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. 2001 Jul;133(5):659-64.
doi: 10.1038/sj.bjp.0704116.

Blockade by ruthenium red of tissue factor-initiated coagulation

A J Chu  1 , Z G Wang, O I Nwobi, S Beydoun
Affiliations

Blockade by ruthenium red of tissue factor-initiated coagulation

A J Chu et al. Br J Pharmacol. 2001 Jul.

Abstract

The ability of ruthenium red (RuR) to inhibit tissue factor (TF)-initiated blood coagulation was demonstrated at the protein and cellular levels as well as in human plasma. In a single-stage clotting assay, RuR concentration-dependently inhibited rabbit brain thromboplastin (rbTF)-induced coagulation and offset bacterial endotoxin (LPS)-induced monocytic TF (mTF) hypercoagulation; the IC(50)s were estimated at 7.5 and 12.3 microM, respectively. A 15-min preincubation of RuR with rbTF or monocyte suspension resulted in the pronounced inhibition with a significantly lowered IC(50) at 1.8 or 7.7 microM for rbTF or mTF procoagulation, respectively. The differences in IC(50)s between rbTF and mTF without or with the preincubation indicated that TF was a primary target for RuR action. The effect of RuR on the physiological function of TF in FVII activation was demonstrated by the proteolytic cleavage of FVII zymogen to its active forms of serine protease on Western blotting analyses. RuR readily blocked TF-catalyzed FVII activation (diminished FVIIa formation), thus down regulating the initiation of blood coagulation. Inclusion of RuR into human plasma samples in vitro significantly prolonged prothrombin time, indicating the depressed coagulation. FVII activity was inhibited by 30 - 60% depending on the dose; as a result, FX activity also decreased. However, RuR showed no effect on thrombin time. Thus, RuR inhibited FVII activation to block the initiation of coagulation.

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Figures

Figure 1
Figure 1
Effect of RuR on mTF hypercoagulation. THP-1 monocytes were challenged with LPS (100 ng ml−1) for 3 h. The cells were harvested and washed. mTF procoagulant activity was assayed as described elsewhere. RuR was included in the assay without or with preincubation with the challenged cells prior to the assay, reaching the indicated final concentration in the reaction mixture. mTF procoagulant activity of resting cells was 6±1 U (106 cells)−1 (n=4). The data are means±s.d. from four independent experiments.
Figure 2
Figure 2
Effect of RuR on rbTF-initiated coagulation. rbTF (2 mg ml−1) procoagulant activity was performed in the single-stage clotting assay as described elsewhere. RuR was included in the assay without or with preincubation with rbTF prior to the assay, reaching the indicated final concentration in the reaction mixture. The data are means±s.d. from six independent experiments.
Figure 3
Figure 3
Effect of RuR on FVII proteolytic activation. FVII (1.5 μg) was incubated with rbTF (2.2 μg) in the absence or presence of 5 μM RuR at 37°C for 30 min. In some cases, FVII (1.5 μg) was incubated with the same amount (0.85×106 cells) of LPS-challenged cells, resting or LPS-challenged cells in the presence or absence of 10 μM RuR. The samples were subjected to 12.5% SDS–PAGE. The blot was immunostained with sheep-anti-hFVII followed by peroxide-conjugated goat anti-sheep IgG as described elsewhere.
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