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In a previous study, we reported that human endothelial cells (ECs) express and produce their own coagulation factors (F) that can activate cell surface FX without the additions of external proteins or phospholipids. We now describe experiments that detail the expression and production in ECs and fibroblasts of the clotting proteins necessary for formation of active prothrombinase (FV-FX) complexes to produce thrombin on EC and fibroblast surfaces. EC and fibroblast thrombin generation was identified by measuring: thrombin activity; thrombin-antithrombin complexes; and the prothrombin fragment 1.2 (PF1.2), which is produced by the prothrombinase cleavage of prothrombin (FII) to thrombin. In ECs, the prothrombinase complex uses surface-attached FV and γ-carboxyl-glutamate residues of FX and FII to attach to EC surfaces. FV is also on fibroblast surfaces; however, lower fibroblast expression of the gene for γ-glutamyl carboxylase (GGCX) results in production of vitamin K-dependent coagulation proteins (FII and FX) with reduced surface binding. This is evident by the minimal surface binding of PF1.2, following FII activation, of fibroblasts compared to ECs. We conclude that human ECs and fibroblasts both generate thrombin without exogenous addition of coagulation proteins or phospholipids. The two cell types assemble distinct forms of prothrombinase to generate thrombin., (© 2021. The Author(s).)

This study aims to investigate the gastrointestinal functions of patients with liver failure (LF) based on gastrointestinal dysfunction (GD) scores and serum gastrointestinal hormone levels.The GD in LF patients was scored using the gastrointestinal dysfunction scoring criteria. Serum gastrin (GAS), cholecystokinin (CCK), and motilin (MTL) levels were determined in LF patients. In addition, liver function and prothrombin activity were detected, and ultrasonography was performed.The GD score was significantly higher in the LF groups than in the control group. Compared with the control group, serum GAS, CCK, and MTL levels significantly increased in the LF groups, and was positively correlated with the severity of LF. Furthermore, in the LF groups, GD was positively correlated with the severity of LF. However, the GD score and serum GAS, CCK, and MTL levels in the acute LF group were not statistically different, when compared with those in the subacute LF group, acute-on-chronic LF group and chronic LF group.LF plays a key role in the development of GD, and may be the main cause of obvious gastrointestinal symptoms, such as abdominal distension, nausea, vomiting and anorexia, in LF patients. The severity of GD is not associated with LF type, but is positively correlated with the severity of LF, suggesting that GD in LF patients may have complicated mechanisms.

Purpose: We previously reported that hepatocellular carcinoma (HCC) changes to a phenotype producing des-γ-carboxy prothrombin (DCP) during epithelial mesenchymal transition (EMT) in vitro. To confirm this change in vivo, we evaluated the association between DCP production and HCC hemodynamics in patients undergoing resection as EMT and hemodynamic changes are closely associated with each other., Methods: We evaluated HCC hemodynamics by employing Sonazoid-enhanced ultrasound (SEUS) before surgical resection, and sought associations with histological grade and immunohistochemical staining of DCP in 19 areas from 11 patients., Results: In 10 HCC areas showing early washout (3 min ≥) using SEUS, three areas corresponded to poorly differentiated HCC and the remaining seven areas corresponded to moderately differentiated HCC, and positive DCP staining was observed in only two of the seven moderately differentiated HCC areas, whereas no staining was observed in poorly differentiated HCC areas. Six HCC areas showing intermediate washout (3-10 min) using SEUS were moderately differentiated, of which five demonstrated positive DCP staining (83.3%, 5/6). However, all HCC areas without enhancement in the arterial phase were well-differentiated and did not show DCP staining., Conclusion: Our preliminary findings suggest that HCC hemodynamics evaluated by SEUS are associated with histological grade and/or DCP production.

Introduction: The objective of this study was to estimate the effects of different doses of oral hormone therapy (HT) on thrombin generation and fibrinolytic activity in postmenopausal women after 12 months of treatment., Material and Methods: Thrombin generation, fibrinolysis activators and inhibitors were determined before and after 12 months of treatment. Participants (180) were assigned (1:1:1) as follows: (1) standard HT group, 17β-estradiol (1 mg/day) with dydrogesterone (5 mg/day); (2) ultra-low dose HT group, 17β-estradiol (0.5 mg/day) with dydrogesterone (2.5 mg/day); (3) control group, no treatment., Results: The standard HT led to a higher concentration of prothrombin 1 + 2 fragment (by 5.8%) with lower antithrombin activity (by 6.1%). Compared with baseline, we observed a reduction in mean antithrombin activity in the standard HT group and increases in mean levels of prothrombin 1 + 2 fragment in two HT groups. We found decreases after treatment in both standard and ultra-low dose HT groups in plasminogen activator inhibitor-1 (PAI-1) activity (-32.4% and -19.6%, respectively) and PAI-1 antigen (-9.9% and -7.8%, respectively). Intergroup analysis revealed reduction in both mean PAI-1 activities and PAI-1 antigen levels in the two treatment groups when compared with the control., Conclusion: Contrary to the standard estrogen plus dydrogesterone treatment, ultra-low dose HT revealed positive effects on hemostasis by intensifying fibrinolysis through a decrease in both PAI-1 activity and antigen levels, and with no impact on thrombin generation., (© 2017 Nordic Federation of Societies of Obstetrics and Gynecology.)

Coagulation factor II (prothrombin; FII) is the pre-proteolyzed precursor to thrombin in the coagulation cascade. It has 10 sites of gamma-carboxylation, which are required for its bioactivity, and is N-glycosylated at three of four putative sites. Production of recombinant human FII (rhFII) using a platform fed-batch process designed for monoclonal antibody production resulted in low levels of gamma-carboxylation and sialylation. There have not been any prior reports of successful process development and clinical manufacture of rhFII with optimal, consistent gamma-carboxylation and sialylation. In order to develop such a fed-batch process, various process parameters were evaluated to determine their impact on product quality. Process temperature and temperature shift timing were important for both sialic acid level and gamma-carboxyglutamate (Gla) level. In addition, vitamin K concentration and the type of surfactant used for preparation of vitamin K stock solution were also important for gamma carboxylation. A fed-batch study performed with various medium additives known to be involved in the N-glycosylation pathway, such as N-acetyl-d-mannosamine (ManNAc), galactose (Gal), dexamethasone, and manganese sulfate, increased the level of sialylation and enabled the elucidation of some potential bottlenecks in the sialylation pathway. The optimized process based on these studies yielded a reduction in the level of missing Gla by 0.4 moles per mole of rhFII in cell culture and a nearly threefold increase in sialic acid level. The process was successfully implemented at the 2000 L scale where a high Gla level and sialylation levels were achieved in all GMP lots. Biotechnol. Bioeng. 2017;114: 1991-2000. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

A 78-year-old man was admitted to our hospital for multiple lung and liver tumors. Initial clinical diagnosis was hepatocellular carcinoma (HCC) with lung metastases because of a high value of serum protein induced by vitamin K absence or antagonist II (PIVKA-II) (6,705 mAU/mL). However, a review of a prior CT showed the lung tumor had existed 6 months before liver tumors were detected. The tumors progressed rapidly and the patient died 37 days after admission. Autopsy revealed that both lung and liver tumors exhibited the histology of large cell neuroendocrine carcinoma (LCNEC). Immunohistochemistry revealed that the tumor cells expressed not only neuroendocrine markers but also PIVKA-II diffusely. Hepatoid differentiation was not detected. Background liver did not show any chronic liver disease. The final diagnosis was PIVKA-II producing LCNEC of the lung with multiple liver metastases. PIVKA-II producing tumors other than HCC are extremely rare. To our best knowledge, this is the first case report of PIVKA-II producing neuroendocrine tumors of the lung., (© 2017 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

Des-γ-carboxyprothrombin (DCP), an abnormal prothrombin produced in human hepatocellular carcinoma (HCC), plays crucial roles in the progression of HCC. DCP binding to cellular mesenchymal-epithelial transition factor (c-Met) is an initial event and consequently stimulates HCC through the increase of c-Met-Janus kinase 1- signal transducers and activators of transcription pathways. DCP stimulates HCC invasion through activation of matrix metalloproteinase via upregulation of extracellular signal-regulated kinase-mitogen-activated protein kinase (MAPK) pathway. DCP stimulates HCC angiogenesis through activation of the DCP-kinase insert domain receptor-phospholipaseC-γ-MAPK pathway. Identification of these pathways is important for designing the therapeutic strategy for HCC.

