We developed a sensitive tissue factor (TF) chromogenic assay on a limited number of endothelial cells (EC), performed in microtiter plates, and which uses a normal pooled human plasma instead of purified concentrates as a source of coagulation factors. Primary cultures of human umbilical vein EC (HUVEC), both unstimulated and stimulated by lipopolysaccharide (LPS) were incubated with 50 microliters of of diluted normal human plasma (NHP) and 50 microliters of Factor Xa-specific chromogenic substrate (CBS 31-39, Stago, France). Hirudin was added at 4 U/ml to the plasma/CBS 31-39 mixture to inhibit thrombin generation. Optical densities were read at 405 nm and corresponding amounts of generated factor Xa were expressed in mU Xa/well using a standard curve established with purified human Factor Xa. The following parameters were then defined: the number of EC to plate (10(4) EC/well of a 96-well plate), the plasma-test dilution (1:20), the concentration of CBS 31-39 (0.50 mM) and the incubation time of reagents with EC (2 hours). The procoagulant activity (PCA) measured was only dependent on TF since it was no longer detectable either when FVII-deficient plasma was tested instead of normal human plasma or when PCA assays were performed in the presence of a blocking anti-human TF monoclonal antibody. This method allowed detection of a TF-dependent PCA on as few as 1000 EC per well. In addition, TF expression equal to 50% of maximal values was measured with LPS concentrations as low as 1 ng/ml, supporting the high sensitivity of the assay.

BACKGROUND

Hemophilia A treatment relies on costly factor VIII (FVIII) replacement that may transmit iatrogenic viral diseases. Viral vectors and cell implants are being developed as improvements. We investigated in vivo electroporation of naked DNA as a safe and simple method for correcting FVIII deficiency.

METHODS

B-domain-deleted murine FVIII cDNA expression plasmids were constructed with CMV and elongation factor 1alpha promoters for characterisation in murine C2C12 myoblasts. The construct conferring highest in vitro FVIII secretion was electroporated into skeletal muscle of FVII null mice in vivo for phenotypic correction using a protocol that minimised tissue injury.

RESULTS

B-domain-deleted murine FVIII cDNA plasmids induced FVIII secretion from stably transfected C2C12 myoblasts (0.54+/-0.20 mU/day/10(5) cells). Phenotypic correction of hemophilic mice was more consistently achieved using a protocol for in vivo electroporation of gastrocnemius muscle with FVIII cDNA that reduced tissue injury by the use of plate electrodes, hyaluronidase pre-treatment and lower field strength. This technique was associated with <10% muscle necrosis. Activated partial thromboplastin time decreased from 51.4+/-3.3 to 34.7+/-1.1 (mean+/-s.e.m.) seconds (p=0.0004) following in vivo electroporation (0.1 mg plasmid/limb; 8x20 ms pulses, 175 V/cm, 1 Hz) of hemophilic mice. All hemophilic mice (8/8) survived hemostatic challenge after muscle electroporation with FVIII cDNA, whereas all (9/9) untreated hemophilic mice died. Plasmid DNA was detectable only in electroporated muscle and not in all other organs tested, including gonads.

CONCLUSIONS

In vivo intramuscular electroporation of naked FVIII plasmid successfully corrects murine hemophilia.

OBJECTIVE

The goal of this study was to determine the levels of factor VII (FVII), factor VIIa-antithrombin complexes (FVIIa-AT), total tissue factor (TF), and tissue factor-bearing microparticles (MPs-TF) in patients with acute ischemic stroke. Further, we sought evidence of an association between hemostatic markers, time of blood sampling, type of treatment, and patient outcomes.

METHODS

Venous blood samples were collected from 33 patients on the first day and on the seventh day after stroke diagnosis. Age-matched controls were also included (n = 20). Plasma levels of FVII, FVIIa-AT, total TF, and MPs-TF were measured by enzyme-linked immunosorbent assay. We divided patients into 2 groups: thrombolysis group (n = 13) and nonthrombolysis group (n = 20). Furthermore, evaluation of the National Institutes of Health Stroke Scale and the Barthel Index was performed on the first day and the seventh day.

