Identification of a second putative receptor of platelet-activating factor from human polymorphonuclear leukocytes.

S B Hwang

doi:10.1016/s0021-9258(18)69059-3

Abstract

Due to multiple molecular species of platelet-activating factor (PAF) and the existence of high affinity binding sites in a variety of cells and tissues, possible existence of PAF receptor subtypes has been suggested. This report shows differences between specific PAF receptors in human leukocytes and platelets. Human polymorphonuclear leukocyte membranes showed high affinity binding sites for PAF with an equilibrium dissociation constant (KD) of 4.4 (+/- 0.3) x 10(-10) M. We compared the relative potencies of several PAF agonists and receptor antagonists between human platelet and human leukocyte membranes. One receptor antagonist (Ono-6240) was found to be 6-10 times less potent in inhibiting the specific [3H]PAF receptor binding, PAF-induced GTPase activity, as well as the PAF-induced aggregation in human leukocytes than in human platelets. Mg2+, Ca2+, and K+ ions potentiated the specific [3H]PAF binding in both systems. Na+ and Li+ ions inhibited the specific [3H]PAF binding to human platelets but showed no effects in human leukocytes. K+ ions decreased the Mg2+-potentiated [3H]PAF binding in human leukocytes but showed no effects in human platelets. PAF stimulates the hydrolysis of [gamma-32P] GTP with an ED50 of about 1 nM, whereas the biological inactive enantiomer shows no activity even at 10 microM in both human platelets and human leukocytes. The PAF-stimulated GTPase in human leukocytes can be abolished by the pretreatment of membranes with pertussis toxin and cholera toxin. However, the PAF-stimulated activity of GTPase in human platelets is insensitive to pertussis toxin and cholera toxin. These results suggest that there exists a second type of PAF receptor in human polymorphonuclear leukocytes, which is structurally different from the one characterized in human platelets, and that the guanine nucleotide-binding protein coupled to PAF receptors in human leukocytes is also different from the one in human platelets.

Highlights

K' ions decreased the Mg+-potentiated['HIPAF bind- types are obtained from two different species, whether the ing in human leukocytes but showed no effects in hu- difference in potency of kadsurenone is due to the species man platelets
There seems no clear evidence to results suggest that there existsa second type of Platelet-activating factor (PAF) demonstrate that there is a difference in the PAF receptors receptorin human polymorphonuclear leukocytes, which is structurally different from tohnee characterized in human platelets, and that the guanine nucleotide-binding protein coupled to PAF receptors in human leukocytes is different from thoene in human platelets
We found that Ono-6240, a PAF receptor antagonist

Summary

EXPERIMENTAL PROCEDURES

Specific PHIPAF Binding-The binding of [3H]PAF to human platelet or human PMN membranes was measured as previously. The gel-filtered human plateleta were prepared exactly solution (150 mM NaC1,lO mM Tris, 2 mM EDTA at pH7.5) [9] and as described for rabbit platelets. For further fractionation induced aggregationof human PMNs prepared from the freshly drawn of membrane fragments, the lysed membrane suspension was layered blood was monitored with a Chrono-LogLumi-Aggregometerat 37 "C. over the top of a discontinuous sucrose density gradient of0.25 M Percent aggregation was calculated as described [24]. The majority of human PMN membranes used in this study were threitol, and either 100 p g of the cholera toxin/ml or 50 pg of the prepared from human blood (aged between 12 and 36 h) purchased pertussis toxin/ml for 30 min at 30 'C. Preparation of Human Platelet Membranes-Human platelet mem- Specific Binding of PHJPAF to Human Leukocyte Mernbranes were prepared from platelet concentrates(age(36 h) obtained branes-To assess the affinity of PAF binding and thenumber

A Human Leukocytes

DISCUSSION

Findings

Human Platelets g - c