Studies on platelet proteins

Biochimica et Biophysica Acta, 328 (1973) 448-455 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

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STUDIES ON P L A T E L E T PROTEINS IX. T H E I D E N T I T Y OF F I B R I N O G E N

HAROLD L. JAMES AND P A N K A J GANGULY

Laboratory of Hematology, St. Jude Children's Research Hospital, 332 North Lauderdale, P.O. Box 318, Memphis, Tenn. 38±oz (U.S.A.) (Received June i8th, 1973)

SUMMARY

The molecular differences between human platelet fibrinogen and plasma fibrinogen observed previously were further investigated to determine whether the platelet protein might be a proteolytic derivative of its plasma counterpart. Purified plasma fibrinogen labeled with 125I was added to washed, intact platelets, and the total fibrinogen was isolated in the presence of proteolytic inhibitors by the same procedure used for platelet fibrinogen alone. Comparison of control ~25I-labeled plasma fibrinogen, total fibrinogen, and platelet fibrinogen by sodium dodecylsulfate gel electrophoretic staining patterns, as well as the radioactivity distribution for control and total fibrinogen, showed no conversion of the added plasma fibrinogen to platelet fibrinogen. Furthermore, the electrophoretic pattern of platelet fibrinogen was different from the slowly clottable plasmin derivative of plasma fibrinogen, termed Fragment X. It is concluded that platelet fibrinogen is not derived, during the course of its isolation, from plasma fibrinogen.

INTRODUCTION

Human blood platelets contain a thrombin-clottable component which comprises about 15% of the total protein1, 2. This material, referred to as platelet fibrinogen, shares some of the properties of plasma fibrinogen such as clottability, electrophoretic mobility under standard conditions, and immunologic characteristics1, 3-6. Fibrinogen is located in two compartments of platelets, the membrane and granular fractions ~-9. Using radioactively or fluorescein-labeled plasma fibrinogen, no exchange between these external and internal fractions was observe&, 8. The membrane-associated material is generally assumed to be adsorbed plasma fibrinogen. Earlier studies have revealed some distinct differences between platelet and plasma fibrinogens. Platel et fibrinogen is not completely coagulated even by high concentrations of thrombin, and its carbohydrate content, sedimentation behavior, and intrinsic viscosity were shown to differ from those of plasma fibrinogen 7,1°,n.

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Fibrinogen is absent from the platelets of some patients with thrombasthenia whose plasma fibrinogen levels are normaF. Furthermore, platelets are capable of synthesizing proteins, including fibrinogenTM. We have recently shown that, compared to plasma fibrinogen, platelet fibrinogen isolated in the presence of suitable proteolytic inhibitors has a lower molecular weight, different characteristics of interaction with thrombin, a different pattern of derivatives formed by complete digestion with plasmin, and a non-identical type of subunit structure~L Platelets are known to contain very active proteolytic enzymes, some of which are not well understood. Thus, the platelet extract represents a precarious milieu for isolation of a protein. In spite of the use of proteolytic inhibitors, the important question remaining after all previous studies was whether isolated platelet fibrinogen is a proteolytically modified product of plasma fibrinogen. The present study was undertaken to answer this question. The approach involved (a) addition of different amounts of purified, 125I-labeled plasma fibrinogen to washed platelets; (b) isolation of the total fibrinogen fraction from the platelet extract using glycine; (c) comparison of the sodium dodecylsulfate gel electrophoretic staining pattern of the total fibrinogen preparations with control plasma and platelet fibrinogen glycine isolates; and (d) comparison of the sodium dodecylsulfate gel electrophoretic distribution of radioactivity for a total fibrinogen preparation with that for control l~5I-labeled plasma fibrinogen. If platelet fibrinogen is actually modified plasma fibrinogen, then the total fibrinogen fraction would behave as purified platelet fibrinogen. If not, plasma fibrinogen would retain its identity. Furthermore, the electrophoretic patterns of Fragment X, the "early" plasmin derivative of plasma fibrinogen~3, and platelet fibrinogen were compared. The results show that platelet fibrinogen has a separate identity and is not a proteolytic product of plasma fibrinogen. M A T E R I A L S AND METHODS

Preparation of platelets Platelet concentrates were prepared by differential centrifugation of acid citrate-dextrose-anticoagulated human blood. Platelets separated from these concentrates by centrifugation were washed with 1% ammonium oxalate-o.I% EDTA (pH 7.5), to remove residual plasma and red cells14. Following a single wash with o.i M Tris buffer (pH 7.5)-o.1 M NaC1, the platelets were finally resuspended in 5 ml of the same buffer containing IOOO kallikrein inhibitor units (o.I ml) of Trasylol (FBA Pharmaceuticals, N.Y.), 5 mg of a-N-CBZ-L-glutamyl-L-tyrosine (Schwarz/ Mann, Orangeburg, N.Y.) and 30 mg of iodoacetamide (Sigma Chemical Co., St. Louis, Mo.) per unit of platelets.

