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Inherited afibrinogenemia and hypofibrinogenemia are rare bleeding disorders characterized by markedly reduced levels of fibrinogen in blood. Thrombotic complications in these disorders have been rarely described. We performed a multicenter retrospective study and reviewed the occurrence of thrombotic complications among patients with inherited fibrinogen deficiency. Cases were identified during a review of medical records of all patients with inherited fibrinogen deficiency followed at three different university hospitals in Israel. Nine patients were included in this study: five were afibrinogenemic and four hypofibrinogenemic. There were seven thrombotic events, mostly venous, that occurred in four out of nine patients (44 %). All thrombotic events occurred in afibrinogenemic patients. Mean age at the time of thrombosis was 45 (range 28-61) years. Thrombophilic evaluation performed was negative in all cases. At the time of thrombosis in five out of seven (71.4 %) events, fibrinogen replacement therapy was concurrently given. Therapeutic approach was different among patients ranging from supportive therapy alone, antiplatelet agents and anticoagulant therapy with the concurrent administration of fibrinogen replacement therapy. This study discloses a high rate of thrombosis in patients with afibrinogenemia. Events were both venous and arterial and may be recurrent. Management is highly problematic due to the precarious balance between bleeding and thrombotic risk in these patients. Fibrinogen replacement therapy should be cautiously used in these patients as most thrombotic events followed the administration of fibrinogen replacement therapy. Larger cohorts are warranted to better characterize the best management strategy in these paradoxical events.

The phenotype of bleeding in patients with severe FXI deficiency is unpredictable and unlike other bleeding disorders, it is not directly correlated with levels of FXI. In this study we analyzed whether the global coagulation assays can serve as a clinical tool in predicting bleeding tendency in patients with severe FXI deficiency undergoing surgery, taking into account the large inter-individual variability of FXI levels and genotypes. Thrombin generation (TG) was measured in 39 platelet-poor plasma with or without tissue factor (TF) and in the presence or absence of corn trypsin inhibitor (CTI). Rotation thromboelastometry (ROTEM) was performed with fresh whole blood of 26 patients applying NATEM and INTEM tests. TG induced by recalcification can distinguish between bleeding and non-bleeding patients with severe FXI deficiency particularly among those with FXI activity of 2-20IU/dl. The addition of TF or TF and CTI to the TG assay masked the ability to differentiate between XI activity, genotype as well as bleeding and non-bleeding patients. ROTEM assays failed to distinguish bleeding from non-bleeding patients but could do so between different FXI activity levels and genotypes. In conclusion, in the current study we found a sensitive tool to distinguish between bleeding and non-bleeding patients. In order to recommend TG as a predictive tool for treatment tailoring, a larger patient group is required.

Summary Treatment of haemophilia A patients with inhibitors is challenging, and may require individually tailored regimens. Whereas low titre inhibitor patients may respond to high doses of factor VIII (FVIII), high-responding inhibitor patients render replacement therapy ineffective and often require application of bypassing agents. Thrombin generation (TG) assays may be used to monitor haemostasis and/or predict patients' response to bypass agents. In this study we defined by TG, the potential contribution of FVIII to recombinant activated factor VII (rFVIIa)-induced haemostasis in inhibitor plasma. Based upon results, prospectively designed individual regimens of coadministration of rFVIIa and FVIII were applied. Plasma samples from 14 haemophilia patients with inhibitors (including high titre inhibitors) were tested. The response to increasing concentrations of FVIII, rFVIIa or both was assayed by TG. Eight patients, chosen following consent and at physician's discretion, comprised the combined FVIII–rFVIIa therapy clinical study cohort. Combined spiking with FVIII/rFVIIa improved TG induced by rFVIIa alone in all inhibitor plasmas. Combined rFVIIa and FVIII therapy was applied during bleeding or immune tolerance to eight patients, for a total of 393 episodes. Following a single combined dose, 90% haemostasis was documented and neither thrombosis nor any complications evolved. During study period decline of inhibitor levels and bleeding frequency were noted. Pre-analytical studies enabled us to prospectively tailor individual therapy regimens. We confirmed for the first time that the in vitro advantage of combining FVIII and rFVIIa, indeed accounts for improved haemostasis and may safely be applied to inhibitor patients.