The opportunistic pathogen Pseudomonas aeruginosa causes a wide range of infections in multiple hosts by releasing an arsenal of virulence factors such as pyocyanin. Despite numerous reports on the pleiotropic cellular targets of pyocyanin toxicity in vivo, its impact on erythrocytes remains elusive. Erythrocytes undergo an apoptosis-like cell death called eryptosis which is characterized by cell shrinkage and phosphatidylserine (PS) externalization; this process confers a procoagulant phenotype on erythrocytes as well as fosters their phagocytosis and subsequent clearance from the circulation. Herein, we demonstrate that P. aeruginosa pyocyanin-elicited PS exposure and cell shrinkage in erythrocyte while preserving the membrane integrity. Mechanistically, exposure of erythrocytes to pyocyanin showed increased cytosolic Ca(2+) activity as well as Ca(2+) -dependent proteolytic processing of μ-calpain. Pyocyanin further up-regulated erythrocyte ceramide abundance and triggered the production of reactive oxygen species. Pyocyanin-induced increased PS externalization in erythrocytes translated into enhanced prothrombin activation and fibrin generation in plasma. As judged by carboxyfluorescein succinimidyl-ester labelling, pyocyanin-treated erythrocytes were cleared faster from the murine circulation as compared to untreated erythrocytes. Furthermore, erythrocytes incubated in plasma from patients with P. aeruginosa sepsis showed increased PS exposure as compared to erythrocytes incubated in plasma from healthy donors. In conclusion, the present study discloses the eryptosis-inducing effect of the virulence factor pyocyanin, thereby shedding light on a potentially important mechanism in the systemic complications of P. aeruginosa infection., (© 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

The purpose of this study was to determine whether trauma-induced coagulopathy is due to changes in 1) thrombin activity, 2) plasmin activity, and/or 3) factors that stimulate or inhibit thrombin or plasmin. Sprague-Dawley rats were anesthetized with 1-2% isoflurane/100% oxygen, and their left femoral artery and vein were cannulated. Polytrauma included right femur fracture, and damage to the small intestines, the left and medial liver lobes, and right leg skeletal muscle. Rats were then bled 40% of blood volume. Plasma samples were taken before trauma, and at 30, 60, 120, and 240 min. Polytrauma and hemorrhage led to a significant fall in prothrombin levels. However, circulating thrombin activity did not change significantly over time. Antithrombin III and α2 macroglobulin fell significantly by 2 h, then rose by 4 h. Soluble thrombomodulin was significantly elevated over the 4 h. Circulating plasmin activity, plasminogen, and D-dimers were elevated for the entire 4 h. Tissue plasminogen activator (tPA) was elevated at 30 min, then decreased below baseline levels after 1 h. Plasminogen activator inhibitor-1 was significantly elevated at 2-4 h. Neither tissue factor pathway inhibitor nor thrombin activatable fibrinolysis inhibitor changed significantly over time. The levels of prothrombin and plasminogen were 30-100 times higher than their respective active enzymes. Polytrauma and hemorrhage in rats lead to a fibrinolytic coagulopathy, as demonstrated by an elevation in plasmin activity, D-dimers, and tPA. These results are consistent with the observed clinical benefit of tranexamic acid in trauma patients.

In this study, we established stable cell lines producing 1.5 mg/mL recombinant human prothrombin in 400-L fed-batch culture, using CHO DG44 cells as a host cell line. And we also established a recombinant human α-thrombin purification process that produces a purified product suitable for use as a biopharmaceutical, by using recombinant ecarin from CHO DG44 cells, achieving a total yield of approximately 27% of prothrombin in culture medium. The establishment of stable cell lines with high expression levels, long-term passage stability and satisfactory scale-up are essential to ensure the stable supply of biopharmaceuticals. Furthermore, biopharmaceuticals must be of high quality to assure safety and effectiveness in target applications. We had previously reported that recombinant human prethrombin-2 expression level in a stable cell line established using the mouse myeloma cells, Sp2/0-Ag14, reached 200 μg/mL using animal-free materials in 50-L fed-batch culture. However, the productivity was insufficient to completely replace α-thrombin in human plasma preparations. By employing CHO DG44 cells as a host cell line, we had established a stable cell line and achieved significant improvements in quality, productivity of recombinant human α-thrombin manufacture suitable for use as a biopharmaceutical., (Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Background: The production of therapeutically relevant proteases typically involves activation of a zymogen precursor by external enzymes, which may raise regulatory issues about availability and purity. Recent studies of thrombin precursors have shown how to engineer constructs that spontaneously convert to the mature protease by autoactivation, without the need for external enzymes., Objectives: Autoactivation is an innovative strategy that promises to simplify the production of proteases of therapeutic relevance, but has not been tested in practical applications. The aim of this study was to provide a direct test of this strategy., Methods: An autoactivating version of the thrombin mutant W215A/E217A (WE), which is currently in preclinical development as an anticoagulant, was engineered., Results and Conclusions: The autoactivating version of WE can be produced in large quantities, like WE made in BHK cells or Escherichia coli, and retains all significant functional properties in vitro and in vivo. The results serve as proof of principle that autoactivation is an innovative and effective strategy for the production of trypsin-like proteases of therapeutic relevance., (© 2014 International Society on Thrombosis and Haemostasis.)

Aim: Venous stasis is a well-known risk factor for the development of venous thromboembolism. It is likely that stasis increases the risk of thrombosis by inducing hypercoagulability via the hypoxic procoagulant activation of endothelial and mononuclear cells and the accumulation of activated clotting factors. However, increased rates of thrombin formation have not been demonstrated in response to venous stasis in vivo., Methods: In this study, we used the venous occlusion (VO) test to determine, if stasis triggers thrombin formation in healthy individuals (n=25) and patients with additional thrombotic risk factors, such as inherited thrombophilia (n=19) and symptomatic atherosclerosis (n=15). Thrombin formation was monitored by measuring plasma levels of free thrombin using a highly sensitive oligonucleotide enzyme capture assay (OECA) in addition to the plasma levels of prothrombin fragment 1+2 (F1+2) and thrombin-antithrombin-complexes (TAT). The plasma levels of activated protein C (APC) were additionally measured using an APC-OECA., Results: VO induced a significant (p<0.05) increase in the levels of tissue-type plasminogen activator and plasmin-α2-antiplasmin-complexes. In all three cohorts, the majority of samples obtained during VO showed no quantifiable thrombin or APC levels. Consistent with these findings F1+2 and TAT did not change., Conclusions: We conclude that short-term venous stasis induces a profibrinolytic response due to the activation of endothelial cells, but not a prothrombotic response, even in the presence of additional thrombophilic risk factors. Furthermore, our results support the hypothesis that the stasis-induced profibrinolytic activation of endothelial cells occurs independently from thrombin formation.

