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Tissue factor pathway inhibitor can interact with platelets
Journal of Immunological Methods 266 (2002) 181 – 184 www.elsevier.com/locate/jim
Technical note
Tissue factor pathway inhibitor can interact with platelets Takuya Nishioka *, Masaharu Yokota, Masayuki Hino, Izumi Tsuda, Noriyuki Tatsumi Department of Clinical and Laboratory Medicine, Osaka City University Medical School, 1-4-3 Asahimachi, Abeno, Osaka 545-8585, Japan Received 14 January 2002; received in revised form 5 April 2002; accepted 23 April 2002
Abstract Tissue factor pathway inhibitor (TFPI) regulates the extrinsic pathway of blood coagulation. However, there is no report on interaction between TFPI and platelets other than that by Tsuji, who found that whole blood anticoagulated with TFPI exhibited remarkable decrease in platelet count. Our study revealed that washed platelets suspended in modified Tyrode’s buffer (8 mM CaCl2) containing TFPI exhibit platelet aggregation. However, platelets aggregation was observed without TFPI, but its increase and intensity were slow and weak, compared to that in the presence of TFPI. This aggregation was inhibited by antiCD41 (anti-GPIIb) antibody. This finding suggested that TFPI promotes platelet aggregation. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Tissue factor pathway inhibitor; Platelet aggregation; GPIIb/IIIa
1. Introduction Tissue factor pathway inhibitor (TFPI) inhibits the intravascular coagulation induced by tissue factor (TF). TFPI is a 40-kDa glycoprotein with 276 amino acids (Wun et al., 1988), and its normal plasma concentration is about 54 –142 ng/ml (Lindahl et al., 1992). TFPI is produced mostly in endothelial cells (Bajaj et al., 1990; Werling et al., 1993) and pooled in the endothelium (50 – 80%), plasma (10 – 50%) (Sandset, 1996) and platelets (less than 2.5%) (Novotny et al., 1989). Most of TFPI in plasma is complexed with low-density lipoproteins and high-density lipoproteins (Novotny et al., 1989; Hansen et al., 1994). Injection of heparins causes release of TFPI into blood, and this release results in two- to four-fold greater increase in TFPI activity than *
Corresponding author. Tel.: +81-6-6645-3881; fax: +81-66645-3880. E-mail address: [email protected] (T. Nishioka).
that in normal concentration, demonstrating strong anticoagulation activity in regulating fibrin clot formation (Hubbard et al., 1994; Novotny et al., 1991). Tsuji et al. (2001) interestingly showed that TFPI could be used as an anticoagulant for clinical laboratory tests, although remarkable decrease in platelet count was observed with it due to platelet aggregation. However, it is still unclear whether TFPI has direct effects on platelets. Therefore, in the present study, we examined such effects.
2. Materials and methods 2.1. Materials Recombinant human TFPI was kindly donated by Chemo Sero Therapeutic Research Institute (Kumamoto, Japan). TFPI was obtained as a solution containing 99.3 Amol/l in 20 mM citrate, 100 mM NaCl
0022-1759/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 ( 0 2 ) 0 0 1 4 5 - X
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T. Nishioka et al. / Journal of Immunological Methods 266 (2002) 181–184
(pH 7.4). Anti-CD41 monoclonal antibody (clone HIP8, BD PharMingen, San Diego, USA) was obtained as a solution containing 0.5 mg/ml in Trisbuffered saline (pH 8.0). 2.2. Preparation of washed human platelets Blood was carefully collected with a plastic syringe and 22-gauge needle from healthy volunteers (aged 30– 50 years). It was carefully transferred immediately to a blood collection tube (NIPRO, Osaka, Japan) with 3.13% sodium citrate as anticoagulant. Platelet-rich plasma (PRP) was obtained from the collected blood by centrifugation at 80 g for 10 min. The PRP was transferred to another plastic tube. Then, the platelets were washed twice with washing buffer (20 mM HEPES, 145 mM NaCl and 9 mM Na2EDTA, pH 7.4) by centrifugation at 2000 g for 10 min. After washing, the platelets were suspended in Ca2 + -free Tyrode’s buffer (137 mM NaCl, 12 mM NaHCO3, 5.5 mM glucose, 2 mM KCl, 1 mM MgCl2 and 0.3 mM Na2HPO4, pH 7.4). The washed platelet solution was counted by an automated blood cell counter SF-3000 (Sysmex, Kobe, Japan) and was finally adjusted to 3 105/Al.
