William R. Church

Antithrombotic Efficacy of a Novel Murine Antihuman Factor IX Antibody in Rats

A murine antihuman factor IX monoclonal antibody (BC2) has been generated and evaluated for its capacity to prolong the activated partial thromboplastin time (aPTT) in vitro and ex vivo and to prevent arterial thrombosis in a rat model in vivo. BC2 extended aPTT to a maximum of 60 to 80 seconds at 100 to 1000 nmol/L in vitro (rat and human plasma, respectively) and ex vivo (rat) after dosing of rats up to 6 mg/kg in vivo. BC2, administered as bolus (1 to 6 mg/kg) followed by infusion (0.3 to 2 mg x kg(-1) x h(-1)), dose-dependently prevented thrombosis of an injured rat carotid artery (FeCl(3)-patch model), increased time to artery occlusion, and reduced incidence of vessel occlusion. BC2 efficacy in preventing arterial thrombosis exceeded that of heparin (bolus 15 to 120 U/kg followed by infusion 0.5 to 4.0 U x kg(-1) x min(-1)), whereas the latter rendered the blood incoagulable (aPTT>1000 seconds). BC2 demonstrated complete antithrombotic efficacy also as a single bolus given either as prevessel or postvessel injury as evidenced by reduction of thrombus mass (from 4.18+/-0.49 to 1.80 +/-0.3 mg, P<0.001), increasing vessel patency time (from 14.9+/-0.9 minutes to 58.3+/-1.7 minutes, P<0.001) and decreasing incidence of vessel occlusion from 100% to 0% in vehicle- versus BC2-treated rats, respectively. BC2 (3 mg/kg, IV) administered in a single bolus resulted in 50% reduction in thrombus mass (P<0.01), extended vessel patency time (P<0.001), extended aPTT only 4-fold, and had no effect on blood loss via a tail surgical wound; heparin, at doses that reduced thrombus mass to a similar extent, extended aPTT beyond 1000 seconds (over 500-fold) and increased blood loss from 1.8+/-0.7 to 3.3 +/-0.6 mL (P<0.001). These data suggest that BC2 may provide enhanced therapeutic efficacy in humans at lesser interference with blood hemostasis than heparin.

Purification of Six Human Vitamin K-Dependent Proteins in a Single Chromatographic Step Using Immunoaffinity Columns1

The major human vitamin K-dependent proteins were purified from plasma using immunoadsorbents made with antibodies specific for each protein. Monoclonal antibodies to Factor VII, Factor IX, Factor X, Protein C, and Protein S were prepared from mice immunized with isolated vitamin K-dependent antigens. Purified monoclonal antibodies and a purified burro polyclonal anti-prothrombin immunoglobulin were individually coupled to Sepharose and used in a tandem series of columns to purify each of the vitamin K-dependent proteins from eluates of barium citrate precipitates of plasma. The proteins were eluted from the columns by sodium thiocyanate and retained functional activity following dialysis. Prothrombin, Factor VII, Factor IX, Factor X and Protein C were essentially homogeneous as judged by NaDodSO4-PAGE; Protein S was isolated as a Protein S-C4b binding protein complex. These results indicate the utility of monoclonal antibody immunoadsorbents for purifying the human vitamin K-dependent proteins and represent a considerable simplification over other purification schemes.

Inhibiting GPIbα Shedding Preserves Post-Transfusion Recovery and Hemostatic Function of Platelets After Prolonged Storage

Objective—The platelet storage lesion accelerates platelet clearance after transfusion, but the underlying molecular mechanism remains elusive. Although inhibiting sheddase activity hampers clearance of platelets with storage lesion, the target platelet protein responsible for ectodomain shedding–induced clearance is not definitively identified. Monoclonal antibody 5G6 was developed recently to bind specifically human platelet receptor glycoprotein (GP)Ib&agr; and inhibit its shedding but not shedding of other receptors. Here, the role of GPIb&agr; shedding in platelet clearance after transfusion was addressed. Approach and Results—Both human leukoreduced apheresis-derived platelets and transgenic mouse platelets expressing human GPIb&agr; were stored at room temperature in the presence and absence of 5G6 Fab fragment. At various time points, aliquots of stored platelets were analyzed and compared. 5G6 Fab inhibited GPIb&agr; shedding in both platelets during storage and preserved higher level of GPIb&agr; on the platelet surface. Compared with age-matched control platelets, 5G6 Fab–stored platelets exhibited similar levels of platelet activation, degranulation, and agonist-induced aggregation. 5G6 Fab–stored human GPIb&agr; platelets exhibited significantly higher post-transfusion recovery and in vivo hemostatic function in recipient mice than control platelets. Consistently, 5G6 Fab–stored, 8-day-old human platelets produced similar improvement in post-transfusion recovery in immunodeficient mice and in ex vivo thrombus formation over collagen under shear flow. Conclusions—Specific inhibition of GPIb&agr; shedding in the stored platelets improves post-transfusion platelet recovery and hemostatic function, providing clear evidence for GPIb&agr; shedding as a cause of platelet clearance. These results suggest that specific inhibition of GPIb&agr; shedding may be used to optimize platelet storage conditions.

Immunochemical techniques for studying coagulation proteins

Publisher Summary This chapter provides and discusses some basic immunochemistry techniques that are applicable to the isolation and characterization of plasma proteins, more specifically the plasma coagulation proteins. It discusses the (1) selection and purification of an appropriate antibody, (2) immunoaffinity purification of coagulation proteins, and (3) several antibody–antigen binding assays that have proven useful in the study of blood plasma proteins. In blood coagulation and fibrinolysis, antibodies are versatile and powerful agents for the immunoaffinity purification of proteins, for protein quantitation by radioimmunoassays and enzyme-linked immunosorbent assays (ELISA), as site-specific probes of metal ion- and activation-dependent epitopes, and as reaction-specific regulators of the macromolecular catalytic complexes of blood. Several factors influence the performance of an antibody with respect to immunochemical applications: (1) the affinity of the antibody–antigen complex, (2) the ability of the antibody to bind the native antigen under conditions of the binding reaction during immunoaffinity purification, and (3) the ability to dissociate the antibody–antigen complex under a set of conditions that are nondestructive to both the antibody and the antigen.

Domain-specific monoclonal antibodies to ovotransferrin indicate conservation of determinants involved in avian transferrin receptor recognition

1. Three of five monoclonal antibodies produced to chicken ovotransferrin bound quail ovotransferrin but none of the antibodies bound human, bovine or equine serum transferrin. 2. Equilibrium binding experiments indicate that both quail and chicken ovotransferrin bind to transferrin receptors on chick reticulocytes although the quail protein binds to 40% fewer sites with an affinity which is three times lower than chicken ovotransferrin. 3. The antibodies that recognize quail ovotransferrin block binding of both radiolabelled chicken and quail ovotransferrin to chick reticulocytes. 4. Quail NH2-terminal half-molecule domain appears to be unable to form a functional hybrid holo-ovotransferrin with chicken C-terminal half-molecule domain.