Pharmacokinetics,clot strength and safety of a new fibrinogen concentrate: randomized comparison with active control in congenital fibrinogen deficiency

Essentials

• Congenital afibrinogenemia causes a potentially life‐threatening bleeding and clotting tendency.
• Two human fibrinogen concentrates (HFCs) were compared in a randomized pharmacokinetic study.
• Bioequivalence was not shown for AUC_norm, which was significantly larger for the new HFC.
• Increases in clot strength were comparable, and no thromboses or deaths occurred in the study.

Summary

Background

Human fibrinogen concentrate (HFC) corrects fibrinogen deficiency in congenital a‐/hypofibrinogenemia.

Objectives

To assess pharmacokinetics (PK), effects on thromboelastometry maximum clot firmness (MCF), and safety of a new double virus‐inactivated/eliminated, highly purified HFC vs. active control.

Patients/Methods

In this multinational, randomized, phase II, open‐label, crossover study in 22 congenital afibrinogenemia patients aged ≥ 12 years, 70 mg kg^?1 of new HFC (FIBRYGA, Octapharma AG) or control (Haemocomplettan^® P/RiaSTAP?, CSL Behring GmbH) were administered, followed by crossover to the other concentrate. Fibrinogen activity, PK and MCF in plasma were assessed.

Results

The concentrates were not bioequivalent for the primary endpoint, AUC_norm (mean ratio, 1.196; 90% confidence interval [CI], 1.117, 1.281). Remaining PK parameters (C_maxnorm, IVR, t_1/2, MRT) reflected bioequivalence between concentrates, except for clearance (mean ratio, 0.836; 90% CI, 0.781, 0.895) and V_ss (mean ratio, 0.886; 90% CI, 0.791, 0.994). Mean AUC_norm was significantly larger for the new HFC (1.62 ± 0.45 vs. 1.38 ± 0.47 h kg g L^?1 mg^?1, P = 0.0001) and mean clearance was significantly slower (0.665 ± 0.197 vs. 0.804 ± 0.255 mL h^?1 kg^?1, P = 0.0002). Mean MCF increased from 0 mm to 9.68 mm (new HFC) and 10.00 mm (control) 1‐hour post‐infusion (mean difference, ?0.32 mm; 95% CI, ?1.70, 1.07, n.s.). No deaths, thromboses, viral seroconversions or serious related adverse events occurred.

Conclusions

Bioequivalence was not demonstrated for AUC_norm, clearance and V_ss. Larger AUC_norm and slower clearance were observed for the new HFC. Remaining pharmacokinetic parameters reflected bioequivalence to control. Safety profiles and increases in clot strength were comparable between concentrates.

     

Effect of SanOrg123781A, a synthetic hexadecasaccharide, on clot-bound thrombin and factor Xa in vitro and in vivo

Summary. Factor (F)Xa and thrombin bound to the clot during its formation contribute to the propensity of thrombi to activate the coagulation system. The aim of this work was to study the inhibition of clot‐bound FXa and clot‐bound thrombin by SanOrg123781A, a synthetic hexadecasaccharide that enhances the inhibition of thrombin and FXa by antithrombin (AT). SanOrg123781A, designed to exhibit low non‐specific binding to proteins other than AT, was compared with heparin. In buffer, heparin and SanOrg123781A inhibited FXa and thrombin at similar concentrations [concentration inhibiting 50% (IC₅₀) of Xa and IIa activity were, respectively: heparin 120 ± 7 and 3 ± 1 ng mL^?1; SanOrg123781A 77 ± 5 and 4 ± 1 ng mL^?1]. In human plasma, the activity of both compounds was reduced, although the activity of heparin was much more affected than that of SanOrg123781A (IC₅₀ values for inhibition of FXa and FIIa activity were, respectively: heparin 100 ± 5 and 800 ± 40 ng mL^?1; SanOrg123781A 10 ± 5 and 30 ± 3 ng mL^?1). We demonstrated, in agreement with our previous results, that the procoagulant activity of the clot is essentially due to clot‐bound FXa and to some extent to clot‐bound thrombin. We showed that heparin and SanOrg123781A were able to inhibit fragment F1+2 generation induced by clot‐bound FXa with IC₅₀ values of 2 ± 0.5 µg mL^?1 and 0.6 ± 0.2 µg mL^?1, respectively. Both compounds also inhibited clot‐bound thrombin activity, the IC₅₀ values of heparin and SanOrg123781A being 1 ± 0.01 µg mL^?1 and 0.1 ± 0.1 µg mL^?1, respectively. Moreover, both heparin and SanOrg123781A significantly inhibited fibrinopeptide A generated by the action of clot‐bound thrombin on fibrinogen but also by free thrombin generated from prothrombin by clot‐bound FXa with IC₅₀ values of 4 ± 0.6 and 1 ± 0.1 µg mL^?1, respectively. As with clot‐bound enzymatic activities, SanOrg123781A was three times more active than heparin in vivo on fibrinogen accretion onto a pre‐existing thrombus and as activators of recombinant tissue‐type plasminogen activator‐induced thrombolysis. In conclusion, due to the specific activities of SanOrg123781A, this compound is much more active than heparin in the presence of plasma proteins, on clot‐bound enzymes and in in vivo models of thrombosis/thrombolysis.     

