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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online October 29, 2001 as doi:10.1096/fj.01-0486fje. |
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Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, The Biomedical Center, 751 23 Uppsala, Sweden
2Correspondence: Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, The Biomedical Center, Box 575, 751 23 Uppsala, Sweden. E-mail: Gunnar.Pejler{at}vmk.slu.se
SPECIFIC AIMS
We have recently characterized a heparin-deficient mouse strain generated by targeting the gene for N-deacetylase/N-sulfotransferase-2 (NDST-2) and demonstrated that the heparin-deficient mice displayed severe defects in the connective tissue-type mast cell (MC) secretory granules, including an almost complete absence of the various heparin binding MC proteases (chymases, tryptases, and carboxypeptidase A). In the present report, we studied the consequences of heparin/MC protease deficiency for extravascular coagulation and fibrinolysis.
PRINCIPAL FINDINGS
1. Thrombin is regulated by MC chymase in a heparin-dependent manner
Peritoneal cells, a mixture of macrophages, lymphocytes, and connective tissue-type MCs, were prepared from both wild-type and NDST-,2-/- mice and cultured under serum-free conditions. Addition of prothrombin to the peritoneal cells resulted in rapid formation of thrombin (Fig. 1
A). Thrombin accumulation was considerably faster in the NDST-2-/- than in wild-type cells. The thrombin generated was subsequently inactivated in the NDST-2+/+ but not in the NDST-2-/- cells (Fig. 1A
). When thrombin was added to the cells, rapid inactivation of the protease was seen in the wild-type cells whereas thrombin inactivation was virtually absent in the heparin-deficient cells. Thrombin inactivation was inhibited by 
1-antichymotrypsin, indicating that MC chymase is primarily responsible for the regulation of thrombin (Fig. 1A
, 1B
). MC chymase is known to occur in vivo in a tight complex with heparin proteoglycan (PG). To study the possible role of the chymase-associated heparin PG in the inactivation of thrombin, we added a polycationic heparin antagonist (protamine) to the cells and studied its effect on thrombin inactivation in the wild-type cells. Thrombin inactivation was completely inhibited by protamine, indicating that chymase is dependent on heparin PG for inactivation of thrombin (Fig. 1A
, 1B
). Protamine did not inhibit the catalytic activity of chymase toward small peptide substrates, indicating that protamine only blocked the heparin moiety of the chymase:heparin PG complex without affecting the actual active site of chymase. The processing of prothrombin in the peritoneal cell cultures was studied further by Western blot analysis using an anti-prothrombin antiserum. Prothrombin was converted to thrombin at equal rates in both wild-type and NDST-2-/- cells (Fig. 1C
). In NDST-2+/+ cell cultures, the band corresponding to generated thrombin was reduced over time, consistent with degradation into fragments that were not detected by the anti-prothrombin antibody. In contrast, the thrombin band was accumulated in samples from NDST-2-/- cell cultures, in agreement with reduced rate of chymase-catalyzed inactivation.
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2. Heparin-dependent chymase regulates plasmin
Addition of plasminogen to peritoneal cells from wild-type and heparin-deficient NDST-2-/- mice resulted in formation of active plasmin. However, higher levels of plasmin activity were observed in the NDST-2-deficient cells than in the normal controls (Fig. 2
A). Plasminogen activation was expressed by the adherent peritoneal cell population and was inhibited by antibodies toward urokinase-type plasminogen activator (uPA) and by amiloride, indicating that plasminogen activation was catalyzed by uPA expressed by macrophages. The generated plasmin was subsequently inactivated in the wild-type but not in the NDST-2-/- cell cultures (Fig. 2A
). When plasmin instead of plasminogen was added to the cells, we observed that plasmin was inactivated in the wild-type cells but not in the heparin-deficient counterparts. Moreover, the inactivation of plasmin was inhibited by protamine an 1-antichymotrypsin (Fig. 2B
). When MCs were activated by either calcium ionophore or anti-IgE, inactivation of plasmin was accelerated. Plasmin inactivation was MC dependent. Taken together, these findings indicate that, similar to thrombin, plasmin is inactivated through the action of MC chymase and that this inactivation process is dependent on heparin PG. Plasminogen processing was also studied by Western blot analysis using an anti-plasminogen antiserum (Fig. 2B
). Plasminogen was converted to lower molecular weight compounds in NDST-2+/+ and NDST-2-/- cell cultures. However, whereas these compounds appeared to be further degraded in the NDST-2+/+ cells, accumulation of 30–50 kDa bands was observed in NDST-2-/- cell cultures. These results are thus consistent with equal rates of plasminogen activation in both NDST-2+/+ and NDST-2-/- cells, but with further degradation into inactive fragments occurring mainly in NDST-2+/+ cultures.
