Bleeding In Hemophilia Research Articles

Multiple bleeds in haemophilia A.

One hundred and eighty-one bleeding episodes involving two sites simultaneously were noted during a survey of 4935 bleeding episodes, an incidence of 3.7%. Elbows, knees and ankles were the commonest sites involved in double bleeds, while the thigh, upper arm and elbow were the commonest sites involved in the double bleeds needing most transfusions. The overall transfusion requirements were less than for single bleeds. The frequency of multiple bleeds correlated significantly with the overall bleeding frequency, but not with the number of days under observation.

Hemostatic effect of recombinant factor VIIa, NN1731 and recombinant factor VIII on needle-induced joint bleeding in hemophilia A mice.

Hemophilia A is the most common serious bleeding disorder, and the hallmark of this disease is joint bleeding episodes. These result in hemophilic synovitis, an inflammatory and proliferative condition of the joint, which progresses into a chronic degenerative arthritis, hemophilic arthropathy. In this paper, we describe the effect of recombinant factor VIIa (rFVIIa), and an analogue NN1731 as well as rFVIII on needle-induced bleeding in hemophilia A mice. Here we show a reducing effect of rFVIIa and NN1731 on bleeding induced in hemophilic mice, and we show that preventive treatment with rFVIII normalizes bleeding.

Operative treatment of hemophilic arthropathy in the knee (author's transl)

Within the knee occur 27.7% of all hemophilic bleeding episodes. Therefore it is the organ in which hemophilia is likely to be manifested. Changed in the sense of hemophilic arthropathy made 117 operative interventions necessary in the years 1973 through 1981. The osteotomies, performed to correct axial deviations (38 high tibial head osteotomies and 22 supracondylar osteotomies) formed the biggest group, followed by 34 hamstring-release operations, 6 synovectomies, 5 temporary epiphysiodeses, 4 ventralisations of the tibial tuberosis. An endoprosthetik replacement was necessary in only one case. A follow-up study after osteotomies and hamstring-release procedures after one to eight years, correspondingly six months to eight years, gives good results, concerning improved motility, subjective satisfaction and reestablishment of stabilizing muscular performance. In presence of sufficiently controlled substitution of the missing clotting factors, there is no increased operative risk, compared with non-hemophiliacs.

Perivascular tissue factor is down-regulated following cutaneous wounding: implications for bleeding in hemophilia

Healing of skin wounds is delayed in hemophilia B (HB) mice. HB mice do not bleed excessively at wounding, yet rebleed hours to days later. Tissue factor (TF) expression is up-regulated by inflammatory cytokines and has been linked to angiogenesis. We hypothesized that impaired thrombin generation in HB leads to impaired TF expression following injury. Punch biopsies were placed on wild-type (WT) and HB mice. Tissues from wound sites were immunostained for TF. Blood vessels are normally surrounded by a coat of pericytes expressing TF. Surprisingly, within a day after wounding TF disappeared from around nearby vessels; returning after 8 days in WT and 10 days in HB mice. The granulation tissue filling the wound during healing also lacked TF around angiogenic vessels. Thus, perivascular TF expression is down-regulated during wound healing. This may prevent thrombosis of neovessels during angiogenesis but renders hemophiliacs vulnerable to hemorrhage during healing.

Understanding the therapeutic action of recombinant factor VIIa in platelet disorders

Recombinant factor VIIa (rFVIIa) is a pro-haemostatic agent that is effective in the treatment of bleeding in haemophilia complicated by inhibitors and some other haemorrhagic disorders. The rFVIIa product promotes haemostasis in these settings by enabling the rapid generation of thrombin on the surface of activated platelets which increases fibrin deposition. rFVIIa also has therapeutic activity in bleeding in platelet function disorders such as Glanzmann's thrombasthenia and thrombocytopenia. This suggests that rFVIIa also has a pro-haemostatic effect on platelets and this may contribute to the overall therapeutic action of this agent. The effects of high-concentration FVIIa on platelets have been studied in detail in experiments using ex vivo model systems of haemostasis under static and flow conditions. These experiments show that FVIIa potentiates multiple functional markers of platelet activation and this effect requires direct binding of FVIIa to the activated platelet membrane and the generation of thrombin. The mechanism of this potentiating effect of FVIIa on platelet activation is complex and is likely to require multiple platelet activation receptors and signalling pathways. These include the GPIb-IX-V glycoprotein complex and the thrombin protease activated receptors. The proposed mechanism of action of high-concentration FVIIa has important implications for the therapeutic activity of rFVIIa in vivo. This review summarizes the clinical and experimental evidence supporting a potentiating effect of FVIIa on platelet activation and discusses the likely mechanism of action.