Comparison of the efficacy of two human fibrinogen concentrates to treat dilutional coagulopathy in vitro

Both congenital and acquired fibrinogen deficiency can be safely treated with administration of fibrinogen concentrate.

The aim of this study was to test the efficacy of a new fibrinogen product (Fibryga) compared to a licensed product (Haemocomplettan) in an in vitro model of dilutional coagulopathy.

Ten blood specimens from healthy volunteers were diluted 1:1 with balanced crystalloid solution and subsequently supplemented with each fibrinogen concentrate at a dose replicating in vivo supplementation (50?mg kg^?1). Changes in clot firmness (FIBTEM and EXTEM assay), as well as changes in the fibrinogen antigen level, fibrinogen activity, factor XIII level and fibronectin levels were assessed at baseline, after dilution and after adding fibrinogen concentrate.

There was no significant difference between the drugs in their in vitro ability to improve clot firmness in the FIBTEM assay (Fibryga: mean MCF 14.4?mm (SD 3.4?mm) vs. Haemocomplettan: MCF 14.1?mm (2.4); p?=?.584). Fibryga led to significantly higher clot firmness in EXTEM MCF: 56.7?mm (3.8) vs. 53.7?mm (3.7); p?p??1 (SD 0.002?g L^?1) vs. 0.002?g L^?1 (SD 0.002?g L^?1; p?This is the first study to demonstrate that Fibryga and Haemocomplettan have similar efficacy in improving clot firmness in a dilutional hypofibrinogenemia model in vitro.     

Factor XIII combined with recombinant factor VIIa: a new means of treating severe hemophilia A

Summary. Background: Abnormal thrombin generation is considered the key defect in hemophilia. Conventional treatment seeks to correct this using coagulation factor replacement or bypassing agents, for example recombinant factor VIIa (rFVIIa). Previous studies demonstrate abnormal FXIII activation in patients with hemophilia. FXIII activation is essential for formation of structurally normal, stable clots. Objectives: The present study challenges the hypothesis that in hemophilia the use of plasma‐derived FXIII (pdFXIII) in combination with rFVIIa will produce a greater improvement in clot stability than promotion of thrombin generation alone. Methods: Fourteen individuals with severe hemophila A were enrolled. Whole blood was spiked ex vivo with buffer, rFVIIa (2 μg mL^?1) or rFVIIa (2 μg mL^?1) plus pdFXIII (10 μg mL^?1). Whole blood thromboelastometry assessed clot stability, after activation with tissue factor (TF) (0.15 pm ) plus tissue‐type plasminogen activator (tPa) (2 nm ). The primary outcome measure of clot stability was area under the elasticity curve (AUEC). Results: The combination of pdFXIII and rFVIIa significantly improved clot stability as measured by AUEC (P < 0.05) compared with rFVIIa alone. Conclusion: The use of pdFXIII resulted in superior clot stability compared with solely enhancing thrombin generation and we suggest that increasing thrombin generation alone fails to fully correct dysregulation of clot‐stabilizing mechanisms associated with bleeding disorders. Hemorrhage control in hemophilia may be improved using clot stabilizing drugs. FXIII shows potential as a novel agent.     

A monoclonal antibody to the fibrinogen γ-chain alters fibrin clot structure and its properties by producing short, thin fibers arranged in bundles

