title: Hydrolysis of human platelet membrane glycoproteins with a Serratia marcescens metalloprotease: effect on response to thrombin and von Willebrand factor.
abstract: Metalloproteases from at least two Gram-negative organisms selectively hydrolyze the surface of human fixed washed platelets. The protease-treated platelets lose their ability to aggregate with bovine von Willebrand factor and human von Willebrand factor plus ristocetin. The present study reports the membrane glycoprotein alterations and loss of platelet function that occur after incubation of fresh washed human platelets with a purified protease produced by Serratia marcescens. The studies were undertaken in order to examine, using the Serratia protease as an enzymatic probe, the relationship between externally oriented glycoproteins and two known aggregating agents, bovine von Willebrand factor and thrombin. Platelet membrane glycoproteins were analyzed after discontinuous NaDodSO(4)/polyacrylamide gel electrophoresis using both staining with periodic acid-Schiff reagent and autoradiography of (3)H-labeled platelets. Both methods of detection demonstrated that at Serratia protease concentrations above 0.6 mug/ml there was hydrolysis within 3 min of a membrane glycoprotein (M(r) 185,000) corresponding to GPIb in control platelets. The loss of GPIb was accompanied by the appearance in the platelet supernatant of a glycopeptide (M(r) 156,000). Under reducing conditions, the hydrolyzed membrane glycopeptide in the supernatant had a consistently faster migration (M(r) 149,000). Platelets treated with Serratia protease at concentrations sufficient to give maximal cleavage of GPIb were unresponsive to bovine von Willebrand factor and did not aggregate or release [(14)C]serotonin at doses of von Willebrand factor up to 0.33 unit/ml. On the other hand, the response to bovine alpha-thrombin was only minimally impaired by treatment with Serratia protease. These results implicate GPIb in the response by platelets to bovine von Willebrand factor but suggest that surface components other than GPIb play a major role in the response by platelets to thrombin.
mesh_terms: D001779:Blood Coagulation Factors / Q000494:pharmacology*; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology*; D002462:Cell Membrane / Q000502:physiology; D010450:Endopeptidases / Q000378:metabolism; D006023:Glycoproteins / Q000502:physiology; D006801:Humans; D008565:Membrane Proteins / Q000502:physiology; D008666:Metalloendopeptidases; D008970:Molecular Weight; D012706:Serratia marcescens / Q000201:enzymology; D013917:Thrombin / Q000494:pharmacology*; D014841:von Willebrand Factor / Q000494:pharmacology*
chemical_list: D001779:Blood Coagulation Factors; D006023:Glycoproteins; D008565:Membrane Proteins; D014841:von Willebrand Factor; D010450:Endopeptidases; D013917:Thrombin; D008666:Metalloendopeptidases