A 78-year-old man was admitted for workup for a liver tumor. Both serum AFP and PIVKA-II levels were high (2260ng/ml and 806mAU/ml, respectively). Contrast-enhanced CT scan and MRI using Gd-EOB-DTPA demonstrated a liver tumor in segment 6 resembling the imaging patterns of hepatocellular carcinoma (HCC), while the upper gastrointestinal endoscopy revealed a type 2 gastric cancer in the gastric antrum. Although the liver metastasis of the gastric cancer was undeniable, we performed partial resection of segment 6 of the liver and distal gastrectomy under a preoperative diagnosis of double cancer. Histopathologically, gastric tumor consisted of two components, such as well differentiated adenocarcinoma and hepatoid adenocarcinoma. The histology of the liver tumor was similar to that of the hepatoid component in the stomach lesion. Immunohistochemical staining revealed both the gastric and the liver tumors to be positive for AFP and PIVKA-II, yielding a definite diagnosis of AFP and PIVKA-II producing gastric cancer with liver metastasis. Because many cases of this disease have liver metastases at presentation with confusing images with HCC, the diagnosis of liver tumors should be carefully differentiated in the gastric cancer patients with liver tumors, high serum AFP and PIVKA-II levels.

Background: Des-γ-carboxy prothrombin (DCP) is an established tumor marker for hepatocellular carcinoma (HCC), but the precise mechanism of its production remains unknown. We have recently demonstrated that cytoskeletal rearrangement during the phenotypic changes involved in epithelial mesenchymal transition (EMT) plays a crucial role in DCP production through the impairment of vitamin K uptake. However, DCP production in long-lasting severe hypoxic conditions with nutrient deprivation-such as transarterial embolization-remains unknown., Methods: We examined the effects of long-lasting hypoxia with nutrient deprivation, as well as the constitutive expression of hypoxia-inducible factor (HIF)-1α, on EMT status, DCP production, and protein synthesis in human hepatoma cell lines by enzyme-linked immunosorbent assay, immunofluorescent studies, and western blotting. Immunohistochemistry findings for DCP, anti-hepatocyte paraffin 1 (Hep Par 1), and vimentin were examined using human resected HCC samples., Results: Both severe hypoxia with nutrient deprivation and HIF-1α transfection led to the cessation of DCP production, by attenuating protein synthesis through the hypophosphorylation of mammalian target of rapamycin and its effector proteins, indicative of a further phenotypic shift involving impaired liver-specific protein synthesis. In immunohistochemistry, the distribution of DCP- and Hep Par 1-positive HCC cells was mostly similar and vimentin-positive HCC cells were frequently observed in the areas that were negative for Hep Par 1 and/or DCP., Conclusions: HCC cells produce DCP when they undergo mild phenotypic changes. However, when HCC cells adopt mesenchymal properties they lose their capacity for protein synthesis, and the production of DCP is attenuated. Building upon our previous works, it appears that DCP could be a unique tumor marker that reflects the stepwise phenotypic changes of HCC.

Des-gamma-carboxy prothrombin (DCP) is an established HCC tumor marker, but the precise mechanism of its production is still unclear. Recently, we demonstrated that cytoskeletal changes during epithelial-to-fibroblastoid conversion (EFC) or epithelial mesenchymal transition (EMT) induced by chemicals plays a critical mechanistic role in DCP production via impairment in vitamin K uptake. Our proposed mechanism of DCP production is consistent with substantial clinical evidence. Supplementary vitamin K2 analogues reduced serum DCP levels in hepatocellular carcinoma (HCC) patients. HCC patients with high serum DCP are associated with vascular invasion, metastasis and tumor recurrence. On the other hand, hypoxia has been reported to induce EMT or cytoskeletal changes. Therefore, we examined whether hypoxia induced DCP production during EFC or EMT in HCC cells. Indeed, hypoxic stimulation induced hepatoma cell lines (HepG2 or PLC/PRF/5 cells) to undergo EFC or EMT and these cells produced DCP. Immunofluorescence study demonstrated that hypoxic stimulation impaired labeled low-density lipoprotein uptake, which was a surrogate for vitamin K uptake. In addition, fine filamentous actin network, which has crucial role for clathrin-mediated endocytosis of vitamin K, was disrupted in DCP producing cells by hypoxic stimulation. Thus, hypoxic stimulation induced HCC cells to produce DCP in the same mechanism as chemicals. Furthermore, immunohistochemical study using surgically resected HCC samples showed that a positive staining of nuclear hypoxia inducible factor (HIF)-1alpha was more frequently observed in HCC cells with stronger staining intensity of DCP. Importantly, clinical observations that DCP as an HCC tumor marker was more useful in larger tumors, which is likely to be exposed with hypoxia during tumor development, support our results.

Des-gamma-carboxy prothrombin (DCP) is a well-established tumor marker for hepatocellular carcinoma (HCC), but the precise mechanism by which HCC cells produce DCP remains unknown. Our preliminary experiments demonstrated that HepG2 cells with chemical induction of epithelial-to-fibroblastoid conversion (EFC) produced DCP through impairment of vitamin K uptake via cytoskeletal rearrangement. Therefore, in this study we further examined this mechanism in vitro and using human HCC samples. Hepatoma cell lines (HepG2 and PLC/PRF/5) were induced EFC or epithelial-mesenchymal transition (EMT) by phorbol 12-myristate 13 acetate (TPA) or transforming growth factor (TGF)-beta1. We analyzed these cells by ELISA, Western blotting and immunofluorescent studies. We also examined DCP production and E-cadherin expression in human surgically resected HCC samples by immunohistochemical studies. Labeled low-density lipoprotein (LDL) uptake (as a surrogate for vitamin K) was significantly impaired in DCP-producing hepatoma cells following induction of EFC or EMT. Further, filamentous actin, which plays a critical role in clathrin-mediated endocytosis, was dissociated in DCP-producing cells. Latrunculin A, an actin depolymerizer, induced naïve hepatoma cells to produce DCP with impairment of labeled-LDL uptake. In addition, an E-cadherin neutralizing antibody did not induce DCP production. Finally, immunohistochemical studies demonstrated that DCP production was inversely correlated with the intensity of E-cadherin expression in human HCC cells. In conclusion, cytoskeletal changes during EFC or EMT plays a critical mechanistic role in DCP production via impairments in vitamin K uptake.

Factors II, V, VII and Xa have materialized as a key enzymes for the intervention of blood coagulation cascade and for the development of new anti thrombotic agents. The combined density functional quantum mechanical/molecular mechanical (QM/MM) approach has been used to access inhibition of prothrombin and thrombin production. The biological activities of coumarin derivatives as clotting factor inhibitors was quantitatively analyzed in terms of physicochemical parameters utilizing the principal component analysis. Structural requirements for maximal potency were derived from the results of a quantitative structure activity relationship analysis.

Measurements of anti-FXa or anti-FIIa (thrombin) activities are conventional tests for biological monitoring of low molecular weight heparin (LMWH) or unfractionated heparin treatment. It was the aim of our study to assess the anticoagulant efficacy of the LMWHs nadroparin and enoxaparin and that of unfractionated heparin not by the above-mentioned isolated measurements but in the physiological environment of clotting plasma or whole blood. The effects of increasing amounts of nadroparin, enoxaparin, or unfractionated heparin on the time-course of FXa or FIIa formation were investigated in tissue factor activated platelet-poor plasma using a subsampling technique and chromogenic substrates. Moreover, the anticoagulant efficacy of these drugs was also investigated in whole blood triggered by the physiological relevant activator collagen/endogenous thrombin using thrombelastometry. Nadroparin is as efficient as enoxaparin concerning suppression of endogenous FXa or FIIa formation. The two LMWHs are capable of suppressing the formation of FIIa as efficient as that of FXa. Compared with equivalent anti-FXa activity, unfractionated heparin is markedly more efficient in suppressing the formation of FXa and FIIa than the LMWHs. Corresponding results were obtained in whole blood. The anticoagulant efficacy of nadroparin was comparable with that of enoxaparin and the influence of unfractionated heparin on thrombelastometry parameters was markedly stronger than that of the two LMWHs. We conclude that LMWHs are efficient inhibitors not only of endogenous FXa formation but also of endogenous FIIa formation. Under our experimental conditions, the anticoagulant efficacy of nadroparin was comparable with that of enoxaparin but markedly lower than that of unfractionated heparin.

