Analyses on molecular mechanisms of activation of intravascular Tissue Factor

Both activated platelets and circulating microparticles were
described to express tissue factor (TF), the principal initiator of
coagulation, on their cell surface (intravascular TF). It is still
not clear whether TF is functionally active on activated platelets.
TF expressed on activated monocytes and various other cell types
has been described to be functionally inactive (encrypted or latent
TF). In the present study, cellular mechanisms are analyzed that
could release the TF procoagulant activity of blood components.
Tissue factor pathway inhibitor-1 (TFPI) represents the main
physiologic inhibitor of the coagulation start. It inhibits the
ternary initiator complex of the extrinsic coagulation pathway by
first binding the circulating factors X / Xa and subsequently
interacting with VII / VIIa. We found that after stimulation with
thrombin and collagen type I, TFPI was recovered in the platelet
releasate and it was degraded by neutrophil elastase (NE) released
from activated neutrophils. TFPI degradation was also induced by NE
on neutrophil microparticles. We found that NE is bound to
negatively charged macromolecules (proteoglycans, RNA) on the
surface of activated neutrophils by polar interactions. Overall, we
could provide substantial experimental evidence that upon
interaction of activated platelets with PMN a microenvironment is
formed, which allows the efficient degradation of TFPI by the
PMN-associated serine protease NE. This cross talk between the
innate immune system and the coagulation system might be of general
importance in pathologies, such as sepsis, arterial and venous
thrombosis and myocardial infarction. TF-encryption has represented
an unsolved problem for several decades. We reveal that
thiol-disulfide exchange in the extracellular C186/C209 disulfide
pair of TF triggers the TF procoagulant activity. Formation of the
intramolecular C186/C209 disulfide activates TF procoagulant
function, whereas reduction of the disulfide to the appropriate
sulfhydryls and the formation of mixed disulfides (protein
S-glutathionylation of TF) were found to suppress its procoagulant
function. TF activation is supported by the thiol isomerase protein
disulfide isomerase (PDI) and it is facilitated by the reactive
oxygen species (ROS) detoxifying enzyme glutaredoxin (GRX). Protein
S-glutathionylation of TF was uncovered as one reversible mechanism
preventing the intravascular coagulation start. We suggest that
thiol isomerases are injury-responsive signals driving coagulation
through posttranslational cysteine modifications of TF. This
mechanism could help to explain the augmented occurrence of
vasoocclusive pathologies during ageing, where increasing
concentrations of ROS might favour TF oxidation.