RESULTS

Patients with ischemic stroke showed significantly lower plasma FVII, FVIIa-AT, and total TF levels than controls (median, 112.25% vs 132.05%, P = .004; 107.97 pmol/L vs 154.94 pmol/L, P < .001; 81.74 pg/mL vs 105.71 pg/mL, P < .001, respectively). In contrast, levels of plasma MPs-TF were significantly higher in patients with stroke compared to healthy controls (1.60 pg/mL vs 0.74 pg/mL, P < .001). Additionally, the thrombolysis group had lower FVII levels on the seventh day compared to the first day (median, 109.80% vs 115.74%, P = .04).

CONCLUSIONS

Factor VII, FVIIa-AT, and total TF are decreased, while MPs-TF are elevated in patients with ischemic stroke. We observed a slight but significant effect of alteplase on FVII plasma levels.

The clinical relevance of thrombophilia screening in stroke patients is still a matter of debate, and descriptions of larger patterns of genetic variability are rare. We assessed the frequency of hereditary hypercoagulability in young patients with cryptogenic stroke (n = 44) and in healthy blood donors (n = 282) without prior cardiovascular event. Furthermore, we focused on the impact of thrombophilia screening on secondary stroke prevention.

RESULTS

Compared to the control group (19-67 years; median 38.5 years; 64% women), there was a lower prevalence of the FVII-R353Q mutation (p = 0.033) in stroke patients (17-52 years; median 36 years; 59.1% women). Of note, the FVII-R353Q mutation lowers FVII plasma levels, probably reducing the risk of cardiovascular events. The prevalence of the remaining 13 gene polymorphisms did not differ significantly. However, the prevalence of FV Leiden mutation tended to be higher among stroke patients.

CONCLUSIONS

Overall, extended screening for inherited thrombophilia had an impact on medical stroke prevention in every sixth patient with cryptogenic stroke.

We explored the interrelationships of coagulation FVII activity levels with obesity, leptin and insulin resistance in diabetes mellitus (DM Type 2) and in non-diabetic control subjects. We found FVII hypercoagulant activity levels in DM not associated with obesity, leptin levels or insulin resistance. It was found independently associated with hypertriglyceridemia.

OBJECTIVE

The aim of this prospective controlled study was to compare the effects of two therapies for menopause on factor VII (FVII) and hemostatic variables.

METHODS

Postmenopausal women were assigned to receive one of the following treatments: transdermal estradiol (TTS E2; 50 microg) combined in a continuous sequential regimen with oral medroxyprogesterone acetate (MPA; 10 mg/day for 12 days) (group A; n = 20), tibolone (2.5 mg/day) (group B; n = 21) or placebo (group C; n = 19). Sixty women completed the 1-year treatment and underwent follow-up examinations after 3, 6 and 12 months.

RESULTS

TTS E2/MPA induced various changes in procoagulatory factors. At 12 months, fibrinogen, activated FVII (FVIIa) and coagulative FVII (FVIIc) had increased by 10.7, 12.9 and 3.7%, respectively. Among the fibrinolytic factors, plasminogen and alpha2-antiplasmin increased by 11.3 and 7.2%, respectively. Lipoprotein(a) [Lp(a)] and antithrombin III (ATIII) did not show any significant variation. Tibolone induced some changes toward a more homogeneous antithrombotic profile. Fibrinogen, FVIIa and FVIIc decreased significantly by 7.5, 8.1 and 21.3%, respectively. Plasminogen increased (by 11.8%) and Lp(a) decreased (by 28.4%). ATIII was unchanged with tibolone therapy.

CONCLUSIONS

Our results show that tibolone induces a significant reduction in FVIIc and Lp(a) and a greater enhancement of factors promoting fibrinolysis than the TTS E2/MPA regimen.

