Yuichi Kamikubo

The Low Density Lipoprotein Receptor-related Protein Is a Motogenic Receptor for Plasminogen Activator Inhibitor-1

Although plasminogen activator inhibitor-1 (PAI-1) is known to stimulate cell migration, little is known about underlying mechanisms. We show that both active and inactive (e.g. cleaved) PAI-1 can activate the Jak/Stat signaling system and stimulate cell migration in chemotaxis, haptotaxis, chemokinesis, and wound healing assays. Moreover, antibodies to the LDL receptor-related protein (LRP) and an LRP antagonist (RAP) blocked these motogenic effects of PAI-1, while a PAI-1 mutant that did not bind to LRP failed to activate the Jak/Stat signaling pathway or to stimulate cell migration. PAI-1 had no chemotactic effect on LRP-deficient cells. These results indicate that LRP is a signaling molecule, that it mediates the migration-promoting activity of PAI-1, and that this activity does not require intact, biologically active PAI-1. Activation of this LRP-dependent signaling pathway by PAI-1 may begin to explain how the inhibitor stimulates cell migration in a variety of normal and pathological processes.

Human recombinant tissue-factor pathway inhibitor prevents the proliferation of cultured human neonatal aortic smooth muscle cells

Tissue‐factor pathway inhibitor (TFPI) inhibits the procoagulant activity of the tissue‐factor/factor VIIa complex. It was recently reported that TFPI prevented restenosis following tissue injury in a rabbit atherosclerotic model. In order to clarify the mechanism behind this successful prevention of restenosis, we investigated the direct effect of human recombinant TFPI (h‐rTFPI) on the proliferation of cultured human neonatal aortic smooth muscle cells (hSMC). We found that h‐rTFPI exhibits inhibitory activity toward hSMC proliferation, while h‐rTFPI‐C which lacks the carboxyl (C)‐terminal region does not. Furthermore, we found that h‐rTFPI binds to hSMCs with K d=526 nM but that this binding is inhibited by the addition of the synthetic C‐terminal peptide, Lys254–Met276, to h‐rTFPI. Thus, the interaction of h‐rTFPI with hSMCs mediated via the C‐terminal region is responsible for the anti‐proliferative action of h‐rTFPI. On the basis of these results, we presume that the anti‐proliferative effect of h‐rTFPI in addition to its anticoagulant function plays a significant role in preventing restenosis following tissue injury.

Human recombinant tissue factor pathway inhibitor induces apoptosis in cultured human endothelial cells

Tissue factor pathway inhibitor (TFPI) is mainly synthesized in vascular endothelial cells and exhibits a strong and specific inhibitory activity against tissue factor‐mediated blood coagulation. In the present study, we demonstrate that human recombinant TFPI (h‐rTFPI) inhibits the growth of cultured human umbilical vein endothelial cells (HUVECs) by inducing apoptosis. In a growth‐rate assay of HUVECs, the growth of the cultured HUVECs is completely abolished by the addition of 1 μM h‐rTFPI to the culture medium containing fetal bovine serum (FBS), basic fibroblast growth factor, and epidermal growth factor. In addition, h‐rTFPI and h‐rTFPI‐C which lacks the carboxyl‐terminal basic region prevent the survival of growth‐arrested HUVECs which are starved in a medium containing 2% FBS alone, suggesting that h‐rTFPI directly induces the death of these HUVECs. This hypothesis is supported by the finding that h‐rTFPI does not inhibit the synthesis of DNA in HUVECs during proliferation, as shown by a 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation assay. Furthermore, Giemsa staining and a gel electrophoretic analysis of DNA fragmentation show that the HUVEC death mediated by h‐rTFPI has the typical characteristics of apoptosis. However, the apoptosis in HUVECs is considerably inhibited in the presence of 1 μg/ml of the protein synthesis inhibitor, cycloheximide. Therefore, the process of apoptosis triggered by h‐rTFPI is, at least in part, actively conducted by the cells.

