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The hemostatic balance in the placenta may be determined by both maternal and fetal factors cooperatively regulating coagulation at the feto-maternal interface. The maternal spiral arteries become remodeled by pregnancy hormones and trophoblast into uteroplacental arteries toward the end of the first trimester. The evidence for pregnancy loss having a thrombotic basis is due to the association between antiphospholipid antibodies and recurrent pregnancy loss. Case-control studies can only show associations between thrombophilias and pregnancy losses. The presumed benefit of antithrombotic therapy and the absence of side effects has led many clinicians to prescribe low molecular weight heparin, aspirin, or both to women with recurrent pregnancy loss and hereditary thrombophilia. Cytokine imbalances have been described in recurrent pregnancy loss, antiphospholipid syndrome, preeclampsia, preterm births, and intrauterine growth restriction. Afibrinogenemia—a defect in hepatic fibrinogen secretion or release—is inherited as an autosomal recessive trait and is associated with bleeding diathesis, impaired wound repair, and recurrent pregnancy loss.

SummaryBackground Factor XIII deficiency is a rare, severe congenital bleeding disorder. Monthly prophylaxis with recombinant factor XIII (rFXIII) has demonstrated favorable safety and efficacy in patients ≥6 years and may similarly benefit younger children. Objective To evaluate the long-term safety and efficacy of rFXIII in children

Tyrosine kinase inhibitors (TKIs) have revolutionized the prognosis of chronic myeloid leukemia. With the advent of highly efficacious therapy, the focus has shifted toward managing TKI adverse effects, such as vascular adverse events (VAEs). We used an in vitro angiogenesis model to investigate the TKI-associated VAEs. Our data show that imatinib, nilotinib, and ponatinib reduce human umbilical vein endothelial cells (HUVECs) viability. Pharmacological concentrations of ponatinib induced apoptosis, reduced migration, inhibited tube formation of HUVECs, and had a negative effect on endothelial progenitor cell (EPC) function. Furthermore, in HUVECs transfected with VEGF receptor 2 (VEGFR2), the effect of ponatinib on tube formation and on all parameters representing normal endothelial cell function was less prominent than in control cells. This is the first report regarding the pathogenesis of ponatinib-associated VAEs. The antiangiogenic effect of ponatinib, possibly mediated by VEGFR2 inhibition, as shown in our study, is another piece in the intricate puzzle of TKI-associated VAEs.

Recombinant factor XIII-A2 (rFXIII-A2) was developed for prophylaxis and treatment of bleeds in patients with congenital FXIII A-subunit deficiency. mentor™2 (NCT00978380), a multinational, open-label, single-arm, multiple-dosing extension to the pivotal mentor™1 trial, assessed long-term safety and efficacy of rFXIII-A2 prophylaxis in eligible patients (patients with severe [

The aim of the study was to further investigate the role of fibrinogen-like protein 2 (FGL-2), a transmembrane prothrombinase that directly cleaves prothrombin to thrombin, in angiogenesis and tumor development and the mechanism(s) underlying these processes. To study angiogenesis HUVEC clones with decreased fgl-2 mRNA were generated by specific siRNA. To study tumorigenesis SCID mice were implanted with intact (wild type) and fgl-2-silenced PC-3 clones. IFN-γ treated HUVEC expressing increased fgl-2 mRNA exhibited significant capillary sprouting that was not inhibited by hirudin, whereas fgl-2 silencing completely inhibited blood-vessel formation. Tumors (poorly differentiated carcinoma) developed in all 12 mice injected with wild type PC-3 compared with 8/12 mice injected with the fgl-2-silenced PC-3 clone. The tumors developed by fgl-2-silenced PC-3 clones were smaller and less aggressive and contained significantly fewer blood vessels (p

Factor XIII (FXIII) is composed of two catalytic A subunits and two carrier B subunits. Following activation by thrombin FXIII becomes plasma transglutaminase, which cross-links the γ-glutamyl-e-lysine residues of fibrin chains stabilizing fibrin clot. Congenital deficiency of factor XIII results in a severe life-long hemorrhagic disorder, abnormal wound healing in about 30% of patients and recurrent abortions. Most of the FXIII deficiency patients have mutations in the F13A gene. Only few mutations in F13B gene have been published. Plasma-derived concentrate of factor XIII used to be the treatment of choice. Recently, recombinant FXIII concentrate has been developed and tested in multinational clinical studies. This new product appears to be safe and appropriate for life-long prophylactic treatment of patients with FXIII A deficiency.

Background: Refractoriness to platelet transfusion, prevalent among 15-20% of hemato-oncological patients, is associated with multitransfusions and inferior outcomes. We evaluated the effectiveness of extended slow-dose transfusion (ESDT) in increasing platelet increments in multitransfused patients. Methods: Patients treated after the implementation of ESDT were compared with historical controls treated with standard single-donor platelet (SDP) transfusions. Cohorts of early and late recipients were assembled for comparison, i.e. the 8th or 9th and 11th platelet unit per patient, respectively. Patients in the ESDT group received transfusions equal to half an SDP unit, administered over 4 h. Effectiveness was defined as a higher corrected count increment (CCI) at 1, 12, and 24 h after transfusion. Results: In the early-recipients cohort, 24-h-posttransfusion increments were available for 29 ESDT patients and 6 standard patients, and did not differ significantly between the groups (p = 0.078). The 24-h-posttransfusion increment was available for 20 ESDT patients and 7 standard patients in the late-recipients cohort. The CCI was significantly higher in the ESDT group (p = 0.042). ABO compatibility improved the CCI (p = 0.01). Conclusions: ESDT demonstrated slightly higher increments at 24 h after transfusion in late recipients, suggesting this could be a cost-effective approach for the treatment of thrombocytopenic multitransfused hemato-oncological patients.

Congenital factor XIII (FXIII) deficiency is a rare, autosomal recessive bleeding disorder with potentially life-threatening consequences. FXIII is composed of two subunits (A and B), and a deficiency or dysfunction of either can result in FXIII deficiency. Traditionally, FXIII deficiency has been managed by infusing plasma-derived products containing FXIII (fresh frozen plasma, cryoprecipitate, and plasma-derived FXIII concentrates), all of which contain both subunits. Despite the increased safety of plasma-derived products, concern remains regarding potential viral safety issues. This review describes the development, from concept to clinical use, of a recombinant FXIII molecule (containing subunit A only; rFXIII-A2) for congenital FXIII-A subunit deficiency. Unmet needs and ongoing challenges in congenital FXIII deficiency are also discussed. Despite the challenges in developing a product for a very rare bleeding disorder, the information gathered on efficacy, safety, and pharmacokinetics of FXIII replacement therapy represents the largest dataset on congenital FXIII-A subunit deficiency in the world. It also provides evidence for the safety and efficacy of monthly prophylaxis with 35 IU/kg of rFXIII-A2 in patients with FXIII-A subunit deficiency. The issues encountered and overcome, along with lessons learned, may be applied to and encourage the development of new recombinant products for other rare bleeding disorders.