Experimental animal use in the study of haemophilic bleeding

Experimental animal use in the study of haemophilic bleeding

Prevention of bleeding in haemophilia: trends, overcoming barriers and future treatment options

Prevention of bleeding in haemophilia: trends, overcoming barriers and future treatment options

Intracranial bleeding in haemophilia beyond the neonatal period – the role of CT imaging in suspected intracranial bleeding

We conducted a review of a single institutional experience of patients with haemophilia presenting with suspected intracranial haemorrhage (ICH) who underwent computed tomographic (CT) neuro-imaging. We found that over a 9-year period (1996-2004) 43 patients with haemophilia presented 73 times with suspected ICH: 10 presented multiple times (range: 2-9 times). The median age at presentation was 3.5 years (range: 0.5-17). Preceding trauma occurred in most (62/73; 85%) episodes. ICH was confirmed in 11 of the 73 (16%) episodes in eight patients. Patients with severe haemophilia accounted for a disproportionate number of episodes of suspected (60/73; 82%) and of confirmed ICH (10/11; 91%). All ICH occurred in patients not on prophylaxis; five occurred in three inhibitor-positive patients. Altered consciousness at presentation was present in 10/11 (91%) cases of confirmed ICH but only in 5/62 (8%) (ICH-negative) episodes. The positive and negative predictive values of altered consciousness to predict/rule out an ICH was 67% and 98%, respectively. The following were associated with an increased risk of presenting with suspected ICH and of having a confirmed ICH: (i) having severe haemophilia; (ii) not being on prophylaxis; (iii) having an inhibitor; and (iv) presenting with an altered level of consciousness. Patients without any of these features may not need to undergo CT imaging when presenting with suspected ICH. Ideally a prospective study to evaluate this hypothesis should be conducted.

Use of Recombinant Factor VIIa During Excision of Vascular Anomalies

The surgical excision of vascular anomalies is often accompanied with significant perioperative bleeding. Novel hemostatic agents, including recombinant factor VIIa (rVIIa), have been shown to reduce bleeding in hemophilia and trauma patients along with decreasing blood loss during various surgical procedures. The role of rVIIa during excision of vascular anomalies has not been examined. A retrospective chart review of patients from 2001 to 2003 who received perioperative rVIIa during excision of vascular anomalies at one institution. Nine patients were identified who received perioperative rVIIa during removal of their vascular anomalies (7 venous malformations, 1 lymphatic-venous malformation, 1 arteriovenous malformation). All patients received at least one dose of rVIIa during the perioperative period (2/9 received 2 doses). An accurate account of hourly blood loss was reported, and could be graphed, in three patients. These patients displayed significant reductions in hourly blood loss after the administration of rVIIa. These cases are detailed in this report. Surgeons reported subjective improvements in operative bleeding, efficiency, and operative time in six of six patients after the administration of rVIIa intraoperatively. Reduced postoperative bleeding was reported in two patients who received rVIIa for persistent drain output. rVIIa may be an effective adjunct in improving the surgical efficiency and outcome of excised vascular anomalies.

Recombinant Activated Factor VII: Current Perspectives and Epworth Experience

Recombinant factor VII was first developed for control and prevention of bleeding in haemophilia with antibodies to factor VIII and IX. Its efficacy in these situations is well established. Over recent years, the 'off-label' use of this substance has become more widespread in trauma, surgery and obstetric bleeding. Here we describe the mechanism of action of recombinant factor VII, review current literature of 'off-label' usage and our initial experience in surgical related cases of bleeding at Epworth Hospital.

