Low Factor VIII Levels Research Articles

All too common: bleeding and genetic variation

Inherited bleeding disorders are both common and challenging to diagnose, particularly when the disorder is mild (reviewed in references [1, 2]). Most mild bleeding disorder patients are not routinely bleeding in their daily lives, and it can be difficult to differentiate bleeding events common in healthy populations from events which rise to the level of pathology. Fundamental to the diagnosis of bleeding disorders is a detailed assessment of the history of the patient and the family. This article is protected by copyright. All rights reserved.

Optimization of the thrombin generation test components to measure potency of factor VIII concentrates.

The thrombin generation test (TGT) is used both as a global haemostasis assay, and to compare activities of coagulation factor concentrates that have been spiked into patient plasma. However, TGT has not been systematically optimized to evaluate factor VIII (FVIII) product potency. To improve the sensitivity of TGT to FVIII and allow a comparative analysis of the thrombin generating capacities of FVIII concentrates against reference preparations with known FVIII activity. Concentrations of TGT components (analytical variables) were assessed to maximize the linearity and range of responses to the concentration of FVIII. We optimized the range and sensitivity of the TGT assay with respect to FVIII through the addition of FXIa to the assay. Other parameters that were adjusted, i.e. tissue factor (TF), procoagulant lipids and plasma concentrations, did not improve the ability of the assay to measure both high and very low levels of FVIII. In the optimized TF/FXIa-activated TGT assay, all thrombin generation curve parameters were suitable for FVIII quantification, but thrombin peak height and maximal velocity demonstrated better linearity in the desired FVIII range. We found that the optimized TF/FXIa-activated TGT has a wider range of sensitivity to FVIII than a commercially available TGT. Additionally, we demonstrated that the TF/FXIa-activated assay performs adequately by comparing potency measurements of five commercially available FVIII products using TGT and traditional chromogenic and one-stage clotting assays. The optimized TGT assay can be used to quantify and compare the thrombin generating capacities of FVIII concentrates.

Assessing Circulating Factor VIIa-Antithrombin Complexes in Acute Ischemic Stroke: A Pilot Study.

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. 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. 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). 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 molecular basis of low activity levels of coagulation factor VII: a Brazilian cohort.

Inherited factor VII (FVII) deficiency is the most common among the rare bleeding disorders. It is transmitted as an autosomal recessive inheritance, due to mutations in the FVII gene (F7). Molecular studies of FVII deficiency are rare in non-Caucasian populations. The aim of the study was to evaluate the molecular basis behind low levels of FVII activity (FVII:C) levelsin a cohort of Brazilian patients. A total of 34 patients with low FVII levels were clinically evaluated and submitted to laboratory tests, among these, prothrombin time and FVII:C, with different thromboplastins. All exons and intron/exon boundaries of F7 were amplified and sequenced. A total of 14 genetic alterations were identified, of which six were described previously, c.1091G>A, c.1151C>T, c.-323_-313insCCTATATCCT, c.285G>A, c.525C>T, c.1238G>A and eight (54.0%) and eight were new, c.128G>A, c.252C>T, c.348G>A, c.417G>A, c.426G>A, c.745_747delGTG, c.843G>A and c.805+52C>T. In addition to the mutation c.1091G>A, known as FVII Padua, the mutation c.1151C>T also presented discrepant FVII:C levels when tested with human and rabbit brain thromboplastin. There was no association between phenotype and genotype. Most of the identified genetic alterations found were polymorphisms. Low levels of FVII:C in this population were mostly related to polymorphisms in F7 and associated with a mild clinical phenotype. Mutation c.1151C>T was associated with discrepant levels of FVII:C using different thromboplastins, such as reported with FVII Padua.

Factor VIII Antigen, Activity, and Mutations in Hemophilia A.

C and Ag levels along with their blood groups to determine their cross-reacting material (CRM) status. In all, 37 cases (18 moderate and 19 mild) were found to have discordant CRM status. Missense mutations (Ala723ThrandLys439Ser) and protein truncating changes (Trp1854*andArg2326*) were observed in 2 each of these cases. Although 37 (70%) of the 53 had discordant antigen-activity ratio, majority of those mutations produced FVIII with low FVIII-specific activity. However, 4 (7.5%) of the 53 mutations produced higher specific activity of FVIII. It is possible that these mutations either produce a secretory defect or an increased metabolic turnover to account for the low levels of FVIII with these mutations.

