OBJECTIVE

We have focused on the role of coagulation factor VII (FVII) Arg353Gln polymorphism as a risk predictor of complications following percutaneous transluminal coronary angioplasty (PTCA), directional coronary atherectomy (DCA), and stenting.

BACKGROUND

The FVII Arg353Gln mutation decreases FVII activity, and presence of the Gln353 allele could be protective against thrombus formation during catheter interventions.

METHODS

A total of 666 consecutive patients with coronary artery disease who had undergone PTCA (n = 280), DCA (n = 104), or stenting (n = 282) were followed up for a 30-day composite end point, which included need for target vessel revascularization, myocardial infarction, and death. The Arg353Gln polymorphism of FVII was determined by PCR/RFLP assay.

RESULTS

Carriers of the Gln353 allele had significantly lower levels of total FVII activity (FVIIc, -20.7%, p < 0.001) and of activated circulating FVII (FVIIa, -32.7%, p = 0.03) compared with Arg353/Arg353. The composite end point occurred in 43 patients: 4 were heterozygous Arg353/Gln353, and 39 were homozygous Arg353/Arg353. The incidence of the composite end point was 2.5% in carriers of the Gln353 allele and 7.7% in Arg353/Arg353 homozygotes (p = 0.013). This corresponds to a 72% risk reduction in carriers of the Gln353 allele (relative risk: 0.28; 95% confidence interval: 0.09-0.81; p = 0.02).

CONCLUSIONS

The Gln353 allele of FVII is associated with substantial risk reduction in adverse events that complicate coronary catheter interventions. With the perspective of active site-blocked activated FVII (FVIIai) as conjunctive medication, the results suggest that the FVII genotype should be taken into due consideration in assessment of FVIIai medication and of its dosage.

Essentials The lack of factor (F) VIIa-endothelial protein C receptor (EPCR) binding in mice is unresolved. A single substitution of Leu4 to Phe in mouse FVIIa (mFVIIa) enables its interaction with EPCR. mFVIIa with a Phe4 shows EPCR binding-dependent enhanced hemostatic function in vivo vs. mFVIIa. Defining the FVIIa-EPCR interaction in mice allows for further investigating its biology in vivo.

Background Human activated factor VII (hFVIIa), which is used in hemophilia treatment, binds to the endothelial protein C (PC) receptor (EPCR) with unclear hemostatic consequences. Interestingly, mice lack the activated FVII (FVIIa)-EPCR interaction. Therefore, to investigate the hemostatic consequences of this interaction in hemophilia, we previously engineered a mouse FVIIa (mFVIIa) molecule that bound mouse EPCR (mEPCR) by using three substitutions from mouse PC (mPC), i.e. Leu4→Phe, Leu8→Met, and Trp9→Arg. The resulting molecule, mFVIIa-FMR, modeled the EPCR-binding properties of hFVIIa and showed enhanced hemostatic capacity in hemophilic mice versus mFVIIa. These data implied a role of EPCR in the action of hFVIIa in hemophilia treatment. However, the substitutions in mFVIIa-FMR only broadly defined the sequence determinants for its mEPCR interaction and enhanced function in vivo. Objectives To determine the individual contributions of mPC Phe4, Met8 and Arg9 to the in vitro/in vivo properties of mFVIIa-FMR. Methods The mEPCR-binding properties of single amino acid variants of mFVIIa or mPC at position 4, 8 or 9 were investigated. Results and conclusions Phe4 in mFVIIa or mPC was solely critical for interaction with mEPCR. In hemophilic mice, administration of mFVIIa harboring a Phe4 resulted in a 1.9-2.5-fold increased hemostatic capacity versus mFVIIa that was EPCR binding-dependent. This recapitulated previous observations made with triple-mutant mFVIIa-FMR. As Leu8 is crucial for hFVIIa-EPCR binding, we describe the sequence divergence of this interaction in mice, now allowing its further characterization in vivo. We also illustrate that modulation of the EPCR-FVIIa interaction may lead to improved FVIIa therapeutics.

