I. Suppression by compound 48/80 of bacterial endotoxin-inducible monocytic tissue factor activity: direct blockade of factor VII binding to THP-1 monocytes1This study was presented in part in abstract form to the American Society for Biochemistry and Molecular Biology annual meeting held in San Francisco, CA, 16–20 May 1999.1

1 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. 2 In a single-stage clotting assay, RuR concentration-dependently inhibited rabbit brain thromboplastin (rbTF)-induced coagulation and oset bacterial endotoxin (LPS)-induced monocytic TF (mTF) hypercoagulation; the IC 50 s were estimated at 7.5 and 12.3 mM, respectively. 3 A 15-min preincubation of RuR with rbTF or monocyte suspension resulted in the pronounced inhibition with a signi®cantly lowered IC 50 at 1.8 or 7.7 mM for rbTF or mTF procoagulation, respectively. The dierences in IC 50 s between rbTF and mTF without or with the preincubation indicated that TF was a primary target for RuR action. 4 The eect 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. 5 Inclusion of RuR into human plasma samples in vitro signi®cantly 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 eect on thrombin time. Thus, RuR inhibited FVII activation to block the initiation of coagulation.

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Blockade by ruthenium red of tissue factor‐initiated coagulation

Chu

Wang

Nwobi

et al. 2001

British J Pharmacology

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Protamine inhibits tissue factor‐initiated extrinsic coagulation

et al. 2001

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Summary. The enhanced extrinsic coagulation in response to inflammation could contribute to disseminated intravascular coagulation, often manifesting cardiovascular complications. The complex mechanism remains unclear and effective management is not well established. The ability of protamine to offset bacterial endotoxin (LPS)-induced tissue factor (TF)-initiated extrinsic coagulation was demonstrated in human peripheral blood monocytes and cultured human leukaemia THP-1 monocytes, which was consistent with the inhibition of rabbit brain thromboplastin (rbTF) procoagulant activity in a cell-free in vitro model. Protamine significantly prolonged prothrombin time, further confirming the downregulation of the extrinsic pathway. However, thrombin time remained unaltered. Chromogenic assays were performed to dissect the extrinsic pathway, identifying inhibitory site(s). Protamine significantly inhibited factor VII (FVII) activation but not the dissected FX activation. The amidolytic activities of FVIIa and FXa were unaffected. The inhibited FVII activation in the presence of protamine was confirmed by the diminished FVIIa formation on Western blot analyses. Protamine preferentially inhibited TF-catalysed FVII activation, downregulating the extrinsic cascade. Protamine could be of anticoagulant significance in the management of the extrinsic hypercoagulation.

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Possible role of Marcks in the cellular modulation of monocytic tissue factor‐initiated hypercoagulation

Chu¹,

Lin

Piasentin

2002

Br J Haematol

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Summary. The enhanced extrinsic tissue factor (TF)-initiated coagulation, often resulting from sepsis, could lead to disseminated intravascular coagulation presenting cardiovascular complications. Using model human leukaemia THP-1 monocytes, we studied monocytic TF (mTF) hypercoagulation and its regulation. After an 8 h exposure to bacterial endotoxin [lipopolysaccharide (LPS); 100 ng/ml], mTF activity was significantly upregulated as the result of the enhanced mTF synthesis. Thereafter, LPS induction declined, exhibiting a Ôquiescent-desensitizingÕ phenomenon. Such diminished LPS induction was, however, associated with sustained LPS-enhanced mTF synthesis, revealing the possible occurrence of a post-translational downregulation. It was noted that LPS desensitization was accompanied by the increased expression of myristoylated alanine-rich C kinase substrate (Marcks). In contrast, A23187 (20 lmol/l) or Quin-2AM (20 lmol/l) drastically activated mTF activity without detectable effect on mTF synthesis; both of which showed that sustained functional upregulation during 24 h culture did not enhance Marcks expression. These inverse correlations between mTF activity upregulation and Marcks expression suggested that Marcks could be inhibitory. Marcks phosphorylation site domain (151-175) (Marcks PSD) readily inhibited mTF-dependent FVII activation and diminished FVIIa formation in LPS-challenged cells. As a result, Marcks PSD offset LPS-induced mTF hypercoagulation upon inclusion in the single-stage clotting assays. The anticoagulant activity was confirmed by showing that Marcks PSD significantly blocked rabbit brain thromboplastin (rbTF) procoagulation and inhibited rbTF-dependent FVII activation as well as FVIIa formation. Our study suggests that Marcks expression plays a role in a novel cellular modulation to downregulate mTF hypercoagulation.

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Cited by 4 publications

References 36 publications

Blockade by ruthenium red of tissue factor‐initiated coagulation

Blockade by ruthenium red of tissue factor‐initiated coagulation

Protamine inhibits tissue factor‐initiated extrinsic coagulation

Possible role of Marcks in the cellular modulation of monocytic tissue factor‐initiated hypercoagulation

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