Intravascular Free Tissue Factor Pathway Inhibitor Is Inversely Correlated With HDL Cholesterol and Postheparin Lipoprotein Lipase but Proportional to Apolipoprotein A-II

To elucidate the distribution and clinical implications of tissue factor pathway inhibitor (TFPI) concentrations, we measured TFPI levels consisting of preheparin free, lipoprotein-bound (Lp-bound), and endothelial cell-anchor pools in 156 patients with coronary artery disease (average age, 61.2+/-9.1 years; range, 32 to 78 years) by heparin infusion (50 IU/kg) and compared them with the preheparin TFPI levels of 229 healthy subjects (average age, 59. 6+/-9.4 years; range, 41 to 80 years). The patients had lower preheparin free TFPI and lower HDL cholesterol (HDL-C) levels than the healthy subjects with equivalent Lp-bound forms (free TFPI, 15. 9+/-6.5 versus 19.2+/-8.1 ng/mL). In a partial correlation analysis, the preheparin free TFPI levels were shown to be inversely correlated with the HDL-C concentrations in both the patients (r=-0. 454, P<0.001) and the healthy subjects (r=-0.136, P<0.05). As determined by comparison of preheparin and postheparin plasma, the patients generally showed preheparin free TFPI <10%, Lp-bound TFPI at 30%, and endothelial cell-anchor TFPI at 60%. When the patients were divided into 4 categories by their LDL cholesterol (LDL-C, 130 mg/dL) and HDL-C (40 mg/dL) levels to specify their coronary risks, the low-HDL-C groups had significantly increased preheparin and postheparin free TFPI levels and decreased postheparin LPL levels, whereas the high-LDL-C groups showed increased levels of Lp-bound TFPI. In a partial correlation analysis, we found a proportional relation between postheparin free TFPI and apolipoprotein A-II (r=0. 5327) and between HDL-C and LPL (r=0.4906), whereas postheparin free TFPI was inversely correlated with HDL-C (r=-0.4280) and postheparin LPL (r=-0.4791). The inverse relationship between TFPI and LPL suggests that increased free TFPI concentrations as a compensatory response of the endothelium to prevent atherothrombotic processes compete with and displace LPL on endothelial surface, resulting in reduced LPL and low HDL-C.

Overexpression of tissue factor pathway inhibitor in aortic smooth muscle cells inhibits cell migration induced by tissue factor/factor VIIa complex.

BRIEF COMMUNICATION Overexpression of Tissue Factor Pathway Inhibitor in Aortic Smooth Muscle Cells Inhibits Cell Migration Induced by Tissue Factor/Factor VIIa Complex Yuichiro Sato1, Hiroaki Kataoka2, Yujiro Asada1, Kousuke Marutsuka1, Yu-ichi Kamikubo3, Masashi Koono2 and Akinobu Sumiyoshi1 1First and 2Second Department of Pathology, Miyazaki Medical College, Miyazaki, Japan; 3The Chemo-Therapeutic Research Institute, Kumamoto, Japan.

Vitronectin inhibits plasminogen activator inhibitor-1-induced signalling and chemotaxis by blocking plasminogen activator inhibitor-1 binding to the low-density lipoprotein receptor-related protein.

We have previously reported that the serpin plasminogen activator inhibitor-1 activates the Janus kinase (Jak)/signal transducer and activator of transcription (Stat) signalling pathway and stimulates cell migration by binding to the low-density lipoprotein receptor-related protein. All the free forms (cleaved, latent or active) of this inhibitor were shown to be motogenic. However, the plasminogen activator inhibitor-1 can also interact with vitronectin which acts as a cofactor by increasing the half-life of the active form of the serpin. Since vitronectin influences most of the biological functions of the plasminogen activator inhibitor-1, we explored the effects of vitronectin on signalling and cell migration induced by this serpin. We found that the interaction between vitronectin and the plasminogen activator inhibitor-1 suppressed signalling and cell migration. In fact, a purified vitronectin(1-97)/plasminogen activator inhibitor-1 complex was not chemotactic. Vitronectin interaction with the plasminogen activator inhibitor-1 blocks the binding of this serpin to its motogenic receptor, the low-density lipoprotein receptor-related protein. Consequently, vitronectin inhibits the activation of the Janus kinase/signal transducer and activator of transcription signalling pathway by the plasminogen activator inhibitor-1 and subsequent cell migration. In conclusion, we have unveiled a new inhibitory role of vitronectin, which turns off the intracellular signalling and migration-promoting activity of the plasminogen activator inhibitor-1. Thus, the motogenic (cleaved, latent or active) and non-motogenic (in complex with vitronectin) forms of the plasminogen activator inhibitor-1 have different properties that may explain the rather contrasting physiological and pathological roles of this serpin.

Contribution of leptin receptor N-linked glycans to leptin binding.

