Human Platelet-Initiated Formation and Uptake

of the C5-9 Complex of Human Complement

THEODoRES. ZIMMERMANand WILLIAM P. KoLs

From the Department of Molecular Immunology, Scripps Clinic and ResearchFoundation, La Jolla, California 92037

A B S T R A CT We have studied the interaction of ra-diolabeled complement components with normal humanplatelets, platelets from a patient with paroxysmal noc-turnal hemoglobinuria, and rabbit platelets in the ab-sence of known complement activators or in the presenceof coba venon factor (CVF). When unwashed plateletsin platelet-rich plasma, or washed platelets suspended inserum or autologous plasma, were incubated for 30 min,C3 and terminal components (C5, C8, and C9) werefound to bind to them. The terminal components wereshown to be bound as the C5-9 complex, rather than asindividual proteins, by eluting them from the plateletmembrane and examining their behavior on ultracentri-fugation. They cosedimented at a rate characteristic of thestable C5-9 complex (22S). As many as 370-1,380 C5-9complexes/platelet were calculated to have been boundduring the incubation period. The complex so formeddid not differ by ultracentrifugational criteria from thatbinding to rabbit platelets after CVF activation of com-plement. Though C3 was not included in the complex,it did not appear to be bound by nonspecific absorption.It could not be removed by washing but rather waseluted by the freeze-thaw technique used to elute theC5-9 complex. Incubation of radiolabeled components inplatelet-free plasma did not result in C5-9 complex for-mation, indicating an initiating role for platelets in thisreaction. In contrast to platelets, erythrocytes incubatedin analogous plasma did not induce detectable C5-9 for-mation. Neither EDTA, phenylmethylsulfonylfluoride,nor epsilon-amino-N-caproic acid prevented platelet-ini-tiated formation of C5-9, suggesting that the reactionmay involve mechanisms of complement activation notpreviously described.

Dr. Zimmerman is recipient of Research Career Develop-ment Award 5 K04 HL-70242. Dr. Kolb is the recipientof Research Career Development Award 1 K04 CA-70841.

Received for publication 9 May 1975 and in revised form15 September 1975.

INTRODUCTIONHuman platelets and platelets of other mammalian spe-cies may respond with the release reaction and/or lysisafter activation of the classical complement pathwaywith anti-platelet antibody (1, 2), immune aggregates(3-5), or activation of the alternative pathway with im-mune aggregates, endotoxin, inulin, cobra venom factor(CVF) ,I or zymosan (6-17). Description of a coagula-tion abnormality in the blood of rabbits congenitally de-ficient in C6 (18) and demonstration of the role ofplatelets in this coagulation defect (2, 19-21) has sug-gested that complement-platelet interaction may proceedin the absence of known complement activators. Morerecently, deficient platelet aggregation in response tocollagen and ristocetin have been demonstrated in humansubjects deficient in C5 (21), suggesting a role for com-plement in normal human platelet function.

Utilizing radiolabeled complement components, wehave now demonstrated that normal human platelets aswell as those of rabbits can themselves initiate formationand binding of the C5-9 complex, the mediator of com-plement-directed membrane damage (22, 23). This reac-tion proceeds in the presence of EDTA and epsilon-amino-N-caproic acid (EACA), suggesting that it isnot initiated by the classical or alternative pathways ofcomplement activation and also differs from C5-cleavingplatelet factor of Weksler and Coupal (24).

METHODSPreparation of platelet-rich plasma, washed platelets,

washed erythrocytes, platelet-free plasma, and serum. Freshwhole blood was anticoagulated with acid citrate dextroseor citrate phosphate dextrose (25). Platelet-rich plasma wasimmediately prepared by centrifugation of whole blood at

1Abbreviations used in this paper: BSA, bovine serumalbumin; CVF, cobra venom factor; DFP, diisopropylfluoro-phosphate; EACA, epsilon-amino-N-caproic acid; PMSF,phenylmethylsulfonylfluoride; PNH, paroxysmal nocturnalhemoglobinuria.

