J. Cell Sci. 4, 763-779 (1969) 763Printed in Great Britain

CHANGES IN THROMBOCYTE ULTRA-

STRUCTURE DURING CLOT RETRACTION*

D. SHEPRO, F. A. BELAMARICH, F. B. MERK and F. C. CHAO

Department of Biology, Graduate School, Boston University,Massachusetts, U.S.A.

SUMMARY

The contraction of the entire thrombocyte appears to be a primary factor in the retractingmechanism of dogfish (Musteltis canis) in vitro blood clots. Microtubules probably account forthe condensation and lobation of the nucleus, and the impetus for cytoplasmic contractionappears to be dependent upon the grouping of microfibres of dimensions difficult to measure.These microfibres show a striking resemblance to materials stored within adjacent vesicles.A second type of fibre (60 A in diameter) extends from the bases of pseudopods into theadjacent matrix. There also appears to be partial engulfment of an extracellular amorphousmaterial by the plasma membrane. The ultrastructure of the thrombocyte during clot retractionshows some striking similarities to the mammalian platelet under similar conditions, and leadsone to believe a phylogenetic relationship exists between these haemostatic cells.

INTRODUCTION

In mammals, platelets are the haemostatic cells and during thrombogenesis thesemegakaryocyte fragments participate in the formation of thrombin, which in turn actsupon the freely circulating cells, causing them to aggregate. The resulting haemostaticplug or clot undergoes retraction and the evidence is quite conclusive that intactplatelets are needed for clot retraction. In the non-mammalian vertebrate, thrombo-cytes are the platelet equivalents and they are likewise involved in clot promotion andretraction. However the phylogenetic relationship of thrombocytes to platelets is stillopen to question, for as we have reported, ADP will cause aggregation of platelets butnot thrombocytes (Belamarich, Fusari, Shepro & Kien, 1966; Belamarich, Shepro &Kien, 1968).

In 1951 Budtz-Olsen published a compilation of data on clot retraction; his con-clusion was that retraction was due to the 'contraction of the cytoplasmic strands andthe merging of the platelets'. This process produced a drawing together of the extra-cellular fibrin meshwork and the secondary compression squeezed out the serum.White, Krivit & Vernier (1965) described an intimate relationship between extra-cellular fibrin strands and the a-granulomere during clot retraction; Erichson, Katz& Cintron (1967), in their study of platelet adhesiveness, reported on the engulfmentof fibrin by platelets; and Zucker-Franklin, Nachman & Marcus (1967) showed thatantiserum to thrombosthenin (the contractile protein extracted from platelets) inter-fered with clot retraction. Notwithstanding these contributions there is a need foradditional precise, structural information related to the physiology of retraction.

• This work was performed primarily at the Marine Biological Laboratories, Woods Hole,Massachusetts, U.S.A.

764 D. Shepro, F. A. Belamarich, F. B. Merk and F. C. Clmo

We reported that the ultrastructure of the non-dividing, non-activated dogfishthrombocyte (Figs. 2-4) was characterized by its size (12-15 /<), oval shape, perinucleargranules, and microtubules that frequently reflected the shape of the lobed nucleusas well as appearing between the lobes (Shepro, Belamarich & Branson, 1966).Recently White & Krivit (1968) commented that the microtubules, which appear afterplatelet activation, may be related to the movement of granules to the centre and thatthe microtubules in the platelet pseudopods may be involved in the mechanism of clotretraction. White (1968), in another discussion, commented that the involvement ofmicrotubule subunits still remains uncertain and they could be involved in clotretraction. In this presentation the relationship between thrombocyte activation, asseen in clot retraction, and the quantity, distribution, and orientation of microtubulesand microfibres is discussed.