Congenital factor XIII (FXIII) deficiency is a rare, autosomal-recessive disorder, with most patients having an A-subunit (FXIII-A) deficiency. Patients experience life-threatening bleeds, impaired wound healing, and spontaneous abortions. In many countries, only plasma or cryoprecipitate treatments are available, but these carry a risk for allergic reactions and infection with blood-borne pathogens. The present study was a multinational, open-label, single-arm, phase 3 prophylaxis trial evaluating the efficacy and safety of a novel recombinant FXIII (rFXIII) in congenital FXIII-A subunit deficiency. Forty-one patients ≥ 6 years of age (mean, 26.4; range, 7-60) with congenital FXIII-A subunit deficiency were enrolled. Throughout the rFXIII prophylaxis, only 5 bleeding episodes (all trauma induced) in 4 patients were treated with FXIII-containing products. The crude mean bleeding rate was significantly lower than the historic bleeding rate (0.138 vs 2.91 bleeds/patient/year, respectively) for on-demand treatment. Transient, non-neutralizing, low-titer anti-rFXIII Abs developed in 4 patients, none of whom experienced allergic reactions, any bleeds requiring treatment, or changes in FXIII pharmacokinetics during the trial or follow-up. These non-neutralizing Abs declined below detection limits in all 4 patients despite further exposure to rFXIII or other FXIII-containing products. We conclude that rFXIII is safe and effective in preventing bleeding episodes in patients with congenital FXIII-A subunit deficiency. This study is registered at http://www..clinicaltrials.gov as number NCT00713648.

The histone deacetylase inhibitor (HDACI) butyroyloxymethyl diethylphosphate (AN-7) has been shown to synergize doxorubicin (Dox) anticancer activity while attenuating its cardiotoxicity. In this study we further explored the selectivity of AN-7's action in several cancer and normal cells treated with anticancer agents. The cells studied were murine mammary 4T1, human breast T47D and glioblastoma U251 cancer cell lines, neonatal rat cardiomyocytes, cardiofibroblasts and astrocytes, and immortalized cardiomyocyte H9C2 cells. Cell death, ROS production and changes in protein expression were measured and in vivo effects were evaluated in Balb-c mice. AN-7 synergized Dox and anti-HER2 cytotoxicity against mammary carcinoma cells with combination indices of 0.74 and 0.79, respectively, while it protected cardiomyocytes against their toxicity. Additionally AN-7 protected astrocytes from Dox-cytoxicity. Cell-type specific changes in the expression of proteins controlling survival, angiogenesis and inflammation by AN-7 or AN-7+Dox were observed. In mice, the protective effect of AN-7 against Dox cardiotoxicity was associated with a reduction in inflammatory factors. In summary, AN-7 augmented the anticancer activity of Dox and anti-HER2 and attenuated their toxicity against normal cells. AN-7 modulation of c-Myc, thrombospondin-1, lo-FGF-2 and other proteins were cell type specific. The effects of AN-7, Dox and their combination were preserved in vivo indicating the potential benefit of combining AN-7 and Dox for clinical use.

SummaryTissue transglutaminase was reported to act as protein disulfide isomerase (PDI). We studied whether plasma transglutaminase – coagulation factor XIII (FXIII) – has PDI activity as well. PDI activity was measured by determining the ability to renature reduced-denatured RNase (rdRNase). We found that FXIII can re-nature rdRNase, with efficiency comparable to commercial PDI. This PDI activity was inhibited by bacitracin. Like tissue transglu-taminase, FXIII-mediated PDI activity is independent of its transglutaminase activity and is located on the A subunit. Surface-associated PDI has been previously shown to catalyse two distinct functions: transnitrosation with subsequent release of intracellular nitric oxide and disulfide bond rearrangement during platelet integrin ligation. Our results imply that FXIII-PDI activity may have a role in platelet function.

Histone deacetylase inhibitory prodrugs that are metabolized to butyric acid and formaldehyde possess antineoplastic properties and low toxicity. We sought to characterize the antiangiogenic and antimetastatic activities of two lead prodrugs, pivaloyloxymethyl butyrate (AN-9) and butyroyloxymethyl-diethyl phosphate (AN-7) in murine cancer models. In the sc implanted human colon carcinoma HT-29 xenograft model AN-7, exhibited superior anticancer activity compared to AN-9, as was evident by the significantly greater inhibition of tumor growth and reduction of serum CEA. AN-7 was also more effective in reducing mean vessel density (MVD) by 7-fold, bFGF, Ki-67 (7-fold) and HIF-1alpha in immunohistochemically stained tumor sections. Semi-quantitative evaluation of the levels of bFGF, HDAC1 and HIF-1alpha by Western blot analysis showed a decrease in expression only in the tumors of mice treated with AN-7. The level of bFGF was reduced 3-fold in the tumor and that of TIMP1 was elevated (by 3-fold) in the serum of AN-7 treated mice. In a 4T1 metastatic breast carcinoma model, AN-7 inhibited the formation of lung lesions by 76% and AN-9 by 47%, further demonstrating the greater efficacy of AN-7 compared to AN-9 (P0.02). Both AN-7 and AN-9 exhibited antimetastatic and antiangiogenic activities by reducing vascularization, bFGF expression and HIF-1alpha. Yet, AN-7 was more potent than AN-9.

Factor XIII is a plasma transglutaminase that participates in the final stage of the coagulation cascade. Thrombin-activated FXIII (FXIIIa) catalyzes the formation of covalent crosslinks between gamma-glutamyl and epsilon-lysyl residues on fibrin molecules to yield the mature clot. In addition to its role in hemostasis, FXIIIa was previously shown by us to stimulate endothelial cells to exhibit pro-angiogenic activity. In this work, we studied the effect of FXIIIa on other cells that participate in angiogenesis and tissue repair, such as monocytes and fibroblasts. FXIIIa significantly enhanced migration and proliferation, and inhibited apoptosis of monocytes and fibroblasts. Similar to our previous observations with endothelial cells, the stimulating effect of FXIIIa on monocytes and fibroblasts was elicited via its binding to alpha (v)beta (3) integrin leading to cJun upregulation and TSP-1 downregulation. Since monocytes and fibroblasts are essential components of the tissue repair process, the results of this study, together with the proangiogenic activity of FXIIIa, further substantiate a significant role of FXIII in tissue repair.

Four missense mutations in the factor XIII A-subunit gene, Arg260Leu, Ala318Val, Thr398Asn and Gly210Arg, were previously reported by us in patients with severe factor XIII deficiency. The objective of our study was to discern the effect of all four mutations on the stability and intracellular localization of the factor XIII A-subunit by their expression in COS cells. In-vitro mutagenesis, transient expression of the mutants in COS cells and subsequent pulse-chase analyses were carried out. Intracellular localization of wild-type and mutant proteins was analyzed by immunohistochemistry using a monoclonal antibody against factor XIII A-subunit. Pulse-chase analyses of metabolically labeled proteins demonstrated rapid intracellular degradation of each mutant protein as compared with wild type. Immunocytochemical and immunofluorescence analyses disclosed that wild-type and all four mutant factor XIII A-subunit proteins were diffusely distributed within the cytoplasm but not in the endoplasmic reticulum of the COS-7 cells. The Arg260Leu, Ala318Val, Thr398Asn and Gly210Arg mutations in FXIII A-subunit cause rapid intracellular degradation of the corresponding mutated protein.

Antithymocyte globulin (ATG) is increasingly used in pre-allogeneic stem cell transplantation (allo-SCT) conditioning regimens to prevent graft rejection and graft-versus-host disease. However, ATG was also found to be associated with increased incidence of thrombosis during organ transplantation. In the present study, we tested the coagulation status of 21 patients with hematologic malignancies undergoing allo-SCT who received ATG-based (11 patients) or non-ATG-based (10) conditioning treatment. We assessed several thrombophilia markers as well as circulating total and endothelial microparticles (TMP/EMP) and soluble CD40 ligand (CD40L). No significant difference in the mean values of prothrombin time, partial thromboplastin time, fibrinogen, antithrombin, protein C, protein S, thrombin-antithrombin III complex, homocysteine levels, prevalence of genetic thrombophilia markers and levels of EMP, TMP or CD40L was observed between the ATG-treated and ATG-untreated patients, as well as before and after conditioning in each group separately. Platelet counts decreased significantly in ATG-treated patients; however, this decrease was not associated with clinical or laboratory evidence of disseminated intravascular coagulation. No patient developed thromboembolic event or veno-occlusive liver disease. Our results suggest that allo-SCT is not associated with increased hypercoagulability and addition of ATG to conditioning regimen has no significant procoagulant effect.