The molecular defects causing CRM+ factor VII deficiency were investigated in seven unrelated subjects and several members of their families. Four missense mutations located in the catalytic domain of factor VII were found. The previously reported 304Arg->>Gln substitution was present in the homozygous and heterozygous forms, with different polymorphic haplotypes, thus demonstrating that it is recurrent and frequent in the Italian population. The 310Cys->>Phe substitution was found in the homozygous form and in the compound heterozygous condition with the nonsense mutation 356Trp->>stop. Two missense mutations, 298Met->>Ile and 342Gly->>Arg, were found in the homozygous and in the heterozygous condition respectively. Molecular heterogeneity was further increased by finding of the 353Arg->>Gln polymorphism in the doubly heterozygous condition with the 304 and 342 mutations. Plausible explanations for loss of FVII function were found by inspecting a model of the serine protease domain of factor VIIa. Inefficient activation of the catalytic site is predicted for 298Met->>Ile. 342Gly->>Arg would directly distort the geometry of the 'oxyanion hole' preventing formation of a substrate enzyme intermediate. 310Cys->>Phe is predicted to have an adverse effect on tissue factor interaction. These mutations point to important regions of the factor VII molecule.

BACKGROUND

Uncontrolled hemorrhage in polytrauma patients usually results in rapid need of blood products. Despite the shorter thawing times of microwave devices for heating fresh frozen plasma (FFP), their use has remained controversial, and just a few laboratory analyses have been published on this topic. The aim of this study was to analyse the quality of clotting factors immediately after thawing FFP with a microwave device and after 48-hour post thaw storage at 4 degrees C.

METHODS

24 FFP units of all four ABO blood groups (six of each blood group) were thawed with a Transfusio-therm 2000 and later stored at 4 degrees C for 48 hours. Samples were drawn aseptically and investigated on various clotting factors and protein proteases (fibrinogen, antithrombin, FII, FV, FVII, FVIII, FIX, FX, FXI, FXIII, vWF antigen and activity, protein S, and protein C) using standard coagulation and chromogenic assays immediately after thawing and again after a 48-hour storage period at 4 degrees C. All units were tested for both anaerobic and aerobic microbial contamination using standard operating procedures immediately after thawing.

RESULTS

After thawing, all coagulation factors and protein protease activities were within normal ranges. Blood group O individuals had approximately 25% lower plasma levels of vWF antigen and activity. After a 48-hour storage period at 4 degrees C, FVIII and FIX activities declined significantly in all blood groups, whereas the remaining clotting factors remained comparably stable.

CONCLUSIONS

Immediately after rapid thawing using a microwave system, all FFP units contained adequate coagulation factor activities to maintain hemostatic activity at the time of product thaw. The post thaw refrigerated storage caused an anticipated decrease in factor VIII and IX activities, but retained normal coagulation factor levels of many plasma proteins. Therefore we conclude that the Transfusio-therm 2000 has no clinically significant influence on the activity of clotting factors and plasma proteases in FFP units.

Inactivation of specific genes in mammals by gene targeting has accelerated our ability to determine gene function. Nearly all genes involved in the blood coagulation system have been knocked out in mice. Tissue factor (TF) is the main initiator of the coagulation system and functions as a cell surface receptor for coagulation factor VII (FVII). Knockout studies have shown that TF deficiency results in lethality around embryonic day (E) 8.5-10.5. The results suggest a role for TF in embryonic blood vessel development and maintenance of vascular integrity in the yolk sac. In addition, TF may be involved in the maintenance of the placental labyrinth. Factor X (FX) deficiency causes partial embryonic lethality between E11.5-12.5. FX-/- mice that were born died from fatal neonatal bleeding. In contrast, FVII deficiency is not embryonic lethal, but FVII-/- neonates died from hemorrhage within the first days after birth. The various lethal phenotypes of deficiencies of the different coagulation factors suggest involvement in processes beyond hemostasis. Both TF/FVIIa and FXa can trigger intracellular signaling events in certain cell types. Signaling by coagulation proteases and protease-activated receptors (PARs) may have important roles in embryonic development.

Although protein Z (PZ) has a domain arrangement similar to the essential coagulation proteins FVII, FIX, FX, and protein C, its serine protease (SP)-like domain is incomplete and does not exhibit proteolytic activity. We have generated a trial sequence of putative activated protein Z (PZa) by identifying amino acid mutations in the SP-like domain that might reasonably resurrect the serine protease catalytic activity of PZ. The structure of the activated form was then modeled based on the proposed sequence using homology modeling and solvent-equilibrated molecular dynamics simulations. In silico docking of inhibitors of FVIIa and FXa to the putative active site of equilibrated PZa, along with structural comparison with its homologous proteins, suggest that the designed PZa can possibly act as a serine protease.