2.3. Measurement of platelet aggregation Platelet aggregation was measured by an aggregometer (NBS HEMATRACER 801, M C Medical, Tokyo, Japan). The washed platelets were placed in the measurement tube and pre-incubated for 1 min in the incubating hole of the aggregometer. The platelets were moved into the measuring hole and measurement was started, and CaCl2 was then applied at a final concentration of 8 mM as modified Tyrode’s buffer (8 mM CaCl2, 137 mM NaCl, 12 mM NaHCO3, 5.5 mM glucose, 2 mM KCl, 1 mM MgCl2 and 0.3 mM Na2HPO4, pH 7.4) in the aggregometer.
3. Results 3.1. Effect of TFPI on platelet aggregation Platelets suspended in modified Tyrode’s buffer (8 mM CaCl2) exhibited aggregation. With lower calcium ion concentration (6 mM), platelet aggregation was delayed and was weak in intensity. Aggregation was accelerated in the presence of TFPI (22.6 Ag/ml) (Fig. 1).
Fig. 1. Platelet aggregation in modified Tyrode’s buffer. Platelets aggregation occurred with 6 mM CaCl2 (A) and 8 mM CaCl2 (B). This aggregation with 8mM CaCl2 was accelerated in the presence of TFPI (22.6 Ag/ml) (C).
T. Nishioka et al. / Journal of Immunological Methods 266 (2002) 181–184
183
Fig. 2. Enhancement of platelet aggregation by TFPI. Platelet aggregation was stronger in the presence of TFPI [(B) 4.52 Ag/ml, (C) 45.2 Ag/ml] than in the saline control (A).
When platelets were suspended in HEPES buffer instead of Tyrode’s buffer, no platelet aggregation was observed. However, when platelets were suspended in
the HEPES buffer and phosphate and calcium ions were added at the same concentration as contained by the modified Tyrode’s buffer, platelet aggregation was
Fig. 3. Inhibition of platelet aggregation with anti-GPIIb antibody. Platelet aggregation was inhibited by HIP8 anti-GPIIb; (A) saline control, (B) 18 Ag/ml, (C) 36 Ag/ml.
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T. Nishioka et al. / Journal of Immunological Methods 266 (2002) 181–184
observed (data not shown). The observed findings indicated that TFPI enhances platelet aggregation in the presence of phosphate and calcium ions. Enhancement was dose dependently observed with TFPI concentrations of 4.52 –45.2 Ag/ml. In particular, the lag time to initiation of aggregation was shortened by the addition of TFPI (Fig. 2). 3.2. Inhibition of platelet aggregation by addition of anti-GPIIb monoclonal antibody When HIP8 anti-GPIIb (36 Ag/ml) was added to the platelet suspension in modified Tyrode’s buffer, platelet aggregation was eliminated (Fig. 3). This inhibition was expressed as prolongation of the lag time that was dose-dependent with respect to antibody concentration.