Mitochondrial myopathy and sideroblastic anemia (MLASA) is a rare, autosomal recessive oxidative phosphorylation disorder specific to skeletal muscle and bone marrow. Linkage analysis and homozygosity testing of two families with MLASA localized the candidate region to 1.2 Mb on 12q24.33. Sequence analysis of each of the six known genes in this region, as well as four putative genes with expression in bone marrow or muscle, identified a homozygous missense mutation in the pseudouridine synthase 1 gene (PUS1) in all patients with MLASA from these families. The mutation is the only amino acid coding change in these 10 genes that is not a known polymorphism, and it is not found in 934 controls. The amino acid change affects a highly conserved amino acid, and appears to be in the catalytic center of the protein, PUS1p. PUS1 is widely expressed, and quantitative expression analysis of RNAs from liver, brain, heart, bone marrow, and skeletal muscle showed elevated levels of expression in skeletal muscle and brain. We propose deficient pseudouridylation of mitochondrial tRNAs as an etiology of MLASA. Identification of the pathophysiologic pathways of the mutation in these families may shed light on the tissue specificity of oxidative phosphorylation disorders.

Factor XIII subunit A (FXIII A) is synthesized by megakaryocytes, and monocytes/macrophages. In addition, the liver has been reported as an extrahaematopoietic source of FXIII A. At present, the extent of contribution of either haematopoietic or extrahaematopoietic sources to the plasma FXIII A level is unknown. We studied the effect of bone marrow aplasia due to high-dose chemotherapy followed by autologous peripheral blood stem cell transplantation (ASCT) on plasma FXIII A activity and concentration in 20 patients with haematological or solid tumour malignancies. A multiple linear regression model was used to assess the effect of gender, age, malignancy and treatment types, platelet and monocyte counts, abnormal liver function tests, prothrombin time, and number of platelet transfusions on FXIII activity measured in plasma before and following ASCT. Significant correlation between platelet counts and FXIII A activity in plasma was observed (r = 0.51, P = 0.0001), which remained after the adjustment for the aforementioned parameters (multiple R = 0.52, P = 0.0001). In contrast, no significant correlation between FXIII A levels and monocyte counts was observed (r = 0.19), and this lack of correlation persisted after the adjustment. These results suggest that in the ASCT model, following myeloablation, platelets but not monocytes are the haematopoietic cells that contribute significantly to plasma FXIII A levels. In addition, extra-haematopoietic sources of FXIII synthesis may also contribute to FXIII levels in plasma.

Objective— Factor XIII (FXIII), a plasma transglutaminase that stabilizes fibrin clots at the final stages of blood coagulation by crosslinking fibrin monomers, is essential for embryo implantation and participates in tissue remodeling and wound healing, processes that involve angiogenesis. The aim of our study was to analyze the effect of FXIII on angiogenesis using in vitro and in vivo models and to examine the role of FXIII in the basic steps of angiogenesis, ie, migration, proliferation, and apoptosis/cell survival. Methods and Results— In the Matrigel tube formation model, only FXIIIa caused a dose-dependent enhancement of array formation. This proangiogenic effect was not associated with alterations in vascular endothelial growth factor (VEGF) protein levels nor VEGF or VEGFR2 mRNA levels. FXIIIa, but not nonactivated or transglutaminase-inactivated FXIII, significantly enhanced endothelial cell migration and proliferation and inhibited apoptosis. After treatment of HUVECs with FXIIIa, almost complete disappearance of mRNA of thrombospondin 1 (TSP-1) and a marked reduction in the secretion of TSP-1 protein were observed. A reduction in TSP-1 protein synthesis, although to a lesser extent, was observed on treatment of microvascular endothelial cells with FXIIIa. In a rabbit cornea model, injection of FXIIIa caused neovascularization associated with almost complete disappearance of TSP-1 in the cornea. Conclusions— These results show that FXIIIa exhibits a novel proangiogenic activity that is associated with downregulation of TSP-1 and also involves stimulation of endothelial cell proliferation and migration and inhibition of apoptosis. These findings might shed light on the mechanism by which FXIII mediates tissue repair and remodeling.

Summary. We have designed a simple test for the early diagnosis and treatment monitoring of thrombotic thrombocytopenic purpura (TTP). We examined plasma from 24 TTP patients and normal plasma using a cone and plate(let) analyser (CPA). Test plasma was mixed with citrated normal whole blood (group O) and subjected to flow at a shear rate of 1800/s. Mixing normal plasma (12·5, 25, 50 or 75 µl) with heterologous normal whole blood (final volume of 200 µl) resulted in a decrease of surface coverage (SC, maximally by 63%) and, to a lesser extent, of average size (AS, maximally by 37%) due to dilution of the blood sample. In contrast, mixing the same quantities of acute TTP plasma with normal blood yielded an increase in both SC (up to 125%) and AS (up to 130%). Increased SC and/or AS were detected in all 15 patients in acute phase and in three out of 14 patients in remission. Following repeated plasmapheresis, the enhanced platelet deposition in five patients with acute TTP returned to almost normal patterns. Mixing plasma from patients with other thrombocytopenic conditions in this way resulted in a decrease in both SC and AS, and did not differ from control subjects. In conclusion, the CPA is a simple and specific laboratory test that can be used for the diagnosis and monitoring of plasma exchange therapy in TTP.

The present study was undertaken to evaluate stability, pharmacokinetic profile and efficacy of continuous infusion of 8Y in patients with different types of von Willebrand disease (vWD). Following reconstitution, 8Y levels of von Willebrand factor ristocetin cofactor (vWF:Rco), vWF antigen and factor VIII coagulant activity (FVIII:C) decreased to about 80% of the baseline levels; addition of low molecular weight heparin decreased the level of FVIII:C even further. Reconstituted 8Y was found to be sterile for up to 6 days postreconstitution. Ten vWD patients (four with type 2A, three with type 3, two with type 1 and one with 2N) underwent pharmacokinetic analysis. The recovery of vWF: RCo was significantly lower in patients with type 3 vWD (1.4 +/- 0.05% U(-1) kg(-1)) compared with that of the patients with types 1 (2.3 +/- 0.52% U(-1) kg(-1)) or 2A (2.0 +/- 0.06% U(-1) kg(-1)) vWD (P = 0.015). Type 3 vWD patients exhibited significantly higher vWF:RCo clearance (5.1 +/- 1.1 mL kg(-1) h(-1)) compared with that of patients with type 2A (2.8 +/- 0.7 mL kg(-1) h(-1)) and type 1 (2.6 +/- 1.0 mL kg(-1) h(-1)) vWD (P = 0.028). Accordingly, terminal half-life was lower in patients with type 3 vWD (8.0 +/- 0.6 h(-1)) compared with type 2A (12.7 +/- 5.9 h(-1)) or type 1 (14 +/- 1.2 h(-1)) vWD patients. Multimeric pattern of vWF from patients' plasma was similar to that of 8Y. In two patients treated with 8Y by continuous infusion for prevention or treatment of bleeding haemostasis was achieved. Thus, 8Y is suitable and haemostatically effective for continuous infusion treatment in patients with vWD.