The extracellular domain of the human leptin receptor (Ob-R) contains 20 potential N-glycosylation sites whose role in leptin binding remains to be elucidated. We found that a mammalian cell-expressed sOb-R (soluble Ob-R) fragment (residues 22-839 of the extracellular domain) bound leptin with a dissociation constant of 1.8 nM. This binding was inhibited by Con A (concanavalin A) or wheatgerm agglutinin. Treatment of sOb-R with peptide N-glycosidase F reduced leptin binding by approximately 80% concurrently with N-linked glycan removal. The human megakaryoblastic cell line, MEG-01, expresses two forms of the Ob-R, of approx. 170 and 130 kDa molecular mass. Endo H (endoglycosidase H) treatment and cell culture with alpha-glucosidase inhibitors demonstrated that N-linked glycans are of the complex mature type in the 170 kDa form and of the high-mannose type in the 130 kDa form. Both isoforms bound leptin, but not after peptide N-glycosidase F treatment. An insect-cell-expressed sOb-R fragment, consisting of the Ig (immunoglobulin), CRH2 (second cytokine receptor homology) and FNIII (fibronectin type III) domains, bound leptin with affinity similar to that of the entire extracellular domain, but this function was abolished after N-linked glycan removal. The same treatment had no effect on the leptin-binding activity of the isolated CRH2 domain. Our findings show that N-linked glycans within Ig and/or FNIII domains regulate Ob-R function, but are not involved in essential interactions with the ligand.

Anti-human factor IX monoclonal antibodies specific for calcium ion-induced conformations

Four monoclonal antibodies have been produced, which are specific for the Ca2+ or Sr2(+)-induced conformation of human factor IX. Certain, but not all, gamma-carboxy-glutamic acid residues in factor IX are involved in the epitope expression together with the conformation stabilized by the adjacent region of Gla-domain and a disulfide bridge. All the antibodies interfered with the binding of factor IX to phospholipids and inhibited the procoagulant activity of factors IX and IXa beta.

Structural Requirements of Human Tissue Factor Pathway Inhibitor (TFPI) and Heparin for TFPI-Heparin Interaction

Heparin affinity chromatography of synthetic peptide fragments mimicking tissue factor pathway inhibitor (TFPI) indicated that the minimal heparin binding sequence consists of 12 amino acid residues located at the C-terminal tail. Within this minimal sequence, Arg-257 and Arg-259 appeared to contribute most significantly to interaction with heparin. Affinity chromatography of TFPI using immobilized heparin derivatives regiospecifically desulfated at O-6 of the glucosamine residue, N-2 of the glucosamine residue, and/or O-2 of the iduronic acid residue indicated that all the sulfate groups in heparin appeared to be required for TFPI-heparin interaction. Among them, however, the 6-O-sulfate groups appeared to make the largest contribution to the interaction, while the 2-O-sulfate groups contributed the least. In vitro experiments on the inhibition of factor Xa by TFPI enhanced with native and chemically modified heparins afforded similar results.

THE CLEARANCE OF PROTEOGLYCAN-ASSOCIATED HUMAN RECOMBINANT TISSUE FACTOR PATHWAY INHIBITOR (H-RTFPI) IN RABBITS: A COMPLEX FORMATION OF H-RTFPI WITH FACTOR XA PROMOTES A CLEARANCE RATE OF H-RTFPI

The very rapid clearance of human recombinant tissue factor pathway inhibitor (h-rTFPI) may result from its binding to vascular proteogly can and LDL receptor-related protein (LRP). To investigate the effect of factor Xa on the clearance of h-rTFPI, we developed a specific ELISA for h-rTFPI/factor Xa complex, and compared the pharmacokinetic parameters of h-rTFPI/factor Xa complex and the clearance rate of the cellular proteogly can-associated h-rTFPI/factor Xa complex with those of h-rTFPI by itself in rabbits. We found that the h-rTFPI/factor Xa complex disappeared from circulation at a rapid rate of clearance, having pharmacokinetic parameters similar to those of non-complexed h-rTFPI. After the rapid disappearance of the h-rTFPI complex from plasma, an intravenous injection of heparin resulted in a release of h-rTFPI/factor Xa complex into plasma. However, the recovery of heparin-releasable h-rTFPI/factor Xa decreased significantly in a time-dependent manner. Therefore, we examined the half-life of proteogly can-associated h-rTFPI/factor Xa and determined it to be 51 min, which was significantly shorter than that of h-rTFPI by itself (107 min). These results suggest that a complex formation of h-rTFPI with factor Xa promotes a clearance of proteogly can-associated h-rTFPI existing in the liver and kidney.