The mechanisms linking prothrombotic changes to endothelial dysfunction and accelerated atheroma formation have yet to be fully defined. Expression of TF (tissue factor) on the endothelium is potentially an initiating event as binding and activation of FVII (factor VII) can result in thrombosis. Although PAR2 (protease-activated receptor-2) is expressed on vascular endothelium, its precise physiological significance and mechanism of activation have yet to be defined. In the present study, we investigated whether PAR2 can be activated by FVIIa (activated FVII) and induce ET-1 (endothelin-1) synthesis. In bovine aortic endothelial cells pretreated with TNF (tumour necrosis factor-alpha) to increase TF expression, FVIIa stimulated ET-1 synthesis via activation of PAR2. Although FX (factor X) alone was inactive, this response was enhanced by using FVII and FX in combination. Inhibition of the proteolytic activity of FVIIa abolished the response. The PAR2 agonist peptide SLIGKV also enhanced ET-1 release on TNF-pretreated cells. The response to FVIIa was inhibited by a PAR2 antagonist peptide FSLLRY. Inhibition of the p38 MAPK (mitogen-activated protein kinase) reduced PAR2 expression and the ET-1 response. In summary, FVIIa can stimulate ET-1 synthesis in endothelial cells by activating PAR2, demonstrating a potential link between thrombotic processes and endothelial cell dysfunction.

Blood coagulation is initiated by Ca(2+)-dependent binding of coagulation factor VIIa (FVIIa) to its cofactor, tissue factor (TF). The TF:FVIIa complex activates factors IX and X, ultimately leading to the formation of thrombin and the coagulation of blood. FVII consists of an N-terminal gamma-carboxyglutamic-acid-containing (Gla) domain followed by two epidermal growth factor (EGF) like domains, the first of which can bind one Ca2+ ion (Kd approximately 150 microM) and a C-terminal serine protease domain. Using 1H nuclear magnetic resonance spectroscopy, we have determined the solution structure of a synthetic N-terminal EGF-like domain (EGF1) of human FVII (residues 45-85) in the absence of Ca2+. A comparison of this structure of apo EGF1 with the Ca(2+)-bound EGF1 in the complex of FVIIa and TF [Banner, D. W., et al. (1996) Nature 380, 41-46] suggests that the structural changes in the EGF1 domain upon Ca2+ binding are minor and are concentrated near the Ca(2+)-binding site, which is facing away from the TF interaction surface. Amino acid side chains that are crucial for the binding of FVII to TF show a similar conformation in both structures and are therefore unlikely to directly influence the Ca(2+)-dependent binding of FVII to TF. As Ca2+ binding to EGF1 does not lead to a conformational change in the residues constituting the interaction surface for binding to TF, our results are consistent with the idea that the altered orientation between the Gla and EGF1 domains that result from Ca2+ binding is responsible for the increased affinity of FVII/FVIIa for TF.

FVII is a vitamin K dependent serine protease that plays a key role in extrinsic coagulation pathway. In this paper, we report the full-length cDNA sequences of rhesus monkey FVII. The full-length cDNA has 2424 bp, and predicts an open reading frame of 1416 bp corresponding to 472 amino acids. The deduced protein sequence of rhesus monkey FVII indicates the functional domains including signal peptide, Gla domain, two EGF domains, and catalytic domain. Rhesus monkey FVII is highly homologous to human FVII with amino acid identity of 91.0%. Comparison of three-dimensional protein structure shows high conservation between them. The important functional sites such as the N-terminal gamma-carboxyglutamic acids of the Gla domain, the Ca(2+) binding region of the EGF I domain, the TF binding region, the active site binding cleft, and the macromolecular substrate binding exosite of trypsin domain are all well conserved in FVII of rhesus monkey. Prothrombin time test shows rhesus monkey FVII has a similar clotting time with that of human. This study of rhesus monkey FVII might be helpful for understanding the function compatibility of human and rhesus monkey FVII, which is beneficial for the study of xenotransplantation.

