Enhanced potency of recombinant factor VIIa with increased affinity to activated platelets.

BACKGROUND: Recombinant factor VIIa (rFVIIa) enhances thrombin generation in a platelet-dependent manner; however, rFVIIa binds activated platelets with relatively low affinity. Triggering receptor expressed on myeloid cells (TREM)-like transcript (TLT)-1 is expressed exclusively on activated platelets. OBJECTIVE: To enhance the potency of rFVIIa via binding TLT-1. METHODS: Recombinant FVIIa was conjugated to a TLT-1 binding Fab. In vitro potency of this platelet-targeted rFVIIa (PT-rFVIIa) was evaluated using factor X activation assays and by measuring viscoelastic changes in whole blood. In vivo potency was evaluated using a tail vein transection model in F8-/- mice expressing human TLT-1. RESULTS: PT-rFVIIa and rFVIIa had similar dissociation constant values for tissue factor binding and similar tissue factor-dependent factor X activation. However, PT-rFVIIa had increased catalytic efficiency on TLT-1-loaded vesicles and activated platelets. The in vitro potency in normal human blood with antibody-induced hemophilia A was dependent on assay conditions used; with maximally activated platelets, the half maximal effective concentration for clot time for PT-rFVIIa was 49-fold lower compared with rFVIIa. In the murine bleeding model, a 53-fold lower half maximal effective concentration was observed for blood loss for PT-rFVIIa, supporting the relevance of the assay conditions with maximally activated platelets. In vitro analysis of blood from subjects with hemophilia A confirmed the data obtained with normal blood. CONCLUSIONS: Increasing the affinity of rFVIIa to activated platelets resulted in approximately 50-fold increased potency both in vitro and in the mouse model. The correlation of in vivo with in vitro data using maximally activated platelets supports that these assay conditions are relevant when evaluating platelet-targeted hemostatic concepts.

Prolonged half-life and preserved enzymatic properties of factor IX selectively PEGylated on native N-glycans in the activation peptide.

Current management of hemophilia B entails multiple weekly infusions of factor IX (FIX) to prevent bleeding episodes. In an attempt to make a longer acting recombinant FIX (rFIX), we have explored a new releasable protraction concept using the native N-glycans in the activation peptide as sites for attachment of polyethylene glycol (PEG). Release of the activation peptide by physiologic activators converted glycoPEGylated rFIX (N9-GP) to native rFIXa and proceeded with normal kinetics for FXIa, while the K(m) for activation by FVIIa-tissue factor (TF) was increased by 2-fold. Consistent with minimal perturbation of rFIX by the attached PEG, N9-GP retained 73%-100% specific activity in plasma and whole-blood-based assays and showed efficacy comparable with rFIX in stopping acute bleeds in hemophilia B mice. In animal models N9-GP exhibited up to 2-fold increased in vivo recovery and a markedly prolonged half-life in mini-pig (76 hours) and hemophilia B dog (113 hours) compared with rFIX (16 hours). The extended circulation time of N9-GP was reflected in prolonged correction of coagulation parameters in hemophilia B dog and duration of effect in hemophilia B mice. Collectively, these results suggest that N9-GP has the potential to offer efficacious prophylactic and acute treatment of hemophilia B patients at a reduced dosing frequency.

Plasma elimination kinetics for factor VII are independent of its activation to factor VIIa and complex formation with plasma inhibitors.

The mechanism for the elimination of factor VII (FVII) from the circulation is unknown, just as it is unclear how activation of FVII to FVIIa and subsequent complex formation with antithrombin III (AT) or alpha2-macroglobulin (alpha2M) affects clearance. The possibility that the clearance mechanism involves activation and inhibitor complex formation as obligatory intermediate reactions is examined in this study. Human and murine sera were spiked with human FVIIa in the absence and presence of heparin and analysed for complex formation. Complex formation in vivo was studied after intravenous injection of (125)I-VIIa in mice; and the pharmacokinetics (PK) of human and murine FVIIa was studied in normal mice. Furthermore, comparative PK studies were performed with FVII, FVIIa, active site blocked FVIIa and a preformed FVIIa-AT complex in normal and alpha2M-deficient mice. The data demonstrated that FVIIa-AT complexes and to a much lesser extent FVIIa-alpha2M-complexes accumulated in vivo after FVIIa administration. FVIIa-AT accounted for about 50% of total FVIIa antigen left in the circulation after 3 hours. All FVII derivatives studied including FVII, FVIIa and FVIIa-AT were cleared with similar rates suggesting an elimination kinetics which is unaffected by FVII activation and subsequent inactivation by plasma inhibitors.

Properties of the co-chaperone protein p23 erroneously attributed to ALG-2 (apoptosis-linked gene 2).

A commercial antibody (clone 22) directed against the apoptosis-linked gene 2 (alg2, pdcd6) encoded protein has been used by several groups. Up-regulated expression of the antigen was observed in primary tumours and in metastatic tissue and also during rat brain ischemia. Furthermore, antigen down-regulation was found in human atherosclerotic plaques. Recently, we found that the clone 22 antibody does not recognise ALG-2. In the present study the antigen of the clone 22 antibody was identified as the heat shock protein 90 (HSP90) co-chaperone protein p23, identical to the cytosolic prostaglandin E2 synthase, by immunoprecipitation followed by tryptic in-gel digests and mass spectrometry of the purified peptides. Moreover, the heterogeneous ribonuclear protein A2/B1 was found to be a part of the p23 co-immunoprecipitated protein complex.