Hydrolysis of human platelet membrane glycoproteins with a Serratia marcescensmetalloprotease: Effect on response to thrombin and von Willebrand factor

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 Serratiaprotease 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 NaDodSO4/polyacrylamide gel electrophoresis using both staining with periodic acid-Schiff reagent and autoradiography of 3H-labeled platelets. Both methods of detection demonstrated that at Serratiaprotease concentrations above 0.6 μg/ml there was hydrolysis within 3 min of a membrane glycoprotein (Mr185,000) corresponding to GPIb in control platelets. The loss of GPIb was accompanied by the appearance in the platelet supernatant of a glycopeptide (Mr156,000). Under reducing conditions, the hydrolyzed membrane glycopeptide in the supernatant had a consistently faster migration (Mr149,000). Platelets treated with Serratiaprotease at concentrations sufficient to give maximal cleavage of GPIb were unresponsive to bovine von Willebrand factor and did not aggregate or release [14C]serotonin at doses of von Willebrand factor up to 0.33 unit/ml. On the other hand, the response to bovine α-thrombin was only minimally impaired by treatment with Serratiaprotease. 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.