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The physiological role of autophagy in the catabolic process of the body involves protein synthesis and degradation in homeostasis under normal and stressed conditions. In hepatocellular carcinoma (HCC), the role of tumor microenvironment (TME) has been concerned as the main issue in fighting against this deadly malignancy. During the last decade, the crosstalk between tumor cells and their TME in HCC extensively accumulated. However, a deeper knowledge for the actual function of autophagy in this interconnection which involved in supporting tumor development, progression and chemoresistance in HCC is needed but still largely unknown. Recent studies have shown that coagulants tissue factor (TF) and factor VII (FVII) has a pathological role in promoting tumor growth by activating protease-activated receptor 2 (PAR2). Autophagy-associated LC3A/B-II formation was selectively suppressed by FVII/PAR2 signaling which mediated by mTOR activation through Atg7 but not Atg5/Atg12 axis. The coagulant-derived autophagic suppression seemed potentiate a vicious circle of malignancy in producing more FVII and PAR2 which facilitate in vivo and in vitro tumor progression of HCC and the investigations are consistent with the clinical observations. In this review, we briefly summarize the current understanding of autophagy and discuss recent evidence for its role in HCC malignancy., (Copyright © 2018 Chang Gung University. Published by Elsevier B.V. All rights reserved.)

Objective: To preliminarily verify the cross talk between tissue factor/active coagulation factor VII (TF/FVIIa) and epidermal growth factor receptor (EGFR) pathways in human colon cancer cells in culture., Methods: FVIIa was treated to HT-29 (KRAS-wild type) and LoVo (KRAS-mutant) colon cancer cells to activate TF/FVIIa pathway, qRT-PCR and Western blot were used to detect the expressions of amphiregulin (AREG) and epiregulin (EREG), ligands of EGFR on mRNA and protein levels, respectively. After knocking down expression of TF by TF-targeted siRNA transfection, FVIIa was treated and mRNA expressions of AREG and EREG were detected to see whether the FVIIa-induced effects were dependent on TF. Expressions of mRNA of TF and FVII were detected by qRT-PCR following the activation of EGFR pathway by treatment with epidermal growth factor (EGF) to HT-29 and LoVo cells., Results: After TF/FVIIa pathway was activated, for HT-29 cells, expressions of AREG (on mRNA level) and EREG (both on mRNA and protein level) were significantly down-regulated versus those of control group, gene expressions of AREG and EREG were 0.55±0.09 vs.0.99 ±0.09, 0.67±0.10 vs.1.02±0.02, protein expressions of EREG were 0.54±0.09 vs.1.04±0.13, all P<0.05. For LoVo cells, expressions of AREG (both on mRNA and protein level) and EREG (on protein level) were significantly up-regulated versus those of control group, gene expression of AREG were 1.87±0.39 vs. 0.93±0.23, protein expressions of AREG and EREG were 3.09±0.73 vs.1.11±0.21, 1.53±0.19 vs.0.97±0.23, all P<0.05. The regulating effect of AREG and EREG mRNA expression by FVIIa in HT-29 and LoVo cells could both be partly blocked by knocking down TF expression. For HT-29 cells, activation of EGFR pathway induced no significant TF mRNA expression, FVII mRNA expression was not detected. However,for LoVo cells, activation of EGFR pathway induced significantly higher mRNA expressions of both TF and FVII, expressions were 1.53±0.23 vs.1.00±0.23, 53.20±6.08 vs.1.00±0.15, all P<0.05., Conclusion: In colon cancer cell LoVo, when activated, TF/FVIIa pathway and EGFR pathway could interact through upregulating the other pathway's effectors, and mutant KRAS might play a critical role in the two pathways' cross talk.

Background: Given the importance of factor VII (FVII) in extrinsic pathway of coagulation cascade, we sought to elucidate the relationship between FVII and traumatic brain injury-induced coagulopathy and progressive hemorrhagic injury (PHI)., Methods: Eighty-one patients with isolated traumatic brain injury, 16 years or older, were recruited between 2010 and 2012. Blood was collected on arrival in the emergency department and analyzed with activated partial thromboplastic time, international normalized ratio, platelet count, and activity of FVII. Coagulopathy was defined as thrombocytopenia (platelet count < 120,000/μL) or elevated international normalized ratio of greater than 1.2 or prolonged activated partial thromboplastin time greater than 40 seconds at admission. PHI was present when the follow-up computed tomographic scan reported any increase in size or number of the hemorrhagic lesions. Logistic regression examined the risks for coagulopathy and PHI., Results: Mean (SD) FVII activity in patients with coagulopathy was 85.69% (34.88%), which was significantly lower than patients without coagulopathy (99.57% [29.37%], p = 0.04). Isolated traumatic brain injury patients with FVII activity less than 77.5% have an odds ratio for coagulopathy of 5.52 (95% confidence interval, 1.82-16.68; p = 0.03) relative to patients with FVII activity of 77.5% or greater. Mean (SD) FVII activity in patients with PHI was 70.76% (18.21%), which was significantly lower than patients without PHI (105.76% [32.27%], p < 0.001). A stepwise logistic regression analysis identified FVII less than 77.5% (odds ratio, 4.53; 95% confidence interval, 1.62-12.67; p = 0.004) as a predisposing risk factors independently associated with the presence of PHI. The overall mortality rate in the study population was 7.4% (n = 6). The plasma FVII in death patients (91.44% [47.19%]) was slightly lower than that in survival patients (92.01% [32.04%]). However, there was no statistical difference between the two groups (p = 0.95)., Conclusion: A decrease of FVII activity significantly contributes to the coagulopathy and PHI in patients with isolated traumatic brain injury., Level of Evidence: Prognostic study, level III.

Objective: To identify and characterize a missence mutation Ser250 Phe underlying coagulation factor Ⅶ (FⅦ) deficiency in a Chinese patient and his family., Methods: The FⅦ gene (F7) was analyzed by DNA sequencing, and the FⅦ levels (including antigen and activity) in patient's plasma were determined with enzyme-linked immunoabsorbent assay (ELISA) and one stage prothrombin time based method. In addition, a FⅦ-250 Phe mutant corresponding to the identified mutation was expressed in HEK293 cells, and a subcellular localization experiment in CHO cells was performed to clarify the molecular mechanism of FⅦ deficiency caused by the FⅦ-250 Phe mutation., Results: The patient had a prolonged prothrombin time (PT: 36.5 s) and low levels of both FⅦ antigen and activity (130.2 ng/mL and 4.0%, respectively). Two heterozygous mutations were identified in the F7 gene (NG-009262.1), which included a g.15975 G>A mutation at the splice receptor site of intron 6 (IVS6-1G>A) and a novel g.16750 C>T mutation in exon 8, which resulted in replacement of Ser (TCC) 250 with Phe (TTC)250 in the vicinity of a charge-stablizing system. By gene expression experiments, the antigen and activity levels of FⅦ-250 Ser and FⅦ-250 Phe in the culture medium were (37.77 ± 2.30) ng/mL and (4.02 ± 0.52) ng/mL, respectively. ELISA and Western blotting analyses indicated that expression of the mutant FⅦ-250 Phe and wild type FⅦ-250 Ser was (130.51 ± 2.32) ng/mL and (172.45 ± 2.25) ng/mL, respectively. FⅦ-250 Phe was found in endoplasmic reticulum and Golgi apparatus, suggesting that the mutant FⅦ-250 Phe could be normally synthesized in the cells but was inefficiently secreted., Conclusion: Compound heterozygous mutations in F7 gene (g.15975G>A and g.16750C>T) may be responsible for the FⅦ deficiency in this patient. The novel FⅦ 250 Phe can be transported from endoplasmic reticulum to Golgi apparatus, but may be degraded or inefficient.