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CONCLUSIONS
MCs are key effector cells in various types of inflammatory reactions, most notably allergic reactions, but have also been implicated in several other pathophysiological conditions such as rheumatoid arthritis, tumor metastasis, and wound healing. When MCs are activated, they release the contents of their secretory granules, including histamine, heparin PG, cytokines, and various heparin binding proteases: chymases, tryptases, and carboxypeptidase A. In a previous study, we examined the biological function of heparin by targeting the gene for NDST-2, a key enzyme in heparin biosynthesis. We found that inactivation of NDST-2 affected the MCs severely, with both altered morphology and an essentially complete absence of the various heparin binding MC proteases at the protein, but not mRNA, level.
The biological function of the mast cell proteases is not known at this time. Many substrates for the various mast cell proteases have been identified, but it is uncertain which, if any, of these substrates are also substrates in vivo. In the present study, we have addressed possible functional consequences of the lack of heparin/mast cell proteases, one of the principal goals being to identify potential physiological substrates for the mast cell proteases.
We demonstrate that inactivation of NDST-2, and thus of the heparin/MC protease system, may affect the regulation of coagulation at extravascular sites, as exemplified by the peritoneal cavity. Previous studies have shown that extravascular activation of the coagulation system, accompanied by extravascular fibrin deposition, is a consistent feature of various inflammatory reactions. Macrophages are thought to be the main cell type responsible for initiation of coagulation under such circumstances by expressing a variety of coagulation factors, including prothrombinase (Fig. 3
). In this report, we show that MC chymase:heparin PG complexes may modulate such extravascular coagulation at the level of inactivating thrombin by cleaving this protease into inactive fragments (Fig. 3)
. Thrombin inactivation was undetectable in cells from NDST-2-/- mice, indicating that the MC chymase:heparin complexes represent the major regulatory system for thrombin.
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Dissolution of extravascular fibrin deposits requires plasmin, which may be formed by action of the macrophage uPA (Fig. 3)
. Plasminogen may be available due to extravasation of plasma components during the inflammatory response. This study shows that MC chymase may also be potentially involved in regulation of plasmin levels by degrading it in a heparin-dependent manner (Fig. 3)
. Our results show that plasmin inactivation was close to undetectable after inactivation of the NDST-2 gene, indicating that the MC chymase:heparin PG system provides the main mechanism for control of formed plasmin in this cellular system. Regulation of plasmin by MC chymase could be relevant to several pathophysiological conditions such as tumor metastasis and wound healing, where the plasmin/plasminogen activator system is known to be involved. It should be noted that MCs show a strong tendency to accumulate at sites of ongoing tumor metastasis and wound healing.
The chymase-catalyzed degradation of thrombin and plasmin was strongly dependent on heparin. Our results suggest a mechanism where the heparin part of the chymase:heparin complexes attracts heparin binding proteins (both thrombin and plasmin are heparin binding), thus presenting them to chymase and resulting in accelerated proteolysis. Based on these findings, we propose a general model for mast cell chymase action (Fig. 3)
.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0486fje; to cite this article, use FASEB J. (October 29, 2001) 10.1096/fj.01-0486fje 
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) or NDST-2-/- mice (•). Protamine (1 µg; 
) or 
) were added to cell cultures from NDST-2+/+ mice 30 min before the addition of prothrombin. B) Thrombin (1 µg) was added to peritoneal cells from NDST-2+/+ (