BACKGROUND: The genetic predispositions to venous thrombosis such as factor V Leiden (FVL) mutation (Arg 506 Gln), prothrombin (FII) gene mutation (G20210A), and mutation of the methylenetetrahydrofolate reductase (MTHFR) gene (C677T) have been reported to be associated with recurrent pregnancy loss. This paper examines the prevalence of markers for genetic thrombophilias in women with recurrent miscarriage. METHODS: The prevalence of FVL, FII G20210A and MTHFR C677T was compared in 108 women with three or more pregnancy losses either exclusively in the first trimester, or mixed first and second trimester losses, with the prevalence found in 82 fertile parous control women without miscarriages. Markers for the thrombophilias were assessed by PCR analysis. RESULTS: Twenty-three of the 108 patients (21.3%), had thrombophilia markers, which was similar to the proportion of patients in the control group (20.7%) with these markers. The prevalences of FVL and FII G20210A were lower in the study group than in the control group (3.7 versus 6.1% for FVL and 4.6 versus 6.1% for FII respectively); however, the difference was not statistically significant. In contrast, the prevalence of MTHFR C677T was higher in the study group than the control population (13 versus 8.5% respectively), but this difference was not statistically significant. There was no statistically significant prevalence of any particular thrombophilia in patients with previous first and second trimester pregnancy losses compared with patients with first trimester losses alone. CONCLUSION: Thrombophilia was not found to be associated with recurrent pregnancy loss.

A 75 year old patient presenting with mucocutaneous bleeding was diagnosed with acquired thrombasthenia. The diagnosis was based on lack of platelet aggregation with adenosine diphosphate (ADP), arachidonic acid and collagen, and normal aggregation induced by ristocetin.To study the mechanism of platelet function inhibition in a patient with acquired thrombasthenia.Aggregation assays of platelets from the patient and healthy controls were performed. In addition, anti-glycoprotein (GP) IIbIIIa antibodies bindingto normal in the presence or absence of the patient's serum was by flow cytometry.Aggregation of normal platelets in the presence of patient's plasma was inhibited four- and 2.5-fold in the presence of ADP and arachidonic acid respectively, while collagen-induced aggregation was completely abolished. Ristocetin-induced aggregation was normal. The patient's serum inhibited binding of commercial anti-glycoprotein IIbIIIa antibodies to normal platelets twofold by flow cytometry. Treatment with anti-CD20 monoclonal antibody (rituximab) normalized the patient's platelet aggregation.These results suggest that the patient developed inhibitory anti-GPIIbIIIa autoantibodies that caused acquired thrombasthenia.

Fibrinogen-like protein 2, FGL-2, was reported to be overexpressed in various cancer tissues, where it acts as a transmembrane prothrombinase. This study aims to determine the prothrombinase activity of FGL-2 in peripheral blood mononuclear cells (PBMC) of patients with B-cell lymphoma. FGL-2 activity was determined in patients with B-cell lymphoma (n = 53), and healthy controls (n = 145). FGL-2 activity in patients at diagnosis increased 3 ± 0.3 fold (p < 0.001). Sensitivity and specificity of the test was established at 73.6% and 80.7%, respectively, using a cutoff of 150% activity over control. Moreover, FGL-2 activity in 10 of 11 patients in remission decreased by 76%. In contrast, no significant difference was observed in expression levels of fgl-2 gene in patients and controls. Taken together, our study indicates that FGL-2 prothrombinase activity in PBMC of lymphoma patients is increased in active disease and normalizes during remission, thus being a potential marker for follow up of lymphoma patients.

Summary Background The use of monthly recombinant factor XIII (rFXIII) recently demonstrated favorable safety and efficacy for congenital FXIII A-subunit deficiency patients aged ≥ 6 years (mentor™1 trial), although the pharmacokinetics (PK) were not fully evaluated. Objectives To comprehensively evaluate the steady-state PK of rFXIII in patients aged ≥ 6 years with congenital FXIII A-subunit deficiency. Patients/methods mentor™2 is an ongoing, multinational safety and efficacy trial in which patients are receiving monthly rFXIII (35 IU kg−1) for ≥ 52 weeks. For this 28-day PK analysis, blood samples were collected immediately predosing, and 1 h, 2 h, 3, 7, 14, 21, and 28 days postdosing. FXIII activity was measured and PK parameters were calculated using non-compartmental analysis, without prior baseline adjustment. Information regarding adverse events and bleeding was collected at each visit. Antibody assessments were performed predosing and at day 28. Results PK analysis in 23 patients revealed first-order elimination of rFXIII with a geometric mean half-life of 13.6 days. Mean FXIII activity was > 0.1 IU mL−1 throughout the 28-day period, with a geometric mean peak activity of 0.87 IU mL−1 and trough of 0.16 IU mL−1. The geometric mean clearance was 0.15 mL h−1 kg−1. No bleeding episodes occurred during the PK session, and no anti-rFXIII antibodies were detected. Peak and trough FXIII activities were constant over time, compared with previous activities (≥ 10 rFXIII doses) in the same patients. Conclusions Clearance of rFXIII is unaffected over time, and monthly prophylaxis with 35 IU kg−1 rFXIII provides FXIII activity > 0.1 IU mL−1 throughout the dosing interval in patients with congenital FXIII A-subunit deficiency.