Tissue factor (TF) initiates the extrinsic coagulation cascade and is a high-affinity receptor for coagulation factor VII. TF also participates in protease-activated receptor (PAR)1 and PAR2 activation. Human epithelial basal cells were previously purified on the basis of TF expression. The purpose of this study was to determine if tracheobronchial epithelial basal cell-associated TF drives coagulation and/or activates PARs to promote basal cell functions. We used human tracheobronchial tissues to isolate human airway epithelial cells using specific cell surface markers by flow cytometry and studied TF expression by immunostaining. TF-dependent fibrin network formation was observed by confocal and scanning electron microscopy. TF knockdown was done using short hairpin RNA, and TF mRNA was measured using quantitative RT-PCR. We found that 97 ± 5% of first-passage human tracheobronchial epithelial cells were basal cells, and 100% of these basal cells expressed TF. Basal cell-associated TF was active, but TF activity was dependent on added extrinsic coagulation cascade factors. TF inhibition caused basal cell apoptosis and necrosis. This was due to two parallel but interdependent TF-regulated processes: failure to generate a basal cell-associated fibrin network and suboptimal PAR1 and PAR2 activity. The data indicate that membrane surface TF mediates airway epithelial basal cell attachment, which maintains cell survival and mitotic potential. The implications of these findings are discussed in the context of basal cell-associated TF activity in normal and injured tissues and of the potential for repair of airway epithelium in lung disease.

Objective: Emerging research suggests links between menopausal hot flashes and cardiovascular risk. The mechanisms underlying these associations are unclear due, in part, to the incomplete understanding of the physiology of hot flashes. We aimed to examine the longitudinal associations between hot flashes/night sweats and both inflammatory and hemostatic markers, controlling for cardiovascular risk factors and estradiol concentrations., Methods: Participants in the Study of Women's Health Across the Nation (n = 3,199), a longitudinal cohort study, were aged 42 to 52 years at cohort entry. Women completed interviews (hot flashes, night sweats: none, 1-5, and ≥6 d in the past 2 weeks), physical measures (blood pressure, height, weight), and a blood draw (high-sensitivity C-reactive protein, plasminogen activator inhibitor-1, factor VIIc, tissue plasminogen activator antigen [tPA-ag], fibrinogen, glucose, serum estradiol) yearly for 8 years. Hot flashes/night sweats were examined in relation to each inflammatory/hemostatic marker in linear mixed models adjusting for demographic factors, cardiovascular risk factors, and medication use, as well as serum estradiol., Results: Compared with experiencing no flashes, reporting hot flashes was associated with higher tPA-aglog (hot flashes 1-5 d: percent change, or % change [95% CI], 3.88 [2.22-5.58]; P < 0.0001; ≥6 d: % change [95% CI], 4.11 [1.95-6.32]; P < 0.001) and higher factor VIIclog (hot flashes ≥6 d: % change [95% CI], 2.13 [0.80-3.47]; P < 0.01) in multivariable models. Findings persisted after adjusting for estradiol. Findings for night sweats were similar but attenuated with adjustment., Conclusions: Frequent hot flashes were associated with higher factor VIIc and tPA-ag. Hemostatic pathways may be relevant to understanding hot flash physiology and links between hot flashes and cardiovascular risk.

The binding of thrombin to its receptor stimulates inflammatory cytokines including IL-6 and monocyte chemoattractant protein-1 (MCP-1); both are associated with the development of insulin resistance. Because increased adiposity enhanced the expression of coagulation factor VII that stimulates the coagulation pathway in adipose tissue, we tested whether the inhibition of thrombin action ameliorates insulin resistance in obese diabetic (Lpr(-/-):db/db) mice. The 4-wk administration of argatroban, a selective thrombin inhibitor, reduced fasting plasma glucose and ameliorated insulin resistance in these mice. It also reduced adipocyte size and macrophage infiltration into adipose tissue. The aberrant gene expression of MCP-1, IL-6, adiponectin, and factor VII and suppressed insulin receptor substrate-1-Akt signaling in adipose tissue of db/db mice were reversed by argatroban treatment. These results demonstrate that increased adiposity enhances the production of thrombin in adipose tissue by stimulating factor VII expression and suggest that increased thrombin activity in adipose tissue plays an important role in the development of insulin resistance via enhancing MCP-1 production, leading to macrophage infiltration and insulin receptor substrate-1-Akt pathway inactivation.

Hemostasis is traditionally defined as the physiologic process whereby bleeding is antagonized and possibly stopped to minimize blood loss. The first medical description of the clinical and inherited features of hemostasis can be dated back more than 1000 years, when Abu al-Qasim Khalaf ibn 'Abbas al-Andalusi al-Zahrawi' medical treatise provided some initial insights into this puzzling process. Since then, continuous and revolutionary scientific developments have contributed to decoding several aspects of this intricate but essential physiologic phenomenon, providing a reliable model to explain the leading mechanisms involved. Although the point at which bleeding stops is commonly referred to as "coagulation," blood coagulation is actually only one part of a two-part hemostatic process that develops through sequential steps referred to as primary and secondary hemostasis. Throughout its activation and development, the coagulation cascade is strictly regulated by a series of natural inhibitors, which prevent unnecessary and excessive clotting. The aim of this article is to provide a concise overview of the major discoveries and past and current perspectives in coagulation and hemostasis.