The Journal of Clinical Investigation Volume 57 January 1976 *203-211 20uv3

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FIGURE 1 Binding of complement components to plateletswith and without CVF activation of complement. Plateletswere suspended in normal human serum containing mI-C3,'I-C5, and 'I-C8, in various combinations or individually.CVF or buffer was then added. After a 30-min incubation,platelets or platelet membrane fragments were collectedon Millipore filters and retained radioactivity was deter-mined. In parallel experiments, platelets were labeled with'Cr and percent release was determined by measuringradioactivity remaining in the supernates after centrifuga-tion. Upper panel: Binding of C3, C5, and C8 to rabbitplatelets and comparison with 'Cr release. Almost 50%o of'Cr release occurred in serum without added CVF. How-ever, increased 6'Cr release and visible lysis as well asmarkedly increased C5 and C8 uptake were seen uponaddition of CVF. No significant increase in C3 uptake withCVF (as compared to serum alone) was seen in this ex-periment though it did occur occasionally. Lower panel:Absence of effect of CVF on uptake of C3 or C8 ontonormal human platelet preparations as compared with amarked enhancement of C8 uptake onto PNH platelets. C3uptake onto PNH platelets (not shown) did not exceedthe control (0.05-0.09%o). On the left ordinate is indicatedthe percent of radiolabeled complement components in theincubation mixture that specifically bound to the platelets.On the right ordinate, top panel, is the percent of 'Crreleased from the platelets.

190 g for 15 min at 22-250C. The platelet-rich plasma wasthen recentrifuged at 620 g for 60 s to reduce the con-taminating red and white cells to less than 1%. The finalplatelet count was between 1.5 and 2.0 X 105/mm'. Washedplatelets were prepared in modified Tyrode's buffer con-taining 0.2% albumin and 0.03 M adenosine, as described

elsewhere (26). Final platelet suspensions contained lessthan 0.1% red or white blood cells.

Erythrocytes were prepared free of platelets in the fol-lowing manner: After removal of platelet-rich plasma, theerythrocyte-containing infranate was placed on a 6-35%albumin gradient. The gradient was constructed by suc-cessively freezing 0.7-ml quantities of 35%o, 28%o, 21%o,14%o, and 6% albumin in 15-ml Corning polystyrene conicalcentrifuge tubes (no. 25310, Corning Glass Works, ScienceProducts Div., Corning, N. Y.). Gradients were thawedbefore the erythrocytes were applied. 1 ml of the erythro-cyte-rich infranate was layered on each gradient. The gradi-ents were centrifuged at 2,300 g for 30 min and the erythro-cytes were removed and washed three times in Tyrode'sbuffer. The erythrocytes so prepared contained less than0.2% leukocytes and no detectable platelets.

Fresh plasma was prepared from citrated whole bloodby centrifugation at 2,300 g for 15 min and recentrifugationof the supernate at 82,500 g for 45 min. Human serum wasprepared by allowing fresh whole blood to clot at 370C for2 h in a glass tube. The clot was removed by centrifugationat 2,300 g for 15 min and the serum recentrifuged at 82,500g for 45 min. The serum was stored at - 70'C.

Complement components and reagents. Complement com-ponents (27-29) and CVF (30) were prepared as describedpreviously. Complement components were radiolabeled with

'I or 'I by the chloramine T method of McConahey andDixon (31). Specific activity was between 0.5 and 1.9 X 106cpm/,ug protein. 50%o or more of the radiolabeled comple-ment proteins were functionally active. After being labeled,each component was dialyzed for 48 h against 4 X 10 litersof 0.15 M sodium chloride containing 50 mMchloramphen-icol (donated by Parke, Davis & Co., Detroit, Mich.) and25 juM kanamycin sulfate (Bristol Laboratories, Inc., Div.Bristol-Myers Co., Inc., Syracuse, N. Y.). The dialyzedpreparations were centrifuged for 2 h at 98,000 g to removeaggregates. Bovine serum albumin (BSA) (Pentex Bio-chemicals, Kankakee, Ill.) was added to a final concentra-tion of 1 mg/ml and the preparations were stored at 0°C.