Smaller and widely available animals such as rats are commonly used to evaluate antithrombotic drug candidates in vivo. However, the isolation and purification of FVII from rats and other species is very challenging because they are present in extremely low levels in plasma (approximately 10 nM). Furthermore, purification of FVII from other coagulation factors present in the plasma such as prothrombin, factor IX and factor X can often be very challenging and labor-intensive. To facilitate studies on the role of the extrinsic pathway of coagulation in rats, a full-length cDNA-encoding rat factor VII was isolated using polymerase-mediated DNA amplification using a rat liver cDNA library. The cDNA codes for a 41-residue signal/propeptide region, followed by a 405-residue mature protein consisting of the light chain with gamma-carboxy glutamic acid (gla) including epidermal growth factor domains (EGF) and the heavy chain with the serine protease catalytic domain. Rat factor VII cDNA was transfected into human embryonic kidney 293 cells and several cell lines that constitutively express rat factor VII were established. The media from the stable lines expressing recombinant rat FVII were rapidly screened for functional activity and were found to normalize clotting time of FVII-depleted human plasma. The supernatants were also functionally active in the presence of tissue factor in chromogenic assays by measuring FVIIa activation using a tripeptide chromogenic substrate and in a two-stage, coupled assay measuring the generation of FXa. Recombinant rat FVII may be an important new tool in the development of novel antithrombotic drugs.

BACKGROUND

Initiation of the coagulation serine protease cascade in mammalian cells is mediated by tissue factor (TF), which is a cell surface receptor and cofactor for coagulation factor VII (FVII) and its activated form FVII (FVIIa). Increasing evidence suggests that TF is expressed in a wide range of cancer cells and plays important roles in cancer progression and metastasis. In this study, we investigated the association between the expression level of TF transcript and histologic features of glioma.

METHODS

RNA was extracted from normal brain tissues and glioma tissues. We developed and validated a real-time quantitative reverse transcription (RT)-PCR assay, based on fluorescent TaqMan methodology, to quantify TF gene expression and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) at the mRNA level in human glioma.

RESULTS

The dynamic range of the assay was 10(3)-10(8) copy/microg RNA. The relationship between Ct and log starting concentration was linear (r2>> or = 0.99). The mean expression of TF in healthy brain tissue was 6.2 x 10(3) copy/microg RNA. Overexpression of TF was found in 42 brain glioma samples, mean value is 2.9 x 10(6) copy/microg RNA.

CONCLUSIONS

TF mRNA transcript is expressed in glioma and the level of expression correlates with histologic grade of malignancy. This new simple, rapid, semiautomated assay is a major alternative to Northern blot and competitive quantitative PCR for gene alteration analysis in human tumors and may be a powerful tool for large randomized, prospective cooperative group trials and support future TF-based clinical applications.

Activated factor (F) VII is a vitamin K-dependent glycoprotein that initiates blood coagulation upon interaction with tissue factor. FVII deficiency is the most common of the rare congenital bleeding disorders. While the mutational pattern has been extensively characterized, the pathogenic molecular mechanisms of mutations, particularly at the intracellular level, have been poorly defined. Here, we aimed at elucidating the mechanisms underlying altered FVII biosynthesis in the presence of three mutation types in the catalytic domain: a missense change, a microdeletion and a frameshift/elongation, associated with severe or moderate to severe phenotypes. Using CHO-K1 cells transiently transfected with expression vectors containing the wild-type FVII cDNA (FVIIwt) or harboring the p.I289del, p.G420V or p.A354V-p.P464Hfs mutations, we found that the secretion of the FVII mutants was severely decreased compared to FVIIwt. The synthesis rate of the mutants was slower than the FVIIwt and delayed, and no degradation of the FVII mutants by proteasomes, lysosomes or cysteine proteases was observed. Confocal immunofluorescence microscopy studies showed that FVII variants were localized into the endoplasmic reticulum (ER) but were not detectable within the Golgi apparatus. These findings suggested that a common pathogenic mechanism, possibly a defective folding of the mutant proteins, was triggered by the FVII mutations. The misfolded state led to impaired trafficking of these proteins causing ER retention, which would explain the low to very low FVII plasma levels observed in patients carrying these mutations.

