Progressive improvement in wound healing with increased therapy in haemophilia B mice.

Previous work has shown that normalized haemostasis only at the time of an injury is not sufficient to promote optimal wound healing in haemophilia B (HB) mice. However, the duration of treatment required for optimal healing has not been established. The goal of these studies was to determine the effect of different durations of replacement or bypassing therapy [factor IX(FIX) or factor VIIa (FVIIa)] on wound healing parameters in a mouse model of HB. A dermal wound was placed on the back of HB mice. Animals were either untreated or pretreated and then subsequently treated for 3 days, 5 days, or 7 days with FIX or FVIIa. Wound area, time to wound healing, haematoma formation and iron deposition were measured. All treated animals showed shortened time to healing relative to untreated animals. Haematoma formation was prevented by treatment and bleeding into the wounds, measured by iron scores, was reduced by treatment. In addition, there was a progressive improvement in healing with 7 days of treatment more effective than 5 days which was more effective than 3 days. Replacement therapy with FIX had slightly shorter healing times than bypassing therapy with FVIIa. HB mice treated with FIX had slightly smaller wound area than untreated animals; by contrast, FVIIa-treated animals had much smaller wound areas that were close to the wound areas seen in wild-type animals. The data suggest that sustained therapy is required for normal wound healing.

Secondary prophylaxis with recombinant activated factor VII improves health-related quality of life of haemophilia patients with inhibitors.

Haemophilia patients with inhibitors characteristically have impaired joint function and reduced health-related quality of life (HRQoL). This analysis examined whether secondary prophylaxis with recombinant activated factor VII (rFVIIa) improves HRQoL vs. conventional on-demand therapy in patients with haemophilia with inhibitors and frequent bleeds. After a 3-month preprophylaxis period, 22 patients received daily rFVIIa prophylaxis (90 or 270 microg kg(-1)) for 3 months, followed by 3 months' postprophylaxis. Days of hospitalization, absence from school/work and mobility aids requirements were recorded. HRQoL was assessed by EuroQoL (EQ-5D) questionnaire, visual analogue scale (VAS), derived Time to Trade-Off (TTO) scores and Quality Adjusted Life Years (QALYs). rFVIIa prophylaxis significantly (P < 0.0001) reduced bleeding frequency vs. prior on-demand therapy. Hospitalization (5.9% vs. 13.5%; P = 0.0026) and absenteeism from school/work (16.7% vs. 38.7%; P = 0.0127) decreased during prophylaxis; these effects tended to be maintained during postprophylaxis. HRQoL (evaluated by EQ-5D) tended to improve during and after rFVIIa prophylaxis. Notably, pain decreased and mobility increased in 40.9% and 27.3% of patients, respectively, at the end of the postprophylaxis period vs. preprophylaxis. Median VAS score increased from 66 to 73 (P = 0.048), and TTO scores suggested better HRQoL (0.62 vs. 0.76; P = 0.054) during postprophylaxis than preprophylaxis. Small to moderate changes in effect sizes were reported for VAS and TTO scores. Median QALYs were 0.68 (VAS) and 0.73 (TTO). Reductions in bleeding frequency with secondary rFVIIa prophylaxis were associated with improved HRQoL vs. on-demand therapy.

Restoring hemostatic thrombin generation at the time of cutaneous wounding does not normalize healing in hemophilia B.

BACKGROUND: We recently reported that wound healing is abnormal in hemophilia B (HB) mice [1]. The wounds show abnormal histology: s.c. hematoma formation; delayed re-epithelialization; delayed macrophage influx; and an increase in wound site angiogenesis. OBJECTIVE: To test the hypothesis that restoring a hemostatic level of thrombin generation at the time of wounding would allow formation of an adequate platelet/fibrin plug and correct abnormalities of wound healing in HB. METHODS: We placed a 3-mm cutaneous wound on the back of each HB or wild-type (WT) mouse. Some HB mice were treated just prior to wounding with either human factor IX (FIX) or FVIIa in a dose sufficient to normalize bleeding in a tail bleed model. RESULTS: The average wound size over time in treated HB animals was intermediate between those in WT and untreated HB mice. However, the time to complete skin closure was not improved by treatment. Hematoma formation was decreased and macrophage influx began earlier in treated than in untreated HB animals. However, treated HB mice had evidence of ongoing low-level bleeding near the wound site, even after closure of the skin defect. Treatment also did not normalize the increased angiogenesis observed in HB mice. CONCLUSIONS: Restoring initial hemostasis can modulate some of the parameters of wound healing. However, an extended period of adequate hemostatic function is necessary to achieve normal healing, probably because the risk of hemorrhage is increased by vascular remodeling and angiogenesis during the healing process.

Thrombin generation in vascular tissue.

