Journal of Clinical InvestigationVol. 46, No. 8, 1967

Platelet Thrombosthenin: Subcellular Localization andFunction *

RALPH L. NACHMAN,t AARON J. MARCUS, AND LENORE B. SAFIER(From the Department of Medicine, New York Hospital-Cornell Medical Center, and the

New York Veterans Administration Hospital, New York)

Summary. Thrombosthenin, an immunologically distinct contractile proteinwas isolated in relatively pure form from human platelets. The protein,which was of high molecular weight appeared to be composed of multiplepolypeptide subunits, probably polymeric in nature.Thrombosthenin had magnesium-dependent ATPase activity, releasing

an average of 3 Jug phosphorus per mg protein in 30 min. After the additionof ATP, there was a reversible alteration in viscosity with calculated ATPsensitivity ranging from 64 to 90%. These biochemical properties of throm-bosthenin resemble those of smooth muscle.

'Specific antisera to thrombosthenin significantly inhibited the ATPase ac-tivity of the protein. Clot retraction of recalcified platelet-rich plasma andclot retraction of clotted fibrinogen-platelet mixtures were also inhibited bythe antisera. The findings suggest that thrombosthenin is an importantcomponent of the clot retraction system.Thrombosthenin was extracted from isolated platelet granule and mem-

brane fractions. The contractile protein derived from the membrane com-partment was more active as an ATPase and appeared to be more homo-geneous on immunologic analysis.

Introduction

Certain biochemical and physiological propertiesof blood platelets suggest that they possess con-tractile activity similar in some respects to thatfound in muscle tissue. Clot retraction requiresthe presence of platelets, and during the formationof a hemostatic plug, platelet aggregates appear toactively contract as part of the phenomenon knownas viscous metamorphosis (1). Actomyosin-like

* Submitted for publication November 28, 1966; ac-cepted May 9, 1967.

Supported by grants from the National Institutes ofHealth (HE-09070-03 and C-1905), the Veterans Ad-ministration, the New York Heart Association, and theR. A. Hummel Memorial Fund of New York Hospital.

Presented in part at the annual meeting of the Ameri-can Society for Clinical Investigation, Atlantic City, May3, 1966. An abstract appeared in the Journal of ClinicalInvestigation 1966, 45, 1050.t Send reprint requests to Dr. Ralph L. Nachman, the

New York Hospital-Cornell Medical Center, 525 East68th Street, New York, N. Y. 10021.

ATPase activity is present in platelets (2) andglycolysis is a major pathway of energy-metabo-lism (3). An actomyosin-like protein, thrombos-thenin, was first extracted from human plateletsby Bettex-Galland and Luscher (2). A similarprotein in pig platelets was extracted and partiallycharacterized by Grette (4). Of further interestis the description of intracellular contractile pro-teins in flagellated protozoans, mitochondria, sar-coma cells (5), and chick embryo fibroblast cells(6). This report describes the isolation and fur-ther characterization of human platelet thrombos-thenin. Furthermore, its distribution in specificsubcellular compartments will be defined.

Methods

Thrombosthenin preparation. The general procedureutilized to separate thrombosthenin was based mainly onthe technique as described by Grette (4) and is pat-terned after methods employed for the extraction ofactomyosin from muscle tissue (7). The starting ma-

1380

PLATELET CONTRACTILE PROTEIN

terials were platelet concentrates1 prepared from 10-20units of freshly collected whole blood, using acid-citrate-dextrose solution as the anticoagulant. Usually the con-centrates were processed to the point of butanol extrac-tion on the day of collection. Rarely they were storedat 40 C overnight before processing. Platelets wereseparated by means of the "oil bottle" centrifugation tech-nique (8), washed three times in Alsever's solution (9),and washed three times more in 0.9%o NaCl containing 0.1volume of 3.14% sodium citrate dihydrate at 40 C. 2 mlof cold 0.6 M KC1 in 0.015 M Tris buffer (pH 7.5) wasadded to each gram (wet weight) of platelets, followedby 25 pIA of n-butanol for each milliliter of final solution.Lysis of the cells occurred upon the addition of butanol(4). The extraction proceeded for 24 hr at 40 C, andafter being warmed to room temperature the materialwas centrifuged at 12,100 g for 15 min at 220 C. Thesupernatant was cooled to 40 C and 2 volumes of cold0.002 M MgSOs were added. The pH was adjusted to 6.5with 0.1% acetic acid, and the mixture was kept at 4° Cfor 1-3 hr, after which it was centrifuged at 8,700 g for10 min at 40 C. The contractile protein precipitated inthe form of a gel and was redissolved in 2 volumes of0.6 M KC1 in 0.015 M Tris. It was then reprecipitatedfive times over the following 24-36 hr. Final solubiliza-tion was carried out in the KCl-Tris solution. In someinstances the thrombosthenin preparation was furtherpurified by column chromatography on polyacrylamidegel. Bio-Gel P 300,2 suspended in KCl-Tris solution,was poured into a column 12 inches long and 1 inch indiameter. The column was then charged with 50-75 mgof thrombosthenin solution and the bulk of the proteinappearing in the void volume was concentrated by ultra-filtration and reprecipitated once with MgSO4.