Summary. Background: We previously reported that hamster monoclonal antibody 7E9, which reacts with the C‐terminus of the γ‐chain of mouse fibrinogen, inhibits factor (F)XIIIa‐mediated cross‐linking, platelet adhesion to fibrinogen, and platelet‐mediated clot retraction; in addition, it facilitates thrombolysis. Objectives: To understand the mechanism(s) by which 7E9 acts, we have now studied the effect of 7E9 IgG, 7E9 F(ab′)₂, and 7E9 Fab on fibrin clot structure using electron microscopy and measurements of clot physical properties. Results: By transmission electron microscopy, 7E9 IgG was found to bind primarily to the ends of the fibrinogen molecule. 7E9 IgG and 7E9 F(ab′)₂, both of which are bivalent, were capable of binding to two fibrinogen molecules simultaneously. Scanning electron microscopy of clots formed in the presence of equimolar concentrations of fibrinogen and 7E9 IgG demonstrated the presence of very short and thin fibers (63% reduction in fiber diameter) arranged in unusual bundles, surrounding large pores. Clots formed in the presence of 7E9 demonstrated a marked increase in permeation (approximately 25‐fold increase in perfusion rate at constant pressure), an approximately 50% reduction in dynamic storage modulus (G'; a reflection of decreased clot stiffness), and an approximately 38% increase in loss tangent (tan δ; a reflection of the clot's ability to undergo irreversible deformation). These clots also showed decreased absorbance at 350 nm, reflecting the clot structure produced by 7E9 IgG. The effects of 7E9 IgG were not observed with control hamster IgG, 7E9 F(ab′)₂, or 7E9 Fab fragments, indicating requirements for both the binding properties and mass of 7E9 IgG. Conclusions: These data indicate that 7E9 antibody affects fibrin clot structure in a way that is consistent with the enhanced fibrinolysis we reported previously. Together with our previous observations, we conclude that 7E9 is directed at a strategically important region of fibrinogen with regard to platelet function, FXIIIa‐mediated cross‐linking, clot retraction, fibrin structure, and fibrinolysis. Thus targeting this region of fibrinogen may have antithrombotic therapeutic potential.     

Fibrinogen substitution improves whole blood clot firmness after dilution with hydroxyethyl starch in bleeding patients undergoing radical cystectomy: a randomized, placebo-controlled clinical trial

Summary.  Background:  Infusion of artificial colloids such as hydroxyethyl starch (HES) induces coagulopathy to a greater extent than simple dilution. Several studies have suggested that the coagulopathy could be corrected by substitution with a fibrinogen concentrate. Objectives:  The aims of the present prospective, randomized, placebo-controlled trial were to investigate the hemostatic effect of a fibrinogen concentrate after coagulopathy induced by hydroxyethyl starch in patients experiencing sudden excessive bleeding during elective cystectomy. Methods:  Twenty patients were included. Blood loss was substituted 1:1 with HES 130/0.4. At a dilution level of 30%, patients were randomly selected for intra-operative administration of a fibrinogen concentrate or placebo. The primary endpoint was maximum clot firmness (MCF), as assessed by thromboelastometry. Secondary endpoints were blood loss and transfusion requirements, other thromboelastometry parameters, thrombin generation and platelet function. Results:  Whole-blood MCF was significantly reduced after 30% dilution in vivo with HES. The placebo resulted in a further decline of the MCF, whereas randomized administration of fibrinogen significantly increased the MCF. Furthermore, only 2 out of 10 patients randomly chosen to receive fibrinogen substitution required postoperative red blood cell transfusions, compared with 8 out of 10 in the placebo group ( P  = 0.023). Platelet function and thrombin generation were reduced after 30% hemodilution in vivo , and fibrinogen administration caused no significant changes. Conclusions:  During cystectomy, fluid resuscitation with HES 130/0.4 during sudden excessive bleeding induces coagulopathy that shows reduced whole-blood maximum clot firmness. Randomized administration of fibrinogen concentrate significantly improved maximum clot firmness and reduced the requirement for postoperative transfusion.     

Pharmacokinetics and pharmacodynamics of a new highly secured fibrinogen concentrate

Summary.  Background:  Inherited afibrinogenemia is a rare autosomal recessive disorder characterized by the absence or trace amounts of plasma fibrinogen inducing varying bleeding tendencies. Little is known about the pharmacokinetics of plasma-derived fibrinogen concentrates used in the treatment of afibrinogenemic patients. Objective: This open, prospective, multicenter study assessed the pharmacokinetic and pharmacodynamic profiles of FIBRINOGENE  T1 (FGT1; LFB, Les Ulis, France), a human fibrinogen concentrate treated with three specific biological safety steps. Patients/methods: Five adult patients with congenital afibrinogenemia received a single infusion of 0.06 g kg⁻¹ of FGT1 . Plasma samples drawn up to day 14 were assayed for fibrinogen antigen and activity and for coagulation parameters in a central laboratory. Results: Fibrinogen antigen and activity were similar and highly correlated, with very low between-patient variability for pharmacokinetic parameters. Fibrinogen levels increased rapidly and significantly, with a mean plasma concentration of 1.39 g L⁻¹ being achieved 1 h after the end of the infusion, leading to almost complete in vivo recovery (94%). The mean half-life was 3.4 days, with slow linear elimination, and the distribution was mainly restricted to the vascular compartment. Coagulation parameters were normalized after the infusion and during the following 6–10 days. FGT1 was well tolerated overall. Conclusions: FGT1 behaves like natural functional fibrinogen, and its pharmacokinetic properties are in line with those expected from a fibrinogen concentrate. Our findings suggest that FGT1 can restore efficient hemostasis in afibrinogenemic patients, and predict good clinical efficacy.     