Specific binding of complement components to plateletmembranes. Platelets were concentrated by centrifugationat 1,000 g and resuspended in normal human serum at afinal concentration of 1-3 X 106 platelets/mm3. 'I or "I-labeled complement components were added to the abovesuspension to give 1-5 X 10' cpm/ml. Complement was acti-vated in each mixture by adding increasing quantities ofCVF (28-110 ,ug/ml). Control incubations consisted of:(a) Tyrode's buffer substituted for CVF, and (b) activa-tion of serum by the indicated amount of CVF in theabsence of platelets. After a 30-min incubation at 37°C,the platelets were examined visually under phase micros-copy for lysis. Platelet fragments were collected on 0.45-,umMillipore filters (Millipore Corp., Bedford, Mass.) andwashed with 40 vol of normal saline, 40 vol of distilledwater, and 40 vol of normal saline. Radioactivity remainingon the filters was determined in a gamma scintillationspectrometer (Packard Instrument Co., Inc., DownersGrove, Ill.). Control serum, which contained radiolabeledcomplement components but no platelets and had otherwisebeen treated like the platelet serum mixtures, was alsofiltered, and counts retained on the filters were subtractedfrom the counts remaining on the appropriate filters fromthe platelet-serum samples.

Sucrose density gradient ultracentrifugation. 10-40o lin-ear sucrose density gradients were made with barbital-buffered saline, pH 7.5, ionic strength 0.15, in 5-ml (1 inchX 2 inch) cellulose nitrate tubes. The sample size was

204 T. S. Zimmerman and W. P. Kolb

100-200 /Al and centrifugation was performed in a BeckmanSW50.1 rotor (Beckman Instruments, Inc., Spinco Div.,Palo Alto, Calif.) for 16 h at 114,000 g. 125-dul fractionswere collected and counted on a gamma scintillation spec-trometer.

Sucrose density gradient ultracentrifugational analysis ofcomplement components bound to platelet membranes orerythrocyte membranes. For these experiments freshlyprepared (less than 3 h from venipuncture) platelet-richplasma (1.5-2.0 X 105/mm3) was used or washed platelets orerythrocytes were suspended in normal human serum or au-tologous plasma. In the initial experiments concentrations ofplatelets of 1-5 X 10'/mm3 were used. However, lower con-centrations, similar to those in platelet-rich plasma, subse-quently proved more efficient in initiating formation of theC5-9 complex, and erythrocytes were therefore used atthose concentrations (1.5-2.0 X 105/mm'). Complement com-ponents were added to give 10-20 X 100 cpm/ml. CVF,when added, was employed at a final concentration of 110Atg/ml. The mixture was incubated at 370C for 30 min andthe platelets separated by centrifugation for 3 h at 123,000 g.Supernatant plasmas were stored at - 70'C before ultra-centrifugational analysis. Platelets or erythrocytes werewashed four times by suspension in phosphate-buffered salinewith a Potter-Elvehjem-type tissue grinder and centrifuga-tion at 123,000 g for 2 h. Radiolabeled complement compo-nents were eluted from the platelet or erythrocyte membranefragments by suspending them in 0.5-0.7 ml of phosphate-buffered saline, freezing to - 70'C, and thawing. The freeze-thaw process was repeated four times. Particulate matterwas removed by centrifugation at 123,000 g for 2 h.

5"Cr release from platelets. Platelets suspended in Ty-rode's buffer at a concentration of 2 X 105/mm' were in-cubated with 10 juCi/ml of 'Cr. The platelets were washedtwice in Tyrode's buffer and resuspended in serum as inthe procedure for quantitating specific uptake of radio-labeled complement components. However, after incubationwith CVF or buffer, platelets were removed by centrifuga-tion at 7,000 g for 15 min and the 'Cr radioactivity re-maining in the supernatant was determined. A 100% valuewas determined by counting a platelet suspension not sub-jected to centrifugation.

