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Related Collections Hemostasis, Thrombosis, and Vascular Biology Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, Vol. 91 No. 11 (June 1), 1998: pp. 4173-4179 Plasmin Can Reduce the Function of Human 2 Glycoprotein I by Cleaving Domain V Into a Nicked Form By Naoki Ohkura, Yoshihisa Hagihara, Tetsuro Yoshimura, Yuji Goto, and Hisao Kato From the Graduate School of Science, Osaka University, Toyonaka, Osaka; Faculty of Engineering, Mie University, Tsu; and National Cardiovascular Center Research Institute, Suita, Osaka, Japan.     ABSTRACT Abstract Introduction Methods Results Discussion References 2-Glycoprotein I (2GPI) is a highly glycosylated plasma protein with the ability to bind negatively charged substances such as DNA, heparin, dextran sulfate, and negatively charged phospholipids. The most relevant physiological role of 2GPI is supposed to be the regulation of the function of anionic phospholipids like cardiolipin (CL). 2GPI consists of a single polypeptide chain (326 amino acid residues) with a molecular mass of about 50 kD and with five tandem repeated domains (I, II, III, IV, and V). In the previous study, we found that factor Xa can produce the nicked form by cleaving Lys 317-Thr 318, using recombinant human domain V (r-Domain V). However, the reaction was extremely slow. In the present paper, we found that plasmin can produce the nicked form of domain V, using recombinant domain V (r-Domain V) and 2GPI from human plasma. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, r-Domain V was rapidly cleaved into a nicked form by plasmin, very slowly by factor Xa, but not by thrombin, tissue-type plasminogen activator, urokinase, and tissue factor/factor VIIa. The cleavage site of r-Domain V and 2GPI by plasmin was proved to be Lys 317-Thr 318 by amino acid sequence analysis of the digest and of the C-terminal peptide isolated by high-performance liquid chromatography. The cleavage was completely inhibited by plasmin inhibitor (2PI). The nicked form was demonstrated to show reduced affinity for CL with a dissociation constant of one order of magnitude larger than that of the intact 2GPI. To determine whether the specific cleavage of 2GPI by plasmin can occur also in plasma, human plasma was first acid-treated to inactivate 2PI and then incubated with urokinase. About 12% of 2GPI in plasma was nicked when 2PI activity decreased to 80%. The nicked form was not generated in plasminogen-depleted plasma. These results suggest that plasmin can produce the nicked form of 2GPI with the reduced ability to bind phospholipids in vivo.     INTRODUCTION Abstract Introduction Methods Results Discussion References -GLYCOPROTEIN I (2GPI) is a highly glycosylated 2plasma protein with the ability to bind negatively charged substances such as DNA, heparin, dextran sulfate, and negatively charged phospholipids.1-4 The most relevant physiological role of 2GPI is thought to be the binding with the anionic phospholipids, including cardiolipin (CL). Anticardiolipin antibodies or lupus anticoagulants are strongly associated with thrombosis. In these autoimmune diseases with anti-phospholipid antibody syndrome, 2GPI is a cofactor in the recognition of the phospholipid antigen, CL, by anti-CL antibodies.5-7 2GPI consists of a single polypeptide chain (326 amino acid residues) with a molecular mass of about 50 kD and with five tandem repeated domains (I, II, III, IV, and V), which have a common motif named as the complement control protein or Sushi domain superfamily.8-10 We reported that both domain I and domain V of 2GPI are important for the interaction with CL, using bovine 2GPI.11 Particularly, the importance of domain V has been proved by several lines of evidence. Steinkasserer et al12 found that recombinant human domain V (r-domain V) inhibited the interaction of 2GPI with CL. Hunt et al13 reported that the commercial preparation of human 2GPI contained a nicked form in domain V, which displayed a lower interaction with CL. The generation of a nicked form of 2GPI in vivo may regulate the function of 2GPI. However, it is unknown what proteases generate the nicked form