Packaging of supplemented urokinase into alpha-granules of in vitro-grown megakaryocytes for targeted nascent clot lysis.

Fibrinolytics delivered into the general circulation lack selectivity for nascent thrombi, reducing efficacy and increasing the risk of bleeding. Urokinase-type plasminogen activator (uPA) transgenically expressed within murine platelets provided targeted thromboprophylaxis without causing bleeding, but is clinically infeasible. Recent advances in generating megakaryocytes prompted us to develop a potentially clinically relevant means to produce "anti-thrombotic" platelets from CD34+ hematopoietic stem cell-derived in vitro-grown megakaryocytes. CD34+-megakaryocytes internalize and store in -granules single-chain uPA (scuPA) and a plasmin-resistant thrombin-activatable variant (uPAT). Both uPAs co-localized with internalized factor V (FV), fibrinogen and plasminogen, low-density lipoprotein receptor-related protein 1 (LRP1), and interferon-induced transmembrane protein 3, but not with endogenous von Willebrand factor (VWF). Endocytosis of uPA by CD34+-megakaryocytes was mediated, in part, via LRP1 and IIb3. scuPA-containing megakaryocytes degraded endocytosed intragranular FV, but not endogenous VWF in the presence of internalized plasminogen, whereas uPAT-megakaryocytes did not significantly degrade either protein. We used a carotid-artery injury model in NOD-scid IL2rnull (NSG) mice homozygous for VWFR1326H (a mutation switching binding VWF specificity from mouse to human glycoprotein Ib) to test whether platelets derived from scuPA- or uPAT-megakaryocytes would prevent thrombus formation. NSG/VWFR1326H mice exhibited a lower thrombotic burden after carotid artery injury compared to NSG mice unless infused with human platelets or megakaryocytes, whereas intravenous injection of uPA-megakaryocytes generated sufficient uPA-containing human platelets to lyse nascent thrombi. These studies describe the use of in vitro-generated megakaryocytes as a potential platform for delivering uPA or other ectopic proteins within platelet -granules to sites of vascular injury.

Factor IXa variants resistant to plasma inhibitors enhance clot formation in vivo.

Factor IXa (FIXa) plays a pivotal role in coagulation by contributing to FX activation via the intrinsic pathway. Although antithrombin (AT) and other plasma inhibitors are thought to regulate FIXa procoagulant function, the impact of FIXa inhibition on thrombin generation and clot formation in vivo remains unclear. Here, we generated FIXa variants with altered reactivity to plasma inhibitors that target the FIXa active site but maintain procoagulant function when bound to its cofactor, FVIIIa. We found that selected FIXa variants (eg, FIXa-V16L) have a prolonged activity half-life in the plasma due, in part, to AT resistance. Studies using hemophilia B mice have shown that delayed FIXa inhibition has a major impact on reducing the bleeding phenotype and promoting thrombus formation following administration of FIX protein. Overall, these results demonstrate that the regulation of FIXa inhibition contributes in a major way to the spatial and temporal control of coagulation at the site of vascular injury. Our findings provide novel insights into the physiological regulation of FIXa, enhance our understanding of thrombus formation in vivo via the intrinsic pathway, and suggest that altering FIXa inhibition could have therapeutic benefits.

Microfluidic hemophilia models using blood from healthy donors.

BACKGROUND: Microfluidic clotting assays permit drug action studies for hemophilia therapeutics under flow. However, limited availability of patient samples and Inter-donor variability limit the application of such assays, especially with many patients on prophylaxis. OBJECTIVE: To develop approaches to phenocopy hemophilia using modified healthy blood in microfluidic assays. METHODS: Corn trypsin inhibitor (4 µg/mL)-treated healthy blood was dosed with either anti-factor VIII (FVIII; hemophilia A model) or a recombinant factor IX (FIX) missense variant (FIX-V181T; hemophilia B model). Treated blood was perfused at 100 s-1 wall shear rate over collagen/tissue factor (TF) or collagen/factor XIa (FXIa). RESULTS: Anti-FVIII partially blocked fibrin production on collagen/TF, but completely blocked fibrin production on collagen/FXIa, a phenotype reversed with 1 µmol/L bispecific antibody (emicizumab), which binds FIXa and factor X. As expected, emicizumab had no significant effect on healthy blood (no anti-FVIII present) perfused over collagen/FXIa. The efficacy of emicizumab in anti-FVIII-treated healthy blood phenocopied the action of emicizumab in the blood of a patient with hemophilia A perfused over collagen/FXIa. Interestingly, a patient-derived FVIII-neutralizing antibody reduced fibrin production when added to healthy blood perfused over collagen/FXIa. For low TF surfaces, reFIX-V181T (50 µg/mL) fully blocked platelet and fibrin deposition, a phenotype fully reversed with anti-TFPI. CONCLUSION: Two new microfluidic hemophilia A and B models demonstrate the potency of anti-TF pathway inhibitor, emicizumab, and a patient-derived inhibitory antibody. Using collagen/FXIa-coated surfaces resulted in reliable and highly sensitive hemophilia models.