Activated recombinant factor VIIa should not be used in patients with refractory variceal bleeding: it is mostly ineffective, is expensive, and may rarely cause serious adverse events.

Activated recombinant factor VIIa should not be used in patients with refractory variceal bleeding: it is mostly ineffective, is expensive, and may rarely cause serious adverse events.

Genetic variants of the vitamin K dependent coagulation system and intraventricular hemorrhage in preterm infants.

BackgroundPathogenesis of intraventricular hemorrhage (IVH) in premature infants is multifactorial. Little is known about the impact of genetic variants in the vitamin K-dependent coagulation system on the development of IVH.MethodsPolymorphisms in the genes encoding vitamin K epoxide reductase complex 1 (VKORC1 -1639G>A) and coagulation factor 7 (F7 -323Ins10) were examined prospectively in 90 preterm infants <32 weeks gestational age with respect to coagulation profile and IVH risk.ResultsF7-323Ins10 was associated with lower factor VII levels, but not with individual IVH risk. In VKORC1-wildtype infants, logistic regression analysis revealed a higher IVH risk compared to carriers of the -1639A allele. Levels of the vitamin K-dependent coagulation parameters assessed in the first hour after birth did not differ between VKORC1-wildtype infants and those carrying -1639A alleles.ConclusionsOur data support the assumption that genetic variants in the vitamin K-dependent coagulation system influence the coagulation profile and the IVH risk in preterm infants. Further studies focussing on short-term changes in vitamin K-kinetics and the coagulation profile during the first days of life are required to further understand a possible link between development of IVH and genetic variants affecting the vitamin K-metabolism.

Common and rare von Willebrand factor (VWF) coding variants, VWF levels, and factor VIII levels in African Americans: the NHLBI Exome Sequencing Project

AbstractSeveral rare European von Willebrand disease missense variants of VWF (including p.Arg2185Gln and p.His817Gln) were recently reported to be common in apparently healthy African Americans (AAs). Using data from the NHLBI Exome Sequencing Project, we assessed the association of these and other VWF coding variants with von Willebrand factor (VWF) and factor VIII (FVIII) levels in 4468 AAs. Of 30 nonsynonymous VWF variants, 6 were significantly and independently associated (P < .001) with levels of VWF and/or FVIII. Each additional copy of the common VWF variants encoding p.Thr789Ala or p.Asp1472His was associated with 6 to 8 IU/dL higher VWF levels. The VWF variant encoding p.Arg2185Gln was associated with 7 to 13 IU/dL lower VWF and FVIII levels. The type 2N-related VWF variant encoding p.His817Gln was associated with 17 IU/dL lower FVIII level but normal VWF level. A novel, rare missense VWF variant that predicts disruption of an O-glycosylation site (p.Ser1486Leu) and a rare variant encoding p.Arg2287Trp were each associated with 30 to 40 IU/dL lower VWF level (P < .001). In summary, several common and rare VWF missense variants contribute to phenotypic differences in VWF and FVIII among AAs.