The potential and benefits of isotope-coded labeling in the context of MS-based glycan profiling are evaluated focusing on the analysis of O-glycans. For this purpose, a derivatization strategy using d0/d5-1-phenyl-3-methyl-5-pyrazolone (PMP) is employed, allowing O-glycan release and derivatization to be achieved in one single step. The paper demonstrates that this release and derivatization reaction can be carried out also in-gel with only marginal loss in sensitivity compared to in-solution derivatization. Such an effective in-gel reaction allows one to extend this release/labeling method also to glycoprotein/glycoform samples pre-separated by gel-electrophoresis without the need of extracting the proteins/digested peptides from the gel. With highly O-glycosylated proteins (e.g. mucins) LODs in the range of 0.4 μg glycoprotein (100 fmol) loaded onto the electrophoresis gel can be attained, with minor glycosylated proteins (like IgAs, FVII, FIX) the LODs were in the range of 80-100 μg (250 pmol-1.5 nmol) glycoprotein loaded onto the gel. As second aspect, the potential of isotope coded labeling as internal standardization strategy for the reliable determination of quantitative glycan profiles via MALDI-MS is investigated. Towards this goal, a number of established and emerging MALDI matrices were tested for PMP-glycan quantitation, and their performance is compared with that of ESI-based measurements. The crystalline matrix 2,6-dihydroxyacetophenone (DHAP) and the ionic liquid matrix N,N-diisopropyl-ethyl-ammonium 2,4,6-trihydroxyacetophenone (DIEA-THAP) showed potential for MALDI-based quantitation of PMP-labeled O-glycans. We also provide a comprehensive overview on the performance of MS-based glycan quantitation approaches by comparing sensitivity, LOD, accuracy and repeatability data obtained with RP-HPLC-ESI-MS, stand-alone nano-ESI-MS with a spray-nozzle chip, and MALDI-MS. Finally, the suitability of the isotope-coded PMP labeling strategy for O-glycan profiling of biological important proteins is demonstrated by comparative analysis of IgA immunoglobulins and two coagulation factors.

Factor VII (FVII) deficiency is one of the two congenital coagulation disorders that was not discovered by the description of a new bleeding patient whose clotting pattern did not fit the blood coagulation knowledge of the time (the other is factor XIII deficiency). The existence of an additional factor capable of accelerating the conversion of prothrombin into thrombin was suspected before 1951, the year in which the first family with FVII deficiency was discovered. As several investigators were involved in the discovery of FVII deficiency from both sides of the Atlantic, several different names were tentatively suggested to define this entity, namely stable factor (in contrast with labile factor or FV), cothromboplastin, proconvertin, serum prothrombin conversion accelerator, prothrombin acceleration, and autoprothrombin I. The last term was proposed by those who denied the existence of this new entity, which was instead considered to be a derivate of prothrombin activation, namely autoprothrombin. The description of several families, from all over the world, of the same defect, however clearly demonstrated the singularity of the condition. Factor VII was then proposed to define this protein. In subsequent years, several variants were described with peculiar reactivity toward tissue thromboplastins of different origin. Molecular biology techniques demonstrated several gene mutations, usually missense mutations, often involving exon 8 of the FVII gene. Later studies dealt with the relation of FVII with tissue factor and activated FVII (FVIIa). The evaluation of circulating FVIIa was made possible by the use of a truncated form of tissue factor, which is only sensitive to FVIIa present in the circulation. The development of FVII concentrates, both plasma derived and recombinant, has facilitated therapeutic management of FVII-deficient patients. The use of FVIIa concentrates was noted to be associated with the occasional occurrence of thrombotic events, mainly venous. Total or partial liver transplants have been performed with success in these patients and have "cured" their deficiencies. Prenatal diagnosis has also been performed and recent research involves the development of inhibitors of FVII + tissue factor complex or of FVIIa. This approach, if successful, could provide another antithrombotic therapeutics tool. The story of FVII well summarizes the efforts of both theoretical and clinical approaches in the characterization of a coagulation disorder, that is, among the rare bleeding conditions, most frequently encountered in clinical practice.