Assessing Blood Clotting and Coagulation Factors in Mice.

The mammalian blood coagulation system was designed to restrict blood loss due to injury as well as keep the blood fluid within the blood vessels of the organism. Blood coagulation activity in inbred mouse strains varies widely among strains, suggesting that many genomic variants affect hemostasis. Some of these molecules have been discovered and characterized; however, many are still unknown. Genetically modified mouse technologies are providing a plethora of new mouse models for investigating the regulation of blood coagulation. Here we provide a protocol for the tail bleeding time as a primary assessment of in vivo blood coagulation, as well as in vitro methods such as the prothrombin time, activated partial thromboplastin time, and thrombin generation assay. We also provide protocols for the assessment of the activities of specific known factors involved in blood coagulation. © 2019 by John Wiley & Sons, Inc.

Design and characterization of an APC-specific serpin for the treatment of hemophilia.

Hemophilia is a bleeding disorder caused by deficiency in factors VIII or IX, the two components of the intrinsic Xase complex. Treatment with replacement factor can lead to the development of inhibitory antibodies, requiring the use of bypassing agents such as factor VIIa and factor concentrates. An alternative approach to bypass the Xase complex is to inhibit endogenous anticoagulant activities. Activated protein C (APC) breaks down the complex that produces thrombin by proteolytically inactivating factor Va. Defects in this mechanism (eg, factor V Leiden) are associated with thrombosis but result in less severe bleeding when co-inherited with hemophilia. Selective inhibition of APC might therefore be effective for the treatment of hemophilia. The endogenous inhibitors of APC are members of the serpin family: protein C inhibitor (PCI) and α1-antitrypsin (α1AT); however, both exhibit poor reactivity and selectivity for APC. We mutated residues in and around the scissile P1-P1' bond in PCI and α1AT, resulting in serpins with the desired specificity profile. The lead candidate was shown to promote thrombin generation in vitro and to restore fibrin and platelet deposition in an intravital laser injury model in hemophilia B mice. The power of targeting APC was further demonstrated by the complete normalization of bleeding after a severe tail clip injury in these mice. These results demonstrate that the protein C anticoagulant system can be successfully targeted by engineered serpins and that administration of such agents is effective at restoring hemostasis in vivo.

A rapid pro-hemostatic approach to overcome direct oral anticoagulants.

Direct inhibitors of coagulation factor Xa (FXa) or thrombin are promising oral anticoagulants that are becoming widely adopted. The ability to reverse their anticoagulant effects is important when serious bleeding occurs or urgent medical procedures are needed. Here, using experimental mouse models of hemostasis, we show that a variant coagulation factor, FXa(I16L), rapidly restores hemostasis in the presence of the anticoagulant effects of these inhibitors. The ability of FXa(I16L) to reverse the anticoagulant effects of FXa inhibitor depends, at least in part, on the ability of the active site inhibitor to hinder antithrombin-dependent FXa inactivation, paradoxically allowing uninhibited FXa to persist in plasma. Because of its inherent catalytic activity, FXa(I16L) is more potent (by >50-fold) in the hemostasis models tested than a noncatalytic antidote that is currently in clinical development. FXa(I16L) also reduces the anticoagulant-associated bleeding in vivo that is induced by the thrombin inhibitor dabigatran. FXa(I16L) may be able to fill an important unmet clinical need for a rapid, pro-hemostatic agent to reverse the effects of several new anticoagulants.