An update of consensus guidelines for warfarin reversal

• Despite the associated bleeding risk, warfarin is the most commonly prescribed anticoagulant in Australia and New Zealand. Warfarin use will likely continue for anticoagulation indications for which novel agents have not been evaluated and among patients who are already stabilised on it or have severe renal impairment. • Strategies to manage over-warfarinisation and warfarin during invasive procedures can reduce the risk of haemorrhage. • For most warfarin indications, the target international normalised ratio (INR) is 2.0-3.0 (venous thromboembolism and single mechanical heart valve excluding mitral). For mechanical mitral valve or combined mitral and aortic valves, the target INR is 2.5-3.5. • Risk factors for bleeding with warfarin use include increasing age, history of bleeding and specific comorbidities. • For patients with elevated INR (4.5-10.0), no bleeding and no high risk of bleeding, withholding warfarin with careful subsequent monitoring seems safe. • Vitamin K1 can be given to reverse the anticoagulant effect of warfarin. When oral vitamin K1 is used for this purpose, the injectable formulation, which can be given orally or intravenously, is preferred. • For immediate reversal, prothrombin complex concentrates (PCC) are preferred over fresh frozen plasma (FFP). Prothrombinex-VF is the only PCC routinely used for warfarin reversal in Australia and New Zealand. It contains factors II, IX, X and low levels of factor VII. FFP is not routinely needed in combination with Prothrombinex-VF. FFP can be used when Prothrombinex-VF is unavailable. Vitamin K1 is essential for sustaining the reversal achieved by PCC or FFP. • Surgery can be conducted with minimal increased risk of bleeding if INR ≤ 1.5. For minor procedures where bleeding risk is low, warfarin may not need to be interrupted. If necessary, warfarin can be withheld for 5 days before surgery, or intravenous vitamin K₁ can be given the night before surgery. Prothrombinex-VF use for warfarin reversal should be restricted to emergency settings. Perioperative management of anticoagulant therapy requires an evaluation of the risk of thrombosis if warfarin is temporarily stopped, relative to the risk of bleeding if it is continued or modified.

Clinical Experience With Three-Factor Prothrombin Complex Concentrate to Reverse Warfarin Anticoagulation in Intracranial Hemorrhage

The effectiveness of prothrombin complex concentrate (PCC) products available in the United States that contain low levels of factor VII (3-factor PCC) has not been tested. The purpose of this study was to review our experience with 3-factor PCC (Profilnine) in the setting of warfarin-associated intracranial hemorrhage (wICH). In November 2007, we implemented a protocol for reversal of anticoagulation in wICH using Profilnine. Additional treatment with fresh-frozen plasma was at the discretion of the treating physician. Medical records of all patients receiving PCC for wICH between November 1, 2007, and December 7, 2011 were reviewed. Correction of the international normalized rate (INR) was defined as an INR <1.4. Seventy wICH patients were treated with Profilnine, including 46 (66%) with intraparenchymal hemorrhage, 22 (31%) with subdural hemorrhage, and 2 (3%) with subarachnoid hemorrhage. Mean INR was reduced from 3.36 to 1.96, and in 44 (62.9%) patients the INR corrected to <1.4. Baseline INR ≥3.0 decreased the likelihood of INR correction. Concomitant administration of fresh-frozen plasma (mean, 2.6 U) did not increase the likelihood of INR correction. Seven (10%) patients had serious adverse events during their hospital course, including 2 sudden deaths from suspected pulmonary embolism. Reversal of coagulopathy in wICH with Profilnine was incomplete and associated with serious adverse events. In the absence of available 4-factor PCC, options for urgent reversal of anticoagulation in wICH remain limited.

Abstract 2752: Incomplete Reversal of Anticoagulation Following Intracranial Hemorrhage with Three-Factor Prothrombin Complex Concentrate