MATERIALS AND METHODS

Clot formation and retraction

Fresh dogfish (Mustelus canis) blood is collected in one-tenth volume o-i M sodiumcitrate by methods previously described (Shepro et al. 1966). The thrombocyte-richplasma (TRP) is prepared by centrifugation at 500 rev/min (50 g) for 5 min at 4 °Cand then brought back to room temperature. Sixty to eighty per cent of the formedelements in the TRP are thrombocytes; lymphocytes are the principal cell contaminant.Clots of TRP are made in non-siliconized clotting tubes by addition of 0-2 M CaCl2 andtissue factor (a saline-urea extract of ground dogfish skin) to TRP in a ratio of o-2 :o-2:i-8 ml respectively. After the formation of a firm clot, the tubes are put in a 22 °Cwater bath without shaking. Clots are removed for fixation at 'zero time', 30 and 60 min.Retractions are measured by quantitating, with a calibrated burette, the fluid expressed.

Fixation

For the light microscope, the clots are fixed in 10% acrolein and prepared forembedding in purified glycol methacrylate using a technique devised by N. Feder(personal communication). One-half micron sections are cut and stained with acidfuchsin-methylene blue.

For the electron microscope, the clots are fixed at room temperature in buffered 3 %glutaraldehyde (total osmolarity, 580 m-osmole) for 1 h. They are washed in buffer at4 °C for 1 h and postfixed in 2% osmium tetroxide for i£ h at 4 °C. Following abuffered rinse, the clots are routinely dehydrated in a graded ethanol series at 4 °Cand embedded in Epon 812. The sodium-phosphate-biphosphate buffer system ofSorensen is used throughout the fixation. Sections of 'silver gold' thickness are cuton a Porter-Blum 1 microtome equipped with a diamond knife, mounted on uncoated300-mesh copper grids, double stained with 4% uranyl acetate followed by lead citrate(Reynolds, 1963), and examined with a Siemens Elmiskop 1.

RESULTS

With formation of the clot, characteristic morphological changes take place inthrombocytes, which we will refer to as the 'activated state'. These changes include

Thrombocyte ultrastructure in clot retraction 765

the following features, (i) Cell shape becomes asymmetric with pseudopod formationand lobation of the nucleus (Figs. 6, 8). In some areas the plasma membrane appearsdiscontinuous (Fig. 11). (ii) There are alterations in appearance and population ofcertain non-fibrous cell inclusions, e.g. an increase in the number of ribosomes, whichare sometimes seen on the outer aspect of the nuclear envelope (Figs. 6, 7). There isalso an increase in the number of vesicles containing varying amounts of electron-dense material and a decrease in the number of glycogen granules (Figs. 6, 7). (iii) Twotypes of microfibres are found, some extending into the extracellular space (Fig. 11),and others of smaller dimensions restricted to the cytoplasm (Fig. 8). Microtubules(mt) are found in both the non-activated thrombocyte (Fig. 3) and activated throm-bocyte (Fig. 5). The microtubules seem to increase in activated cells and they appearintimately associated with nuclear furrowing (Fig. 9).

As activation progresses, portions of the plasma membrane, granules, mitochondria,ribosomes, microtubules, and microfibres coalesce toward the cell centre (Fig. 6). Inthis study particular attention is directed to the microfibres and microtubules, andto cell shape.

The activated thrombocytes are characterized by a large population of microfibres,which are long, well defined, about 60 A in diameter, and they maintain their linearintegrity for a considerable distance into the extracellular matrix (Figs. 11-15). Hence-forth these microfibres shall be designated as/^ As seen in Fig. 13, t h e ^ generallyhave the same orientation as the microtubules. Although the possibility is not elimin-ated that the plane of section is tangential along the membrane, a distinct plasmamembrane in the region of the origin of the/! is never observed. It is not possible todetermine from the micrographs whether contiguous fx actually pass through themembrane. However, indistinct microtubules are often seen at the point of contactwhere/i appears to touch the cell (Fig. 11).

An intracellular microfibre (to be designated/2) is observed primarily at the basesof the pseudopods and is never found in their distal portions. These microfibres are ofdimensions difficult to measure, but can be distinguished by their grainy texture andfilamentous orientation (Figs. 6-8). This/2 material bears a striking resemblance to thesubstances within the filled vesicles of the non-activated cell (Fig. 3) and activatedcell (Fig. 6, inset). The fibres are often located adjacent to ruptured vesicle membranesin activated cells (Figs. 6, 7); in the non-activated thrombocyte, the/2 are not seen.The content of the intact filled vesicles is separated from the limiting unit membraneby an electron-translucent space of varying width (Figs. 2-4, 6, 7). As activationprogresses, the membranes of some vesicles appear to break down and the content ofeach vesicle is individually discharged into the cell matrix (Fig. 7).

