British Journal ofHuematology, 1975, 31, 51.

Distribution of Fibrinogen, and Platelet Factors 4 and XIII in Subcellular Fractions of Human Platelets M. JOHAN BROEKMAN, ROBERT I. HANDIN AND PHINCOHEN Department of Nutrition, Harvard School of Public Health, Division of Hematology, Prter Bent Brighaw Hospital and Department of Medicine, Harvard Medical School, Boston, Massachusetts (Received 16 January 1975; acceptedfor publication

25

January 1975)

SUMMARY. The distribution of fibrinogen, platelet factor 4 (heparin-neutralizing factor) and factor XI11 amongst subcellular fractions of human platelets was determined. It was found that fibrinogen and platelet factor 4 peaked sharply in the region of the density gradient previously shown to be heavily enriched with agranules. By contrast, factor XIII, fibrin-stabilizing factor, was found exclusively in the supernatant.

We have recently described a new method for subcellular fractionation of human platelets which, in addition to separating microsomal and lysosomal activities, provides the purest yields of a-granules and mitochrondria yet achieved (Broekman et al, 1974). The purity of the a-granules, in particular, afforded an opportunity to establish which, if any, of the procoagulant activities they contain (Day & Solum, 1973; Day et al, 1973). The present report is concerned with the distribution of fibrinogen, platelet factor 4 (PF 4, and platelet factor XI11 amongst the nine subcellular fractions which can be separated by our technique. MATERIALS AND METHODS Platelet procurement andfractionation. Washed platelets were derived in a plastic pack system (Fenwal Laboratories, Deerfield, IL, U.S.A.) and homogenized by the nitrogen decompression technique (Cell Disruption Bomb, Parr Instrument Co., Moline, IL, U.S.A.) with 1200 lb/in2 (80 atm) at 1°C as recently described (Broekman et al, 1974).The density gradient centrifugation method, which had been developed using a SW36 rotor, was adapted for use with a SW40 rotor (Beckman Instruments, Palo Alto, CA, U.S.A.). Ultracentrifugation was for go min at 154 400 g. Nine fractions were obtained, and protein and enzyme activities were monitored as previously reported (Broekman et al, 1974). Procoagulant activities. Fibrinogen content was measured by the staphylococcal clumping technique; serial dilutions of a plasma fibrinogen standard were tested in parallel and the results were expressed as fibrinogen equivalents in pg/ml (Leavelle et al, 1971). PF 4 activity was measured by a modification of the technique of Harada & Zucker (1971). Platelet fractions were diluted in platelet-poor plasma derived from blood anticoagulated with 0 . 3 8% Correspondence: Dr Phin Cohen, Department of Nutrition, 66s Huntington Avenue, Boston, Massachusetts OZIIS, U.S.A.

M.]. Broekman, R. I. Handin and P. Cohen

52

sodium citrate. The amount of heparin neutralized before addition of any platelet material was subtracted in calculating experimental results. Platelet-poor plasma contributed 0.25 -0.50 units of heparin-neutralizing activity (USP, heparin) per ml. Platelet factor XIII activity was measured by the incorporation of [' 4C]putrcscine into casein (Dvilansky et al, 1970). 5-Hydroxytryptamine (gHT) uptake. The localization of [14C]gHTuptake was used as a benchmark for comparison with the localization of clotting factors. Platelet-rich plasma was

Zone number I

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FIG I. Distribution of platelet factor XIII, PF 4, fibrinogen and ['4C]sHT uptake among human platelet subcellular fractions. See Methods for details of (I) platelet factor XIII, PF 4, and fibrinogen assays, and (2) incubation of platelet rich plasma with ['4C]gHT and subsequent isolation and fractionation of platelets prior to radioactivity assay of harvested fractions. Data are plotted as relative specific activities (Broekman el a/, 1974). For gHT, results are expressed as a ratio: % total radioactivity recovered/% total protein recovered. Shown are the means of four separate studies for PF 4, fibrinogen and gHT,and three studies for platelet factor XIII.

incubated for 20 min at 37°C with 2.5 ptci [~-~~C]j-hydroxytryptarnine binoxalate (Hcyssel, 1961) and then cooled to 4°C bcfore deriving a platelet pellet which was then washed, homogenized and fractionated (Broekman et al, 1974).Radioactivity in an aliquot of each zone was then determined using Aquasol as scintillation liquid. Cheniicals. All chemicals were of reagent grade. Radiochemicals and Aquasol were obtained from New England Nuclear (Boston, MA, U.S.A.) ; bovine thrombin, from Parke Davis (Detroit, MI, U.S.A.) ; and heparin, from Organon (West Orange, NJ, U.S.A.).