Treatment of Bleeding in Hemophilia

Treatment of Bleeding in Hemophilia

Tissue Factor Is Absent from Sites of Cutaneous Wounds: Implications for Bleeding in Hemophilia.

Tissue factor (TF) plays an important role in hemostasis. Its expression is upregulated by a variety of proinflammatory cytokines and thus can link inflammation with thrombosis. We have been studying wound healing in hemophilia B mice. Many of the hemophilic mice do not bleed more than wild type mice at the time of wounding. However, hemophilia B mice show a pattern of bleeding 12 to 24 hours after wounding; in some cases this bleeding is quite severe. We hypothesized that the level and pattern of TF expression might be a major determinant of the bleeding response to cutaneous wounding, since enhanced activity of the TF pathway could overcome the lack of the intrinsic factor X-activating complex (FVIIIa/FIXa) in hemophilia. Briefly, the wound healing model is carried out as follows: a single three mm punch biopsy was placed on the dorsal skin of each C57BL/6 wild-type or FIX knockout mouse. Tissue from around the wound was collected at different days, examined histologically and immunostained with a rabbit polyclonal antibody against the extracellular domain of mouse TF (anti-whole TF, the kind gift of Dr. Mirella Ezban, Novo Nordisk A/S) and a rabbit polyclonal antibody to desmin, a marker for pericytes (Abcam Inc, Cambridge, MA). Under normal conditions, non-capillary blood vessels are surrounded by a prominent TF coat due to expression on pericytes and adjacent adventitial tissues. However, TF surprisingly disappeared from the pericyte coat around blood vessels near the wound by one day after wounding. The TF expression returned around these vessels by six to eight days after wounding. One possible explanation is that the TF-bearing pericytes disappeared, either by migrating away or by apoptosis. We found that desmin-positive cells were present around the vessels in both the absence and presence of TF, indicating that pericytes were present even when TF expression was absent. Furthermore, we found that the highly vascular granulation tissue that grew in to fill the defect at the punch biopsy site was also devoid of TF antigen - through the latest time points sampled at 20 days after wounding - even though the new vessels were surrounded by a sheath of pericytes. We propose two hypotheses that might account for these findings:Pericytes may downregulate TF expression by an as yet undetermined pathway during angiogenesis and wound healing; and/orthe inflammatory cell infiltrate migrating through the vessel walls to the wound area may proteolytically cleave TF antigen from the cell membrane.Thus, tissues in the area of a skin wound have a profound depression of TF expression that may serve to prevent thrombosis of the fragile neovessels as they develop and remodel during healing. Previous studies in this model (Blood 2006; DOI 10.1182/blood-2006-05-020495) have shown that hemophilia B mice have evidence of significant ongoing bleeding around the wound site and in the granulation tissue, while wild type mice have only occasional extravasation of red cells in the granulation tissue. Thus we speculate that absence of TF around new vessels is not a significant liability to hemostatically competent individuals. However, the lack of TF renders hemophilic subjects highly vulnerable to recurrent hemorrhage during this critical period of healing.

Immune response after neonatal transfer of a human factor IX-expressing retroviral vector in dogs, cats, and mice

