SYNERGISTIC STIMULATION OF FIBRINOGEN GELATION BY CASEIN KINASE II AND POLYCATIONIC COMPOUNDS. Kyoungho Suk, Jin-Young Lee, and Seung-Ho Kim*

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1 Vol. 42, No. 3, July 1997 Pages SYNERGISTIC STIMULATION OF FIBRINOGEN GELATION BY CASEIN KINASE II AND POLYCATIONIC COMPOUNDS Kyoungho Suk, Jin-Young Lee, and Seung-Ho Kim* Protein Function Research Unit, Korea Research Institute of Bioscience and Biotechnology, KIST, Taejon, 35-6 Korea Received April 1, 1997 SUMMARY The processes of thrombin-induced gelation of the fibrinogen may be influenced by a variety of factors including phosphorylational state of the fibrinogen. Casein kinase II (CKII) has been shown to phosphorylate fibrinogen in vitro and accelerate gelation of the fibrinogen. In this work, we have demonstrated that CKII and polycationic compounds such as polylysine, spermine, and spermidine synergistically stimulate thrombininduced gelation of the fibrinogen. These polycationic compounds were also found to increase the stimulatory effects of CKII on the platelet aggregation. Current findings suggest potential role of CKII and polycationic compounds in the process of hemostasis. Keywords: casein kinase II, polycationic compound, fibrinogen, hemostasis INTRODUCTION Human fibrinogen is a phosphoprotein composed of six polypeptide chains with the structure of A~2Bf~272 (1). Fibrinogen contains most of its phosphates in the m-chain on various serine residues. When thrombin proteolytically removes the A and B peptides from the fibrinogen, fibrin monomers are formed which polymerize to form a fibrin mesh (2). Fibrinogen's protein-bound phosphate has been recently shown to reversibly affect the thickness of the gel fibres in the thrombin-induced gelation assay (3). Several protein kinases have been shown to phosphorylate fibrinogen in vitro. These include protein kinase A (4), protein kinase C (5), casein kinase I (CKI) (6), and casein kinase II (CKII) (7). Phosphorylation of fibrinogen by these kinases differentially affected sensitivity of fibrinogen to plasmin degradation as well as fibrin fibre *To whom correspondence should be addressed (Tel: , Fax: ) /97/3487~955./ Copyright by Academic Press Australi~. All rights of reproduction in any form reserved.

2 thickness. Among these kinases, CKII has been shown to phosphorylate fibrinogen at Ser 523, Ser 59 and serine and threonine residues between amino acids 259 and 268 in As chain (8). This phosphorylation led to a slight but consistent increase of the turbidity in a gelation assay compared to the unphosphorylated (8). Phosphorylation of the fibrinogen by these kinases appears to be an extracellular event, and the importance of this extracellular phosphorylation event in the blood has been previously suggested (9-11). CKII, a widely expressed eukaryotic kinase directed to phosphorylate Ser/Thr residues upstream of a cluster of acidic residues, is an ~2~ 2 complex whose activity is high in the cytosol and the nucleus of proliferating and differentiating cells (12). The catalytic ~ subunit is stimulated several-fold by regulatory f~ subunit, which undergoes autophosphorylation. The enzyme is unusual in utilizing either ATP or GTP efficiently as phosphoryl donor. The activity of the kinase is stimulated in vitro by polycationic compounds and inhibited by polyanions, but the mechanism of this regulation is not clearly understood (13). The kinase phosphorylates a number of substrates including cytoplasmic as well as nuclear proteins (12). Here we examined how polycationic compounds as an activator of CKII participate in the processes of thrombin-induced gelation of fibrinogen as well as platelet aggregation. CKII synergistically stimulate these processes. Our results indicated that polycations and MATERIALS AND METHODS Materials Fibrinogen, thrombin, CKII, polylysine, spermine, and spermidine were purchased from Sigma (St. Louis, MO, U.S.A.). Venous blood was obtained from healthy volunteers (age range years) who had not taken any drugs two weeks prior to the study. Assessment of whole blood coagulation The.8 ml of thrombin (.25 NIH-U/ml), either with or without CKII (33 nu/ml), dissolved in 1 mm Tris-HC1 buffer (ph 7.4) containing 12 mm MgC12 and 1 mm ATP was added to 1 ml of whole blood, then incubated at 25~ for 5 min to monitor blood coagulation. Fibrinogen gelation assay Fibrinogen (1.2 mg/ml) was dissolved in 1 mm Tris-C1 buffer (ph 7.4) containing 12 mm MgC12. The.7 ml of this fibrinogen solution was mixed with.3 ml of various combinations of thrombin (1 NIH-U/ml), CKII (4 nu/ml), polylysine (.5 to.25 mg/ml), spermine (.2 mg/ml), and spermidine (.2 mg/ml). The mixture was then incubated at 25~ and absorbance at 633 nm measured to monitor turbidity changes. 488