Factor VII (FVII) deficiency is the most frequent among rare congenital bleeding disorders, accounting for one symptomatic individual per 500,000 population, apparently without any racial/ethnic predilection. FVII deficiency prevalence in the general population is probably higher because of the presence of asymptomatic and poorly symptomatic individuals. In accordance with the role of FVII as part of the initiating complex of the extrinsic coagulation pathway, laboratory diagnosis is easy, because FVII deficiency is the only congenital bleeding disorder characterized by isolated prolonged prothrombin time. Molecular diagnosis is available, and a broad spectrum of mutations has been characterized in the FVII gene, which is located in chromosome 13. Clinical manifestations are heterogeneous, ranging from severe life-threatening haemorrhages, such as cerebral, gastrointestinal, and joint haemorrhages, to miscellaneous minor bleeding. The main clinical features in our database (International Registry on Congenital FVII Deficiency database, n = 515) are as follows: (i) the absence of a clear-cut and consistent correlation between bleeding symptoms and FVII clotting levels; (ii) an excess of symptomatic women compared with men; (iii) frequent surgery-related bleeding, which is often a diagnostic tool in previously asymptomatic individuals. Several therapeutic options are possible, including plasma-derived and recombinant products, but therapeutic schedules, optimal dosages, and administration times still have to be precisely defined, and clinical studies, including online registries such as the Seven Treatment Evaluation Registry, are actually ongoing to achieve in a better manner a safe, rational and standardized substitution treatment for this congenital disorder.

Study Objective: To validate a Thromboelastograph (Haemoscope Corporation, Niles, IL) assay for functional fibrinogen., Design: Correlation study of the Thromboelastograph assay with two conventional fibrinogen assays by the standard Clauss method., Setting: Research laboratory of a university medical center., Participants and Interventions: Blood samples were obtained from 19 healthy volunteers., Measurement and Main Results: Thromboelastograph assays, using heparinized whole blood from 19 healthy donors, indicated that reptilase-XIIIa mixture (Activatorf)-generated clot shear elasticity in dynes per square centimeter (Gf) correlated with fibrinogen (mg/dL). Blood from four donors was used to define the contribution of hematocrit (Hct) to Gf by titration with platelet-rich plasma. The Gf versus Hct gave linear correlations (r2 = 0.746) with Gf = 1258 - 17.8 x % Hct. A commercial collection of 19 normal, 10 borderline, and one deficient for functional fibrinogen-citrated plasmas was assayed for Gf after recalcification using Activatorf. Of the 30 plasma samples, four were from factor X- or factor VII-deficient donors and one was from a coumadin-treated donor. There was a linear correlation of Activatorf Gf with functional fibrinogen (r2 = 0.940) with Gf = -730 + 9.21 x fibrinogen (mg/dL)., Conclusion: Thrombelastography with Activatorf may be used to determine fibrinogen levels in whole blood.

Recombinant activated factor VII (rFVIIa) was originally developed for the treatment of spontaneous and surgical bleeding of hemophiliacs with inhibitors. Along with the elucidation of its molecular mechanism of action, rFVIIa has been successfully used over the last few years in a wide range of non-hemophilic bleeding conditions. The aim of this review is to summarize the current clinical experience on the use of rFVIIa in the management of bleeding associated with congenital or acquired platelet disorders.

Introduction: 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., Materials and 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, prothrombin fragment 1+2) 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 prothrombin fragment 1+2 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<0.001). Factor X and antithrombin III were both reduced in chronic kidney disease and correlated (r=0.58; P<0.001). 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.

The FVII level is considered a risk factor for cardiovascular disease. Some of the polymorphic differences in the promoter of the F7 gene have been associated with variations in FVII levels. However, linkage disequilibrium among those polymorphisms has made it difficult to pinpoint the true functional variants, so contradictory results have often appeared among various studies. We provide new findings of the effect of the polymorphisms in the promoter region of F7. In vitro transfection of 15 plasmids containing different combinations of F7 promoter polymorphisms was performed in HepG2 cells. We found that allelic variants -323ins10 and -122C strongly reduced promoter activity and that allelic variant -402A significantly increased promoter activity. We report the effect of a novel variant (-2989A) that significantly increases F7 expression levels. However, this novel allelic variant is in strong linkage disequilibrium with the -323ins10 variant in our Spanish population, which has a clear dominant effect over the -2989A variant and completely masks its effect. Our results have important implications for mapping genes affecting complex diseases using association studies. That is, they imply that true functional variants should be chosen to confirm the analyses and to ensure that the results can be reproduced in other populations. In addition, our results suggest that it would be informative to screen for the -2989A variant in other populations, since it may well be a risk factor for cardiovascular disease in populations where it does not appear with the decanucleotide insertion.

A hypothesis: thrombin is a "Universal Enzyme of Energy Transduction" that employs ATP energy in flowing blood to activate biochemical reactions and cell effects in both hemostasis and tissue repair. All cells possess PAR-1 (thrombin) receptors and are affected by thrombin elevations, and thrombin effects on individual cell types are determined by their unique complement of PAR-1 receptors. Disruption of the vascular endothelium (VE) activates a tissue repair mechanism (TRM) consisting of the VE, tissue factor (TF), and circulating Factors VII, IX and X that governs localized thrombin elevations to activate clot formation and cellular effects that repair tissue damage. The culmination of the repair process occurs with the restoration of the VE followed by declines in thrombin production that causes Apoptosis ("programmed cell death") in wound-healing fibroblasts, which functions as a mechanism to draw wound edges together. The location and magnitude of TRM activity governs the location and magnitude of Factor VIII activity and clot formation, but the large size of Factor VIII prevents it from penetrating the clot formed by its activity, so that its effects are self-limiting. Factors VII, IX and X function primarily as tissue repair enzymes, while Factor VIII and Factor XIII are the only serine protease enzymes in the "Coagulation Cascade" that are exclusively associated with hemostasis.

Thrombosis superimposed on a disrupted plaque is the proximate event that triggers most acute ischemic syndromes and episodes of sudden cardiac death. A significant number of acute ischemic events occur in individuals without traditional atherosclerosis-related risk factors. In an attempt to pinpoint additional risk factors, researchers are examining the thrombotic cascade and the cellular components, plasma proteins, and endothelium-derived mediators, as well as their genetic polymorphisms, that may affect this system. This article enumerates a number of potential hemostatic risk factors and discusses the evidence linking them to atherothrombotic events.

Blood coagulation factor VII (fVII) is physiologically synthesized in the liver and released into the blood. Binding of fVII to tissue factor (TF) at sites of vascular injury triggers coagulation and hemostasis. TF/fVIIa complex formation on the surface of cancer cells plays important roles in cancer biology. Although fVII is synthesized by hepatocellular carcinoma, it remained unclear how TF/fVIIa complex formation and promigratory signaling can occur for most other cancers in extravascular locations. Here, we show by reverse transcription-PCR analysis that nonhepatic cancer cell lines constitutively express fVII mRNA and that endogenously synthesized fVIIa triggers coagulation activation on these cells. fVIIa expression in cancer cells is inducible under hypoxic conditions and hypoxia-inducible factor-2 alpha bound the promoter region of the FVII gene in chromatin immunoprecipitation analyses. Constitutive fVII expression in an ovarian cancer cell line enhanced both migration and invasion. Enhanced motility was blocked by anti-TF antibodies, factor Xa inhibition, and anti-protease-activated receptor-1 antibody treatment, confirming that TF/fVIIa stimulated migration by triggering cell signaling. This study shows that ectopic synthesis of fVII by cancer cells is sufficient to support proinvasive factor Xa-mediated protease-activated receptor-1 signaling and that this pathway is inducible under hypoxia.