Introduction: Warfarin-associated intracranial hemorrhage (wICH) is common and associated with worse outcomes than spontaneous intracranial hemorrhages. National guidelines for treatment are vague and consensus-driven. Prothrombin complex concentrates (PCC) have been advocated as a safe and rapid means for reversing anticoagulation but the effectiveness of formulations with low levels of factor VII (three-factor PCC) have not been tested. The purpose of this study was to determine the safety and effectiveness of a three-factor PCC (Profilnine) to reverse anticoagulation in the setting of wICH. Methods: In November, 2007, a protocol for reversal of anticoagulation in wICH using Profilnine was instituted at our instituion. All patients with wICH received 10 mg IV vitamin K and 50 IU/kg of PCC. If the INR remained &gt;1.4 after 30 minutes, a second dose of PCC of 25 IU/kg was infused. Additional treatment with plasma was at the discretion of the treating physician. Charts of all patients receiving PCC between November 1, 2007 and May 31, 2010 were reviewed. Patient demographics, hemorrhage subtype [intracerebral (ICH), subarachnoid (SAH) or subdural (SDH)], plasma usage, complications, and clinical outcome were determined from the chart review. Adequate correction of the INR was defined as an INR &lt;1.4. Kaplan-Meier survival analysis examined the time to INR correction. Results: Fifty-one wICH patients were treated with Profilnine including 28 (55%) with ICH, 21 (41%) with SDH and 2 (4%) with SAH. The mean age was 70.3 (SD 12.7), 32 (63%) were male, 37 (74%) patients were white and the remaining African-American. The mean INR was 3.56 (SD 2.71) at baseline [30 (61%) &lt;3.0, 11 (23%) 3.0-4.9, 8 (16%) &gt;4.9]. Seventeen (33%) patients received plasma along with the first dose of PCC. Mean INR was 2.13 post-PCC and only 33 (67.4%; 95% CI, 54.3% - 80.5%) were corrected to an INR &lt;1.4. Eleven (22%) patients received a second dose of PCC and 22 (43%) received subsequent plasma. The likelihood of correction was not increased by concomitant plasma (p=0.77). However, those with INRs &lt;3.0 and those with INRs between 3.0-4.9 were more likely to have an INR that corrected to &lt;1.4 than those with and INR &gt;4.9 before PCC dosing (p=0.02). The median time to INR correction following initial PCC dose was 11.5 hours. If a second dose of PCC was needed, the median time to INR correction was 17.6 hours. No arterial and one (2%) venous thromboembolic adverse events occurred. Twenty-four (47%) patients died or were discharged to hospice. Conclusions: A three-factor PCC (Profilnine) was incompletely effective in INR correction in wICH presumably due to the relatively low concentration of factor VII. The addition of plasma to PCC did not reduce the time to effective INR correction. Presumed superiority of three-factor PCC to plasma for wICH appears premature.

A Multiscale Model of Venous Thrombus Formation with Surface-Mediated Control of Blood Coagulation Cascade

A combination of the extended multiscale model, new image processing algorithms, and biological experiments is used for studying the role of Factor VII (FVII) in venous thrombus formation. A detailed submodel of the tissue factor pathway of blood coagulation is introduced within the framework of the multiscale model to provide a detailed description of coagulation cascade. Surface reactions of the extrinsic coagulation pathway on membranes of platelets are studied under different flow conditions. It is shown that low levels of FVII in blood result in a significant delay in thrombin production, demonstrating that FVII plays an active role in promoting thrombus development at an early stage.

Endothelial Cell Processing and Alternatively Spliced Transcripts of Factor VIII: Potential Implications for Coagulation Cascades and Pulmonary Hypertension

BackgroundCoagulation factor VIII (FVIII) deficiency leads to haemophilia A. Conversely, elevated plasma levels are a strong predictor of recurrent venous thromboemboli and pulmonary hypertension phenotypes in which in situ thromboses are implicated. Extrahepatic sources of plasma FVIII are implicated, but have remained elusive.Methodology/Principal FindingsImmunohistochemistry of normal human lung tissue, and confocal microscopy, flow cytometry, and ELISA quantification of conditioned media from normal primary endothelial cells were used to examine endothelial expression of FVIII and coexpression with von Willebrand Factor (vWF), which protects secreted FVIII heavy chain from rapid proteloysis. FVIII transcripts predicted from database mining were identified by rt-PCR and sequencing. FVIII mAb-reactive material was demonstrated in CD31+ endothelial cells in normal human lung tissue, and in primary pulmonary artery, pulmonary microvascular, and dermal microvascular endothelial cells. In pulmonary endothelial cells, this protein occasionally colocalized with vWF, centered on Weibel Palade bodies. Pulmonary artery and pulmonary microvascular endothelial cells secreted low levels of FVIII and vWF to conditioned media, and demonstrated cell surface expression of FVIII and vWF Ab–reacting proteins compared to an isotype control. Four endothelial splice isoforms were identified. Two utilize transcription start sites in alternate 5′ exons within the int22h-1 repeat responsible for intron 22 inversions in 40% of severe haemophiliacs. A reciprocal relationship between the presence of short isoforms and full-length FVIII transcript suggested potential splice-switching mechanisms.Conclusions/SignificanceThe pulmonary endothelium is confirmed as a site of FVIII secretion, with evidence of synthesis, cell surface expression, and coexpression with vWF. There is complex alternate transcription initiation from the FVIII gene. These findings provide a framework for future research on the regulation and perturbation of FVIII synthesis, and of potential relevance to haemophilia, thromboses, and pulmonary hypertensive states.

