A monoclonal antibody that recognizes the GPIIb/IIIa antagonist DMP 728. Reversal of the effects of DMP 728 on platelet aggregation and bleeding time in the dog.

Since hemorrhagic events represent a major safety concern associated with the use of new antithrombotic therapies such as glycoprotein (GP) IIb/IIIa receptor blockade, we evaluated the ability of a monoclonal antibody recognizing DMP 728 (cyclic [D-2-aminobutyryl-N2-methyl-L-argininyl-glycyl-L-aspartyl-3- aminomethyl-benzoic acid] methanesulfonic acid salt), a potent GPIIb/IIIa receptor antagonist, to reverse the pharmacological actions of DMP 728 in the dog. DC11 was chosen for in vivo evaluation based on its ability to inhibit the binding of [3H]DMP 728 to activated platelets and to attenuate the inhibition of ADP-induced aggregation on platelet-rich plasma ex vivo by DMP 728. After anesthesia mongrel dogs were given DMP 728 (20 micrograms/kg body wt IV) infused into the femoral vein, bleeding times were determined using a Simplate device from incisions on the backside of the tongue, and platelet aggregation was determined ex vivo. Nearly complete inhibition of platelet aggregation was observed for the dogs treated with DMP 728 (20 ug/kg IV) for up to 210 minutes, and bleeding times were prolonged > 15 minutes for 2 hours and remained elevated for more than 4 hours. DC11 (0.2 or 1.0 mg/kg body wt IV) given to dogs 10 minutes after DMP 728 resulted in 50% attenuation of the effect of DMP 728 on aggregation at 3 hours. Approximately 34% inhibition of the DMP 728-mediated bleeding time was achieved at 1 hour with the 0.2 mg/kg dose, whereas approximately 50% inhibition of the bleeding time was observed for the 1 mg/kg dose at 1 hour.(ABSTRACT TRUNCATED AT 250 WORDS)

Platelet GPIIb/IIIa receptor occupancy studies using a novel fluoresceinated cyclic Arg-Gly-Asp peptide.

DMP 728 is a potent and specific platelet GPIIb/IIIa antagonist. Like all GPIIb/IIIa antagonists, DMP 728 has a steep dose-response relationship in inhibiting platelet aggregation. In this study the relationships between receptor occupancy, platelet aggregation and bleeding time was determined in anesthetized dogs after intravenous infusion of DMP 728 (0.01 and 0.1 mg/kg/2h). Receptor occupancy was determined by flow cytometry using XL086, a novel fluorescent cyclic RGD peptide that binds to GPIIb/IIIa with high specificity and affinity (kd approximately 55 nM). Mean number of GPIIb/IIIa as determined by flow cytometric assay was approximately 53,8000 and 79,000 on unactivated and ADP-activated platelets respectively. After DMP 728 intravenous infusion, there was a dose- and time-dependent increase in receptor occupancy, inhibition of platelet aggregation and bleeding time. The two methods of receptor occupancy determination correlate with each other with an r2 = 0.78. The present data suggest that blockade of only 40-60% (approximately 40,000 receptors) of the total platelet GPIIb/IIIa was required to achieve > 90% inhibition of platelet aggregation and > 15 min bleeding time. Our results showed the potential clinical utility of this approach in the study of GPIIb/IIIa dose-response relationship.

Characterization of a panel of monoclonal antibodies against human tissue-type plasminogen activator.

Five murine monoclonal antibodies have been produced against the 1-chain form of human tissue plasminogen activator (t-PA). Affinity constants, calculated from data generated in a solid phase radioimmunoassay, ranged from 4.9 x 10(8)M-1 to 2.3 x 10(9) M-1 for these antibodies. This panel was classified into three groups based on the antibodies' effects on both the plasminogen activation and amidolytic activities of t-PA: complete inhibitors (CD2), partial inhibitors (DB10), and those with limited effects (AE5, BA10 and EG2). This same pattern for the five antibodies was observed in an assay measuring the binding of 125I-t-PA to its fast-acting inhibitor, PAI-1. It is concluded that this panel of antibodies defines at least three domains on the t-PA molecule, including its catalytic site.

A monoclonal anti-idiotypic antibody which mimics angiotensin II in inducing a population of anti-hormone antibodies.

IG12, a syngeneic monoclonal anti-idiotypic antibody, was raised against an idiotype on a monoclonal antibody, ICH2, with specificity for the octapeptide hormone angiotensin II (AII). Competitive radioimmunoassays, and ELISAs utilizing AII antiserum raised in different species (rats and rabbits), indicate that IG12 detects a private idiotypic determinant at or near the paratopic region of the ICH2 combining site. Immunization of syngeneic mice with either IG12 or IG12 coupled to keyhole limpet hemocyanin induced a population of anti-anti-idiotypic antibodies that shared two properties with ICH2: the binding of AII and the binding to IG12. Antibodies from these anti-anti-idiotypic populations were also partially active in inhibiting the interaction between AII and ICH2. To our knowledge, this is the first demonstration of a monoclonal anti-idiotypic antibody mimicking a physiological hormone in inducing a population of anti-hormone antibodies.