Using GGCX gene-specific real-time PCR, exon 2 deletion splice variant of vitamin K-dependent gamma-glutamyl carboxylase (GGCX) mRNA was identified in HCC cell lines. Expressions of wild type and exon 2 deletion variant of GGCX were analyzed with relevance to DCP production in HCC cell lines. Hep3B, HepG2, HuH1, HuH7, and PLC/PRF/5 produced DCP, while SK-Hep-1, HLE, HLF, and JHH1 produced no detectable level of DCP. DCP-producing cells expressed exon 2 deletion variant of GGCX mRNA and protein, while DCP-negative cells expressed no detectable level of exon 2 deletion variant of GGCX. These results suggest that exon 2 deletion splice variant of GGCX causes dysfunction of GGCX enzyme activity resulting in DCP production in HCC cell lines.

Background: Haplotypes A1 and A3 in the endothelial protein C receptor (EPCR) gene are tagged by 4678G/C and 4600A/G respectively. We assessed whether these haplotypes modify the risk of venous thromboembolism in carriers of the prothrombin 20210A allele., Design and Methods: We genotyped 4678G/C and 4600A/G in 246 20210A carriers: 84 venous thromboembolism propositi and 162 relatives (13 symptomatic), and in 140 relatives not carrying the 20210A variant. Prothrombin and soluble EPCR (sEPCR) levels were also measured., Results: Among propositi, the mean age at first onset was lower in carriers (35 +/- 8 years) than non-carriers of the 4600G allele (44 +/- 14 years) (p = 0.004). The probability of being free of thrombosis at age 40 was lower in 20210A carriers with the EPCR 4600G allele (p = 0.015). The frequency of the 4600G allele (p=0.002) and the levels of prothrombin antigen (p = 0.002) and sEPCR (p < 0.001) were higher in the propositi than in their asymptomatic relatives. Multivariate analyses showed that the presence of the 4600G allele (OR = 2.5, 95% confidence interval 1.3-5.0), sEPCR > 147 ng/mL (2.8, 1.5-5.2) and prothrombin > 129% (3.8, 1.8-8.3) all increased the thrombotic risk. In bivariate analysis, including the 4600G allele and sEPCR > 147 ng/mL, only the latter remained associated with risk., Conclusions: These results show that in 20210A carriers the venous thromboembolism risk is influenced both by the actual prothrombin levels and by the EPCR A3 haplotype, via its effect on sEPCR levels.

Prothrombin complex concentrates (PCCs) are widely administered for emergency oral anticoagulation reversal and for coagulation defects in liver disease. Pharmacokinetic data may help to optimize treatment. The objective of this study was to characterize the pharmacokinetics of a PCC (Beriplex P/N) containing coagulation factors II (FII), VII (FVII), IX (FIX) and X (FX) and anticoagulant proteins C and S. Fifteen healthy volunteers received a single rapid 50 IU/kg infusion of PCC and underwent frequent blood sampling until 144 hours (h) after infusion. Coagulation factors and anticoagulant protein pharmacokinetic parameters were estimated by non-linear regression. The mean infusion rate of PCC was 7.9 ml/min, equivalent to 196.4 IU/min. By the earliest post-infusion sampling point at 5 minutes (min), plasma FIX concentration increased by a median of 73%. Median increases in FII, FVII and FX at 5 min were 122%, 62% and 158%, respectively. Proteins C and S also increased rapidly. The median terminal half-life of FIX was 16.7 h, FII 59.7 h, FVII 4.2 h and FX 30.7 h. The median in-vivo recovery of FIX was 1.57 %/IU/kg and that of the other three coagulation factors > 2 %/IU/kg. Plasma concentration of thrombogenicity marker D-dimer did not increase, and there was no clinical evidence of thrombosis. Through up to 12 weeks follow-up there were no laboratory findings indicating PCC-related viral exposure. Rapid PCC infusion produced prompt sustained increases in coagulation factors and anticoagulant proteins with no clinical evidence of thrombosis or viral transmission.

Background: Coagulation tests, such as activated partial thromboplastin time and activated clotting time, are used to monitor the effects of unfractionated heparin and the direct thrombin inhibitor, bivalirudin. These tests reflect only the initial phase of blood clotting, when <5% of thrombin has been formed. In this study, we sought to determine if similar increases in activated partial thromboplastin time or activated clotting time due to heparin or bivalirudin would reflect the same degree of inhibition of thrombin formation., Methods: Thrombin formation was evaluated in platelet-poor plasma activated in the presence of heparin (0-5 U/mL) or bivalirudin (0-30 microg/mL) using a thrombin generation assay (Thrombinoscope). Prothrombin activation was measured by prothrombin fragment 1.2 (F1.2) formation. Thrombus formation was further evaluated in kaolin-activated whole blood samples containing heparin (1.5 or 2.5 U/mL) or bivalirudin (12.5 or 25 microg/mL) using Sonoclot and thromboelastography., Results: Based on the Thrombinoscope results, increasing concentrations of bivalirudin and heparin progressively delayed the onset of thrombin formation, but only heparin dose-dependently decreased the amount of thrombin generated. Heparin and bivalirudin delayed the onset of F1.2 formation, but there was no difference in peak F1.2 levels between bivalirudin and non-anticoagulated samples (206 +/- 28.2 vs 182 +/- 23.9 nmol/L, P = 0.09). In heparinized samples, F1.2 levels were significantly lower (75.7 +/- 29.8 nmol/L, P < 0.05) than controls. Heparin and bivalirudin prolonged the onset of clotting on viscoelastic monitors, but only heparin decreased the rate of thrombus formation., Conclusion: Thrombus formation kinetics differs between heparin and bivalirudin despite similar prolongation of clotting test values.

Regular multilaboratory surveys of laboratories by the Royal College of Pathologists of Australasia Quality Assurance Program (QAP) have been conducted to assess proficiency in tests of hemostasis for the last 40 years. This article focuses primarily on specialized assays of hemostasis, for which surveys have been conducted for some 10 years. For von Willebrand disease (vWD) evaluations, a total of 47 plasma samples have been dispatched to survey participants, including representative samples from normal individuals plus all of the major vWD subtypes (i.e., types 1, 2A, 2B, 2M, 2N, and 3). These surveys have focused partly on the issue of diagnostic interpretive error rates associated with different assays and test panels. In this context, considerable improvement is seen when laboratories incorporate the vWF:collagen-binding assay into the test panel. Thrombophilia-associated tests assessed by the program and discussed in this review include activated protein c resistance, lupus anticoagulant, and deficiencies of protein C, protein S, and antithrombin. Other tests briefly reviewed here include factor assays and inhibitors, D-dimer, and heparin/anti-Xa assays. Anticardiolipin antibody and anti-beta(2)-glycoprotein I antibody (aB(2)GPI) testing, assessed by the Immunology QAP, is also reviewed briefly, as are genetic tests associated with thrombophilic markers such as factor V Leiden and the prothrombin gene.

We compared an antisense-oligodeoxynucleotide and four DNAzymes directed to the prothrombin mRNA for their efficiency to reduce prothrombin transcript level in HepG2 cells. The DNAzymes have different binding arm symmetry and cleavage sites, but are directed to the identical target site of the antisense-oligodeoxynucleotide. The nucleic acid-based inhibitors were transfected into HepG2 cells and prothrombin transcript level was quantified and normalized to the beta-actin transcript level by multiplex PCR. All nucleic acid-based inhibitors reduced prothrombin transcript level and the effect was in almost all cases, strongest 24 h after transfection, but still remarkable up to 68 h after transfection. The antisense-oligodeoxynucleotide was more effective than the DNAzymes tested.