In this paper are reported the basal results of a multidisciplinary, multicenter study designed to explore in a population with ischemic disease the relation between hemostatic variables, conventional risk factors and atherothrombotic sequelae. 953 patients less than or equal to 69 yrs with documented coronary, cerebral or peripheral atherosclerotic disease were studied and followed-up for 24 months. Examinations included hemostatic and lipid laboratory assays, arterial Doppler examination, cerebral computerized tomography and nuclear magnetic resonance, exercise electrocardiogram and coronary angiography. Fibrinogen (301.4 +/- 71.52 mg/dl) correlated positively with antithrombin III (r = 0.27) and leukocytes (r = 0.25), negatively with HDL-cholesterol (r = 0.18) and tended to increase with smoking. Heavy smokers had higher leukocyte counts than non-smokers (8.0 +/- 2.0 vs. 7.2 +/- 2.1 x 10(3)/microliters), higher triglycerides (1.87 +/- 1.12 vs. 1.53 +/- 1.35 mmol/l) and lower HDL-cholesterol (0.93 +/- 0.27 vs. 1.00 +/- 0.25 mmol/l). FVII correlated positively with triglycerides (r = 0.16) and protein C (r = 0.45). vWF:Ag (145.4 +/- 70.58%) ad FVII:C (139.7 +/- 59.10%) were positively correlated (r = 0.44). FVIII:C correlated positively with fibrinogen (r = 0.21). Myocardial infarction survivors with associated cerebral and peripheral vascular lesions had higher FVIII:C, FVII, fibronogen and vWF:Ag. These findings suggest that hemostatic factors may enhance and/or mediate the effects of conventional risk factors in atherothrombotic ischemic events.

OBJECTIVE

To study the effect of arsenic trioxide (As2O3) or all-trans retinoic acid (ATRA) on coagulopathy in patients with acute promyelocytic leukemia (APL), and the mechanism of hemorrhage in these patients.

METHODS

Thrombomodulin (TM) or tissue factor (TF) transcription of mRNA of freshly isolated bone marrow blast from APL patients was detected by semi-quantitative RT-PCR. The parameters of coagulation and cell procoagulation activity (PCA) were assessed in plasmic levels. Bleeding symptom was observed during As2O3 or ATRA treatment.

RESULTS

TM expression in the APL cell surface was significantly upregulated from (14.31 +/- 1.60) ng/10(7) to (21.61 +/- 6.82) ng/10(7) cells. The levels of P-selectin, soluble fibrin monomer complex (SFMC) and D-dimer (D-D) decreased after ATRA or As2O3 treatment. Abnormal high expression of TF in APL cell was downregulated in patients treated with ATRA or As2O3. The expression level was (14.81 +/- 6.23) ng/L before treatment, but undetected after 20 days of treatment. In addition, the membrane PCA of fresh APL cells was predominantly FVII-dependent after ATRA or As2O3 treatment. Bleeding symptom was ameliorated during As2O3 or ATRA treatment.

CONCLUSIONS

Bleeding symptom was controlled in patients with APL after As2O3 or ATRA treatment.

OBJECTIVE

Arginine 315 in factor VII (FVII) belongs to a solvent-exposed loop involved in direct interaction with the co-factor (tissue factor, TF), in transmission of TF-induced effects and potentially in FVIIa inactivation. Natural FVII variants at position 315 provide peculiar models for structure-function studies.

METHODS

We characterized a mild coagulation FVII deficiency associated with reduced FVII activity (26%) and antigen (67%). Mutations were searched by FVII gene sequencing. FVII variants were created by mutagenesis of FVII cDNA and characterized through expression in HEK293 cells followed by functional studies. FVII antigen in media was estimated by immunoassay while FVII activity was assessed by prothrombin-time based and FXa generation assays. FVII variants were injected into mice to investigate their recovery and half-life. One-way ANOVA was used to test statistical significance.

RESULTS

The patient was double heterozygous for a novel R315W mutation and for the R304Q substitution (FVII Padua) previously demonstrated to impair TF binding. The recombinant 315W-FVII was normally expressed in medium but showed a markedly reduced coagulant function (52%) and activity towards factor X (FX) in plasma (34%). Moreover, the 315W-FVII showed significantly decreased recovery of the protein (20%) and a slightly shorter half-life (8.6 min) as compared to wt-FVII (50% and 10.7 min). We also studied the conservative R315K change that was responsible for low recovery (20%) and a decreased half-life (7 min) of a FVII variant with virtually normal FVII antigen and activity levels.

CONCLUSIONS

These findings suggest a dual role of R315 for FVII function and clearance, and indicate that substitutions at this position have appreciable effects on human FVII biology, compatible with residual FVII function and thus with mild FVII deficiency.