RESULTS

Comparison of uptake of C3 and C8 onto human andrabbit platelets with and without activation of the alter-native pathway by CVF. Normal human platelets from10 different donors were suspended in serum and C3 andC8 uptake compared in the presence or absence of addedCVF. No increase in uptake of C8, and usually no in-crease in uptake of C3, could be discerned with CVFpresent, as compared to uptake in its absence (Fig. 1,lower panel). CVF did not induce visible lysis or re-lease detectable amounts of 'Cr from normal humanplatelets. In contrast, platelets from individuals withparoxysmal nocturnal hemoglobinuria (PNH platelets)did show a marked increase in C8 uptake when comple-ment was activated with CVF, though C3 uptake was

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FIGURE 2 Photomicrograph of PNH and normal human platelets after exposure to CVF-activated complement. PNH or normal human platelets were suspended in serum and CVFto a concentration of 110 pg/ml was added. (a) Marked clumping and lysis of PNH plateletswas seen. (b) Normal human platelets remained freely suspended without visible lysis.(X 1,000)

Platelet-Initiated CS-9 Complex Formation 205

Fraction Number

FIGURE 3 Ultracentrifugational demonstration of Cplex in eluates from rabbit platelets with and with(activation of complement. Platelets were incubate(min in normal human serum with and without CV]lets were removed by centrifugation and the superitaining CVF was subjected to ultracentrifugation40%o linear sucrose density gradient at 114,00 g fDirection of sedimentation was to the left. Thewere homogenized and washed and the membracomplement components eluted by freeze-thawinticulate material was removed by centrifugationeluates then subjected to sucrose density ultracetional analysis. Upper panel: The sedimentation EC8 and C9 in whole human serum after incubatCVF and platelets. Middle panel: The C8 and Cof eluates from rabbit platelets subjected to CVItion. Lower panel: The C8 profile of eluates fromincubated in serum without CVF. In the lower panlabeled albumin has been included in the incubationas a control.

only minimally increased (from 0.05 to 0.09%)dition, the PNH platelets were visibly lysed (IRabbit platelets, on the other hand, reacted likePNH platelets. Addition of increasing quaniCVF to platelet serum mixtures resulted in ationate increase in C8 uptake, visible lysis, and rc'mCr (Fig. 1, upper panel). Increased C3 uptakethe usual concomitant of C8 uptake after CVFtion of the alternative pathway (Fig. 1), thougloccasionally observed.

Demonstration that terminal components bounpresence or absence of CVF were incorporatedC5-9 complex. Rabbit and human platelets, ir

for 30 min in human serum with or without CVF, werehomogenized and washed. The membrane-bound comple-ment components that remained were then eluted byfreeze-thawing and the eluates analyzed by sucrose den-sity gradient ultracentrifugation. Terminal components(C8 and C9) eluted from CVF-treated rabbit plateletsnot unexpectedly cosedimented with a sedimentation co-efficient of 22S, the same rate as C5-9 formed in fluidphase by CVF activation (Fig. 3). Surprisingly, how-ever, the terminal components eluted from rabbit and

I human platelets incubated in serum without CVF alsocosedimented with a sedimentation-rate of 22S (Figs. 3and 4). Examination of eluates from platelets incubatedin serum with or without CVF usually revealed theincorporation of terminal components into a heaviercomplex as well (28S). Though the quantity of terminalcomponents bound to rabbit platelets in the presenceof CVF was usually greater than that seen in its ab-

! sence, no qualitative differences in the C5-9 complexformed by either method could be detected by ultracentri-fugational analysis.