by cleaving domain V of 2GPI. Hunt et al13 found two cleavage sites in domain V of commercial human 2GPI, one major site as Lys 317-Thr 318 and another minor site as Ala 314-Phe 315. We expressed human r-domain V with a signal peptide containing factor Xa recognition site and prepared the intact domain V by removing the signal peptide, using factor Xa.14 During the course of that study, we found that a nicked form of domain V was produced by a long incubation with factor Xa by the cleavage of Lys 317-Thr 318 bond and that the nicked form lost the ability to interact with CL by the specific cleavage. Although the study presented clearly the evidence that factor Xa can produce a nicked form of domain V, the reaction was extremely slow. In the present study, to examine the protease responsible for producing the nicked form in vivo, we incubated r-Domain V with various proteases from plasma and found that plasmin produced the nicked form by cleaving Lys 317-Thr 318. Here, we report the evidence that plasmin can produce the nicked form of 2GPI by the incubation of purified 2GPI with plasmin and also by the activation of plasminogen in plasma by urokinase.     MATERIALS AND METHODS Abstract Introduction Methods Results Discussion References Materials.   Human proteases were purchased from the following manufacturers; thrombin from Protogen AG (Laüfelfingen, Switzerland), plasmin from American Diagnostica (Greenwhich, CT), UK from BioPur AG (Bubendorf, Switzerland), t-PA from Chromogenix (Mölndal, Sweden), 2PI from Biopool AB (Umea, Sweden), and factor Xa from Enzyme Research Laboratories, Inc (South Bend, IN). Platelin (rabbit brain phospholipids) was purchased from Organon Teknika Co (Durham, NC). HiTrap Heparin was purchased from Pharmacia Biotechnology (Uppsala, Sweden). Soluble tissue factor (sTF) was kindly supplied by Dr Toshiyuki Miyata (National Cardiovascular Center Research Institute). Factor VIIa was kindly supplied by Dr Tomohiro Nakagaki (Chemo-Sero-Therapeutic Research Institute, Kumamoto, Japan). CL and PC were purchased from Avanti Polar Lipids (Alabaster, AL). Preparation of human 2 GPI and r-Domain V.   r-Domain V with signal peptide, which consists of extra amino acid sequence of Tyr-Val-Glu-Phe-Met-Ile-Glu-Gly-Arg-Thr with the amino terminal Lys 242 of human 2GPI, was expressed in methylotrophic yeast, Pichia pastoris, and then purified using anion-exchange and reversed-phase chromatographies, as described in the previous paper.14 r-Domain V was obtained by treatment with factor Xa, giving it an extra Thr at the N-terminus. The numbers of amino acid residues of intact 2-GPI were employed for the numbering of the amino acid residues of r-Domain V, neglecting the extra Thr at the N-terminus. Human 2GPI was prepared as described by Polz et al.2 Briefly, fresh human plasma was treated with 1.3% perchloric acid at 4°C for 15 minutes and centrifuged at 5,000g for 10 minutes. The supernatant was neutralized by adding 1/3 vol of 1.0 mol/L Tris-HCl (pH 8.0) and concentrated by Centricon-30 (Amicon Co, Beverly, MA). The concentrate was applied to a column of HiTrap Heparin. After washing the column with 10 mmol/L Tris-HCl (pH 8.0), 2GPI was eluted with a linear gradient of 10 mmol/L Tris-HCl and the same buffer containing 1 mol/L NaCl. Cleavage of human 2GPI and r-Domain V by various proteases.   All enzymatic reactions were performed in 0.1 mol/L Tris-HCl (pH 7.5) containing 20 mmol/L NaCl and 0.3 mmol/L CaCl2 at 37°C. About 1 µg of r-Domain V was reacted with factor Xa, human thrombin, human plasmin, UK, t-PA, and sTF/ VIIa. In the case of sTF/VIIa, the reaction was performed in the presence of 60 µg/mL of platelin. The molar ratios of enzymes to substrates were 1:50. At the specified times, aliquots were removed from the reaction mixture. These samples were reduced by 2-mercaptoethanol and subjected to 10% to 20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).15 Separation of the peptide chains of plasmin-treated r-Domain V was carried out using reversed-phase high-performance liquid chromatograpyy (HPLC). r-Domain V (50 µg) was incubated with 7.2 µg of plasmin, with molar ratio of plasmin to substrate