3 Vol. 42, No, 3, 1997 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL Measurement of platelet aggregation Washed human platelets were obtained from freshly drawn blood using an albumin density gradient method (14). The.4 ml of washed platelet suspension (7 x 18 platelets/ml) in Tris-saline buffer (ph 7.4) was prewarmed at 37~ for 5 min. To this,.1 ml of thrombin (4 NIH-U/ml), CKII (4 nu/ml), polylysine (.2 mg/ml), or spermidine (.2 mg/ml) was added. To measure platelet aggregation, the decreases in absorbance at 69 nm were recorded by aggregometer (Chronoqog Co.). Absorbance values were converted to percent aggregation as described (15). The results of platelet aggregation were also expressed as the slope of aggregation curve. Slope was determined by drawing a tangent to the steepest part of the curve. A right triangle was then constructed over an interval of one minute. The height of the triangle was the rate of change of aggregation in one minute, which was defined as the slope (16). RESULTS AND DISCUSSION Stimulation of blood coagulation by CKII CKII is known to phosphorylate fibrinogen in vitro and this phophorylation is believed to enhance gelation of the fibrinogen, which may lead to stimulated blood clot formation (8). Thus, we first sought to determine whether CKII could influence the process of whole blood coagulation. The blood treated with both thrombin and CKII showed much faster coagulation compared to the blood treated with thrombin alone (Fig. 1). The blood treated with both thrombin and CKII coagulated within 5 min incubation, whereas the blood treated with thrombin alone remained uncoagulated within this time frame, demonstrating the stimulatory effect of CKII on the blood coagulation processes. This suggested that CKII may be involved in the regulation of hemostatic system. Synergistic stimulation of thrombin-induced gelation of fibrinogen by CKII and polycationic compounds Polymerization of the fibrin is one of the critical steps of blood coagulation process (2). CKII has been reported to enhance fibrinogen gelation (8). Also, polylysine and naturally occurring polyamines, spermine and spermidine have been shown to increase the activity of CKII (13, 17, 18). Thus, we next tested whether these polycationic compounds affect the activity of CKII to stimulate gelation of the fibrinogen. CKII alone, as previously reported (8), stimulated thrombin-induced gelation of the fibrinogen (Fig. 2). While neither polylysine nor polyamines alone had 489

4 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL A B : r F , ~ ~. Figure 1. CKII stimulation of'whole blood coagulation. Freshly drawn whole blood was incubated either with thrombin alone (A) or with CKII and thrombin (B) for 5 min, then the degree of blood coagulation was compared. While the blood treated with both CKII and thrombin coagulated, the blood treated with thrombin alone remained uncoagulated at 5 min incubation. The result is a representative of three separate experiments. Similar results were obtained from all three experiments..3- t~ E.2".1.. I I I I Time (rain) Figure 2. Effects of CKII or polycatinic compounds on thrombin-induced gelation of the fibrinogen. Thrombin-treated fibrinogen was incubated alone (O) or mixed with CKII (4 nu/ml) (O), spermine (.2 mg/ml) (A), polylysine (.2 mg/ml) (A), or spermidine (.2 mg/ml) (!"3) before incubation at 25~ and absorbance measurement at 633 nm. Error bar represents standard error of mean. 49

5 significant effects on thrombin-induced gelation of the fibrinogen (Fig. 2), these compounds enhanced the stimulatory effects of CKII on fibrinogen gelation (Fig. 3). Although there were differences in the magnitude of enhancing effect among polycationic compounds tested, all of them showed synergistic effects with CKII. Furthermore, as represented by the experiment with polylysine, the synergistic effects of polycationic compounds and CKII were exerted in a polycation-dose-dependent manner (Fig. 4). Synergistic effects of CKH and polycationic compounds on the platelet aggregation Based on our findings that CKII and polycations synergistically stimulate fibrinogen gelation, we extended our studies by investigating the effects of these compounds on the platelet aggregation. Like fibrinogen gelation, platelet aggregation was similarly stimulated by these compounds (Fig. 5). The effect of CKII and polycationic compounds on the platelet aggregation was synergistic and the degree of stimulatory effect of each.4.3 t~ 3.2.I. L l I I Time (min) Figure 3. Synergistic stimulation of thrombin-induced fibrinogen gelation by CKII and polycationic compounds. In order to examine the effects of CKII and polycationic compounds on fibrinogen gelation, thrombin-treated fibrinogen was incubated alone (O) or mixed with CKH (4 nu/ml) (O), CKII plus spermine (.2 mg/ml) (A), CKII plus polylysine (.2 mg/ml) (A), or CKII plus spermidine (.2 mg/ml) (I-q) before incubation at 25~ and absorbance measurement at 633 nm. Error bar represents standard error of mean. 491

