BACKGROUND:While replacement therapy in hemophilia (H)A and B (HB) has provided remarkable improvements, the development of neutralizing antibodies remains a significant complication, with rational engineering of bypassing (BP) molecules being a relevant issue. OBJECTIVES:This study developed a human factor (F)X variant containing a FIX-derived chimeric activation peptide (AP) driving FVIII or FIX deficiency bypass. METHODS:Engineered variants were expressed and purified, and activation/activity properties were evaluated through functional assays, also in plasma with inhibitors from patients with HA and HB. RESULTS:After screening a panel of recombinant variants, the most promising were purified for functional characterization. Unlike wild-type FX (FXWT), the BP variants FXBP4b and FXBP4c at physiological concentrations effectively normalized the clotting time in HA and HB plasma. Chromogenic assays confirmed that both variants preserved activation via the extrinsic tenase complex or by Russell viper venom X, while the intrinsic activation pathway was impaired, thus favoring their selective dose-dependent FXIa activation. In thrombin generation assays, FXBP4b-c restored the peak and the endogenous thrombin potential in FX-deficient plasma. Remarkably, the FXBP4b outperformed FXWT in HA and HB plasma and showed a comparable or superior activity in comparison with B-domainless FVIII and FIX. Notably, FXBP4b significantly shortened the clotting time in plasma from patients with high-titer anti-FVIII or anti-FIX inhibitory antibodies, with efficacy levels comparable with those of well-established BP agents. CONCLUSION:These results support FXBP4b as a promising and versatile BP agent able to sustain coagulation across multiple hemophilic contexts including inhibitors presence, while maintaining favorable activation profiles.

Background:The development of acquired factor (F)V with inhibitor (AFVwI) is rare, resulting mainly in bleeding complications, although sporadic cases of thrombosis in adults have been reported. Key Clinical Question:How do you diagnose and manage a pediatric case of acute deep venous thrombosis associated with the concurrent finding of AFVwI? Clinical Approach:A 13-year-old female with Crohn's Disease and May-Thurner anatomy developed extensive deep venous thrombosis of the left lower extremity, complicated by the finding of AFVwI, discovered during the evaluation of a prolonged prothrombin time and a low FV activity. Anticoagulation was initiated with low-molecular-weight heparin followed by a direct oral anticoagulant, rivaroxaban, without any complications. AFVwI was undetectable after 5 months with normalization of FV activity. Conclusion:Our case highlights the first pediatric case of thrombosis with a rare finding of AFVwI, successfully managed with anticoagulation therapy with complete resolution.

BACKGROUND:Factor (F)V plays a pivotal role in coagulation, having both pro- and anticoagulant functions. Conserved acidic and basic regions (AR and BR) within FVs central B-domain play a key role in regulating procoagulant function. Removing the BR (residues 963-1008) through proteolysis or splicing, as in FV-short, produces an active cofactor, which is partly regulated by tissue factor pathway inhibitor-α (TFPIα) through its C-terminal BR. Although AR2 (residues 1493-1537) is known to be involved, other FV regions that may interact with the FV-BR or TFPIα-BR have not been defined. OBJECTIVES:To uncover binding interactions between the BRs and other regions on FV-short. METHODS:We used a unique chemical cleavage agent, FeBABE (Fe-p-bromoacetamidobenzyl-ethylenediaminetetraacetic acid) tethered to the FV-BR or TFPIα-BR (BR-FeBABE) to footprint areas of engagement with FV-short using biochemical methods and western blotting. RESULTS:BR-FeBABE bound FV-short and FV-810, another B-domainless form of FV, with high affinity and blocked procoagulant activity, similar to untethered BR. When BR-FeBABE was stimulated, it cut FV-short and FV-810 at 3 sites including the beginning of AR2, near residues 678 to 692 comprising part of AR1 (residues 659-697), and at the beginning of the A2 domain. Cleavage by BR-FeBABE was specific as untethered FV-BR, TFPIα-BR, or TFPIα blocked proteolysis, and forms of FV that do not bind BR-FeBABE, such as FV, FVa, and any form of FV lacking AR2, were not cut. CONCLUSION:These results define areas of interaction between the BR and FV-short and advance our knowledge about how FV is regulated by the B-domain and TFPIα.

