Interaction between Histidine-Rich Glycoprotein and Platelet Factor 4 with Dermatan Sulfate and Low-Molecular-Weight Dermatan Sulfate

Giuseppe Cella, M.D. Giuseppe Boeri, Ph.D. Graziella Saggiorato, Ph.D. Rossella Paolini, M.D. Guido Luzzatto, M.D. and

Vittorio Ilario Terribile, M.D.

PADUA, ITALY

Abstract There is a controversy about whether or not histidine-rich glycoprotein (HRG), the most abundant plasma protein with glycosaminoglycans-neutralizing capacity, is able to prevent the inhibition of human thrombin by heparin cofactor II (HC II) in the presence of dermatan sulfate (DS). The authors studied the interaction of DS and low molecular weight DS, in a purified system with HRG, platelet factor 4 (PF 4), and with HC II. Their results show that HRG, like PF 4, has an affinity, not only for heparin, but also for DS. However, this affinity seems very weak. In fact, HRG is 10 times less effective than PF 4 in neutralizing the 50% antithrombin activity of HC II in the presence of DS.

Introduction Dermatan Sulfate (DS), located mainly in the subendothelium, plays an important role maintaining the structural integrity of the tissue.’ It is able to activate heparin cofactor II (HC II), a second heparin-dependent inhibitor of thrombin . Among proteins with glycosaminoglycans (GAGs)-binding and GAGs-neutralizing capacity, it is still unsettled in

From the Institute of Medical Semeiotics, Second Chair of Medicine, University of Padua, Medical School, Padua, This study was supported in part by grants 60% (M.U.R.S.T., Rome) and from the Veneto Region, Venice, Italy

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60 whether or not histidine-rich glycoprotein (HRG) is able to neutralize DS. In fact, it has been shown by Tollefsen and Pestka3 that between HRG and platelet factor 4 (PF 4)another protein with GAGs-neutralizing activity-only the latter is able to prevent the inhibition of human thrombin by heparin cofactor II (HC II) in the presence of dermatan sulfate. In contrast, Niwa at al4 found that HRG inhibited the antithrombin activity of HC II also in the presence of dermatan sulfate. The aim of our study was to see whether or not HRG is able to prevent the inhibitive of human thrombin by HC II in the presence of DS.

Materials and Methods We studied the interaction of dermatan sulfate (DS) and low molecular weight dermatan sulfate (LMWDS), in a purified system, with PF 4 or HRG and with HC II. Highly purified PF 4 was obtained as described by us’; purified HRG, DS, and LMWDS were purchased. LMWDS had a declared molecular weight average of 8,700 daltons. The inhibition of antithrombin activity of HC II by PF 4 or HRG was evaluated

by a commercially available chromogenic substrate method to measure dermatan sulfate at submicrogram concentration. Since HRG-neutralizing properties are divalent-cation dependent. HRG was dissolved in the presence of CaC 12 (0.02 M, final concentration). Briefly: dermatan sulfate (1 ~g), HC II (0.052 PEU) and PF 4 or HRG (different concentrations) were allowed to react for 120 seconds. Human thrombin (1.2 NIH) was then added, and sixty seconds later its residual activity was measured by chromogenic substrate CBS 34.47. The calibration curves were prepared with both DS and LMWDS. Results Our results (Fig. 1 panel A) seem to support the findings of Niwa et a14 indicating that HRG, like PF 4, has an affinity, not only for heparin, but also for dermatan sulfate. This affinity seems, however, to be very weak. In fact, HRG is 10 times less effective than PF 4 in neutralizing the 50070 antithrombin activity of HC II in the presence of DS. In contrast with this observation and with the fact that by decreasing the chain length of heparin there is a progressive resistance to neutralization,’ LMWDS seems to be more neutralizable by HRG (Fig. 1, panel B). Discussion Dermatan sulfate, located mainly in the subendothelium, plays an important role in maintaining the structural integrity of the tissue.’ It has been suggested that the normal role of HC II is not to inhibit intravascular thrombin . However, under conditions of endothelial cell disruption that lead to contact between subendothelial structures and plasma, thrombin formation may be inhibited by HC II in the presence of dermatan sulfate.3 Although HRG does not interfere with interaction between antithrombin III and heparin-like compounds on vascular endothelial celIS7 that, together with PF 4, penetrate extravascular space,8 it could play a role in the modulation of the antithrombotic activity of dermatan sulfate in vivo.

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FIG. 1. Inhibitory effect of platelet factor 4 (PF 4) and histidine-rich

glycoprotein (HRG) on heparin cofactor II activity in the presence of dermatan sulfate (A) and low molecular weight dermatan sulfate (B).

Conclusion It seems likely that HRG of other proteins with high

inhibit DS although its affinity for GAGs.

can

activity is less powerful than that

Acknowledgmentt We thank Mediolanum Farmaceutici for the generous (MF 701) and low molecular weight dermatan sulfate.

gift

of dermatan sulfate

Giuseppe Cella, M.D. Istituto di Semeiotica Medica Seconda Cattedra di Patologia Speciale Medica Università degli Studi di Padova Via Ospedale Civile 105 35100 Padova, Italy

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References and their binding to biological macromolecules. Ann Rev Biochem 47:385-417, 1978. 2. Tollefsen DM, Maimone MM, McGuire EA, et al: Heparin Cofactor II activation by dermatan sulfate. Ann NY Acad Sci 556:116-122, 1989. 3. Tollefsen DM, Pestka CA: Modulation of heparin cofactor II activity by histidine-rich glycoprotein and platelet factor 4. J Clin Invest 75:496-501, 1985. 4. Niwa M, Yamagishi R, Kondo S-I, et al: Histidinerich glycoprotein inhibits the antithrombin activity of heparin cofactor II in the presence of heparin or dermatan sulfate. Thromb Res 37:237-240, 1985. 1. Lindal U, Hook M:

Glycosaminoglycans

et al: Improved purification of human platelet factor 4 by affinity and ion-exchange chromatography.

5. Medici

I, Di Martino A, Cella G,

method for

Thromb Res 54: 277-287, 1989. 6. Lane DA, Pejler G, Flynn AM, et al: Neutralization of heparin-related saccharides by histidine-rich glycoprotein and platelet factor 4. J Biol Chem 261:3980-3986, 1986. 7. Shimada K, Kawamoto A, Matsubayashi K, et al: Histidine-rich glycoprotein does not interfere with interactions between antithrombin III and heparin-like compounds on vascular endothelial cells. Blood 73:191-193, 1989. 8. Adelman B, Stemerman MB, Mennell D, et al: The interaction of platelet with aortic subendothelium: Inhibition of adhesion and secretion by prostaglandin I . Blood 58:198-205, 1981. 2

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Interaction between histidine-rich glycoprotein and platelet factor 4 with dermatan sulfate and low-molecular-weight dermatan sulfate.

There is a controversy about whether or not histidine-rich glycoprotein (HRG), the most abundant plasma protein with glycosaminoglycans-neutralizing c...
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