Viscosity experiments. Solutions of thrombosthenin inKCl-Tris buffer were used for relative viscosity deter-minations. They were carried out in a Cannon-ManningSemi-Micro Viscometer.8 Specific viscosity values werecomputed according to the formula: Z= 2.3 log ?lrel/concentration (grams/liter) (2). Sensitivity to ATPwas calculated according to the method of Portzehl (10).The concentrations of membrane thrombosthenin solutionswere 0.45%o in 0.6 M KCl, 0.015 M Tris (pH 7.5).Granule thrombosthenin was studied in 0.3%o solutionsunder the same conditions. The times obtained in eachviscosity experiment represent the average of triplicatedeterminations which were then used to calculate rela-tive viscosity.

Superprecipitation studies. In a low magnesium en-vironment and in the presence of ATP, muscle proteinsflocculate and contract to small volume. This phenome-non is known as superprecipitation (2). Superprecipi-tation experiments on whole platelet thrombostheninwere carried out in the presence and absence of 10' M

'Generously supplied by the New York Blood Center,New York, N. Y.2Bio-Rad Laboratories, Richmond, Calif.s No. 150, 1 ml capacity at 22° C, Cannon Instrument

Co., State College, Pa.

ATP. Magnesium sulfate (6 X 10' M) was alwayspresent in the test system. The protein concentrationswere adjusted to 0.2%, ionic strength 0.08, and the pHwas 6.3. In the studies involving thrombosthenin de-rived from the platelet membranes and granules, EDTA(4 X 10' M) was added. The membrane thrombostheninsolutions contained 0.18% protein in a final volume of 1ml of 0.12 M KCl containing MgSO4 and EDTA. Thesolutions of granule thrombosthenin contained 0.15%protein in a final volume of 1 ml of 0.12 M KCl, withthe same concentrations of Mg", ATP, and EDTA.ATPase experiments. 1 mg of reprecipitated thrombos-

thenin in a final volume of 1 ml was incubated at 220 Cwith 5 X 10' M ATP. The medium also included KCl5 X 10'2 M; MgSO4 1 X 10' M; and borate buffer 0.2 M(pH 7.0) (4). After a 30 min incubation period, the re-action was stopped by the addition of 1 ml of 10%o trichlo-roacetic acid (TCA) and the mixture allowed to stand for30 min at room temperature. The tubes were then cen-trifuged at 3,000 g for 15 min and inorganic phosphorusin the supernatant fraction was determined by the methodof Marsh (11). Tissue blanks, ATP blanks, and re-agent blanks were included in all determinations. Fur-ther ATPase experiments were carried out in the presenceof the following: (a) mersalyl 4 in a final concentrationof 2.5 X 10' M; (b) ouabain 1 X 10-a M; (c) rabbitantithrombosthenin globulin, 1 mg/ml borate buffer; and(d) rabbit antifibrinogen globulin, 1 mg/ml borate buffer.Finally, another group of experiments was performed inthe absence of MgSO4. Mersalyl was prepared in boratebuffer (0.2 M, pH 7.0). Mersalyl acid was converted tothe sodium salt by dissolving initially in 0.2 M borax(calculated as sodium borate) with heating at 500 C.The pH was adjusted to 7.0 with 0.2 M boric acid andthe solution brought to volume with borate buffer.Ouabain was dissolved directly in borate buffer withheating at 370 C.Immunologic experiments. For the preparation of

antisera, thrombosthenin extracts were reprecipitated sixtimes. A single intravenous injection of 1-2 mg of thethrombosthenin in KCl-Tris buffer was given to two rab-bits at four weekly intervals. The animals were then"boosted" with subcutaneous and intramuscular injectionsof 2-4 mg of protein in complete Freund's adjuvant atbiweekly intervals for 8-12 wk. After inactivation at560 C for 30 min, the sera were stored at - 200 C untilused. Specimens from individual blood collections werenot pooled.

Antisera to isolated human platelet granules, mem-branes, and the soluble supernatant fractions were pro-duced in individual rabbits by repeated subcutaneous andintramuscular injections of washed subcellular fractions(see below) in Freund's adjuvant. Antihuman plateletserum was prepared as previously described (8). Anti-sera to human albumin, human IgG, whole human serum,and human fibrinogen were obtained commercially.5

4 Winthrop Laboratories, Special Chemical Department,New York, N. Y.

5 Lloyd Bros., Inc., Cincinnati, Ohio.

1381

R. L. NACHMAN, A. J. MARCUS, AND L. B. SAFIER

Antisera to actomyosin derived from human striated andsmooth muscle were kindly provided by Dr. Carl Becker.6Antithrombosthenin and antifibrinogen globulin were pre-pared by precipitation with equal volumes of 30% sodiumsulfate at room temperature, followed by washing with15% sodium sulfate. Before use in ATPase inhibitionstudies, the globulins were dialyzed in borate buffer.For studies involving fluoresceination, the antithrombos-

thenin globulin was prepared from antithrombosthenin se-

rum which was previously adsorbed with 10 mg oflyophilized human fibrinogen7 and 5 mg of lyophilizednormal plasma per ml of antiserum. The antiserum was

incubated for 1 hr at 37° C and overnight at 40 C andthen cleared by centrifugation. The adsorbed antiserumdid not react in immunodiffusion with purified fibrinogenat antigen concentrations ranging from 1 to 10 mg/ml.The adsorbed antithrombosthenin globulin was then di-alyzed into a 1:10 dilution of bicarbonate buffer (0.05 M,pH 9) in saline. Fluorescein isothiocyanate 8 (0.05 mg/mg protein) was dissolved slowly with stirring for 18 hrat 40 C. After dialysis into phosphate buffer (0.01 M,pH 7.2), the dialyzate was passed through a SephadexG-25 9 column in phosphate buffer and ultrafiltered to theoriginal volume of the serum. Washed human plateletswere prepared for examination by application to a glassslide with a Pasteur pipette and fixation in acetone for10 min. Bone marrow spicules were prepared in a simi-lar fashion. 2 drops of fluoresceinated antithrombostheninglobulin (diluted 1: 4) were added to the slide which was