Study of the Role of Factor VII in Venous Thrombus Formation Using Combination of a Multiscale Model and Experiment

To prevent the loss of blood following a break in blood vessels, components in blood and the vessel wall interact rapidly to form a venous thrombus to limit hemorrhage. Combination of extended multiscale model, new image processing algorithms and biological experiments is used for studying the role of Factor VII (FVII) in venous thrombus formation. A detailed sub-model of the tissue factor (TF) pathway of blood coagulation is introduced within the framework of the multiscale model to provide detailed description of coagulation cascade. Macro scale dynamics of the blood flow is described by the continuum Navier-Stokes equations. Micro scale interactions between activated platelets, platelets and fibrin(ogen) and platelets and vessel wall are modeled using an extended stochastic discrete model. The novelty of the approach is in representing each platelet as an extended object with a boundary and modeling in detail the production of thrombin by each individual platelet. Also, clot is treated as a porous medium. Surface reactions of the extrinsic coagulation pathway on membranes of platelets are studied under different flow conditions. It is shown that low levels of FVII in blood result in a significant delay in thrombin production leading to changes in the surface composition of developing thrombi. The changes likely alter the mechanism and dynamics of thrombus stabilization which we are now studying in computational and experimental models.Xu, Z., Chen, N., Shadden, S., Marsden, J.E., Kamocka, M.M., Rosen, E.D., and M.S. Alber [2009], Study of Blood Flow Impact on Growth of Thrombi Using a Multiscale Model, Soft Matter 5, 769-779.Xu, Z., Chen, N., Kamocka, M.M., Rosen, E.D., and M.S. Alber [2008], Multiscale Model of Thrombus Development, Journal of the Royal Society Interface 5 705-722.

Combined Factor V and Factor VIII Deficiency

Combined deficiency of factor V (FV) and factor VIII (FVIII) (F5F8D, or FV+FVIII) is a autosomal recessive bleeding disorder caused by mutations in genes encoding two components of the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC-53), that is, lectin mannose binding protein ( LMAN1) and multiple coagulation factor deficiency 2 ( MCFD2), involved in the FV and FVIII intracellular transport rather than by DNA defects in the genes that encode the corresponding coagulation factors. F5F8D is estimated to be extremely rare (1:1,000,000) in the general population, but an increased frequency is observed in regions where consanguineous marriages are practiced. F5F8D is characterized by concomitantly low levels (usually between 5% and 20%) of both FV and FVIII and is associated with a mild to moderate bleeding tendency. Treatment of bleeding episodes requires a source of both FV and FVIII; replacement of FV is achieved only through use of fresh-frozen plasma (FFP) and replacement of FVIII by FFP and desmopressin or specific FVIII concentrates (plasma-derived or recombinant FVIII products).

Mapping of Conformational Epitopes, and Analyze of the Distribution of Ig Isotypes and Subclasses Using a Semi-Quantitative Multiplex Assay in 6 Cases of Postpartum Acquired Hemophilia.

