Annette J. Eckardt

RGD induces conformational transition in purified platelet integrin GPIIb/IIIa-SDS system yielding multiple binding states for fibrinogen γ-chain C-terminal peptide

Fibrinogen γ‐chain C‐terminal peptide HHLGGAKQAGDV (γ12) and α‐chain peptide GRGDSP are known to inhibit fibrinogen‐mediated platelet cell aggregation via competitive interactions with platelet integrin receptor GPIlb/IIIa. NMR studies of γ12 in the presence of purified GPIIb/IIIa in SDS/water solution have demonstrated the presence of two γ12 binding states, one of which is eliminated by GRGDSP (RGD) up to a RGD: γ12 ratio of 2:1. RGD: γl2 ratios greater than 2:1 produce multiple sets of γ12 NMR signals in TOCSY spectra. At a ratio of 4:1, two to four such resonance sets can be resolved for A405, Q407, A408, G409, D410 and V411 spin systems. The number of multiple resonances remains unchanged at ratios of 6:1 and 8:1. Addition of γ12 to reverse the ratio to 8:8 (1::1) has no apparent effect on the RGD‐induced distribution. Results suggest that RGD irreversibly induces a conformational transition(s) in GPIIb/IIIa to produce multiple γ12 binding sites on the receptor.

Separation of different receptor-mediated effects of a prostaglandin H2 analogue (U46619) on human platelets by means of human granulocytic elastase and chymotrypsin

Previous investigations indicated two classes of thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors on human platelets and suggested that shape change and myosin light chain phosphorylation correlated with the occupancy of high affinity receptors while serotonin release was related to a putative low affinity binding component (Morinelli TA et al., Am J Physiol 253: H1035-H1043, 1987). The current study shows that chymotrypsin destroyed three receptor-mediated responses of platelets to U46619 (a TXA2/PGH2 agonist), i.e. shape change, myosin light chain phosphorylation and serotonin release. Human granulocyte elastase selectively inactivated platelet ability to release serotonin following stimulation with U46619, but it did not affect significantly shape change and myosin light chain phosphorylation. In conclusion, it is possible to separate different receptor-mediated effects of U46619 on human platelets by means of human granulocytic elastase and chymotrypsin.

Characterization of an antibody raised against reduced glycoprotein IIIa of human platelets

A polyclonal antibody against reduced and vinylpyridylethylated human glycoprotein IIIa was raised in rabbits. Its reactivity with reduced GPIIIa was about 500 times higher than that of the antibody against native GPIIIa. The lowest amounts of purified reduced and native GPIIIa recognized by the antibody against reduced GPIIIa were 25 and 400 ng, respectively. The antibody did not recognize native GPIIIa (about 1-2 micrograms) in platelet extracts and chymotryptic degradation products of GPIIIa. It inhibited ADP-induced platelet aggregation but it did not inhibit fibrinogen binding to ADP-stimulated platelets. Our experiments suggest that the antigenicity of GPIIIa (beta 3 integrin) depends on the conformation of the molecule determined by numerous S-S bridges between cysteine residues.

Studies on the Localization of Fibrinogen Binding Sites on Platelet Glycoprotein IIIa

The domains of various integrins involved in the binding to adhesive proteins are poorly characterized. The approaches to localize ligand binding sites on the adhesive receptors include studies on the genetic characterization of the defective β 2 subunits in leukocyte adhesion deficiency and the defective β 1 subunit in Glanzmann thrombasthenia, studies on the chemical and photoaffinity crosslinking of the adhesive ligands to the receptors, proteolytic degradation of the integrins, attempts to recognize binding sites with monoclonal antibodies, and studies on the synthetic peptides derived from glycoprotein IIIa (GPIIIa).