Pathogen-induced coagulopathy: the role of pathogen-derived virulence factors in the development of clinically significant coagulopathy

Authors:
Andrew Benjamin, Ellen Cohn, Jessica Cao, John Alverdy, Olga Zaborina, Rebecca Meltzer, Robert Keskey

Body of Abstract:
Background: Trauma-induced coagulopathy (TIC) results in significant morbidity and mortality despite improved understanding of specific TIC phenotypes that occur after injury. While individual patient factors (sex, genetics, injury severity, etc.) can influence a patient’s coagulation profile, there remains poorly understood within-group differences. Life-threatening injury requiring surgery, antibiotics, and prolonged hospitalization often predisposes trauma patients to colonization by pathogenic bacteria (i.e. Pseudomonas aeruginosa), but current TIC research focuses almost exclusively on host factors. Host-pathogen interactions may play an underappreciated role in the development of coagulopathy, given the intricate interplay between infection, inflammation, and coagulation. Bacterial pathogens are known to produce virulence factors that degrade key components of the clotting cascade as a strategy for subverting the host immune system, but the extent to which these mechanisms contribute to clinically significant coagulopathy remains unclear.

Hypothesis: We hypothesize that Pseudomonas aeruginosa has the capacity to induce clinically significant coagulopathy via secreted virulence factors.

Methods: Pseudomonas aeruginosa (Pa) isolated from an ICU patient was grown in liquid media. Supernatant (Pa-sup) was collected at stationary phase, filtered, and added to citrate-anticoagulated whole blood from 5 healthy, human volunteers at 1:10 and 1:15 dilutions and compared to the addition of liquid media alone (Ctrl). Coagulation profiles were analyzed using the ROTEM sigma (Werfen), a functional assay of the intrinsic (int)/extrinsic (ext) clotting cascade and fibrin contribution to clot formation. Also, collagenolytic activity of Pa-sup was assessed in vitro using fluorescein-conjugated gelatin (Invitrogen).

Results: Pa-sup caused significant coagulopathy in both the intrinsic and extrinsic clotting cascades in a dose-dependent manner. Pa-sup significantly decreased maximum clot firmness (MCF) (MCFint: 10.2 vs 26.7 vs 53.4; 1:10 vs 1:15 vs Ctrl; p<0.001 | MCFext: 17.6 vs 39.7 vs 58.5; 1:10 vs 1:15 vs Ctrl; p<0.001) and amplitude at 10 minutes (A10) (A10int: 4.8 vs 24.3 vs 49.1; 1:10 vs 1:15 vs Ctrl; p<0.001 | A10ext: 16.4 vs 34.2 vs 51.7; 1:10 vs 1:15 vs Ctrl; p<0.001). Additionally, 1:10 Pa-sup caused a significant increase in clotting time (CT) (CTint: 438.2s vs 168.1s Ctrl; p<0.001 | CText: 191.2s vs 61.1s Ctrl p=0.06). Pa-sup-induced coagulopathy correlated with increased collagenolytic activity. Conclusion: Pseudomonas aeruginosa yields exoproducts that more than double clotting time and weaken clot strength by almost 50%. Taken together, these data demonstrate that certain pathogens can secrete virulence factors that cause clinically significant coagulopathy. Further work is ongoing to determine the extent to which pathogen-induced coagulopathy is amplified in the setting of trauma and to determine its generalizability to other pathogens.