Purpose: We investigated the differences in clinical features between alpha-fetoprotein (AFP)-predominant hepatocellular carcinoma (HCC) and protein induced by vitamin K absence or antagonist-II (PIVKA-II)-predominant HCC, especially regarding host factors thought to contribute to hepatocarcinogenesis in chronic hepatitis C virus (HCV) infection., Methods: HCV-related HCC patients (n=306) were divided into four groups according to median AFP (48.1 ng/ml) and PIVKA-II (60 mAU/ml). Host factors, tumor factors, survival, and risk factors affecting survival were compared., Results: Aspartate aminotransferase (AST; IU/L), alanine aminotransferase (ALT; IU/L), and platelet count (x 10(4)/ml) were, respectively, 81, 67, and 8.2 in AFP-predominant HCC (group A; n=66) vs. 50, 42, and 11.4 in PIVKA-II-predominant HCC (group P; n=52). Tumor sizes (mm) in groups A and P were 20 and 37, respectively. Significant differences were evident. Survival was identical between the two groups. Factors affecting survival were total bilirubin, portal tumor thrombus and number of nodule in group A, and albumin and tumor distribution in group P., Conclusions: PIVKA-II-predominant HCC had a milder hepatitis and a better-preserved platelet count compared with AFP-predominant HCC. Considering the strong relation between hepatocarcinogenesis and hepatic inflammation with chronic HCV infection, these differences indicate that hepatocarcinogenic mechanisms in PIVKA-II-predominant HCC may differ from those in AFP-predominant HCC.

A 71-year-old man was admitted to our hospital with edema of the ambilateral crura and liver dysfunction. Serum levels of AFP and PIVKA-II were both abnormally high. An abdominal CT revealed many low density masses in both lobes of the liver. Endoscopic examination and gastric radiography showed a Type 1 tumor in the fornix of the stomach. Biopsy specimen showed poorly differentiated adenocarcinoma. Immunohistochemical stains demonstrated the presence of AFP and PIVKA-II in tumor cells. A diagnosis of AFP and PIVKA-II producing gastric carcinoma with liver metastasis was made and the internal use of S-1 was started. The result of treatment was determined to be partial response. However, the condition of his entire body gradually worsened and he died of hepatic insufficiency.

The prothrombin gene mutation G20210A is a common risk factor for thrombosis and has been reported to cause APC resistance. However, the inhibition of thrombin formation by APC not only limits fibrin formation but also stimulates fibrinolysis by reducing TAFI activation. We evaluated the influence of prothrombin G20210A mutation on the anticoagulant and fibrinolytic activities of APC (1 microg/ml). Thirty-two heterozygous carriers and 32 non carriers were studied. APC anticoagulant activity was assessed by aPTT prolongation whereas APC fibrinolytic activity was determined by a microplate clot lysis assay. APC-induced aPTT prolongation was markedly less pronounced in carriers than in non carriers. On the contrary, fibrinolysis time was shortened by APC to a comparable extent in both groups. Accordingly, prothrombin levels were strongly correlated with APC-induced aPTT prolongation but not with APC-induced shortening of lysis time. The addition of purified prothrombin to normal plasma (final concentration 150%) caused APC resistance in the clotting assay over the whole range of tested APC concentrations (0.125-1.5 microg/ml). In the fibrinolytic assay, instead, prothrombin supplementation made the sample resistant to low but not to high concentrations of APC (>0.5 microg/ml). Thrombin and TAFIa determination in the presence of 1 microg/ml APC revealed that hyperprothrombinemia, although capable of enhancing thrombin generation, was unable to induce detectable TAFIa formation. It is suggested that APC resistance caused by hyperprothrombinaemia does not translate in impaired fibrinolysis, at least in the presence of high APC levels, because the increase in thrombin formation is insufficient to activate the amount of TAFI required to inhibit plasminogen conversion. These data might help to better understand the relationship between thrombin formation and fibrinolysis down-regulation.

This study describes a generic biological screening assay designed to detect anticoagulant rodenticides based on their inhibitory action on the vitamin K epoxide reductase protein complex, resulting in an accumulation of under-carboxylated prothrombin or proteins induced by vitamin K antagonism (PIVKA-II). A combined cell culture/ELISA assay was optimized to measure PIVKA-II production by the human hepatoma HepG2 cell line cultured in the presence of anticoagulant rodenticides. The specificity and sensitivity of the assay was validated using 41 grain extracts containing representative concentrations of rodenticide or appropriate nonrodenticide control compounds. In all cases, PIVKA-II produced by HepG2 cells in response to grain extracts spiked with rodenticides was detected by ELISA, while PIVKA-II was not detected in supernatants collected from cells exposed to nonrodenticide controls. This represents a novel, class-specific biological assay for the detection of anticoagulant rodenticides present in contaminated grain.

Background and Purpose: Treatment doses of heparins are not recommended for acute ischemic stroke. Despite this, their use in this setting is widespread. We investigated whether subgroups of patients with acute ischemic stroke and atrial fibrillation, identified by clinical, hemostatic (d-dimer, prothombin fragments(1+2) [F(1+2)], soluble fibrin monomer), or inflammatory (C-reactive protein [CRP]) variables might have a differential response to low molecular weight heparin (LMWH) over aspirin. In addition, we sought to identify factors associated with a poor clinical outcome at 3 months., Methods: We conducted a post hoc subgroup analysis of a randomized, placebo-controlled, double-blind trial (Heparin in Acute Embolic Stroke Trial) designed to test the hypothesis that treatment doses of LMWH (dalteparin; 100 IU/kg BID) would be superior to aspirin (160 mg per day) in patients with acute ischemic stroke and atrial fibrillation. For the current analysis, 431 participants were included. The primary outcome measure was a poor outcome at 3 months, defined as death or dependency in activities of daily living. Using regression analysis, we determined whether any of the chosen variables were associated with a differential response to dalteparin (treatment interaction) or with poor outcome., Results: In the multivariable logistic regression model, none of the clinical, hemostatic, or inflammatory variables were associated with a significant treatment interaction. Stroke severity (odds ratio [OR], 1.09 [95% CI, 1.07 to 1.12]), increasing age (OR, 1.09 [CI, 1.05 to 1.14]), CRP level (OR, 1.32 [CI, 1.04 to 1.66]), and F(1+2) level (OR, 1.77 [CI, 1.07 to 2.91]) were independently associated with a poor outcome at 3 months., Conclusions: Our study does not support the use of treatment doses of LMWH in any of the studied subgroups of patients with acute ischemic stroke and atrial fibrillation. Age, stroke severity, CRP, and F(1+2) were predictive of poor outcome at 3 months.

Retinal vein occlusion (RVO) is a multifactorial disease involving vessel damage, stasis, viscosity and thrombosis. Conflicting findings on hereditary thrombophilic risk factors have been reported and their impact on RVO features remains to be defined. The aim of the present study was to evaluate the prevalence of hereditary thrombophilic risk factors (HTRF) and characteristics of RVO in patients with or without HTRF. The design of the study was a prospective, observational case series. Two hundred and thirty-four patients with RVO were included consecutively. A French healthy population of the same region was studied as control group. The HTRF studied were protein C (PC), protein S (PS) and antithrombin (AT) deficiencies, factor V Leiden (FVL) and factor II 20210A polymorphisms. Chi-Square was used for comparison with the healthy subjects and between RVO patient with and without HTRF according to localisation (branch vs. central), type of RVO (ischemic or non-ischemic), recurrence, age at first event and classical vascular risk factors. Twenty-two patients had HTRF (12 FV Leiden heterozygotes, 9 FII 20210A heterozygotes and 1 PS deficiency). No AT or PC deficiency was detected. Frequencies of PS deficiency, FVL and FII 20210A allele were similar to the reference population as well as to published data in the general caucasian population. Eighty-six patients experienced their first episode before the age of 60 years. Systemic hypertension, glaucoma and angina were significantly less frequent in patients with RVO before 60 years. Fourteen of the 22 patients with one HTRF (64%) experienced their first episode of RVO before the age of 60 years compared to 72 of 212 without HTRF (34%) (p = 0.006). Heterozygote status for FV Leiden was significantly more frequent in patients who had experienced their first episode of RVO before 60 years (p = 0.027). In conclusion, this study suggests a role of FV Leiden in the occurrence of RVO in patients younger than 60 years who exhibit fewer acquired vascular risk factors than in older patients.