4. Discussion Our results clearly show that TFPI enhances platelet aggregation. Platelets are very deformable and are easily activated not only by agonists but even by simple shear stress (Ikeda et al., 1991). Our results indicate that platelet aggregation was induced by shear stress from stirring. Phosphate and calcium ions were essential for this aggregation. Platelet aggregation was inhibited by HIP8 anti-GPIIb antibody. von Willebrand factor, fibrinogen, and fibronectin are known to play roles in platelet aggregation. TFPI is thought to promote interaction among platelets and release of intracellular binding molecules from platelets via GPIIb receptors. This promotion of aggregation by TFPI was observed at a concentration of 4.52 Ag/ml in this study. Although the concentration of TFPI in normal plasma is about 54– 125 ng/ml, it is reported that heparin can induce release of TFPI. Therefore, promotion by TFPI of platelet aggregation may occur in vivo. It is currently thought that TF and TFPI are released from damaged vascular endothelial cells and that interaction between TF and TFPI may regulate thrombus formation in the human body. TF induces thrombosis and TFPI prevents arterial reocclusion after thrombolysis (Haskel et al., 1991). Our
demonstration that TFPI affects not only the coagulation pathway but also platelets may lead to a new concept of blood coagulation in the future.
References Bajaj, M.S., Kuppuswany, M.N., Saito, H., Spitzer, S.G., Bajaj, S.P., 1990. Cultured normal human hepatocytes do not synthesize lipoprotein-associated coagulation inhibitor: evidence that endothelium is the principal site of its synthesis. Proc. Natl. Acad. Sci. U. S. A. 87, 8869. Hansen, J.-B., Huseby, N.-E., Sandset, P.M., Svensson, B., Lyngmo, V., Nordoy, A., 1994. Tissue-factor pathway inhibitor and lipoproteins: evidence for association with and regulation by LDL in human plasma. Arterioscler. Thromb. 14, 223. Haskel, E.J., Torr, S.R., Day, K.C., Palmier, M.O., Wun, T.C., Sobel, B.E., Abendschein, D.R., 1991. Prevention of arterial reocclusion after thrombolysis with recombinant lipoproteinassociated coagulation inhibitor. Circulation 84, 821. Hubbard, A.R., Weller, L.J., Gray, E., 1994. Measurement of tissue factor pathway inhibitor in normal and post-heparin plasma. Blood Coagul. Fibrinolysis 5, 819. Ikeda, Y., Handa, M., Kawano, K., Kamata, T., Murata, M., Araki, Y., Anbo, H., Kawai, Y., Watanabe, K., Itagaki, I., Sakai, K., Ruggeri, Z.M., 1991. The role of von Willebrand factor and fibrinogen in platelet aggregation under varying shear stress. J. Clin. Invest. 87, 1234. Lindahl, A.K., Sandset, P.M., Abildgaard, U., 1992. The present status of tissue factor pathway inhibitor. Blood Coagul. Fibrinolysis 3, 439. Novotny, W.F., Girard, J.P., Miletich, J.P., Broze, G.J., 1989. Purification and characterization of the lipoprotein associated coagulation inhibitor from human plasma. J. Biol. Chem. 264, 18832. Novotny, W.F., Brown, S.G., Miletich, J.P., Rader, D.J., Broze, G.J., 1991. Plasma antigen levels of the lipoprotein associated coagulation inhibitor in patient samples. Blood 78, 387. Sandset, P.M., 1996. Tissue factor pathway inhibitor (TFPI)—an update. Haemostasis 26 (Suppl. 4), 154. Tsuji, R., Tatsumi, N., Hino, M., Nishioka, T., Takubo, T., 2001. Tissue factor pathway inhibitor as a universal anticoagulant for use in clinical laboratory tests. Tohoku J. Exp. Med. 194, 165. Werling, R.W., Zacharski, L.R., Kisiel, W., Bajaj, S.P., Memoli, V.A., Rousseau, S.M., 1993. Distribution of tissue factor pathway inhibitor in normal and malignant human tissues. Thromb. Haemost. 69 (4), 366. Wun, T.-C., Kretzmer, K.K., Girard, T.J., Miletich, J.P., Broze, G.J., 1988. Cloning and characterization of a cDNA coding for the lipoprotein-associated coagulation inhibitor shows that it consists of three tandem Kunitz-type inhibitory domains. J. Biol. Chem. 13, 6001.