Blood coagulation factor IX plays a crucial role in the intrinsic pathway of coagulation by generating factor Xa, ultimately leading to thrombin formation. Over the past 50 years, extensive research has deepened our understanding of the biology, physiology, pathology, biochemistry, and molecular genetics of factor IX. This wealth of knowledge has revealed how the factor IX gene and protein evolved, how factor IX is regulated, how it functions within the coagulation cascade, and how structural changes affect its function. In this review, we will summarize current knowledge on the biology of factor IX, with a focus on its structure-function relationships, gene structure, and regulation.

Background Factor V (FV) plays a central role in the coagulation cascade, acting in both a procoagulant and anticoagulant manner. The majority of FV is produced by the liver hepatocytes. In humans, FV is endocytosed by megakaryocytes, whereas in mice, FV is synthesized by megakaryocytes. Little is known about the genomic factors regulating FV transcription in humans and mice. Objective To investigate genomic regulatory mechanisms for coagulation FV levels in the hepatocytes and megakaryocytes of inbred mice. Methods Plasma and platelet FV levels were measured via ELISA in 5 mouse strains. A cross between the CAST/EiJ and DBA/2J strains was performed to generate 146 genetically informative F2 mice for analysis of circulating and platelet FV. Plasma and platelet FV levels were measured by ELISA for the F2 mice and whole genome genotyping for each F2 was performed using the TransnetYX MiniMUGA genotyping array. The genotyping and phenotyping data collected from these mice were then analyzed using quantitative trait loci (QTL) analysis. Results and Conclusions We identified one significant locus controlling plasma FV levels on Chromosome 1, ∼57.7 million base pairs upstream of the FV structural gene. We also identified a significant QTL for platelets on Chromosome 14 when sex was included as an interactive covariate, with additional suggestive loci present on Chromosomes 15 and 2. Our findings provide foundational information regarding the cell-type and sex-specific control of FV expression, establishing the basis for further investigations aimed at fine-mapping these loci and understanding how FV expression is regulated. Essentials ### Competing Interest Statement The authors have declared no competing interest. American Heart Association, https://ror.org/013kjyp64, 23AIREA1055261, 23IAUST1034173, 25ISFRNCP1501445, 24SFRNCCN1276092 National Science Foundation, https://ror.org/021nxhr62, 2152007 National Institutes of Health, https://ror.org/01cwqze88, TL1DK136046, R15HL172202