of 1 to 50. The reaction was stopped by adding twice volume of 8 mol/L guanidium hydrochloride at each reaction time. Next, the samples were reduced by the final concentration of 3 mmol/L of dithiothreitol for 3 hours. The reduced samples were pyridylethylated by the final concentration of 10 mmol/L of 4-vinylpyridine for 2 hours. Each reduced-pyridylethylated sample was subjected to 5C18-AR reversed-phase HPLC column (4.6 × 20 mm; Nacalai Tesque, Kyoto, Japan). Peptides were eluted with a linear gradient from 7% to 55% acetonitrile. Separation of the nicked form of 2GPI from the intact protein was performed using HiTrap Heparin column. 2GPI (660 µg) was incubated with 19 µg of plasmin with molar ratio of plasmin to substrate of 1 to 56 in the presence or absence of 5.4 µg/mL of 2PI. The reaction was stopped by adding twice volume of 8 mol/L guanidium hydrochloride at each reaction time. Then, the samples were diluted 10 times with 10 mmol/L Tris-HCl buffer (pH 8.0) and subjected to HiTrap Heparin column (1 mL). Proteins were eluted with a linear concentration gradient formed by the solutions containing 0.1 mol/L and 1 mol/L NaCl with a flow rate of 1.0 mL/min. The peak areas were calculated using a Waters 911J Photodiode array system controller (Millipore, Milford, MA). The N-terminal amino acid sequences of the samples were analyzed by a protein sequencer, model 473A (Applied Biosystem, Foster City, CA). Liposome preparation and binding assay.   Large unilamellar vesicles consisting of CL and PC at molar ratio of 1:1 were prepared, and analysis of the binding affinity for liposome membranes was performed as described previously.11 The dissociation constant (kd) and the number of phospholipids in the binding site (n) were estimated by curve-fitting of the plot of percentage bound versus phospholipid concentration according to the method described previously.11 UK activation of human plasma.   Plasminogen-depleted plasma was prepared by applying 20 mL of normal plasma to a column (1.2 × 5 cm) of Lysine-Sepharose 4B (Pharmacia Biotechnology), which had been equilibrated with 50 mmol/L sodium phosphate buffer, pH 7.5, and by collecting the nonadsorbed fraction. Normal plasma or plasminogen-depleted plasma (3.8 mL) was dialyzed against 2 L of 10 mmol/L sodium acetate buffer, pH 4.0, at 4°C for 12 hours, and then against 5 L of 3 mmol/L Tris-HCl buffer, pH 8.0, containing 50 mmol/L NaCl for 12 hours. The dialysates were mixed with 30 IU of UK (Midori Jyuji, Osaka, Japan) and incubated at 37°C for 12 hours. To examine the effect of 2PI in plasma on the cleavage of 2GPI by plasmin, the acid-treated plasma was mixed with nontreated plasma in a various ratio and the mixtures were incubated with UK. 2PI activity in the mixtures was estimated according to the method described previously.16,17 The intact and nicked forms of 2GPI were separated by the treatment with perchloric acid and by heparin affinity column chromatography as described above.     RESULTS Abstract Introduction Methods Results Discussion References Degradation of r-Domain V and 2GPI by plasmin.   Figure 1A shows a profile of SDS-PAGE of r-Domain V incubated with factor Xa, thrombin, UK, t-PA, and sTF/VIIa with enzyme to substrate mole ratio of 1 to 50, for 8 hours or 24 hours. Lanes 1 and 2 are the intact r-Domain V and nicked r-Domain V prepared as described in the previous report,14 respectively. As reported in the previous paper,14 very slow cleavage of r-Domain V with factor Xa was observed (lanes 6 and 11). Thrombin, t-PA, UK, and sTF/VIIa did not cleave r-Domain V under this condition. Figure 1B shows SDS-PAGE of r-Domain V incubated with plasmin at an enzyme to substrate mole ratio of 1 to 50. After a 30-minute incubation, more than 50% of r-Domain V band was decreased and a new band that corresponded to the nicked r-Domain V was appeared. After 1-hour incubation, the intact r-Domain V was almost disappeared. These results indicate that plasmin can convert r-Domain V into a nicked Domain V very quickly. The cleavage of r-Domain V by plasmin was not significantly affected in the presence of phospholipid and calcium (data not shown). View larger version (35K): [in this window] [in a new window]   Fig 1. (A) SDS-PAGE of r-Domain V incubated with various proteases at 37°C for 8 hours (lanes 3 to 7) and 24 hours (lanes 8 to 12). r-Domain V was incubated with UK (lanes 3 and 8), t-PA (lanes 4 and 9), thrombin (lanes 5 and 10), factor Xa (lanes 6 and 11), and sTF/VIIa (lanes 7 and 12) in 0.1 mol/L Tris-HCl (pH 7.5) containing 20 mmol/L NaCl and 0.3 mmol/L CaCl2. Molar ratios of r-Domain V to proteases were 50:1. (B) SDS-PAGE of r-Domain V incubated with plasmin. r-Domain V was incubated with plasmin with a molar ratio of r-Domain V to plasmin was 50:1 at 37 °C. Lanes 3 to 6 are samples taken at 0.25, 0.5, 1, and 2 hours, respectively. For comparison, intact r-Domain V and nicked r-Domain V as prepared in the previous paper14 are shown in lanes 1 and 2 in (A) and (B), respectively. The positions of molecular-weight markers are shown at the left of the gel. The r-Domain V was incubated with plasmin for 2 hours with enzyme/substrate molar ratio of 1:50; the N-terminal amino acid sequence of the digest was examined (Fig 2[1]), comparing with that of intact r-Domain V (Fig 2[7]). From plasmin-digest of r-Domain V, a new amino acid sequence was found which started from Thr 318, in addition to the N-terminal amino acid sequence of r-Domain V. Figure 3 shows reversed-phase HPLC patterns of r-Domain V and plasmin-digest of Domain V after reduction and pyridylethylation. The peak shown by the arrow indicates the position of a new peptide generated by plasmin digestion. The N-terminal amino acid sequence of the peptide from the plasmin digest was found to be Thr-Asp-Ala-Ser-Asp-Val-Lys-Pro, as shown in Fig 2(2), confirming that this peptide was derived from the C-terminal part of the nicked r-Domain V. These results indicate that plasmin cleaved a peptide bond of Lys 317-Thr 318 in r-Domain V, which was the same cleavage site by factor Xa as reported previously.14 View larger version (30K): [in this window] [in a new window]   Fig 2. Amino acid sequences of plasmin digests of 2GPI and r-Domain V. Each value under the amino acid sequence identified shows the amount of PTH-amino acid (in picomoles) recovered using a gas-phase sequencer. Any other sequence was not observed: (1), plasmin digest of r-Domain V; (2), C-terminal peptide of plasmin-treated r-Domain V (see Fig 3); (3), Peak 1 from plasmin-treated 2GPI (see Fig 4A); (4), peak A of 2GPI from acid-treated and UK-activated plasma (see Fig 7); (5), peak B of 2GPI from acid-treated and UK-activated plasma; (6), intact 2GPI; and (7), intact r-Domain V. View larger version (16K): [in this window] [in a new window]   Fig 3. Reversed-phase HPLC patterns of plasmin-treated r-Domain V. The digest of r-Domain V (1.4 µg) was subjected to reversed-phase HPLC, as described in Materials and Methods. Arrow indicates the peak corresponding to the C-terminal peptide of the digest. 2GPI was then incubated with plasmin at enzyme/substrate mole ratio of 1:56 for each time. When the digests of 2GPI by plasmin were subjected to HiTrap Heparin column chromatography, two distinct peaks were separated (Fig 4A). Peak 1 was found to have a new N-terminal amino acid sequence that started from Thr 318, in addition to the N-terminal amino acid sequence of 2GPI as shown in Fig 2(3), confirming that peak 1 corresponded to the nicked 2GPI. Peak 2 was shown to be the intact 2GPI by the amino acid sequence analysis. View larger version (21K): [in this window] [in a new window]   View larger version (16K): [in this window] [in a new window]   Fig 4. (A) HiTrap Heparin column chromatography patterns of plasmin-treated 2GPI. A total of 660 µg of 2GPI was incubated with 19 µg of plasmin at 37°C, where molar ratio of plasmin to substrate was 1:56. A total of 60 µg of the digest of 2GPI was subjected to HiTrap Heparin column chromatography, as described in Materials and Methods. (B) Time course of the increase of nicked 2GPI (peak 1 in [A]). Horizontal axis and vertical axis correspond to the reaction time (hours) and area of peaks of nicked 2GPI. The experiment was performed in the presence () or absence of 2PI (). Figure 4B shows the increases of the nicked 2GPI after the incubation with plasmin, which was calculated by the area of peak 1 shown in Fig 4A. The half-time of the cleavage reaction of 2GPI was about 30 minutes. The maximum cleavage was obtained about at 2-hour incubation. The slow decrease of the nicked 2GPI after 2-hour incubation indicates that the other peptide bonds in 2GPI were cleaved by plasmin after the longer incubation. In the presence of 2PI, 2GPI was not cleaved at all (closed circles in Fig 4B). Interaction of intact and nicked forms of 2GPI with CL.   