We have investigated the feasibility of continuous infusion of undiluted factor IX (F IX) over several days using minipumps. The stabilities of seven different reconstituted F IX products were substantially better than those declared by the manufacturers. Several concentrates maintained factor activities 80% of baseline for the entire period of 4 weeks at 4-8d̀C as did one product at 20-23d̀A. At 37d̀C the latter concentrate was stable for at least 1 week. The stability seemed to correlate with the purity of the product. Analysis of two prothrombin comples concentrates by gel electrophoresis demonstrated degradation of prothrombin to prethrombin-1 and fragment 1 at 37d̀C and in one of the concentrates also at 20-23d̀C. In two F IX concentrates the corresponding analysis did not reveal any degradation. Four patients were treated with continuous infusion with a pure F IX concentrate (Mononine™, Armour) after surgery or for serious haemorrhage (two each) with good haemostatic effect, an initial progressive decrease of the F IX clearance, and no side-effects. Continuous infusion with F IX, using a minipump and undiluted reconstituted factor, is therefore feasible and effective, and can be conveniently prepared for several days at a time. Pure F IX products are more stable and probably safer for this purpose.

Factor X (FX) is one of the vitamin K-dependent serine proteases, which forms the prothrombinase complex converting prothrombin into thrombin. To search for mutations in F10 gene giving rise to severe FX deficiency and to study the contribution of thrombin generation and thromboelastometry as a tool for evaluation of hemostasis. Mutations in the F10 gene were sought by direct sequencing of all the eight exons and intron/exon boundaries. Thrombin generation and thromboelastometry were performed. Three unrelated Palestinian patients had undetectable FX level (

See also Duga S, Asselta R. Mutations in disguise. This issue, pp 1973–6. Summary. Background: Point mutations within exons are frequently defined as missense mutations. In the factor (F)XI gene, three point mutations, c.616C>T in exon 7, c.1060G>A in exon 10 and c.1693G>A in exon 14 were reported as missense mutations P188S, G336R and E547K, respectively, according to their exonic positions. Surprisingly, expression of the three mutations in cells yielded substantially higher FXI antigen levels than was expected from the plasma of patients bearing these mutations. Objectives: To test the possibility that the three mutations, albeit their positions within exons, cause splicing defects. Methods and results: Platelet mRNA analysis of a heterozygous patient revealed that the c.1693A mutation caused aberrant splicing. Platelet mRNA of a second compound heterozygote for c.616T and c.1060A mutations was undetectable suggesting its degradation. Cells transfected with a c.616T minigene favored production of an aberrantly spliced mRNA that skips exon 7. Cells transfected with a mutated minigene spanning exons 8–10 exhibited a significant decrease in the amount of normally spliced mRNA. In silico analysis revealed that the three mutations are located within sequences of exonic splicing enhancers (ESEs) that bind special proteins and are potentially important for correct splicing. Compensatory mutations created near the natural mutations corrected the putative function of ESEs thereby restoring normal splicing of exons 7 and 10. Conclusions: The present findings define a new mechanism of mutations in F11 and underscore the need to perform expression studies and mRNA analysis of point mutations before stating that they are missense mutations.

SummaryFactor XI (FXI) plays a dual role in haemostasis and thrombosis. It contributes to thrombin generation and promotes inhibition of fibrinolysis. Severe FXI deficiency was shown to confer protection against arterial and venous thrombosis in animal models without compromising haemostasis. We have previously shown that patients with severe FXI deficiency have a low incidence of ischaemic stroke, but display the usual incidence of myocardial infarction. In the present study, we compared the incidence of deep-vein thrombosis (DVT) in 219 unrelated patients with severe FXI deficiency aged 20–94 to the incidence in a large population-based study. No cases of DVT were observed in the FXI-deficient cohort, a result that is significantly lower than the expected number (4.68) computed from the population-based study. The low incidence remains statistically significant when compared to three other population-based studies. These data suggest that severe FXI deficiency provides protection against DVT.

Venous thromboembolism (VTE) is a well-recognized complication of Acute Traumatic Spinal Cord Injury (ATSCI). Despite prophylaxis by heparins, VTE occurs in a substantial number of ATSCI patients without an obvious explanation. In this matched case-control study we examined whether thrombophilia and other risk factors are associated with failure of thromboprophylaxis. Cases and controls receiving heparin thromboprophylaxis were selected from consecutively admitted ATSCI patients. Patients who developed a new, objectively confirmed, symptomatic VTE despite prophylaxis at hospital were matched by gender, age, level and mechanism of ATSCI with 2-3 controls without VTE. Patients were interviewed about VTE risk factors and tested for factor V Leiden (FVL), prothrombin G20210A (PT), methylenetetrahydrofolate reductase C677T homozygosity (MTHFR), lupus anticoagulant, homocysteine (Hcy) and plasma factor VIII (FVIII) levels. Twenty-two patients with new VTE episodes and 64 controls were ascertained. The total number of gene alterations for MTHFR, FVL and PT or elevated levels of Hcy or FVIII was significantly more common in patients compared to controls (82% vs. 48%, p = 0.006). Multiple logistic regression proved the PT mutation, a positive family history of thrombosis and elevated levels of either FVIII or Hcy to be predictors of thrombosis. Conclusion A positive family history of VTE, carriership of the prothrombin mutation and elevated FVIII or Hcy levels were significantly associated with failure to prevent VTE by heparin therapy following ATSCI. Testing for thrombophilia in patients with ATSCI and possibly a more intense thromboprophylactic regimen seem desirable but need to be verified by a prospective study.