INTRODUCTION Congenital FXIII A-subunit deficiency is a rare, autosomal recessive coagulation disorder with a high risk of life-threatening bleeding complications such as intracranial hemorrhages (Muszbek & Katona. Semin Thromb Hemost 2016;42:429-439). The true prevalence is estimated to be ~1 in 1 to 2 million people worldwide (Anwar R, et al. Pediatrics 2002;109:E32), however, there are only ~1300 diagnosed patients in the world (World Federation of Haemophilia. Report on the Annual Global Survey 2014), suggesting significant under-diagnosis. When severe congenital FXIII deficiency is confirmed, prophylactic replacement therapy is strongly recommended as without prophylaxis, FXIII deficiency places patients at high risk of spontaneous fatal or devastating intracranial bleeding, and because FXIII prophylaxis is reasonably easy and infrequent (once monthly) due to the long half-life of FXIII concentrates. Recombinant FXIII (rFXIII) is a new treatment possibility (Dorey E. Nat Biotechnol 2014;32:210), and here we report long-term safety and efficacy results from mentor™2, the largest clinical trial performed to date in patients with congenital FXIII A-subunit deficiency treated with rFXIII. METHODS The mentor™2 trial was a multinational, open-label, single-arm, multiple-dosing safety extension to the pivotal mentor™1 trial (Inbal A, et al. Blood 2012;119:5111-5117). The trial period was from September 2009 to October 2015. Eligible patients were aged ≥6 years, weighed ≥20 kg, and were diagnosed with congenital FXIII A-subunit deficiency. The Berichrom® FXIII activity assay was used to measure FXIII activity. The trial aimed to assess the long-term safety of replacement therapy with rFXIII (35 IU/kg once monthly) when used for the prevention of bleeding episodes and the treatment of breakthrough bleeds. RESULTS Sixty patients (64% male) were enrolled and exposed to rFXIII; 34 from the mentor™1 trial, and 26 newly recruited patients. Median age of patients at enrollment was 26.0 years (range 7.0-77.0) and 16 (27%) were Safety: a total of 920 treatment-emergent adverse events (AEs) were reported in 56 of the 60 (93.3%) patients: headache and nasopharyngitis occurred most commonly. No anti-rFXIII neutralizing or non-neutralizing antibodies were detected. In the mentor™1 trial 4 patients developed non-neutralizing antibodies, this was not seen in the current mentor™2 trial. There were no thromboembolic events, fatal AEs, or AEs leading to withdrawal, and no anaphylactic or allergic reactions to rFXIII. Serious AEs were few (19 events in 12 patients) and were evaluated as unlikely to be related to rFXIII. There were 7 AEs in 7 patients that were evaluated as possibly/probably related to the trial product (incorrect dose, overdose, arthralgia, leukopenia, limb injury, alanine aminotransferase increase, and blood alkaline phosphatase increase); all patients recovered. The case of overdose was 2.3 times the planned dose; the patient did not experience any adverse consequences. Efficacy: in all, 8 FXIII treatment-requiring bleeds occurred in 7 patients: 6 were trauma induced and 2 were spontaneous (Table). Three of the 8 bleeds occurred within the first 14 days post rFXIII infusion when FXIII levels would have been predicted to be >30% in all cases, while 5 bleeds occurred between days 17 and 24 post rFXIII infusion. No internal organ bleeds or severe gastrointestinal bleeds occurred. One trauma-induced muscular bleed was treated with rFXIII with an excellent response. The annual mean bleeding rate was 0.043 bleeds/patient/year, implying 1 bleed per patient, per ~23 years (0.032 for traumatic bleeds and 0.011 for spontaneous bleeds). The geometric mean FXIII trough level was 0.17 IU/mL (coefficient of variation of 0.37). CONCLUSION This is the largest dataset on congenital FXIII A-subunit deficiency, both in terms of number of patients and number of patient-years. Over the 6-year trial duration, no safety issues were identified and very few FXIII treatment-requiring bleeding episodes occurred, mainly trauma-related. These data all point to the safety and efficacy of a prophylactic regimen of 35 IU/kg of rFXIII given every 4 weeks to manage congenital deficiency of FXIII A subunit. Disclosures Carcao: Biogen/Idec/Sobi: Honoraria, Research Funding; Baxalta: Honoraria; Bayer: Honoraria; CSL Behring: Honoraria, Research Funding; Novo Nordisk: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Octapharma: Honoraria. Altisent:Octapharma: Consultancy; CSL Behring: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Grifols: Consultancy; Novo Nordisk: Consultancy, Research Funding; Bayer: Consultancy, Research Funding; Baxalta: Consultancy, Research Funding. Castaman:Kedrion: Membership on an entity's Board of Directors or advisory committees; Baxalta-Shire: Speakers Bureau; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Sobi: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees. Fukutake:simic: Research Funding; Sekisui Medical: Consultancy, Honoraria, Speakers Bureau; Roche Diagnostics: Honoraria, Speakers Bureau; Biogen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbott: Honoraria, Speakers Bureau; Kaketsuken: Honoraria; Japan Blood Products Organization: Honoraria, Research Funding; Torii: Speakers Bureau; Bayer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; LSI Medience: Consultancy; SRL Inc: Consultancy; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Baxalta: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novo Nordisk: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; CSL Behring: Honoraria; EPS: Research Funding; Siemens: Speakers Bureau. Kerlin:Bayer Healthcare US: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees; CSL Behring Foundation: Research Funding. Kessler:Pfizer: Consultancy; Octapharma: Consultancy, Research Funding; Novo Nordisk: Consultancy, Research Funding; Grifols: Consultancy; Genentech: Consultancy, Research Funding; Biogen: Consultancy; Bayer: Consultancy, Research Funding; LFB: Other: Member of DSMB; Baxalta: Consultancy, Research Funding. Lassila:Aplagon Oy, Finland: Consultancy, Patents & Royalties. Rosholm:Novo Nordisk: Employment, Equity Ownership. Garly:Novo Nordisk: Employment, Equity Ownership.

Activated, but not resting, platelets are capable of adhering to intact endothelial cells (ECs). We evaluated the effect of a recombinant von Willebrand factor (VWF) fragment AR545C, which inhibits glycoprotein Ib (GPIb)/VWF binding, on platelet adhesion to human ECs under static or flow conditions. Incubation of resting platelets with intact endothelium under flow conditions (350/s) resulted in minimal platelet adhesion. The adhesion was enhanced two- to threefold after either platelet activation by thrombin receptor agonist peptide (TRAP) or EC pretreatment with thrombin. The enhancing effect of thrombin was abolished by addition of either hirudin (10 u/ml) or PGE1 (1 microg/ml). Preincubation of resting platelets with increasing concentrations of AR545C under static or flow conditions resulted in a dose-dependent inhibition of thrombin-induced enhanced adhesion to ECs. AR545C (0.3 microM) completely abolished the effect of thrombin, reducing platelet adhesion to the control level observed with non-treated ECs. In contrast, the same concentration of AR545C had no effect on the adhesion of TRAP-activated platelets to ECs. AR545C also inhibited thrombin-induced platelet aggregation and binding in a dose-dependent manner. In addition, 0.3 microM of AR545C reduced thrombin-induced serotonin release by 57%, whereas monoclonal antibody AN51, which inhibits ristocetin-induced platelet aggregation, had no effect on either thrombin-induced platelet aggregation or binding or on serotonin release. Similarly, AR545C had no effect on TRAP-induced serotonin release. These findings suggest that (i) AR545C inhibits platelet activation mediated by thrombin and this inhibition occurs through blocking the high-affinity thrombin binding sites on the GPIb/IX complex and (ii) AR545C has no effect on the moderate affinity thrombin receptor (seven transmembrane domain thrombin receptor; STDR).

Abstract —In a previous report by Freedman et al ( J Clin Invest. 1996;97:979–987), plasma from 2 brothers with stroke or transient ischemic attack inactivated the antiplatelet effects of nitric oxide (NO), and this effect was found to be a consequence of a deficiency of plasma glutathione peroxidase (GSH-Px). In this study, we attempted to define the generalizability of this deficiency by studying NO-mediated antiplatelet effects in 7 families with familial childhood stroke. Seven families with familial childhood stroke that consecutively presented to a large referral center were included in the study. We monitored ADP-induced aggregation of normal gel-filtered platelets (GFP) in platelet-poor plasma (PPP) from normal individuals and from patients in the presence or absence of an NO donor (S-nitroso–glutathione). Surface P-selectin expression of normal GFP in patients’ PPP was analyzed by flow cytometry after incubation with a P-selectin–specific monoclonal antibody in the presence or absence of the NO donor. We also measured GSH-Px activity in plasmas from family members and normal controls using standard methods. In 6 of 7 families, NO failed to inhibit platelet P-selectin expression and platelet aggregation in PPP from the affected family members and some of their relatives. Of 4 families studied, 3 probands and their corresponding affected parent had 50% decrease in plasma GSH-Px activity. In some patients with childhood stroke, impaired metabolism of reactive oxygen species as a result of reduced GSH-Px activity results in NO insufficiency that affects normal platelet inhibitory mechanisms and predisposes to arterial thrombosis.

Activated protein C resistance (APCR) in the absence of alterations in the factor V gene has been observed during pregnancy, in patients on oral contraceptives, in the presence of antiphospholipid antibodies, and in patients with ischemic stroke. We report a 49-year-old woman with recurrent major venous and arterial thromboses who displayed pronounced APCR, yet no changes in the activated protein C (APC) cleavage sites of factor V. The APCR values determined by four different assays were similar to those obtained in plasma from a homozygote for factor V Q506. Addition of IgG isolated from the patient’s serum to normal plasma lowered the APCR ratio from 2.4 to 1.6. Incubation of patient’s IgG with normal APC resulted in a profound change in the mobility of APC in crossed immunoelectrophoresis. APC was also shown to bind to patient’s IgG immobilized on a protein A agarose column. Factor Va inactivation by APC was inhibited by patient’s IgG, but not by control IgG in the presence or absence of either phospholipids or protein S. These results provide evidence for the existence of an acquired antibody against APC in the patient’s plasma, which gave rise to the APCR phenotype and was probably responsible for the major thrombotic events. We suggest that acquired APCR due to anti-APC antibodies be considered a potential cause for severe venous and arterial thromboses.

