plasma thrombomodulin in health and diseases

Plasma Thrombomodulin in Health and Diseases

By Shogo Takano, Shigeru Kimura, Shinichi Ohdama, and Nobuo Aoki

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblot analysis of plasma thrombomodulin concentrate revealed that four degraded forms of thrombomodulin with different molecular weights a r e present in plasma. Plasma concentrations of thrombomodulin in patients with various diseases were measured by two methods of enzyme-linked immunosorbent assay using monoclonal antibodies. One method measures intact thrombomodulin and degraded forms of thrombomodulin; the other does not detect the two smaller degraded forms of thrombomodulin present in plasma. The results indicated that thrombomodulin was increased in the circulating blood of patients with disseminated intravascular coagulation

H E ENDOTHELIAL CELL surface forms an effective T thromboresistant surface against clot formation in the vasculature. Under physiological conditions, the thromboresistant properties of endothelium are intact. When the procoagulant or thrombogenic factors outweigh the anticoagulant or control factors, a major breakdown in the mecha- nism for thromboresistance occurs, leading to thrombus formation. Thrombus formation resulting in vessel obstruc- tion causes tissue ischemia and necrosis, and may also cause secondary bleeding. The thromboresistance is mediated by several mechanisms, including inhibition of platelet aggrega- tion (secretion of prostacyclin) and fibrinolysis (release of plasminogen activators). In addition, endothelial cells have heparinlike molecules on their luminar surface that can accelerate antithrombin 111-mediated inactivation of thrombin and other activated coagulation factors. Endothelial cells also express a surface glycoprotein called thrombomodulin that neutralizes thrombin clotting activity and accelerates thrombin-catalyzed activation of protein C. Activated protein C in turn functions as an anticoagulant by proteolyti- cally degrading factor Va and factor VIIIa in the presence of protein S . Thus, thrombomodulin plays a major role in the regulation of intravascular coagulation.’ Although thrombomodulin is mainly present on endothelial cell surfaces, thrombomodulin is also found in circulating blood plasma.*

In the present study, we measured the concentration of

From the First Department of Internal Medicine. Tokyo Medical and Dental University. Tokyo and Tokyo Research Laboratories, Kowa Ltd, Tokyo.

Submitted March 14.1990: accepted July 23,1990. Supported in part by a grant-in-aid for scientific research on

priority areas from the Ministry of Education, Science, and Culture of Japan: a research grant for cardiovascular diseases: and a research grant for intractable diseases from the Ministry of Health and Welfare of Japan.

Address reprint requests to Nobuo Aoki. MD. The First Depart- ment of Internal Medicine, Tokyo Medical and Dental University. Yushima 1-5-45, Bunkyo-ku. Tokyo 113, Japan.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 8 I990 by The American Society of Hematology. 0006-4971 f90~7610-0005$3.00~0

syndrome, pulmonary thromboembolism, adult respiratory distress syndrome, chronic renal failure, or acute hepatic failure. The different values obtained by the two methods indicate that the increase of plasma thrombomodulin found in these patients was mainly due to an increase of the smaller fragments of degraded forms, suggesting that the release of thrombomodulin from endothelial cells was accelerated in various disease states by proteolytic activity generated on the surface of the endothelium and may be removed from the circulation mostly by the kidneys and liver. 0 1990 by The American Society of Hematology.

thrombomodulin antigen in the plasma from patients with or without intravascular coagulation due to a variety of causes with two different methods, and our results suggest that the increase of plasma thrombomodulin found in various diseases is mainly due to an increase of the degraded forms of thrombomodulin, which may be removed from the circulation mostly by the kidneys and liver.

MATERIALS AND METHODS

Venous blood was collected into 3.2% sodium citrate (9 parts blood: 1 part anticoagulant) using a 10-mL syringe with a 21-gauge needle. Plasma was obtained by centrifuging the blood at 1,500g for 30 minutes. The plasma samples were frozen and stored at -8OOC until the assay.

After informed consent was obtained, plasma samples were obtained from normal healthy donors (12 male, 10 female) and from patients with disseminated intravascular coagulation syndrome (DIC, n = 19). adult respiratory distress syndrome (ARDS, n = 9), pulmonary thromboembolism (n = 1 l) , stable interstitial pneumonitis (n = 4), lung cancer (n = 14), chronic renal failure (n = lo), compensated liver cirrhosis (n = 1 l) , and hepatic failure (n = 8; two cases of fulminant hepatitis and six cases of advanced liver cirrhosis with hepatocellular carcinoma).

