Background: Thrombin generation is central to haemostasis. Although tissue factor (TF) initiates coagulation, thrombin generation is amplified and propagated on cell membranes, particularly phosphatidylserine-exposing platelets. Decreasing platelet counts in thrombocytopenia are associated with reduced thrombin generation. Recently, some studies have suggested that neutrophils also enhance thrombin generation.  

Aims: To investigate the contribution of neutrophils to thrombin generation in the presence and absence of platelets.

Methods: Blood from healthy drug-free donors was drawn into sodium citrate with informed, written consent. Platelets and platelet-poor plasma (PPP) were isolated by differential centrifugation. PPP was filtered through a 0.22 µm filter to remove the majority of circulating microparticles. Neutrophils were isolated using a Ficoll gradient. Neutrophils and platelets were added to PPP to achieve different cell counts. Thrombin generation following recalification of plasma was monitored using a fluorogenic thrombin substrate (zGGR-AMC) in a microplate reader. TF (Dade, Innovin) was added to some samples.

Results: TF (1 or 5pM) initiated thrombin generation in PPP. This was significantly enhanced when neutrophils were also present. In contrast, no thrombin generation was detected with neutrophils in the absence of TF. Neutrophils also enhanced thrombin generation in the presence of platelets up to 1×10⁸ platelets/ml, although at 5×10⁸ platelets/ml, neutrophils had little additional effect.  Neutrophils had no effect on thrombin generation if they were fixed with paraformaldehyde prior to being added to PPP, indicating that neutrophil activation is required. In a screen of potential activators, stimulation of PAR2 by the peptide agonist, LIGRLO, also enhanced TF-initiated thrombin generation in PPP in the absence of TF. In contrast, SFLLRN (agonist of PAR1), AYPGKF (PAR4), lipopolysaccharide (TLR4) or fMLP (FPR1) had no effect.

Conclusions: Neutrophils enhance TF-initiated thrombin generation in plasma in the absence of platelets or at low platelet counts. This effect might contribute to thrombin generation during thrombocytopenia.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/neutrophils-promote-tissue-factor-dependent-thrombin-generation-in-the-absence-of-platelets/