BACKGROUND:Platelet cross-linking during arterial thrombosis involves von Willebrand Factor (VWF) multimers. Therefore, proteolysis of VWF appears promising to disaggregate platelet-rich thrombi and ...restore vessel patency in acute thrombotic disorders such as ischemic stroke, acute coronary syndrome, or acute limb ischemia. N-Acetylcysteine (NAC, a clinically approved mucolytic drug) can reduce intrachain disulfide bonds in large polymeric proteins. In the present study, we postulated that NAC might cleave the VWF multimers inside occlusive thrombi, thereby leading to their dissolution and arterial recanalization.
METHODS:Experimental models of thrombotic stroke induced by either intra-arterial thrombin injection or ferric chloride application followed by measurement of cerebral blood flow using a combination of laser Doppler flowmetry and MRI were performed to uncover the effects of NAC on arterial thrombi. To investigate the effect of NAC on larger vessels, we also performed ferric chloride–induced carotid artery thrombosis. In vitro experiments were performed to study the molecular bases of NAC thrombolytic effect, including platelet aggregometry, platelet-rich thrombi lysis assays, thromboelastography (ROTEM), and high-shear VWF string formation using microfluidic devices. We also investigated the putative prohemorrhagic effect of NAC in a mouse model of intracranial hemorrhage induced by in situ collagenase type VII injection.
RESULTS:We demonstrated that intravenous NAC administration promotes lysis of arterial thrombi that are resistant to conventional approaches such as recombinant tissue-type plasminogen activator, direct thrombin inhibitors, and antiplatelet treatments. Through in vitro and in vivo experiments, we provide evidence that the molecular target underlying the thrombolytic effects of NAC is principally the VWF that cross-link platelets in arterial thrombi. Coadministration of NAC and a nonpeptidic GpIIb/IIIa inhibitor further improved its thrombolytic efficacy, essentially by accelerating thrombus dissolution and preventing rethrombosis. Thus, in a new large-vessel thromboembolic stroke model in mice, this cotreatment significantly improved ischemic lesion size and neurological outcome. It is important to note that NAC did not worsen hemorrhagic stroke outcome, suggesting that it exerts thrombolytic effects without significantly impairing normal hemostasis.
CONCLUSIONS:We provide evidence that NAC is an effective and safe alternative to currently available antithrombotic agents to restore vessel patency after arterial occlusion.
There are no specific recommendations for the management of patients with bleeding disorders (BD), such as haemophilia A (HA), haemophilia B (HB), or von Willebrand disease (WD), in urology surgery.
...We conducted a retrospective study of 32 patients with HA, HB, or WD of any severity. Fifty-seven procedures were performed between January 2017 and September 2023. Surgical interventions were divided into two groups: those with and without electrocoagulation. The control patients were successively matched in a 2:1 ratio.
The study group consisted of 30 men and 2 women, with 23 HA, 2 HB, and 7 WD. The median age of the patients was 69 years. The BD group had a longer hospital stay of 4 days compared to 1 day (
< 0.0001). The incidence of bleeding events was 21% versus 2% (
< 0.0001), and the incidence of complications was 21% versus 7% (
= 0.0036) for Clavien 1-2 respectively. In the subgroup with intraoperative coagulation, the readmission rate at 30 days was higher (17% vs. 3%,
= 0.00386), as was the transfusion rate (17% vs. 3%,
= 0.0386).
This study showed that urological procedures in patients with bleeding disorders were associated with a higher risk of bleeding and complications.
