Redistribution of glycoprotein Ib within platelets in response to protease-activated receptors 1 and 4: roles of cytoskeleton and calcium.
Thrombin activates human platelets by hydrolyzing the protease-activated receptors PAR-1 and PAR-4, exposing new N-terminal sequences which act as tethered ligands, and binding to glycoprotein (GP) Ib, whose surface accessibility transiently decreases when platelets are stimulated by the enzyme. In an attempt to better understand this latter process, we used the peptides SFLLRNPNDKYEPF (PAR-1-AP or TRAP) and AYPGKF (PAR-4-AP) to study whether hydrolysis of both PAR receptors leads to GPIb redistribution. Both peptides induced surface clearance of GPIb with a maximum at 2 min and 5 min for PAR-1-AP and PAR-4-AP, respectively, followed by a slow return to the surface with levels normalizing between 30 and 60 min. Translocation was associated with the formation of clusters of GPIb as revealed by fluorescence microscopy. This transient redistribution of GPIb was blocked by cytochalasin D and in large part by the membrane permeable Ca2+ chelator, BAPTA. The inhibitor of phosphatidylinositol 3-kinase and myosin light chain kinase, wortmannin, did not significantly modify internalization of GPIb, although its return to the surface was delayed for PAR-1-AP. PAR receptor-mediated association of GPIb to the insoluble cytoskeleton was blocked by cytochalasin D, while BAPTA alone increased and stabilized the presence of GPIb. Globally, immunoprecipitation experiments and analysis of the cytoskeleton confirmed that GPIb translocation is powered by a contractile mechanism involving Ca2+ mobilization, actin polymerization, and myosin incorporation into the cytoskeleton and that both PAR-1 and PAR-4 can activate this process.
Han
Y
Y
UMR 5533 CNRS, Hôpital Cardiologique du Haut-Lévêque, Avenue Magellan, Pessac, France.
Nurden
A
A
Combrié
R
R
Pasquet
J-M
JM
D000199:Actins / Q000378:metabolism; D000730:Androstadienes / Q000494:pharmacology; D001792:Blood Platelets / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism*; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D006868:Hydrolysis; D008024:Ligands; D008856:Microscopy, Fluorescence; D009218:Myosins / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000096:biosynthesis*; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D044463:Receptor, PAR-1 / Q000378:metabolism*; D018179:Receptors, Thrombin / Q000378:metabolism*; D013997:Time Factors; D000077191:Wortmannin
D000199:Actins; D000730:Androstadienes; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D008024:Ligands; D010455:Peptides; D019038:Platelet Glycoprotein GPIb-IX Complex; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; D015638:Cytochalasin D; D004533:Egtazic Acid; D019869:Phosphatidylinositol 3-Kinases; D009218:Myosins; C112908:protease-activated receptor 4; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium; D000077191:Wortmannin