IntroductionGene therapy could prevent bleeding in hemophilia. However, antibodies could inhibit coagulation, while cytotoxic T lymphocytes could destroy modified cells. The immaturity of the newborn immune system might prevent these immune responses from occurring after neonatal gene therapy. Materials and methodsNewborn dogs, cats, or mice were injected intravenously with a retroviral vector expressing human Factor IX. Plasma was evaluated for antigen and anti-human Factor IX antibodies. Cytotoxic T lymphocyte responses were evaluated indirectly by analysis of retroviral vector RNA in liver. Lymphocytes were evaluated for cytokine secretion and the ability to suppress an immune response to human Factor IX in mice. Results and conclusionsHemophilia B dogs that achieved 942±500 ng/ml (19% normal) or 5±0.4 ng/ml (0.1% normal) of human Factor IX in plasma only bled 0 or 1.2 times per year, respectively, and were tolerant to infusion of human Factor IX. Normal cats expressed human Factor IX at 118+/−29 ng/ml (2% normal) in plasma without antibody formation. However, plasma human Factor IX disappeared at late times in 1 of 4 cats, which was probably due to a cytotoxic T lymphocyte response that destroyed cells with high expression. C3H mice were tolerant to human Factor IX after neonatal gene therapy, which may involve clonal deletion of human Factor IX-responsive cells. These data demonstrate that neonatal gene therapy does not induce antibodies to human Factor IX in dogs, cats, or mice. The putative cytotoxic T lymphocyte response in one cat requires further study.

Recombinant factor VIIa in paediatric bleeding disorders – a 2006 review

Recombinant factor VIIa (rFVIIa) has been used in haemophilia bleeding since its introduction in 1996. It has been found to be safe and effective in the majority of patients with haemophilia who have developed inhibitors. There is increasing use of rFVIIa in many off-label bleeding conditions, but there is a paucity of randomized studies regarding the use of rFVIIa in children. This review will attempt to address and summarize the studies focusing on the role of rFVIIa in both haemophilia and non-haemophilia bleeding conditions in children. rFVIIa has been administered as both bolus and continuous infusions, and at varying doses. Furthermore, adverse events have not reportedly increased in children despite growing experience with its use in the paediatric population.

Low Molecular Weight Activated Protein C Inhibitors as a Potential Treatment for Hemophilic Disorders

The synthesis and evaluation of inhibitors of activated protein C (aPC) are reported. This serine protease is partly responsible for the degradation of factor VIIIa, involved in the regulation of bleeding in hemophilia A. Benzamidine-containing derivatives were found to be potent aPC inhibitors, some of them showing selectivity against the procoagulant protease thrombin. Moreover, compound 1 significantly restored the generation of thrombin in hemophiliac plasma.

Manipulation of prothrombin concentration improves response to high‐dose factor VIIa in a cell‐based model of haemophilia

Clinical reports suggest that treatment regimens employing both activated prothrombin complex concentrates (aPCCs) and recombinant activated factor VII (rFVIIa) may control the bleeding in patients with haemophilia who fail to respond to either agent alone. We hypothesised that increased concentrations of prothrombin, as may be observed after the infusion of aPCCs, favourably influence parameters of thrombin generation in haemophilia treated with high-dose rFVIIa. We examined the effect of varied prothrombin and rFVIIa concentrations on thrombin generation in a model of haemophilia. At all concentrations of rFVIIa, increased prothrombin concentrations led to increases in the peak and rate of thrombin generation. In assays with the highest concentrations of prothrombin and rFVIIa, peak thrombin actually equalled that measured in the model of normal haemostasis. The significant impact of prothrombin concentration on the effect of rFVIIa in vitro may explain the improved haemostasis reported with concurrent use of aPCCs and rFVIIa. These results imply that persons with plasma prothrombin levels at either end of the 'normal' range could have significantly different responses to similar rFVIIa doses. Furthermore, these results suggest that increasing plasma prothrombin concentration prior to rFVIIa administration may offer advantages over the use of rFVIIa alone in the treatment of haemophilic bleeding.

Liver haemorrhage in haemophilia – a case report and review of the literature

We present two patients with severe haemophilia A, who presented with haemorrhagic shock, secondary to a liver haematoma in the neonatal period. Both required emergency treatment and diagnosis of the hereditary coagulation defect. Major organ bleeding in severe haemophilia A in the newborn period is rare, and this unusual complication is not well recognized. Failure to recognize that the bleeding is due to a bleeding disorder, particularly in the absence of a family history, may lead to delay in appropriate management.