Thrombomodulin prepared from human placenta by the previously described method’ was further purified by high-performance liquid ~hromatography.~ The purified thrombomodulin appeared as a single peak in a region of 100- to 150-Kd proteins in gel filtration with Ultrogel AcA 34 (Pharmacia, Uppsala, Sweden) using buffer containing 0.05% Tween 20 or 0.01% Lubrol. Increase of the concentrations of the detergents used did not appreciably change the elution volume, suggesting that the thrombomodulin was monomeric in the buffer used for the assay in the present study.

Polyclonal antibody to human thrombomodulin was kindly supplied by Dr T. Yamaji, Bio-Medical Research Laboratories, Teijin Ltd, Iwakuni, Japan.

Three monoclonal antibodies (MoAbs)-KA-2, -3, and -&to human thrombomodulin were used for the assay of thrombomodulin. The functional properties of these antibodies were previously de~cribed.~ The antibodies bind to different epitopes on the thrombomodulin molecules and do not interfere with each other’s bindings. KA-3 and -4 bind to protease (elastase or trypsin)-treated thrombomodulin as well as native intact thrombomodulin, but KA-2 does not bind to protease-treated thrombomodulin. Calcium ions are required for the maximum binding of KA-4 to native thrombomodulin.

Horseradish peroxidase (HRPJ-conjugated antibody. Conjugation of the antibodies with HRP (Sigma Chemical Co, St Louis) was performed by the method of Nakane and Kawaoi.’

Collection of blood samples.

Thrombomodulin.

Antibodies to thrombomodulin.

2024 Blood, Vol76, No 10 (November 15). 1990: pp 2024-2029

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PLASMA THROMBOMODULIN 2025

Antibody-coupled Sepharose. Sepharose-4B (Pharmacia, Swe- den) was activated with cyanogen bromide (CNBr) by the method of Cuatrecasas,6 and MoAb KA-3 or polyclonal antibody to thrombomodulin was linked to the agarose to give a final IgG concentration of around 5 mg/mL of agarose.

Five milliliters of plasma was mixed and incubated with 1 mL of polyclonal antibodyaupled Sepharose for 12 hours, and thrombomodulin-depleted plasma was separated by centrifugation.

Concentration of plasma thrombomodulin by immunoafinity chromatography. p- Aminodiphenyl methane sulfonyl fluoride-HCl (Wako Chemicals, Tokyo) was added to 15 mL plasma to obtain a final concentration of 5 mmol/L. After incubation for 30 minutes, the plasma was dialyzed against 3 L of Tris-buffered saline (TBS) (0.02 mol/L Tris-HC1,O.l mol/L NaCI, pH 7.4). After the dialysis, the plasma sample was diluted to 100 mL with TBS and applied onto a 2-mL column of KA-3 or polyclonal antithrombomodulin antibody- conjugated Sepharose 4B (5 mg IgG/mL) equilibrated with TBS. The column was washed with 100 mL each of TBS, Tris-buffered (pH 7.4) 1 mol/L NaCl containing 0.05% Tween 20, and TBS, sequentially. Thrombomodulin was finally eluted from the column with 20 mL of Tris-buffered 1 mol/L NaCl containing 2 mol/L sodium thiocynate (NaSCN). The eluted thrombomodulin was concentrated to 0.5 mL by means of Millipore CX-10 (Millipore Corp, MA) ultrafiltration or by evaporation under vacuum.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) on a slab gel was carried out using a separating gel of acrylamide (10%) containing 0.1% SDS and a stacking gel of 4% acrylamide containing 0.1% SDS according to the method of Laemmli.' Molecular weight standards (rabbit muscle phosphory- lase b, 97.4 Kd; bovine serum albumin, 66.2 Kd; hen egg white ovalbumin, 42.7 Kd; bovine carbonic anhydrase, 31 Kd; and soybean trypsin inhibitor, 21.5 Kd) were obtained from Bio-Rad Laborato- ries (Richmond, CA).