Background: A reduced sensitivity for activated protein C (APC) is associated with an increased risk of venous thrombosis even in the absence of the factor (F)V Leiden mutation. This risk has been demonstrated with two APC sensitivity tests, which quantify the effects of APC on the activated partial thromboplastin time (APTT) and the endogenous thrombin potential (ETP), respectively., Objectives: We examined determinants of both APC sensitivity tests in the control group of the Leiden Thrombophilia Study (LETS)., Methods: Multiple linear regression analysis was performed with normalized APC-SR(APTT) or APC-SR(ETP) as dependent variable and putative determinants [levels of FII, FV, FVII, FVIII, FIX, FX, FXI, FXII, FXIII A subunit, FXIII B subunit, protein S total, protein S free, protein C, tissue factor pathway inhibitor (TFPI) total, TFPI free, antithrombin and fibrinogen] as independent variables., Results and Conclusions: The major determinant of the APTT-based test was FVIII level, followed by FII level. The ETP-based test was influenced most by free protein S and free TFPI levels. In both tests FXa formation plays a major role, as the effect of FVIII and TFPI on the tests seems to be executed via FXa. The ETP-based test was also strongly influenced by oral contraceptive use, even when we adjusted for all the clotting factors listed above. This means that the effect of oral contraceptives on the ETP-based test is not fully explained by the changes of coagulation factor levels investigated in this study, and that the molecular basis of acquired APC resistance during use of oral contraceptives remains to be established.

Prothrombin deficiency is a rare (1:200 000) autosomal recessive disorder caused by diverse mutations in prothrombin gene. We have studied the molecular basis of this disorder in four unrelated Indian patients. The diagnosis was based on prolonged prothrombin (PT) and activated partial thromboplastin times and low factor II coagulant activity (FII: C) measured using a PT based assay. FII: C levels ranged between 4.7% and 17.5%. Mutations were identified in all the four patients. Five different causative mutations including four (80%) missense and an in-frame deletion (20%) were identified. One of them was a novel, Ala362 --> Thr amino acid change affecting 'B' chain of -thrombin. This mutation was present in a compound heterozygous state with a previously reported Arg-1 --> Gln missense change affecting pro-peptide cleavage site. Ala362 --> Thr occurred at a codon, evolutionarily conserved in all the 24 different prothrombins or its related serine proteases studied. Molecular modeling of this mutation was found to cause a conformational change around the region involving a catalytic triad residue His363 and a cysteine residue at codon 364. The FII: C level in this patient was 17.5%. Three other previously reported mutations were also detected in the homozygous state: Arg271 --> Cys in Kringle-2 region, a Glu309 --> Lys in "A" chain of -thrombin and an in-frame deletion of 3 bp (AAG) leading to Del Lys301/302 in "A" chain of -thrombin. This is the first report of the molecular basis of prothrombin deficiency in Indian patients and we suggest the eponym 'Prothrombin Vellore 1' for Ala362 --> Thr mutation.

From 1998 to 2003, 133 Caucasian women aged 17-40 years (median 29 years) suffering from unexplained recurrent miscarriage (uRM) were consecutively enrolled. In patients and 133 age-matched healthy controls prothrombotic risk factors (factor V (FV) G1691A, factor II (FII) G20210A, MTHFR T677T, 4G/5G plasminogen activator inhibitor (PAI)-1, lipoprotein (Lp) (a), protein C (PC), protein S (PS), antithrombin (AT), antiphospholipid/anticardiolipin (APA/ACA) antibodies) as well as associated environmental conditions (smoking and obesity) were investigated. 70 (52.6%) of the patients had at least one prothrombotic risk factor compared with 26 control women (19.5%; p<0.0001). Body mass index (BMI; p=0.78) and smoking habits (p=0.44) did not differ significantly between the groups investigated. Upon univariate analysis the heterozygous FV mutation, Lp(a) > 30 mg/dL, increased APA/ACA and BMI > 25 kg/m(2) in combination with a prothrombotic risk factor were found to be significantly associated with uRM. In multivariate analysis, increased Lp(a) (odds ratio (OR): 4.7/95% confidence interval (CI): 2.0-10.7), the FV mutation (OR:3.8/CI:1.4-10.7), and increased APA/ACA (OR: 4.5/CI: 1.1-17.7) had independent associations with uRM.

We determined anticoagulant parameters that depend on protein S function in plasma, i.e. the APC-independent anticoagulant activity of protein S (expressed as pSR) and APC resistance determined with thrombin generation-based tests (expressed as APCsr) as well as plasma levels of total and free protein S and prothrombin in men, women not using oral contraceptives (OC), and in women using second or third generation OC. Thrombin generation in the APC resistance assays was initiated either with factor Xa (Xa-APCsr) or tissue factor (TF-APCsr). The APC-independent anticoagulant activity of protein S was highest in men (pSR=1.69) and gradually decreased from women not using OC (pSR=1.49) via women using second generation (pSR=1.35) to women using third generation OC (pSR=1.27). The pSR correlated inversely with nAPCsr determined with the tissue factor-based APC resistance test (TF-APCsr) but not with nAPCsr determined with the factor Xa-based assay (Xa-APCsr). Multiple linear regression analysis in which sex, OC use, and protein S and prothrombin levels were included as independent variables and the pSR, TF-APCsr or Xa-APCsr as dependent variables indicated that plasma protein S levels poorly predict the pSR and the TF-APCsr, but are the main determinant of the Xa-APCsr. This indicates that OC use alters the expression of protein S activity. This phenomenon can be caused by differences in modulation of the activity of protein S by other plasma proteins that change during OC use or by OC-induced changes in the protein S molecule that impair its anticoagulant activity. Functional impairment of protein S as a result of hormonal influence may, at least in part, contribute to the thrombotic risk of OC users.

The human prothrombin G20210A polymorphism located at the 3' cleavage site of the mRNA results in elevated plasma prothrombin levels and increased risk of venous thrombosis. This polymorphism has been shown to directly influence a variety of processes related to prothrombin mRNA metabolism. We have constructed plasmids that express the full-length prothrombin mRNA that is polyadenylated at its natural site. The A allele prothrombin variant was more efficient than the G allele at promoting cleavage at this site in the presence of a competing poly (A) sequence. In the absence of competition, both allelic variants give rise to a similar level of cleavage site heterogeneity. An upstream sequence element (USE) was also identified within the prothrombin 3'-UTR. When placed upstream of two competing poly (A) sites, the USE directed cleavage preferentially to the proximal poly (A) site. In the absence of competition, the USE had no effect on cleavage site selection. This study suggests that the basis for the increase in prothrombin expression in A allele carriers is not due to allelic changes in cleavage site selection per se. In addition, the functionality of USEs needs to be considered within the context of endogenous sequence architecture.