To find if there is a relation between levels of haemostatic variables at low and high hormonal levels (oestradiol and progesterone) in an individual, blood samples were drawn from 12 women repeatedly during one menstrual cycle (Study I) and from 14 women undergoing in vitro fertilization, before hormonal stimulation and daily during the periovulatory period (Study II). Regression coefficients were calculated between minimum (independent) and maximum (dependent) values in both studies. In Study II highly significant regression coefficients were found between oestradiol minimum (pretreatment) and maximum (median 105 and 4730 pmol/l, respectively) for coagulation factors FVIII, von Willebrand Factor (antigen), FVII (activity and antigen), fibrinogen, protein C, protein S (free), antithrombin, plasminogen and plasminogen activator inhibitor-1; furthermore, between progesterone-minimum at day -3 or -2 (related to ovum pick up) and maximum (median 4.7 and 98 nmol/l, respectively) for FVIII, von Willebrand Factor, FVII (activity and antigen), protein C, protein S (free), and plasminogen. In Study I, where much lower hormonal levels were obtained at maximum (oestradiol median 297 pmol/l and progesterone 47 nmol/l), the same pattern was observed especially for FVII, FX, fibrinogen, plasminogen and plasmin inhibitor. Thus, the concentration of a haemostatic variable at a low oestradiol or progesterone level can predict the level at a high hormonal level.

A missense mutation at codon 100 in the second epidermal growth factor-like domain, resulting in Gln100->>Arg, was detected in 19 out of 21 available severely factor VII (FVII) deficient patients in Norway. Seventeen patients were homozygous, and the two remaining were compound heterozygotes. In the homozygous patients, FVII antigen was measured to 10-28%, and activity to 0.6-6.5% of that in normal pooled plasma. Recombinant FVII containing the mutation was expressed transiently in CHO cells to a mean antigen level of 57% of the wild type FVII protein, and with a specific activity of 6% of wild type. The mutant protein had a 14-fold reduction in affinity for tissue factor (TF), whereas binding of FX seemed unaffected. In line with the experimental data, molecular modelling of the mutation based on the coordinates of the tissue factor/FVIIa complex showed that substituting arginine for glutamine disrupts the interface between the catalytic and second epidermal growth factor-like domains.

Diabetes mellitus (DM) is a complex progressive disease characterized by hyperglycemia and a high risk of atherothrombotic disorders affecting the coronary, cerebral, and peripheral arterial trees. Oxidative stress is reported in diabetic patients. We investigated the hemostatic functions and oxidative stress in streptozotocin (STZ)-induced diabetic rats and the effects of warfarin and L-carnitine on those parameters. Forty male Sprague-Dawley rats were divided into four groups: control, DM, and DM received warfarin or L-carnitine. In all rats, blood glucose, insulin, hemoglobin A1c (HbA1c), fibrinogen, factor VII (FVII), plasminogen activator inhibitor-1 (PAI-1), fibrin degradation products (FDP), protein C, antithrombin III (ATIII), malondialdehydes (MDA), and antioxidants (superoxide dismutase, catalase, glutathione peroxidase, glutathione) were measured. Also, prothrombin time (PT), activated partial thromboplastin time (aPTT), coagulation time, and platelet aggregation were evaluated. In diabetic rats, plasma glucose, HbA1c, MDA, fibrinogen, FVII, FDP, PAI-1, and platelet aggregation increased while insulin, PT, aPTT, coagulation time, protein C, ATIII, and antioxidants decreased. Warfarin administration to diabetic rats decreased FVII and FDP and increased PT, aPTT, and coagulation time with no effect on MDA, antioxidants, PAI-1, protein C, ATIII, and platelet aggregation. On the other hand, L-carnitine decreased fibrinogen, FVII, FDP, PAI-1, MDA, and platelet aggregation and increased PT, aPTT, coagulation time, protein C, ATIII, and antioxidants in diabetic rats. Therefore, we concluded that hyperglycemia plays an important role in hypercoagulation state and oxidative stress in STZ-induced DM. While L-carnitine improves oxidative stress and decreases the hypercoagulation state in DM, warfarin normalizes the hypercoagulation state with no effect on oxidative stress.