Procoagulants in Human Platelet Fractions

53

RESULTS Fig I shows the distribution of platelet factor XIII, platelet factor 4, fibrinogen and [14C]5HT uptake, plotted as relative specific activities according to the method of de Duve et al(1955). Platelet factor XI11 was almost exclusively located in the supernatant, with only insignificant amounts in zones 2 and 5-7, and none at all in zones 3,4,8 and 9. PF 4 activity peaked in zone 7 with lesser amounts in zones 5, 6 and 8, but was not detectable in zones 3 and 4. Only small amounts of activity were found in zones I, 2 and 9. Fibrinogen had a broader peak which centred on zones 6-8, but included substantial spillover into zone 9. Zones I and 2 contained only small amounts of fibrinogen. [14C]~HTuptake was highest in zone 9, the only zone where electron microscopy had shown dense bodies (Broekman et al, 1974). However, a considerable amount of activity also appeared in the supernatant, zone I. DISCUSSION Fibrinogen. Day & Solum (1973) described coincidental peaking of fibrinogen and acid hydrolases, including pnitrophenylphosphatase, in zones where electron-lucent granules and mitochondria predominated. By contrast, our method separates pnitrophenylphosphatase from other acid hydrolases, and clearly associates electron-dense a-granules with fibrinogen. Thus, we would agree with the suggestion by Day & Solum (1973) that disruption of granules during their homogenization procedure could have released and redistributed some fibrinogen to the lighter zones. In view of the similarity of the density gradient procedures used by the two laboratories (Day & Solum, 1973;Broekman et al, 1g74), we conclude that, under the reported conditions, nitrogen decompression is less disruptive for a-granules than teflon-pestle homogenization. The localization of fibrinogen within granules is in keeping with studies that have shown secretion of platelet fibrinogen during the release reaction (Grette, 1962; Davey & Liischer, 1968;Keenan & Solum, 1g7z),the absence of detectablefibrinogen on the surface of carefully washed platelets (Grette, 1962; Gokcen & Yunis, 1963; Castaldi & Caen, 1965; Keenan & Solum, 1972)~and the presence of fibrinogen in granule-derived protein (Nachman et al, 1967; Nachman & Marcus, 1968). The contribution of platelet fibrinogen to haemostasis has not been determined although its possible role as a necessary cofactor for aggregation has been suggested (Solum, 1970). Platelet factor 4. Barber et a1 (1972) showed 55% of PF 4 activity in the ‘granule rich’ fraction following disruption by glycerol lysis. Subsequently,Day et al (1973) found most of the PF 4 activity where they had previously localized gHT and the metabolically inert pool of adenine nucleotides; however, as with fibrinogen, they also found a considerable amount of PF 4 in the lighter zones suggesting that it had been released from disrupted granules. By contrast, we found a separation of 5HT and PF 4 peaks, with the latter sharply localizing in zone 7, the purest collection ofa-granules, and showing very little spillover into lighter zones. We interpret this as further evidence that our method not only concentrates a-granules to a greater degree than previously reported, but also leaves more of them intact as well as separate from 5HT storage organelles. In common with platelet fibrinogen, the function of PF 4 is incompletely understood.

54

M. I. Broekman, R. I. Handin and P. Cohen

PF 4 is recognized by its ability to neutralize heparin. Although it is released during platelet aggregation, in association with a high molecular weight mucopolysaccharide carrier (Barber et at, 1972), PF 4 does not apparently affect aggregation, in contrast to other granuleassociated components-ADP, gHT, or calcium. Moreover, PF 4 may interact with polymerized fibrinogen and fibrin degradation products (Niewiarowskiet al, 1965) and other acid mucopolysaccharides, in addition to heparin (Barber et al, 1972). Plateletfactor XIII. As opposed to plasma and platelet fibrinogen which are reported to be identical (Doolittle et a), 1974), plasma and platelet factor XI11 are structurally distinguishable in that the latter lacks the b chain, while both have fibrin and fibrinogen cross-linking properties (Schwartz et ai, 1973). Platelets contain approximately half of the blood's factor XI11 activity (Dvilansky et al, 1970); however, its functional releasability has not clearly been established. McDonagh & McDonagh (1972) showed release of platelet factor XI11 by ADPinduced aggregation of gel filtered platelets, whereas Joist & Niewiarowski (1973) found it to be retained after the thrombin-induced release reaction and subsequent clot retraction. Our data show that platelet factor XIII may be quantitativelyreleased by cavitation forces which do not disrupt those granules that hold fibrinogen and platelet factor 4. Clearly, further studies are needed to establish whether there is, in fact, cooperative interaction between the fibrinogen and factor XI11 of human platelets. ACKNOWLEDGMENTS

The technical assistance of Vera Martin and Jeffrey Goldman is gratefully acknowledged. The platelets were obtained from freshly donated blood at the Blood Bank of the Massachusetts General Hospital. This work was supported in part by grants HL 13584, HL 13802 and HL 17513 from the National Institutes of Health, by a contract DADA 17-70-C-0083 from the United States Army, and by the Hematology Research Fund of the Peter Bent Brigham Hospital. Dr Handin is a Cancer Research Scholar of the Massachusetts Division of the American Cancer Society. REFERENCES