Technical note
Tissue factor pathway inhibitor can interact with platelets Takuya Nishioka *, Masaharu Yokota, Masayuki Hino, Izumi Tsuda, Noriyuki Tatsumi Department of Clinical and Laboratory Medicine, Osaka City University Medical School, 1-4-3 Asahimachi, Abeno, Osaka 545-8585, Japan Received 14 January 2002; received in revised form 5 April 2002; accepted 23 April 2002
Abstract Tissue factor pathway inhibitor (TFPI) regulates the extrinsic pathway of blood coagulation. However, there is no report on interaction between TFPI and platelets other than that by Tsuji, who found that whole blood anticoagulated with TFPI exhibited remarkable decrease in platelet count. Our study revealed that washed platelets suspended in modified Tyrode’s buffer (8 mM CaCl2) containing TFPI exhibit platelet aggregation. However, platelets aggregation was observed without TFPI, but its increase and intensity were slow and weak, compared to that in the presence of TFPI. This aggregation was inhibited by antiCD41 (anti-GPIIb) antibody. This finding suggested that TFPI promotes platelet aggregation. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Tissue factor pathway inhibitor; Platelet aggregation; GPIIb/IIIa
1. Introduction Tissue factor pathway inhibitor (TFPI) inhibits the intravascular coagulation induced by tissue factor (TF). TFPI is a 40-kDa glycoprotein with 276 amino acids (Wun et al., 1988), and its normal plasma concentration is about 54 –142 ng/ml (Lindahl et al., 1992). TFPI is produced mostly in endothelial cells (Bajaj et al., 1990; Werling et al., 1993) and pooled in the endothelium (50 – 80%), plasma (10 – 50%) (Sandset, 1996) and platelets (less than 2.5%) (Novotny et al., 1989). Most of TFPI in plasma is complexed with low-density lipoproteins and high-density lipoproteins (Novotny et al., 1989; Hansen et al., 1994). Injection of heparins causes release of TFPI into blood, and this release results in two- to four-fold greater increase in TFPI activity than *
Corresponding author. Tel.: +81-6-6645-3881; fax: +81-66645-3880. E-mail address: [email protected] (T. Nishioka).
that in normal concentration, demonstrating strong anticoagulation activity in regulating fibrin clot formation (Hubbard et al., 1994; Novotny et al., 1991). Tsuji et al. (2001) interestingly showed that TFPI could be used as an anticoagulant for clinical laboratory tests, although remarkable decrease in platelet count was observed with it due to platelet aggregation. However, it is still unclear whether TFPI has direct effects on platelets. Therefore, in the present study, we examined such effects.
2. Materials and methods 2.1. Materials Recombinant human TFPI was kindly donated by Chemo Sero Therapeutic Research Institute (Kumamoto, Japan). TFPI was obtained as a solution containing 99.3 Amol/l in 20 mM citrate, 100 mM NaCl
0022-1759/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 ( 0 2 ) 0 0 1 4 5 - X
182
T. Nishioka et al. / Journal of Immunological Methods 266 (2002) 181–184
(pH 7.4). Anti-CD41 monoclonal antibody (clone HIP8, BD PharMingen, San Diego, USA) was obtained as a solution containing 0.5 mg/ml in Trisbuffered saline (pH 8.0). 2.2. Preparation of washed human platelets Blood was carefully collected with a plastic syringe and 22-gauge needle from healthy volunteers (aged 30– 50 years). It was carefully transferred immediately to a blood collection tube (NIPRO, Osaka, Japan) with 3.13% sodium citrate as anticoagulant. Platelet-rich plasma (PRP) was obtained from the collected blood by centrifugation at 80 g for 10 min. The PRP was transferred to another plastic tube. Then, the platelets were washed twice with washing buffer (20 mM HEPES, 145 mM NaCl and 9 mM Na2EDTA, pH 7.4) by centrifugation at 2000 g for 10 min. After washing, the platelets were suspended in Ca2 + -free Tyrode’s buffer (137 mM NaCl, 12 mM NaHCO3, 5.5 mM glucose, 2 mM KCl, 1 mM MgCl2 and 0.3 mM Na2HPO4, pH 7.4). The washed platelet solution was counted by an automated blood cell counter SF-3000 (Sysmex, Kobe, Japan) and was finally adjusted to 3 105/Al.
