Rodney M. Camire

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.

Tissue factor pathway inhibitor-alpha inhibits prothrombinase during the initiation of blood coagulation

Significance The generation of thrombin by prothrombinase, a complex composed of activated (a) factors X (FXa) and V (FVa), is a final step in blood coagulation. We demonstrate that tissue factor pathway inhibitor (TFPI) blocks thrombin generation by prothrombinase at physiologically relevant rates and concentrations, but only during the initiation of clot formation. TFPI mediates this inhibitory activity through two high-affinity interactions, one with FXa and one with FVa. This is the first description of an endogenous human protein that inhibits prothrombinase under physiological conditions and may prevent a full thrombotic response to subthreshold coagulant stimuli that otherwise could occlude blood vessels. It provides unique understanding of thrombotic disorders and has important implications for development of anti-TFPI agents to treat hemophilia. Tissue factor (TF) pathway inhibitor (TFPI) is a well-characterized activated factor X (FXa)-dependent inhibitor of TF-initiated coagulation produced in two alternatively spliced isoforms, TFPIα and TFPIβ. The TFPIα C terminus has a basic sequence nearly identical to a portion of the factor V (FV) B domain necessary for maintaining FV in an inactive conformation via interaction with an acidic region of the B domain. We demonstrate rapid inhibition of prothrombinase by TFPIα mediated through a high-affinity exosite interaction between the basic region of TFPIα and the FV acidic region, which is retained in FXa-activated FVa and platelet FVa. This inhibitory activity is not mediated by TFPIβ and is lost upon removal of the acidic region of FVa by thrombin. The data identify a previously undescribed, isoform-specific anticoagulant function for TFPIα and are a unique description of physiologically relevant inhibition of prothrombinase. These findings, combined with previous descriptions of differential expression patterns of TFPIα and TFPIβ in platelets and endothelial cells, suggest that the TFPI isoforms may act through distinct mechanisms to inhibit the initial stages of intravascular coagulation, with TFPIβ acting to dampen TF expressed on the surface of vascular cells, whereas TFPIα dampens the initial prothrombinase formed on the activated platelet surface.

Factor V Leiden improves in vivo hemostasis in murine hemophilia models

Summary.u2002 The role of factor V Leiden (FVL) as a modifier of the severe hemophilia phenotype is still unclear. We used mice with hemophilia A or B crossed with FVL to elucidate in vivo parameters of hemostasis. Real‐time thrombus formation in the microcirculation was monitored by deposition of labeled platelets upon laser‐induced endothelial injury using widefield microscopy in living animals. No thrombi formed in hemophilic A or B mice following vascular injuries. However, hemophilic mice, either heterozygous or homozygous for FVL, formed clots at all injured sites. Injection of purified activated FV into hemophilic A or B mice could mimic the in vivo effect of FVL. In contrast to these responses to a laser injury in a microvascular bed, FVL did not provide sustained hemostasis following damage of large vessels in a ferric chloride carotid artery injury model, despite of the improvement of clotting times and high circulating thrombin levels. Together these data provide evidence that FVL has the ability to improve the hemophilia A or B phenotype, but this effect is principally evident at the microcirculation level following a particular vascular injury. Our observations may partly explain the heterogeneous clinical evidence of the beneficial role of FVL in hemophilia.

Zymogen-like factor Xa variants restore thrombin generation and effectively bypass the intrinsic pathway in vitro

Inhibitory antibodies to factors VIII or IX represent a serious complication for hemophilia patients. Treatment involves products that bypass the intrinsic pathway and promote thrombin generation. Direct infusion of factor Xa should also restore hemostasis; however, it has a short half-life in plasma and could activate systemic coagulation in an uncontrolled fashion. Here we show that factor Xa mutants with zymogen-like properties (FXa(I16L) and FXa(V17A)) circumvent these limitations. In the absence of factor Va, the FXa variants are poor enzymes for a range of physiological ligands and are resistant to inactivation by antithrombin III and tissue factor pathway inhibitor. Notably, assembly of FXa(I16L) and FXa(V17A) on activated platelets with factor Va to form prothrombinase completely restores biologic activity. In hemophilic plasma, FXa(I16L) and FXa(V17A) have prolonged half-lives compared with wild-type factor Xa (approximately 60 minutes vs approximately 1 minute) and promote robust thrombin generation that bypasses the intrinsic pathway. The variants require factor Va generated in situ for procoagulant function, and cofactor inactivation by the protein C pathway regulates their activity. The efficacy, extended half-life, and mechanism of action suggest that novel zymogen-like forms of factor Xa might prove useful as new therapeutic procoagulants to treat deficiencies upstream of the common pathway.

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.