Figure 5 shows the effect of NaCl concentration on the bindings of 2GPI and r-Domain V and of their nicked forms to liposomes containing CL. The affinity of nicked forms decreased markedly with the increase of NaCl concentrations, whereas intact proteins retained their affinity as reported previously.11 These results indicate that the affinity of 2GPI for CL was reduced after the cleavage of Lys 317-Thr 318 in domain V by plasmin. Figure 6 shows the bindings of intact and nicked forms of 2GPI with liposomes containing CL as a function of the phospholipid concentration. The dissociation constant (kd) and the number of phospholipids in the binding sites (n) in the interaction of 2GPI with CL were estimated by the curve-fitting procedure as described previously.11 As shown in Table 1, the approximate kd value of the nicked form was one order of magnitude larger than that of the intact 2GPI and n value of the nicked form was three times larger than that of the intact protein. These values of human intact and nicked 2GPI were similar to those of bovine intact and nicked 2GPI, as reported respectively.11 View larger version (22K): [in this window] [in a new window]   Fig 5. Effect of NaCl concentration on the bindings of 2GPI and r-Domain V and of their nicked forms to liposomes containing CL. Solutions containing intact 2GPI (), nicked 2GPI (), intact r-Domain V (), or nicked () and various concentrations of NaCl in 10 mmol/L Tris-HCl, pH 7.5, were mixed with liposomes containing CL and PC. After the incubation at 25°C for 30 minutes, the mixtures were centrifuged at 120,000g for 1 hour. The amount of proteins in the supernatant was measured as described by Hagihara et al.11 The amount of proteins in the absence of liposomes at the indicated NaCl concentration was taken to be 100%. View larger version (16K): [in this window] [in a new window]   Fig 6. Plots of the binding of 2GPI to liposome membranes containing CL as a function of the concentration of CL. One micromolar of intact () or nicked () 2GPI was incubated with CL/PC (1:1)-LUV at 25°C in 10 mmol/L Tris-HCl buffer containing 10 mmol/L NaCl. The amounts of proteins bound to liposomes were measured as described by Hagihara et al.11   View this table: [in this window] [in a new window]   Table 1. Dissociation Constant (kd) and the Number of the Phospholipids in the Binding Site (n) in the Interaction of Intact and Nicked 2GPI With CL Cleavage of 2GPI by plasmin in plasma.   To prove that the same specific cleavage of 2GPI by plasmin can occur also in plasma, acid-treated plasma was incubated with UK, and intact and nicked form of 2GPI were separated using heparin affinity column chromatography, as described in Materials and Methods. As shown in Fig 7(1), a small peak (peak A) and a large peak (peak B) were separated. The amino acid sequence of peak A was found to be the same as the plasmin-digest of 2GPI, as shown in Fig 2. The amino acid sequence of peak B was found to be the same as intact 2GPI (Fig 2). Without the addition of UK to the acid-treated plasma, peak A was not detected (data not shown). When plasminogen-depleted plasma was used, instead of normal plasma, the peak A was also not detected (Fig 7[2]). To examine the effect of 2PI in plasma on the cleavage of 2GPI, the acid treated plasma was mixed with normal plasma in a various ratio and treated with UK. The acid-treated plasma retained 22.9% of 2PI activity. As shown in Fig 8, a nicked form of 2GPI was generated with the decrease of 2PI activity in plasma. On the plasma with 80% of 2PI activity of normal plasma, about 12% of 2GPI was detected as nicked 2GPI after 16 hours incubation with