Immunoblotting technique. After separation by SDS-PAGE, the proteins were electrophoretically transferred to polyvinylidene difluoride membranes (Immobilon Transfer, Millipore) as described by Matsudaira. After the transfer, the membranes were blocked by incubating them in Tris-buffered saline containing 1% gelatin for 1 hour, washed with the washing buffer (0.05 mol/L Tris-HC1, 0.5 mol/L NaCI, 0.05% Tween 20, 5 mmol/L CaCI,, pH 7.9, and incubated with HRP-conjugated polyclonal or MoAbs (1 fig/mL) dissolved in the washing buffer for 16 hours at 4"C. The membranes were then washed with the washing buffer and subjected to color development by soaking the membranes in a solution of 0.01% 3-amino-9-ethylcarbazol (Sigma), 4% dimethyl-formamide, and 0.05% H,02 in 0.05 mol/L acetate buffer, pH 5.

Measurement of thrombomodulin. An enzyme-linked immunosorbent assay (ELISA) was used to measure the concentration of plasma thrombomodulin. Polyvinyl chloride microtiter plates (Nunc- Immuno Plate, Nunc, Denmark) were coated with KA-3 at 10 pg/mL in 0.05 mol/L carbonate buffer, pH 9.6, for 2 hours at 37OC. The plates were washed three times with TBS containing 0.05% Tween 20 and 5 mmol/L CaCI,, pH 7.4. (TBS-Tween-Ca).

Standard purified thrombomodulin was diluted with TBS- Tween-Ca containing 1% bovine serum albumin (Sigma) or thrombomodulin-depleted plasma to obtain various concentrations of thrombomodulin (from 0.01 ng/mL to 5 ng/mL) and 100-pL aliquots of these solutions were added to the coated wells. Plasma was diluted various times with TBS-Tween-Ca, and a 100-pL aliquot of the diluted plasma was added to the coated wells of the same plate. The plate was incubated for 16 hours at 37OC and then washed three times with TBS-Tween-Ca.

Thrombomodulin-depleted plasma.

Subsequently, 100-pL aliquots of HRP-conjugated KA-2 or KA-4 (0.1 pg/mL) was applied to each well and incubation performed for 4 hours at 37OC. After washing with TBS-Tween-Ca three times, 100-pL aliquots of the solution containing 0.01% H,O, and 0.4 mg/mL orthophenylenediamine (Sigma), dissolved in 0.075 mol/L citrate-sodium phosphate buffer pH 5.0, were added to each well and incubation carried out for 30 minutes at 37OC. The reaction was stopped by adding 50 pL of 4.5 mol/L H,SO, to each well, and the absorbance at 492 nm was measured by a Micro Plate Reader (Toyo Soda, Tokyo). The plasma thrombomodulin concentration was estimated according to the thrombomodulin standard curve constructed with purified thrombomodulin. When dilutions of purified thrombomodulin were made with thrombomodulin-depleted plasma for the construction of the standard curve, the absorbance obtained was dependent on the concentration of plasma protein and the absorbance was low when the plasma used was not properly diluted. When thrombomodulin-depleted plasma had been diluted more than 20 times with TBS-Tween-Ca, the standard curve obtained was identical with that constructed by dilution of purified thrombomodulin with TBS-Tween-Ca containing I% albumin (Fig 1). Accord- ingly, plasma was diluted 25-fold with TBS-Tween-Ca for the assay.

Although no clot formation was observed in plasma diluted with TBS-Tween-Ca, it may be possible that activation of the blood coagulation enzyme cascade by calcium ions present in TBS- Tween-Ca takes place during the incubation, and activated coagulation factors influence the assay. This possibility was ruled out when an assay was performed using TBS-Tween-Ca containing enough heparin to suppress activation of the coagulation cascade; the addition of heparin did not alter the assay results.

Because the epitope recognized by KA-4 is located in proximity to a thrombin-binding site, there was a possibility that thrombomodulin bound to thrombin may not be measured by the present method using KA-4. To test this possibility, various amounts of thrombin were added to a fixed amount of purified thrombomodulin, and the concentration of thrombomodulin was measured by ELISA using KA-4. The values obtained were the same regardless of the amounts of thrombin added, indicating that thrombomodulin in the form of a complex with thrombin can be measured equally by the method using KA-4. The thrombin-thrombomodulin complex, if present, probably was dissociated by the dilution buffer or the washing buffer containing 0.05% Tween, and thrombomodulin thus liberated from the complex may have reacted with KA-4.