To obviate a possible role of isoantibodies or aggre-gated immunoglobulin as complement activators in theabsence of CVF, human platelets were incubated in

5-t cVF fresh autologous plasma and the components so boundd for 30 eluted and examined by ultracentrifugation. Results ofF. Plate- four such experiments are summarized in Figs. 4 and 5.nate con- As with the human or rabbit platelets incubated in se-in a 10- rum, the eluted terminal components (C5, 8, and 9) co-tor io n.

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FIGURE 4 Demonstration of a C5-9 complex in eluatesfrom human platelets incubated in fresh autologous plasma.In the upper panel radiolabeled C5 had been added to theincubation mixture and in the lower panel both C8 and C9had been added. Small quantities of C5 and C9 were ob-served in their native position.

206 T. S. Zimmerman and W. P. Kolb

sedimented as the C5-9 complex, with a sedimentationcoefficient of 22S (Fig. 4). A heavier complex, with asedimentation coefficient and 28S, was described in mostexperiments. C3, on the other hand, was not included inthe complex and sedimented at its native rate of 8.4S(Fig. 5). However, binding of C3 did not appear to bedue to nonspecific absorption because C3 was presenton platelet membranes that had been washed entirely

Platelet Free Plasma-8

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Washed Platelets in Plasma15 C

Platelet Rich Plasma 215

8 16 24 32 40Fraction Numher

FIGURE 5 Comparative sedimentation velocities of C3 andC8 incubated in plasma with C3 and/or C8 eluted fromprewashed or unwashed platelets. Radiolabeled 'I-C3 and131C8 were incubated with previously washed human plate-lets suspended in autologous plasma and platelet-free plasma,and 12I-C8 was incubated with human platelet-rich plasma.Platelet-rich plasma was used to provide a source of un-washed platelets. The platelet-free plasma (upper panel )and the freeze-thaw eluates from the platelets (middle andlower panels) were examined by sucrose density ultra-centrifugation as described for Fig. 3. In thbe eluates fromboth preparations of platelets, most or all of the CS sedi-mented at a rate characteristic of the C5-9 complex. In theplatelet eluate in which no native C8 could be detected, allof the C3 sedimented at its native rate (middle panel). Inthe platelet-free plasma, C8 sedimented at its native rate,with none sedimenting at the rate of the C5-9 complex.

Control

co 0.2M EACAX

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FIGURE 6 Failure of EACAor EDTA to inhibit formationof the 22S C5-9 complex. Normal human platelets werepreincubated for 30 min at 370C in either Tyrode's bufferalone, or Tyrode's buffer containing 02 M EACA or 0.02M EDTA. They were then suspended in normal plasmacontaining these inhibitors and C5-9 elution was conductedas in Fig. 3. It can be seen that there was no inhibitionof formation of the 22S C5-9 complex. The 28S complexwas not seen in this experiment. PMSF, 0.001 M also failedto inhibit formation of the C5-9 complex.

free of native C8 and could only be removed by the sametechniques used to elute the C5-9 complex. The forma-tion of platelet-bound C5-9 complex did not appear to bethe result of platelet injury induced by the washingprocess. The same reaction was demonstrated with theunwashed platelets present in platelet-rich plasma (Fig.5, lower panel).

Evidence for platelet-initiated CS-9 formation. Todetermine if C5-9 binding in the absence of CVF wasthe result of a platelet-initiated process, or rather re-sulted from fluid phase activation of complement byanother mechanism, platelet-free plasmas were incubatedin parallel with platelet-plasma mixtures and were sub-jected to sucrose density ultracentrifugation. Terminalcomponents sedimented in their native position (Fig. 5),with no evidence for spontaneous fluid phase formationof the C5-9 complex in the absence of platelets (32).

Platelet-Initiated C5-9 Complex Formation 207

Supernatant plasmas from the platelet-plasma mixtureswere also subjected to sucrose density gradient ultra-centrifugation and little or no C5-9 could be detected,suggesting that most, if not all, C5-9 formed in thepresence of platelets was bound to the platelet surface.