Several recent studies evaluated a possible effect of the prothrombotic polymorphisms such as 5,10 methylenetetrahydrofolate reductase (MTHFR) nt 677C → T, factor V (F V) nt 1691G → A (F V Leiden), and factor II (F II) nt 20210 G → A on the risk of myocardial infarction. In the present study, we analyzed the effect of these prothrombotic polymorphisms, as well as apolipoprotein (Apo) E4, smoking, hypertension, diabetes mellitus, and hypercholesterolemia, on the risk of myocardial infarction in young males. We conducted a case-control study of 112 young males with first acute myocardial infarction (AMI) before the age of 52 and 187 healthy controls of similar age. The prevalences of heterozygotes for F V G1691A and F II G20210A were not significantly different between cases and controls (6.3% v 6.4% and 5.9% v 3.4% among cases and controls, respectively). In contrast, the prevalence of MTHFR 677T homozygosity and the allele frequency of Apo E4 were significantly higher among patients (24.1% v 10.7% and 9.4% v5.3% among cases and controls, respectively). Concomitant presence of hypertension, hypercholesterolemia, or diabetes and one or more of the four examined polymorphisms increased the risk by almost ninefold (odds ratio [OR] = 8.66; 95% confidence interval [CI], 3.49 to 21.5) and concomitant smoking by almost 18-fold (OR = 17.6; 95% CI, 6.30 to 48.9). When all atherogenic risk factors were analyzed simultaneously by a logistic model, the combination of prothrombotic and Apo E4 polymorphisms with current smoking increased the risk 25-fold (OR = 24.7; 95% CI, 7.17 to 84.9).The presented data suggest a synergistic effect between atherogenic and thrombogenic risk factors in the pathogenesis of AMI, as was recently found in a similar cohort of women.

A patient with a severe bleeding tendency due to acquired von Willebrand disease (VWD) is presented. Although no underlying disorder has emerged during 6 years of follow-up, an immune-mediated mechanism was responsible for acquired VWD in this patient as demonstrated by detection of von Willebrand factor (VWF)/anti-VWF complexes in the patient's plasma and their removal by protein A-sepharose beads and resumption of normal haemostasis with correction of VWF antigen, VWF activity and VWF multimeric pattern after treatment of the patient with high-dose gammaglobulin. Detection of anti-VWF antibodies in the patient's plasma had a significant impact on the choice of therapeutic intervention to control bleeding.

SummaryPurpura fulminans is associated with homozygous protein C and homozygous protein S deficiency or may follow bacterial or viral infections. We present 2 children from 2 unrelated Arab families with purpura fulminans who were double heterozygotes for factor V Leiden inherited from their fathers and protein S deficiency inherited from their mothers. No previous thrombotic events have occured in either patient or their respective family members. In one patient sepsis accompanied by disseminated intravascular coagulation appeared to be the trigger of purpura fulminans. In the other patient varicella infection preceded purpura fulminans and was also associated with disseminated intravascular coagulation. This report emphasizes the need for evaluation of hereditary defects in the inhibitory mechanisms of blood coagulation in patients with purpura fulminans at any age.

article i nfo Background: Factor XIII (FXIII), a plasma pro-transglutaminase, consists of two A subunits and two B subunits (FXIIIA2B2). Following activation by thrombin, it cross-links fibrin chains at the final step of coagulation. We previously reported that FXIII subunit A (FXIIIA) serves as a protein disulfide isomerase (PDI), and that PDI promotes platelet adhesion and aggregation. Objective: This study sought to examine possible mechanistic effect of FXIII on platelet adhesion to fibrinogen; specifically, the role of its PDI activity. Methods:Exvivoexperiments: Blood platelets derived from five patients with hereditary FXIIIA deficiency before and after treatment with Fibrogammin-P (FXIIIA2B2 concentrate) were washed and incubated on immobilized fibrinogen. Bound platelets were stained and counted by microscopy. In vitro experiments: Platelets derived from patients before treatment and five healthy controls were washed and analyzed for adhesion in the pres- ence or absence of Fibrogammin-P or recombinant FXIII (FXIIIA2 concentrate). Results:Inexvivoexperiments, one hour afterFibrogammin-Ptreatment, mean (±SEM) plateletadhesion to fibrin- ogen increased by 27±2.32% (pb0.001). In in vitro experiments, treatment with Fibrogammin-P or recombinant FXIII (10 IU/mL each) enhanced platelet adhesion to fibrinogen (in patients, by 29.95±6.7% and 29.05±5.3%, respectively; in controls, by 26.06±3.24% and 26.91±4.72, respectively; pb0.04 for all). Iodoacetamide-treated FXIII (I-FXIII), where transglutaminase activity is blocked, showed similar enhanced adhesion as untreated FXIII. By contrast, addition of an antibody that specifically blocks FXIIIA-PDI activity inhibited FXIII-mediated platelet adhesion to fibrinogen by 65%. Conclusion: These findings indicate that FXIII-induced enhancement of platelet adhesion is mediated by FXIII-PDI activity.

The histone deacetylase inhibitor (HDACI) butyroyloxymethyl diethylphosphate (AN-7) synergizes the cytotoxic effect of doxorubicin (Dox) and anti-HER2 on mammary carcinoma cells while protecting normal cells against their insults. This study investigated the concomitant changes occurring in heart tissue and tumors of mice bearing a subcutaneous 4T1 mammary tumor following treatment with AN-7, Dox, or their combination. Dox or AN-7 alone led to inhibition of both tumor growth and lung metastases, whereas their combination significantly increased their anticancer efficacy and attenuated Dox- toxicity. Molecular analysis revealed that treatment with Dox, AN-7, and to a greater degree, AN-7 together with Dox increased tumor levels of γH2AX, the marker for DNA double-strand breaks and decreased the expression of Rad51, a protein needed for DNA repair. These events culminated in increased apoptosis, manifested by the appearance of cytochrome-c in the cytosol. In the myocardium, Dox-induced cardiomyopathy was associated with an increase in γH2AX expression and a reduction in Rad51 and MRE11 expression and increased apoptosis. The addition of AN-7 to the Dox treatment protected the heart from Dox insults as was manifested by a decrease in γH2AX levels, an increase in Rad51 and MRE11 expression, and a diminution of cytochrome-c release. Tumor fibrosis was high in untreated mice but diminished in Dox- and AN-7-treated mice and was almost abrogated in AN-7+Dox-treated mice. By contrast, in the myocardium, Dox alone induced a dramatic increase in fibrosis, and AN7+Dox attenuated it. The high expression levels of c-Kit, Ki-67, c-Myc, lo-FGF, and VEGF in 4T1 tumors were significantly reduced by Dox or AN-7 and further attenuated by AN-7+Dox. In the myocardium, Dox suppressed these markers, whereas AN-7+Dox restored their expression. In conclusion, the combination of AN-7 and Dox results in two beneficial effects, improved anticancer efficacy and cardioprotection.

Acquired thrombotic thrombocytopenic purpura (TTP) is an uncommon disease in adults, characterized by fever, neurological manifestations, microangiopathic hemolytic anemia, thrombocytopenia, renal dysfunction, and the presence of antibodies against the enzyme ADAMTS13. Treatment with plasmapheresis has increased the survival from 10% to more than 90%. Still, there is a subset of patients with resistant TTP who fail to respond to plasmapheresis or remain dependent on this procedure. There is mounting evidence that rituximab may play an important role in remission induction of resistant/relapsing TTP, but the extent of the remission is unknown. We present here four patients with chronic-relapsing TTP who responded favorably to rituximab. All four patients achieved prolonged remission of 23 to 82 months after the treatment. One patient relapsed 6 years afterthe initial treatment with rituximab and re-entered remission following retreatment.