BACKGROUND: Classically, it is thought that the vast majority of thrombin is generated on the surface of platelets, however, thrombotic events occur in patients despite treatment with potent antiplatelet agents. METHODS AND RESULTS: In freshly harvested left internal mammary artery (IMA) sections, addition of CaCl2 and platelet-poor plasma (PPP) were sufficient to stimulate a profound burst of thrombin and this effect was inhibited by antitissue factor antibodies. Ultracentrifugation of PPP to remove platelet microparticles had no effect on thrombin generation. Both the extrinsic and factor VIII-dependent pathways were necessary for IMA-supported thrombin generation as PPP derived from individuals deficient in factors V, VII, VIII or X did not support thrombin production. Small amounts of thrombin were generated utilizing factor IX (FIX)-deficient plasma, however, thrombin was not generated by aorta from FIX-deficient mice when FIX-deficient plasma was used. The addition of non-lipidated tissue factor (0.6 pM) and CaCl2 to actively proliferating cultured human aortic smooth muscle cells (SMC) resulted in a pronounced burst of thrombin generation occurring between 3 and 15 min after treatment. In the absence of tissue factor, thrombin was generated but at a slower rate and with a peak value 26% of that observed in the presence of tissue factor. CONCLUSION: Significant thrombin generation can occur on vascular tissue in the absence of platelets or platelet microparticles and on the surface of non-apoptotic SMC.

Cellular immune responses in hemophilia: why do inhibitors develop in some, but not all hemophiliacs?

Advances in molecular immunology over the past two decades permit a better understanding of why antibodies develop to peptide antigens like factor VIII and the events that lead to the development of these antibodies. Two important variables that are critical in antibody formation are (i) the molecular defect in FVIII and the consequences of that defect on translation and protein production, and (ii) the major histocompatibility complex (MHC) molecules which bind specific peptide sequences and present those peptides to CD4 T lymphocytes to initiate the cellular cascade leading to B-cell stimulation and differentiation, and ultimately to antibody formation. Inhibitors develop in hemophilia because transfused FVIII can be seen as a foreign protein and elicits an immune response in much the same way that any other foreign protein might elicit an immune response. However, not all hemophiliacs generate an immune response, either because they do not recognize FVIII as foreign or because their MHC phenotype is such that a cellular immune response is not initiated. In this model, it is the combination of molecular defect and MHC phenotype that determines inhibitor formation. The interplay of these two variables in the context of why some but not all hemophiliacs develop antibodies after treatment with replacement factor is reviewed.

Recombinant human factor VIIa (rFVIIa) can activate factor FIX on activated platelets.

The studies reported here show that factor (F)VIIa can activate factor (F)IX on activated platelets in the absence of tissue factor. Both FIX and FIXa bind to the activated platelet surface with a K(d) of 8 nM and 2 nM, respectively. With factor (F)VIIIa, FIXa binds more tightly to platelets (K(d) 0.6 nM). At rFVIIa concentrations < 100 nm, no direct binding to the activated platelet surface can be detected with electrophoretic light scattering. However, in the presence of FIX, rFVIIa binding to platelets at concentrations as low as 10 nm rFVIIa can be detected. This is reflected by a decrease in the FIX K(d) from 8 to 1.6 nM. When rFVIIa is added to activated platelets in the presence of both FIX and FVIIIa, the K(d) for FIX decreases to 0.6, suggesting that rFVIIa activates FIX on the surface of activated platelets in the absence of tissue factor. The activation of FIX by FVIIa on activated platelets can also be demonstrated by a functional assay for FIXa. These data show that pharmacological doses of rFVIIa result in the direct activation of FIX by rFVIIa to form additional tenase complexes ultimately resulting in improved thrombin generation. These results may explain, at least in part, the mechanism of action of rFVIIa in hemorrhagic conditions seen in otherwise normal patients who develop an acquired coagulopathy due to trauma, surgery or a variety of other events in which rFVIIa has been found to be effective.

Impact of procoagulant concentration on rate, peak and total thrombin generation in a model system.

Using a cell-based model system of coagulation, we performed a systematic examination of the effect of varying individual procoagulant proteins (over the range of 0-200% of pooled plasma levels) on the characteristics of thrombin generation. The results revealed a number of features unique to the different coagulation factors, as well as common features allowing them to be grouped according to the patterns observed. Variation of those factors contributing to formation of the tenase complex, factor (F)VIII, factor (F)IX and factor (F)XI, primarily affected the rate and peak of thrombin production, but had little to no effect on total thrombin production. The effect of decreased FXI was milder than seen with decreased FVIII or FIX, and more variable between platelet donors. In contrast, varying the concentration of factors that contribute to formation of the prothrombinase complex, prothrombin or factor (F)V (with FV-deficient platelets), significantly affected all three measures of thrombin production: rate, peak and total. Additionally, while no thrombin generation was observed with no factor X, only very small amounts (between 1% and < 10% of normal plasma levels) were required to normalize the measured parameters. Finally, our results with this cell-based system highlight differences in thrombin generation on cell surfaces (platelets) compared with phospholipids, and suggest that platelets contribute more than simply a surface for the generation of thrombin.