The lymphatic system is a network of vessels, tissues, and organs whose main function is to maintain fluid balance. Lymphatic vessels form a unidirectional conduit system that traffics fluid derived from cell exudate and blood capillary leakage back to the bloodstream via the subclavian veins. This exchange introduces the potential for proteins from the blood to infiltrate the lymphatic system. Accordingly, previous analyses of lymphatic fluid, albeit limited, have established the presence of coagulation factors. Depending on their composition and concentration, these coagulation factors could present a risk for thrombosis in lymphatic fluid, especially when concurrent with other pathological conditions. For instance, although reports of thrombosis within the lymphatic system are rare, disruptions of blood flow, like those in cardiac conditions, can cause lymphedema and contribute to a hypercoagulable state. However, the current body of research lacks a comprehensive evaluation of coagulation within lymphatic fluid, thereby leaving our understanding of lymphatic thrombosis largely undefined. Here we evaluated coagulation in thoracic duct lymphatic fluid collected from pediatric patients at the Children's Hospital of Philadelphia. Coagulation of lymphatic fluid samples was evaluated using a thrombin generation assays (TGAs) as well as prothrombin time (PT) and activated partial thromboplastin time (aPTT) clotting assays. We also characterized antigen concentrations of major clotting factors using ELISAs. In TGAs initiated with low tissue factor (TF; 0.1 pM), most lymphatic fluid from patient samples (n= 52) had thrombin generation (TG) profiles comparable to pooled normal plasma (PNP). Average peak thrombin (IIa) and endogenous thrombin potential (ETP) were around 75% of PNP at 166.2 +/- 17.27 nM and 3185.7 +/- 314.3 nM*min, respectively. Lag times of lymphatic samples were slightly prolonged with an average of 14.1 +/- 1.9 min compared to PNP (10.2 +/- 1.4 min). Consistent with these results, lymphatic fluid samples also demonstrated appreciable clotting activity in PT assays, with most samples having a twofold increase in clot time in comparison to PNP (31.7 +/- 4.3 sec lymph vs. 13.6 +/- 0.6 sec PNP). In contrast to TF-initiated TGAs, initiation via the intrinsic pathway using a dilute aPTT reagent generally produced a weak TG profile (peak IIa: 86.9 +/- 15.4 nM, ETP: 2474.2 +/- 303.8 nM*min, lag time: 22.4 +/- 2.2 min) compared to PNP (peak IIa: 281.2 +/- 20.2 nM, ETP: 4195.58 +/- 279.1 nM*min, lag time: 21.25 +/- 3.75). These lymphatic fluid samples also had markedly prolonged aPTT clotting times compared to both PNP (32.6 +/- 0.4 sec) and FVIII deficient plasmas (96.1 +/- 3.0 sec). These findings suggest concentrations of one or more clotting factors involved in the intrinsic pathway may be low in lymphatic fluid compared to normal plasma. Coagulation assays of lymphatic fluid were supported by measurement of several key coagulation factors. ELISA data for FVIII revealed levels at 30% of PNP concentrations, which is consistent with antigen levels observed in moderate hemophilia A plasma (a FVIII deficiency with 5-40% of PNP antigen levels). Fibrinogen levels were also notably low at 30% of plasma concentrations. In contrast, FV antigen concentrations were just below the normal range of 50-150% antigen level at 43% while lymphatic FIX and FX antigen were above 50%, suggesting that none of these factors is limiting for coagulation in lymphatic fluid. Because lymphatic endothelial cells are known to express thrombomodulin, we also tested lymphatic samples for evidence of a functional Protein C (PC) pathway. The addition of soluble thrombomodulin to lymphatic fluid in TF-initiated TGAs produced a robust anti-thrombogenic effect comparable to a PNP control, confirming the presence of functional PC anticoagulant activity in lymphatic fluid. These data support further investigation of other anticoagulant regulators (e.g. antithrombin, tissue factor pathway inhibitor) in lymphatic fluid. Together these data confirm that human lymphatic fluid supports thrombin generation and clot formation in vitro, and this procoagulant activity is primarily TF-dependent. Further analysis of additional components of the pro- and anticoagulant pathways will provide a more complete understanding of coagulation in the lymphatic system and its potential to contribute to lymphatic thrombosis.

Mechanisms of proteostasis in anucleate circulating platelets are unknown and may regulate platelet function. We investigated the hypothesis that plasma-borne growth factors/hormones (GFHs) maintain constitutive translation in circulating platelets to facilitate reactivity. Bio-orthogonal noncanonical amino acid tagging (BONCAT) coupled with liquid chromatography-tandem mass spectrometry analysis revealed constitutive translation of a broad-spectrum translatome in human platelets dependent upon plasma or GFH exposure, and in murine circulation. Freshly isolated platelets from plasma showed homeostatic activation of translation-initiation signaling pathways: phosphorylation of p38/ERK upstream kinases, essential intermediate MNK1/2, and effectors eIF4E/4E-BP1. Plasma starvation led to loss of pathway phosphorylation, but it was fully restored with 5-minute stimulation by plasma or GFHs. Cycloheximide or puromycin infusion suppressed ex vivo platelet GpIIb/IIIa activation and P-selectin exposure with low thrombin concentrations and low-to-saturating concentrations of adenosine 5'-diphosphate (ADP) or thromboxane analog but not convulxin. ADP-induced thromboxane generation was blunted by translation inhibition, and secondary-wave aggregation was inhibited in a thromboxane-dependent manner. Intravenously administered puromycin reduced injury-induced clot size in cremaster muscle arterioles, and delayed primary hemostasis after tail tip amputation but did not delay neither final hemostasis after subsequent rebleeds, nor final hemostasis after jugular vein puncture. In contrast, these mice were protected from injury-induced arterial thrombosis and thrombin-induced pulmonary thromboembolism (PE), and adoptive transfer of translation-inhibited platelets into untreated mice inhibited arterial thrombosis and PE. Thus, constitutive plasma GFH-driven translation regulates platelet G protein-coupled receptor reactivity to balance hemostasis and thrombotic potential.

