Identification of the 1.4 KB and 4.0 KB messages for the lipoprotein associated coagulation inhibitor and expression of the encoded protein

Girard, Thomas; Warren, Louise A.; Novotny, William; Bejcek, B; Miletich, Joseph P.; Broze, George J.

doi:10.1016/0049-3848(89)90454-4

Cited by 44 publications

(55 citation statements)

References 25 publications

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“…Clearly the cultured endothelial cells and the lung tissue rich (6). The difference between the two mRNA sizes has been primarily attributed to the presence of "-2.6 kb of additional 3' untranslated sequence in the 4-kb band (8 (35), platelets were found to contain LACI. ConsistentIwith the previous report, the amount of LACI released from 3 x 108 platelets (average number present in 1 ml of whole blood) by three freeze-thaw cycles (-700C/370C) was -6% of that present in 1 ml of plasma.…”

Section: Resultsmentioning

confidence: 99%

“…The inhibitor (henceforth referred to as LACI) has been purified from the conditioned medium of a hepatoma cell line HepG2 (4) and recently from human plasma where essentially all of it is associated with lipoproteins (7). LACI cDNAs have been identified in a human fetal liver cDNA library (6) and have been isolated from placenta (6) and endothelial cell (8) cDNA libraries. The translated amino acid sequence revealed that LACI consists of three tandem Kunitz-type inhibitory domains with three N-linked potential glycosylation sites.…”

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confidence: 99%

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Cultured normal human hepatocytes do not synthesize lipoprotein-associated coagulation inhibitor: evidence that endothelium is the principal site of its synthesis.

Bajaj

Kuppuswamy

Saito

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

272

206

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Human plasma contains a factor Xadependent inhibitor of tissue factor/factor VI~a complex termed lipoprotein-associated coagulation inhibitor (LACI). The present study examines the site(s) of LACI synthesis. In this study, cultured hepatocytes isolated from normal human liver were found to be essentially negative in LACI mRNA as revealed by Northern blot analysis using a full-length LACI cDNA as probe. The conditioned media from these cultures were also essentially negative for LACI activity. Similarly, poly(A)+ RNA obtained from normal human liver did not contain detectable LACI mRNA. In contrast, cultured human umbilical vein endothelial cells and human lung tissue (rich in endothelium) both contained abundant amounts of LACI mRNA. Moreover, erythrocyte lysates and culture media from normal monocytes, lymphocytes, or neutrophils did not contain measurable LACI activity; these cells were also negative for LACI mRNA. Platelets, however, contained LACI activity. The likely source of platelet LACI is the megakaryocyte cell since a megakaryocyte cell line (MEG-01) was found to contain LACI mRNA and to secrete small amounts of LACI activity.Additionally, human vascular smooth muscle cells and lung fibroblasts were also found to synthesize only small amounts of LACI. From these observations, we conclude that normal liver does not synthesize LACI and that endothelium is the principal source of plasma LACI. The undegraded LACI synthesized by endothelial cells had a molecular weight of 41,000.

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Cultured normal human hepatocytes do not synthesize lipoprotein-associated coagulation inhibitor: evidence that endothelium is the principal site of its synthesis.

Bajaj

Kuppuswamy

Saito

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

272

206

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“…As illustrated in Figure 6b, we identified by Northern blot, for TFPI mRNA in ECs, the 2 transcripts of 4.0 and 1.4 kb originally described by Girard et al 37 The expression of TFPI was analyzed at different time points during incubation of ECs with UFH or LMWH for 24 hours. After densitometric scanning of the gels, the ratio between the band intensity for each TFPI transcript and S26 was calculated and represented as a percentage of the appropriate value obtained for control cells at the same time point.…”

Section: Expression Of Tfpi Messagementioning

confidence: 99%

Cellular Effects of Heparin on the Production and Release of Tissue Factor Pathway Inhibitor in Human Endothelial Cells in Culture