Chronic hypertriglyceridemia is thought to be atherogenic and is associated with an elevated thrombotic potential, both of which may be improved with aerobic exercise training. Eight subjects were tested for aerobic capacity, body composition, and postprandial lipemia (PPL), followed by 6 mo of exercise training and final testing. Blood samples were obtained for measurement of free fatty acid (FFA), triglycerides (TG), insulin (Ins), and glucose (Glu). Hemostatic variables including factor VII activity (FVIIa), tissue factor pathway inhibitor-factor Xa complex (TFPI/Xa), and plasminogen activator inhibitor-1 (PAI-1) antigen/activity as well as leukocyte tumor necrosis factor-alpha (TNF-alpha) gene expression were determined among four subjects. We found that the exercise training was of sufficient intensity to increase aerobic capacity (P < 0.0001) and improve body composition (P = 0.04). There were no differences between tests among PPL responses of FFA, TG, Ins, or Glu; however, the mean TG response and fat oxidation rate improved. PAI-1 antigen/activity, FVIIa, TFPI/Xa, and TNF-alpha gene expression were all improved after exercise training after adjusting for confounders. We conclude that aerobic exercise training reduces the potential for coagulation, improves fibrinolytic potential, and reduces leukocyte TNF-alpha gene expression after the ingestion of a high-fat meal.

The extrinsic coagulation is recognized as an 'inducible' signalling cascade resulting from tissue factor (TF) upregulation by exposure to clotting zymogen FVII upon inflammation or tissue injury. Following the substantial initiation, an array of proteolytic activation generates mediating signals (active serine proteases: FVIIa, FXa and FIIa) that lead to hypercoagulation with fibrin overproduction manifesting thrombosis. In addition, TF upregulation plays a central role in driving a thrombosis-inflammation circuit. Coagulant mediators (FVIIa, FXa and FIIa) and endproduct (fibrin) are proinflammatory, eliciting tissue necrosis factor, interleukins, adhesion molecules and many other intracellular signals in different cell types. Such resulting inflammation could ensure 'fibrin' thrombosis via feedback upregulation of TF. Alternatively, the resulting inflammation triggers platelet/leukocyte/polymononuclear cell activation thus contributing to 'cellular' thrombosis. TF is very vulnerable to upregulation resulting in hypercoagulability and subsequent thrombosis and inflammation, either of which presents cardiovascular risks. The prevention and intervention of TF hypercoagulability are of importance in cardioprotection. Blockade of inflammation reception and its intracellular signalling prevents TF expression from upregulation. Natural (activated protein C, tissue factor pathway inhibitor, or antithrombin III) or pharmacological anticoagulants readily offset the extrinsic hypercoagulation mainly through FVIIa, FXa or FIIa inhibition. Therefore, anticoagulants turn off the thrombosis-inflammation circuit, offering not only antithrombotic but anti-inflammatory significance in the prevention of cardiovascular complications., (Copyright 2005 John Wiley & Sons, Ltd.)

Aim: To investigate the major steps of thrombogenesis and to identify the differences in these steps between idiopathic patient group and control group., Methods: Fibrinogenesis was studied by measuring the activated factor VII, total and free levels of tissue factor pathway inhibitor (TFPI). The fibrinolysis step was investigated by determining the global fibrinolytic capacity. The endothelial function was assessed by measuring the levels of soluble adhesion molecules, namely soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular cell adhesion molecule 1 (sVCAM-1) and soluble E-selectin molecule. The exclusion criteria from "idiopathic" patient group were abdominal surgery, pregnancy, use of oral contraceptives, anti-phospholipid syndrome, Behçet's disease, cancer, myeloproliferative diseases. The congenital factors like mutations of factor-V Leiden and prothrombin, deficiencies of proteins C and S, antithrombin, hyperhomocysteinemia and hyperfibrinogenemia were ruled out. The total number of patients was reduced from 96 to 9 (7 with portal vein thrombosis, 2 Budd Chiari syndrome) by exclusion criteria., Results: The levels of adhesion molecules sICAM-1, sVCAM-1, free TFPI levels and global fibrinolytic capacity were significantly different (P<0.05) in the patient group indicating an endothelial dysfunction and a lower fibrinolytic activity., Conclusion: These results show that this patient group should be tested by means of endothelial dysfunction and managed differently.

Perhaps it is not surprising that in the critical care environment, where lives are frequently on the line, off-label use of certain drugs is relatively common. In general, there are two camps of opinion on this type of utilization. One camp would suggest that potentially life saving products cannot ethically be withheld from patients who may benefit. The other camp would counter that it is inappropriate to administer products if the risk/benefit ratio has not been clearly defined in clinical trials. Off-label use of factor VII is debated in this issue of Critical Care for a patient with uncontrolled nontraumatic hemorrhage. Perhaps this product promotes additional discussion given that its ability to control bleeding can be dramatic, yet its costs and potential for complications high.

We report a case of transient acquired and isolated factor VII deficiency associated with severe head trauma. A 16-year-old boy was involved in a motor vehicle accident. CT scan showed frontal brain contusion and a cerebral haematoma (5 cm). First prothrombine time (PT) was normal. Rapidly, a severe coagulopathy developed, unresponsiving to fresh frozen plasma and vitamin K. Haemostatic markers analysis showed an isolated deficiency of factor VII at 15%. No inhibitory activity against factor VII could be detected. We successfully treated the deficiency with intermittent intravenous human factor VII (factor VII-LFB) during 10 days. Factor VII return to normal at 84%. Physiopathological and therapeutic aspects of this rare pathology are presented.

Polymorphic variants in the gene encoding factor VII (F7) affect the plasma levels of this coagulation protein and modify the clinical phenotype of FVII deficiency in some patients. In this study we report the in vitro functional analysis of a novel polymorphic variant located in the 3' untranslated region of F7: g.11293&#95;11294insAA. To determine whether this variant regulates FVII expression, we initially compared an expression vector containing FVII cDNA with g.11293&#95;11294insAA with the FVII wild-type (WT) construct. The kinetics of mRNA production showed that the insertion decreases the steady-state FVII mRNA levels. To assess whether the insertion influences the phenotype of FVII-deficient patients, we evaluated its effect on the expression of FVII in a patient with severe FVII deficiency (undetectable FVII activity and antigen) carrying two additional homozygous missense variations (p.Arg277Cys and p.Arg353Gln). The two substitutions alone reduced the expression of FVII activity and antigen in vitro, but with the insertion polymorphism in our expression vector the patient's phenotype of undetectable plasma FVII was recapitulated. The insertion polymorphism in the 3' untranslated region of F7 is another modifier of FVII expression that might explain the poor genotype-phenotype correlation in some FVII-deficient patients., (Copyright 2005 Wiley-Liss, Inc.)