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 not clinically feasible. Recent advances in generating megakaryocytes prompted us to develop a potentially clinically relevant means to produce "antithrombotic" platelets from CD34+ hematopoietic stem cell-derived in vitro-grown megakaryocytes. CD34+ megakaryocytes internalize and store in alpha granules (alpha-granules) single-chain uPA (scuPA) and a plasmin-resistant thrombinactivatable variant (uPAT). Both uPAs colocalized with internalized factor V (FV), fibrinogen and plasminogen, low-density lipoprotein receptor-related protein 1 (LRP1), and interferoninduced transmembrane protein 3, but not with endogenous von Willebrand factor (VWF). Endocytosis of uPA by CD34+ megakaryocytes was mediated, in part, via LRP1 and alpha IIb beta 3. scuPA-containing megakaryocytes degraded endocytosed intragranular FV but not endogenous VWF in the presence of internalized plasminogen, whereas uPATmegakaryocytes did not significantly degrade either protein. We used a carotid artery injury model in nonobese diabetic-severe combined immunodeficiency IL2r gamma null (NSG) mice homozygous for VWFR1326H(a mutation switching binding VWF specificity from mouse to human glycoprotein Ib alpha) to test whether platelets derived from scuPA- or uPATmegakaryocytes would prevent thrombus formation. NSG/VWFR1326H mice exhibited a lower thrombotic burden after carotid artery injury compared with NSG mice unless infused with human platelets or megakaryocytes, whereas intravenous injection of uPAmegakaryocytes 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 alpha-granules to sites of vascular injury.

Our prior finding that uPA endogenously expressed and stored in the platelets of transgenic mice prevented thrombus formation without causing bleeding, prompted us to develop a potentially clinically relevant means of generating anti-thrombotic human platelets in vitro from CD34 + hematopoietic cell-derived megakaryocytes. CD34 + -megakaryocytes internalize and store in α-granules single-chain uPA (scuPA) and a uPA variant modified to be plasmin-resistant, but thrombin-activatable, (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 (IFITM3), but not with endogenous von Willebrand factor (VWF). Endocytosis of uPA by CD34 + -\megakaryocytes was mediated in part via LRP1 and αIIbβ3. 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 IL2rγnull (NSG) mice homozygous for VWF R1326H (a mutation switching binding VWF specificity from mouse to human glycoprotein IbmlIX) to test whether platelets derived from scuPA-MKs or uPAT-Mks would prevent thrombus formation. NSG/VWF R1326H mice exhibited a lower thrombotic burden after carotid artery injury compared to NSG mice unless infused with human platelets or MKs, whereas intravenous injection of either uPA-containing megakaryocytes into NSG/VWF R1326H generated sufficient uPA-containing human platelets to lyse nascent thrombi. These studies suggest the potential to deliver uPA or potentially other ectopic proteins within platelet α-granules from in vitro- generated megakaryocytes. Key points:Unlike platelets, in vitro-grown megakaryocytes can store exogenous uPA in its α-granules.uPA uptake involves LRP1 and αIIbβ3 receptors and is functionally available from activated platelets.