BACKGROUND

Scientific and technical advances made in transfusion medicine sustain the need for more comprehensive understanding of the impact of collection procedures on the quality of plasma for fractionation and for transfusion. This prospective work evaluated protein composition and markers of activation in plasma donations collected with three different automatic collection procedures (performed on Haemonetics machines), including a new procedure using a high-separation core-molded bowl.

METHODS

A total of 90 collection procedures have been performed from a population of 37 donors, under comprehensively standardized conditions. Plasma aliquots were taken from the plasma units within 30 minutes of the end of the collection procedures and immediately frozen at -70 degrees C. Content in an extended range of proteins and of markers of activation of the coagulation and fibrinolytic systems has been measured using standard in vitro testing methods.

RESULTS

Plasma donations had normal mean total protein, IgG, IgM, and fibrinogen content. The mean levels in coagulation FV, FVII, FVIII, and FXI and in antithrombin were above the standard international requirements. There was no sign of activation of the hemostasis system, as assessed by activated FVII, thrombin antithrombin complex, Prothrombin fragment 1+2, and D-dimers. Activated complement component C3 and C5 were low.

CONCLUSIONS

Data indicates the good and consistent protein composition of plasma obtained by those automatic apheresis procedures. In particular, the new high-separation core procedure yields a high-quality plasma meeting requirements for transfusion and fractionation.

Genetic factors are involved in the individual predisposition to develop ischemic stroke (IS). In the present study we tested the role of the Factor VII G10976A and -C122T polymorphisms on the susceptibility to develop IS in a genetically homogenous and clinically well ascertained case-control study including 294 cases (median age 75 years; 176 males/118 females) and 286 controls (median age 73 years; 163 males/123 females) in Sardinia, Italy. In addition, we carried out an exploratory analysis with respect to other frequently studied polymorphisms of haemostatic factor genes:Factor II G20210A, Factor V G1691A,,Fibrinogen alpha-chain Thr312Ala, Fibrinogen beta-chain -C148T, Factor XIII G185T, GPIIb/IIIa T1565C. Among all the genes tested, FVII -C122T showed a significant, independent contribution to IS predisposition both in crude and adjusted analyses (crude OR 1.52, 95% CI 1.09-2.10, P=0.013; adjusted OR 1.48, 95% CI 1.04-2.09, P=0.028, respectively). Haplotype analyses revealed a conserved population structure with high linkage disequilibrium between both FVII mutations tested. Blood levels of FVII had an inverse relationship with the polymorphism involved. Apart from genetic influence, there was a significant role for hypertension (OR=1.7, 95% CI 1.19-2.43, P=0.003), hypercholesterolemia (OR=2.21, 95% CI 1.38-3.54, P=0.001) and atrial fibrillation (OR=1.66, 95% CI 1.06-2.58, P=0.026) on IS occurrence. In summary, we describe evidence for a possible direct association of FVII gene molecular variants with the occurrence of IS in a genetically homogenous human sample.

Direct injection of plasmid DNA into skeletal muscle has been proposed as a method of effecting somatic gene therapy. This article describes the construction and testing of a plasmid derived expression cassette believed to confer skeletal muscle specific expression. Expression constructs were designed containing the full-length cDNAs for both coagulation factor VIII and factor VII. The engineered genes were flanked by two muscle specific regulatory elements from different myosin isoforms and by an artificial polyadenylation signal sequence. In vitro transfection of C2-myoblasts led to expression of the factor VIII gene, shown by reverse transcription and polymerase chain reaction, upon differentiation of the myoblasts. The expression of the FVII construct was tested in a C2 cell culture system and also when injected directly into mouse muscle. It was found that in cell culture the level of factor VII antigen outside the cell, ie in the cell culture medium was two- to three-fold higher than inside the cell, ie in the cell lysate. This level of expression was found to continue for the duration of cell culture maintenance and a fully functional protein was produced. In vivo transfection experiments in mice showed a substantial increase in factor VII antigen compared with the background level 4-5 days after injection. An anti-human factor VII antibody was detected 7-10 days after injection. We conclude that muscle cells in vitro secrete and efficiently carry out post-translational modifications of the engineered gene product and in vivo secrete the gene product resulting in elevation of systemic levels. The data provide the basis for the use of muscle cells as an in vivo expression system for coagulation proteins in the treatment of inherited haemostatic and thrombotic disorders.

