PAR4 antagonists in inflammation

Slofstra and colleagues employ the Shwartzman model of LPS-induced inflammation in mice to show that inhibition of the thrombin receptor PAR4 reduces neutrophil infiltration into inflamed tissue but does not reduce mortality or prolong survival.

It is a commonly held notion that inflammation-induced, excessive activation of coagulation contributes to morbidity and mortality in severe inflammatory disease by causing thrombotic damage to the microvasculature and by exacerbating the inflammatory response. Targeting critical molecular links between inflammation and coagulation has been a rationale for therapeutic intervention in animal studies and in human clinical trials on patients with severe inflammatory response syndromes. Protease-activated receptors (PARs 1, 2, 3, and 4) for activated coagulation factors and related proteases are prime candidates for the crosstalk between inflammation and coagulation.¹  In an extensive study, Camerer et al²  examined how individual PAR receptors modify the outcome of severe inflammation triggered by administration of a single lethal dose of LPS to mice. This study showed that activation of PARs, including PAR4, is not required for the extent of coagulation activation, the magnitude of systemic cytokine elaboration, platelet consumption, or the ultimate end point of mortality. Camerer and colleagues went on to demonstrate that thrombin inhibition, genetic elimination of fibrinogen-dependent hemostasis, or complete elimination of platelets did not protect against lethality triggered by endotoxemia in the absence of live microorganisms. Altogether, these findings seem a convincing demonstration that the extent of coagulation activation and/or platelet consumption has minor, if any, effect on the survival of murine endotoxemia.

In this issue of Blood, Slofstra and colleagues revisit this issue by testing the effect of pharmacologic PAR4 inhibition by using the Shwartzman reaction to high-dose LPS in low-dose, endotoxin-primed mice. They show that blocking the PAR4 receptor with cell-penetrating peptides (pepducins) that disrupt G-protein coupling prevents organ damage. The authors provide circumstantial evidence that the primary cellular target of the PAR4 antagonist may be neutrophils, rather than platelets or endothelial cells. The beneficial effect of PAR4 inhibition seems to be based on reduced neutrophil infiltration into sites of inflammation. Al-though the relevant cell type targeted by the PAR4 antagonist, the actual ligand engaging PAR4 on the target cell (PAR4 is activated not only by thrombin, but by other proteinases as well^3,4 ), is not entirely clear from the present work, the authors' hypothesis is in line with earlier findings on PAR4 effects in a model of localized, neutrophil-driven inflammation in the hind paw of rats.⁵  Also, it remains to be stringently resolved whether improved organ function is due to reduced—possibly neutrophil-dependent—thrombosis. In acute endotoxin models, TF expression by monocytes is the immediate trigger of coagulation activation. LPS priming of innate immune cells leads to marked phenotypic changes and LPS tolerance, raising the issue of whether alternative cell types acquire the ability to initiate coagulation in the Shwartzman model. For example, priming may lead to a transfer of TF-positive microparticles to neutrophils or lead to formation of platelet-neutrophil aggregates with translation-controlled TF synthesis in platelets.^6,7  Such events may induce neutrophil-dependent microthrombotic organ damage that is specific for this model.

Interestingly, the choice of the Shwartzman reaction as a model was based on a previous side-by-side comparison with single-dose endotoxemia, which suggested that both models elicit comparable inflammatory responses, but only the Shwartzman reaction is associated with significant microvascular thrombosis and therefore might more accurately reflect the situation in patients with severe sepsis complicated by disseminated intravascular coagulation (DIC).⁷  Yet the current study convincingly shows that PAR4 inhibition, despite moderately improving organ function, does not translate into a reduction of mortality or prolonged survival, consistent with the results obtained with PAR4 deficient mice in the single-dose LPS model.²  While minimal effects on survival may suggest that the Shwartzman model requires additional refinement to become predictive for clinical outcome, these results beg the question of whether the concept of thrombosis-driven lethality in mouse models of endotoxemia should finally be put to rest.