2.3. Measurement of platelet aggregation Platelet aggregation was measured by an aggregometer (NBS HEMATRACER 801, M C Medical, Tokyo, Japan). The washed platelets were placed in the measurement tube and pre-incubated for 1 min in the incubating hole of the aggregometer. The platelets were moved into the measuring hole and measurement was started, and CaCl2 was then applied at a final concentration of 8 mM as modified Tyrode’s buffer (8 mM CaCl2, 137 mM NaCl, 12 mM NaHCO3, 5.5 mM glucose, 2 mM KCl, 1 mM MgCl2 and 0.3 mM Na2HPO4, pH 7.4) in the aggregometer.
3. Results 3.1. Effect of TFPI on platelet aggregation Platelets suspended in modified Tyrode’s buffer (8 mM CaCl2) exhibited aggregation. With lower calcium ion concentration (6 mM), platelet aggregation was delayed and was weak in intensity. Aggregation was accelerated in the presence of TFPI (22.6 Ag/ml) (Fig. 1).
Fig. 1. Platelet aggregation in modified Tyrode’s buffer. Platelets aggregation occurred with 6 mM CaCl2 (A) and 8 mM CaCl2 (B). This aggregation with 8mM CaCl2 was accelerated in the presence of TFPI (22.6 Ag/ml) (C).
T. Nishioka et al. / Journal of Immunological Methods 266 (2002) 181–184
183
Fig. 2. Enhancement of platelet aggregation by TFPI. Platelet aggregation was stronger in the presence of TFPI [(B) 4.52 Ag/ml, (C) 45.2 Ag/ml] than in the saline control (A).
When platelets were suspended in HEPES buffer instead of Tyrode’s buffer, no platelet aggregation was observed. However, when platelets were suspended in
the HEPES buffer and phosphate and calcium ions were added at the same concentration as contained by the modified Tyrode’s buffer, platelet aggregation was
Fig. 3. Inhibition of platelet aggregation with anti-GPIIb antibody. Platelet aggregation was inhibited by HIP8 anti-GPIIb; (A) saline control, (B) 18 Ag/ml, (C) 36 Ag/ml.
184
T. Nishioka et al. / Journal of Immunological Methods 266 (2002) 181–184
observed (data not shown). The observed findings indicated that TFPI enhances platelet aggregation in the presence of phosphate and calcium ions. Enhancement was dose dependently observed with TFPI concentrations of 4.52 –45.2 Ag/ml. In particular, the lag time to initiation of aggregation was shortened by the addition of TFPI (Fig. 2). 3.2. Inhibition of platelet aggregation by addition of anti-GPIIb monoclonal antibody When HIP8 anti-GPIIb (36 Ag/ml) was added to the platelet suspension in modified Tyrode’s buffer, platelet aggregation was eliminated (Fig. 3). This inhibition was expressed as prolongation of the lag time that was dose-dependent with respect to antibody concentration.