Bleeding is a rare manifestation of antiphospholipid syndrome, unless associated with reduced clotting factors or severe thrombocytopenia. Accurate assessment of the autoantibodies in plasma is very important since the autoantibodies can lead to bleeding or thrombosis. The objective of the present study was to define the inhibitors causing reduced clotting activity in a patient with antiphospholipids antibodies and to assess the potential of thrombin generation assay to assist in establishment of optimal treatment in case of major bleeding. Levels of clotting factors as well as inhibitors to factors II, V, VII, VIII, IX, X and XI were defined. For detection of inhibitors to prothrombin crossed immunoelectrophoresis was used. IgG was purified by commercial protein A column. Thrombin generation was measured using a fluorometric assay in platelet-poor and platelet-rich plasma. Inhibitors toward the activity of factors V, VII, VIII, IX, X and XI were defined and also an inhibitor to prothrombin antigen. No thrombin generation was induced in the patient's plasma by recalcification even in the presence of recombinant factor VIIa or factor VIII inhibitor bypassing activity. In contrast, addition of platelets from either donor or patient or synthetic phospholipids normalized the thrombin generation. The thrombin generation model showed that the addition of platelets and no recombinant factor VIIa or factor VIII inhibitor bypassing activity would correct thrombin generation in vitro. On this basis, platelet concentrates were administered to a patient with bleeding caused by lupus anticoagulant and low clotting factors activity.

Patients with severe haemophilia A and an inhibitor may become refractory to FEIBA and/or recombinant factor VIIa (rFVIIa). Sequential therapy with both products has been reported in such patients. In this pilot study, we examined the safety and efficacy of combined rFVIIa and FEIBA therapy in patients with haemophilia A and inhibitors during bleeding episodes. We also tried to evaluate whether thrombin generation (TG), by various mixtures of these agents, can serve as a guide for tailoring therapy. TG was measured in plasma taken from eight haemophilia A patients. Increasing concentrations of rFVIIa, FEIBA or both were added ex vivo to the plasmas, and TG was induced by recalcification. Since low concentrations of rFVIIa and FEIBA had either an additive or a synergistic effect in all patients, the lowest combination, yielding TG comparable or lower than TG achieved with either FEIBA 100 U kg(-1) or rFVIIa 160 microg kg(-1) alone, was selected for the treatment of bleeding episodes. Five patients with a high titre of an inhibitor (8-1300 BU), including one previously refractory to infusions of rFVIIa at doses up to 400 microg kg(-1) X4 daily, were treated with combinations of 30-70 microg kg(-1) rFVIIa and 20-30 U kg(-1) FEIBA during a total number of 400 bleeding episodes with excellent haemostatic effect. No adverse events and no DIC were observed following these infusions. Concomitant infusion of low-dose rFVIIa and low-dose FEIBA, seems to be safe, efficacious and economical in patients refractory to rFVIIa and probably other haemophilia A patients with an inhibitor.

Summary. Inherited factor VII (FVII) deficiency is a rare autosomal recessive disorder with a wide heterogeneous clinical pattern. Intracranial haemorrhage in infants has been previously reported in the severe form of the FVII deficiency and it has a high fatality rate. We report a family with high consanguineous relations, who experienced death of two baby girls, the first with prenatal manifestation of foetal hydrocephalus secondary to intracranial bleeding and the second with postnatal intracranial bleeding, both with less than 1% activity of FVII. Genetic analysis revealed that both parents are heterozygous and both daughters homozygous for a point mutation gG9639A in exon 7, predicting Gly180Arg substitution. This mutation was described previously in a compound heterozygous patient with mild bleeding manifestation. It seems that in this family, the mutation in its homozygous state is fatal and the lethal clinical expression can appear in utero at an early stage of gestation.