Background and objectives: Acquired hemophilia (AH) is a severe life-threatening autoimmune disease due to the development of polyclonal autoantibodies (autoAbs) which neutralize the procoagulant activity of coagulation factor VIII (FVIII). The most common epitopes for autoAbs to FVIII are located in the A2, A3 and C2 domains of FVIII. We have recently developed a multiplex assay based on the Luminex technology that allows simultaneously the detection and epitope mapping of anti-FVIII antibodies (Lavigne-Lissalde et al, Thromb; Haemost. 2008). This technology is fast and requires only 100 μl of plasma and we showed that most autoAbs to FVIII are monospecific and preferentially bind to the light chain (LC) of FVIII. The aim of the present study was to evaluate the epitope specificity and IgG isotype/subclasses of autoAbs to FVIII detected in the post partum period.Methods: Acquired hemophilia was diagnosed in six women (median age: 28 years) included in a French cohort (SACHA) and who exhibited prolonged activated partial thromboplastin time, low FVIII levels (range = 0.5–21 IU/dL) and inhibitor titers between 1.5 and 40 Bethesda units (BU). The epitope mapping was studied in each patient by testing 5 plasma samples collected (at inclusion and after 1, 3, 6 and 12 months of follow up) using a multiplex assay as previously described (Lavigne-Lissalde et al, Thromb; Haemost. 2008). A panel of monoclonal antibodies that bind to LC, heavy chain (HC) and the C2 domain of FVIII was employed and the use of specific immunoconjugates also allowed to define the isotype (IgG, A, M) and subclass (IgG1 to IgG4) distributions of autoAbs to FVIII. Results were expressed in mean fluorescence intensity (MFI).Results: The autoAbs detected in the post partum in the six women studied recognized both and simultaneously HC and LC with predominant epitopes located in the LC and the C2 domain in 5 cases. At the inclusion, the autoAbs were mixtures of IgG1 and IgG4 in all patients, but the latter subclass was predominant in most cases. In addition, IgM Abs were also present in 3 women. No correlation between the titer of inhibitor (in BU) and the level of autoAbs binding (in MFI) could be evidenced. The evolution was good in all patients but one case for whom an increase in IgG4 binding to the FVIII C2 domain was demonstrated whereas inhibitor titer decreased (from 40 to 2.6 BU). Importantly, no modification in the epitope specificity between inclusion and 12 months of follow-up was found.Conclusion: The study shows that autoAbs to FVIII developed in the context of pregnancy are mainly IgG4 specific to the C2 domain. In addition, the epitope mapping of anti-FVIII antibodies by multiplex approach could contribute to a better understanding of the pathophysiology of acquired hemophilia.

Factor V deficiency: a concise review

Factor V (FV; proaccelerin or labile factor) is the plasma cofactor for the prothrombinase complex that activates prothrombin to thrombin. FV deficiency can be caused by mutations in the FV gene or in genes encoding components of a putative cargo receptor that transports FV (and factor VIII) from the endoplasmic reticulum to the Golgi. Because FV is present in platelet alpha-granules as well as in plasma, low FV levels are also seen in disorders of platelet granules. Additionally, acquired FV deficiencies can occur in the setting of rheumatologic disorders, malignancies, and antibiotic use and, most frequently, with the use of topical bovine thrombin. FV levels have limited correlation with the risk of bleeding, but overall, FV-deficient patients appear to have a less severe phenotype than patients with haemophilia A or B. The most commonly reported symptoms are bleeding from mucosal surfaces and postoperative haemorrhage. However, haemarthroses and intramuscular and intracranial haemorrhages can also occur. Because no FV-specific concentrate is available, fresh frozen plasma remains the mainstay of treatment. Antifibrinolytics can also provide benefit, especially for mucosal bleeding. In refractory cases, or for patients with inhibitors, prothrombin complex concentrates, recombinant activated FVIIa, and platelet transfusions have been successfully used. Some patients with inhibitors may also require immunosuppression.

The Relation Between Cytokines, Soluble Endothelial Protein C Receptor, and Factor VIII Levels in Turkish Pediatric Stroke Patients

