On the interaction of F-actin with fibrin

Biochimica et Biophysica Acta, 371 (1974) 519-525

© Elsevier Scientific Publishing Company, Amsterdam - - Printed in The Netherlands BBA 36866 ON T H E I N T E R A C T I O N OF F-ACTIN W I T H FIBRIN

K. LAKI and L. MUSZBEK* National Institute of Arthritis, Metabolism and Digestive Diseases National Institutes of Health, Public Health Service U.S. Department of Health, Education and Welfare Bethesda, Md. 20014 (U.S.A.)

(Received May 2nd, 1974)

SUMMARY It was found that F-actin combined with fibrin but not with fibrinogen. At saturating concentrations, two actin molecules were found to combine with one fibrin molecule. These experiments also revealed that under certain conditions thrombin was capable of fragmenting actin.

INTRODUCTION According to prevailing theory, blood clot retraction, a contractile process, involves platelet actomyosin [1-3], though the actual interaction between contractile proteins and the fibrinogen-fibrin system has never been investigated. Since platelets contain fibrous actin, we undertook to explore whether F-actin interacts with fibrin. In this paper, we present experiments which demonstrate that F-actin combines with fibrin, but not with fibrinogen. MATERIALS AND METHODS In the experiments reported below, bovine fibrinogen was prepared by the method of Laki [4] and kept frozen at --10 °C. The acetone powder from rabbit muscle was made by the method of Feuer et al. [5]. G-Actin was obtained from the acetone powder by centrifuging F-actin and dialyzing the pellet against water containing ATP in 3-fold excess. The G-actin was then lyophilized and kept below --5 °C. Purer preparations were obtained from the acetone powder by the method of Spudich and Watt [6]. The polymerized F-actin pellets were converted to G-actin by dissolving and dialyzing them in 2 mM Tris-HCl, pH 8.0, buffer containing mercaptoethanol (0.2 mM) ATP (0.5 mM) with or without CaCI2 (0.2 raM). Parke-Davis bovine thrombin was purified by the chromatographic procedure of Rasmussen [7]. The final thrombin solution contained 0.49 mg/ml protein and the active site titration method [8] revealed these thrombin preparations to be 76 7o pure. Sodium dodecylsulfate polyacrylamide (7.5 ~ ) gel electrophoresis was carried out according to the procedure of Weber and Osborn [9]. * Visiting Associate. Permanent Address: Institute of Pathophysiology, Medical School, University of Debrecen, Hungary.

520 P r o t e i n c o n c e n t r a t i o n s w e r e d e t e r m i n e d b y t h e b i u r e t m e t h o d . F u r t h e r experim e n t a l d e t a i l s a r e g i v e n i n t h e l e g e n d s t o t h e figures. RESULTS A N D DISCUSSION W h e n a c t i n a n d f i b r i n o g e n a r e m i x e d i n 0.15 M K C l s o l u t i o n , a c t i n p o l y m e r i z e s and can be sedimented in the preparative ultracentrifuge leaving fibrinogen in the s u p e r n a t a n t u n a l t e r e d . T h e c l o t t i n g t i m e o f t h e f i b r i n o g e n a n d t h e u r e a s o l u b i l i t y of t h e t h r o m b i n - p r o d u c e d c l o t r e m a i n e d u n c h a n g e d . T h i s is a c l e a r i n d i c a t i o n t h a t F-actin and fibrinogen do not interact. O n t h e o t h e r h a n d , i f t h r o m b i n is a d d e d t o t h e f i b r i n o g e n - a c t i n m i x t u r e , t h e A !