Recombinant activated factor VII (rFVIIa) is currently licensed in the United States for treatment of bleeding episodes in patients with deficiencies of factor VIII (FVIII) or IX (FIX) who are refractory to factor replacement because of circulating inhibitors. A 1999 report of its successful use to stop what was deemed to be lethal hemorrhage after an abdominal gunshot wound in a young soldier without pre-existing coagulopathy has prompted exploration of other uses for rFVIIa. The virtual explosion of proposed uses of rFVIIa raises issues not only regarding our understanding of the coagulation system, but also regarding its efficacy, cost-effectiveness, and safety.

As interest in the use of activated recombinant factor VII (rFVIIa) in trauma grows, questions arise regarding how best to monitor rFVIIa therapy and when rFVIIa may be expected to improve hemostasis. Knowledge of the mechanisms of action may be combined with available data on laboratory monitoring and efficacy in various coagulopathic states in coming to clinically relevant conclusions. This review addresses the physiology of hemostasis, placing emphasis on how rFVIIa influences the process by both tissue factor dependent and tissue factor independent mechanisms. This is extended to a mechanistic consideration of how rFVIIa may function under acidotic, hypothermic, and hemodilutional and/or consumptive conditions of trauma related coagulopathy. When these considerations are viewed alongside the available clinical data, it becomes apparent that rFVIIa has potential to improve hemostasis during trauma coagulopathy, within limitations. Common laboratory procedures are discussed with reference to mechanisms of action of rFVIIa and the available clinical data. Although there is no single assay that can predict rFVIIa efficacy in trauma, the prothrombin time (PT) is recommended as a minimum. Although a shortened PT does not predict success, correction of PT into the normal range may be a better indicator. A nonresponding PT appears to indicate that rFVIIa alone will not lead to hemostasis, and that additional blood products and other measures must be applied. Once the patient is more stable, PT and thromboelastography are recommended.

The extrinsic hypercoagulation often resulting from sepsis could contribute to disseminated intravascular coagulation and cardiovascular complications. The effective prevention and intervention remained largely complex and unclear. In a cell model of human leukemia THP-1 monocytes following bacterial endotoxin (LPS) exposure, we show the novel anticoagulant ability of polyamino acid (polyAA) to suppress the extrinsic hypercoagulation. LPS-induced monocytic tissue factor (mTF) procoagulation was readily offset by poly-L-lysine (PLK), poly-L-arginine (PLR), or poly-L-ornithine (POR) included in single-stage clotting assays. IC50 was estimated at 0.35, 0.30, or 0.58 microM for PLR, POR, or PLK, respectively, whereas, poly-L-asparatic acid (PLD) remained ineffective. In a separate approach, inclusion of cationic polyAA in human plasma significantly prolonged prothrombin time, confirming the depressed extrinsic coagulation. In chromogenic assays dissecting the extrinsic pathway, we further determined the inhibitory site(s). PLK, PLR, or POR significantly inhibited LPS-induced FVII activation, which was consistent with the diminished FVIIa formation shown on Western blotting analysis. In contrast, polyAA did not show any additional effect on either FVIIa/FXa amidolytic activities or mTF/FVIIa-catalyzed FX activation. Nor did polyAA show any effect on FVII activation directly catalyzed by FXa. Taken together, PLK, PLR, or POR preferentially inhibited mTF-dependent FVII activation, accounting for their novel anticoagulant activities. PolyAA might present the specific antagonists to arrest the extrinsic hypercoagulation following inflammation.

Tissue factor (TF) is a transmembrane glycoprotein, currently considered as being the major regulator of the coagulation cascade and the initiator of thrombogenesis in vivo. When TF comes in contact with blood, it forms a high-affinity complex with factors VII/VIIa, activating factors IX and X and thus leading to the formation of an insoluble fibrin clot. The regulation of TF-VIIa activity plays a key role in blood-vessel wall interactions. Selective patterns of cellular expression of TF are observed in tissues. TF is constitutively localized only on the surface of cells anatomically separated from the blood, where it plays an essential role in hemostasis by limiting hemorrhage after vessel wall injury. A number of pathophysiologic stimuli are capable of inducing TF transcription and activity in endothelial cells and monocytes. An aberrant TF expression in contact with blood is implicated in thrombotic complications of atherosclerosis, including acute myocardial infarction. Recent findings have demonstrated cell-derived microparticles containing TF in the circulating blood of patients with acute coronary syndromes, capable of triggering and propagating thrombus growth. This observation suggests a new view of thrombosis that does not necessarily require the exposure of vessel wall-derived TF at the site of vascular injury to initiate and propagate thrombosis.

Blood coagulation normally occurs when factor VII interacts with its specific cellular receptor, tissue factor, which is exposed when a blood vessel is severed. The factor VII/tissue factor complex then initiates a cascade of proteolytic reactions involving factors IX, X, prothrombin and fibrinogen, culminating in the formation of a fibrin clot. The role of platelets in the initiation phase of blood coagulation is still unclear. It has been postulated that platelets bind activated factor VIIa independently of tissue factor, and that this interaction forms the basis of the usefulness of high-dose recombinant factor VIIa in treating hemophiliacs with inhibitory antibodies, and other thrombocytopenia-like syndromes. In this review, we will examine the evidence for and against such an hypothesis, as well as discuss an alternative mechanism for the efficacy of high-dose factor VIIa in treating hemophilic patients with inhibitors.

Objective: To determine the cellular origin of synovial microparticles, their procoagulant properties, and their relationship to local hypercoagulation., Methods: Microparticles in synovial fluid and plasma from patients with rheumatoid arthritis (RA; n = 10) and patients with other forms of arthritis (non-RA; n = 10) and in plasma from healthy subjects (n = 20) were isolated by centrifugation. Microparticles were identified by flow cytometry. The ability of microparticles to support coagulation was determined in normal plasma. Concentrations of prothrombin fragment F(1+2) (by enzyme-linked immunosorbent assay [ELISA]) and thrombin-antithrombin (TAT) complexes (by ELISA) were determined as estimates of the coagulation activation status in vivo., Results: Plasma from patients and healthy controls contained comparable numbers of microparticles, which originated from platelets and erythrocytes. Synovial microparticles from RA patients and non-RA patients originated mainly from monocytes and granulocytes; few originated from platelets and erythrocytes. Synovial microparticles bound less annexin V (which binds to negatively charged phospholipids) than did plasma microparticles, exposed tissue factor, and supported thrombin generation via factor VII. F(1+2) (median 66 nM) and TAT complex (median 710 microg/liter) concentrations were elevated in synovial fluid compared with plasma from the patients (1.6 nM and 7.0 microg/liter, respectively) as well as the controls (1.0 nM and 2.9 microg/liter, respectively)., Conclusion: Synovial fluid contains high numbers of microparticles derived from leukocytes that are strongly coagulant via the factor VII-dependent pathway. We propose that these microparticles contribute to the local hypercoagulation and fibrin deposition in inflamed joints of patients with RA and other arthritic disorders.