Effect of combined treatment with immunoadsorption and membrane filtration on plasma coagulation—Results of a randomized controlled crossover study

The combined use of immunoadsorption (IA) and membrane filtration (MF) may markedly enhance removal of IgM and complement component C1q, supporting its use as an element of recipient desensitization in antibody‐incompatible transplantation. However, coagulation factor removal may contribute to altered hemostasis, posing a risk of bleeding in the perioperative setting. This secondary endpoint analysis of standard coagulation assays and rotational thromboelastometry ( ROTEM® ) was performed in the context of a randomized controlled crossover study designed to assess the effect of combined IA (GAM‐146‐peptide) and MF on levels of ABO antigen‐specific IgM. Fourteen patients with autoimmune disorders were randomized to a single treatment with IA+MF followed by IA alone, or vice versa. MF was found to markedly enhance fibrinogen depletion (57% vs. 28% median decrease after IA alone, P < 0.001), whereby four patients showed post‐treatment fibrinogen concentrations below 100 mg dL^?1. In support of a critical contribution of fibrinogen depletion to impaired coagulation, extrinsically activated ROTEM^® analysis revealed a marked reduction in fibrinogen‐dependent clot formation upon IA+MF (59% median decrease in FIBTEM mean clot firmness (MCF) as compared to 24% after IA alone, P < 0.001). Moreover, the addition of MF led to a substantial prolongation of activated partial thromboplastin time, possibly due to depletion of macromolecular coagulation factors contributing to intrinsically activated coagulation. Our study demonstrates substantial effects of combined IA+MF on clot formation, which may be mainly attributable to fibrinogen depletion. We suggest that the use of combined apheresis in the setting of transplant surgery may necessitate a careful monitoring of coagulation. J. Clin. Apheresis 31:29–37, 2016. © 2015 Wiley Periodicals, Inc.     

The role of thrombin activatable fibrinolysis inhibitor and factor XI in platelet‐mediated fibrinolysis resistance: a thromboelastographic study in whole blood

Summary. Background: The resistance of platelet‐rich thrombi to fibrinolysis is generally attributed to clot retraction and platelet PAI‐1 release. The role of TAFI in platelet‐mediated resistance to lysis is unclear. Objective: We investigated the contribution of TAFI to the antifibrinolytic effect of platelets in whole blood by thromboelastography. Methods: Platelet‐poor (PP‐WB, < 40 × 10³ μL^?1) and platelet‐rich (PR‐WB, > 400 × 10³ μL^?1) blood samples were obtained from normal human blood (N‐WB, 150–220 × 10³ μL^?1). Clot lysis time was measured by thromboelastography in recalcified blood supplemented with t‐PA (100 ng mL^?1) and tissue factor (1:1000 Recombiplastin). Results: t‐PA‐induced lysis time increased in parallel with platelet concentration (up to 3‐fold). Neutralization of TAFI, but not of PAI‐1, shortened the lysis time by ～ 50% in PR‐WB and by < 10% in PP‐WB. Accordingly, prothrombin F1+2 and TAFIa accumulation was greater in PR‐WB than in PP‐WB. A similar TAFI‐dependent inhibition of fibrinolysis was observed when clot retraction was prevented by cytochalasin D or abciximab, or when platelet membranes were tested. Moreover, in blood with an intact contact system, platelet‐mediated fibrinolysis resistance was attenuated by an anti‐FXI but not by an anti F‐XII antibody. Finally, platelets made the clots resistant to the profibrinolytic effect of heparin concentrations displaying a strong anticoagulant activity. Conclusions: Our data indicate that TAFI activation is one major mechanism whereby platelets make clots resistant to fibrinolysis and underscore the importance of TAFI inhibitors as new antithrombotic agents.     