Recombinant human FVIIa (rhFVIIa) corrects the coagulopathy in hemophilia A and B as well as FVII deficiency. This is also the case in dogs until canine anti-human FVIIa antibodies develop (~2 weeks). Recombinant canine factor VIIa (rcFVIIa), successfully over-expressed by gene transfer in haemophilia dogs, has provided long-term haemostasis (>2 years). However, pharmacokinetics (PK), pharmacodynamics (PD) and safety of rcFVIIa after pharmacological administration have not been reported. We therefore wanted to explore the safety, PK and PD of rcFVIIa in dogs. A pilot study was set up to evaluate the safety as well as PK and PD of rcFVIIa after a single intravenous dose of 270 μg kg(-1) to one HA and one haemostatically normal dog and to directly compare rcFVIIa with rhFVIIa in these two dogs. Single doses of rcFVIIa and rhFVIIa were well tolerated. No adverse events were observed. Pharmacokinetic characteristics including half-life (FVIIa activity: 1.2-1.8 h; FVIIa antigen 2.8-3.7 h) and clearance were comparable for rcFVIIa and rhFVIIa. Kaolin-activated thromboelastography approached normal in the HA dog with the improvement being most pronounced after rcFVIIa. This study provided the first evidence that administering rcFVIIa intravenously is feasible, safe, well tolerated and efficacious in correcting the haemophilic coagulopathy in canine HA and that rcFVIIa exhibits pharmacokinetic characteristics comparable to rhFVIIa in haemophilic and haemostatically competent dogs. This strengthens the hypothesis that rcFVIIa can be administered to dogs to mimic the administration of rhFVIIa to humans.

FVII Padua is a Type 2 defect owing to an Arg304Gln substitution in exon 8. The defect was originally discovered in an isolated valley in northeastern Italy. Subsequently, it was described in several other countries of the Mediterranean basin and Middle East. Recently, several proven or suspected cases have been described among Afro-Americans in the USA. This study has demonstrated the existence of at least a two-founder effect for this FVII abnormality, Mediterranean countries, and USA Afro-Americans. Patients are usually asymptomatic or only paucisymptomatic. The defect is characterized by low FVII activity when rabbit brain thromboplastins are used in the assay system. On the contrary, FVII levels are normal when ox-brain thromboplastins are used. FVII antigen is always normal.

BACKGROUND

We have recently described an experimental animal model of non-overt disseminated intravascular coagulation (DIC) in the rabbit in which the induction of tissue factor (TF) mRNA and TF antigen expression in peripheral blood leukocytes (PBL) was demonstrated to occur within 2 h of administration of low-dose endotoxin [Hematol. J. 2 (2001) 188]. In the present study, we demonstrate that the leukocyte TF expressed has procoagulant activity leading to a rapid decline in the concentration of factor VII (FVII) in rabbit plasma.

METHODS

Total plasma FVII antigen and FVIIa were quantitated by rabbit FVII-specific immunoassay and FVIIa-specific clotting assays, respectively. Plasma samples from either saline-injected rabbits or rabbits administered a single bolus of 10 microg/kg Salmonella lipopolysaccharide were compared over a 24-h period.

RESULTS

Total plasma FVII antigen decreased progressively post-endotoxin injection, reaching 71% of the baseline concentration at 8 h (p<0.001, n=18), and remained low (78%) at 24 h post-injection (p<0.01, n=16), returning to normal by 48 h. Plasma FVIIa levels increased to 120% within 2 h of endotoxin injection, fell to 73% of the baseline concentration at 8 h (p<0.05, n=18) and returned to normal by 24 h post-endotoxin administration. Procoagulant activity of rabbit peripheral blood leukocytes was enhanced at 2 h (p<0.01, n=6) and 4 h (p<0.05, n=6) post-endotoxin injection. The prothrombin time (PT) was increased by <3 s, and thrombin-antithrombin (TAT) complex formation was not significantly increased in the plasma of endotoxin-treated rabbits. No significant changes in total plasma FVII antigen, FVIIa or leukocyte procoagulant activity were observed in rabbits treated with saline.