Failure of EDTA, EACA, phenylmethylsulfonylfluoride(PMSF), or diisopropylfluorophosphate (DFP) to in-hibit platelet-initiated formation of the CS-9 complex.Preincubation of platelets with 0.02 M EDTA and 0.2M EACA and subsequent inclusion of these inhibitorsin the platelet-plasma incubation mixtures failed to pre-vent platelet-initiated formation of the C5-9 complex(Fig. 6). Similarly, no inhibition was seen if plateletswere preincubated with 0.02 MDFP and 0.02 MPMSFwas included in the platelet-plasma incubation mixtures.

Absence of C5-9 complex formation on erythrocytesincubated in homologous plasma. When erythrocyteswere incubated with radiolabeled C8 in autologousplasma, no C8 with the 22S sedimentataion character-istics of the C5-9 complex could be subsequently elutedfrom them. In contrast, a parallel experiment run withplatelets and plasma from the same blood sample showedcharacteristic formation of platelet-bound C5-9 complex.

C5-9 complexes bound per platelet. The number ofC5-9 complexes bound per platelet during the 30-minincubation was estimated at 370-1,380 in three separateexperiments. This figure was determined by calculatingthe number of C8 molecules bound per platelet which,when subsequently eluted, sedimented at a rate of22S and were therefore included in the complex. Ithas previously been shown that there is one C8 mole-cule per C5-9 complex (22). These calculations weremade for those experiments in which the platelet con-centrations similar to those normally seen in plate-rich plasma were used.2 These concentrations of platelets

'A typical calculation was performed as follows: Thespecific radioactivity of the 'I-C8 used was 1.6 X 10' cpm/,ug. 15% of the platelet-bound 'I-C8 could be eluted and11.28 X 10' cpm sedimented in the 22S peak on ultra-centrifugation. Therefore, (11.28 X 108)/0.15 = 7.5 X 10' cpmwere incorporated into the C5-9 complexes bound tothe 2.4 X 109 platelets in the 15-ml incubation mixture.Therefore, cpm/platelet = (7.5 X 10' cpm/2.4 X 109 plate-lets) = 3.125 X 10' cpm/platelet and ug of 'I-C8/plate-let = (3.125 X 10-5 cpm/platelet/1.6 X 106 cpm/,ug) = 1.953X 10-11 jug/platelet. Total (cold plus 'I-labeled) C8 perplatelet was calculated as follows: 100 ug of 'I-C8 (70%of which was hemolytically active) was initially addedto 15 ml of platelet-rich plasma containing 80 ,ug/ml X 15ml = 1,200 ,g of cold C8. Thus the ratio of hemolyticallyactive 12'I-C8 to total hemolytically active C8 was (100,ug X 0.7/1,200.ug + (100 X 0.7),ug) = 0.055. And total boundC8 = (1.953 X 10`1 Ag/platelet/0.055) = 3.54 X 10"1 ug/platelet. Molecule of C8/g = [6.02 X 101 molecule/g-mol(Avogadro's number) /1.53 X 106 g/g-mol (mol wt of CS)]= 3.9 X 10"8 molecules/g or 3.9 X 1012 molecules/pg. Thus,molecules of C8 per platelet = 3.54 X 10-° Ag C8/platelet X3.9 X 10's molecules/.Mg = 1.38 X 106 molecules/platelet.

were far more efficient in generating the C5-9 complexthan the 10-fold higher concentrations used in earlierexperiments (compare Fig. 5, bottom panel, with otherexperiments). In making these calculations it was as-sumed that the 'I-C8 used was 70% active. A lowerpercentage of active C8 would give a higher number ofcomplexes bound per platelet.

DISCUSSIONThe experiments reported here demonstrate that normalhuman and rabbit platelets can initiate formation of theC5-9 complex in the absence of known activators of thecomplement system. Platelets appeared essential for theformation of C5-9 in this fresh plasma system as therewas no evidence for C5-9 complex formation in plasmafrom which they had been removed. It is not certainwhether other plasma factors are also required. Thepresence of C3 on the platelet membrane suggests a pos-sible role for this protein in the activation process. How-ever, the sucrose density gradient ultracentrifugationtechniques employed are not sensitive enough to de-termine if the bound radiolabeled C3 was in its nativeform or had been converted to C3b.