UK. These results indicate that a nicked domain V of 2GPI could be generated in plasma by the action of plasmin after activation of plasminogen, particularly when 2PI activity in plasma decreased. View larger version (15K): [in this window] [in a new window]   Fig 7. Heparin affinity chromatography of 2GPI from acid-treated and UK-activated plasma. Both normal and plasminogen-depleted human plasmas were treated with acid and activated with UK as described in Materials and Methods. After the treatment with perchloric acid, sample was applied to a column of HiTrap Heparin and eluted with a linear gradient of NaCl. (1), normal plasma; (2), plasminogen-depleted plasma. View larger version (16K): [in this window] [in a new window]   Fig 8. Relationship between the formation of nicked 2GPI and 2PI activity in plasma. After 16-hour incubation of samples with 120 IU of UK at 37°C, the intact and nicked form of 2GPI were separated as described in Materials and Methods. Horizontal axis and vertical axis correspond to 2PI activity and amount of nicked 2GPI (percentage of total 2GPI) in the plasma. 2PI activity was expressed as the percentage of normal plasma.     DISCUSSION Abstract Introduction Methods Results Discussion References In the present work we showed that plasmin generated quickly a nicked form of 2GPI by the limited cleavage of a peptide bond of domain V, Lys 317-Thr 318, by using recombinant domain V and 2GPI from human plasma. Cleavage of the peptide bond by factor Xa was extremely slow. Cleavage by thrombin, UK, t-PA, and sTF/VIIa was not detected. The nicked form of 2GPI was shown to have a reduced affinity for CL by the quantitative analysis of the binding with liposomes containing CL. The importance of domain V in 2GPI has been demonstrated for the interaction with CL and other phospholipids.18 Sheng et al19 demonstrated that Lys 284, Lys 286, and Lys 287 in domain V are critical for the binding with phospholipid by using mutant proteins. We elucidate that cleavage of Lys 317-Thr 318 by plasmin causes the conformational change of the domain V, leading to the reduced interaction with phospholipid, although the conformational change was not significantly detected by circular dichroism and fluorescent measurements.14 Cleavage of the peptide bond in domain V by plasmin was also demonstrated in plasma. The nicked form of 2GPI was isolated from UK-treated plasma. We could not detect the cleavage of Ala 314-Phe 315 in 2GPI isolated from plasma after the activation of plasminogen, as reported in commercial preparation of human 2GPI by Hunt et al.13 Other proteases in plasma or from cells may participate in the cleavage of the peptide bond. The activation of plasminogen occurs after the formation of fibrin, because both plasminogen and plasminogen activator associate with fibrin.20,21 Then, plasmin can degrade fibrin and other plasma proteins. In the fluid phase, most of plasmin generated is inactivated by 2 plasmin inhibitor. This study demonstrated the significant cleavage of 2GPI in acid-treated plasma, which exhibited 23% of 2PI activity of normal plasma. Our study also indicated that about 12% of 2GPI was nicked when 2PI activity in plasma decreased to 80% (Fig 8). Therefore, the in vivo limited proteolysis of 2GPI by plasmin could occur significantly when plasmin inhibitor level decreases. Brighton et al22 demonstrated that 2GPI levels in plasma were significantly reduced in patients with thrombosis or DIC. It is highly probable that plasmin inhibitor decreased in these patients and then plasmin could generate the nicked 2GPI. The nicked form of 2GPI may be rapidly cleared from circulation than native 2GPI. It has been reported that plasminogen binds to extracellular matrix (ECM) synthesized by endothelial cell monolayers and activated by t-PA or UK on the matrix.23,24 Plasmin generated on the matrix is protected from inhibition by 2 plasmin inhibitor. It has been also reported that 2GPI binds to heparin.2 The extracellular matrix produced by endothelial cell is a complex array of glycoproteins and glycosaminoglycans. Therefore, it is conceivable that 2GPI binds to heparin-like glycosaminoglycans and cleaved by