The molecular basis of factor V and VIII procofactor activation

Summary.u2002 Activation of precursor proteins by specific and limited proteolysis is a hallmark of the hemostatic process. The homologous coagulation factors (F)V and FVIII circulate in an inactive, quiescent state in blood. In this so‐called procofactor state, these proteins have little, if any procoagulant activity and do not participate to any significant degree in their respective macromolecular enzymatic complexes. Thrombin is considered a key physiological activator, cleaving select peptide bonds in FV and FVIII which ultimately leads to appropriate structural changes that impart cofactor function. As the active cofactors (FVa and FVIIIa) have an enormous impact on thrombin and FXa generation, maintaining FV and FVIII as inactive procofactors undoubtedly plays an important regulatory role that has likely evolved to maintain normal hemostasis. Over the past three decades there has been widespread interest in studying the proteolytic events that lead to the activation of these proteins. While a great deal has been learned, mechanistic explanations as to how bond cleavage facilitates conversion to the active cofactor species remain incompletely understood. However, recent advances have been made detailing how thrombin recognizes FV and FVIII and also how the FV B‐domain plays a dominant role in maintaining the procofactor state. Here we review our current understanding of the molecular process of procofactor activation with a particular emphasis on FV.

Recombinant canine B-domain–deleted FVIII exhibits high specific activity and is safe in the canine hemophilia A model

Production of recombinant B-domain-deleted canine factor VIII (cFVIII-BDD) unexpectedly revealed superior protein yields with 3-fold increased specific activity relative to human FVIII-BDD (hFVIII-BDD). We also determined that activated cFVIII-BDD is more stable than activated hFVIII-BDD. Furthermore, cFVIII-BDD is efficient at inducing hemostasis in human plasma containing FVIII inhibitors. Infusion of cFVIII-BDD in hemophilia A dogs resulted in correction of the disease phenotype with a pharmacokinetic profile similar to clinical experience with hFVIII-BDD. Notably, immune tolerance challenges with cFVIII-BDD in young and adult hemophilia A dogs did not induce the formation of neutralizing or nonneutralizing antibodies to cFVIII. These data establish the framework to quantitatively investigate the efficacy and safety in preclinical studies of novel therapies for hemophilia A.

Minimal modification in the factor VIII B-domain sequence ameliorates the murine hemophilia A phenotype

Recombinant canine B-domain deleted (BDD) factor VIII (FVIII) is predominantly expressed as a single-chain protein and exhibits greater stability after activation compared with human FVIII-BDD. We generated a novel BDD-FVIII variant (FVIII-RH) with an amino acid change at the furin cleavage site within the B domain (position R1645H) that mimics the canine sequence (HHQR vs human RHQR). Compared with human FVIII-BDD, expression of FVIII-RH protein revealed a 2.5-fold increase in the single-chain form. Notably, FVIII-RH exhibited a twofold increase in biological activity compared with FVIII-BDD, likely due to its slower dissociation of the A2-domain upon thrombin activation. Injection of FVIII-RH protein in hemophilia A (HA) mice resulted in more efficacious hemostasis following vascular injury in both the macro- and microcirculation. These findings were successfully translated to adeno-associated viral (AAV)-based liver gene transfer in HA mice. Expression of circulating FVIII-RH was approximately twofold higher compared with AAV-FVIII-BDD-injected mice. Moreover, FVIII-RH exhibits superior procoagulant effects compared with FVIII-BDD following a series of hemostatic challenges. Notably, the immunogenicity of FVIII-RH did not differ from FVIII-BDD. Thus, FVIII-RH is an attractive bioengineered molecule for improving efficacy without increased immunogenicity and may be suitable for both protein- and gene-based strategies for HA.

In vivo efficacy of platelet-delivered, high specific activity factor VIII variants

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.

Venom factor V from the common brown snake escapes hemostatic regulation through procoagulant adaptations.

Venomous snakes produce an array of toxic compounds, including procoagulants to defend themselves and incapacitate prey. The Australian snake Pseudonaja textilis has a venom-derived prothrombin activator homologous to coagulation factors V (FV) and Xa (FXa). Here we show that the FV component (pt-FV) has unique biologic properties that subvert the normal regulatory restraints intended to restrict an unregulated procoagulant response. Unlike human FV, recombinant pt-FV is constitutively active and does not require proteolytic processing to function. Sequence comparisons show that it has shed a large portion of the central B-domain, including residues that stabilize the inactive procofactor state. Remarkably, pt-FV functions in the absence of anionic membranes as it binds snake-FXa with high affinity in solution. Furthermore, despite cleavage in the heavy chain, pt-FV is functionally resistant to activated protein C, an anticoagulant. We speculate this stability is the result of noncovalent interactions and/or a unique disulfide bond in pt-FV linking the heavy and light chains. Taken together, these findings provide a biochemical rationale for the strong procoagulant nature of venom prothrombinase. Furthermore, they illustrate how regulatory mechanisms designed to limit the hemostatic response can be uncoupled to provide a sustained, disseminated procoagulant stimulus for use as a biologic toxin.