CONCLUSIONS

We conclude that the activation of FVII to FVIIa and rapid consumption of total FVII/FVIIa occur very early and likely are integral events linked to the initiation and propagation of non-overt DIC induced by endotoxin.

A chimeric cDNA, encoding residues 1-46 (the gamma-carboxyglutamic acid module and its trailing helical stack) of human coagulant factor (f) VII, bound to residues 47-419 of human anticoagulant protein C (PC), was constructed and expressed. The resulting protein, r-[delta GD-HSPC/[symbol: see text] GD-HSfVII]PC, was properly processed with regard to signal/propeptide release, cleavage of the K156R dipeptide, Gla and Hya contents, and the presence of glycosylation. The mutant protein displayed normal dependencies on Ca2+ for adoption of its metal ion-dependent conformation and for binding to acidic phospholipid vesicles. The chimera failed to recognize a monoclonal antibody (MAb) specific for the Ca(2+)-induced conformation of the Gla domain (GD) of PC, but did react with another MAb directed in part to the Ca(2+)-dependent conformation of the GD of fVII. Further, this chimeric protein possessed similar steady state constants as wild-type r-PC toward activation by thrombin and thrombin/thrombomodulin. The activated form of the chimera was very similar to that of its wild-type counterpart in its whole plasma anticoagulant activity, as well as its activity toward inactivation of coagulation factor VIII. The chimeric protein did not bind to the fVII cofactor, tissue factor, showing that the GD/HS domain region of fVII is insufficient for that particular interaction. The results demonstrate that the GD/HS of fVII, when present in the PC and APC background, serves to maintain the Ca2+/PL-related functions of these latter proteins, and suggest that the Ca2+ and PL-dependent interactions of the GD-HS of PC are sufficiently general in nature such that the GD-HS regions of other proteins of this type can satisfy most of the requirements of PC and APC. The data presented also offer support for the independent nature of the domain unit consisting of the GD/HS module.

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

BACKGROUND

Patients affected by hemophilia A often require frequent prophylactic and therapeutic self-infusion. For those who develop inhibitors, treatment options are limited and mortality is increased. Emicizumab, a bispecific antibody to Factors IXa and X that carries out the function of Factor VIII (FVIII), represents a novel therapeutic approach. Areas covered: We review the clinical trials and key laboratory assay research for emicizumab. Emicizumab reduced the annualized bleeding rate by 87% compared to placebo in patients with inhibitors. For patients without inhibitors, emicizumab reduced the annualized bleeding rate 96-97% compared to no prophylaxis and 68% compared to prior FVIII prophylaxis. Three patients developed a thrombotic microangiopathy (TMA) and two patients had thrombotic events while on emicizumab in combination with activated prothrombin complex concentration (aPCC) alone or concurrent with activated recombinant factor FVII (rFVIIa). Expert opinion: Emicizumab represents a much-needed alternative approach to managing Factor VIII deficiency, especially for those with inhibitors or limited ability to self-infuse. For patients with inhibitors, thrombotic complications including TMA, not seen with other bypassing agents, raises concern about the use of emicizumab in combination with aPCC and how patients who have breakthrough bleeding can be safely managed.

Factor XI (FXI) deficiency is a rare inherited coagulation disorder characterized by infrequent spontaneous bleeding, but increased risk of hemorrhagic complications especially after trauma or surgery. Treatment options for FXI-deficient patients include virus-inactivated fresh frozen plasma, plasma-derived FXI concentrates, and activated recombinant FVII. Inhibitors of fibrinolysis, such as tranexamic acid, and desmopressin (DDAVP) have also been used in these patients, especially in mild cases. The current knowledge on the use of the latter agent in this congenital bleeding condition is systematically reviewed here. Although limited, the available literature data suggest the potential role of DDAVP for either treatment of bleeding episodes or the prevention of postoperative bleeding in patients with milder FXI defects. However, these findings need to be supported by further trials on large population of patients.