Student's t analysis for unpaired samples was used with the two-tailed test. A P value of r.05 was considered to represent a statistically nonsignificant change.

Statistical analysis.

RESULTS AND DISCUSSION

Thrombomodulin was separated and concentrated from plasma by immunoaffinity chromatography using KA-3 coupled to Sepharose 4B. The concentrate was then analyzed by SDS-PAGE followed by immunoblot analysis using the monoclonal antibody KA-2 or -4. As shown in Fig 2, four protein bands were recognized by KA-4. To exclude the possibility that degradation of thrombomodulin occurred during the procedures, purified thrombomodulin was added to plasma to achieve a concentration of 1 pg/mL plasma, which is 100 times higher than the normal plasma level, and thrombomodulin was separated and analyzed by the same procedures without prior concentration. Only one band corresponding to the original purified thrombomodulin was visible by staining (Fig 2), indicating that the four bands recognized by KA-4 was not due to degradation during the procedures. Plasma thrombomodulin was also concentrated




2026

o.8r TAKANO ET AL

by immunoaffinity chromatography using polyclonal antibody to thrombomodulin, and analyzed in the same way using polyclonal or MoAbs (Fig 3). Four bands with the same mobilities as those of the bands seen in Fig 2 were recognized by KA-4 as well as polyclonal antibody, indicating that the four molecular forms represent nearly all the thrombomodulin present in plasma. All four protein bands were recognized by KA-4, whereas only two bands with larger molecular sizes were recognized by KA-2 (Figs 2 and 3). These results are in accordance with the previous findings that KA-2 recognizes intact thrombomodulin but does not recognize protease (trypsin or elastase)-degraded major fragments, whereas KA-4 recognizes protease-degraded forms as well as intact thr~mbomodulin.~

Fig 2. Immunoblotting of thrombomodulin. Pu- rified thrombomodulin incubated with normal p lasm was isolated by immunoaffinity with KA-3- conjugated sepharose and subjected to SDS-PAGE without reduction. followed by immunoblotting with HAP-conjugated antibody K A 4 (lane 1). Plasma thrombomodulin concentrate, obtained by immunoaffinity with KA-3-conjugated sepharose, was subjected to SDS-PAGE without reduction, followed by immunoblotting with HRPconjugated antibody KA-2 (lane 21 or K A 4 (lane 3). Arrows on the right indicate the bands corresponding to the four molecular forms of thrombomodulin. On the left is a molecular weight calibration scale constructed using molecular weight standards stained with coomassie brilliant blue.

97,4 00

66,200

42,7 00

31,000

21,500

Fig 1. Standard curve for the ELSA of plasma t h r o m b modulin. Standard purified thrombomodulin waa diluted with TBS-Tween-& containing 1% bovine serum albumin to obtain various concentrations of thrombomodulin. The diluted samples were assayed for thrombomodulin by ELlSA using MoAb KA-3 as a solid-phase antibody and HRP-conjugated MoAb KA-2 (A) or K A 4 (6) as a fluid-phase antibody (see Mate- rials and Methods).

Molecular weights of the four molecular forms estimated by nonreducing SDS-PAGE were 64. 60. 52, and 47 Kd. Since the molecular weight of intact thrombomodulin, estimated by nonreducing SDS-PAGE, was 71 Kd, all of these four molecular forms must be major fragments of partially degraded thrombomodulin. Particularly, the smallest form might represent a protease (trypsin or elastase)-degraded major fragment, because its molecular weight (47 Kd) is close to those of degraded fragments (45 and 42 Kd) obtained by elastase or trypsin digestion. respectively.' Be- cause the epitope recognized by KA-2 is likely located in the 0-linked glycosylation site-rich domain or the transmem- brane domain: the two smaller molecular forms may be devoid of these domains. These domains are located in the

1 2

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Fig 3. lmmunoblotting of plasma thrombomodulin. Plasma thrombomodulin concentrate, obtained by immunoaffinity w i t h polyclonal antithrombomodulin antibody- conjugated sepharose, was subjected to SDS-PAGE without reduction, followed by immunoblotting with HRP-conjugated antibody KA-2 (lane 1). K A 4 (lane 2). or polyclonal antithrombomodulin antibody (lane 3). Arrows on the right indicate the bands corresponding to the four molecular forms of thrombomodulin. On the left is a molecular weight calibration scale constructed using molecular weight standards stained with coomassie brilliant blue.