then incubated at room temperature for 30 min. Theslides were washed in phosphate buffer and mounted inphosphate-buffered glycerol. The specimens were exam-

ined in a Leitz Ortholux fluorescent microscope con-

taining a mercury vapor lamp (CS 150), a BG 0.12 ex-

citor filter, a dark field condensor, and a Leitz barrierfilter.'0

Immunoelectrophoresis and immunodiffusion studieswere carried out by methods previously described (12,13). Agar was prepared in the presence of 0.6 M KCl.

Clot retraction was evaluated by a modification of thetechnique of Shulman (14). Studies on clot retractioninhibition were carried out as follows: tubes contain-ing 0.9 ml of platelet-rich plasma and 0.1 ml of the vari-ous antisera were recalcified with 0.1 ml of 0.5 M CaCl2and incubated at 370 C for 1 hr. Clot retraction inhibi-tion was also studied in a plasma-free system by incu-bating 0.5 ml of washed platelets in Tris-buffered saline(15) with 0.1 ml of purified fibrinogen (2 g/100 ml) and0.2 ml of glucose (200 mg/100 ml). 0.1 ml of antiserumwas added and the system was clotted with 0.1 ml ofthrombin" (10 units in 0.025 M calcium chloride).

8 Department of Pathology, Cornell University MedicalCollege, New York, N. Y.

7 Mann Research Biochemicals, New York, N. Y.8 Dajac Laboratories, The Borden Chemical Co., Phila-

delphia, Pa.9 Pharmacia Fine Chemicals, Inc., Piscataway, N. J.10 Leitz, Inc., New York, N. Y.11 Bovine thrombin, Parke, Davis & Co., Detroit, Mich.

Starch urea gel electrophoresis in glycine buffer wascarried out by the method of Cohen (16). Peptide map-ping of thrombosthenin purified by column chromatog-raphy was performed as previously described (17).When pronase12 was used for digestion, 2 units of en-zyme were added for every 10 mg of protein. The incu-bation was carried out in 0.2 M sodium bicarbonate (pH8.0) for 24 hr at 370 C.

Studies on subcellular platelet fractions. The tech-nique for preparing platelet homogenates and their sub-sequent separation on a continuous sucrose density gradi-ent has been described in detail (18). Two subcellular"compartments" and the soluble fraction ("cell sap")were studied. Fractions to be used for ATPase experi-ments were washed in borate buffer (0.2 M, pH 7.0), andthose from which thrombosthenin was extracted werewashed in citrate-saline solution (4). The granules werewashed by ultracentrifugation at 36,220 g for 20 min.The membranes were washed at 198,400 g for 60 min.Prior to ATPase studies, the washed pellets were resus-pended in borate buffer and stored overnight in ice orfrozen at -85° C. Washed pellets for thrombostheninextraction were stored at - 850 C. The starting materialfor these extractions was 1.5 - 2.7 g (wet weight) ofgranules and 0.6-1.4 g (wet weight) of membranes.This was the equivalent of fractions derived from plate-lets isolated from 5-10 units of whole blood. The extrac-tion procedure was essentially the same as for wholeplatelet thrombosthenin except for modifications necessi-tated by the quantity and nature of the material. Foreach gram (wet weight) of granules, 2 ml of KCl-Trisbuffer was added. For comparable membrane material,3.3 ml of KCl-Tris buffer was added. After the additionof MgSO4, the mixture was kept at 40 C overnight andthen centrifuged at 12,100 g for 15 min at 40 C. The re-sulting precipitate was redissolved in 1-2 ml of KCl-Trissolution and used for ATPase assay. For some viscosityand superprecipitation studies, a single reprecipitation stepwas carried out. When whole fractions were assayedfor ATPase activity, about 1 mg of protein was used.Slightly less was employed when the extracted thrombos-thenin was similarly assayed. The ATP concentration inthe assay mixture was 1 X 10' M. After the reactionwas stopped with cold TCA, the tubes were centrifugedat 10,000 rpm for 15 min at 4° C. Inorganic phosphatein the clear supernate was determined by the methodof Dryer, Tammes, and Routh (19). Tissue blanks, ATPblanks, and reagent blanks were included in all assays.

Protein determinations were carried out by the Folinmethod (20). Human striated muscle actomyosin wasprepared by the method of Levy and Fleisher (7).

ResultsWhole platelet thrombostheninUnless otherwise stated, the following results

were obtained on thrombosthenin that was six-fold reprecipitated.

12 Calbiochem., Los Angeles, Calif.

1382

PLATELET CONTRACTILE PROTEIN

Viscosity. A 0.3%o solution of soluble throm-bosthenin in KCI-Tris buffer had a relative vis-cosity of 1.8. This value was not appreciably af-fected by the addition of 2% (by volume) of 0.6 MKCL. A typical experiment is shown in Figure 1.Addition of ATP (final concentration 108 M)resulted in a marked drop in relative viscositywhich returned to the original value after 20 min.The calculated sensitivity to ATP was 74%, andthe specific viscosity was 0.13. The results ofthree additional, separate extraction experimentsare shown in Table I. The calculated ATP sen-

sitivity characterizes the presumed degree of dis-sociation of the macromolecular subunits of throm-bosthenin in the presence of ATP. The values ob-served are comparable to those obtained withsmooth muscle (5).