Prothrombin complex concentrate and recombinant prothrombin alone or in combination with recombinant factor X and FVIIa in dilutional coagulopathy: a porcine model

Summary. Background: This study was conducted to assess whether newly developed recombinant clotting factor concentrates enable the reversal of dilutional coagulopathy. Methods: In 50 anesthetized pigs, ～ 60% of the blood volume was withdrawn and replaced with hydroxyethyl starch. Pigs were randomized to receive either 200 mg kg^?1 fibrinogen (n = 10), fibrinogen and 35 IU kg^?1 prothrombin complex concentrate (PCC) (n = 10), fibrinogen and 4 mg kg^?1 recombinant human factor II (rhFII) concentrate (n = 10), fibrinogen and a three‐factor combination (3F) of 4 mg kg^?1 rhFII, 0.006 mg kg^?1 recombinant human FVIIa and 0.32 mg kg^?1 recombinant human FX (n = 10), or saline (n = 10). Thereafter, a standardized liver laceration was performed to induce uncontrolled hemorrhage. Survival time and blood loss were determined, and standard coagulation tests and thrombelastometry were performed. Results: Fibrinogen combined with rhFII or PCC improved survival. Blood loss was significantly decreased in all groups as compared with the animals receiving saline. Clotting time was significantly shortened in the animals treated with fibrinogen and PCC, as well as in those treated with fibrinogen and 3F. One animal died after administration of fibrinogen and PCC. Conclusion: Following hemodilution, a combination of fibrinogen and PCC, rhFII or 3F enhances coagulation and final clot strength. Mortality was reduced statistically significantly only in the animals treated with fibrinogen and rhFII or PCC, whereas administration of the combination of fibrinogen and PCC caused a fatal thromboembolic complication. The combination of fibrinogen and rhFII might be effective in reversing dilutional coagulopathy and may reduce blood loss in cases of dilutional coagulopathy.     

Modified clot waveform analysis to measure plasma coagulation potential in the presence of the anti‐factor IXa/factor X bispecific antibody emicizumab

Essentials

• The activated partial prothrombin time (aPTT) cannot predict the activity of emicizumab (Emi).
• Adjusted clot waveform analyses using a prothrombin time (PT)/aPTT initiator were developed.
• Activity of Emi in the co‐presence of factor VIII or bypassing agents was quantified.
• This assay is useful for assessing coagulation potential in Emi‐treated hemophilia A.

Summary

Background

Emicizumab is an anti‐activated factor IX/FX bispecific antibody that mimics activated FVIII cofactor function. Emicizumab does not require activation by thrombin, and its effect on shortening the activated partial thromboplastin time (APTT) is much greater than that of FVIII. Therefore, the APTT has limited utility in hemophilia A (HA) patients treated with emicizumab.

Aim

To evaluate the global coagulation potential of emicizumab.

Methods

Clot waveform analysis (CWA) with prothrombin time (PT)/APTT mixed reagents was used to define hemostatic monitoring protocols in HA patients. A modified parameter, adjusted‐|min1| (Ad|min1|), was developed. Maximum and minimum percentage transmittance were defined as 100% and 0% in the precoagulation and postcoagulation phases, respectively. Ad|min1| was calculated as an index of the maximum velocity of the coagulation process.

Results

Ad|min1| obtained with mixed‐trigger reagent (PT/APTT/buffer, 1 : 15 : 135) in the presence of emicizumab optimally corresponded to the conversion rate estimated in animals; 0.2–0.4 IU dL^?1 equivalent FVIII per 1 μg mL^?1 emicizumab). Ex vivo addition of emicizumab to HA plasma with or without inhibitors resulted in concentration‐dependent increases in Ad|min1|, with some individual variations. The addition of various concentrations of FVIII to HA plasma mixed with emicizumab resulted in dose‐dependent increases in Ad|min1|. Similarly, mixtures of activated prothrombin complex concentrate and emicizumab added to HA plasma resulted in dose‐dependent increases in Ad|min1|. In contrast, enhanced coagulation potential appeared to be better defined by the clot time than by Ad|min1| in experiments using recombinant activated FVII.

Conclusion

The PT/APTT reagent‐triggered adjusted CWA could provide a useful means of assessing global coagulation potential in emicizumab‐treated HA patients, with enhanced activity neither masking nor being masked by FVIII or bypassing agents.