Background: Tissue factor (TF) combined with its ligand FVII initiates blood coagulation and intracellular signaling. Obese and type 2 diabetic subjects have increased TF expression in their adipose tissue and an increased risk for thrombotic complications. Here we address the role of TF/FVII on adipocyte functions. Materials and methods: Subcutaneous fat was obtained by means of needle aspiration from healthy volunteers, and adipocytes were isolated after collagenase digestion. 3T3-L1 fibroblasts kept in culture were differentiated into adipocytes by addition of IBMX, dexamethasone, rosiglitazone, and insulin to the media. Proteins and mRNA were analyzed by western blot and RT-PCR. Coagulation activity was determined by a colorimetric FX-assay. Lipolysis was measured as free glycerol using a colorimetric method. Glucose uptake was evaluated by scintillation counting of D-[U-¹⁴C] glucose. Results: In isolated human primary adipocytes we found expression of TF and FVII. TF expression was confirmed in 3T3-L1 adipocytes, and both cell types were found to be procoagulant in a TF/FVIIa-dependent manner. FXa was generated without FVIIa added to the coagulation assay, and active site-inhibited FVIIa blocked FXa formation, supporting our finding of FVII production by human primary adipocytes. There was no evidence for a role of TF in either lipolysis or glucose uptake in our experimental settings. Conclusion: Human primary adipocytes express active TF and FVII, and the TF/FVIIa complex formed on the adipocyte surface can activate substrate FX. Whether the TF/FVIIa complex conveys signaling pathways leading to biological functions and has any biological activity in adipocytes beyond coagulation remains to be elucidated.

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_11294insAA. To determine whether this variant regulates FVII expression, we initially compared an expression vector containing FVII cDNA with g.11293_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.

OBJECTIVE

Photochemical treatment (PCT) based on amotosalen and ultraviolet A light (UVA) demonstrated a wide range of pathogen inactivation. However, coagulation proteins are affected by this treatment. The aim of this study was to evaluate the coagulation parameters in apheresis plasma units after thawing and processing by PCT.

METHODS

Thirty apheresis plasma units were rapidly frozen at </= -30 degrees C after collection. Plasma units were thawed after 7 days for PCT with amotosalen and UVA light. Treated apheresis units were refrozen and stored at </= -30 degrees C for 1 month. Samples were collected for each plasma units at several times of process. Coagulation times (prothrombin time, activated partial thromboplastin time), coagulation factors (fibrinogen, Factor [F] II, FV, <em>FVII</em>, <em>FVII</em>I, FIX, FX, FXI), prothrombin fragments 1 and 2, antithrombotic proteins (protein C, protein S, antithrombin) and total protein content were measured. Functionality of ADAMTS-13 was also tested.

RESULTS

After thawing, coagulation times were slightly increased and a decrease of FV, FVIII and protein C activity was found. The mean recovery for all proteins, except one, ranged from 81% to 97% of the baseline activity in plasma units after thawing and PCT. FVIII was more affected with a mean recovery of 69 +/- 8%. ADAMTS-13 function was also preserved after the whole process. The effect of an additional 1-month frozen storage on coagulation parameters was minimum.

CONCLUSIONS

Coagulation protein levels after thawing and processing of plasma by PCT with amotosalen and UVA were preserved well in the physiological ranges.

BACKGROUND

We assessed the haemostatic capacity of thawed plasma produced after ambient storage of whole blood for 24 h (RTFP24), and the supernatant of buffy-coat derived platelet concentrates (PC).

METHODS

Platelet concentrates (n = 20) were tested on days 1, 5 and 7 of storage at 22°C and RTFP24 (n = 10) immediately following thawing and after 4 and 6 days storage at 4°C. Coagulation factor activity, thrombin generation ± an activator of protein C (PROTAC) and rotational thromboelastometry (ROTEM) were assessed.

RESULTS

In plasma and buffy-coat derived PC, there was a < 10% loss of factors II, IX and FX, but much higher loss of factors FV, FVII and FVIII. In plasma, the total or peak amount of thrombin generated was unaffected by storage for 6 days, with or without Protac, but there was an increase in lag time and decreased rate of clot formation by ROTEM. In PC, but not plasma, there was a 16% increase in FXII activity and increase in resistance to activated protein C, co-incidental to 30% loss of free protein S.

CONCLUSIONS

These data suggest thrombin generation is relatively unaltered when RTFP24 is thawed and stored for 6 days, and that the supernatant of PC has significant haemostatic capacity.