Antiphospholipid antibodies are a wide and heterogeneous group of immunoglobulins that include, among others, lupus anticoagulants and anticardiolipin antibodies. Their presence in patients with arterial and venous thrombosis, and/or obstetrical complications, defines the antiphospholipid syndrome. Antiphospholipid antibodies do not recognize anionic phospholipids, but recognize plasma proteins bound to suitable anionic surfaces: beta2-glycoprotein I and prothrombin are the most common and most frequently investigated antigens. We systematically reviewed published articles on the antiphospholipid syndrome to investigate the association between thrombosis and some antiphospholipid antibodies. Lupus anticoagulants were a clear risk factor for thrombosis, irrespective of the site and type of thrombosis, the presence of systemic lupus erythematosus, and the methods used to detect them. Anticardiolipin and anti-beta2-glycoprotein I antibodies were possible risk factors of thrombosis, at least in some selected situations. The measurement of antiprothrombin antibodies is not helpful to define the patient's risk of thrombosis. These results are mainly due to the still far from optimal standardization of the methods to detect antiphospholipid antibodies; the lack of standardized reference materials; the heterogeneity in reagents, calibrators, and assay conditions; and the methods used to calculate the results. Many efforts are currently being made to improve assay standardization and harmonization, which should help to clarify the clinical relevance of antiphospholipid antibodies.

Background: Thrombin promotes angiogenesis and cell proliferation in cancer. Whether thrombin turnover influences cancer incidence is unknown., Objectives: To explore the relation between the status of the coagulant pathway and cancer incidence by population survey., Methods: Of 4,009 middle-aged men clinically free of malignancy, 3052 (76.1%) were recruited. Measurements of hemostatic status were made annually for 4 years, and follow-up for morbidity and mortality was maintained thereafter. Persistent activation of the coagulant pathway was diagnosed when prothrombin fragment 1+2 and fibrinopeptide A concentrations exceeded the upper quartiles of the population distribution in two consecutive annual examinations. Cancer incidence rates in men developing persistent activation (taking the time of onset of activation as baseline) were compared with those in men remaining free of this condition., Results: Persistent activation of the hemostatic pathway was a distinct entity found in 111 men [43 expected by chance alone (P <0.001)], and associated with activation throughout the coagulation pathway. Total mortality (/1000 person-years) was higher in those with persistent activation than in others (17.1 and 9.7, respectively, P=0.015), owing to a higher mortality from all cancers (11.3 and 5.1, respectively, P=0.01), due in turn largely to a higher mortality from cancers of the digestive tract (6.3 and 1.9, respectively, P=0.004). Trends were similar for non-fatal cancers., Conclusions: Persistent activation of the coagulant pathway plays a role in the preclinical phase of cancer and is associated with an increased incidence of clinical malignancy, especially of the digestive tract.

Platelets play a key role in (sub)acute thrombotic occlusion after stenting. We examined the possible differences between biodegradable polylactide (PLA) and stainless steel (SS) stents in platelet attachment and morphology after whole blood perfusion. PLA stents of different configurations (spiral/braided) and polycaprolactone-polylactide (PCL-PLA)-coatings, or SS stents were implanted into a PVC tube (Ø 3.2 mm), with or without precoating of the tube with type-I collagen. PPACK (30 microM)-anticoagulated blood with (3)H-serotonin prelabeled platelets was perfused (flow rate: 30 ml/min, 90 s) over the stents. Platelet deposition was assessed by scintillation counting and morphology by scanning electron microscopy (SEM). To examine coagulation activation, plasma prothrombin fragments (F1 + 2) were measured before and after the perfusion. Protein deposition on PLA/SS stents was assessed at augmented shear forces mimicking coronary flow (rate: 60 ml/min, 60 s) under minimal anticoagulation (PPACK 1 microM). More platelets deposited on PLA stents than on SS stents under all study conditions (p < 0.03). Under anticoagulation (PPACK 30 microM) the generation of F1 + 2 remained unaltered. Under higher flow rate and limited anticoagulation SS stents accumulated 3.27 +/- 0.75 microg and PLA stents 5.25 +/- 1.74 microg of protein (Mean +/- SD, p <0.95). Among all biodegradable stents, the braided PLA stent coated with PCL-PLA-heparin accumulated the fewest platelets (p < 0.02). In SEM, signs of platelet activation on braided heparin-coated PLA stents, when compared with uncoated braided PLA/SS stents, appeared modest. In conclusion, PCL-PLAheparin coating of biodegradable stents may enhance their hemocompatibility, expressed by less platelet deposition. Nevertheless, materials, design, and coating techniques of biodegradable stents must be further developed.

Recombinant human activated protein C (rhAPC) has recently been demonstrated to be a promising candidate to improve the outcome for patients with severe sepsis. Plasma-derived activated protein C and unfractionated heparin (UH) exert anticoagulant synergy due to mechanisms that simultaneously decrease thrombin generation. Melagatran, a new direct thrombin inhibitor, does not bind to plasma proteins or requires antithrombin as a cofactor. The latter is often consumed in patients with severe sepsis. We investigated the anticoagulant efficiency in combined administration of rhAPC and UH or melagatran in terms of prolongation of the standard clotting assays activated partial thromboplastin time (aPTT) and prothrombin time (PT) in pooled plasma samples in vitro. RhAPC dose-dependently prolonged the aPTT but not the PT. The ability of UH and melagatran to prolong the aPTT was significantly enhanced in combination with rhAPC. The combined administration of rhAPC and melagatran, but not UH, resulted in additive prolongation of the PT. In control measurements the capability of rhAPC to suppress prothrombin fragment 1.2 generation dose-dependently increased in combination with heparin and melagatran. Our study demonstrates the respective effects of rhAPC, UH, melagatran and further different additive effects in combined administration of rhAPC and UH or melagatran on the prolongation of the aPTT and PT clotting assays usually used to monitor anticoagulant treatment.

The liquid chromatography-mass spectrometry (LC-MS) following on from the two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) technique was applied for the analysis of proteins in a renal stone found in a hyperuricemic patient. This technique was sensitive enough to detect small quantities of proteins even in a renal stone.

We describe the case of a 68-year-old man with primary advanced adenocarcinoma of the stomach, who displayed extremely high plasma levels of protein induced by vitamin K antagonist (PIVKA)-II (15 600 mAU/ml) and normal levels of alphafetoprotein (AFP) (4 ng/ml). Ultrasonography and dynamic computed tomography ruled out hepatocellular carcinoma (HCC) or liver metastasis. After preoperative chemotherapy, pancreatico-spleno total gastrectomy with D2 lymphadenectomy was performed. Postoperatively, plasma levels of PIVKA-II returned to within the normal range (29 mAU/ml). Microscopic examination revealed stomach adenocarcinoma showing various histological types, such as moderately to poorly differentiated mucinous adenocarcinoma, but hepatoid differentiation of gastric adenocarcinoma was not detected. Localization of PIVKA-II and AFP within tumor cells was demonstrated by immunohistochemical staining using monoclonal antibodies. These results indicate that tumor cells from gastric cancer may produce PIVKA-II. Some cases of PIVKA-II- and AFP-producing advanced gastric cancer with liver metastasis have been reported, but this is the first report of gastric cancer without liver metastasis producing PIVKA-II alone.

In a recent experiment, we discovered that liver tissue adjacent to HCC can also produce des-gamma-carboxy prothrombin (DCP). The goal of this study was to advance measurements of DCP levels in hepatocellular carcinoma (HCC) and non-cancer tissues using an electro-chemiluminescence immunoassay (ECLIA) and immunohistochemistry, and to assess their clinical significance. DCP levels in HCC tissues ranged from 0.7 to 209862.4 mAU/0.1 g tissue weight, with a median of 492.6 mAU/0.1 g. DCP levels in non-cancer tissues ranged from 0 to 2329.9 mAU/0.1 g tissue weight, with a median of 88.8 mAU/0.1 g. DCP levels in cell membranes were significantly higher than in the cytoplasm (p<0.001). DCP levels in HCC tissue were significantly higher than in non-cancer tissue (p<0.001). The logarithm of serum DCP levels correlated not only with that of DCP levels in HCC tissues (p=0.019), but also with that in non-cancer tissues (p=0.020), and the total DCP level of liver tissues (p=0.016). The logarithm of DCP levels in HCC tissues correlated with that of DCP levels in non-cancer tissues (p=0.011). DCP levels in HCC tissue with portal vein invasion were significantly greater than in HCC tissues without portal vein invasion (p=0.028). DCP levels in non-cancer tissues with intrahepatic metastatic lesions were significantly higher than in non-cancer tissues without intrahepatic metastatic lesions (p=0.023). Our results suggest that the origin of elevated serum DCP may lie not only in HCC tissue, but in non-cancer tissue. The existence of HCC may influence production of DCP in non-cancer tissue. Tissue DCP may be a prognostic factor, while increased DCP levels in non-cancer tissues may play an important role in hepatocarcinogenesis.