Haemostatic efficacy and pharmacokinetic analysis of solvent/detergent (S/D) treated, virus inactivated plasma (OctaplasTM, Germany) was evaluated in eight patients with hereditary factor VII, X and XI deficiency and in three patients with acquired coagulation disorders due to liver disease. The patients received the S/D plasma for treatment of haemarthrosis, menorrhagia or before surgical procedures. In all the patients the S/D plasma was sufficient to prevent or stop bleeding. Side effects included urticaria (one patient) and moderate anaphylactoid reaction (one patient). No evidence of plasma-born viral infections was observed up to 12 months after the treatment (95% confidence limits 0–22%). Calculated mean half-life of coagulation factors VII, X and XI was 4.36 h, 49.21 h and 44.5 h, respectively, similar to that observed with fresh-frozen plasma. Because of retained coagulation factor integrity and improved viral safety, S/D plasma could be considered a superior alternative to standard fresh-frozen plasma.

von Willebrand factor (vWF) is synthesized almost exclusively by endothelial cells and is stored there as ultra-high-molecular-weight multimers. The vWF multimers that are detected in the plasma are smaller than those stored within the endothelium. In two previous studies, comprising small series of cases with classic Kaposi's sarcoma (KS), an endothelium-derived tumor, increased levels of plasma von Willebrand factor antigen (VWF:Ag, the antigenic structure) were reported, suggesting that vWF:Ag may be a marker of endothelium proliferation.Our purpose was to investigate the quantitative as well as qualitative alterations of plasma vWF in a large series of patients with classic KS at various stages of the disease.Levels of plasma vWF:Ag were studied in 38 patients with classic KS confined to the skin at various stages of the disease and compared with a control group. Thirty-three patients had active KS (i.e., with skin lesions) and five were in remission. In five patients with active KS multimeric analysis of plasma vWF was also performed.The levels of vWF:Ag were significantly higher among KS patients than in the control group (n = 29, p0.01). Levels of vWF:Ag were also significantly higher in patients with active disease as compared with those in remission (p0.05). No correlation was found between vWF:Ag levels and the extent of KS. Analysis of the multimeric pattern of plasma vWF showed enhanced staining of all bands, particularly the intermediate and high molecular weight forms, which resemble the endothelial forms as opposed to normal circulating vWF multimers.Quantitative as well as qualitative alterations in plasma vWF were found in patients with KS, which may reflect the destruction or activation of endothelial cells within the lesions. vWF:Ag may serve as a marker of disease activity in classic KS; however, it is not a good marker for the extent of the disease.

Von Willebrand disease (VWD) is the most common hereditary bleeding disorder, characterized by skin and mucosaL bleeding in most of the cases. In the Western world, the approximate number of bleeding VWD patients is 6.9 to 17 million. The disease is represented by several types with specific treatment for each type. Diagnosis can be troublesome and, therefore, n order to prevent bleeding complications, a professional clinical/laboratory team is required.

Three intragenic restriction fragment length polymorphisms (RFLPs) were used to study linkage and analyse the mode of inheritance in type I and type III von Willebrand's disease (vWD). In two families linkage was established between Sac I RFLPs and the inheritance of type I vWD. RFLP analysis of amniocyte DNA from a potentially affected foetus enabled us to establish a prenatal diagnosis of vWD in a third family with type I vWD. Linkage was also established in four families between the Sac I and two Taq I RFLPs and the inheritance of type III vWD. All type III probands were homozygotes and inherited the same mutant vWF allele from both parents. Heterozygous carriers from one type III family were phenotypically normal and could be detected only by linkage analysis, whereas carriers from the remaining three type III families were asymptomatic but had decreased values of vWF antigen and activity. RFLP-based linkage analysis of vWD alleles provides a way to improve the diagnostic precision, detect carriers, and may be useful for prenatal diagnosis of type III vWD.

Von Willebrand disease (vWD) type IIA is characterized by decreased ristocetin-induced platelet aggregation, and by the absence from plasma of high molecular weight multimers of von Willebrand factor (vWF). Most mutations causing vWD type IIA are clustered within the A2 domain of the mature vWF subunit that is encoded by exon 28. Using the polymerase chain reaction (PCR), the entire exon 28 from patients with vWD type IIA and normal controls was amplified and sequenced. Three missense mutations were detected that result in the amino acid substitutions were detected that result in the amino acid substitutions Arg(834)----Trp, Gly(742)----Glu, and Ser(743)----Leu. The first mutation occurred independently in three unrelated families; each of the latter mutations was found in one family. By restriction endonuclease analysis and allele-specific oligonucleotide (ASO) hybridization the mutations were confirmed in affected family members and excluded in unaffected members and 50 normal controls. The apparently high frequency of identical independent mutations among patients with vWD type IIA suggests that a precise diagnosis may be possible in a majority of patients using relatively simple recombinant DNA screening assays.

Hereditary thrombocythaemia (HT) is an inherited autosomal dominant disorder. Recent studies reported six different mutations, four within the thrombopoietin (TPO) gene and two within c-Mpl (TPO receptor) gene in six unrelated families with HT. This study investigated the molecular basis of hereditary thrombocythaemia in an Israeli-Jewish family. We screened the genes for TPO and c-Mpl by amplification and sequencing of all the corresponding exons including exon/intron boundaries and promoters. In addition, plasma levels of TPO and erythropoietin (EPO) were measured. No abnormality in the TPO/c-Mpl genes has been identified in affected HT family members. Plasma TPO and EPO levels were found to be normal/low or normal respectively in the individuals affected. In conclusion, lack of a molecular lesion within either TPO or cMpl genes indicate that HT may be caused by factors other than TPO-cMpl axis in this family.

SummaryThrombin-activated Factor XIII (FXIIIa),a plasma transglutaminase, stabilizes fibrin clots by crosslinking fibrin chains. FXIIIa was previously shown by us to exhibit proangiogenic activity associated with downregulation of thrombospondin-1, phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR-2), and upregulation of c-Jun. In the current study, we evaluated the proangiogenic effect of FXIIIa in two murine models: a neonatal heterotopic cardiac allograft model in normal mice, and a Matrigel plug model in FXIII-deficient mice. In the neonatal cardiac allograft model, the number of new vessels as well as graft viability (contractile performance) was significantly higher in FXIIIa-injected animals than in controls. A significant increase in the level of c-Jun mRNA and a significant decrease in the level of TSP-1 mRNA were observed in heart allografts treated with FXIIIa. A marked decrease in TSP-1 protein expression was observed within the endothelial cells of hearts treated with FXIIIa. In the Matrigel plug model, FXIII-deficient mice showed a significantly decreased number of new vessels compared to that of the control mice,and the number of vessels almost reached normal levels following addition of FXIIIa. The results of this study provide substantial in vivo evidence for the proangiogenic activity of FXIIIa.