Activated prothrombin complex concentrates (APCCs) are effective in the therapy of bleeding episodes in hemophilic patients with inhibitors. We investigated the respective roles of factor II, factor VII, factor IX, and factor X in the procoagulant activity of the APCC FEIBA. Factor II, factor VII, factor IX, and factor X were reduced in platelet-poor plasma, and the thrombin potential (TP) was determined using a chromogenic substrate in the absence or presence of FEIBA. Reduction of factor II resulted in a significant decrease of the TP without influencing the lag phase until the onset of thrombin generation. The reduction of factor VII showed no effect on the TP, but resulted in a prolongation of the lag phase. Changes of factor IX or factor X concentrations showed neither an effect on the TP nor on lag phases. Our study demonstrates that thrombin generation in the presence of FEIBA mainly depends on prothrombin.

The enhanced extrinsic tissue factor (TF)-initiated coagulation, often resulting from sepsis, could lead to disseminated intravascular coagulation presenting cardiovascular complications. Using model human leukaemia THP-1 monocytes, we studied monocytic TF (mTF) hypercoagulation and its regulation. After an 8 h exposure to bacterial endotoxin [lipopolysaccharide (LPS); 100 ng/ml], mTF activity was significantly upregulated as the result of the enhanced mTF synthesis. Thereafter, LPS induction declined, exhibiting a "quiescent-desensitizing' phenomenon. Such diminished LPS induction was,however,associated with sustained LPS-enhanced mTF synthesis, revealing the possible occurrence of a post-translational downregulation. It was noted that LPS desensitization was accompanied by the increased expression of myristoylated alanine-rich C kinase substrate (Marcks). In contrast, A23187 (20 micromol/l) or Quin-2AM (20 micromol/l) drastically activated mTF activity without detectable effect on mTF synthesis; both of which showed that sustained functional upregulation during 24 h culture did not enhance Marcks expression. These inverse correlations between mTF activity upregulation and Marcks expression suggested that Marcks could be inhibitory. Marcks phosphorylation site domain (151-175) (Marcks PSD) readily inhibited mTF-dependent FVII activation and diminished FVIIa formation in LPS-challenged cells. As a result, Marcks PSD offset LPS-induced mTF hypercoagulation upon inclusion in the single-stage clotting assays. The anticoagulant activity was confirmed by showing that Marcks PSD significantly blocked rabbit brain thromboplastin (rbTF) procoagulation and inhibited rbTF-dependent FVII activation as well as FVIIa formation. Our study suggests that Marcks expression plays a role in a novel cellular modulation to downregulate mTF hypercoagulation.

Obese patients are at risk for the development of cardiovascular diseases, which can in part be explained by disturbances in the haemostatic and fibrinolytic systems. Indeed, obese subjects tend to have higher values of fibrinogen, factor VII, factor VIII, von Willebrand factor and plasminogen activator inhibitor compared to non-obese subjects. Abdominal obesity, in particular, has been shown to be associated with disturbances in fibrinogen, factor VIII and von Willebrand factor, while less consistent results have been found for factor VII. Recently it has been demonstrated that the adipocyte itself is able to produce plasminogen activator inhibitor-1, possibly explaining the high levels found in obesity. Different studies have investigated the association between haemostatic and fibrinolytic parameters and the insulin resistance syndrome, often present in obese subjects. Fibrinogen has been found to be related to insulin, but it has been suggested that this relationship is not independent of the accompanying inflammatory reaction. Results from studies on the relationship between insulin resistance and factor VII, factor VIII and von Willebrand factor levels are inconsistent. In contrast, plasminogen activator inhibitor-1 has been found to correlate with all components of the insulin resistance syndrome, and can be considered as a true component of this metabolic syndrome. Weight loss seems to have a beneficial effect on factor VII--probably mediated through a reduction in triglycerides. Data on factor VIII and von Willebrand factor are scarce but weight loss does not seem to have an effect. Fibrinogen does not seem to be reduced by modest weight loss and a more substantial weight loss seems necessary to lower fibrinogen levels. In contrast, both modest and substantial weight loss have been found to significantly reduce plasminogen activator inhibitor-1 levels. In conclusion, the increased cardiovascular risk observed in obesity could in part be explained by the association between insulin resistance and components of the fibrinolytic and haemostatic systems. Whether this relationship is truly causal or indirect needs to be elucidated further.

Vulnerable atherosclerotic plaques, those that are prone to rupture, are soft and lipid rich. Other factors that contribute to destabilizing these plaques are inflammation and vasoconstriction. Although less information is available regarding noncoronary vulnerable plaques than the more frequently studied coronary plaques, it appears that the amount of soft lipid "gruel" as well as in situ inflammation plays a crucial role in destabilizing peripheral vascular atherosclerotic plaque, eventually causing its rupture. As in the coronary circulation, it is likely that the majority of acute plaque rupture events are clinically silent. Nevertheless, preventing plaque rupture in the noncoronary circulation is of the utmost importance because this is the presumed mechanism of progression of peripheral vascular atherosclerotic lesions., (Copyright 2002, Elsevier Science (USA). All rights reserved.)

Recent observations document that many patients with acute coronary syndromes harbor multiple complex plaques by angiography, which correlate with multiple plaque ruptures and clots at necropsy. Multifocal plaque instability is evident not only in coronary vessels but also in peripheral vessels where peripheral and coronary plaque instability may exist concomitantly. These observations support the concept that plaque instability is not merely a local vascular accident but instead reflects more systemic pathophysiological processes with potential to destabilize atherosclerotic plaques throughout the cardiovascular system., (Copyright 2002, Elsevier Science (USA). All rights reserved.)

The response to injury in the vasculature and the heart is inflammation. Atherosclerosis is often the result of injury followed by inflammation and atherosclerosis. Vascular and myocardial infections from various pathogens, including viruses, bacteria, chlamydia, and other infections result in vascular inflammation and almost certainly play a role in the development of atherosclerosis and acute coronary heart disease syndromes in at least some patients. Current evidence favors prior exposure to multiple pathogens as most likely playing a role in initiating inflammation and contributing to atherosclerosis. Genetic predisposition is almost certainly an important factor in the development of inflammation, impaired endothelial vascular repair, vascular infection, thrombosis, and atherosclerosis. The aging process itself is most likely associated with altered vascular and myocardial defense mechanisms predisposing to inflammation. The oxidation of cholesterol and low-density lipoprotein (LDL) leads to the production of oxidized radicals that promote vascular inflammation. Interventional injury, including angioplasty and stenting, causes endothelial inflammation, thrombosis, and fibroproliferation. Systemic evidence of inflammation identifies patients at high risk of future coronary events, including those who appear to be healthy initially as well as those with stable and unstable coronary heart disease syndromes. Increases in serum C-reactive protein (CRP) identify individuals at risk for future vascular events, including unstable angina, acute myocardial infarction, acute cerebrovascular accident, and sudden death. Similarly, systemic elevations in serum troponin 1, serum amyloid-like protein, fibrinogen, and interleukins-1, 2, 6, 8, and 18 identify patients with unstable angina and non-Q-wave myocardial infarction at increased risk for future coronary events. The presence of vascular inflammation may be detected by identifying temperature heterogeneity within plaques that demonstrate inflammation. In the future, the local evaluation of atherosclerotic plaques to detect the presence of inflammation coupled to measurements of systemic markers of inflammation, such as C-reactive protein, may help identify patients at increased risk and allow both local and systemic therapies that reduce their risk and prevent the development of acute coronary syndromes in at least some patients., (Copyright 2002, Elsevier Science (USA). All rights reserved.)