Older donors are a growing part of the total donor pool but no definite consensus exists on the limit of age for their acceptance. From November 1998 to January 2003, in a retrospective case-control multicenter study, we compared the outcome of 30 orthotopic liver transplantations (OLTs) with octogenarian donors and of 60 chronologically correlated OLTs performed with donors <40 years. The percentage of refusal was greater among older than younger donors (48.2 vs. 14.3%; p < 0.001). Cold ischemia was significantly shorter in the older than younger groups. Recipients with hepatocarcinoma and older age received octogenarian grafts more frequently. No differences were seen in post-operative complications and 6-month graft and patient survival. However, long-term survival was lower in patients transplanted with octogenarian donors (p = 0.04). Interestingly, the mortality related to hepatitis C recurrence was greater in patients with octogenarian donors. Accordingly, the long-term survival of HCV-positive patients who received older grafts was lower than those receiving younger grafts (p = 0.05). Octogenarian livers can be used safely but a careful donor evaluation and a short cold ischemia are required to prevent additional risk factors. However, hepatitis C recurrence is associated with a greater mortality in patients who received octogenarian grafts raising concerns whether to allocate these livers to HCV-positive recipients.

Polymerases with proofreading activity provide high fidelity PCR amplifications. In this study we examined the consequences of using a Taq polymerase with proofreading activity, such as Optimase Taq polymerase, in combination with 4 different mutagenic reverse primers for the amplification of a 345-bp FII PCR product. The amplifications were performed with Optimase Taq polymerase (Transgenomic), and Taq DNA polymerase-recombinant (Invitrogen), without proofreading activity. Mutation screening was carried out by DHPLC and restriction fragment analysis. The usage of Optimase Taq polymerase results in complete reversion of the first and second mutated nucleotide introduced at the 3' end of the mutagenic reverse primer. It also partially reverses the missense nucleotide introduced in the third position of the mutagenic primer and leads to misleading DHPLC and restriction fragment analysis patterns. Nevertheless it cannot perform such an activity when an abnormal nucleotide is introduced in the fourth position.

The prothrombinase complex consists of the protease factor Xa, Ca2+, and factor Va assembled on an anionic membrane. Factor Va functions both as a receptor for factor Xa and a positive effector of factor Xa catalytic efficiency and thus is key to efficient conversion of prothrombin to thrombin. The activation of the procofactor, factor V, to factor Va is an essential reaction that occurs early in the process of tissue factor-initiated blood coagulation; however, the catalytic sequence leading to formation of factor Va is a subject of disagreement. We have used biophysical and biochemical approaches to establish the second order rate constants and reaction pathways for the activation of phospholipid-bound human factor V by native and recombinant thrombin and meizothrombin, by mixtures of prothrombin activation products, and by factor Xa. We have also reassessed the activation of phospholipid-bound human prothrombin by factor Xa. Numerical simulations were performed incorporating the various pathways of factor V activation including the presence or absence of the pathway of factor V-independent prothrombin activation by factor Xa. Reaction pathways for factor V activation are similar for all thrombin forms. Empirical rate constants and the simulations are consistent with the following mechanism for factor Va formation. alpha-Thrombin, derived from factor Xa cleavage of phospholipid-bound prothrombin via the prethrombin 2 pathway, catalyzes the initial activation of factor V; generation of factor Va in a milieu already containing factor Xa enables prothrombinase formation with consequent meizothrombin formation; and meizothrombin functions as an amplifier of the process of factor V activation and thus has an important procoagulant role. Direct activation of factor V by factor Xa at physiologically relevant concentrations does not appear to be a significant contributor to factor Va formation.

Attempts to quantify renal prothrombin (PT) have failed due to interference of its hepatic counterpart. In order to gauge PT synthesized by the kidney, the expression of PT mRNA was compared in renal and hepatic tissues of rats. PT mRNAs were quantified, using quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR), from RNA extracts of kidneys and livers of ten hooded Wistar rats. To control variations in sample preparation, the amount of PT transcript in each sample was normalized to the amount of its beta-actin transcript. The median ratio of PT to beta-actin transcript of 0.0011 (with a mean +/- standard deviation (SD) of 0.0010 +/- 0.0002, range 0.0007-0.0014) in the renal tissues was significantly (p < or = 0.001) lower than its corresponding value of 0.3669 (with a mean +/- SD of 0.3729 +/- 0.0716, range 0.2718-0.4675) in the hepatic tissues. Thus, expression of PT mRNA in the rat kidney was almost 300-fold less as compared with its expression in hepatic tissue.

After an episode of unstable angina or myocardial infarction, a high proportion of patients show biochemical signs of coagulation activation, expressed as persistently elevated thrombin generation, in their blood. It is not known whether this has any influence on long-term outcome. In this prospective multicenter cohort study, we assessed the relation of persistently elevated thrombin generation to outcome in 319 consecutive patients with acute coronary syndromes enrolled in the Global Use of Strategies To Open occluded coronary arteries (GUSTO) IIb trial. Plasma prothrombin fragment 1 + 2 levels, an index of "in vivo" thrombin generation, was measured during the acute phase and after 1, 6, and 12 months, and its relation to outcome was assessed during a median 29-month follow-up period. The primary end point of cardiac death or myocardial (re)infarction occurred in 61 patients (19%). There was a U-shaped relationship between plasma prothrombin fragment 1 + 2 levels and the risk of developing the primary end point; intermediate levels (1.5-1.9 nM) were associated with the lowest risk, whereas both higher (> 1.9 nM) and lower (< 1.5 nM) values were associated with an increased risk (relative risk [RR] 1.56 and 95% confidence interval [CI], 1.25-2.28; RR, 1.35 and 95% CI, 1.11-1.86, respectively). After an episode of acute coronary syndrome, both high and low levels of thrombin generation are predictors of an increased risk of an unfavorable outcome.

Background and Objectives: Protein induced by vitamin K absence or antagonist II (PIVKA-II) has been used as a tumor marker of hepatocellular carcinoma (HCC). In this study, we examined the expressions of PIVKA-II in HCC tissues., Methods: Expression of PIVKA-II in HCC was evaluated with the avidin-biotin-peroxidase complex method. Paraffin-embedded HCC tissues obtained from 48 patients were subjected to immunohistochemical staining using the anti-PIVKA-II antibody monoclonal antibody (MU-3) (Eisai, Tokyo, Japan)., Results: The tissue PIVKA-II positivity rate of 12/23 (52.2%) in HCC with vascular infiltration was significantly higher than the rate of 4/25 (16.0%) in HCC without such infiltration. Further, the tissue PIVKA-II positivity rate of 13/28 (46.4%) in HCC of tumor stage III or IV was significantly higher than the rate of 3/20 (6.0%) in HCC of tumor stage I or II. Overall survival rate tended to be lower in the tissue-PIVKA-II-positive than -negative patients., Conclusions: PIVKA-II may play an important role in progression of HCC and is useful not only as a tumor marker but also as a prognostic factor., (Copyright 2003 Wiley-Liss, Inc.)