A dense amorphous material is found closely associated with the external surface ofthe activated thrombocyte. This material, which is generally accepted to be fibrin, ispoorly defined and randomly oriented except at specific loci of the cell membrane,where it aggregates into fairly large bundles within pockets of the plasma membrane(Figs. 10, 13).

It appears evident, for reasons unknown, that all of the thrombocytes are notactivated simultaneously. Once an individual thrombocyte is activated it undergoes

766 D. Shepro, F. A. Belamarich, F. B. Merk and F. C. Chao

Fig. i For legend see opposite page.

Tkrombocyte ultrastructure in clot retraction 767

a sequential change that is apparently independent of the clot-retraction time. At30 min clot retraction one sees individual thromboeytes with alterations that are nogreater than alterations seen in some thromboeytes at 'zero time'. However, by 60 minpost clotting, practically all of the thromboeytes appear to undergo maximum change.

DISCUSSION

In essence, the overall alterations of thrombocyte ultrastructure following activa-tion parallel the platelet changes described by Rodman, Painter & McDevitt (1963),and by White et al. (1965). In many respects the fibrous areas within the thromboeytesresemble the a-granulomere of platelets described by White et al. (1965) but we wereunable to see filament-granule connexions; in addition, the thrombocyte granulesappear to discharge their contents individually. The close relationship of 'fibrin' to thethromboeytes resembles the platelet engulfment of 'fibrin' reported by Erichson,Katz & Cintron (1967). However, the identification of this material as fibrin is notfully established.

The role of the thrombocyte microtubule-microfibre population during clotretraction still eludes explanation, but some generalizations regarding their activityduring clot retraction seem appropriate from the data presented. We would add to theexisting theoretical models of clot retraction these main points.

The nuclear indentation and pseudopod formation are characteristic changes ofthromboeytes in the first stage of clot retraction (zero time). Whether the cellularalterations (including the unusual extension of the nuclear material well into thepseudopod) are specific adaptations to facilitate the exchange of nuclear-cytoplasmicmaterials cannot be determined from the present data. The activated thrombocyteproduces /2 that are seen primarily at the bases of the pseudopods but never withinthe shafts or the tips of the cell extensions. The possibility exists that the/2 may beinvolved in an intrinsic contractile mechanism which would account for the throm-bocyte's contracted appearance following activation. Lastly, the extracellular fx

produced upon activation may 'bridge' adjacent thromboeytes. Our data do notexclude the possibility that the linear-oriented fx are fibrin.

A theoretical presentation of thrombocyte changes during in vitro retraction of adogfish TRP is presented in Fig. 1. Each drawing is a diagrammatic representation ofa cell as seen in an electron micrograph. We would suggest from the observations that

Fig. 1. A schematic presentation of the sequential changes of a thrombocyte activatedby clotting. Each drawing is a diagrammatic representation of a thrombocyte as seenin an electron micrograph. The various stages were taken from different cells, A, non-activated thrombocyte. B, the onset of activation is characterized by the appearanceof/2 in the cytoplasm and microtubules (mi) located in nuclear indentations. A faintsuggestion of pseudopod formation is also present. There is some vesicle breakdown.C, an intermediate stage showing further nuclear lobation and pseudopod formation,and the definite appearance of the / 2 . D, at this stage the pseudopod formation andfurrowing of the nucleus (seen extending into the pseudopods) are maximum. The / S

is concentrated at the bases of the pseudopods (the central portion of the cell). Anenlargement of a pseudopod (E) illustrates the relationship of the / x to the base of thepseudopod. F, the 'bridging' of two thromboeytes by the / j .