Superprecipitation. Solutions of thrombosthe-nin showed marked superprecipitation in the pres-

ence of ATP. The superprecipitation response

varied from batch to batch, but in general maxi-mal effects were observed 15 min after exposure

to ATP. These experiments were performed on

three separate occasions with three separate

2.0

1.5

'q rel

1.0

0.5

KCI4I ATP

I I20 40 60

Minutes

FIG. 1. REVERSIBLE CHANGES IN VISCOSITY OF THROM-

BOSTHENIN AFTER THE ADDrrION OF ATP.

TABLE I

Viscosity changes of whole platelet thrombosthenin*

17relExperi- Peel 30 min ATPment atel ATP after ATP sensitivity

1 1.9 1.4 1.8 902 1.9 1.45 1.7 733 1.8 1.43 1.7 64

*0.3% solutions of thrombosthenin in KCl-Tris buffer.10-3 ATP (2% by volume) added. Each experiment wasperformed in triplicate.

batches of extracted protein. The results weresimilar on all occasions.ATPase studies. In four separate control ex-

periments the thrombosthenin preparations hy-drolyzed ATP, releasing inorganic phosphate atan average rate of 3 pug of phosphorus per mg ofprotein in 30 min. Various conditions underwhich the ATPase activity of extracted thrombos-thenin was inhibited were then studied (Table II).Deletion of magnesium resulted in a significantbut incomplete loss of ATPase activity. Virtuallycomplete inhibition was noted with mersalyl andslight inhibition was found with ouabain. Signifi-cant loss of ATPase activity occurred when anti-thrombosthenin globulin was added to the assaysystem. Antifibrinogen globulin had no effect onthe enzymatic activity of thrombosthenin.Immunologic studies. A preparation of repre-

cipitated thrombosthenin, further purified by chro-matography, was subjected to immunoelectropho-resis using a potent rabbit antithrombosthenin se-rum. As shown in Figure 2, a single precipitin

TABLE II

Effect of inhibitory conditions on A TP hydrolysisby thrombosthenin

Per cent ofPreparation control value*

Controlt 100 (3,u g P/mg pro-tein per 30 min)

Control without magnesium (2)§ 30Control with mersalyl 10

2.5 X 10-4 M (2)Control with ouabain 80

1 X 10-3 M (2)Control with anti- 17thrombosthenin (2)

Control with antifibrinogen (1) 94

* Average of indicated number of experiments.t 1 mg of thrombosthenin in 1 ml of borate buffer,

pH 7.0, 0.2 M, containing KCl, 5 X 10-' M; MgSO4,1 X 10-4 M; ATP, 5 X 10- M. (Repeated three times.)

§ Number of experiments.

1383

R. L. NACHMAN, A. J. MARCUS, AND L. B. SAFIER

+

FIG. 2. IMMUNOELECTROPHORESIS OF CHROMATOGRAPHEDTHROMBOSTHENIN IN 0.6 M KCI AGAR.

line in the beta globulin region was observed.The procedure was repeated using the thrombos-thenin preparation against antifibrinogen, anti-whole human serum, anti-albumin, and anti-gammaglobulin. In no instance was reactivity observed.However, when less purified, unchromatographedsamples of thrombosthenin were used as antigen,the antiserum to thrombosthenin precipitated twoantigens, one of which appeared to be identicalwith human fibrinogen. The antithrombostheninin the unadsorbed state thus appeared to have anti-fibrinogen activity; however in no case did anti-fibrinogen react with purified preparations ofthrombosthenin. Antisera to striated and smoothmuscle actomyosin of human origin failed to reactwith thrombosthenin. Similarly, antithrombos-

thenin failed to react with a preparation of striatedmuscle actomyosin.A preparation of washed human platelets was

exposed to fluoresceinated antithrombosthenin thathad been previously adsorhed with human fibrino-gen and normal human plasma. A diffuse,"speckled" type of staining was observed (Figure3). In contrast, erythrocytes and buffy coat prep-arations were not stained with the antiserum. In-tense fluorescence was noted in megakaryocytes intwo normal bone marrow preparations.

Clot retraction experiments. The effect ofthrombosthenin antiserum on clot retraction of re-calcified platelet-rich plasma was examined. Atypical experiment is shown in Figure 4, in whichit is seen that antisera to human platelets andthrombosthenin completely inhibited clot retrac-tion. This phenomenon was abolished by prioradsorption of the antisera with thrombosthenin.If the antisera were adsorbed with normal humanplasma or purified human fibrinogen, clot retrac-tion inhibition was still observed. No inhibition ofclot retraction was observed when anti-albumin,

Anti- Anti- Anti- Anti- Anti- Anti-human alburnin throiw- IgO whole fibrinogenplateets bosthenIn) serum

FIG. 3. FLUORESCEIN STAINING OF WASHED HUMAN

PLATELETS WITH FLUORESCEINATED ANTITHROMBOSTHENIN.

