(370008-40) Platelet Protein Phosphatase-1 Modulates Fibrin Polymerization: Implication in Sepsis-Induced TAMOF

Introduction: Thrombocytopenia-associated multiple organ failure (TAMOF) is a sepsis-induced MOF phenotype with disseminated microvascular thrombosis, acute kidney injury, and high mortality. Protein phosphatase 1 transcripts are differentially expressed platelet genes in sepsis. We previously showed in a TAMOF model, platelets mitigate LPS-induced death of kidney cells in part via protein phosphatase 1 alpha (PP1a). Since plasma fibrinogen is phosphorylated and fibrin polymerization is intrinsic to thrombosis, we hypothesize that extracellular PP1a engages and modulates fibrin clot formation, and TAMOF patients have reduced plasma PP1a.

Methods: In a swine model of Escherichia Coli (E.coli) sepsis-induced TAMOF, we collected blood from 8 piglets at pre and 70h post E.coli. Fibrin polymerization assays were done to assess fibrin clot formation potential +/- purified PP1. Extracellular PP1a on ex vivo swine fibrin clots were visualized with fluorescent anti-PP1a Ab using Laser Scanning Confocal Microscopy. Wildtype (WT) mice or conditional deletion of platelet PP1a (PP1a-/-) mice were challenged with 20 mg/kg LPS for 18h, and renal injury assessed in harvested kidneys with mRNA of kidney injury marker 1 (KIM1) and neutrophil gelatinase associated lipocalin (NGAL). PP1a levels were measured by ELISA in plasma of septic TAMOF and non-TAMOF pts.

Results: After E.coli, fibrin clot formation potential was significantly increased (50%; p< 0.05). Extracellular PP1a were seen incorporated into ex vivo fibrin clots from septic piglets. KIM1 and NGAL were significantly increased in LPS-treated PP1a-/- mice (110 & 24% respectively; p< 0.05) compared to WT. Plasma PP1a were significantly lower (23%; p< 0.05) in TAMOF (n=6) compared to non-TAMOF (n=6) septic pts. Purified PP1 at 0.5, 1, and 2 units/100 mcl decreased fibrin formation potential by 36, 43, 50% respectively in ex vivo plasma of septic pts (n=2).

Conclusions: Plasma PP1a decreased, while fibrin clot formation potential was increased in TAMOF. Endogenous PP1a engaged fibrin clots, and exogenous supplementation of PP1 decreased fibrin clot formation potential. These studies suggest that thrombocytopenia-associated organ injury may stem from the loss of a protective role played by platelet PP1a. Recombinant PP1a may have therapeutic implication in TAMOF patients