Abstract

The mechanisms that regulate the incorporation and release of tissue factors (TFs) into cell-derived microparticles are as yet unidentified. In this study, we have explored the regulation of TF release into microparticles by the phosphorylation of serine residues within the cytoplasmic domain of TF. Wild-type and mutant forms of TF, containing alanine and aspartate substitutions at Ser253 and Ser258, were overexpressed in coronary artery and dermal microvascular endothelial cells and microparticle release stimulated with PAR2 agonist peptide (PAR2-AP). The release of TF antigen and activity was then monitored. In addition, the phosphorylation state of the two serine residues within the released microparticles and the cells was monitored for 150 min. The release of wild-type TF as procoagulant microparticles peaked at 90 min and declined thereafter in both cell types. The TF within these microparticles was phosphorylated at Ser253 but not at Ser258. Aspartate substitution of Ser253 resulted in rapid release of TF antigen but not activity, whereas TF release was reduced and delayed by alanine substitution of Ser253 or aspartate substitution of Ser258. Alanine substitution of Ser258 prolonged the release of TF following PAR2-AP activation. The release of TF was concurrent with phosphorylation of Ser253 and was followed by dephosphorylation at 120 min and phosphorylation of Ser258. We propose a sequential mechanism in which the phosphorylation of Ser253 through PAR2 activation results in the incorporation of TF into microparticles, simultaneously inducing Ser258 phosphorylation. Phosphorylation of Ser258 in turn promotes the dephosphorylation of Ser253 and suppresses the release of TF.

Highlights

  • The cytoplasmic domain of tissue factors (TFs) is not required for the procoagulant activity of TF or de-encryption of this protein [12, 13]

  • By aspartate substitution [16] or alanine substitution of two of the serine residues within the cytoplasmic domain of TF, we investigated the contribution of these residues to the regulation of TF incorporation and release into cell-derived microparticles

  • Time Course of TF Release as Microparticles upon PAR2 Activation—Stimulation of HCAEC or HDMEC overexpressing TFWT with either PAR2 agonist peptide (PAR2-AP) (20 ␮M) or FXa (10 nM) resulted in the transient incorporation and release of TF antigen into microparticles, which peaked at 90 min post-stimulation (Fig. 1A) but not in the untransfected cells, PAR1-activated cells, or PAR2-AP-activated HUVEC

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Summary

To whom correspondence should be addressed

The cytoplasmic domain of TF is not required for the procoagulant activity of TF or de-encryption of this protein [12, 13]. It has been reported that substitution of the cytoplasmic domain of TF with the cytoplasmic domain of decayaccelerating factor does not alter the release of TF in cells that are capable of releasing TF-containing microparticles without activation [14]. It is known that cells spontaneously release decay-accelerating factor under standard culture conditions [15]. By aspartate substitution (to mimic phosphoserine) [16] or alanine substitution (to prevent phosphorylation) of two of the serine residues within the cytoplasmic domain of TF, we investigated the contribution of these residues to the regulation of TF incorporation and release into cell-derived microparticles

EXPERIMENTAL PROCEDURES
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