Introduction For patients with hemophilia A, the generation of inhibitors (neutralizing antibodies against FVIII) during treatment remains a significant complication. Clinical studies have demonstrated that patients treated with plasma-derived FVIII (pdFVIII) have a lower incidence of inhibitor development compared to those treated with recombinant FVIII (rFVIII) products. Different post-translational modifications on rFVIII proteins expressed in various cell lines may contribute to the differences in FVIII immunogenicity. Our recent study showed that N-glycosylation affects the development of inhibitors. On the other hand, the presence, and effects of O-glycans on FVIII inhibitor development are less studied, though they are found on >80% of the mammalian surface and secreted proteins. We recently identified highly and differentially expressed O-glycans on various FVIII products. This work aims to investigate how additional O-linked glycosylation might impact the immunogenicity of recombinant FVIII. Method FVIII-α2HS and FVIII-FibA plasmids were constructed by incorporating sequences encoding 6 repeats of short peptide of α-2-HS and Fibrinogen A (FibA), respectively into B-domain deleted FVIII (BDD-FVIII) plasmid. These short peptides are known to be highly O-glycosylated. The FVIII expression and immunogenicity were evaluated by introducing the plasmids into hemophilia A mice through hydrodynamic injection. Subsequently, the FVIII-FibA and BDD-FVIII proteins were expressed in BHK cells and purified for LC-MS/MS-based glycomics analysis. We assessed the activity and half-life of FVIII proteins using an aPTT assay with mouse plasma samples collected at various time points within 96 hours after a single intravenous injection. The immunogenicity of FVIII proteins was evaluated using the Bethesda assay from plasma samples collected from hemophilia A mice after weekly intravenous injection for four weeks. Results Mice treated with plasmid containing α-2-HS and FibA sequences had lower inhibitor titers, especially mice with FVIII-FibA plasmid showing significantly lower inhibitor titers than FVIII-BDD group at week 4 and week 8 after the injection, despite no difference in FVIII activity. Glycosylation analysis revealed that the FVIII-FibA protein displayed a relatively higher abundance of O-glycans throughout all domains compared to the BDD-FVIII protein. The inserted FibA sequence itself is O-glycosylated. Similar to plasmid treatment, mice injected with FVIII-FibA protein had significantly lower inhibitor titers (1.3±1.0 BU) than mice injected with BDD-FVIII protein (37.5±37.2 BU) and commercial rFVIII products (19.8±20.2 BU) with no observed difference in in vivo activity and protein half-life. Conclusion The introduction of extra O-linked glycosylated peptides between A2 and A3 domains of FVIII altered O-glycosylation pattern and substantially reduced the immunogenicity without affecting FVIII activity. Our findings underscore the significant impact of post-translational modification on protein immunogenicity, particularly in the case of FVIII. Additionally, FVIII-FibA shows promising potential for clinical applications, not only in the development of new recombinant FVIII products but also in gene therapy approaches.

Despite over 80 years of clinical experience with coagulation factor (F)VIII inhibitors, surprisingly little is known about the in vivo mechanism of this most serious complication of replacement therapy for hemophilia A. These neutralizing anti-drug alloantibodies arise in ~30% of patients. Inhibitor formation is T cell-dependent, but events leading up to helper T cell activation have been elusive due in part to the complex anatomy and cellular makeup of the spleen. Here, we show that FVIII antigen presentation to CD4+ T cells critically depends on a select set of several anatomically distinct antigen presenting cells, whereby marginal zone B cells, marginal zone and marginal metallophilic but not red pulp macrophages (RPMFs) participate in shuttling FVIII to the white pulp where conventional dendritic cells (DCs) prime helper T cells, which then differentiate into follicular helper T (Tfh) cells. Toll-like receptor 9 (TLR9) stimulation accelerated Tfh responses, Germinal Center and inhibitor formation, while systemic administration of FVIII alone in hemophilia A mice increased frequencies of monocyte-derived and plasmacytoid DCs. Moreover, FVIII enhanced T cell proliferation to another protein antigen (ovalbumin), and inflammatory signaling-deficient mice were less likely to develop inhibitors, indicating that FVIII may have intrinsic immunostimulatory properties. Ovalbumin, which, unlike FVIII, is absorbed into the RPMF compartment, fails to elicit T cell proliferative and antibody responses when administered at the same dose as FVIII. Altogether, we propose that an antigen trafficking pattern that results in efficient in vivo delivery to DCs and inflammatory signaling, shape the immunogenicity of FVIII.