plasmin in the extracellular matrix. The possibility that 2GPI binds to fibrin and then proteolyzed by plasmin also intrigues us. We have shown that radiolabeled 2GPI was incorporated into fibrin (Ohkura N, Kato H, unpublished observations), although further investigation should be performed. Recently, Kochl et al25 showed the interaction of 2GPI with apolipoprotein (a) by using the yeast two-hybrid interaction trap system. These investigators also demonstrated the interaction in plasma by using co-immunoprecipitation experiments. These results suggest that lipoprotein (a) potentiates the association of 2GPI with fibrin, as apolipoprotein (a) has been shown to associate with fibrin. Although further investigation is needed to demonstrate the in vivo significance of the limited cleavage of 2GPI by plasmin, the present data indicate the possibility that plasmin can produce the nicked form of 2GPI and reduce the function of 2GPI in vivo. 2GPI has been shown to interact with phospholipid and many other negatively charged substances and to modulate the activation of the intrinsic pathway, prothrombinase activity and phospholipid-dependent protein Ca activity.26-29 The phopholipid-bound 2GPI can be cleaved by plasmin, since the cleavage of 2GPI by plasmin was not affected in the presence of phospholipid. The cleavage of 2GPI by plasmin demonstrated in the current study may give a new insight into the function of 2GPI.     FOOTNOTES    Submitted December 26, 1997; accepted January 21 1998.    N.O. and Y.H. contributed equally to this work.    Supported by Grants-in-Aid for Scientific Research from the Ministry of Education Science, Sports and Culture of Japan, and by Fellowships of the Japan Society for the Promotion of Science for Japanese Junior Scientists.    Address reprint requests to Hisao Kato, PhD, National Cardiovascular Center Research Institute, Fujishirodai-5, Suita, Osaka 565, Japan.    The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.     REFERENCES Abstract Introduction Methods Results Discussion References 1. Kroll J, Larsen JK, Loft H, Ezban M, Wallevik K, Faber M: DNA binding proteins in Yoshida ascites fluid. Biochim Biophys Acta 434:490, 1976[Medline] [Order article via Infotrieve] 2. Polz E, Wurm H, Kostner GM: Investigation on 2-glycoprotein-I in the rat: Isolation from serum and demonstration in lipoprotein density fractions. Int J Biochem 11:265, 1980[Medline] [Order article via Infotrieve] 3. Schousboe I, Rasmussen MS: The effect of 2-glycoprotein I on the dextran sulfate and sulfatide activation of the contact system (Hageman factor system) in the blood coagulation. Int J Biochem 20:787, 1988[Medline] [Order article via Infotrieve] 4. Wurm H: 2-Glycoprotein-I (apolipoprotein H) interactions with phospholipid vesicles. Int J Biochem 16:511, 1984[Medline] [Order article via Infotrieve] 5. Galli M, Comfurius P, Maassen C, Hemker HC, de Baets MH, van Breda Vriesman PJ, Barbui T, Zwaal RF, Bevers EM: Anticardiolipin antibodies (ACA) directed not to cardiolipin but to a plasma protein cofactor [see comments]. Lancet 335:1544, 1990[Medline] [Order article via Infotrieve] 6. Matsuura E, Igarashi Y, Fujimoto M, Ichikawa K, Koike T: Anticardiolipin cofactor(s) and differential diagnosis of autoimmune disease. Lancet 336:177, 1990[Medline] [Order article via Infotrieve] 7. McNeil HP, Simpson RJ, Chesterman CN, Krilis SA: Anti-phospholipid antibodies are directed against a complex antigen that includes a lipid-binding inhibitor of coagulation: Beta2-glycoprotein I (apolipoprotein H). Proc Natl Acad Sci USA 87:4120, 1990[Abstract/Free Full Text] 8. Ichinose A, Bottenus RE, Davie EW: Structure of transglutaminase. J Biol Chem 265:13411, 1990[Free Full Text] 9. Kato H, Enjyoji K: Amino acid sequence and location of the disulfide bonds in bovine beta 2 glycoprotein I: The presence of five Sushi domains. Biochemistry 30:11687, 1991[Medline] [Order article via Infotrieve] 10. Steinkasserer A, Estaller C, Weiss EH, Sim RB, Day AJ: Complete nucleotide and deduced amino acid sequence of human beta 2-glycoprotein I. Biochem J 277:387, 1991 11. Hagihara Y, Goto Y, Kato H, Yoshimura T: Role of the N- and C-terminal domains of bovine 2 glycoprotein I in its interaction with cardiolipin. J Biochem. 