Abstract

There are several hypotheses which attempt to explain changes in tissue factor (TF) activity on cell surfaces upon treatment with various agents (“encryption-de-encryption”). However, these hypotheses are mostly based upon conjectural data interpretation. We evaluated the influence of lipopolysaccharide (LPS) stimulation on the surface and total expression of TF antigen on/in THP-1 monocytic cells and the corresponding TF activity. A cell-based immunoassay was used for the quantitation of TF antigen on the surface of monocytic cells, coupled with contact pathway-inhibited (corn trypsin inhibitor; CTI) plasma and blood dilute PT, extrinsic factor (F)Xase and synthetic coagulation proteome assays. Prior to stimulation, the concentration of TF was 240±60 molecules/cell on the cell surface and 510±180 total molecules/cell in cell lysates (n=8). Upon stimulation with LPS (1μg/ml), the TF antigen concentration steadily increased reaching a maximum at 4 hr of stimulation (2400±360 molecules/cell on the cell surface and 8400±360 molecules/cell total). Coincidently, the intact cell FXa generation rate increased from 4.2 pM/s to 46.3 pM/s. The clotting time of multi-donor plasma decreased from 192s to 80s corresponding to a 35.7±4.9-fold increase in TF activity. Thus all assays are consistent with increased surface expression of TF. Kinetic studies showed that the KM of the complex enzyme (TF/FVIIa) formed on the surface of LPS-stimulated monocytes for FX is 0.73±0.07 μM and the kcat 59.4±1.8 s−1. The kinetic properties for the FXase formed in lysates of LPS-stimulated THP-1 cells are similar to those observed for the cell surface TF (see Table 1). However, when LPS-stimulated monocyte TF was purified to homogeneity and relipidated using synthetic phospholipid vesicles (PCPS), the efficiency for FX activation was significantly lower due to both an increased KM and decreased kcat. As a consequence, the second order rate constant for FX activation by the extrinsic FXase on the surface of PCPS was only 8±1% of that observed on stimulated intact cells or lysates. A comparison of purified and relipidated monocyte TF with that cell-bound in situ in the synthetic coagulation proteome with platelets at mean physiologic concentration (2x108/ml) showed that thrombin generation profiles were similar for the reactions initiated with 5 pM relipidated TF and 0.05 pM TF expressed on the cell surface; a difference in specific activity of approximately 100-fold. Similar results were observed in CTI-inhibited blood. CTI blood, triggered with 5 pM relipidated monocyte TF clotted in 310 s, whereas initiation with 1.2 pM monocyte TF in situ led to a 40 s clotting time with maximum rates of thrombin generation of 1.3 and >7.0 nM/s, respectively. These data indicate that the increase in monocyte TF activity upon stimulation with LPS is related to an increased expression of TF protein on/in monocytic cells and that there are components of the cell membrane which substantially enhance TF activity and these accessories maintain their function(s) in cell lysates. These components are not represented by synthetic (PCPS) phospholipid vesicles. Our data emphasize the importance of cell membrane composition on TF activity and are in agreement with observations published by

Pendurthi et al. (
Blood
2007
;
110
:
3900
)
.

Table 1. Monocytic TF in the extrinsic FXase

Preparation of TF KM(μM) kcat(s1) kcat/KM((μM•s1) 
In situ 0.73±0.0 59.4±1.8 81.4 
In lysates 0.41±0.0 44.6±0.1 108.8 
On PCPS 1.54±0.14 12.0±0.4 7.8 
Preparation of TF KM(μM) kcat(s1) kcat/KM((μM•s1) 
In situ 0.73±0.0 59.4±1.8 81.4 
In lysates 0.41±0.0 44.6±0.1 108.8 
On PCPS 1.54±0.14 12.0±0.4 7.8 

Disclosures: No relevant conflicts of interest to declare.

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