AAV capsid CD8+ T-cell epitopes are highly conserved across AAV serotypes.

Adeno-associated virus (AAV) has become one of the most promising vectors in gene transfer in the last 10 years with successful translation to clinical trials in humans and even market approval for a first gene therapy product in Europe. Administration to humans, however, revealed that adaptive immune responses against the vector capsid can present an obstacle to sustained transgene expression due to the activation and expansion of capsid-specific T cells. The limited number of peripheral blood mononuclear cells (PBMCs) obtained from samples within clinical trials allows for little more than monitoring of T-cell responses. We were able to identify immunodominant major histocompatibility complex (MHC) class I epitopes for common human leukocyte antigen (HLA) types by using spleens isolated from subjects undergoing splenectomy for non-malignant indications as a source of large numbers of lymphocytes and restimulating them with single AAV capsid peptides in vitro. Further experiments confirmed that these epitopes are naturally processed and functionally relevant. The design of more effective and less immunogenic AAV vectors, and precise immune monitoring of vector-infused subjects, are facilitated by these findings.

Hemostatic agents of broad applicability produced by selective tuning of factor Xa zymogenicity.

There is a clinical need to develop safe therapeutic strategies to mitigate bleeding. Previously, we found that a novel zymogen-like factor Xa variant (FXa-I16L) was effective in correcting the coagulation defect in hemophilic mice. Here we expand the mutational framework to tune the FX(a) zymogen-like state. Alteration of FXa zymogenicity yields variants (V17M, I16L, I16M, V17T, V17S, and I16T) with a wide range (≤1000-fold) of reduced function toward physiologic substrates and inhibitors. The extent of zymogen-like character, including resistance to antithrombin III, correlates well with plasma half-life (<2 minutes to >4 hours). Importantly, biologic function, including that of the most zymogen-like variant (FXa-I16T), was greatly enhanced when bound to FVa membranes. This resulted in improvement of clotting times and thrombin generation in hemophilic plasma. The FXa variants were remarkably effective in mouse injury models. In these systems, the data show that the more active the protease, the more difficult it is to overcome the protective mechanism of circulating inhibitors to achieve a therapeutic benefit. Depending on the treatment situation, the more zymogen-like variants (V17S and I16T) were most useful when given before injury whereas variants exhibiting greater activity but shorter half-lives (I16L and I16M) were most effective when administered after injury. This new class of FXa variants provides a useful and flexible platform for selectively bioengineering biologic function and half-life to target different clinical bleeding scenarios.

An RNAi therapeutic targeting antithrombin to rebalance the coagulation system and promote hemostasis in hemophilia.

Hemophilia A and B are inherited bleeding disorders characterized by deficiencies in procoagulant factor VIII (FVIII) or factor IX (FIX), respectively. There remains a substantial unmet medical need in hemophilia, especially in patients with inhibitory antibodies against replacement factor therapy, for novel and improved therapeutic agents that can be used prophylactically to provide effective hemostasis. Guided by reports suggesting that co-inheritance of prothrombotic mutations may ameliorate the clinical phenotype in hemophilia, we developed an RNA interference (RNAi) therapeutic (ALN-AT3) targeting antithrombin (AT) as a means to promote hemostasis in hemophilia. When administered subcutaneously, ALN-AT3 showed potent, dose-dependent, and durable reduction of AT levels in wild-type mice, mice with hemophilia A, and nonhuman primates (NHPs). In NHPs, a 50% reduction in AT levels was achieved with weekly dosing at approximately 0.125 mg/kg, and a near-complete reduction in AT levels was achieved with weekly dosing at 1.5 mg/kg. Treatment with ALN-AT3 promoted hemostasis in mouse models of hemophilia and led to improved thrombin generation in an NHP model of hemophilia A with anti-factor VIII inhibitors. This investigational compound is currently in phase 1 clinical testing in subjects with hemophilia A or B.

The endothelial protein C receptor enhances hemostasis of FVIIa administration in hemophilic mice in vivo.