Preparation of 125I-labeled human plasma fibrinogen Purified human plasma fibrinogen [Grade L (AB Kabi, Sweden)l with a clottability of at least 90% was labeled with 1~I by the IC1 method of McFarlane 15. Approx. 50 mg (2.5 ml of a 2% solution) of fibrinogen was reacted with io mCi of carrier-free Na 1~I (Schwarz/Mann, Orangeburg, N.Y.) in the presence of working IC1 solution 15 equivalent to io/,moles of iodine. Unreacted ~2~I was removed by dialysis. Radioactivity assays of the dialysis medium and labeled fibrinogen revealed that approx. 50% of the tyrosine residues per molecule had bound radioactivity. The

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observed specific activity was 11.43" Io 7 cpm/mg protein. Attachment of radioactivity to this extent was considered necessary in order to detect even a small modification of the added fibrinogen. The labeled fibrinogen was found to be identical to the unlabeled starting material with respect to sodium dodecylsulfate gel electrophoretic pattern, immunologic reaction against anti-human fibrinogen rabbit serum, and percent clottability. In order to retard radiation damage, 5 ml (IOO mg) of cold fibrinogen was added and the solution was stored in polypropylene tubes at - - 2 0 °C. The observed specific activity of this solution, which was used in the present experiments, was 4.04. lO 7 cpm/mg protein.

Isolation of fibrinogen The platelet extract 11 was prepared in the absence and presence of l~5-I-labeled plasma fibrinogen(5 and IO~'o of the theoretical amount of platelet fibrinogen per platelet unit) with inhibitors as described above. The extract was centrifuged at 25 ooo rev./min in the Beckman 30 rotor for 20 min at 5 °C to remove residual cellular debris. Iodoacetamide, 30 mg/platelet unit, was added to the extracts at room temperature and the fibrinogen fraction was precipitated using a modification of the glycine method of Kazal et al. ~6. Addition of solid glycine with gentle stirring to a final concentration of o.21 g/ml was achieved over a 2o-min period at p H 7.0. After an additional 4 ° rain the precipitate was recovered by centrifugation and dissolved in i.o ml per platelet unit of o.i M Tris-o.I M NaCl-o.o25 M sodium citrate-o.oo25 M E D T A (pH 7-5) containing IOOO kallikrein inhibitor units of Trasylol and 3 ° mg of iodoacetamide. After brief dialysis of the dissolved fibrinogen in the same buffer to remove excess glycine, the dialysates were centrifuged for removal of insoluble material. Any a t t e m p t at further purification of these isolates was considered undesirable since this might lead to disproportionate recoveries of the platelet and plasma fibrinogens. In this communication, the term "total fibrinogen" denotes isolates containing a mixture of platelet fibrinogen and added plasma fibrinogen. Radioactivity recovered in these isolates equalled 85°/0 ± 5~o, of that originally added to the washed platelets. Carrying the labeled fibrinogen through the same isolation procedure as control resulted in a comparable recovery.

Preparation of Fragment X The high molecular weight derivative of plasma fibrinogen, Fragment X (ref. 13), was prepared b y incubating plasma fibrinogen (AB Kabi, Grade L, 9O~o clottable) at 37 °C with standard human plasmin (American Red Cross, io CTA units/ml in 50% glycerol) added to a final concentration of 0.25 CTA unit/ml per Io mg fibrinogen. The reaction was stopped at selected time intervals b y addition of Trasylol to a final concentration of 200 kallikrein inhibitor units/ml. Fragment X was identified by the thrombin clotting time ~7 and standard disc gel electrophoresis ~s. The Fragment X preparation referred to in these studies was slowly clottable by thrombin and contained no unmodified fibrinogen.