BACKGROUND

Haemostatic factors are suspected to be involved in the aetiology of cerebrovascular events.

METHODS

In a case-control study of 105 cases of transient ischaemic attack and minor ischaemic stroke, and 241 controls, data were available on levels of the haemostatic factors-von Willebrand factor (vWF), plasminogen activator inhibitor-1 (PAI), tissue plasminogen activator (TPA) and factor VII (FVII). These are subject to measurement error and within-person fluctuation of true levels, which may bias relative risk estimates. For all subjects, two determinations were performed on the same blood sample, which allowed estimation of pure measurement error. For estimation of within-person fluctuation, levels were measured from a repeat blood sample on 81 of the controls one year later.

RESULTS

The pure measurement error accounted for a very small proportion of the total variation in all cases. Uncorrected for within-person fluctuation, the odds ratio estimates associated with exceeding the median of vWF, PAI, TPA and FVII respectively were 1.88, 0.87, 1.30 and 0.93. After correction for within-person fluctuation odds ratios were 3.56, 0.80, 1.41 and 0.91. Because the PAI determination was not robust to storage conditions, it was estimated that 75% of the variation in this factor was within-person rather than between-persons. Thus, estimates of relative risk relation to PAI cannot be regarded as reliable in this study.

CONCLUSIONS

It is likely that elevated levels of vWF are associated with increased risk of ischaemic stroke, but interpretation must be tentative, due to relatively large within-person fluctuation of vWF levels.

OBJECTIVE

To determine frequencies of genetic polymorphisms of coagulation factor VII (FVII), coagulation factor FXII (FXII), fibrinogen (FBG) and 9p21 in ethnic Han Chinese from Yunnan province, and to assess the association between such polymorphisms and onset of myocardial infarction (MI).

METHODS

One hundred and forty-two patients with MI and 192 healthy controls were analyzed. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and pyrosequencing were used to determine the genotypes of FVII, FXII, FBG and 9p21.

RESULTS

No significant difference was found in the frequencies of R353Q, 5'F7, C46T, -148C/T, rs1333049 and rs4977574 loci between the two groups (P> 0.05). However, the frequencies of AA of -455G/A, T and TT of rs1333040, T and TT of rs10116277 and G and GG of rs2383207 were significantly higher in MI group compared with the controls (P< 0.05), whilst the frequencies of CT of rs1333040 and GT of rs10116277 were significantly lower in MI group compared with the controls (P<0.05).

CONCLUSIONS

Polymorphisms of FVII, FXII, -148C/T of FBG and rs1333049 of 9p21 were not associated with myocardial infarction. Polymorphisms of -455G/A of FBG and rs1333040, rs10116277 and rs2383207 of 9p21 may be associated with MI in ethnic Han Chinese from Yunnan province.

OBJECTIVE

To investigate whether coagulation factor VII (FVII) polymorphisms play a role in the pathogenesis of coronary artery disease (CAD) and/or myocardial infarction (MI) in a series of Hans.

METHODS

The Arg(353)Gln and HVR4 polymorphisms of FVII gene were determined in 374 patients undergoing selective coronary angiography by PCR and restriction fragment length polymorphism assay.

RESULTS

The FVII genotype distribution was in accordance with Hardy-Weinberg equilibrium. The frequencies of FVII genotypes or alleles did not show significant differences between the CAD group and the controls or between the males and the females. The frequencies of carriers of the Gln(353) allele and (Arg/Gln + Gln/Gln) genotypes were significantly higher in the CAD patients without MI than in those with MI (P = 0.031, odds ratio 0.37, 95% CI: 0.15 - 0.94). However, HVR4 polymorphisms were not significantly different between the two groups (P>> 0.05).

CONCLUSIONS

Carrying the F VII Gln(353) gene may be a protective factor against MI in the Chinese Hans.

