Related Experiment Videos
Proteolysis of human fixed, washed platelets by gram-negative bacterial metalloproteases: effect on von Willebrand
Abstract:
Fixed, washed platelets (FWP) are usually stable to aggregation with von Willebrand factor (vWF) from human and certain animal plasmas over several months of storage. When one lot of FWP lost its stability in less than 1 week, studies demonstrated contamination with Serratia marcescens. Extracellular proteases produced by S. marcescens, as well as by Pseudomonas aeruginosa and Escherichia coli, were found to cause loss of FWP aggregability. Purified proteases were prepared from cell-free culture filtrates of S. marcescens and two different strains of P. aeruginosa. They were used to study the effect on the interaction of FWP and vWF. All three purified proteases destroyed FWP aggregability in a time- and concentration-dependent fashion. The protease produced by S. marcescens (SP) was found to be at least eight times more potent against FWP as a substrate than either of the two P. aeruginosa enzymes. The ability of SP to destroy FWP aggregability was prevented by EDTA and could be restored by the addition of Zn2+ in slight molar excess. When compared with trypsin and chymotrypsin, SP was found to be highly selective in digesting the FWP membrane, even at concentrations greater than that established to give a similar loss of FWP aggregability. SP does not induce aggregation of fresh, washed platelets or PRP, but renders them unaggregable with vWF. These proteases may be useful research tools for studying membranes and vWF-platelet interactions.
Insights
Bacterial proteases, particularly from Serratia marcescens, can degrade fixed, washed platelets (FWP), causing loss of aggregation with von Willebrand factor (vWF). This protease activity may offer new research tools for platelet and vWF interaction studies.
Area of Science:
- Biochemistry
- Hematology
- Microbiology
Background:
- Fixed, washed platelets (FWP) are typically stable for platelet aggregation with von Willebrand factor (vWF).
- Contamination with Serratia marcescens led to a loss of FWP stability within a week.
Purpose of the Study:
- To investigate the impact of bacterial proteases on FWP stability and aggregation with vWF.
- To characterize the properties of proteases from Serratia marcescens, Pseudomonas aeruginosa, and Escherichia coli.
Main Methods:
- Purified proteases from cell-free culture filtrates of S. marcescens and P. aeruginosa were used.
- The effect of these proteases on FWP and vWF interaction was studied.
- Protease activity was compared to trypsin and chymotrypsin.
Main Results:
- All tested bacterial proteases degraded FWP aggregability in a time- and concentration-dependent manner.
- Serratia marcescens protease (SP) was significantly more potent than P. aeruginosa proteases.
- SP's activity was inhibited by EDTA and restored by Zn2+, indicating a metalloprotease nature.
- SP selectively digested the FWP membrane without inducing platelet aggregation.
Conclusions:
- Bacterial proteases, especially SP, can compromise FWP stability and vWF-mediated aggregation.
- These proteases may serve as valuable tools for investigating platelet membrane structure and vWF-platelet interactions.