It is widely accepted that blood coagulation in vivo is initiated during normal hemostasis, as well as during intravascular thrombus formation, when the cell-surface protein, tissue factor (TF), is exposed to the blood as a consequence of vascular injury. In addition to its essential role in hemostasis, tissue factor may be also implicated in several pathophysiological processes, such as intracellular signaling, cell proliferation, and inflammation. For these reasons, the tissue factor:factor VIIa complex has been the subject of intense research focus. Many experimental studies have demonstrated that inhibition of tissue factor:factor VIIa procoagulant activity are powerful inhibitors of in vivo thrombosis and that this approach usually results in less pronounced bleeding tendency, as compared to other "more classical" antithrombotic interventions. Alternative approaches may be represented by transfecting the arterial wall with natural inhibitors of tissue factor:factor VIIa complex, such as tissue factor pathway inhibitor (TFPI), which may result in complete inhibition of local thrombosis without incurring in potentially harmful systemic effects. Additional studies are warranted to determine the efficacy and safety of such approaches in patients., (Copyright 2002 Elsevier Science Ltd.)

Genetic variants of key hemostatic mediators increasingly have been proposed as risk factors for atherothrombosis. The Hormone and Estrogen/Progestin Replacement Study group recently reported that the initiation of estrogen replacement in postmenopausal women with known coronary heart disease is associated with an early increase in cardiovascular events. A putative genetic susceptibility factor has been proposed a potential mediator of this increased event risk. This review outlines the recent literature to support the premise for this important proposal. Genetic profiling has great potential to improve the safety and efficacy of individualized pharmacotherapy in postmenopausal women and other at-risk populations for the prevention of cardiovascular disease.

Oral combined hormone replacement therapy (HRT) with oestradiol and norethisterone increases plasma levels of prothrombin fragment 1+2 (F1+2), indicating an increase in thrombin generation, but the mechanisms underlying this increase are uncertain. The aim of this randomized, placebo-controlled study was to determine whether an increase in factor VII, a factor that combines with tissue factor to activate the extrinsic pathway, or a decrease in tissue factor pathway inhibitor (TFPI), an inhibitor of extrinsic pathway activation, may contribute to increases in thrombin generation occurring with HRT. Healthy postmenopausal women aged 50-75 years received placebo (n=19) or oral combined HRT (n=18) and had blood collected for measurement of factor VII coagulation activity (VIIc), activated factor VII (VIIa) and TFPI at baseline and at 6 weeks. Baseline characteristics were similar in the two groups, including age, body mass index and cholesterol levels. As reported previously, HRT increased the F1+2 concentration by 20%. Placebo had no effect on VIIc, VIIa or TFPI, but 6 weeks of combined HRT decreased VIIc [from 1.11+/-0.06 (mean+/-S.E.M.) to 1.03+/-0.06 i.u./ml; P<0.03], VIIa [from 43.9; 10.8-198.3 (median; range) to 35.0; 6.3-66.8 m-units/ml; P<0.03] and TFPI [from 81.3+/-6.5 to 60.4+/-5.5 ng/ml; P<0.0001]. The decrease in TPFI with HRT was not correlated with the elevation in F1+2 levels. In conclusion, the increase in thrombin generation seen with HRT is not due to an effect on factor VII; in addition, while a contribution from the decrease in TFPI is possible, increased thrombin generation is not directly related to the decrease in TFPI.

Tissue factor (TF) is the essential cofactor for the coagulation protease factor VIIa (FVIIa), initiating the coagulation cascade. The role of TF in thrombotic diseases is becoming increasingly evident. Recent findings suggest that inhibition of TF/FVIIa activity could be important in the prevention of clinical sequelae associated with plaque rupture or vessel damage that exposes TF to blood. Furthermore, selective inhibitors of TF/FVIIa may be associated with less bleeding risk than other antithrombotic agents. Several TF/FVIIa inhibitors are in development, including the protein-based inhibitors (such as NAPc2, Corsevin M, FFR-FVIIa, and Tifacogin). Research into the development of small molecule inhibitors is on-going, but is at a less advanced stage.

Acetaminophen may increase International Normalized Ratio (INR) in patients taking anticoagulation medication, and in patients with acetaminophen poisoning without hepatic injury. The objective of this study was to describe and investigate the effect of acetaminophen on INR. The authors studied patients admitted to a regional toxicology treatment center with acetaminophen poisoning with INR and without potentially confounding coingestion or hepatic injury. Exposed and nonexposed (control) cohorts were recruited from admissions with acetaminophen poisoning and psychotropic drug poisoning, respectively. From 1,437 acetaminophen poisonings, after exclusions, there were 143 admissions with 205 estimations of INR. INR showed a time-dependent increase. Fifty percent of all patients and 66% of those with an extrapolated 4-hour acetaminophen concentration > or = 150 mg/L had an abnormal INR at some time. Dose ingested (p = 0.01) and nomogram-based risk (p for trend = 0.005) were correlated with the effect. N-acetylcysteine had a protective effect. Functional factor VII was lower (p = 0.005) in exposed patients (n = 30) than controls (n = 8), and less than antigenic factor VII in exposed patients (p = 0.03). Factor IX was lower (p = 0.02). Factor VIIIc was not significantly different. The authors concluded that an isolated, small rise in INR is common after acetaminophen poisoning without hepatic injury. It appears to be caused by inhibition of Vitamin K-dependent activation of coagulation factors. This effect suggests a possible mechanism for the observed interaction between acetaminophen and warfarin.