Recombinant activated human factor VII (rhFVIIa) is an established hemostatic agent in hemophilia, but its mechanism of action remains unclear. Although tissue factor (TF) is its natural receptor, rhFVIIa also interacts with the endothelial protein C receptor (EPCR) through its γ-carboxyglutamic acid (Gla) domain, with unknown hemostatic consequences in vivo. Here, we study whether EPCR facilitates rhFVIIa hemostasis in hemophilia using a mouse model system. Mouse activated FVII (mFVIIa) is functionally homologous to rhFVIIa, but binds poorly to mouse EPCR (mEPCR). We modified mFVIIa to gain mEPCR binding using 3 amino acid changes in its Gla domain (L4F/L8M/W9R). The resulting molecule mFVIIa-FMR specifically bound mEPCR in vitro and in vivo and was identical to mFVIIa with respect to TF affinity and procoagulant functions. In macrovascular injury models, hemophilic mice administered mFVIIa-FMR exhibited superior hemostatic activity compared with mFVIIa. This was abolished by blocking mEPCR and was absent in ex vivo whole blood coagulation assays, implicating a specific mFVIIa-FMR and endothelial mEPCR interaction. Because mFVIIa-FMR models the TF-dependent and EPCR binding properties of rhFVIIa, our data unmask a novel contribution of EPCR on the action of rhFVIIa administration in hemophilia, prompting the rational design of improved and safer rhFVIIa therapeutics.

New insights into the spatiotemporal localization of prothrombinase in vivo.

The membrane-dependent interaction of factor Xa (FXa) with factor Va (FVa) forms prothrombinase and drives thrombin formation essential for hemostasis. Activated platelets are considered to provide the primary biological surface to support prothrombinase function. However, the question of how other cell types may cooperate within the biological milieu to affect hemostatic plug formation remains unaddressed. We used confocal fluorescence microscopy to image the distribution of site-specific fluorescent derivatives of FVa and FXa after laser injury in the mouse cremaster arteriole. These proteins bound to the injury site extend beyond the platelet mass to the surrounding endothelium. Although bound FVa and FXa may have been present on the platelet core at the nidus of the injury, bound proteins were not evident on platelets adherent even a small distance from the injury site. Manipulations to drastically reduce adherent platelets yielded a surprisingly modest decrease in bound FXa and FVa with little impact on fibrin formation. Thus, platelets adherent to the site of vascular injury do not play the presumed preeminent role in supporting prothrombinase assembly and thrombin formation. Rather, the damaged/activated endothelium and possibly other blood cells play an unexpectedly important role in providing a procoagulant membrane surface in vivo.

Imaging coagulation reactions in vivo.

Significant gaps remain in the understanding of how blood cells and the vasculature differentially support coagulation enzyme complex function leading to regulated thrombus formation in vivo. While studies employing knock-out or transgenic mice have proved useful many of these scientific gaps partly result from the lack of molecular approaches and analytic tools with appropriate sensitivity for incisive conclusions. Over the past decade, studies employing state of the art videomicroscopy to image hemostasis in vivo following laser injury to the mouse cremaster arteriole have begun to bridge these gaps and provide remarkable insight into the early events of the hemostatic process. Many of these new insights have started to question some of the long-standing concepts that were driven by in vitro approaches. This review provides an overview of this technology, describes insights that have been made using it, and discuss limitations and future directions.

Engineered factor IX variants bypass FVIII and correct hemophilia A phenotype in mice.

The complex of the serine protease factor IX (FIX) and its cofactor, factor VIII (FVIII), is crucial for propagation of the intrinsic coagulation cascade. Absence of either factor leads to hemophilia, a disabling disorder marked by excessive hemorrhage after minor trauma. FVIII is the more commonly affected protein, either by X-chromosomal gene mutations or in autoimmune-mediated acquired hemophilia. Whereas substitution of FVIII is the mainstay of hemophilia A therapy, treatment of patients with inhibitory Abs remains challenging. In the present study, we report the development of FIX variants that can propagate the intrinsic coagulation cascade in the absence of FVIII. FIX variants were expressed in FVIII-knockout (FVIII-KO) mice using a nonviral gene-transfer system. Expression of the variants shortened clotting times, reduced blood loss after tail-clip assay, and reinstalled clot formation, as tested by in vivo imaging of laser-induced vessel injury. In addition, we confirmed the therapeutic efficacy of FIX variants in mice with inhibitory Abs against FVIII. Further, mice tolerant to wild-type human FIX did not develop immune responses against the protein variants. Our results therefore indicate the feasibility of using variants of FIX to bypass FVIII as a novel treatment approach in hemophilia with and without neutralizing FVIII Abs.