Gel electrophoresis To samples of plasma fibrinogen, platelet fibrinogen and the total fibrinogen solid sodium dodecylsulfate and /5-mercaptoethanol were added to final concentrations of 2 and I}~, respectively. After heating at I00 °C for 2 min, digestion was

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further continued overnight at 37 °C. The procedure for electrophoresis has been previously described19, ~°. The samples were applied in 50% glycerol and electrophoresis was carried out at 5 mA/gel for 14-16 h. The gels were stained with i % Coomassie Brilliant Blue in methanol-water-acetic acid, (5:5 :i, by vol.) for 4 h or longer and subsequently destained by diffusion in methanol-water-acetic acid (3:5:1, by vol.). Miscellaneous

Assay of radioactivity was carried out in a Packard auto-gamma scintillation spectrometer, Model 578, with counting vials for accommodation of 12 m m × 75 mm plastic tubes. Gel segments for assay of radioactivity were produced from frozen, unstained gels with a fine-wire I-ram slicer. Protein determinations were made by measurements of absorbance at 280 nm assuming for fibrinogen an EIi %~m value of 15.1 (ref. 13). All chemicals and solvents used in these studies were of reagent grade. RESULTS AND DISCUSSION

Fig. I illustrates the sodium dodecylsulfate gel electrophoretic patterns of reduced samples of control lzSI-labeled plasma fibrinogen (Gel A), two total fibrinogen preparations (Gels B and C), and platelet fibrinogen (Gel D). At present, the subunit

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Fig. I. Electrophoretic staining patterns of reduced fibrinogen samples. The electrophoresis time was 16 h with an aclylamide concentration of lO% in the presence of sodium dodecylsulfate. Gel A, control 12SI-labeled plasma fibrinogen; gels B and C, total fibrinogen isolates from platelet extracts containing 5 and io%, respectively, of the theoretical amount of platelet fibrinogen, and gel D the glycine isolate of platelet fibrinogen. The Greek symbols denote subunit polypeptide chains ; dashed lines indicate the positions of those for plasma fibrinogen and the solid lines those for platelet fibrinogen.

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H . L . JAMES. P. GANGULY

nomenclature for plasma fibrinogen 21 has been adopted for platelet fibrinogen. The net mobility of the platelet fibrinogen pattern (Gel D) is higher than that for plasma fibrinogen (Gel A). This is consistent with our earlier finding that platelet fibrinogen has a lower molecular weight n. The proportionately greater degree of separation of the platelet al and a2 chains with respect to those for plasma fibrinogen is indicative of a discrete molecular difference not explained by present knowledge of fibrinogen heterogeneities 22. The combined pattern of platelet fibrinogen and added plasma fibrinogen, as seen in the total fibrinogen preparation (Gels B and C) results from nearly complete overlapping of the platelet fibrinogen al and fl chains with the plasma fibrinogen/~ and y chains. This produces an apparent increase in relative intensity of the latter chains. The staining intensity of the subunit bands as presented in Fig. I, gel D, differs somewhat from that published for purified platelet fibrinogenn. It is well known that platelet fibrinogen is very labile 5. It was considered crucial to the purpose of this study to isolate platelet fibrinogen as rapidly as possible involving a minimal number of steps, even sacrificing some of its purity. Thus tile difference in relative intensities is due perhaps to the differences in methodology followed for the isolation of the protein. All fibrinogen samples presented in this study were obtained using the single procedure of glycine precipitation. This procedure obviously favors the precipitation of plasma fibrinogen more than platelet fibrinogen. The yield of platelet fibrinogen may be increased by changing the conditions of precipitation or by using (NH4)2S04. However, such preparations contain considerable amounts of impurities, particularly platelet albumin (tool. wt. 65 ooo) and platelet fibrin-stabilizing factor (subunit mol. wt 80 ooo), which overlap and interfere with the fibrinogen pattern. This would make realization of the objectives of this study practically impossible. The most important point illustrated by Fig. I is the typical plasma fibrinogen a-chains found in the total fibrinogen preparations, even though added plasma fibrinogen was only 5 (Gel B) and IO% (Gel C) of the theoretical amount of platelet fibrinogen in the starting material. It is evident that the plasma fibrinogen was not affected proteolytically during the isolation procedure, since the plasma fibrinogen a-chains are known to be very sensitive to proteolytic modificationS3, 22-26. Note, finally, in Fig. I that while there is an increase in intensity of the plasma fibrinogen pattern, especially the a-chains, in the two total fibrinogen preparations, no change is seen in the clearly observable, non-overlapping, v-chain of platelet fibrinogen. These results strongly indicate that plasma fibrinogen and platelet fibrinogen are two distinct proteins and that plasma fibrinogen is not converted to platelet fibrinogen during the isolation procedure. The availability of intensely labeled plasma fibrinogen made it possible to further substantiate the conclusions made from the electrophoretic staining patterns. Fig. 2 illustrates a typical radioactivity distribution pattern for sodium dodecylsulfate gel containing control 125I-labeled plasma fibrinogen (Part A) and a total fibrinogen sample (Part B). These gels, with staining patterns illustrated schematically, were obtained in the same way as Gels A and B, respectively, of Fig. I. Approx. 35o ooo and 450 ooo total cpm were present, respectively, in the samples applied for electrophoresis. Within an experimental variation of ~ IO%, the total radioactivity recovered from both gels accounted for the total quantity applied. In Part A of Fig. 2 the a, t5, and Y bands correspond well with three peaks of radioactivity, the quantity