Factor VII circulates as a single chain inactive zymogen (10 nmol/L) and a trace ( approximately 10-100 pmol/L) circulates as the 2-chain form, factor VIIa. Factor VII and factor VIIa were studied in a coagulation model using plasma concentrations of purified coagulation factors with reactions initiated with relipidated tissue factor (TF). Factor VII (10 nmol/L) extended the lag phase of thrombin generation initiated by 100 pmol/L factor VIIa and low TF. With the coagulation inhibitors TFPI and AT-III present, factor VII both extended the lag phase of the reaction and depressed the rate of thrombin generation. The inhibition of factor Xa generation by factor VII is consistent with its competition with factor VIIa for TF. Thrombin generation with TF concentrations >100 pmol/L was not inhibited by factor VII. At low tissue factor concentrations (<25 pmol/L) thrombin generation becomes sensitive to the absence of factor VIII. In the absence of factor VIII, factor VII significantly inhibits TF-initiated thrombin generation by 100 pmol/L factor VIIa. In this hemophilia A model, approximately 2 nmol/L factor VIIa is needed to overcome the inhibition of physiologic (10 nmol/L) factor VII. At 10 nmol/L, factor VIIa provided a thrombin generation response in the hemophilia model (0% factor VIII, 10 nmol/L factor VII) equivalent to that observed with normal plasma, (100% factor VIII, 10 nmol/L factor VII, 100 pmol/L factor VIIa). These results suggest that the therapeutic efficacy of factor VIIa in the medical treatment of hemophiliacs with inhibitors is, in part, based on overcoming the factor VII inhibitory effect. (Blood. 2000;95:1330-1335)

Recently, high-dose factor VIIa has been used to correct bleeding in patients with various thrombocytopathias including Glanzmann's thrombasthenia, Bernard-Soulier syndrome, and uremia. High-dose factor VIIa is postulated to act on platelets in the absence of tissue factor to activate factors IX and X and thus enhance thrombin generation. This enhanced thrombin generation might help provide hemostasis in patients with thrombocytopathias through several mechanisms. Enhanced thrombin generation would provide a strong signal for recruitment of other platelets. Also, enhanced fibrin deposition might provide mechanisms for bypassing the specific defect in thrombocytopathias. Thus, platelets from a patient with Bernard-Soulier syndrome might associate with fibrin by a glycoprotein IIb-IIIa-mediated mechanism. Also, platelets from a patient with Glanzmann's thrombasthenia might associate with fibrin through von Willebrand factor-mediated interactions with glycoprotein Ib-V-IX. Finally, enhanced thrombin generation on platelets would mean that fewer platelets are required for hemostasis.

We have explored the molecular basis of the clinical therapeutic effect of factor VIIa in hemophilia A using empirical reconstituted in vitro thrombin generation models. Tissue factor acts as a receptor and activator of preexistent but virtually inactive two-chain plasma factor VIIa. However, most of the factor VII circulates as a single-chain inactive zymogen (10 nM) and a trace (approximately 10-100 pM) circulates in the active two-chain form. Empirical reconstitution (purified factors VIIa, X, IX, VIII, V, prothrombin, and relipidated tissue factor) showed that plasma concentrations of factor VII (10 nM) prolong the initiation phase of thrombin generation significantly at low concentrations of tissue factor and 100 pM factor VIIa. Thus, we show for the first time that the zymogen factor VII may have a very significant inhibitory action on thrombin generation at physiologic ratios of factor VII to factor VIIa. The inhibition kinetics of factor Xa generation by low concentrations of tissue factor indicate that factor VII inhibits the reaction by competition for the initial binding of factor VIIa to tissue factor. Physiological concentrations of factor VII also inhibit the maximal rate of thrombin generation by 100 pM factor VIIa in the absence of factor VIII. Increasing the concentration of factor VIIa to 2 nM in this hemophilia A model overcame the inhibition of thrombin generation by 10 nM factor VII. Increasing the concentration up to 10 nM factor VIIa in the absence of factor VIII completely normalized the thrombin generation profile to that observed in the presence of factor VIII and 10 nM factor VII/100 pM factor VIIa. The levels of factor VIIa that overcome the inhibitory effect of factor VII and that normalize thrombin generation in our model are consistent with the observed plasma levels of factor VIIa needed to manage hemophilia A. Our data strongly indicate that the therapeutic mechanism of factor VIIa in the medical treatment of hemophiliacs with inhibitors is in large part based on overcoming the inhibitory effect of factor VII on thrombin generation.

Background: Hypercoagulability is one of the commonly exhibited endotoxemia septic symptoms; it could contribute to the manifestation of disseminated intravascular coagulation presenting threats to cardiovascular functions. The underlying mechanism, however, remains largely complex and unknown., Objectives: We herein determine whether bacterial endotoxin (LPS) upregulates the activities of clotting factors in plasma, contributing to extrinsic hypercoagulation. Compound 48/80 (48/80) is also tested for its ability to suppress hypercoagulation., Methods: In an in vitro infection model, we exposed whole blood to LPS (Escherichia coli 0111:B04; 100 ng/ml) for 2 h. Thrombin time (TT), prothrombin time (PT), and the activities of clotting factors ( FVII, FIX, FX ) in plasma contributing to the extrinsic coagulation were determined. Peripheral blood monocytes were isolated from Histoplaque 1077 gradient centrifugation, and the procoagulant activity was determined by a single-stage clotting assay on a Fibrometer., Results: LPS drastically activated monocytic procoagulant activity which was defined as tissue factor (TF) activity, whereas LPS had no effect on TT, PT, and the activities of clotting factors in plasma. 48/80 not only instantaneously offset LPS-induced monocytic TF activation, but also significantly inhibited PT including the activities of clotting factors (FVII, FIX, and FX) in plasma, whereas TT remained unaffected., Conclusions: Monocytic TF activation was solely responsible for the extrinsic hypercoagulation in response to LPS. 48/80 effectively suppressed LPS-induced monocyticTF-initiated extrinsic coagulation at multiple sites, possibly presenting a new therapy for an instantaneous relief of hypercoagulation under septic conditions., (Copyright 1999 Academic Press.)

The first metabolite of ethanol, acetaldehyde, has the ability to form adducts with proteins and alter their function. It has been shown that acetaldehyde reacts with various proteins of the blood coagulation pathway and, subsequently, produces a prolongation of the clotting time. This study evaluated the function of clotting proteins from the extrinsic coagulation pathway (factor VII) and the intrinsic coagulation pathway (factor IX) when preincubated with acetaldehyde as compared to a control and compared to preincubation with ethanol. Prior to use in a clotting assay, incubation times with acetaldehyde, ethanol, and the control were the same for both factors VII and IX. An automatic fibrometer measured the clotting times. Factor VII preincubated with acetaldehyde prolonged the clotting time. However, factor IX preincubated with acetaldehyde actually decreased the clotting time. Of interest, both factors VII and IX preincubated with acetaldehyde produced statistically significant results when compared to the control and ethanol. This experiment indicates that acetaldehyde, in forming an adduct with proteins of the blood coagulation pathway, may induce a conformational change of factors VII and IX so as to either increase or decrease the clotting time. Therefore, it is possible that some of the deranged coagulation in alcohol abusers may be a final net result of the interaction of acetaldehyde and proteins of the coagulation pathway.