that could influence the development of diabetic angiopathy.

METHODS

Circulating levels of von Willebrand factor (vWF) and tissue-plasminogen activator (tPA), defining endothelial perturbation, were measured in 40 young patients with type <em>1</em> diabetes. Patients were divided into two groups according to the duration of diabetes (group A, (<em>1</em> year; group B,>><em>1</em> year) and compared with a control group of age- and sex-matched healthy individuals. <em>Prothrombin</em> <em>fragment</em> <em>1</em> and <em>2</em> (F(<em>1</em>+<em>2</em>)), tumor necrosis factor-alpha (TNF-alpha), and C-reactive protein (CRP) levels were also determined as markers of a prothrombotic state and inflammatory response. A total of <em>1</em>6 of the <em>2</em>0 children in group A were re-examined after <em>1</em><em>2</em> months.

RESULTS

Compared with either normal subjects or patients in group B, children in group A showed increased levels of vWF, tPA, F(<em>1</em>+<em>2</em>), TNF-alpha, and CRP. Significant direct correlations between TNF-alpha or CRP and either vWF, tPA, or F(<em>1</em>+<em>2</em>) were observed. Endothelial perturbation was shown in 70% of group A and <em>2</em>0% of group B. After <em>1</em> year, <em>1</em>6 of the <em>2</em>0 patients in group A showed a significant reduction in vWF, tPA, F(<em>1</em>+<em>2</em>), TNF-alpha, and CRP levels, whereas endothelial perturbation was reversed in 5 of these patients.

CONCLUSIONS

Endothelial perturbation represents an early and, in some cases, reversible event in the chronology of type <em>1</em> diabetes in children. A correlation might exist between the initial inflammatory reaction and the appearance of endothelial perturbation.

Activation of <em>prothrombin</em>, as catalyzed by the <em>prothrombin</em>ase complex (factor X(a), enzyme; factor V(a) and phosphatidylserine (PS)-containing membranes, cofactors), involves production and subsequent proteolysis of two possible intermediates, meizothrombin (MzII(a)) and prethrombin <em>2</em> plus <em>fragment</em> <em>1</em>.<em>2</em> (Pre<em>2</em> & F<em>1</em>.<em>2</em>). V(max), K(m), or V(max)/K(m) for all four proteolytic steps was determined as a function of membrane-phospholipid concentration. Proteolysis was monitored using a fluorescent thrombin inhibitor, a chromogenic substrate, and SDS-PAGE. The kinetic constants for the conversion of MzII(a) and Pre<em>2</em> & F<em>1</em>.<em>2</em> to thrombin were determined directly. Pre<em>2</em> & F<em>1</em>.<em>2</em> conversion was linear in substrate concentration up to 4 microm, whereas MzII(a) proteolysis was saturable. First order rate constants for formation of MzII(a) and Pre<em>2</em> & F<em>1</em>.<em>2</em> could not be determined directly and were determined from global fitting of the data to a parallel, sequential model, each step of which was treated by the Michaelis-Menten formalism. The rate of direct conversion to thrombin without release of intermediates from the membrane-V(a)-X(a) complex (i.e. "channeling") also was adjusted because both the membranes and factor V(a) have been shown to cause channeling. k(cat), K(m), or k(cat)/K(m) values were reported for one lipid concentration, for which all X(a) was likely incorporated into a X(a)-V(a) complex on a PS membrane. Comparing previous results, which were obtained either with factor V(a) (Boskovic, D. S., Bajzar, L. S., and Nesheim, M. E. (<em>2</em>00<em>1</em>) J. Biol. Chem. <em>2</em>76, <em>2</em>8686-<em>2</em>8693) or with membranes individually (Wu, J. R., Zhou, C., Majumder, R., Powers, D. D., Weinreb, G., and Lentz, B. R. (<em>2</em>00<em>2</em>) Biochemistry 4<em>1</em>, 935-949), with results presented here we conclude that both factor V(a) and PS-containing membranes induce similar rate increases and pathway changes. Moreover, we have determined: <em>1</em>) factor V(a) has the greatest effect in enhancing rates of individual proteolytic events; <em>2</em>) PS-containing membranes have the greatest role in increasing the preference for the MzII(a) versus Pre<em>2</em> pathway; and 3) PS membranes cause approximately 50% of the substrate to be activated via channeling at 50 microm membrane concentration, but factor V(a) extends the range of efficient channeling to much lower or higher membrane concentrations.