BACKGROUND:Coagulation factor VIII (FVIII) and von Willebrand factor (VWF) circulate as a noncovalent complex, but each has its distinct functions. Binding of FVIII to VWF results in a prolongation of FVIII's half-life in circulation and modulates FVIII's immunogenicity during hemophilia therapy. However, the biological effect of FVIII and VWF interaction on VWF homeostasis is not fully understood.OBJECTIVES:To determine the effect of FVIII in VWF proteolysis and homeostasis in vivo.METHODS:Mouse models, recombinant FVIII infusion, and patients with hemophilia A on a high dose FVIII for immune tolerance induction therapy or emicizumab for bleeding symptoms were included to address this question.RESULTS:An intravenous infusion of a recombinant B-domain less FVIII (BDD-FVIII) (40 and 160 μg/kg) into wild-type mice significantly reduced plasma VWF multimer sizes and its antigen levels; an infusion of a high but not low dose of BDD-FVIII into Adamts13+/- and Adamts13-/- mice also significantly reduced the size of VWF multimers. However, plasma levels of VWF antigen remained unchanged following administration of any dose BDD-FVIII into Adamts13-/- mice, suggesting partial ADAMTS-13 dependency in FVIII-augmented VWF degradation. Moreover, persistent expression of BDD-FVIII at ∼50 to 250 U/dL via AAV8 vector in hemophilia A mice also resulted in a significant reduction of plasma VWF multimer sizes and antigen levels. Finally, the sizes of plasma VWF multimers were significantly reduced in patients with hemophilia A who received a dose of recombinant or plasma-derived FVIII for immune tolerance induction therapy.CONCLUSION:Our results demonstrate the pivotal role of FVIII as a cofactor regulating VWF proteolysis and homeostasis under various (patho)physiological conditions.

Introduction: Coagulation factor VIII (FVIII) and von Willebrand factor (VWF) circulate as a noncovalent complex. This interaction helps increase the half-life of circulating FVIII and modulates FVIII immunogenicity. Our previous studies demonstrate that FVIII accelerates VWF proteolysis by ADAMTS13 in vitro under fluidic shear or under certain pathophysiological conditions. However, the physiological relevance of the FVIII-enhanced VWF proteolysis remains questionable. Here, we sought to determine if addition of recombinant FVIII at various concentrations to mice with or without circulating ADAMTS13 or to patients with hereditary thrombotic thrombocytopenic purpura (TTP) affects plasma VWF multimer distribution and its antigen levels. Methods: Recombinant human B-domain deleted FVIII (rFVIII) was intravenously administered into wild-type (WT),Adamts13+/-(A13+/-) and Adamts13-/- (A13-/-) mice. Blood samples were collected prior to and 24 hours following rFVIII infusion. In addition, FVIII was stably expressed in fviii-/-mice via administration of AAV8-BDDFVIII. Moreover, plasma was collected from patients with hereditary TTP who were treated with plasma derived FVIII concentrates. Plasma VWF multimers and antigen levels were determined by SDS-agarose gel electrophoresis followed by Western blotting and enzyme-linked immunoassay (ELISA), respectively. Paired T and Mann Whitney tests were performed to determine the statistical significance of the difference between two groups. Results: Our results demonstrated that an infusion of rFVIII at a low dose (40 ug/kg) and a high dose (160 ug/kg) into WT mice resulted in a dramatic reduction in plasma VWF multimer sizes (Fig. 1) and VWF antigen levels (not shown). FVIII at high dose but not at low dose could also significantly reduce the sizes of VWF multimers in Adamts13+/- and Adamts13-/- mice (Fig. 1). The enhanced degradation of VWF multimers in Adamts13-/- mice suggests that other VWF-cleaving enzymes may participate in the degradation of VWF-FVIII complex. In either Adamts13+/- or Adamts13-/- mice, administration of rFVIII at both low and high doses didn't result in significant changes in VWF antigen levels (not shown). Furthermore, a simultaneous infusion of both rFVIII and recombinant murine ADAMTS13 (rADAMTS13) into Adamts13-/- mice resulted in similar changes in plasma VWF multimer distribution (Fig. 1) and antigen levels (not shown). Importantly, all mice tolerated well to the high dose of rFVIII infusion without major thrombotic events during weeks of follow-up. The restoration of plasma FVIII to 100-200% of normal levels in the fviii-/- mice with AAV8-mediated gene therapy also led to a profound degradation of plasma VWF multimers and significantly reduced plasma levels of VWF antigen (not shown). Finally, a similar pattern of dramatically reduced plasma VWF multimers was observed in 3 out of 4 patients with hereditary TTP following treatment with plasma-derived FVIII concentrates (not shown). Conclusions: Our results demonstrate that supplementation of FVIII at physiological or super-physiological concentrations dramatically accelerates the degradation of VWF multimers in mice and human. These findings further support our hypothesis that FVIII is a physiological cofactor of ADAMTS13 in regulating VWF homeostasis. Our findings may also offer a scientific explanation how FVIII concentrates or perhaps rFVIII might work for the treatment of hereditary TTP and other arterial thrombosis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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.

