Proteolytic susceptibility of platelet low density lipoprotein receptor.
In order to further characterize low density lipoprotein (LDL)-platelet interaction, we investigated the effect of protease pretreatment of human platelets on the subsequent binding of iodinated LDL (125I-LDL). Our results showed that the platelet LDL receptor had a proteolytic susceptibility different from that of both classical LDL receptors and the fibrinogen receptor. Platelet pretreatment with chymotrypsin, trypsin, and pronase (at 50 micrograms/mL) had no effect on 125I-LDL binding, whereas fibroblast 125I-LDL binding was markedly reduced. Mild proteolytic digestion, however (up to 1 mg/mL), was helpful in characterizing the platelet LDL receptor. Scatchard analysis showed that chymotrypsin did not modify LDL binding characteristics, whereas trypsin and pronase altered maximal number of binding sites (Bmax) without variation in dissociation constant. Trypsin increased Bmax approximately twofold (2156 +/- 327 binding sites on control platelets vs. 5246 +/- 296 on treated platelets, P < 0.001, mean +/- SEM, n = 5), but pronase decreased Bmax about 50% (2017 +/- 275 control vs. 1153 +/- 195 treated, P < 0.001). A minimum of 30 min preincubation was required to detect significant effects, and apparent equilibrium was reached by 60 min. Maximal increase in platelet LDL binding sites induced by trypsin was observed at a protein concentration of 1 mg/mL at 37 degrees C, whereas at 4 degrees C no effect was found. In contrast, maximal pronase-inhibitory effect also was observed at 37 degrees C but at higher protein concentration (10 mg/mL). Aprotinin, phenylmethylsulfonylfluoride, and soybean trypsin inhibitor were capable of fully blocking both the stimulation and the inhibition of platelet LDL binding induced by trypsin and pronase, respectively. Platelet pretreatment with both chymotrypsin and pronase (0.5 mg/mL) activated fibrinogen binding sites to a similar extent as ADP (100 microM). Furthermore, LDL (at a protein concentration of 0.3 mg/mL) increased by 81 +/- 6% the binding of fibrinogen to both protease- and ADP-stimulated platelets, but was unable to activate fibrinogen binding sites in unstimulated platelets. Overall, the results suggest that platelet LDL receptor presents a different proteolytic susceptibility in comparison with both "classical" LDL receptor and fibrinogen receptor.
Department of Biochemistry, Hospital Universitario Son Dureta, Palma de Mallorca, Spain.
J
J
Pedreño
R
R
Fernández
D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism*; D002918:Chymotrypsin / Q000494:pharmacology; D010450:Endopeptidases / Q000494:pharmacology*; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D008077:Lipoproteins, LDL / Q000378:metabolism; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism; D011402:Pronase / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D011973:Receptors, LDL / Q000378:metabolism*; D014357:Trypsin / Q000494:pharmacology; D014361:Trypsin Inhibitors / Q000494:pharmacology
D007457:Iodine Radioisotopes; D008077:Lipoproteins, LDL; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011480:Protease Inhibitors; D011973:Receptors, LDL; D014361:Trypsin Inhibitors; D010664:Phenylmethylsulfonyl Fluoride; D005340:Fibrinogen; D010450:Endopeptidases; D002918:Chymotrypsin; D014357:Trypsin; D011402:Pronase