A zymogen-like factor Xa variant corrects the coagulation defect in hemophilia.

Effective therapies are needed to control excessive bleeding in a range of clinical conditions. We improve hemostasis in vivo using a conformationally pliant variant of coagulation factor Xa (FXa(I16L)) rendered partially inactive by a defect in the transition from zymogen to active protease. Using mouse models of hemophilia, we show that FXa(I16L) has a longer half-life than wild-type FXa and does not cause excessive activation of coagulation. Once clotting mechanisms are activated to produce its cofactor FVa, FXa(I16L) is driven to the protease state and restores hemostasis in hemophilic animals upon vascular injury. Moreover, using human or murine analogs, we show that FXa(I16L) is more efficacious than FVIIa, which is used to treat bleeding in hemophilia inhibitor patients. FXa(I16L) may provide an effective strategy to enhance blood clot formation and act as a rapid pan-hemostatic agent for the treatment of bleeding conditions.

Catalytic domain modification and viral gene delivery of activated factor VII confers hemostasis at reduced expression levels and vector doses in vivo.

Catalytic domain variants of activated factor VII (FVIIa) with enhanced hemostatic properties are highly attractive for the treatment of bleeding disorders via gene-based therapy. To explore this in a hemophilic mouse model, we characterized 2 variants of murine activated FVII (mFVIIa-VEAY and mFVIIa-DVQ) with modified catalytic domains, based on recombinant human FVIIa (rhFVIIa) variants. Using purified recombinant proteins, we showed that murine FVIIa (mFVIIa) and variants had comparable binding to human and murine tissue factor (TF) and exhibited similar extrinsic coagulant activity. In vitro in the absence of TF, the variants showed a 6- to 17-fold enhanced proteolytic and coagulant activity relative to mFVIIa, but increased inactivation by antithrombin. Gene delivery of mFVIIa-VEAY resulted in long-term, effective hemostasis at 5-fold lower expression levels relative to mFVIIa in hemophilia A mice or in hemophilia B mice with inhibitors to factor IX. However, expression of mFVIIa-VEAY at 14-fold higher than therapeutic levels resulted in a progressive mortality to 70% within 6 weeks after gene delivery. These results are the first demonstration of the hemostatic efficacy of continuous expression, in the presence or absence of inhibitors, of a high-activity gene-based FVIIa variant in an animal model of hemophilia.

Complement inhibition decreases the procoagulant response and confers organ protection in a baboon model of Escherichia coli sepsis.

Severe sepsis leads to massive activation of coagulation and complement cascades that could contribute to multiple organ failure and death. To investigate the role of the complement and its crosstalk with the hemostatic system in the pathophysiology and therapeutics of sepsis, we have used a potent inhibitor (compstatin) administered early or late after Escherichia coli challenge in a baboon model of sepsis-induced multiple organ failure. Compstatin infusion inhibited sepsis-induced blood and tissue biomarkers of complement activation, reduced leucopenia and thrombocytopenia, and lowered the accumulation of macrophages and platelets in organs. Compstatin decreased the coagulopathic response by down-regulating tissue factor and PAI-1, diminished global blood coagulation markers (fibrinogen, fibrin-degradation products, APTT), and preserved the endothelial anticoagulant properties. Compstatin treatment also improved cardiac function and the biochemical markers of kidney and liver damage. Histologic analysis of vital organs collected from animals euthanized after 24 hours showed decreased microvascular thrombosis, improved vascular barrier function, and less leukocyte infiltration and cell death, all consistent with attenuated organ injury. We conclude that complement-coagulation interplay contributes to the progression of severe sepsis and blocking the harmful effects of complement activation products, especially during the organ failure stage of severe sepsis is a potentially important therapeutic strategy.

Endothelial cell O-glycan deficiency causes blood/lymphatic misconnections and consequent fatty liver disease in mice.

