Tissue factor activity in whole blood

Abstract

Tissue factor (TF) is an integral membrane protein essential for hemostasis. During the past several years, a number of studies have suggested that physiologically active TF circulates in blood at concentrations greater than 30 pM either as a component of blood cells and microparticles or as a soluble plasma protein. In our studies using contact pathway-inhibited blood or plasma containing activated platelets, typically no clot is observed for 20 minutes in the absence of exogenous TF. An inhibitory anti-TF antibody also has no effect on the clotting time in the absence of exogenous TF. The addition of TF to whole blood at a concentration as low as 16 to 20 fM results in pronounced acceleration of clot formation. The presence of potential platelet TF activity was evaluated using ionophore-treated platelets and employing functional and immunoassays. No detectable TF activity or antigen was observed on quiescent or ionophore-stimulated platelets. Similarly, no TF antigen was detected on mononuclear cells in nonstimulated whole blood, whereas in lipopolysaccharide (LPS)-stimulated blood a significant fraction of monocytes express TF. Our data indicate that the concentration of physiologically active TF in non-cytokine-stimulated blood from healthy individuals cannot exceed and is probably lower than 20 fM.

Investigation of procoagulant activity in extracellular vesicles isolated by differential ultracentrifugation

“…The removal of 20K pellets and 100K pellets revealed a reduced activity of PPL in the subsequent supernatants (Figure 5), similar to findings by Marchetti et al [15]. Our findings indicated that EVs from healthy persons carry no or a very low amounts of TF, but contain some PPL which is also in agreement with the findings of others [28,29]. …”

No evidence for tissue factor on platelets

“…Expression of cell surface-and microparticle-associated TF by LPS-stimulated THP-1 cells was confirmed by flow cytometry (data not shown). The percentage P-selectin-positive platelets that stained positively for TF when incubated in the presence of THP-1 cells and LPS (1.2% Ϯ 0.6%) was virtually identical to that observed in the absence of LPS (2.4% Ϯ 1.1%; Figure 1B), confirming previous observations made in whole blood 6,7 and suggesting that TF on monocytes or monocyte-derived microparticles is not transferred to platelets.…”

“…This paradigm has been challenged by studies suggesting that platelets express active TF after de novo synthesis, 2,3 transfer from monocytes, 4 or release from ␣-granules. 5 In other studies, 6,7 however, the existence of platelet TF could not be demonstrated.…”

“…6,8 Flow cytometric analyses indicated that no TF was expressed by protease-activated receptor (PAR)1-and PAR4-activated platelets (1.6% Ϯ 1.2%) compared with unactivated platelets (1.5% Ϯ 0.6%; Figure 1A) despite maximal ␣-granule release (Ͼ 98% P-selectin-positive platelets), consistent with previous studies demonstrating a lack of platelet TF expression after A23187-induced platelet activation and ␣-granule release. 6 Consistent with studies performed in whole blood, 6,7 no TF antigen (Ͻ 0.2pM) was detected by immunoassay 8 after prolonged stimulation of platelets with lipopolysaccharide (LPS). In contrast, LPS-stimulated THP-1 cells expressed 6.6 plus or minus 2.0pmol/L TF/10 6 cells.…”

“…These observations were confirmed in 2 TF-dependent activity-based assays. 6 No factor Xa (FXa) (Ͻ 0.1pmol/L FXa/second) was generated by extrinsic FXase using unstimulated or stimulated platelets as a possible TF source. Similarly, no clot was formed in a plasma-based clotting assay (Ͼ 999 seconds).…”