FIG. 4. CLOT RETRACTION INHIBITION STUDIES USINGCITRATED PLATLET-RICH PLASMA AND VARIOUS ANTISERA.Calcium was added after 5 min incubation at 370 C.

anti-gamma globulin, anti-whole serum, or anti-fibrinogen were used. This series of experimentswas repeated on four occasions with two sepa-rate batches of antisera. Identical results wereobtained. Finally, antisera to actomyosin derivedfrom smooth and striated muscle were completelyineffective as inhibitors of clot retraction. In or-der to determine whether plasma proteins in thissystem competed with platelets for antiserum bind-ing, thereby masking the potential inhibitory ef-fect of antiserum, we repeated these experimentsin a purified fibrinogen (plasma-free) system. Noinhibition of clot retraction was observed whenanti-gamma globulin or anti-albumin were addedto the fibrinogen-platelet mixtures. Antiserawhich did not inhibit clot retraction remained

1384

PLATELET CONTRACTILE PROTEIN

iheffective after dilution with buffered saline inranges from 1: 4 to 1: 64.

Structural studies. A chromatographically puri-fied thrombosthenin extract was subjected tostarch gel electrophoresis in urea glycine. Twomajor bands and three to four less discrete minorcomponents were observed. Peptide map "finger-prints" of pronase-digested thrombosthenin re-

vealed only 12-13 clear spots. When an extract ofstriated muscle actomyosin was similarly treated,over 40 different spots were observed. A peptide

ern-nr r- fe-rx7rwca;,_Airaa+aeIf rm achrr cbhkor%,L7Amaldp Ui i1ypsnII-uigeLsu LI]

only 8 spots.

Thrombosthenin associatedpartmnents

The distribution of throrthree main subcellular platenlows. The contractile prot

Anti- Anti-membranes granules

FIG. 5. CLOT RETRACTION IN]

PLATLET-RICH PLASMA WITH A

antisera were exposed to 5 mg

bosthenin for 1 hr at 370 C.

membranes and granules, btthe soluble (cell sap) compathese conclusions is preset

TABLE IIIA TPase activity of platelet subcellular fractions and throm-

bosthenin extracted from the granulesand membranes

No. of ;,g P/mgexperi- protein per

Preparation ments 30 min

Granules 3 1.03 i 0.14Membranes 3 1.16 d 0.14Soluble layer 3 1.34 1 0.42Granule thrombosthenin 2 2.78 0.51Membrane thrombosthenin 2 4.57 :1 0.91

LirumIuostneniIL1 s wcu Thrombosthenin extraction from subcellularfractions. It was possible to extract thrombos-thenin from isolated platelet granules and mem-

corn- branes, as described in the methodology section.An extensive effort was made to extract thrombos-

nbosthenin among the thenin from the soluble (cell sap) fraction de-et fractions was as fol- rived from six ultracentrifugal runs. The pooledLein was found in the fractions were concentrated to a small volume in a

rotary evaporator and ultrafiltered. They were

then dialyzed against Tris buffer to remove thesucrose, and finally against KCI-Tris buffer. Theaddition of MgSO4 and subsequent adjustment to

appropriate pH left no precipitate after standingAnti- Anti- overnight at 40 C and centrifugation (12,000 g formembranes granules 15 min). It was concluded that thrombostheninabsorbed absorbed was not present in the soluble subcellular platelet

HIBITION USING CITRATED compartment.LNTISERA. The adsorbed ATPase activity of subcellular platelet particles.chromatographed throm- In these experiments, the granules, membranes,

and soluble layer were tested first. This was fol-lowed by studies on thrombosthenin isolated from

it none was present in the granules and membranes. ATPase activityLrtment. Evidence for was present in all three subcellular compartments

ated in the following and in the thrombosthein extracted from the gran-

series of experiments.Clot retraction studies. Antiserum to platelet

membranes and granules completely inhibited clotretraction of recalcified platelet-rich plasma. Ifthe antiserum was adsorbed with a thrombosthe-nin extract, this property was abolished. Adsorp-tion with lyophilized human plasma or purifiedhuman fibrinogen did not remove this activity. Atypical experiment is shown in Figure 5. Identicalresults were obtained in three separate experi-ments. In contrast, antiserum to the soluble frac-tion present at the top of the sucrose density gradi-ent after ultracentrifugation had no effect on clotretraction.

TABLE IV

Effect of inhibitory conditions on A TP hydrolysis by plateletfractions and extracted thrombosthenin

Per cent of control values

Mersalyl Ouabain No Mg++Preparation (2.5 X 10-4 M) (1 X 10-3 M)*

Granules 48 73 40Membranes 45 87 61Soluble 100 100 100Granule 53 97thrombosthenin

Membrane 57 100thrombosthenin

* 1 X 10-' M ouabain was used for the thrombostheninexperiments.

1385

R. L. NACHMAN, A. J. MARCUS, AND L. B. SAFIER

ules and membranes (Table III). Attempts werethen made to distinguish this ATPase activityfrom that of the sodium-dependent, potassium-stimulated "pump" ATPase. Ouabain producedpartial inhibition of the ATPase activity in thegranule and membrane fractions, suggesting thepresence of "pump" ATPase activity in these par-ticles. However, ouabain had no inhibitory effecton the ATPase activity of the thrombosthenin ex-tracted from the subcellular particles. Mersalylinhibited the ATPase activity of the subcellularparticles and the thrombosthenin extracted fromthem. None of the inhibitory conditions influ-enced the ATPase activity of the soluble compart-ment. The results are summarized in Table IV.

Superprecipitation and viscosity. The experi-ments were performed with thrombosthenin ex-tracted from isolated membranes and granules.Solutions of membrane and granule thrombostheninshowed clear-cut superprecipitation properties inthe presence of ATP in two separate batches ofextracted material.