R., JAKABOVA, M. BARBER, A.J., KKSBR-GLANZMANN, & LUSCHBR, E.F. (1972) Characterization of a chondroitin +sulfate proteoglycan carrier for heparin neutralizing activity (platelet factor 4) released from human blood platelets. Biochimica et Biophysica Acta, ~86,312. BROEKMAN, M.J., WESTMORELAND. N.P. & COHBN, P. (1974) An improved method for isolating alpha granules and mitochondria from human platelets. Journal of Cell Biology, 60, 507. CASTALDI, P.A. & CABN,J. (1965) Platelet fibrinogen. Journal oJ'Clinical Pathology, 18, 579. DAVEY, M.G.& LUSCHER,E.F. (1968) Release reactions of human platelets induced by thrombin and other agents. Biochimica et Biophysica Acta, 165, 490. DAY,H.J. & SOLUM,N.O. (1973) Fibrinogen associated with subcellular platelet particles. Scandinaviun Joumaf oftfaemutofogy, 10, 136.

DAY,H.J., STORMORKBN, H. & HOLMSEN, H. (1973) Subcellular localization of platelet factor 3 and platelet factor 4. Scandinavian Journal of Haenlatolog y, 10. 254. DB DWE,C.. PRESSMAN, B.C., GIANETTO, R., WATTIAUX, R. & APPELMANS, F. (1955) Tissue fractiona-

tion studies. 6. Intracellular distribution patterns of enzymes in rat-liver tissue. Biochemical Journal, 60, 604.

DOOLIITLE,R.F., TAKAGI, T. & COTTRELL, B.A. (1974) Platelet and plasma fibrinogens are identical genc products. Science, 185,368. DVILANSKY, A,, BRITTEN,A.F.H. & LOEWY, A.G. (1970) Factor XI11 assay by an isotope method. I. Factor XI11 (transamidase) in plasma, serum, leucocytes, erythrocytes and platelets and evaluation of screening tests of clot solubility. British Jorrrnnl of Haematoiogy, 18, 399.

Procoagulants in Human Platelet Fractions GORCEN, M. & YUMS,E. (1963)Fibrinogen as a part of 590. platelet structure. (Letter). Nature, GRETTE, K. (1962) Studies on the mechanism of thrombin-catalyzed hemostatic reactions in blood platelets. Acfa Physiologica Scandinavica, 56, Suppl. 195. K. & ZUCKER, M.B. (1971)Simultaneous HARADA, development of platelet factor 4 activity and release of l*C-serotonin. Thrombosis ef Diathesis Haemorrhagica, 25, 41. HEYSSEL, R.M. (1961)Determination of human platelet survival utilizing C''-labeled serotonin. Journal of Clinical Investigation, 40, 2134. JOIST,J.H. & NIEWIAROWSKI, S. (1973)Retention of platelet stabilizing factor during the platelet release reaction and clot retraction. Thrombosis et Diafhesis Haemonhagica, 2% 679. KEENAN,J.P. & SOLUM,N.O. (1972) Quantitative studies on the release of platelet fibrinogen by thrombin. BritishJournal ojHaematology, 23.461. LEAVELLE, D.E., MERTENS,B.F.,BOW, E.J.W. & OWN,C.A., JR(1971)Staphylococcal clumping on microtiter plates: a rapid, simple method for measuring fibrinogen split products. American Journal of Clinical Pathology, 55,452. MCDONAGH, J. & MCDONAGH,R.P. (1972) Factor XI11 from human platelets: effect on fibrin crosslinking. Thrombosis Research, I, 147.

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NACHMAN,R,L. & MARCUS,A.J. (1968)Immunological studies of proteins associated with the subcellular fractions of thrombasthenic and afibrinogenaemic platelets. BritishJournal OfHaematology, 15, 181.

NACHMAN,R.L., MARCUS, A.J. & ZUCKBR-FRANKLIN, D. (1967)Immunologic studies of proteins associated with subcellular fractions of normal human platelets. Journal of taboratory and Clinical Medicine, 6g, 651. NIBWIAROWSKI, S.. FARBISZIIWSKI, R.& POPLAWSKI, A. (1965)Neutralization of antithrombin VI (fibrinogen breakdown products) with platelet antiheparin factor (platelet factor 4). Thrombosis et Diathesis Haemorrhagica, 14, 490. SCHWARTZ, M.L.. PIZZO.S.V.,HILL,R.L.& MCKBB, P.A. (1973) Human factor XI11 from plasma and platelets. Molecular weights, subunit structures, proteolytic activation, and cross-linking of fibrinogen and fibrin. Journal OfBiological Chemistry, y8, 1395. SOLUM,N.O. (1970) ADP-induced aggregation of washed platelets. Effect of platelet and plasma fibrinogen. Scandinavian Journal of Haematology , 7 , 236.