4. Discussion Our results clearly show that TFPI enhances platelet aggregation. Platelets are very deformable and are easily activated not only by agonists but even by simple shear stress (Ikeda et al., 1991). Our results indicate that platelet aggregation was induced by shear stress from stirring. Phosphate and calcium ions were essential for this aggregation. Platelet aggregation was inhibited by HIP8 anti-GPIIb antibody. von Willebrand factor, fibrinogen, and fibronectin are known to play roles in platelet aggregation. TFPI is thought to promote interaction among platelets and release of intracellular binding molecules from platelets via GPIIb receptors. This promotion of aggregation by TFPI was observed at a concentration of 4.52 Ag/ml in this study. Although the concentration of TFPI in normal plasma is about 54– 125 ng/ml, it is reported that heparin can induce release of TFPI. Therefore, promotion by TFPI of platelet aggregation may occur in vivo. It is currently thought that TF and TFPI are released from damaged vascular endothelial cells and that interaction between TF and TFPI may regulate thrombus formation in the human body. TF induces thrombosis and TFPI prevents arterial reocclusion after thrombolysis (Haskel et al., 1991). Our
demonstration that TFPI affects not only the coagulation pathway but also platelets may lead to a new concept of blood coagulation in the future.
References Bajaj, M.S., Kuppuswany, M.N., Saito, H., Spitzer, S.G., Bajaj, S.P., 1990. Cultured normal human hepatocytes do not synthesize lipoprotein-associated coagulation inhibitor: evidence that endothelium is the principal site of its synthesis. Proc. Natl. Acad. Sci. U. S. A. 87, 8869. Hansen, J.-B., Huseby, N.-E., Sandset, P.M., Svensson, B., Lyngmo, V., Nordoy, A., 1994. Tissue-factor pathway inhibitor and lipoproteins: evidence for association with and regulation by LDL in human plasma. Arterioscler. Thromb. 14, 223. Haskel, E.J., Torr, S.R., Day, K.C., Palmier, M.O., Wun, T.C., Sobel, B.E., Abendschein, D.R., 1991. Prevention of arterial reocclusion after thrombolysis with recombinant lipoproteinassociated coagulation inhibitor. Circulation 84, 821. Hubbard, A.R., Weller, L.J., Gray, E., 1994. Measurement of tissue factor pathway inhibitor in normal and post-heparin plasma. Blood Coagul. Fibrinolysis 5, 819. Ikeda, Y., Handa, M., Kawano, K., Kamata, T., Murata, M., Araki, Y., Anbo, H., Kawai, Y., Watanabe, K., Itagaki, I., Sakai, K., Ruggeri, Z.M., 1991. The role of von Willebrand factor and fibrinogen in platelet aggregation under varying shear stress. J. Clin. Invest. 87, 1234. Lindahl, A.K., Sandset, P.M., Abildgaard, U., 1992. The present status of tissue factor pathway inhibitor. Blood Coagul. Fibrinolysis 3, 439. Novotny, W.F., Girard, J.P., Miletich, J.P., Broze, G.J., 1989. Purification and characterization of the lipoprotein associated coagulation inhibitor from human plasma. J. Biol. Chem. 264, 18832. Novotny, W.F., Brown, S.G., Miletich, J.P., Rader, D.J., Broze, G.J., 1991. Plasma antigen levels of the lipoprotein associated coagulation inhibitor in patient samples. Blood 78, 387. Sandset, P.M., 1996. Tissue factor pathway inhibitor (TFPI)—an update. Haemostasis 26 (Suppl. 4), 154. Tsuji, R., Tatsumi, N., Hino, M., Nishioka, T., Takubo, T., 2001. Tissue factor pathway inhibitor as a universal anticoagulant for use in clinical laboratory tests. Tohoku J. Exp. Med. 194, 165. Werling, R.W., Zacharski, L.R., Kisiel, W., Bajaj, S.P., Memoli, V.A., Rousseau, S.M., 1993. Distribution of tissue factor pathway inhibitor in normal and malignant human tissues. Thromb. Haemost. 69 (4), 366. Wun, T.-C., Kretzmer, K.K., Girard, T.J., Miletich, J.P., Broze, G.J., 1988. Cloning and characterization of a cDNA coding for the lipoprotein-associated coagulation inhibitor shows that it consists of three tandem Kunitz-type inhibitory domains. J. Biol. Chem. 13, 6001.