     

Discordant fibrin formation in hemophilia

Summary. Background: The conversion of fibrinogen to fibrin and its crosslinking to form a stable clot are key events in providing effective hemostasis. Objectives: To evaluate the relationship of fibrinopeptide (FP) release and factor (F) XIII activation in whole blood from hemophiliacs. Patients/Methods: We investigated FPA and FPB release, FXIII activation and fibrin mass in tissue factor‐initiated coagulation in whole blood from individuals with hemophilia and healthy subjects. Results: In hemophiliacs, the rates of fibrin formation were delayed as compared to healthy individuals. FPA/FPB release and FXIII activation were decreased in hemophiliacs vs. healthy individuals: 5.4 ± 0.7 μm min^?1 to 1.7 ± 0.4 μm min^?1 (P = 0.003), 2.3 ± 0.6 μm min^?1 to 0.5 ± 0.1 μm min^?1 (P = 0.025), and 12.1 ± 0.7 nm min^?1 to 3.1 ± 0.7 nm min^?1 (P < 0.0005), respectively. More FPA was released in hemophiliacs (6.6 ± 1.2 μm ) prior to clot time (CT) than in healthy individuals (2.6 ± 0.4 μm , P = 0.013), whereas FPB and activated FXIII levels remained comparable. FXIII activation, which normally coincides with FPA release, was delayed in hemophiliacs. At CT in normal blood, the FPA concentration was 2.6‐fold higher than that of FPB (P = 0.003), whereas in hemophiliacs this ratio was increased to 6.6‐fold (P = 0.001). Conclusions: These data suggest that essential dynamic correlations exist between the presentations of fibrin I, fibrin II, and FXIIIa. The ‘discordance’ of fibrin formation in hemophiliacs results in clots that are more soluble than normal (43% lower mass; P = 0.02). The resulting poor physical clot strength probably plays a crucial role in the pathology of hemophilia.     

Efficacy and safety of secondary prophylactic vs. on-demand sucrose-formulated recombinant factor VIII treatment in adults with severe hemophilia A: results from a 13-month crossover study

Summary. Background: Hemarthroses in severe hemophilia precipitate physical, psychosocial and financial difficulties. Objective: To compare the effects of secondary prophylaxis with on‐demand sucrose‐formulated recombinant factor VIII (rFVIII‐FS) therapy in severe hemophilia A. Patients and methods: This open‐label study included patients aged 30–45 years with factor VIII (FVIII) coagulant activity < 1 IU dL^?1 who were using on‐demand FVIII treatment. Patients were treated with rFVIII‐FS on demand for 6 months, followed by 7 months prophylaxis (20–40 IU kg^?1, three times per week, with the first month considered a run‐in). The primary endpoint was the number of hemarthroses. Results: Twenty patients were enrolled (n = 19 completed); the mean age was 36.4 years, and 16 had target joints. The median (25–75%) number of joint bleeds decreased significantly with prophylaxis [0 (0–3)] vs. on‐demand [15 (11–26); P < 0.001] therapy. The number of all bleeds was 0 (0–3) vs. 20.5 (14–37; P < 0.001), respectively. Median (range) total Gilbert scores improved after prophylaxis [18 (3–39)] compared with on‐demand [25 (4–46)] therapy, predominantly reflecting the improved bleeding score. Median time from last prophylactic infusion to bleed was 2 days; 82.5% of bleeds occurred 2–3 days after the last infusion. Median 48‐h and 72‐h FVIII trough levels measured during months 10 and 13 were consistently > 6 and > 4 IU dL^?1, respectively. Treatment was well tolerated, and no inhibitor formation was observed. Conclusion: Secondary prophylaxis with rFVIII‐FS significantly reduced the frequency of hemarthroses compared with on‐demand therapy in adult patients with severe hemophilia A.     

Fibrinogen replacement therapy for congenital fibrinogen deficiency

Summary. This review of published studies was conducted to derive data on patients with congenital fibrinogen deficiency (CFD), including dosing of fibrinogen replacement therapy, outcome, and adverse events, either temporally related or distant to fibrinogen replacement, in order to assist clinicians in developing treatment plans for patients with CFD. A systematic review was performed of case reports identified by a MEDLINE search between 1961 and 2010. Eligible studies included subjects with a diagnosis of CFD who received fibrinogen replacement. An attempt was made to extract dose, frequency, duration, hemostatic efficacy and adverse events such as thrombosis or allergic reactions. Reported thrombotic events distant from fibrinogen replacement were also recorded. From 104 papers reviewed, a total of 50 cases were identified: afibrinogenemia (35), hypofibrinogenemia (6), and dysfibrinogenemia (9). Fibrinogen replacement therapy was generally effective in preventing or treating bleeding in doses adequate to achieve and maintain fibrinogen activity above 50–100 mg dL^?1 (non‐surgical and obstetric use) or 100–200 mg dL^?1 (surgical prophylaxis). Increased fibrinogen clearance was observed with massive hemorrhage, major surgery, and advanced pregnancy. Obstetric outcomes were optimized when fibrinogen replacement was initiated prior to conception. Uncontrolled hemorrhage, allergic reactions and antibody formation were rare events. However, thromboses, both related and unrelated to fibrinogen replacement, occurred in 15 of 50 (30%) patients overall, and in eight of 12 (67%) adult non‐obstetric patients with afibrinogenemia. Published fibrinogen replacement regimens are presented for 50 CFD patients. Fibrinogen replacement therapy requires careful monitoring of fibrinogen levels. Afibrinogenemia is associated with thromboembolic complications with or without treatment.     