Background Desmopressin (DDAVP) is used in patients with moderate/mild hemophilia A (PWMHs) to increase their factor VIII (FVIII) level and, if possible, normalize it. However, its effectiveness ...varies between individuals. The GIDEMHA study aims to investigate the influence of F8 gene variants. Material and Methods The study collected the trajectory of FVIII levels from therapeutic intravenous DDAVP tests in four French hemophilia treatment centers. A pharmacological analysis was performed associated with efficacy scores according to F8 variants: absolute and relative responses, as well as new scores: absolute duration (based on duration with FVIII ≥ 0.50 IU.mL−1) and relative duration (based on half-life). Results From enrolled 439 PWMHs, 327 had a hot-spot F8 variant (with ≥5 PWMHs). For these, the median (min–max) basal and peak FVIII were 0.20 (0.02–0.040) and 0.74 (0.14–2.18) IU.mL−1 respectively, with FVIII recovery being 3.80 IU.ml−1 (1.15–14.75). The median FVIII half-life was 3.9 hours (0.7–15.9 hours). FVIII was normalized (≥0.50 IU.mL−1) in 224/327 PWMHs (69%) and the median time with normalized FVIII was 3.9 hours (0.0–54.1 hours). Following the response profiles to DDAVP defined by the four efficacy scores, four groups of F8 variants were isolated, and then compared using survival curves with normalized FVIII (p < 0.0001): “long-lastingly effective” p.(Glu739Lys), p.(Ser2030Asn), p.(Arg2178His), p.(Gln2208Glu), and T-stretch deletion in intron 13; “moderately effective” p.(Ser112Phe), p.(Ala219Thr), p.(Thr2105Ile), p.Phe2146Ser), and p.(Asp2150Asn); “moderately ineffective” p.Ala81Asp), p.(Gln324Pro), p.(Tyr492His), p.(Arg612Cys), p.(Met701Val), p.(Val2035Asn), and p.(Arg2178Cys); and “frequently ineffective” c.-219C > T, p.(Cys2040Tyr), p.(Tyr2169His), p.(Pro2319Leu), and p.(Arg2326Gln). Conclusion In view of our data, we propose indications for DDAVP use in PWMH based on F8 variants for minor and major invasive procedures.
Background
Efmoroctocog alfa, the first recombinant factor VIII fusion protein with extended half‐life (rFVIII‐Fc), has been hypothesized to lower FVIII consumption in patients with severe ...Haemophilia A (pwSHA), without reducing clinical efficacy. What about real life?
Method
MOTHIF‐II was a noninterventional, multicentre, before/after study, via the collection of retrospective data from July 2015 to June 2016 (called T1), and from July 2017 to June 2018 (called T2), in 7 French haemophilia treatment centres. We examined the prescriptions and dispensations of factor VIII and the Annual Bleeding Rate (ABR), in pwSHA without current inhibitors on prophylaxis, before and after the introduction of rFVIII‐Fc. The data gathered from the BERHLINGO research database and from the French Healthcare claims database with a determinist pairing process based on the national unique identification number.
Results
A total of 156 pwSHA were included in the prescription cohort and 83 in the ABR cohort. For switched patients, the mean amounts of prescribed FVIII were significantly higher during T1 compared to T2 (4333 (2052) vs. 3921 (2029) IU/kg/year/patient, p: 0.028); a significant decrease in their ABR was also observed between T1 and T2 (6.3 (6.0) vs. 4.4 (5.4), p: 0.047). These patients had a more severe bleeding profile centred on haemarthrosis.
Conclusion
The results are related to those of the pivotal clinical trials for the reduction in FVIII consumption following the switch to rFVIII‐Fc, with a significant improvement in the haemorrhagic phenotype for pwSHA.
Induction of heme oxygenase-1, a stress-inducible enzyme with anti-inflammatory activity, reduces the immunogenicity of therapeutic factor VIII in experimental hemophilia A. In humans, heme ...oxygenase-1 expression is modulated by polymorphisms in the promoter of the heme oxygenase-1-encoding gene (HMOX1). We investigated the relationship between polymorphisms in the HMOX1 promoter and factor VIII inhibitor development in severe hemophilia A. We performed a case-control study on 99 inhibitor-positive patients and 263 patients who did not develop inhibitors within the first 150 cumulative days of exposure to therapeutic factor VIII. Direct sequencing and DNA fragment analysis were used to study (GT)n polymorphism and single nucleotide polymorphisms located at -1135 and -413 in the promoter of HMOX1. We assessed associations between the individual allele frequencies or genotypes, and inhibitor development. Our results demonstrate that inhibitor-positive patients had a higher frequency of alleles with large (GT)n repeats (L: n≥30), which are associated with lesser heme oxygenase-1 expression (odds ratio 2.31; 95% confidence interval 1.46-3.66; P<0.001. Six genotypes (L/L, L/M, L/S, M/M, M/S and S/S) of (GT)n repeats were identified (S: n<21; M: 21≤n<30). The genotype group including L alleles (L/L, L/M and L/S) was statistically more frequent among inhibitor-positive than inhibitor-negative patients, as compared to the other genotypes (33.3% versus 17.1%) (odds ratio 2.21, 95% confidence interval 1.30-3.76; P<0.01). To our knowledge, this is the first association identified between HMOX1 promoter polymorphism and development of anti-drug antibodies. Our study paves the way towards modulation of the endogenous anti-inflammatory machinery of hemophilia patients to reduce the risk of inhibitor development.