Mucin-type O-glycans (O-glycans) are highly expressed in vascular ECs. However, it is not known whether they are important for vascular development. To investigate the roles of EC O-glycans, we generated mice lacking T-synthase, a glycosyltransferase encoded by the gene C1galt1 that is critical for the biosynthesis of core 1-derived O-glycans, in ECs and hematopoietic cells (termed here EHC T-syn(-/-) mice). EHC T-syn(-/-) mice exhibited embryonic and neonatal lethality associated with disorganized and blood-filled lymphatic vessels. Bone marrow transplantation and EC C1galt1 transgene rescue demonstrated that lymphangiogenesis specifically requires EC O-glycans, and intestinal lymphatic microvessels in EHC T-syn(-/-) mice expressed a mosaic of blood and lymphatic EC markers. The level of O-glycoprotein podoplanin was significantly reduced in EHC T-syn(-/-) lymphatics, and podoplanin-deficient mice developed blood-filled lymphatics resembling EHC T-syn(-/-) defects. In addition, postnatal inactivation of C1galt1 caused blood/lymphatic vessel misconnections that were similar to the vascular defects in the EHC T-syn(-/-) mice. One consequence of eliminating T-synthase in ECs and hematopoietic cells was that the EHC T-syn(-/-) pups developed fatty liver disease, because of direct chylomicron deposition via misconnected portal vein and intestinal lymphatic systems. Our studies therefore demonstrate that EC O-glycans control the separation of blood and lymphatic vessels during embryonic and postnatal development, in part by regulating podoplanin expression.

Sepsis-induced coagulation in the baboon lung is associated with decreased tissue factor pathway inhibitor.

Increased tissue factor (TF)-dependent procoagulant activity in sepsis may be partly due to decreased expression or function of tissue factor pathway inhibitor (TFPI). To test this hypothesis, baboons were infused with live Escherichia coli and sacrificed after 2, 8, or 24 hours. Confocal and electron microscopy revealed increased leukocyte infiltration and fibrin deposition in the intravascular and interstitial compartments. Large amounts of TF were detected by immunostaining in leukocytes and platelet-rich microthrombi. TF induction was documented by quantitative reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and coagulation assays. Lung-associated TFPI antigen and mRNA decreased during sepsis, and TFPI activity diminished abruptly at 2 hours. Blocking antibodies against TFPI increased fibrin deposition in septic baboon lungs, suggesting that TF-dependent coagulation might be aggravated by reduced endothelial TFPI. Decreased TFPI activity coincided with the release of tissue plasminogen activator and the peak of plasmin generation, suggesting that TFPI could undergo proteolytic inactivation by plasmin. Enhanced plasmin produced in septic baboons by infusion of blocking antibodies against plasminogen activator inhibitor-1 led to decreased lung-associated TFPI and unforeseen massive fibrin deposition. We conclude that activation of TF-driven coagulation not adequately countered by TFPI may underlie the widespread thrombotic complications of sepsis.

Temporal dynamics of gene expression in the lung in a baboon model of E. coli sepsis.

BACKGROUND: Bacterial invasion during sepsis induces disregulated systemic responses that could lead to fatal lung failure. The purpose of this study was to relate the temporal dynamics of gene expression to the pathophysiological changes in the lung during the first and second stages of E. coli sepsis in baboons. RESULTS: Using human oligonucleotide microarrays, we have explored the temporal changes of gene expression in the lung of baboons challenged with sublethal doses of E. coli. Temporal expression pattern and biological significance of the differentially expressed genes were explored using clustering and pathway analysis software. Expression of selected genes was validated by real-time PCR. Cytokine levels in tissue and plasma were assayed by multiplex ELISA. Changes in lung ultrastructure were visualized by electron microscopy. We found that genes involved in primary inflammation, innate immune response, and apoptosis peaked at 2 hrs. Inflammatory and immune response genes that function in the stimulation of monocytes, natural killer and T-cells, and in the modulation of cell adhesion peaked at 8 hrs, while genes involved in wound healing and functional recovery were upregulated at 24 hrs. CONCLUSION: The analysis of gene expression modulation in response to sepsis provides the baseline information that is crucial for the understanding of the pathophysiology of systemic inflammation and may facilitate the development of future approaches for sepsis therapy.