768 D. Shepro, F. A. Belamarich, F. B. Merk and F. C. Chao

the contraction of the thrombocyte, possibly facilitated by the lobation of the nucleusand by some intrinsic contractile element, e.g. the /2, is involved in clot retraction.Budtz-Olsen (1951) theorized that in mammals the withdrawal of the pseudopodsplayed the important role in the clot-retracting mechanism; in our model the contrac-tion of the entire cell would be equivalent to pseudopod withdrawal. How the throm-bocyte contraction is structurally connected to the compression of the extracellularfibrin webwork cannot be conclusively determined from the present data. However,we are inclined to agree with White et al. (1965) that an intimate relationship existsbetween the intracellular and extracellular fibres in clot retraction. We also agree withErichson et al. (1967) that 'fibrin' engulfment could be a factor in forming an inter-locking network of cells in clotting.

In conclusion, the similarities of the structural changes in thrombocytes andplatelets during clot retraction lead one to believe that a phylogenetic relationshipmay exist between the non-mammalian thrombocyte and the mammalian platelet.

We wish to thank Mr David A. Lang for drawing the schematic diagram and Mrs FrancesSilvia for assistance in preparing the manuscript.

The investigation was supported in part by grants HE 5411, HE 10002, HE 06214, NationalInstitutes of Health, and by 67-690, American Heart Association.

REFERENCES

ANDERSON, W. A., WEISSMAN, A. & ELLIS, R. A. (1967). Cytodifferentiation during spermio-genesis in Lumbricus terrestris. J. Cell Biol. 32, 11-26.

AUBER, J. (1962). Mode d'accroissement des myofibrilles au cours de la nymphose de Calliphoraerythrocephala (Mg). C. r. hebd. Se'anc. Acad. Set., Paris 254, 4074.

BELAMARICH, F. A., FUSAKI, M. H., SHEPRO, D. & KIEN, M. (1966). In vitro studies of aggrega-tion of non-mammalian thrombocytes. Nature, Lond. 21.2., 1579-1580.

BELAMARICH, F. A., SHEPRO, D. & KIEN, M. (1968). ADP is not involved in thrombin-induced aggregation of thrombocytes of a non-mammalian vertebrate. Nature, Land. 220,509-510.

BUDTZ-OLSEN, O. E. (1951). Clot Retraction. Springfield, Illinois: Thomas.ERICHSON, R. B., KATZ, A. J. & CINTRON, J. R. (1967). Ultrastructural observations on platelet

adhesion reactions. I. Platelet-fibrin interaction. Blood 29, 385-400.REYNOLDS, E. S. (1963). Use of lead citrate at high pH as an electron-opaque stain in electron

microscopy. J. Cell Biol. 17, 208-212.RODMAN, N. F., JR., PAINTER, J. C. & MCDEVITT, N. B. (1963). Platelet disintegration during

clotting. J. Cell Biol. 16, 225-241.SHEPRO, D., BELAMARICH, F. A. & BRANSON, R. (1966). The fine structure of the thrombocyte

in the dogfish (Mustelus canis) with special reference to microtubule orientation. Anat.Rec. 156, 203-214.

WHITE, J. G. (1968). Effects of colchicine and vinca alkaloids on human platelets. Am.J. Path.S3 (2), 281 -291 .

WHITE, J. G., KRIVIT, W. & VERNIER, R. L. (1965). The platelet-fibrin relationship in humanblood clots: An ultrastructural study utilizing ferritin-conjugated antihuman fibrinogenantibody. Blood 25, 241-257.

WHITE, J. G. & KHIVIT, W. (1968). Changes in platelet microtubules and granules during earlyclot development. In Platelets: Their Role in Hemostasis and Thrombosis (ed. K. M.Brinkhous). Stuttgart: Schattauer-Verlag.

ZUCKER-FRANKLIN, D., NACHMAN, R. L. & MARCUS, A. J. (1967). Infrastructure of throm-bosthenin, the contractile protein of human plood platelets. Science, N. Y. 157, 945-946.

