Regulation of N-formyl-methionyl-leucyl-phenylalanine receptor recycling by surface membrane neutral endopeptidase-mediated degradation of ligand.

Neutrophil responses to alpha-N-formyl-L-Met-L-Leu-L-Phe (fMLF) are modulated by inhibitors of surface membrane neutral endopeptidase (NEP), such as phosphoramidon (PPAD). Because receptor recycling is presumably required for a sustained cellular response, the effect of PPAD on receptor reexpression was examined. After down-regulation of surface fMLF receptors by fMLF, PPAD blocked the normal reexpression of surface receptors in a manner that was related to the time of prior exposure to fMLF. Internalized fML[3H]F was hydrolyzed by NEP at a rate comparable to the rate of receptor reexpression at the cell surface, suggesting that ligand hydrolysis is rate limiting. To test this hypothesis, cells were incubated with fluorescein-labeled formyl-Met-Leu-Phe-Nle-Tyr-Lys at 15 degrees C. After binding was complete, but before internalization of receptor-ligand complexes, high-affinity antifluorescein antibody F(ab')2 fragments were added and the cells incubated at 37 degrees C for 60 min in the presence of PPAD. Under these conditions, the inhibitory effects of PPAD were largely reversed and nonimmune F(ab')2 fragments were without effect.

Effects of OKM5, a monoclonal antibody to glycoprotein IV, on platelet aggregation and thrombospondin surface expression.

The monoclonal antibody, OKM5, recognizes an 88-Kd monocyte membrane protein and also binds to the platelet membrane protein, GPIV (GPIIIb, CD36). In this study, we have found that the OKM5 target epitope is present at approximately 12,000 copies per platelet and that interaction with the antibody has both stimulatory and inhibitory effects on platelet function. In the absence of other stimuli, OKM5 induced platelet aggregation, secretion, and expression of fibrinogen receptors. These stimulatory responses required intact antibody as F(ab')2 fragments were not active but blocked the stimulatory activity of the intact antibody. In contrast, exposure of platelets to OKM5 followed by another strong stimulus such as thrombin resulted in a marked suppression of fibrinogen, fibronectin, and von Willebrand factor binding to the cells. This effect was not noted when a weak stimulus, adenosine diphosphate, was the second agonist. At OKM5 concentrations that interfered with fibrinogen binding to thrombin-stimulated platelets by 80% to 90%, platelet binding of exogenous thrombospondin, or surface expression of endogenous thrombospondin was not affected. The inhibitory effect of OKM5 on fibrinogen binding to thrombin-stimulated platelets was related to the formation of massive platelet aggregates in the samples. These results show that interaction of OKM5 with its target antigen on platelets can elicit diverse functional responses from the cells.

Divalent cation-dependent and independent surface expression of thrombospondin on thrombin-stimulated human platelets.

Thrombospondin (TSP), a platelet alpha-granule protein, becomes expressed on the surface of thrombin-stimulated platelets. The surface expression of this protein occurs through two distinct mechanisms. At low platelet concentrations (1 X 10(8)/mL), a divalent ion-dependent, low-capacity mechanism predominates. At higher cell concentrations, a divalent ion-dependent, higher capacity mechanism prevails that can account for greater than 90% of all the TSP surface expression measured. This mechanism requires the presence of both calcium and magnesium (Ca + Mg). The dependence of the divalent ion-dependent surface expression on platelet concentration suggests that release of the molecule from the cell followed by its binding to the cell surface mediates this component of the endogenous TSP-platelet interaction. These data are consistent with a two-receptor model for the platelet-surface expression of the endogenous TSP pool.

Identification of a new class of inducible receptors on platelets. Thrombospondin interacts with platelets via a GPIIb-IIIa-independent mechanism.

Thrombospondin with fibrinogen, fibronectin, and von Willebrand factor binds to platelets stimulated with agonists and support platelet adhesive functions. The receptors for the latter three proteins are associated with membrane glycoprotein GPIIb-IIIa. Thrombasthenic platelets deficient in GPIIb-IIIa have been utilized to examine the role of this membrane protein in the interactions of thrombospondin with platelets. Radioiodinated thrombospondin bound to thrombin-stimulated platelets from normal and thrombasthenic donors with a similar affinity and capacity. As monitored with a monoclonal antibody to thrombospondin, the divalent ion-dependent and -independent pathways for the expression of the endogenous pool of thrombospondin on the surface of thrombin-stimulated platelets from normal and thrombasthenic donors were also qualitatively and quantitatively similar. GPIIb-IIIa or ligands associated with GPIIb-IIIa thus are not essential for the binding of thrombospondin to platelets. Therefore, thrombospondin interacts with unique receptors on platelets.