Respiratory compensation point during incremental test in overweight and normoweight boys: is it useful in assessing aerobic performance? A longitudinal study

The purpose of this study was to investigate the characteristics of the respiratory compensation point (RCP) in overweight and normoweight boys and to clarify changes in the RCP over 4 years. This study was conducted with 11 overweight boys and 14 boys with normal weight. The boys performed the graded test every 2 years (three series) beginning at the age of 9–10 years and finishing at the age of 13–14 years. During the test, the RCP was detected. In every series, the RCP occurred earlier in the overweight boys than in the normoweight boys and at a significantly (P<0·05) lower rate relative to body mass power output (P kg^?1). Relative oxygen uptake (VO₂ kg^?1) at the RCP in all studies was also significantly (P<0·05) lower in the group of overweight boys. The maximum level of analysed indicators (VO₂max; Pmax) differentiated both groups in similar ways as their level noted at RCP. This study showed significant (P<0·05) correlation between the values VO₂max kg^?1 and VO₂ kg^?1 at RCP in each series of the test and between Pmax kg^?1 and P kg^?1 at RCP. The respiratory compensation point seems to be a good method for evaluating aerobic performance in children (also overweight). During puberty, a decreasing tendency in aerobic performance was observed in both groups.     

Evaluation of fibrinogen self‐assembly: role of its αC region

Summary. Background: Exposure of cryptic, functional sites on fibrinogen upon its adsorption to hydrophobic surfaces of biomaterials has been linked to an inflammatory response and fibrosis. Such adsorption also induces ordered fibrinogen aggregation which is poorly understood. Objective: To investigate hydrophobic surface‐induced fibrinogen aggregation. Methods: Contact and lateral force scanning probe microscopy, yielding topography, image dimensions and fiber elastic modulus measurements were used along with transmission and scanning electron microscopy. Fibrinogen aggregation was induced under non‐enzymatic conditions by adsorption on a trioctyl‐surface monolayer (trioctylmethylamine) grafted onto silica clay plates. Results: A more than one molecule thick coating was generated by adsorption on the plate from 100 to 200 μg mL^?1 fibrinogen solutions, and three‐dimensional networks formed from 4 mg mL^?1 fibrinogen incubated with uncoated or fibrinogen‐coated plates. Fibrils appeared laterally assembled into branching and overlapping fibers whose heights from the surface ranged from approximately 3 to 740 nm. The elastic modulus of fibrinogen fibers was 1.55 MPa. No fibrils formed when fibrinogen lacking αC‐domains was used as a coating or was incubated with intact fibrinogen‐coated plates, or when the latter plates were sequentially incubated with anti‐Aα529–539 mAb and intact fibrinogen. When an anti‐Aα241–476 mAb was used instead, fine, long fibers formed. Similarly, sequential incubations of fibrinogen‐coated plates with recombinant αC‐domain (Aα392–610 fragment) or αC‐connector (Aα221–372 fragment) and fibrinogen resulted in distinctly fine fiber networks. Conclusions: Adsorption‐induced fibrinogen self‐assembly is initiated by a more than one molecule‐thick surface layer and eventuates in three‐dimensional networks whose formation requires fibrinogen with intact αC‐domains.     

The associations between peak O2 consumption and leptin in 10‐ to 12‐year‐old boys

The aim of this study was to assess the associations of circulating levels of leptin with the peak O₂ consumption (VO_2peak) in 10 ‐ to 12‐year‐old boys of different BMI selected by Cole et al. (BMJ, 320,2000,1–6): total group (n = 248), normal (n = 190), overweight (n = 34) and obese (n = 24). We hypothesized that there is a close relationship in overweight and obese subgroups of boys with relative VO_{2peak kg}^?1(ml min^?1 kg^?1) and leptin. Most of the subjects were Tanner stage 2. Peak O₂ consumption was measured directly using an increasing incremental protocol until volitional exhaustion on an electronically braked cycle ergometer. The expired gas was sampled continuously breadth‐by‐breadth mode for the measurement of oxygen consumption (MetaMax, Germany). Blood samples were obtained after an overnight fast from an antecubital vein for leptin measurements. Peak O₂ consumption (l min^?1) was higher or lower (ml min^?1 kg^?1) in overweight and obese groups, compared with normal BMI group. Leptin was higher in overweight and obese groups, compared with normal BMI group. Peak O₂ consumption (l min^?1) correlated significantly with leptin only in total group (n = 248, r   =   0·196). Contrary, relative VO_2peak kg^?1 correlated significantly and negatively with leptin. The relationship was highest on the total group (r   =  ?0·674). We can conclude that leptin first of all correlated negatively with relative peak O₂ consumption. Absolute VO_2peak correlated with leptin only in total group.     