Hemophilia is a bleeding disorder that results from a deficiency of factor VIII (Hemophilia A; HA) or factor IX (Hemophilia B; HB). Protein replacement therapy ameliorates bleeding but can result in the development of neutralizing antibodies. Innovative nonfactor therapies are in development for hemophilia which target anticoagulant proteins such as tissue factor pathway inhibitor (TFPI), antithrombin, and activated protein C (APC). While these approaches show promise, they influence multiple coagulation factors and pathways which could raise safety concerns. Considering its essential role in coagulation in enhancing thrombin generation (TG), we hypothesized that altering the functional properties of FV/FVa could be an attractive approach for HA and HB treatment. Activated FV (FVa) assembles with FXa on activated cellular surfaces and enhances the kinetics of TG by several orders of magnitude. Here we assess whether monoclonal antibodies targeting all forms of FV are effective in promoting TG in hemophilia using both in vitro and in vivo models in hemophilic mice. Using an initial TG assay screen, we identified a procoagulant antibody (GB5) that binds with high affinity (Kd <0.5 nM) to human (h)FV, FVa, and FV-short and investigated its mechanism of action. Using proteolytic fragments of FV and western blotting experiments, we found that GB5 likely binds in the middle of the light chain region. To evaluate the functional effects of GB5 in vitro, we first employed a plasma-based TG assay using HA or HB plasma triggered with low (0.2 pM) tissue factor. The presence of saturating GB5 increased TG about 3-fold in HA plasma. Similar results were observed with HB plasma. Interestingly, when TG was essentially eliminated in HA and HB plasma with addition of exogenous APC, GB5 restored TG. These results suggest that the procoagulant effect of GB5 neutralizes the anticoagulant effect of APC and enhanced TG in hemophilic plasma To begin to assess the mechanism, the effect of GB5 on FV activation in the TG assay was evaluated. TG assay samples were removed over time, quenched, and analyzed by immunoblotting using labeled FV specific antibodies. In the presence of GB5, ~95% of the starting full-length FV was converted to FVa within 3 min compared to 5 min in the absence of GB5. These results show that FV is activated faster in the presence of GB5 in the TG assay. To extend these data, we next assessed the influence of GB5 in the TG assays using FV-deficient plasma supplemented with either FV or FVa and TFPI⍺. The data show that GB5 had essentially no effect on the TG assay when FV deficient plasma was reconstituted with FVa, but enhanced TG at least 3-fold when supplemented with FV. Consistent with this, the kinetics of prothrombin activation with FXa-FVa-membranes in the presence or absence of GB5 were the same. These data suggest that GB5 impacts reactions that influence FV, with no direct effect on FVa procoagulant function. To evaluate whether GB5 has a procoagulant effect in vivo, a tail vein transection (TVT) model was employed in HA mice. In this model, one lateral tail vein is transected, and blood is collected over 40 min with clots at the vessel wall disrupted every 10 min as needed. Since GB5 interacts with human and not mouse FV, plasma derived human FV was infused into HA mice. We found that blood loss in the TVT model using HA mice infused with human FV alone (5 μg/mouse) or GB5 alone (20 μg/mouse) was no different than mice infused with PBS (n = 3-5 mice/group except GB5 alone where n = 1: ~600 μl blood loss). In contrast to these data, when human FV and GB5 were co-administered, blood loss was reduced ~3-fold (~200 μl) which was statistically significant relative to the controls but did not reach the level of wild-type mice (blood loss ~50 μl). Overall, the results show that GB5 is a procoagulant monoclonal antibody that binds with high affinity to human FV and influences reactions that lead to its activation and inactivation. We hypothesize that the net effect of GB5 is the enhanced production of FVa which has a major impact on TG. In plasma-based systems with and without the protein C system and an in vivo hemophilic model, GB5 is procoagulant and can reduce bleeding. Our work shows that targeting the common coagulation pathway at the level of FV has benefit in the context of hemophilia and may be a viable and safe therapeutic approach.