Recombinant factor VIIa (Novoseven) and the safety of treatment.

Recombinant factor VIIa (rFVIIa; NovoSeven), Novo Nordisk, Bagsvaerd, Denmark) has been used to treat more than 6,500 patients with hemophilia or other bleeding disorders, and by May 2001, more than 180,000 standard doses had been administered. Clinical experience to date suggests that rFVIIa is safe and effective, with a very low incidence of thrombotic events being reported. Moreover, in the majority of cases, thrombotic events appear to be caused by improvements in the hemostatic system, rather than a direct effect of the agent itself.

Rationale for the use of high dose rFVIIa in a high-titre inhibitor patient with haemophilia B during major orthopaedic procedures.

Inhibitor development is a serious complication in patients with haemophilia A and B. Historically, a lack of optimal therapies and factor products for treating inhibitor patients resulted in many patients developing chronic haemophilic arthropathies and flexion contractures of the involved joints. The introduction of immune-tolerance protocols to eradicate high-titre inhibitors has greatly diminished the incidence of these types of complications but as in the case reported here, immune tolerance is not always successful. Various elective surgical procedures were often delayed or not even considered in patients with inhibitor because of the variability in achieving adequate haemostasis and the thrombotic risks involved with the use of activated prothrombin-complex concentrates (APCCs) over extended periods of time. The development of recombinant factor VIIa (rFVIIa; NovoSeven) and its demonstrated safety and efficacy in treating inhibitor patients has opened new possibilities for addressing severe arthropathy with flexion contracture. This case report demonstrates that the use of rFVIIa in such a situation must include dosing flexibility that is both patient-specific and related to the potential for bleeding; the ability to maintain clinical haemostasis with a prophylactic dose of rFVIIa given as little as once daily; and the capacity for higher doses of rFVIIa, particularly in children because their kinetic profiles differ from adults.

High-dose factor VIIa increases initial thrombin generation and mediates faster platelet activation in thrombocytopenia-like conditions in a cell-based model system.

Clinical experience has shown that high doses of recombinant factor VIIa (rFVIIa) may ensure haemostasis in thrombocytopenic patients. We have used a cell-based model system to mimic thrombocytopenia and analyse the effect of rFVIIa. Lowering the platelet density from 200 x 10(9)/l (reflecting normal conditions) to 100, 50, 20 and 10 x 10(9)/l revealed a platelet density-dependent decrease in the maximal rate of thrombin generation, a prolongation in the time to maximal thrombin activity and a lower maximal level of thrombin formed. The platelet activation, measured as the time to half-maximal P-selectin (CD62) exposure, was not significantly dependent on the platelet density in the range of 200 x 10(9)/l to 10 x 10(9)/l, although there was a tendency for slower platelet activation at 20 x 10(9) and 10 x 10(9) platelets/l than at the higher platelet densities. Addition of 50--500 nmol/l rFVIIa to samples with 20 x 10(9) or 10 x 10(9) platelets/l shortened the lag phase of thrombin generation as well as the time to half-maximal platelet activation. Our data indicate that high doses of rFVIIa may help to provide haemostasis in thrombocytopenic patients by increasing the initial thrombin generation, resulting in faster platelet activation and thereby compensating for the lower number of platelets present.

Hantavirus transmission: potential role of ectoparasites.

Hantavirus pulmonary syndrome is an often-fatal disease thought to be transmitted exclusively by rodents. We report the first evidence of hantavirus-specific RNA (Bayou) from two trombiculid mites (chiggers) and an ixodid tick parasitizing wild-caught rodents at a field site in Texas and also from a trombiculid mite in the free-living predatory stage of the chigger life cycle collected from the same site.

The factor VII-platelet interplay: effectiveness of recombinant factor VIIa in the treatment of bleeding in severe thrombocytopathia.

Recently, high-dose factor VIIa has been used to correct bleeding in patients with various thrombocytopathias including Glanzmann's thrombasthenia, Bernard-Soulier syndrome, and uremia. High-dose factor VIIa is postulated to act on platelets in the absence of tissue factor to activate factors IX and X and thus enhance thrombin generation. This enhanced thrombin generation might help provide hemostasis in patients with thrombocytopathias through several mechanisms. Enhanced thrombin generation would provide a strong signal for recruitment of other platelets. Also, enhanced fibrin deposition might provide mechanisms for bypassing the specific defect in thrombocytopathias. Thus, platelets from a patient with Bernard-Soulier syndrome might associate with fibrin by a glycoprotein IIb-IIIa-mediated mechanism. Also, platelets from a patient with Glanzmann's thrombasthenia might associate with fibrin through von Willebrand factor-mediated interactions with glycoprotein Ib-V-IX. Finally, enhanced thrombin generation on platelets would mean that fewer platelets are required for hemostasis.