118:129, 1995 12. Steinkasserer A, Barlow PN, Willis AC, Kertesz Z, Campbell ID, Sim RB, Norman DG: Activity, disulphide mapping and structural modelling of the fifth domain of human beta 2-glycoprotein I. FEBS Lett 313:193, 1992[Medline] [Order article via Infotrieve] 13. Hunt JE, Simpson RJ, Krilis SA: Identification of a region of beta 2-glycoprotein I critical for lipid binding and anti-cardiolipin antibody cofactor activity. Proc Natl Acad Sci USA 90:2141, 1993[Abstract/Free Full Text] 14. Hagihara Y, Enjyoji K, Omasa T, Katakura Y, Suga K, Igarashi M, Matsuura E, Kato H, Yoshimura T, Goto Y: Structure and function of the recombinant fifth domain of human 2-glycoprotein: Effects of specific cleavage between Lys 77 and Thr 78. J Biochem 121:128, 1997[Abstract/Free Full Text] 15. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680, 1970[Medline] [Order article via Infotrieve] 16. Matsuda T, Ogawara M, Miura R, Seki T, Matsumoto T, Teramura Y, Nakamura K: Selective determination of 2-plasmin inhibitor activity in plasma using chromogenic substrate. Thromb Res 33:379, 1984[Medline] [Order article via Infotrieve] 17. Naito K, Aoki N: Assay of 2-plasmin inhibitor activity by means of a plasmin specific tripeptide substrate. Thromb Res 12:1147, 1978[Medline] [Order article via Infotrieve] 18. Igarashi M, Matsuura E, Igarashi Y, Nagae H, Ichikawa K, Triplett DA, Koike T: Human 2-glycoprotein I as an anticoagulant cofactor determined using deleted mutants expressed by a baculovirus system. Blood 87:3262, 1996[Abstract/Free Full Text] 19. Sheng Y, Sali A, Herzog H, Lahnstein J, Krilis SA: Site-directed mutagenesis of recombinant human 2-glycoprotein I identifies a cluster of lysine residues that are critical for phospholipid binding and anti-cardiolipin antibody activity. J Immunol 157:3744, 1996[Abstract] 20. Wu HL, Chang BI, Wu DH, Chang LC, Gong CC, Lou KL, Shi GY: Interaction of plasminogen and fibrin in plasminogen activation. J Biol Chem 265:19658, 1990[Abstract/Free Full Text] 21. Hoylaerts M, Rijken DC, Rijken HR, Collen D: Kinetics of the activation of plasminogen by tissue plasminogen activator: Role of fibrin. J Biol Chem 257:2912, 1982[Abstract/Free Full Text] 22. Brighton TA, Hogg PJ, Dai Y-P, Murray BH, Chong BH, Chesterman CN: Beta2-glycoprotein I in thrombosis: Evidence for a role as a natural anticoagulant. Br J Haematol 93:185, 1996[Medline] [Order article via Infotrieve] 23. Kundsen BS, Silverstein RL, Leung LL, Harpel PC, Nachman RL: Binding of plasminogen to extracellular matrix. J Biol Chem 261:10765, 1986[Abstract/Free Full Text] 24. Stack MS, Rinehart AR, Pizzo SV: Comparison of plasminogen binding and activation on extracellular matrices produced by vascular smooth muscle endothelial cells. Eur J Biochem 226:937, 1994[Medline] [Order article via Infotrieve] 25. Kochl S, Fresser F, Lobentanz E, Baier G, Utermann G: Novel interaction of apolipoprotein (a) with 2-glycoprotein I mediated by the Kringle IV domain. Blood 90:1482, 1997[Abstract/Free Full Text] 26. Schousboe I: 2-glycoprotein I: A plasma inhibitor of the contact activation of the intrinsic blood coagulation pathway. Blood 66:1086, 1985[Abstract/Free Full Text] 27. Nimpf J, Wurm H, Kostner GM: Interaction of 2-glycoprotein-I with human blood platelets: Influence upon the ADP-induced aggregation. Thromb Haemost 54:397, 1985[Medline] [Order article via Infotrieve] 28. Nimpf J, Bevers EM, Bomans PHH, Till U, Wurm H, Kostner GM, Zwaal RFA: Prothrombinase activity of human platelets is inhibited by 2-glycoprotein-I. Biochim Biophys Acta 884:142, 1986[Medline] [Order article via Infotrieve] 29. Mori T, Takeya H, Nishioka J, Gabazza EC, Suzuki K: 2-glycoprotein-I modulates the anticoagulant activity of activated protein C on the phospholipid surface. Thromb Haemost 75:49, 1996[Medline] [Order article via Infotrieve] © 1998 by The American Society of Hematology.   0006-4971/98/91-0015$3.00/0 CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: T. Sakai, K. Balasubramanian, S. Maiti, J. B. Halder, and A. J. 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