The aim of the authors is to examine the relationship between the cytokine levels that are thought to be involved in stroke etiopathogenesis (tumor necrosis factor [TNF]-alpha, interleukin [IL]-2, IL-6, IL-8, IL-11), soluble protein C receptor (sEPCR), and factor VIII (FVIII) levels. The study included 27 patients with stroke and 30 healthy controls, aged 0 to 18. In the comparison of the sEPCR, cytokine, and FVIII levels between patient and control groups, median levels of TNF-alpha, IL-2, IL-6, and IL-8 are found to be high in the patient group when compared with controls, whereas there is no difference in sEPCR, IL-11, and FVIII levels. In the patient group, a positive correlation is seen between TNF-alpha levels and IL-2 and IL-6 levels, between IL-2 and IL-6 levels, and between IL-6 and IL-8 levels, whereas a negative relationship is seen between sEPCR and FVIII. In the control group apart from the patient group, a negative relationship is seen between TNF-alpha and FVIII, whereas there is a positive relationship between IL-11 and sEPCR levels. Median sEPCR levels in patients who have normal or low FVIII levels are significantly high when compared with those with high FVIII levels. In conclusion, in the pediatric population, an increase in TNF-alpha, IL-2, IL-6, and IL-8 levels is seen. Also, an inverse relationship of sEPCR and FVIII levels is shown for the first time. This study provides a basis for ongoing studies that aim to clarify stroke etiopathogenesis. Studies with larger series of patients are warranted to confirm this hypothesis.

Corn trypsin inhibitor in fluorogenic thrombin-generation measurements is only necessary at low tissue factor concentrations and influences the relationship between factor VIII coagulant activity and thrombogram parameters

The fluorogenic calibrated automated thrombin-generation assay is influenced by contact pathway activation in platelet-rich and platelet-poor plasma. This influence lessens with increasing tissue factor (TF) concentrations and is inhibited by corn trypsin inhibitor (CTI). CTI is expensive and at what TF concentration its influence becomes irrelevant is unclear. Spiking of factor VIII (FVIII)-depleted plasma with FVIII, in samples without CTI, shows a plateau of thrombin generation at low normal FVIII levels. Given the association with thrombosis at high levels, a continuing increase in thrombin generation would be expected. We studied the effect of CTI on this relation by spiking experiments up to 4.8 IU/ml at 1 pmol/l TF and compared the influence of CTI at 1 and 5 pmol/l in platelet-poor plasma. CTI significantly influences thrombin generation in platelet-poor plasma at 1 pmol/l TF (difference of means for endogenous thrombin potential of 232.5 nmol/l per min, P<0.0001) and peak of 48 nmol/l (P<0.0001)) but not at 5 pmol/l. Spiking experiments without CTI confirm the hyperbolic relation between FVIII coagulant activity (FVIII:C) and endogenous thrombin potential with a plateau at 0.70-1.40 IU/ml. With CTI, a near-linear response up to 1.0 IU/ml was found with a plateau at 2.4-4.8 IU/ml. For peak thrombin, no plateau was reached with CTI. The present study confirms and extends previous data on CTI and the relationship between FVIII:C and thrombin generation. CTI is not necessary at 5 pmol/l TF, and thrombin generation remains dependent on FVIII:C up to 4.8 IU/ml at 1 pmol/l with CTI. Higher levels than previously thought may be needed to normalize thrombin generation.

Sustained FVIII Expression and Phenotypic Correction of Hemophilia A in Neonatal Mice Using an Endothelial-Targeted Sleeping Beauty Transposon

Hemophilia A, deficiency of coagulation factor VIII (FVIII), is an attractive candidate for gene therapy as expression of modest amounts of FVIII can provide therapeutic benefit. Most gene transfer approaches for hemophilia have focused on the liver, as this is the major source of endogenous FVIII; however, increasing evidence suggests that endothelial cells are capable of synthesis and release of FVIII. Here the Sleeping Beauty (SB) transposon is employed to target long-term expression of the human B-domain-depleted FVIII gene (approved gene symbol F8) within lung endothelia of hemophilic mice. As the formation of inhibitory antibodies to FVIII has been a significant impediment toward achieving therapeutic plasma levels after gene or protein therapy, we chose to perform gene transfer in neonatal mice, which are more likely to be immune tolerant. Using this approach, low therapeutic levels of FVIII (∼10%), as well as phenotypic correction of the bleeding disorder, were achieved in all animals that received the FVIII transposon and functional transposase throughout the duration of the study (24 weeks). Rechallenge of these animals with additional gene transfer did not result in significant increases in FVIII levels, due mainly to increases in inhibitory antibodies. These studies demonstrate the feasibility of using endothelial-targeted SB transposons for the treatment of hemophilia A.