Platelet numbers are intricately regulated to avoid spontaneous bleeding or arterial occlusion and organ damage. The growth factor thrombopoietin (TPO) drives platelet biogenesis by inducing megakaryocyte production. A recent study in mice identified a feedback mechanism by which clearance of aged, desialylated platelets stimulates TPO synthesis by hepatocytes. This new finding generated renewed interest in platelet clearance mechanisms. Here, different established and emerging mechanisms of platelet senescence and clearance will be reviewed with specific emphasis on the role of posttranslational modifications.
Essentials Factor VIII inhibitors are the most serious complication in patients with hemophilia A. Aggregates in biopharmaceutical products are an immunogenic risk factor. Aggregates were identified in recombinant full-length factor VIII products. Aggregates in recombinant factor VIII products are identified by analytical ultracentrifugation. Summary: Background The development of inhibitory anti-factor VIII antibodies is the most serious complication in the management of patients with hemophilia A. Studies have suggested that recombinant full-length FVIII is more immunogenic than plasma-derived FVIII, and that, among recombinant FVIII products, Kogenate is more immunogenic than Advate. Aggregates in biopharmaceutical products are considered a risk factor for the development of anti-drug antibodies. Objective To evaluate recombinant full-length FVIII products for the presence of aggregates. Methods Advate, Helixate and Kogenate were reconstituted to their therapeutic formulations, and subjected to sedimentation velocity (SV) analytical ultracentrifugation (AUC). Additionally, Advate and Kogenate were concentrated and subjected to buffer exchange by ultrafiltration to remove viscous cosolvents for the purpose of measuring s 20,w values and molecular weights. Results The major component of all three products was a population of ~7.5 S heterodimers with a weight-average molecular weight of ~230 kDa. Helixate and Kogenate contained aggregates ranging from 12 S to at least 100 S, representing ≈ 20% of the protein mass. Aggregates greater than 12 S represented < 3% of the protein mass in Advate. An approximately 10.5 S aggregate, possibly representing a dimer of heterodimers, was identified in buffer-exchanged Advat e and Kogenate. SV AUC analysis of a plasma-derived FVIII product was confounded by the presence of von Willebrand factor in molar excess over FVIII. Conclusions Aggregate formation has been identified in recombinant full-length FVIII products, and is more extensive in Helixate and Kogenate than in Advate. SV AUC is an important method for characterizing FVIII products.
Extracellular ATP is a signal of tissue damage and induces macrophage responses that amplify inflammation and coagulation. Here we demonstrate that ATP signaling through macrophage P2X7 receptors uncouples the thioredoxin (TRX)/TRX reductase (TRXR) system and activates the inflammasome through endosome-generated ROS. TRXR and inflammasome activity promoted filopodia formation, cellular release of reduced TRX, and generation of extracellular thiol pathway- dependent, procoagulant microparticles (MPs). Additionally, inflammasome-induced activation of an intracellular caspase-1/ calpain cysteine protease cascade degraded filamin, thereby severing bonds between the cytoskeleton and tissue factor (TF), the cell surface receptor responsible for coagulation activation. This cascade enabled TF trafficking from rafts to filopodia and ultimately onto phosphatidylserine-positive, highly procoagulant MPs. Furthermore, caspase-1 specifically facilitated cell surface actin exposure, which was required for the final release of highly procoagulant MPs from filopodia. Together, the results of this study delineate a thromboinflammatory pathway and suggest that components of this pathway have potential as pharmacological targets to simultaneously attenuate inflammation and innate immune cell-induced thrombosis.
Ectodomain shedding of glycoprotein (GP) Ibα is thought to mediate the clearance of activated, aged or damaged platelets. A monoclonal antibody, 5G6, has been developed recently to specifically bind to the GPIbα shedding cleavage site and to inhibit its shedding. However, the molecular mechanism underlying antigen recognition and inhibitory specificity is not clear. To elucidate the structural basis for 5G6 binding to GPIbα, we determined the crystal structure of 5G6 Fab fragment in complex with its epitope peptide KL10 (GPIbα residues 461-470, KLRGVLQGHL), to 2.4-Å resolution. Key residues in both 5G6 and KL10 were mutated to validate their effects in antibody binding by using isothermal titration calorimetry. The 5G6 Fab-KL10 peptide complex structure confirmed the direct association of 5G6 with its target GPIbα residues and elucidated the molecular basis underlying its binding specificity and high affinity. The similar binding properties of 5G6 Fab fragment to GPIbα on human platelets as those to KL10 suggests that such an interaction may not be affected by the plasma membrane or nearby GPIbβ. This structural information may facilitate further antibody optimization and humanization.
The glycoprotein (GP)Ib-IX-V complex is the platelet receptor for von Willebrand factor and many other molecules critically involved in he most as is and thrombosis. The lack of functional GPIb-IX-V complexes on the platelet surface is the cause of Bernard-Soulier syndrome, a rare hereditary bleeding disorder also associated with macro thrombocytopenia. The GPIb-IX-V complex contains GPIbα, GPIbβ, GPIX and GPV subunits, all of which are type I trans membrane proteins containing leucine-rich repeat domains. Although all the subunits were identified decades ago, not until recently did the mechanism of complex assembly begin to emerge from a systematic characterization of inter-subunit interactions. This review summarizes forces driving the assembly of the GPIb-IX-V complex, discusses their implication on the pathogenesis of Bernard-Soulier syndrome, and identifies questions that remain about the structure and organization of GPIb-IX-V.
Summary
Objective
The pathogenicity of anti-human fVIII monoclonal antibodies (MAbs) was tested in a murine bleeding model.
Methods
MAbs were injected into the tail veins of hemophilia A mice to a peak plasma concentration of 60 nM, followed by injection of human B domain-deleted factor VIII (fVIII) at 180 U/kg, producing peak plasma concentrations of ∼2 nM. At 2 hours, blood loss following a 4 mm tail snip was measured. The following MAbs were tested: 1) 4A4, a type I anti-A2 fVIII inhibitor, 2) I54 and 1B5, classical type I anti-C2 inhibitors, 3) 2-77 and B45, non-classical type II anti-C2 inhibitors, and 4) 2-117, a non-classical anti-C2 MAb with inhibitory activity less than 0.4 Bethesda Units/mg IgG.
Results
All MAbs except 2-117 produced similar amounts of blood loss that were significantly greater than control mice injected with fVIII alone. Increasing the dose of fVIII to 360 U/kg overcame the bleeding diathesis produced by the type II MAbs 2-77 and B45, but not the type I antibodies, 4A4, I54, and 1B5. These results were consistent with the in vitro Bethesda assay in which 4A4 completely inhibited both 1 U/ml and 3 U/ml fVIII, while there was 40% residual activity at saturating concentrations of 2-77 at either concentration of fVIII.
Conclusions
For patients with an inhibitor response dominated by non-classical anti-C2 antibodies both the in vivo and in vitro results suggest that treatment with high-dose fVIII rather than bypassing agents may be warranted.