856. Improvements on Safety and Efficacy of Nonviral Gene Therapy of Factor VII for Hemophilia A with Inhibitors

Recombinant activated factor VII (rFVIIa) has been used as an effective alternative treatment for hemophilia patients who have developed inhibitors. We have successfully modeled a nonviral gene therapy approach to deliver factor VII (FVII) in a hemophilia A mouse model to decrease the cost and frequency of rFVIIa infusions. An engineered murine FVII (mFVIIa) cDNA was inserted into our gene transfer vector to generate a liver_specific construct pBS_ HCRHPI_mFVIIa_A encoding a modified protein, which can be cleaved by intracellular proteases of the furin family to secrete the activated form of mFVII. Partial correction of bleeding was achieved following nonviral gene transfer of this construct into hemophilia A mice. To increase the safety and efficacy of FVII gene therapy, we devised two approaches. The first was to use a mFVII wild_type cDNA (mFVII) encoding the zymogen FVII to reduce the potential thrombotic risk associated with prolonged, high level FVIIa expression. The second was to use a mutant FVIIa or FVII molecule (mFVIIa_DVQ or mFVII_DVQ) encoding a superactive FVII molecule with amino_acid substitutions (V158D/E296V/M298Q; Persson et al., 2003) to increase the efficacy of nonviral gene transfer of FVII. Fifty mg of the respective plasmids encoding wild_type or modified proteins including mFVII, mFVIIa, mFVII_DVQ, and mFVIIa_DVQ, were delivered into 4 groups of hemophilia A mice by a rapid, high volume (hydrodynamics_based) infusion method (n=8 mice/group). Phenotypic correction was measured via tail_clip assays. A 30% reduction in hemoglobin loss was observed in mice treated with mFVII, and a 50% reduction in mice treated with mFVIIa. Significant and long_term correction in PT using 1:60 dilution of mouse plasma was observed in both mFVII and mFVIIa treated mice, with clotting values consistently lower than wild_type and hemophilia A controls (wild_type: 26.4 s, hemophilia A: 25.4 s, mFVII: 23.6 s, mFVIIa: 23.3 s). APTT performed on plasma diluted 1:3 similarly reflects the results of the PT assay, with lowered clotting times for both groups of treated mice (wild_type: 60.2, hemophilia A: 121.3, mFVII: 102.8, mFVIIa: 100.9). Both plasma_ based and platelet_based thrombin generation (TG) assays indicated that both mFVII and mFVIIa have a positive effect on the three measured parameters with a lowered lag phase, lowered peak time and elevated peak thrombin levels. In addition, all these effects lasted long_term in plasmid treated mice for at least six months (experimental duration). Preliminary experiments using our most recently built constructs mFVII_DVQ and mFVIIa_DVQ showed reversion to wild_type phenotype in plasmid_treated mice as indicated by significantly reduced hemoglobin loss via tail clipping and substantial reduction in APTT. These results clearly demonstrate that nonviral gene therapy of either zymogen FVII or activated FVII can rescue bleeding in hemophilia A mice, and mFVII_DVQ/mFVIIa_ DVQ may potentially recover wild_type clotting activity in treated mice.

Efficacy of Zymogen Factor VII Is Comparable to Activated Factor VII for Nonviral Gene Therapy Treatment of Hemophilia A.