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Fig. 1. The reaction mixtures (final volume 1.5 ml) contained 5 mg fibrinogen and/or various amounts of actin (in the range of 0-7.6 mg) in 1.0 ml of Ca:+-free or Ca2+-containing buffers. The final salt concentration of each solution was adjusted to 0.1 M by the addition of KCI. 20/d of thrombin or appropriate phosphate buffer was added to the mixtures and were mixed immediately by a vortex mixer. The final pH of the solutions was 7.2. Each of the fibrinogen-containing samples clotted within 5 s. After incubation for 1 h at room temperature (23 °C), fibrin clots were loosened and centrifuged at 9000 x g for 30 min or carefully compressed by a spatula. Samples containing actin without fibrin were centrifuged as described or ultracentrifuged at 144 000 x g for 1 h. Supernatants or clot liquors were analyzed for protein content. Pellets or the compressed clots were dissolved in 2.5 ml of denaturing solution (6 M urea 3 ~ sodium dodecylsulfate and 3 ~ mercaptoethanol in 0.04 M phosphate buffer, pH 7.1) and 10 #1 aliquots of the samples were applied to polyacrylamide gel electrophoresis. A : Fibrin-actin-thrombin interaction in the presence of Ca 2+. A1 : The a, fl and the dimer of the 7chains of fibrin (showing the presence of Ca 2+ activated Factor XIII). A2: The same as A1 but containing actin, a-polymers appear on account of the diminution of the a-chain. This is probably due to actin promoting Factor XIII or the actin preparation containing traces of tissue transglutaminase. This effect is very small and we have disregarded it in the interpretations of the experiments. A3 and A4: Actin alone and actin incubated with thrombin (A2, A3 and A4 contained 7.6 mg actin in the incubation mixture). B: Fibrin-actin-thrombin interaction without Ca 2+ in the buffer. BI: The 3 chains of fibrinogen. B2: The a,/3 and 7 chains of fibrin. B3: The 3 chains of fibrin, together with actin and heavy (H) and light (L) split products of actin. B4: Actin alone. B5: Actin incubated with thrombin showing the thrombin-induced split products of actin (B3, B4 and B5 contained 4.4 mg actin in the incubation mixture).

521 situation is different (legend to Fig. 1). Two different tests show that F-actin becomes part o f the clot. (a) W h e n the mixed clot is compressed or separated f r o m the clot liquor by centrifugation at 9000 × g, the adherence of actin to the fibrin network is revealed by the absence o f protein in the clot liquor. (b) W h e n the compressed, mixed clots are dissolved in the "denaturing medium" (see legend to Fig. 1) and subjected to sodium dodecylsulfate gel electrophoresis, not only the bands corresponding to fibrin, but also the band corresponding to actin, were evident. The following experiments provide further evidence that F-actin is b o u n d to fibrin, not just occluded in the clot. The mixed clot m a y be dissolved in 6 M urea solution, and when the clot reforms after dialyzing out the urea, the restituted clot again contains the a m o u n t o f actin (e.g. 4 mg actin per 15 mg fibrin) added to it. In urea solution both F-actin and fibrin depolymerize, yet when the clot reforms, it contains actin, suggesting that the association between monomeric F-actin and fibrin molecules is not broken. The specificity o f the binding o f actin is indicated by the observation that ovalbumin o f the size of actin m o n o m e r s is not b o u n d to fibrin. This is seen when fibrinogen mixed with varying amounts of ovalbumin is clotted. All the ovalbumin remains in the clot liquor after compressing the clot. A study of the effect of varying a m o u n t s o f F-actin on the combination indicates a stoichiometric interaction between the two proteins. It is seen from the two experiments presented in Fig. 2 that actin remains in the clot liquor only if more than 0.03 #mole o f actin were added to 0.015 pmole of fibrinogen. Similarly, at higher

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Fig. 2. Estimation of the stoichiometry of actin-fibrin combination. Various concentrations of Factin (in 0.1 M KCI) were added to 0.015~tM fibrinogen and subsequently were clotted by 20pl thrombin (final volume 1.5 ml). Two approaches were used to obtain the stoichiometry of actinfibrin combination. (1) After adding 2.5 ml water to the clots, they were dispersed by vortex mixer and dialyzed against water. (2) The clots were compressed and rinsed with 0.1 M KCI. Then they were placed into 4 ml water and compressed repeatedly with glass rods. In both cases after various time intervals, the samples were centrifuged and protein content of the supernatants was determined. Since fibrin under these conditions does not release protein, the protein content of the supernatants was taken to be actin. The stoichiometry of actin-fibrin association was calculated after no further release of protein could be detected. F-Actin originally present in the incubation mixture was plotted against/~M F-actin released (O I ) and against actin remaining in the clot; i.e. total amount of actin minus released actin (© D). Squares with dotted lines represent results obtained by Method l; circles with solid lines by Method 2. Arrows indicate the concentration of fibrin monomers.

522 actin concentrations (if enough time is given to the excess actin to leak out) again 0.03 #mole remain in the clot. Apparently F-actin bound directly to the fibrin in stoichiometric proportion is withheld while what is unbound is released into the clot liquor. These data indicate that 2 actin molecules bind strongly to 1 fibrin molecule (taking 42 000 for the molecular weight of actin and 330 000 for the duplex fibrin molecule). This stoichiometry of the actin-fibrin combination again suggests a specific combination of the two proteins and from this point of view resembles the actomyosin complex where 2 actin molecules bind to a duplex myosin molecule. In order to settle whether thrombin is needed only for the generation of fibrin

Fig. 3a.