9 7 , 4 0 6 6,2 0

4 2.7 0

3 1.00

2 1.50

0 0

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region following the C-terminal side of the epidermal growth factor (EGF) domain that contains the thrombin binding site recognized by KA-4.'

Thrombomodulin concentrations in plasma were measured by ELISA using KA-3 as a solid-phase antibody. HRP-conjugated KA-2 or -4 was used as a liquid-phase antibody. For convenience, the procedure using conjugated KA-2 or -4 was designated as the KA-2 or -4 method, respectively. The two larger molecular forms were assayed together by the KA-2 method, and all four of the molecular forms were assayed together by the KA-4 method.

2 SD of plasma levels of thrombomodulin in normal healthy adult individuals (n = 22) was 11.8 k 5.2 ng/mL by the KA-4 method and 7.5 k 5.3 ng/mL by the KA-2 method. As shown in Fig 4, thrombomodulin levels measured by the KA-4 method, which represent the total thrombomodulin levels, were markedly increased in chronic renal failure as compared with the level in the control group.

The mean

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A moderate but significant increase of the levels measured by the KA-2 method, which represent the levels of the larger molecular forms, was also noted in chronic renal failure. Therefore, patients with renal dysfunction were excluded from the studies on the other disease groups. The total thrombomodulin levels measured by the KA-4 method were remarkably increased in patients with DIC, pulmonary thromboembolism, ARDS. and acute hepatic failure as compared with normal values. The larger molecular forms measured by the KA-2 method were also significantly increased in DIC, ARDS, and acute hepatic failure, but not in pulmonary thromboembolism.

In contrast to ARDS and pulmonary thromboembolism, there was no increase of the total thrombomodulin measured by the KA-4 method in interstitial pneumonitis and lung cancer, suggesting that the increases observed in ARDS and pulmonary thromboembolism are unique among the pulmonary diseases and caused by the accelerated release of

Fig 4. Plasma thrombamodulin concsntrations in patients with various diseases measured by the two methods of ELISA using MoAbs: KA-2 (striped columns) and K A 4 (open columns). Abbreviations: NC. normal control: DIC, disseminated intravascular coagulation: ARDS, adult respiratory distress syndrome; PTE. pulmonary thromboembolism: IP, interstitial pneumonitis (stable); LCa, lung cancer: CRF, chronic renal failure: LC, liver cirrhosis: HF, acute hepatic failure. Vertical bars indicate the mean f SE. Asterisks represent significant differ-

HF ences from the normal control ( . P < .01:

l * A **




2028 TAKANO ET AL

thrombomodulin from injured pulmonary capillary endothelial cells occurring in these disease states.

In patients with stable interstitial pneumonitis, lung cancer, or compensated liver cirrhosis, the levels measured by the KA-4 method were not statistically different from the level of the control. However, a significant (P < .01) decrease of the levels measured by the KA-2 method was noted in patients with lung cancer, and a less significant (P < .OS) decrease was noted in patients with interstitial pneumonitis and liver cirrhosis.

Because the difference between the values obtained by the two methods represents the concentration of the two smaller molecular forms of thrombomodulin (Fig 2), the proportion of the two smaller molecular forms to the total thrombomodulin was calculated by dividing the difference by the value obtained by the KA-4 method, which measures the total thrombomodulin, and was expressed as a percentage (Fig 5 ) . In the control group, the two smaller molecular forms constituted an average of 43% of the total thrombomodulin in the plasma. In various disease states, however, the two smaller molecular forms constituted a significantly (P < .01) higher percentage of the total than in the normal state: 55% in DIC, 60% in ARDS, 66% in interstitial pneumonitis, 69% in lung cancer, 68% in chronic renal failure, and 62% in compensated liver cirrhosis. The value of 58% in pulmonary thromboembolism was less significantly (P < .OS) higher than normal.

An increase of percentage of the smaller molecular forms in the total thrombomodulin in DIC, ARDS, pulmonary thromboembolism, and chronic renal failure indicates that the increase of the plasma thrombomodulin observed in these patients was mainly caused by the increase of the smaller molecular forms.