Factor VIII (FVIII) is a glycoprotein that plays an important role in the intrinsic pathway of coagulation. In circulation, FVIII is protected upon binding to von Willebrand factor (VWF), a chaperone ...molecule that regulates its half-life, distribution, and activity. Despite the biological significance of this interaction, its molecular mechanisms are not fully characterized. We determined the equilibrium and activation thermodynamics of the interaction between FVIII and VWF. The equilibrium affinity determined by surface plasmon resonance was temperature-dependent with a value of 0.8 nM at 35 °C. The FVIII–VWF interaction was characterized by very fast association (8.56 × 106 M–1 s–1) and fast dissociation (6.89 × 10–3 s–1) rates. Both the equilibrium association and association rate constants, but not the dissociation rate constant, were dependent on temperature. Binding of FVIII to VWF was characterized by favorable changes in the equilibrium and activation entropy (TΔS° = 89.4 kJ/mol, and –TΔS ⧧ = −8.9 kJ/mol) and unfavorable changes in the equilibrium and activation enthalpy (ΔH° = 39.1 kJ/mol, and ΔH ⧧ = 44.1 kJ/mol), yielding a negative change in the equilibrium Gibbs energy. Binding of FVIII to VWF in solid-phase assays demonstrated a high sensitivity to acidic pH and a sensitivity to ionic strength. Our data indicate that the interaction between FVIII and VWF is mediated mainly by electrostatic forces, and that it is not accompanied by entropic constraints, suggesting the absence of conformational adaptation but the presence of rigid “pre-optimized” binding surfaces.
BACKGROUND AND PURPOSE—Tissue-type plasminogen activator (tPA) is the only acute treatment for ischemic stroke. Unfortunately, the benefit of tPA-driven thrombolysis is not systematic, and ...understanding the reasons for this is mandatory. The balance between beneficial and detrimental effects of tPA might explain the limited overall efficiency of thrombolysis. Here, we investigated whether this balance could be influenced by excessive alcohol intake.
METHODS—We used a murine model of thromboembolic stroke, coupled to an array of biochemical assays, near-infrared or magnetic resonance imaging scans, 2-photon microscopy, hydrodynamic transfections, and immunohistological techniques.
RESULTS—We found that 6 weeks of alcohol consumption (10% in drinking water) worsens ischemic lesions and cancels the beneficial effects of tPA-induced thrombolysis. We accumulate in vivo and in vitro evidence showing that this aggravation is correlated with a decrease in lipoprotein receptor–related protein 1–mediated hepatic clearance of tPA in alcohol-exposed mice.
CONCLUSIONS—An efficient liver-driven clearance of tPA might influence the safety of thrombolysis after stroke.
Together with our observations, the data suggest that the generation of a novel FVIII therapeutic compromised in its capability to interact with DCs upon the removal of selected N-glycosylation sites ...may be a novel strategy to produce a hemostatically proficient drug with potentially reduced immunogenicity in patients with hemophilia A. We thank Joelle Treton (INSERM U662, Hospital Saint-Louis, Paris, France) for providing us with blood from healthy MHC-matched donors. mAb 77IP52H7 and Factane were gifts from LFB.
Acquired hemophilia is a rare bleeding disorder characterized by the spontaneous occurrence of inhibitory antibodies against endogenous factor VIII (FVIII). IgG from some patients with acquired ...hemophilia hydrolyze FVIII. Because of the complex etiology of the disease, no clinical parameter, including the presence of FVIII-hydrolyzing IgG, has been associated with patient's survival or death. Here, we demonstrate the presence of anti-FIX antibodies in acquired hemophilia patients. IgG from some patients were found to hydrolyze FIX. In most cases, IgG-mediated FIX-hydrolysis resulted in FIX activation. IgG-mediated hydrolysis of FIX thus led to the significant generation of activated FIX in 25 of 65 patients. Based on the estimated kinetic parameters, patients' IgG activated up to 0.3nM FIX in 24 hours, an amount that restored thrombin generation in vitro provided the presence of more than or equal to 3% residual FVIII activity in plasma. This work identifies proteolytic IgG as novel molecules able to activate FIX under pathologic conditions. IgG-mediated FIX activation is a prevalent phenomenon among acquired hemophilia patients. The presence of FIX-activating IgG may partly compensate for the antibody-mediated inhibition of endogenous FVIII in restoring thrombin generation. This clinical trial was registered at www.clinicaltrials.gov as #NCT00213473.
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