Soluble thrombosthenin was extracted fromplatelet membranes in two separate experiments.The relative viscosity of these preparations was1.6 and 1.7, respectively. In the presence of ATP(10-3M) there was a moderate fall in relative vis-cosity (Table V). In 30 min the relative viscosityreturned to the approximate starting values. ATPsensitivity of the membrane thrombosthenin solu-tions was 48 and 45%o respectively. Solublethrombosthenin extracted from platelet granulesshowed similar but less striking changes in vis-cosity following the addition of ATP.Immunologic studies. Antithrombosthenin, ad-

sorbed with lyophilized human plasma and human

TABLE V

Viscosity changes of thrombosthenin prepared from plateletsubcellular compartments

'irel ATPExperi- trel 30 min sensi-.

Preparation ment ,prel ATP after ATP tivity

Membranes 1 1.6 1.4 1.54 482 1.7 1.44 1.6 45

Granules 1 1.3 1.19 1.28 502 1.3 1.16 1.25 76

Membrane protein concentrations were 0.45%. Granuleprotein concentrations were 0.3%. 10- M ATP (2% byvolume) was added. Each experiment was performed intriplicate.

FIG. 6. IMMUNODIFFUSION ANALYSIS IN 0.6 M KC1AGAR OF THROMBOSTHENIN PREPARATIONS EXTRACTED

FROM WHOLE PLATELETS, GRANULES, AND MEMBRANES.Soluble platelet protein derived from the top layer of thesubcellular gradient was dialyzed into 0.6 M KCI andultrafiltered. The protein concentrations in the antigenswere equal. The middle well contained antithrombos-thenin adsorbed with fibrinogen and plasma.

fibrinogen, reacted with thrombosthenin prepara-tions from granules, membranes, and whole plate-lets (Figure 6). The reaction against the solublesubcellular compartment was negligible.

Discussion

There is little doubt that proteins associatedwith the plasma membrane of the platelet, as wellas those located intracellularly, are of great func-tional significance. In these studies, the contrac-tile protein thrombosthenin was first isolated fromwhole platelets and subsequently from plateletgranules and membranes. A variety of experi-ments on its in vitro properties were then carriedout.The results corroborate previous reports that

thrombosthenin has physicochemical propertiessimilar to those of contractile protein of muscletissue. The protein was soluble only at relativelyhigh ionic strengths. In the presence of ATP itshowed the phenomenon of superprecipitation andunderwent reversible alterations in viscosity. The

1386

PLATELET CONTRACTILE PROTEIN

ATP sensitivity of whole platelet thrombostheninpreparations was similar to that reported for acto-myosin derived from smooth muscle. In addition,purified thrombosthenin showed magnesium-de-pendent ATPase activity which was slightly in-hibited by ouabain and almost totally inhibited bymersalyl, a mercurial compound which is a specificinhibitor of contractile proteins of the actomyosingroup (5). The finding that ouabain exerted amild inhibitory effect on the ATPase activity ofwhole platelet thrombosthenin may have reflectedcontamination of the extracted protein with otherATPase moieties or could have been due to therelatively high concentration of ouabain used in theexperiments (10- M). Ouabain at a concentra-tion which completely inhibited "pump" ATPaseactivity of the subcellular fractions (1O-5 M) hadno inhibitory effect on thrombosthenin from theseparticles. This suggested that the Na-dependent,K-stimulated ATPase activity of platelets wasdistinct from that associated with thrombosthenin.These experiments were carried out on one batchof extracted subcellular thrombosthenin sincequalitative rather than precise quantitative effectswere sought, as a means of distinguishing be-tween the types of ATPase activity present. Ex-periments on the "pump" ATPase of platelets willbe reported elsewhere. The physical properties ofplatelet thrombosthenin closely resembled those ofcontractile protein extracted from rat sarcoma cells(5).The immunologic studies indicated that throm-

bosthenin could not be detected in normal humanplasma or serum and was a protein apparentlyunique to platelets. Furthermore, there was nocross-reactivity with actomyosin derived from hu-man smooth and striated muscle. In general, thethrombosthenin extracts which were not furtherpurified chromatographically contained varyingamounts of platelet fibrinogen and possibly othercontaminants. Therefore, the thrombosthenin anti-sera were adsorbed with fibrinogen and plasmabefore use. When washed normal human plate-lets were exposed to previously adsorbed fluores-ceinated antithrombosthenin, a diffuse "speckled"type of staining was observed. There did not ap-pear to be a preferential binding of the fluoresceinlabel to the plasma membrane. Similar intensestaining was noted in the megakaryocytes of nor-mal bone marrow. The fluoresceinated antiserum

did not stain normal leukocytes or erythrocytes,suggesting that it was specific for platelets.Anti-whole platelet and antithrombosthenin sera

completely inhibited clot retraction of recalcifiedplatelet-rich plasma. This inhibitory activity couldbe completely abolished by adsorption of the anti-sera with a thrombosthenin extract. Other anti-sera, particularly antifibrinogen, did not signifi-cantly affect the ability of platelets to support clotretraction. Prior adsorption of the thrombosthe-nin antiserum with either fibrinogen or humanplasma did not remove its inhibitory properties.The fact that anti-whole platelet sera inhibitedclot retraction, which could be abolished by prioradsorption with extracted thrombosthenin, sug-gested that this property of anti-whole plateletsera was probably related to a population of throm-bosthenin antibodies.