Effect of long‐chain triglyceride lipid emulsion on bupivacaine‐induced changes in electrophysiological parameters of rabbit Purkinje cells

Lipid emulsions are used in the reversal of local anesthetic toxicity. The aim of this study was to investigate the cellular electrophysiological effects of long‐chain triglyceride lipid emulsion (LCTE) on cardiac action potential characteristics and conduction disturbances induced by bupivacaine. Purkinje fibers were dissected from the left ventricle of New Zealand white rabbit hearts and superfused with either Tyrode's solution during 30 min (control group), with bupivacaine 10^?6 m , 10^?5 m , and 5.10^?5 m alone, or in the presence of LCTE 0.5%, in addition, LCTE at 0.1%, 0.5%, and 1% was perfused alone. Electrophysiological parameters were recorded using the conventional microelectrode technique (37 °C, 1 Hz frequency). Bupivacaine 5.10^?5 m ‐induced conduction blocks (8/8 preparations): LCTE 0.5% suppressed the bupivacaine 5.10^?5 m ‐induced conduction blocks (1/8 preparations). Exposure to bupivacaine 10^?6 m , 10^?5 m , and 5.10^?5 m resulted in a significant decrease in the maximal rate of depolarization (V_max) (respectively, 25%, 55%, 75%; P < 0.002 vs. control group). In the presence of LCTE 0.5%, bupivacaine 10^?6 m did not significantly decreased V_max (13%; P = 0.10 vs. control group). The decrease in V_max resulting from bupivacaine 10^?5 m alone was significantly less in the presence of LCTE 0.5% (P < 0.01 vs. bupivacaine 10^?5 m alone). Exposure to bupivacaine 10^?6 m , 10^?5 m , and 5.10^?5 m alone or in the presence of LCTE 0.5% resulted in a significant decrease in action potential duration measured at 50% and 90% repolarization (APD₅₀ and APD₉₀; P < 0.01 vs. control group). LCTE inhibited the Purkinje fibers conduction blocks induced by bupivacaine. Moreover, LCTE 0.5% attenuates the decrease in V_max induced by bupivacaine 10^?6 m and 10^?5 m .     

Safety of catheter‐delivered plasmin in patients with acute lower extremity arterial or bypass graft occlusion: phase I results

Summary. Background: Current treatment of acute peripheral artery or bypass graft occlusion utilizes catheter‐directed thrombolysis of a plasminogen activator (PA). Plasmin is a direct‐acting thrombolytic with a striking safety advantage over PA in preclinical models. Objectives: To report the first use of purified plasmin for acute lower extremity arterial or bypass graft thrombosis in a phase I dose‐escalation study of a catheter‐delivered agent. Methods: Eighty‐three patients with non‐embolic occlusion of infrainguinal native arteries or bypass grafts were enrolled (safety population) into seven sequential dose cohorts to receive 25–175 mg of plasmin by intrathrombus infusion over 5 h. Arteriograms were performed at baseline, 2 h, and 5 h, and subjects were monitored for 30 days for clinical outcomes and laboratory parameters of systemic fibrinolysis. Results: Major bleeding occurred in four patients (4.8%), and minor bleeding alone in 13 (15.7%), with no trend towards more bleeding at higher dosages of plasmin. There was a trend towards lower plasma concentrations of fibrinogen, α₂‐antiplasmin and α₂‐macroglobulin with increasing doses of plasmin, but the nadir fibrinogen concentration was > 350 mg dL^?1 at the highest plasmin dose. Individual nadir values were above 200 mg dL^?1 in 82 of 83 subjects, and were not different in patients with or without bleeding. Thrombolysis (≥50%) occurred in 79% of subjects receiving 125–175 mg of plasmin, as compared with 50% who received 25–100 mg. Conclusions: Catheter‐delivered plasmin can be safely administered to patients with acute lower extremity arterial occlusion at dosages of 25–175 mg.