These findings suggest that the release of the smaller molecular forms from endothelial cells was accelerated in these disease states. In chronic renal failure, retention of thrombomodulin may be an additional factor contributing to the increase of circulating thrombomodulin if plasma thrombomodulin is cleared mainly by the kidneys. In DIC or pulmonary thromboembolism, activated proteolytic enzymes in the coagulation-fibrinolysis system may be responsible for the release of thrombomodulin. In ARDS, proteases released from leukocytes that had adhered to the endothelial cells9 might have split the surface thrombomodulin and released it into the circulation.

In contrast to the aforementioned disease states, the percentage of the smaller molecular forms in the total thrombomodulin in acute hepatic failure was not statistically different from that in the normal state (Fig 5 ) , indicating that the larger molecular forms and the smaller molecular forms were increased in parallel in acute hepatic failure. It is speculated that the liver is an organ partially contributing to the clearance of plasma thrombomodulin, and a rapid and severe deterioration of liver function causes a retention of thrombomodulin in the plasma, although chronic and moder-

T

:a : 14)

Fig 6. Proportions of the smaller molecular forms to the total plasma thrombomodulin 1%). Abbreviations and signs used are the same as those in Fig 4.

ate liver dysfunction does not affect thrombomodulin levels as seen in compensated liver cirrhosis (Fig 4). In this connection it is of interest to note that the liver was the major site of clearance of intravenously injected thrombomodulin in mice."

In interstitial pneumonitis, lung cancer, and liver cirrhosis, there was no significant change of the total thrombomodulin, but the larger molecular forms measured by the KA-2 method were significantly decreased (Fig 4), resulting in an increase of the percentage of the smaller molecular forms (Fig 5 ) . Different mechanisms of release or removal of the two classes of molecular forms may presumably be involved in changing the proportion of the two classes of molecular forms in these disease states, but the detailed mechanisms are not known.

From the results obtained in the present study, we suggest that the measurement of plasma thrombomodulin can be used to assess the state of endothelial cell injuries if renal function is normal and liver function is not severely deterio- rated, but further studies are needed to support this proposal.

ACKNOWLEDGMENT

We thank Eri Yahagi for expert technical assistance and Mitsuko Ito for preparing the manuscript.

ADDENDUM

After submission of this paper, a report addressing enzyme immunoassay of thrombomodulin has been published." We have confirmed the findings of Ishii et a1 that six molecular forms of plasma thrombomodulin were detected by SDS-PAGE under re- duced conditions, whereas four molecular forms were found under nonreduced condition in the present study. They reported that the mean plasma level of thrombomodulin was 35.2 ng/mL, whereas our estimate was 11.8 ng/mL. The cause of the discrepancy is not known.

REFERENCES

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3. Kurosawa S, Aoki N: Preparation of thrombomodulin from human placenta. Thromb Res 37:353, 1985

4. Kimura S, Nagoya T, Aoki N: Monoclonal antibodies to human thrombomodulin whose binding is calcium dependent. J Biochem 105:478,1989

5. Nakane PK, Kawaoi A: Peroxidase-labeled antibody. A new method of conjugation. J Histochem Cytochem 22:1084,1974

6. Cuatrecasas P Protein purification by affinity chromatography. J Biol Chem 245:3059, 1970

7. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680, 1970

8. Matsudaira P Sequence from picomole quantities of proteins

electroblotted onto polyvinylidine difluoride membranes. J Biol Chem 262:10035,1987

9. Warshawski FJ, Sibbald WJ, Driedger AA, Cheung H: Abnor- mal neutrophil-pulmonary interaction in the adult respiratory distress syndrome. Am Rev Respir Dis 133:797, 1986

10. Kumada T, Dittman WA, Majerus P W A role for thrombomodulin in the pathogenesis of thrombin-induced thromboembolism in mice. Blood 71:728,1988

1 1 . Ishii H, Nakano M, Tsubouchi J, Ishikawa T, Uchiyama H, Hiraishi S, Tahara C, Miyajima Y, Kazama M: Establishment of enzyme immunoassay of human thrombomodulin in plasma and urine using monoclonal antibodies. Thromb Haemost 63:157, 1990




1990 76: 2024-2029

S Takano, S Kimura, S Ohdama and N Aoki Plasma thrombomodulin in health and diseases

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