Antisera to the soluble subcellular "compart-ment" of platelets had no effect on clot retraction.This was taken as further evidence of the speci-ficity of the clot retraction inhibition reaction.The soluble (cell sap) subcellular component didnot contain thrombosthenin, but was previouslyfound to contain practically the entire complex ofsoluble platelet protein (21). It is possible thatthe antigen-antibody complex involving thrombos-thenin in or on the platelet membrane caused non-specific damage, thereby destroying other unde-fined components of the clot retraction system.However, if this were true, one might expect adeleterious effect on clot retraction from other non-specific immune reactions. No such effects wereobserved in experiments with anti-albumin, anti-IgG, anti-whole serum and antifibrinogen. Anti-IgG and anti-albumin were also ineffective in aplasma-free system, which is significant since al-bumin and gamma globulin are identifiable inplatelets even after extensive washing (22).Therefore, not all antigen-antibody reactions in-volving platelet components impair clot retraction.This favors the specificity of the thrombosthenin-antithrombosthenin reaction. Perhaps immunedrug reactions which affect clot retraction may bedeleterious to the steric and functional integrity ofthrombosthenin.

Since disrupted platelets do not support clotretraction, it would appear that thrombostheninmust retain a specific steric relationship in the cellin order to participate in the retraction process.

1387

R. L. NACHMAN, A. J. MARCUS, AND L. B. SAFIER

Platelet fibrinogen is another necessary componentfor clot retraction (23) and a recent report byMorse, Jackson, and Conley indicated that enzy-matic splitting of fibrinopeptide B from fibrinogenby thrombin is a necessary prerequisite for clotretraction (24). Clot retraction probably repre-sents the culmination of a complex series of physio-logical events, requiring at least a source of energyin an intact platelet, proteolytic digestion of plate-let fibrinogen, and the presence of the contractileprotein thrombosthenin. The capacity of throm-bosthenin to hydrolyze ATP may be an importantpart of its function in mediating clot retraction.The observation that antithrombosthenin signifi-cantly blocks the ATPase activity of thrombos-thenin suggests that an enzymatic site(s) of themolecule may be necessary for its activity. How-ever, the antiserum probably also contains anti-bodies to nonenzymatic portions of the molecule.Chambers, Salzman, and Neri have proposed thatthe ATPase activity of thrombosthenin on theouter platelet membrane may be related to the ac-tion of ADP in aggregating platelets (25).Our structural studies on purified thrombos-

thenin suggest that the molecule may be composedof subunit polypeptide chains, possibly of poly-meric nature as illustrated by multiple bands instarch urea gel electrophoresis. The molecularweight was greater than 300,000, as evidenced byexclusion of the active material by a P-300 Bio-Gelcolumn. It was of interest that only eight peptideswere identifiable after tryptic digestion of thepurified protein. Pronase digestion revealed 12-13 spots on fingerprinting, which was in markedcontrast to over 40 spots when striated muscle ac-tomyosin was similarly treated. The small numberof peptides in the presence of a large molecularweight suggests the presence of repeating units.

Studies on the subcellular platelet fractions indi-cated that thrombosthenin was associated withplatelet granules and membranes. Anti-granuleand anti-membrane sera, previously adsorbed withfibrinogen' and human plasma, inhibited clot re-traction. Furthermore, it was possible to directlyextract KCl-soluble, magnesium-precipitable pro-tein from isolated granules and membranes. Thismaterial showed potent ATPase activity partiallyinhibited by mersalyl and not inhibited by ouabain.In similar fashion, the isolated granules and mem-branes themselves actively hydrolyzed ATP. That

these fractions contained other ATPase activitiessuch as the cation "pump" was evident by partialinhibition of enzyme activity by ouabain. Thespecific activity of the extracted subcellular throm-bosthenin was greater than that associated with thewhole particle fractions (Table III). It appearedthat thrombosthenin derived from the plateletmembrane was more active than that extractedfrom the granules. These materials reactedstrongly with the adsorbed antithrombostheninserum. The granule thrombosthenin showed twoto three precipitin lines with the antiserum, whilewhole platelet and membrane thrombostheninshowed single precipitin arcs in agar. Both thegranule and membrane thrombosthenin underwentsuperprecipitation in the presence of ATP andATP caused reversible changes in their relativeviscosity (Table V). However, these changeswere less marked than those observed with wholeplatelet thrombosthenin, possibly due to the in-tricate series of manipulations involved in proces-sing the material as well as to its inherent lability(2). In general, the properties of thrombostheninextracted from the membranes more closely re-sembled those of whole platelet thrombostheninthan did the granule preparation. The reasonsfor these differences are not clear, but it is pos-sible that hydrolytic enzymes known to be presentin platelet granules (18) may have altered someof the properties of thrombosthenin during theseparation procedure. Finally, the granule throm-bosthenin may have contained more contaminantsthan the membrane thrombosthenin.The specificity of the subcellular distribution of

thrombosthenin may still require further examina-tion. For example, thrombosthenin made avail-able after platelet homogenization might denature,precipitate, and then nonspecifically sediment inthe sucrose gradient (22). However, the absenceof extractable thrombosthenin in the soluble (cellsap) layer of the sucrose gradient and its relativelyintact physicochemical and immunological char-acter in the subcellular fractions suggest that theprotein may be a normal constituent of plateletmembranes. The significance of the differencesbetween granule and membrane thrombostheninnoted in the viscosity and immunochemical stud-ies remains to be determined. It is of interest thata contractile protein has recently been isolatedfrom the membrane of liver cells (26).