[Graphic][1] Introduction Recombinant FXIII (rFXIII) represents a new treatment opportunity for patients with congenital FXIII A-subunit deficiency. Monthly prophylaxis with 35 IU/kg rFXIII was shown to effectively control bleeding with an annualized bleeding rate (ABR) of 0.138 bleeds requiring treatment per patient per year and an excellent safety profile (Inbal A, et al. Blood 2012;119:5111-7). PK analysis revealed first-order elimination of rFXIII with a geometric mean half-life of 13.6 days. All patients had a mean FXIII trough activity level of >0.1 IU/mL (Kerlin B, et al. JTH 2013;11:235). The PK profile was independent of age, supporting that monthly dosing with a fixed 35 IU/Kg rFXIII regimen for all patients with FXIII A-subunit deficiency (regardless of age) was adequate for prophylaxis (Brand-Staufer B, et al . Blood 2013;122:3613. Williams M, et al. Haemophilia 2014;20:99–105). Due to the novel nature of this recombinant molecule, a phase 3b safety extension program was offered to bridge to product availability, the extensive interim results of which are analyzed here. Methods The mentorTM2 trial is an ongoing safety extension trial to the pivotal mentorTM1 trial (Figure). All patients were dosed with 35 IU/kg rFXIII every 4th week. ![Figure.][2] Figure. The Novo Nordisk clinical trial program for recombinant Factor 13 A planned interim analysis for mentorTM2 trial was performed (data cut-off: 31-DEC-2013). The Berichrom® FXIII activity assay was used for measurement of FXIII activity. Results Sixty patients have been enrolled into mentorTM2; baseline patient demographics are presented in the Table. Of these 60, 34 patients were enrolled from the mentorTM1 trial, and 26 new patients were enrolled. | Age, median (range) | 26 (7-77) | || | Age, mean (range) | 31 (7-77) | | Male sex, n (%) | 38 (63) | | Race, n (%) Black or African American American Indian or Alaska Native White Asian* Other Unknown** | 6 (10) 1 (2) 34 (57) 9 (15) 6 (10) 4 (7) | | Body Mass Index, median (range) | 23.7 (12.8-36.9) | | Height in cm, median (range) | 167.0 (131.0-187.5) | | Weight in kg, median (range) | 67.5 (22.0-119.4) | Table. * Including 5 Japanese ** French patients are marked as unknown as per the French authorities guideline In mentorTM2, 60 patients had 2,157 exposures (monthly dosing), corresponding to a total of 168 patient years. The ABR was 0.042 bleeds/patient/year overall, 0.012 bleeds/patient/year for spontaneous bleeds, and 0.030 bleeds/patient/year for traumatic bleeds. In total 6 patients experienced 7 bleeds requiring FXIII treatment (5 trauma-induced and 2 spontaneous). Of these 7 bleeds, 1 trauma-induced muscular bleed was treated with rFXIII with excellent hemostatic response. No intracranial, internal organ or severe gastrointestinal bleeds occurred during the trial period. Mean FXIII trough levels were greater than 0.10 IU/mL in all patients. No thromboembolic events, fatal adverse events or adverse events leading to withdrawal were reported. Serious adverse events were few (16 events in 10 patients) and were evaluated by the investigators as unlikely related to trial drug. No anti-rFXIII antibodies were detected. Discussion Prophylaxis of bleeding of patients with congenital FXIII A-subunit deficiency with rFXIII in the mentor™ trials program has demonstrated very effective bleed control, with an excellent safety profile. The ABR in the ongoing mentorTM2 safety extension trial was 0.042, which is lower than the rate of 0.138 seen in the mentor™1 pivotal study. A bleeding rate of 0.042 corresponds to an average patient having 1 bleed approximately every 24 years. One patient who had a traumatic breakthrough bleed was treated with rFXIII with excellent outcome. These efficacy data, combined with comprehensive PK- and safety data, represent the largest data collection in congenital FXIII A-subunit deficiency in the world, and provide extensive evidence for the safety and efficacy of monthly prophylaxis with 35 IU/kg rFXIII. Disclosures Fukutake: Novo Nordisk: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Baxter: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bayer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Biogen Idec: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kaketsuken: Consultancy, Honoraria, Research Funding, Speakers Bureau; CSL Behring: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; SRL, Inc: Consultancy; LSI Medience Corporation: Consultancy, Honoraria, Speakers Bureau; Chugai Pharm: Honoraria, Membership on an entity's Board of Directors or advisory committees; TORII PHARMACEUTICAL CO., LTD.: Honoraria; Sekisui Medical CO., LTD: Honoraria, Research Funding, Speakers Bureau. Carcao: Baxter, Bayer, Biogen, Novo Nordisk, Pfizer, CSL Behring, Octapaharma : Honoraria, Research Funding, Speakers Bureau. Kerlin: Bayer HealthCare US: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees. Oldenburg: Baxter: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biogen Idec: Honoraria, Membership on an entity's Board of Directors or advisory committees; Biotest: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Grifols: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Swedish Orphan Biovitrum: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Rosholm: Novo Nordisk: Employment, Equity Ownership. Garly: Novo Nordisk: Employment, Equity Ownership. Nugent: Novo Nordisk: Membership on an entity's Board of Directors or advisory committees, Research Funding. [1]: /embed/inline-graphic-2.gif [2]: pending:yes

Background: Refractoriness to platelet transfusion is prevalent among 15-25% of hemato-oncology patients. Refractoriness has been linked to inferior clinical outcomes, including bleeding and mortality, as well as higher health care costs. Suggested etiologies to refractoriness include both non-immune and immune causes. Methods to manage refractoriness include leuko-reduction, HLA- and HPA- matched platelets, use of ABO compatible transfusions and platelet cross-matching. Leuko-reduction has been proven to decrease the rates of allo-immunization and is widely used in hemato-oncology units. Other methods have demonstrated modest effectiveness in some studies, but some of these (HLA- and HPA- matched platelets) are not widely available. There are only a few reports in the literature of continuous platelet transfusion (CPT) as an alternative method for increasing post-transfusion platelet increments. Those reports prompted the current analysis of CPT in platelet refractory patients. Aim: To evaluate the effectiveness of CPT in producing satisfactory platelet increments in refractory patients compared to the standard care of routinely prepared single donor platelet transfusions. Patients and Methods: Patients included in this study were treated for hematological malignancies in our Institution between January 2007 and December 2013. A retrospective analysis of the increment of platelets achieved in refractory patients was performed. Refractoriness was defined as a corrected calculated increment (CCI) less than 10,000 platelets per micro-liter following two successive platelet transfusions. The CCI was calculated as PPI (post-transfusion platelet count minus pre-transfusion platelet count) x BSA (body surface area measured in square meters m2) x 1011/number of platelets transfused. All refractory patients included in this analysis received single donor platelet transfusions. The practice of CPT was adopted by us in March 2008. All refractory patients who received CPT between March 2008 and December 2013 were included. These patients received a continuous 24 hour transfusion of single donor platelet units, each dose given over 4 to 6 hours, comprising of 1.5 x 1011 platelets and equaling to half a single donor platelet unit. Their outcome was compared with that of our refractory patients treated with single donor transfusions in the routine manner, between January 2007 and March 2008 (i.e. before the introduction of CPT). The CCI was used to monitor the effectiveness of each platelet unit at 12 and 24 hours post-transfusion intervals. To account for prior antigen-exposure of the patients, we chose to include for each patient the 11th consecutive platelet transfusion. Factors known to contribute to the development of refractoriness, such as infection, disseminated intravascular coagulation (DIC) and splenomegaly were evaluated for impact on the CCI. The statistical analysis was generated using SAS Software, Version 9.4. Continuous variables were presented as mean ± STD, Categorical variables were presented by (N, %). t-tests and non-parametric Wilcoxon tests were used to compare the value of continuous variables between study groups. Results: Twenty eight patients with hematologic malignancies received at least eleven single-donor platelet transfusions during 2007-2013. Twenty one of the 28 patients received CPT due to refractoriness and seven patients were treated before the introduction of this approach. CPT resulted in a significantly higher post-transfusion mean increment at 24 hours (1.16 vs. 0.37, p Conclusions: CPT results in significantly higher increments at 24 hours post-transfusion, and therefore suggest an effective approach to the treatment of platelet refractoriness in patients treated for hematological malignancies. Disclosures No relevant conflicts of interest to declare.