(Received 28 July 1968)

Thrombocyte ultrastructure in clot retraction 769

Fig. 2. Non-activated thrombocyte seen in longitudinal section (from pellet prepara-tion). Alpha-granules and glycogen granules are found scattered throughout thecytoplasm, x 13000.Fig. 3. Detail of Fig. 2. Content of the a-granules is granular. None of this granularmaterial is found in the cytoplasm. Note peripheral location of microtubules. x 30000.Fig. 4. Non-activated thrombocyte seen in cross-section (from a pellet preparation).Note characteristic indentation of nucleus, x 10000.

77© D. Shepro, F. A. Belamarich, F. B. Merk and F. C. Chao

Fig. 5. Thrombocyte from a clot in early stage of activation (zero time, no retraction).Nuclear furrowing is taking place (arrows). Microtubules may completely surroundthe nucleus like a collar. Cytoplasmic invaginations (seen just above the arrows) mayrepresent the beginning of pseudopod formation, x 30000.

Thrombocyte ultrastructure in clot retraction

• * # • #

1

'*>$. • : . * *

Fig. 6. Thrombocyte in later state of activation (zero time, no retraction). The pseudo-pods are more developed than in Fig. 5. A dense granular material (/s) is seen in thebasal regions of the pseudopods but is not found in their more distal areas. Surround-ing the nucleus are many vesicles, some empty and others partly filled (see arrow atinset). Compare / , material (bottom of inset) with content of the filled vesicle.x 16000; inset, x 25000.

772 D. Shepro, F. A. Belamarich, F. B. Merk and F. C. Chao

Fig. 7. Central portion of an activated thrombocyte, showing an aggregation ofvesicles and microtubules (zero time, no retraction). Ribosomes are seen associatedwith the nuclear envelope and also in the cytoplasm. The content of the vesicles isseparated from the limiting unit membrane (small arrow) by an electron-translucentspace of varying width. As activation progresses, the membranes of some vesiclesappear to be broken down and the vesicle contents are found in the cell matrix (largearrows), x 51 000.

Fig. 8. Detail of completely formed pseudopods (30 min, 42 % retraction). The seg-mentation of the nucleus permitting its occupation in each of the pseudopods isillustrated here, x 16000.

Thrombocyte ultrastructure in clot retraction 773

774 D- Shepro, F. A. Belamarich, F. B. Merk and F. C. Chao

Fig. 9. Illustrations of nuclear furrowing by microtubules (zero time, no retraction).The microtubules are seen in longitudinal and cross-section, x 30000.Fig. 10. Example of partial 'fibrin' engulfment (arrow) (60 min, 64% retraction).Note the close conformity to the thrombocyte membrane. 'Fibrin' is separated fromthe plasma membrane by a space of approximately 150 A. x 30000.

Thrombocyte ultrastructure in clot retraction 775

776 D. Shepro, F. A. Belamarich, F. B. Merk and F. C. Chao

Fig. i i . Relationship of microtubules to fl (60 min, 6 4 % retraction). Small arrowindicates area of microtubules which lies partly in a furrow of the nucleus (n). Notethat the plasma membrane at the point of contact of/] fibres is not distinct. Comparethe linear appearance of fl fibres and amorphous material (large arrow), x 47000.

Fig. 12. View showing fx relationship to the base of the pseudopod (60 min, 6 4 %retraction), x 16000.

Fig. 13. Another view of microtubule//i relationship (60 min, 6 4 % retraction).Intact microtubules (small arrows) are in the same axis as the fx found in the extra-cellular space. Large arrow marks an example of dense material partly surroundedby the membrane, x 24000.

Thrombocyte ultrastructure in clot retraction 777

Cell Sci. i

778 D. Shepro, F. A. Belamarkh, F. B. Merk and F. C. Chao

Fig. 14. Illustration shows the relationship of the/i and two thrombocytes (30 min,42 % retraction). The/X appear to emerge from the base of the pseudopod (in upperleft). Note the difference in appearance between the /x and the surrounding fibrin.x 18000.

Fig. 15. Lower magnification of plate used in Fig. 14 (30 min, 42% retraction).X4000.

Fig. 16. Light micrograph of three activated thrombocytes 'joined' together in triadformation (15 min, 35 % retraction), x 1300.

Thrombocyte ultrastructure in clot retraction 779

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