In vivo efficacy of platelet-delivered, high specific activity factor VIII variants.
ABSTRACT: Ectopically expressed, human B-domainless (hB) factor 8 (F8) in platelets improves hemostasis in hemophilia A mice in several injury models. However, in both a cuticular bleeding model and a cremaster laser arteriole/venule injury model, there were limitations to platelet-derived (p) hBF8 efficacy, including increased clot embolization. We now address whether variants of F8 with enhanced activity, inactivation resistant F8 (IR8) and canine (c) BF8, would improve clotting efficacy. In both transgenic and lentiviral murine model approaches, pIR8 expressed at comparable levels to phBF8, but pcBF8 expressed at only approximately 30%. Both variants were more effective than hBF8 in cuticular bleeding and FeCl(3) carotid artery models. However, in the cremaster injury model, only pcBF8 was more effective, markedly decreasing clot embolization. Because inhibitors of F8 are stored in platelet granules and IR8 is not protected by binding to von Willebrand factor, we also tested whether pIR8 was effective in the face of inhibitors and found that pIR8 is protected from the inhibitors. In summary, pF8 variants with high specific activity are more effective in controlling bleeding, but this improved efficacy was inconsistent between bleeding models, perhaps reflecting the underlying mechanism(s) for the increased specific activity of the studied F8 variants.
Project description:Ischemia which leads to heart attacks and strokes is one of the major causes of death in the world. Whether an occlusion occurs or not depends on the ability of a growing thrombus to resist flow forces exerted on its structure. This manuscript provides the first known in vivo measurement of how much stress a clot can withstand, before yielding to the surrounding blood flow. Namely, Lattice-Boltzmann Method flow simulations are performed based on 3D clot geometries, which are estimated from intravital microscopy images of laser-induced injuries in cremaster microvasculature of live mice. In addition to reporting the blood clot yield stresses, we also show that the thrombus "core" does not experience significant deformation, while its "shell" does. This indicates that the shell is more prone to embolization. Therefore, drugs should be designed to target the shell selectively, while leaving the core intact to minimize excessive bleeding. Finally, we laid down a foundation for a nondimensionalization procedure which unraveled a relationship between clot mechanics and biology. Hence, the proposed framework could ultimately lead to a unified theory of thrombogenesis, capable of explaining all clotting events. Thus, the findings presented herein will be beneficial to the understanding and treatment of heart attacks, strokes and hemophilia.
Project description:HNG, a highly potent mutant of the anti-Alzheimer peptide-humanin, has been shown to protect against ischaemia-reperfusion (I/R) injury. However, the underlying mechanism related to platelet activation remains unknown. We proposed that HNG has an effect on platelet function and thrombus formation. In this study, platelet aggregation, granule secretion, clot retraction, integrin activation and adhesion under flow conditions were evaluated. In mice receiving HNG or saline, cremaster arterial thrombus formation induced by laser injury, tail bleeding time and blood loss were recorded. Platelet microtubule depolymerization was evaluated using immunofluorescence staining. Results showed that HNG inhibited platelet aggregation, P-selectin expression, ATP release, and ?IIb ?3 activation and adhesion under flow conditions. Mice receiving HNG had attenuated cremaster arterial thrombus formation, although the bleeding time was not prolonged. Moreover, HNG significantly inhibited microtubule depolymerization, enhanced tubulin acetylation in platelets stimulated by fibrinogen or microtubule depolymerization reagent, nocodazole, and inhibited AKT and ERK phosphorylation downstream of HDAC6 by collagen stimulation. Therefore, our results identified a novel role of HNG in platelet function and thrombus formation potentially through stabilizing platelet microtubules via tubulin acetylation. These findings suggest a potential benefit of HNG in the management of cardiovascular diseases.
Project description:New therapies are needed to control bleeding in a range of clinical conditions. This review will discuss the biochemical properties of zymogen-like factor Xa, its preclinical assessment in different model systems, and future development prospects.Underlying many procoagulant therapeutic approaches is the rapid generation of thrombin to promote robust clot formation. Clinically tested prohemostatic agents (e.g., factor VIIa) can provide effective hemostasis to mitigate bleeding in hemophilia and other clinical situations. Over the past decade, we explored the possibility of using zymogen-like factor Xa variants to rapidly improve clot formation for the treatment of bleeding conditions. Compared to the wild-type enzyme, these variants adopt an altered, low activity, conformation which enables them to resist plasma protease inhibitors. However, zymogen-like factor Xa variants are conformationally dynamic and ligands such as its cofactor, factor Va, stabilize the molecule rescuing procoagulant activity. At the site of vascular injury, the variants in the presence of factor Va serve as effective prohemostatic agents. Preclinical data support their use to stop bleeding in a variety of clinical settings. Phase 1 studies suggest that zymogen-like factor Xa is safe and well tolerated, and a phase 1b is ongoing to assess safety in patients with intracerebral hemorrhage.Zymogen-like factor Xa is a unique prohemostatic agent for the treatment of a range of bleeding conditions.