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Fig. 2. Sodium dodecylsulfate gel (zo% acrylamide) r a d i o a c t i v i t y distribution p a t t e r n of control t*SI-labe|ed p l a s m a fibrinogen (A) and total fibrinogen (B). The gels represented in this figure are comparable to Gels A a n d B of Fig. I, respectively. The positions are shown of the plasma (PLS) a n d platelet (PLT) fibrinogen p a t t e r n s a n d also of a c o n t a m i n a t i n g protein (CON) p r e s e n t in the fibrinogen p r e p a r a t i o n utilized for labeling with l=sI. Dashed and solid lines indicate t h e mobilities respectively, of t h e p o l y p e p t i d e chains of p l a s m a and platelet fibrinogen. The arrows indicate t h e mobility of t h e b r o m o p h e n o l blue B P B tracer dye.

in each peak corresponding roughly to the known tyrosine content in the respective chains 27. A fourth peak corresponds to a contaminating protein (referred to as "CON") present in the plasma fibrinogen preparation used for labeling. The distribution of label in the total fibrinogen sample illustrated in Part B is virtually identical to that of the control. The locations of the platelet fibrinogen bands do not correspond to the observed radioactivity peaks, These results further show that the added plasma fibrinogen remained unchanged during the isolation steps which yield platelet fibrinogen. A number of investigators have described modifed forms of plasma fibrinogen produced by limited digestion with plasmin 13,z~-26. These "early" derivatives are slowly clottable with thrombin and have molecular weights lower than intact fibrinogen. These properties, similar to those reported for platelet fibrinogenn, might

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H . L . JAMES, P. GANGULY

suggest that platelet fibrinogen is such a derivative. This point is particularly important in view of the report that plasminogen is present in platelets 4. To examine this possibility the "early" derivative Fragment X (ref. I3) was isolated and compared following reduction of disulfide bonds in the sodium dodecylsulfate gel electrophoretic system with platelet fibrinogen. The pattern for Fragment X was similar to that reported in the literature 2s and was distinctly different from that of platelet fibrinogen. There is now increasing evidence that the proteins of blood platelets play an important role in their physiological function. Some of these proteins which are present in platelets also occur in plasma. However, a critical evaluation is necessary before two proteins derived respectively from platelets and plasma can be considered to be identical. This is clearly seen from the recent developments concerning fibrinstabilizing factor, a protein which is closely associated with fibrinogen. It is now well established that platelet fibrin-stabilizing factor is a molecule different from its plasma counterpart 29-32. Our present findings for platelet fibrinogen become more significant in view of other closely related studies. Nachman et al. s and Day and Solum 9 have shown that the major part of fibrinogen is associated with the granular fraction of platelets, specifically, with the low density granules. Fibrinogen found in the platelet soluble fraction may have been derived from disruption of granules during the isolation procedure s. Internally bound fibrinogen has been reported to be very rapidly transferred to the extracellular phase by an active process during viscous metamorphosis 5m, and although the mechanism of ADP-induced aggregation of platelets has not yet been explained, fibrinogen is known to be a cofactor s4 necessary in this process. Once platelet fibrinogen has been brought to the surface, it then may play a role in aggregation. This function might involve covalent bonding between y-chains of the fibrinogen molecules or between fibrinogen and another protein such as thrombosthenin 35. The latter possibility is particularly interesting in view of the observation that fibrinogen was shown to be absent from platelets of some patients with thrombasthenia whose plasma fibrinogen levels were normal 7. The origin and specific role of platelet fibrinogen in platelet function remain to be defined. The present studies confirm that platelet fibrinogen, although having some properties in common with plasma fibrinogen, is neither identical to nor derived from plasma fibrinogen during the isolation procedure. ACKNOWLEDGEMENT

This study was supported in part by research grant HLI5o9o from N.I.H. and by A.L.S.A.C. REFERENCES I 2 3 4 5 6

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