6 .4 fi.3 c~ r.2.1. i i i i Time (min) Figure 4. Dose-dependent effects of polylysine on CKII stimulation of fibrinogen gelation. Thrombin-treated fibrinogen was mixed with CKII (4 nu/ml) and increasing concentrations of polylysine (.5 mg/ml, /k;.1 mg/ml, A;.25 mg/ml, 1"7), followed by incubation and absorbance measurement. As a comparison, thrombin-treated fibrinogen was incubated alone (O) or mixed only with CKII () before gelation assay. Error bar represents standard error of mean. compound on the platelet aggregation correlated with that of its effect on fibrinogen gelation. Platelet aggregation is the process by which platelets interact with one another to form a hemostatic plug or thrombus. The essential role of fibrinogen in platelet aggregation has been recognized for more than 3 years (19). The dimeric structure and twofold symmetry render fibrinogen uniquely suited to serve as a molecular bridge from platelet to platelet. It has been also demonstrated that platelets interact specifically with polymerizing fibrin (2). Thus, the effect of CKII and polycationic compounds on the platelet aggregation may be due to their effects on fibrinogen gelation. This is supported by our findings that the processes of fibrinogen gelation and platelet aggregation were similarly modulated by CKII and polycationic compounds. It is becoming clearer that protein phosphorylation is not merely an intracellular process, but can also occur extracellularly in the blood. The presence of plasma protein kinases and ~tm levels of plasma ATP have been previously demonstrated (1, 21). Furthermore, platelets have been shown 492

7 Time (min) Time (min) Figure 5. Synergistic stimulation of platelet aggregation by CKII and polycationic compounds. Washed platelet suspension containing thrombin (4 NIH-U/ml) was analyzed alone (A) or mixed with CKII (4 nu/ml) (B), CKII plus polylysine (.2 mg/ml) (C), or CKII plus spermidine (.2 mg/ml) (D) before the analysis of turbidity change by measuring absorbance at 69 nm. The result shown is a representative of three independent experiments. The mean slopes of aggregation curve from three experiments were: (A), ; (B), ; (C), 74 +_ 5; (D), , respectively. Slopes were determined by drawing a tangent to steepest part of the curve. Values are mean + standard deviation. Z O m "T" r'n bo --I O r'n O o O Z --t m Z o Z = a~ A 25 B l= 25 ~ 5 "~ 5 :i;:: e~ 75 ; ~ :: 75 2 C ~ Time (min) Time (min) D ~ 25 ~ ~ 5 "~

8 to release protein kinase when activated by thrombin (22), and cell surface CKII activity has been reported to phosphorylate both fibrinogen and fibrin (9). Thus, as observed in the current work, stimulation by CKII and polycationic compounds of fibrinogen gelation as well as platelet aggregation suggests physiological importance of CKII as an extracellular regulator of hemostasis. And, this activity of CKII could be further regulated by polycationic compounds that may be present in the blood. Although it has been reported that polylysine may stimulate CKII activity by a polylysine-induced aggregation of substrates (18), the precise mechanism of CKII activation by polycationic compounds remains to be elucidated. Whatever the mechanism of this activation is, modulation of CKII activity may lead to the alteration in the phosphorylational state of the fibrinogen. This in turn may influence fibrin polymerization, platelet aggregation, and ultimately the hemostatic system. ACKNOWLEDGMENTS This work was supported by a grant from the Ministry of Science and Technology, Korea. REFERENCES 1. McKee, P. A., Rogers, L. A., Marler, E., and Hill, R. L. (1966) Arch. Biochem. Biophys. 116, Davie, E. W., Fujikawa, K., and Kisiel, W. (1991) Biochemistry 3, Forsberg, P.-O. (1989) Thromb. Res. 53, Engstrom, L., Edlund, B., Ragnarsson, U., Dahlqvist-Edberg, U., and Humble, E. (198) Biochem. Biophys. Res. Commun. 96, Papanikolaou, P., Humble, E., and Engstrom, L. (1982) FEBS Lett. 143, Itarte, E., Plana, M., Guash, M. D., and Martos, C. (1983) Biochem. Biophys. Res. Commun. 117, Humble, E., Heldin, P., Forsberg, P.-O., and Engstrom, L. (1985) Arch. Biochem. Biophys. 241, Heldin, P. (1987) Arch. Biochem. Biophys. 257, Sonka, J., Kubler, D., and Kinzel, V. (1989) Biochim. Biophys. Acta 997, Chiang, T. M., and Kang, A. H. (1985) Arch. Biochem. Biophys. 243, Walter, J., Schnolzer, M., Pyerin, W., Kinzel, V., and Kubler, D. (1996) J. Biol. Chem. 271, Allende, J. E., and Allende, C. C. (1995) FASEB J. 9, Gatica, M., Jacob, G., Allende, C. C., and Allende, J. E. (1995) Biochemistry 34,

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