Project description:BACKGROUND:Treatments for major internal bleeding after injury include permissive hypotension to decrease the rate of blood loss, intravenous infusion of plasma or clotting factors to improve clot formation, and rapid surgical hemostasis or arterial embolization to control bleeding vessels. Yet, little is known regarding major internal arterial hemostasis, or how these commonly used treatments might influence hemostasis. OBJECTIVES:(i) To use a swine model of femoral artery bleeding to understand the perivascular hemostatic response to contained arterial hemorrhage. (ii) To directly confirm the association between hemodynamics and bleeding velocity. (iii) To observe the feasibility of delivering an activated clotting factor directly to internal sites of bleeding using a simplified angiographic approach. METHODS:Ultrasound was used to measure bleeding velocity and in vivo clot formation by elastography in a swine model of contained femoral artery bleeding with fluid resuscitation. A swine model of internal pelvic and axillary artery hemorrhage was also used to demonstrate the feasibility of local delivery of an activated clotting factor. RESULTS:In this model, clots formed slowly within the peri-wound hematoma, but eventually contained the bleeding. Central hemodynamics correlated positively with bleeding velocity. Infusion of recombinant human activated factor VII into the injured artery near the site of major internal hemorrhage in the pelvis and axillae was feasible. CONCLUSIONS:We rediscovered that clot formation within the peri-wound hematoma is an integral component of hemostasis and a feasible target for the treatment of major internal bleeding using activated clotting factors delivered using a simplified angiographic approach.
Project description:Intraosseous (IO) infusion of lentiviral vectors (LVs) for in situ gene transfer into bone marrow may avoid specific challenges posed by ex vivo gene delivery, including, in particular, the requirement of preconditioning. We utilized IO delivery of LVs encoding a GFP or factor VIII (FVIII) transgene directed by ubiquitous promoters (a MND or EF-1?-short element; M-GFP-LV, E-F8-LV) or a platelet-specific, glycoprotein-1b? promoter (G-GFP-LV, G-F8-LV). A single IO infusion of M-GFP-LV or G-GFP-LV achieved long-term and efficient GFP expression in Lineage(-)Sca1(+)c-Kit(+) hematopoietic stem cells and platelets, respectively. While E-F8-LV produced initially high-level FVIII expression, robust anti-FVIII immune responses eliminated functional FVIII in circulation. In contrast, IO delivery of G-F8-LV achieved long-term platelet-specific expression of FVIII, resulting in partial correction of hemophilia A. Furthermore, similar clinical benefit with G-F8-LV was achieved in animals with pre-existing anti-FVIII inhibitors. These findings further support platelets as an ideal FVIII delivery vehicle, as FVIII, stored in ?-granules, is protected from neutralizing antibodies and, during bleeding, activated platelets locally excrete FVIII to promote clot formation. Overall, a single IO infusion of G-F8-LV was sufficient to correct hemophilia phenotype for long term, indicating that this approach may provide an effective means to permanently treat FVIII deficiency.
Project description:Hemophilia A (HA) is an X-linked bleeding disorder caused by deleterious mutations in the coagulation factor VIII gene (F8). To date, F8 mutations have been documented predominantly in European subjects and in American subjects of European descent. Information on F8 variants in individuals of more diverse ethnic backgrounds is limited.To discover novel and rare F8 variants, and to characterize F8 variants in diverse population backgrounds.We analyzed 2535 subjects, including 26 different ethnicities, whose data were available from the 1000 Genomes Project (1000G) phase 3 dataset, for F8 variants and their potential functional impact.We identified 3030 single nucleotide variants, 31 short deletions and insertions (Indels) and a large, 497 kb, deletion. Among all variants, 86.4% were rare variants and 55.6% were novel. Eighteen variants previously associated with HA were found in our study. Most of these 'HA variants' were ethnic-specific with low allele frequency; however, one variant (p.M2257V) was present in 27% of African subjects. The p.E132D, p.T281A, p.A303V and p.D422H 'HA variants' were identified only in males. Twelve novel missense variants were predicted to be deleterious. The large deletion was discovered in eight female subjects without affecting F8 transcription and the transcription of genes on the X chromosome.Characterizing F8 in the 1000G project highlighted the complexity of F8 variants and the importance of interrogating genetic variants on multiple ethnic backgrounds for associations with bleeding and thrombosis. The haplotype analysis and the orientation of duplicons that flank the large deletion suggested that the deletion was recurrent and originated by homologous recombination.