A major problem for clinical treatment of hemophilia A using factor replacement therapy is the high frequency formation of inhibitory antibodies against factor VIII. This problem is also predicted to occur following strategies currently aimed at targeted genetic correction of this disease. Recombinant activated factor VII (rFVIIa, NovoSeven®, Novo Nordisk, Bagsvaerd, Denmark) has been successfully used as an effective alternative treatment for hemophilia patients who have developed inhibitors. In order to decrease the cost and fluctuation of FVIIa levels associated with frequent infusions of rFVIIa, nonviral gene transfer of factor VII (FVII) was attempted in a hemophilia A mouse model. In addition, to investigate the potential thrombotic risks associated with prolonged, high level of FVIIa expression following gene transfer, we compared the effects of gene transfer vectors encoding zymogen FVII with an engineered secreted FVIIa in hemophilia A mice. We inserted murine FVII (mFVII) cDNA into a liver-specific vector developed recently in our lab to generate pBS-HCRHPI-mFVII-A. The mFVII cDNA sequence was then modified by site-directed mutagenesis to insert a protease cleavage site in between Arg152-Ile153. The resulting construct of murine FVIIa (mFVIIa) encodes a modified protein, which can be cleaved by intracellular proteases of the furin family to secrete the activated form of mFVII. Fifty mg of either mFVII or mFVIIa the plasmid was delivered into hemophilia A mouse liver by a rapid, high volume (hydrodynamics-based) infusion method (n=8 mice/group). Phenotypic correction was evaluated using tail-clip assays by measuring the hemoglobin levels in the collected blood. This assay indicated that vectors carrying either mFVII or mFVIIa can partially correct clotting following gene transfer. Significant correction in PT and APTT was observed in mFVII or mFVIIa-treated mice. Plasma-based and platelet-based thrombin generation (TG) assays indicated that either mFVII or mFVIIa significantly shorten peak time and peak thrombin levels of TG. In addition, these effects lasted long-term in plasmid treated mice for at least three months (experimental duration). We also constructed vectors carrying human FVII (hFVII) or FVIIa (hFVIIa) to test their efficacy for correcting hemophilia A. Constructs produced supra-physiological levels (800–1200ng/ml) of hFVII or hFVIIa assayed by ELISA at days 1 and 4 post-injection. However, the levels of hFVII and hFVIIa dropped precipitously at day 14 post-injection shown by both ELISA and Western blot, most likely due to the formation of species-specific antibodies against hFVII. These results clearly demonstrate that nonviral gene therapy of either zymogene FVII or activated FVII can rescue bleeding in hemophilia A mice.

Systematic Review of Randomized Controlled Trials of Prophylactic Clotting Factor Concentrate in Hemophilia.

Introduction: One of the current approaches to managing persons with hemophilia entails the use of prophylactic treatment with clotting factor concentrates (CFC) to prevent bleeds and bleeding related complications. However, this process is not practiced in all countries, or is based on the synthesis of evidence from data of all trials.Objective: To conduct a systematic review/meta-analysis (SR/MA) regarding the effectiveness of clotting factor concentrate prophylaxis in the management of people with hemophilia A or B.Methods: We conducted a SR of all randomized controlled trials (RCTs) that studied the effect of prophylactic CFCs in people with hemophilia A or B. We searched all major electronic databases (MEDLINE, LILACS, EMBASE, and the Cochrane Cystic Fibrosis and Genetic Disorders Group's Controlled Trials Register) as well as performing handsearches of journals and relevant meeting abstract books. We included RCTs that that compared the use of prophylactic CFCs to a control group.Results: We identified 29 studies, of which four (including 37 participants) were eligible for inclusion. Three studies evaluated hemophilia A; one showed a decrease in frequency of joint bleeds with prophylaxis compared to placebo (non-physiological dose), with a rate difference (RD) −10.80 (95% confidence interval (CI) −16.33 to −5.27) bleeds per year. The remaining two studies evaluating hemophilia A compared two prophylaxis regimens, one study showed no difference in joint bleed frequency, RD −5.04 (95%CI −17.02 to 6.94) bleeds per year and another failed to demonstrate an advantage of factor VIII dosing based on individual pharmacokinetic data over the standard prophylaxis regimen with RD −0.14 (95% CI −1.34 to 1.05) bleeds per year. The fourth study evaluated hemophilia B and showed fewer joint bleeds with weekly (15 IU/kg) versus bi-weekly (7.5 IU/kg) prophylaxis, RD −3.30 (95% CI −5.50 to − 1.10) bleeds per year.Conclusion: There is insufficient evidence to determine whether prophylactic clotting factor concentrates decrease bleeding and bleeding-related complications in hemophilia A or B, compared to placebo, on-demand treatment, or prophylaxis based on pharmacokinetic data from individuals. Well-designed RCTs are needed to assess the effectiveness of prophylactic clotting factor concentrates.