Determination of the Microbicidal Capacity of Platelets By a Biomonitoring System

Introduction: Various antimicrobial polypeptides are contained in platelet α-granules and collectively termed platelet microbicidal proteins. Remarkably, these constituents identified by purification and structural determination of platelet-derived antimicrobial functions were found to be identical with or derived from proteins previously known for other functions, among them thymosin β-4 (Tβ-4), a ubiquitous actin-binding protein. Others are the fibrinopeptides A and B (FPA and FPB), which are released upon proteolytic activation of fibrinogen by thrombin. Two other platelet microbicidal proteins are CXC chemokines, namely platelet factor 4 (PF4; new designation CXCL4) and platelet basic protein (PBP/CXCL7). Another platelet microbicidal proteins was found to be identical with the CC chemokine RANTES (renamed CCL5). Two proteolytic products of PBP known as connective tissue-activating peptide-3 (CTAP-3) and neutrophil-activating peptide-2 (NAP-2), were also isolated as platelet microbicidal proteins. Apparently, the most potent antimicrobial activity is associated with C-terminal truncation products of NAP-2 and CTAP-3, both lacking two amino acids at the N-terminus. These products were are designated thrombocidin-1 (TC-1) and thrombocidin-2 (TC-2) (Virulence 2010; 1:5; 440-464).

The antimicrobial functions of platelets are donor-dependent, agonist-induced, and influenced by target microorganisms. It is cost intensive and time consuming to determine the microbicidal capacity of platelets in vitro. We therefore developed a biomonitoring system to assess the microbicidal capacityof platelets in response to pathogenic gram-negative strains.

Methods: Platelets, obtained by apheresis from healthy volunteers, were washed repeatedly in HEPES-buffered (pH 7.3) Tyrode solution (Cold Spring Harb Protoc 2007). For specific experiments, platelet-poor plasma or serum, incubated after preparation at 37°C for 1 hour, was used. Washed platelets (5x10⁸/ml) were activated by 10 U/ml thrombin and incubated at 37°C for 1 hour. After adding 1 % agarose, blood-derived samples were casted onto wedge-shaped low concentrated lysogeny broth (low LB) agar (1 g/l tryptone, 0.5 g/l yeast extract, 1 g/l NaCl, and 1.8 % agar) into square dishes (12 cm x 12 cm) to obtain a linear agar gradient. Mid-log cultures of acinetobacter baumanni, aeromonas caviae, citrobacter freundii, klebsiella oxytoca, pseudomonas aeroginosa, pseudomonas fluorescence, stenotrophomonas maltophilia grown in LB-Miller medium (10 g/l tryptone, 5 g/l yeast extract, 10 g/l NaCl, and 1.8 % agar) were washed and the OD was adjusted to 0.4. To incubate the bacteria over night at 37°C, lines of diluted bacterial cultures were distributed along to the agar gradients. Photographs were taken to measure the growth inhibition (n=7) longitudinal to the increasing concentration of a blood-derived sample agar gradient in comparison to the negative control (plasma agar gradient) or in comparison to the agar gradient without blood-derived samples.

Results: Apart from S. maltophilia, overall growth of the bacterial strains was inhibited by (mean+SD) 25.9+10,5 % (p<0.05) on the serum agar gradient compared to the plasma control agar gradient, specifically 25.0% (A.baumanni), 38.5 % (A. caviae), 37.5 % (C. freundii), 25.0 % (K. oxytoca), 17.0 % (P. aeroginosa), and 12.5 % (P. fluorescence). Testing of thrombin-activated platelets revealed an inhibition of S. maltophilia by 15.0 % and of A. caviae by 8.8 % compared to the HEPES-buffered control agar (mean 11.9 + 4.4 % inhibition of growth, p>0.05). Other bacterial strains were not inhibited by thrombin-activated platelets.

Conclusion: Inhibition of bacterial growth is measurable longitudinal a linear agar gradient containing microbicidal proteins released by activated platelets. Due to their strain-specific biochemistry not all gram-negative strains tested are sensitive to platelet microbicidal proteins. Serum and thrombin-activated platelet convey different antimicrobial effects. Thus, distinct bacterial strains can resist to platelet microbicidal proteins to different extents.

Biomonitoring allows monitoring of inhibition of bacterial growth caused by platelet microbicidal proteins. The bacterial strains evaluated in this study may be used as bioindicators to determine the microbicidal capacity of platelets.