Lupu

Poulsen

Roquefeuil

et al. 1999

ATVB

112

113

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Abstract-Tissue factor pathway inhibitor (TFPI), the major downregulator of procoagulant activity of the tissue factor-factor VIIa complex (TF ⅐ FVIIa), is synthesized and constitutively secreted by endothelial cells (ECs). Here we describe the in vitro effects of heparin on the cellular localization, gene expression, and release of TFPI in human ECs in culture. Both unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH; Fragmin) time-dependently induced a significant enhanced secretion of TFPI, paralleled by a redistribution and increase of TFPI on the cell surface and a decrease of intracellular TFPI. Immunogold electron microscopy showed the presence of clusters of TFPI, both on the plasmalemma proper and within cell-surface opened caveolae/enlarged caveolar profiles. Activation of FX by TF ⅐ FVIIa on ECs treated with endotoxin was inhibited by both heparins but to a higher extent by LMWH. Inhibition of protein synthesis by cycloheximide did not reduce the release of TFPI induced by heparin. Long-term incubation (48 hours) resulted in a time-dependent enhanced production of TFPI. After the first 4 to 8 hours, depletion of intracellular TFPI was observed, more significantly with UFH. Northern blot analysis of TFPI mRNA also showed a decrease of the 1.4-kb transcript after 4 hours of incubation with UFH, followed by recovery and an increase over the control level after 24 hours. Incubation of ECs with phorbol ester (PMA) significantly enhanced the secretion of TFPI and increased its activity on the cell surface, probably by preventing invagination of caveolae. Heparin-stimulated release of TFPI decreased significantly in the presence of PMA to a level that was 2.4 times lower than the expected additive value for PMA and UFH separately. Pretreatment of ECs with PMA suppressed a subsequent response to heparin. Altogether, our results suggest that the heparin-induced release of TFPI might involve a more specific mechanism(s) than the previously hypothesized simple displacement of TFPI from the cell surface glycocalyx. We assume that the increased secretion and redistribution of cellular TFPI induced by heparins in ECs in culture can play an important role in the modulation of the anticoagulant properties of the endothelium. Key Words: tissue factor pathway inhibitor Ⅲ human endothelial cells Ⅲ unfractionated heparin Ⅲ low-molecular-weight heparin Ⅲ caveolae E ndothelial cells (ECs) play a central role in the regulation of hemostasis by ensuring the cellular control of both procoagulant and anticoagulant mechanisms. The anticoagulant and profibrinolytic functions predominate in the quiescent state of the endothelium, thus maintaining blood fluidity (for a review, see References 1 and 2). Blood coagulation is initiated when factor VII/VIIa (FVII/VIIa) in plasma gains access to tissue factor (TF) at sites of blood vessel injury, and the resulting TF ⅐ FVIIa complex activates FX to Xa and FIX to IXa, leading to thrombin generation and the formation of a fibrin clot. 3 Although healthy ECs do not express ...

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“…Alteplase (tPA) was purchased from Genentech, and FXIa was obtained from Enzyme Research Laboratories. Plasmin, pKLK, immunodepleted FV-deficient plasma, mouse monoclonal anti-TFPI-1 N-terminal antibody, anti-human FV-light chain monoclonal antibody resin (AHV-5101-Seph), and sheep antihuman FV polyclonal antibody were obtained from Hemato- (59). SK9 monoclonal antibody was coupled to CNBr-activated Sepharose at 1 mg/ml settled resin using the protocol outlined by GE Healthcare.…”

Section: Methodsmentioning

confidence: 99%

“…The secondary antibody used was HRP-conjugated rabbit anti-mouse IgG (left) or donkey anti-rabbit IgG (right). The protocol used was as outlined by the manufacturer for the mouse monoclonal antibody, and by Broze and co-workers (47,59) for the rabbit polyclonal antibody. The blots were developed with the SuperSignal Femto chemiluminescent substrate for 5 min and scanned using Alpha Innotech FluorChem FC2 digital imaging system.…”

Section: Expression Of Cd41 and Tfpi-2 In Meg-01 Cells Using Rt-pcr Amentioning

confidence: 99%

Platelets Contain Tissue Factor Pathway Inhibitor-2 Derived from Megakaryocytes and Inhibits Fibrinolysis

Vadivel

Ponnuraj

Kumar

et al. 2014

Journal of Biological Chemistry

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Background: TFPI-2 inhibits plasma kallikrein, FXIa, and plasmin, but its concentration in normal plasma is insufficient to inhibit clotting or fibrinolysis. Results: Platelets contain TFPI-2 derived from megakaryocytes and binds to platelet FV/Va and circulating FV in late pregnancy when plasma TFPI-2 is ϳ7 nM. Conclusion: Platelet-derived TFPI-2 regulates intrinsic coagulation and tPA-induced fibrinolysis. Significance: Platelet-derived TFPI-2 promotes clot stabilization while attenuating intrinsic clotting.

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Identification of the 1.4 KB and 4.0 KB messages for the lipoprotein associated coagulation inhibitor and expression of the encoded protein

Cited by 44 publications

References 25 publications

Cultured normal human hepatocytes do not synthesize lipoprotein-associated coagulation inhibitor: evidence that endothelium is the principal site of its synthesis.

Cultured normal human hepatocytes do not synthesize lipoprotein-associated coagulation inhibitor: evidence that endothelium is the principal site of its synthesis.

Cellular Effects of Heparin on the Production and Release of Tissue Factor Pathway Inhibitor in Human Endothelial Cells in Culture

Platelets Contain Tissue Factor Pathway Inhibitor-2 Derived from Megakaryocytes and Inhibits Fibrinolysis

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