Congenital factor VII (FVII) deficiency is a rare autosomal recessive bleeding disorder. Its clinical manifestation and mutational spectrum are highly variable. The purpose of this study was to identify and characterize the mutation causing the FVII deficiency in a Chinese patient and his family. The FVII gene was analyzed by genomic DNA sequencing, and the FVII levels in patient's plasma were measured with an enzyme-linked immunoabsorbent assay (ELISA) and one-stage prothrombin time based method. In addition, the FVII-Phe190 mutant identified in the pedigree was expressed in the HEK293 cells, and the subcellular localization experiments in the Chinese hamster ovary (CHO) cells were performed. The patient had a prolonged prothrombin time and low levels of both FVII antigen and activity, and two heterozygous mutations were identified in F7 gene (NG-009262.1): a g.15975 G>A in the splice receptor site of intron 6 and a novel g.16750 C>T in exon 8 resulting in Ser190 to Phe190 replacement. In expression experiments, the reduced antigen and activity levels of FVII-Phe190 in the culture medium were found, whereas an ELISA and Western blotting analysis of FVII revealed that mutant FVII-Phe190 was synthesized in the cells as the wild-type FVII-Ser190. And FVII-Phe190 was found in endoplasmic reticulum and Golgi apparatus. Compound heterozygous mutations in F7 gene should be responsible for the FVII deficiency in this patient. The FVII-Phe190 can normally be synthesized and transported from endoplasmic reticulum to Golgi apparatus, but degraded or inefficiently secreted.

Factor VII deficiency, although rare, is now recognized as the most common autosomal recessive inherited factor deficiency. It is usually considered to be associated with bleeding only in the severely affected subject and heterozygotes (>10%) are not considered at risk. The general recommendation for surgery is to achieve a FVII level in excess of 15% (0.15 1U/mL). We present three cases of severe factor VII deficiency, each of whom appeared hemostatically competent based on clinical history. Subject 1 is a 33 year-old African-American female with a baseline FVII of <1%, who had a fractured tibia requiring open reduction with internal fixation without any FVII replacement and subsequently underwent successful laparoscopic knee surgery with a factor VII level measured at 6%. Subject 2 is a 58 year-old African-American female with a factor VII level of 9% who underwent an elective left total hip replacement without any factor replacement and had no excessive bleeding, but who sustained a pulmonary embolism postoperatively. Subject 3 is a 19-year-old African-American male with a baseline FVII of 1% with a history of active participation in football without noticeable injury and who underwent an emergent appendectomy without bleeding. These three cases represent individuals with the severe form of FVII deficiency who did not exhibit excessive bleeding when challenged with surgical procedures. The clinical history would appear the most valuable tool in predicting the likelihood of bleeding in these patients, and we suggest that the presumption that all patients with severe FVII deficiency should receive replacement therapy before surgical procedures may not be valid in all cases.

Pregnancy, childbirth, and the puerperium are hemostatically challenging to women with bleeding disorders. This article provides general recommendations for the management of pregnant women with inherited coagulation disorders. Each factor deficiency is discussed, providing an up-to-date review of the literature and, where possible, guidance about how to manage patients throughout pregnancy, delivery, and the puerperium. The factor deficiencies covered are inherited abnormalities of fibrinogen; deficiencies of prothrombin, factor (F)V, FVII, FX, FXI, FXIII; combined deficiencies of FV and FVIII; and the inherited deficiency of vitamin K-dependent clotting factors. The management of carriers of hemophilia A and B is also discussed.

Rare bleeding disorders (RBDs) include the inherited deficiencies of fibrinogen, factor (F)II, FV, FV+FVIII, FVII, FX, FXI and FXIII. There have been remarkable advances in understanding the molecular profiles that lead to each type of coagulation factor deficiency. However, as a consequence of their rarity, clinical data regarding the characteristics of bleeding symptoms and their management remain limited. The clinical manifestations in different RBDs are heterogeneous, and the residual plasma coagulant factor level does not always predict bleeding tendency. In this review, we describe the general features and recent advances in understanding three such deficiencies: FXI, FVII and fibrinogen deficiencies.