523

Fig. 3. For the electron microscopy, two reaction mixtures were prepared : one containing fibrinogen, the other containing both fibrinogen and actin. The total volume of the reaction mixtures was 2 ml, the KC1 concentration 0.13 M. The mixtures contained 8.3 mg fibrinogen (1A and B) and 4.8 mg actin (B). The fibrinogen-containing mixtures were clotted with 10/~1 of thrombin. After standing at room temperature for about 2 h, the samples were prepared for electron microscopy the following way: The clots were fixed in 2.5 or 3 ~ glutaraldehyde in cacodylate buffer for 45 min. The samples were then rinsed three times in cacodylate buffer and post-fixed for 1 h in Dalton's chrome osmium. A drop of 2 % agar was used to hold the very diffuse pellets together just before post-fixation. Tissue was then rinsed in 1 ~ aqueous uranyl acetate for 1 h, dehydrated in alcohol and embedded in eponaraldite. Blocks were sectioned on an LKB ultra microtome III and screened and photographed on an Hitachi HU-11-E electron microscope. A. Fibrin alone. B. Fibrin + F-actin.

524 and not for the fibrin-actin combination, fibrinogen was first clotted (15 mg fibrinogen and 60 #l thrombin in 0.3 ml, 0.15 M KC1 solution) and after inactivating thrombin with diisopropylphosphorofluoridate [10], the clot was dissolved in 6 M urea containing actin (4.4 mg actin in 3.7 ml 6 M urea). When the mixture was dialyzed to remove urea, the clot reformed and retained the actin added to it showing that thrombin was not involved in the binding of actin to fibrin. A different type of experiment gives further indication that fibrin and F-actin interact. When the electron-microscopic pictures (Fig. 3) of the fibrin clot and the clot in the presence of F-actin are compared, it is clearly evident that in the mixed clot, the fibrin strands are narrower. When we compared the distribution of the width of the strands on several of our electron-microscopic pictures, we found that in the presence of F-actin, the majority of the strands had widths around 270 and 570 A. In contrast, in fibrin clots, the strands having widths 580, 760 and 1020/k were predominant. Apparently the presence of F-actin hinders the aggregation of the fibrin strands. It seems likely that as fibrin molecules are formed by thrombin, these become attached to pairs of actin in the actin filaments. This would suggest that the primary fibrin strands in the presence of F-actin arise through side-to-side association guided by the actin filaments. The thin filaments seen on Fig. 3B do not resemble the electron microscopic pattern of F-actin gel treated similarly (not shown on Fig. 3). We suspect that these represent protofilaments of end-to-end aggregating fibrin molecules loosely attached to F-actin filaments. During the experiments presented in this paper, we noticed that thrombin was capable of fragmenting actin (see Fig. IB). The proteolytic action of thrombin on actin was also observed by Shainoff [11]. We are presenting the details of the actinthrombin interaction in a separate communication [12]. The experiments discussed above clearly indicate an interaction between fibrin and F-actin. The specificity of this interaction is evident from the observation that only fibrin but not fibrinogen binds F-actin and that ovalbumin, of the size of actin monomers, does not bind to fibrin. The binding of F-actin to fibrin molecules but not to fibrinogen may be connected to the role of the platelets in clot retraction. It may be important for platelets no to have their actin filaments tied up to fibrinogen before these participate in clot retraction. ACKNOWLEDGEMENTS The electron microscopic studies were carried out with the help of Dr William T. Hall and Mrs Barbara Hobbs at Electro-Nucleonics Laboratories, Inc., Bethesda, Md. REFERENCES 1 Bettex-Galland, M. and Lfischer, E. F. (1959) Nature 184, 276-277 2 Kuhnke, E. (1962) Naturwissenschaften 49, 60-61 3 Lfischer, E. F. and Bettex-Galland, M. (1971) In Circulating Platelets, (Johnson, S. A., ed.), pp. 225-239, Academic Press, New York 4 Laki, K. (1951) Arch. Biochem. Biophys. 32, 317-324 5 Feuer, G., Molnar, F., Pettko, E. and Straub, F. B. (1948) Hung. Acta Physiol. l, 150-163

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