Project description:Hemophilia A is a common X chromosome-linked genetic bleeding disorder caused by abnormalities in the coagulation factor VIII gene (F8). Hemophilia A patients suffer from a bleeding diathesis, such as life-threatening bleeding in the brain and harmful bleeding in joints and muscles. Because it could potentially be cured by gene therapy, subhuman animal models have been sought. Current mouse hemophilia A models generated by gene targeting of the F8 have difficulties to extrapolate human disease due to differences in the coagulation and immune systems between mice and humans. Here, we generated a porcine model of hemophilia A by nuclear transfer cloning from F8-targeted fibroblasts. The hemophilia A pigs showed a severe bleeding tendency upon birth, similar to human severe hemophiliacs, but in contrast to hemophilia A mice which rarely bleed under standard breed conditions. Infusion of human factor VIII was effective in stopping bleeding and reducing the bleeding frequency of a hemophilia A piglet but was blocked by the inhibitor against human factor VIII. These data suggest that the hemophilia A pig is a severe hemophilia A animal model for studying not only hemophilia A gene therapy but also the next generation recombinant coagulation factors, such as recombinant factor VIII variants with a slower clearance rate.
Project description:Normally factor (F) VIII is not expressed in megakaryocytes, but when human FVIII was transgenically expressed in murine megakaryocytes, it was stored in platelet alpha-granules and released at sites of injury. This platelet FVIII (pFVIII) is effective in correcting hemostasis, even in the presence of circulating inhibitors, so it offers a potential gene therapy strategy for hemophilia A. To understand clot development by pFVIII, we have examined clot response to laser injury in both cremaster arterioles and venules in FVIII(null) mice either infused with FVIII or transgenic for pFVIII. In both sets of vessels, pFVIII is at least as effective as infused FVIII. However, there are temporal and spatial differences in fibrin and platelet accumulation within clots depending on how FVIII is delivered. These differences may be related to the temporal and spatial distribution of the alpha-granular-released FVIII within the developing clot, and may explain the increased frequency and size of embolic events seen with pFVIII. These observations may not only have implications for the use of pFVIII in gene therapy for hemophilia A, but may also have physiologic consequences, explaining why many procoagulant factors are delivered both in the plasma and in platelet alpha-granules.
Project description:Hemothorax is a common occurrence after blunt or penetrating injury to the chest. Posterior intercostal vessel hemorrhage as a cause of major intrathoracic bleeding is an infrequent source of massive bleeding. Selective angiography with trans-catheter embolization may provide a minimally invasive and efficient method of controlling bleeding refractory to surgical treatment.A 19 year-old male sustained a gunshot wound to his left chest with massive hemothorax and refractory hemorrhage. He was emergently taken to the operating room for thoracotomy and was found to have uncontrollable bleeding from the chest due to left posterior intercostal artery transection. The bleeding persisted despite multiple attempts with sutures, clips and various hemostatic agents. Thoracic aortography was undertaken and revealed active bleeding from the left 7th posterior intercostal artery, which was coil-embolized. The patient's hemodynamic status significantly improved and he was transferred to the intensive care unit.Posterior intercostal bleeding is a rare cause of massive hemothorax. Bleeding from these arteries may be difficult to control due to limited exposure in that area. Transcatheter-based arterial embolization is a reliable and feasible option for arresting hemorrhage following failed attempts at hemorrhage control from thoracotomy.Massive hemothorax from intercostal arterial bleeding is a rare complication after penetrating chest injury (Aoki et al., 2003). Selective, catheter-based embolization is a useful therapeutic option for hemorrhage control and can be expeditiously employed if a hybrid operating room is available.
Project description:Abdominopelvic trauma (APT) remains a leading cause of morbidity and mortality in the 15- to 44-year-old age group in the Western World. It can be life-threatening as abdominopelvic organs, specifically those in the retroperitoneal space, can bleed profusely. APT is divided into blunt and penetrating types. While surgery is notably considered as a definitive solution for bleeding control, it is not always the optimum treatment for the stabilization of a polytrauma patient. Over the past decades, there has been a shift toward more sophisticated strategies, such as non-operative management of abdominopelvic vascular trauma for haemodynamically stable patients. Angiographic embolization for bleeding control following blunt and/or penetrating intra- and retroperitoneal injuries has proven to be safe and effective. Embolization can achieve hemostasis and salvage organs without the morbidity of surgery, and the development and refinement of embolization techniques has widened the indications for non-operative treatment in solid organ injury. Moreover, advances in computed tomography provided more efficient scanning times with improved image quality. While surgery is still usually recommended for patients with penetrating injuries, non-operative management can be effectively used as well as an alternative treatment. We review indications, technical considerations, efficacy, and complication rates of angiographic embolization in APT.