1388

PLATELET CONTRACTILE PROTEIN

Acknowledgment

We wish to acknowledge the technical assistance ofH. L. Ullman and E. Henry.

References

1. Marcus, A. J., and M. B. Zucker. The Physiologyof Blood Platelets: Recent Biochemical, Morpho-logic and Clinical Research. New York, Gruneand Stratton, 1965, p. 57.

2. Bettex-Galland, M., and E. F. Liischer. Thrombos-thenin-a contractile protein from thrombocytes.Its extraction from human blood platelets and someof its properties. Biochim. biophys. Acta 1961,49, 536.

3. Gross, R. Metabolic aspects of normal and'patho-logical platelets in Blood Platelets. S. A. Johnson,R. W. Monto, J. W. Rebuck, and R. C. Horn, Jr.,Eds. Boston, Little, Brown and Co., 1961, p. 407.

4. Grette, K. Studies on the mechanism of thrombin-catalyzed hemostatic reactions in blood platelets.Oslo, Norwegian Universities Press, 1962.

5. Hoffmann-Berling, H. Other mechanisms producingmovements in Comparative Biochemistry. M.Florkin and H. S. Mason, Eds. New York, Aca-demic Press Inc., 1960, p. 341.

6. Knight, V. A., B. M. Jones, and P. C. T. Jones. In-hibition of the aggregation of dissociated embryo-chick fibroblast cells by adenosine triphosphate.Nature 1966, 210, 1008.

7. Levy, H. M., and M. Fleisher. Studies on the super-precipiation of actomyosin suspensions as mea-sured by the change in turbidity. I. Effects ofadenosine triphosphate concentration and tempera-ture. Biochim. biophys. Acta 1965, 100, 479.

8. Nachman, R. L. Immunologic studies of plateletprotein. Blood 1965, 25, 703.

9. Siqueira, M., and R. A. Nelson, Jr. Platelet ag-glutination by immune complexes and its possiblerole in hypersensitivity. J. Immunol. 1961, 86,516.

10. Portzehl, H., G. Schramm, and H. H. Weber. Akto-myosin und seine Komponenten. Z. Naturforsch.1950, 5b, 61.

11. Marsh, B. B. The estimation of inorganic phos-phate in the presence of adenosine triphosphate.Biochim. biophys. Acta 1959, 32, 357.

12. Korngold, L., G. van Leeuwen, and R. L. Engle, Jr.Diagnosis of multiple myeloma and macroglobu-

linemia by the Ouchterlony gel diffusion technique.Ann. N. Y. Acad. Sci. 1962, 101, 203.

13. Korngold, L. The distribution and immunochemicalproperties of human tissue and tumor antigens.Ann. N. Y. Acad. Sci. 1957, 69, 681.

14. Shulman, N. R. Immunoreactions involving plate-lets. III. Quantitative aspects of platelet agglu-tination, inhibition of clot retraction, and other re-actions caused by the antibody of quinidine purpura.J. exp. Med. 1958, 107, 697.

15. Morse, E. E., D. P. Jackson, and C. L. Conley. Roleof platelet fibrinogen in the reactions of plateletsto thrombin. J. clin. Invest. 1965, 44, 809.

16. Cohen, S., and R. R. Porter. Heterogeneity of thepeptide chains of -y-globulin. Biochem. J. 1964, 90,278.

17. Nachman, R. L., R. L. Engle, Jr., and L. Copeland.Correlation of immunologic and structural hetero-geneity of Bence Jones proteins. J. Immunol.1966, 97, 356.

18. Marcus, A. J., D. Zucker-Franklin, L. B. Safier, andH. L. Ullman. Studies on human platelet granulesand membranes. J. clin. Invest. 1966, 45, 14.

19. Dryer, R. L., A. R. Tammes, and J. I. Routh. Thedetermination of phosphorus and phosphatase withN-phenyl-p-phenylenediamine. J. biol. Chem. 1957,225, 177.

20. Kabat, E. A., and M. M. Mayer. Experimental Im-munochemistry, 2nd ed. Springfield, Ill., CharlesC Thomas, 1961, p. 557.

21. Nachman, R. L., A. J. Marcus, and D. Zucker-Franklin. Immunologic studies of proteins as-sociated with subcellular fractions of normal hu-man platelets. J. lab. clin. Med. 1967, 69, 651.

22. Davey, M., and E. F. Luscher. Platelet proteins.Proc. 3rd Federation European Biochem. Soc.,Warsaw, 1966. In press.

23. Schmid, H. J., D. P. Jackson, and C. L. Conley.Mechanism of action of thrombin on platelets.J. clin. Invest. 1962, 41, 543.

24. Morse, E. E., D. P. Jackson, and C. L. Conley. Dif-ferential effects of reptilase and thrombin on hu-man blood platelets and clot retraction. Fed. Proc.1966, 25, 553 (Abstract).

25. Chambers, D. A., E. W. Salzman, and L. L. Neri.Characterization of "ecto ATPase" of human bloodplatelets. Arch. Biochem. 1967, 119, 173.

26. Neifakh, S. A., J. A. Avramov, V. S. Gaitskhoki,T. B. Kazakova, N. K. Monakhov, V. S. Repin,V. S. Turovski, and I. M. Vassiletz. Mechanismof the controlling function of mitochondria. Bio-chim. biophys. Acta 1965, 100, 329.

1389