The failure of 0.02 M EDTA to block platelet-ini-tiated C5-9 formation suggests that participation ofeither the classical or alternative pathways of comple-ment activation, in the usual sense, is unlikely. It is pos-sible that one or more components of these pathwaysmay play a role, however. It is also possible that one ormore of the constituents of the clotting or fibrinolyticsystems play a role in platelet-initiated C5-9 formation,even though the serine esterase inhibitor, PMSF, andthe plasmin inhibitor, EACA, failed to block the re-action.

Weksler and Coupal (24) have demonstrated that afactor present in an acid-soluble extract of human plate-lets can release chemotactically active C5a from C5. Atthe present time it is not certain whether the property ofhuman platelets that initiates formation of the C5-9 com-plex is related to the C5-splitting activity described bythese investigators. In their experiments the evolutionof chemotactic activity was blocked by as little as 0.02M EACA. Yet 0.2 MEACA failed to block the abilityof human platelets to induce formation of the C5-9 com-plex, suggesting that a different mechanism is involved.

C3 activation, without the participation of C5-9, mayinduce the release reaction in rabbit or human plateletsafter complement activation by such agents as zymosanor immune aggregates (1, 14-17). Nevertheless, it isclear from these studies that binding of C3 to PNHorrabbit platelets is not necessary for C5-9 binding (withresultant lysis) after alternative pathway activation ofcomplement with CVF. These findings agree with thoseof Gotze and Muller-Eberhard (23), who have shown

208 T. S. Zimmerman and W. P. Kolb

that the C423 enzyme can effect binding of C5, 6, and 7to the surfaces of cells distinct from those to which theC423 enzyme is bound.

The existence of a coagulation defect in the blood ofrabbits congenitally deficient in C6 has been previouslyrelated to the action of the terminal components onplatelet coagulant activity. Activation of the alternativecomplement pathway with such agents as inulin andendotoxin leads to lysis of rabbit platelets with markedenhancement of their coagulant activity, an effect notseen in the absence of one or more of the terminal com-ponents (2, 20, 21). The observations reported heresuggest a mechanism by which complement-inducedenhancement of platelet coagulant activity might occurin the absence of such extrinsic activators. The plate-let, by activating complement at its own surface, couldenhance its own coagulant activity, thus acceleratingthe rate of whole-blood clotting. In C6-deficient bloodthe clotting time would, therefore, be relatively pro-longed.

Though normal human platelets, like rabbit platelets,can initiate formation of the C5-9 complex, they differfrom rabbit platelets in being considerably less sensitiveto the effects of C5-9. It is therefore not surprising thatin human C6 (33) and C7 (34) deficiency, no significantprolongation of the whole-blood clotting time is seen.On the other hand, PNH platelets are more sensitivethan normal platelets to the effect of complement (35)and behave much like those of the normal rabbit. Themechanism we have described might therefore play arole in the thrombotic tendency known to complicatePNH (36, 37).

We have calculated that as many as 370-1,380 C5-9complexes bind per platelet during the 30-min incuba-tions in these experiments. It therefore seems un-likely that a small subpopulation of platelets could beresponsible for this reaction. As little as one C5-9 com-plex has been found sufficient to induce lysis in sheeperythrocytes (38), thus emphasizing the relative resis-tance of human platelets to the lytic effects of C5-9.

The formation of the C5-9 complex on unwashedplatelets in freshly prepared platelet-rich plasma sug-gests that this phenomenon may be an ongoing in vivoprocess. A small degree of activation of the clotting sys-tem may occur as the blood is drawn and the clottingenzymes so formed participate in platelet-initiated C5-9formation. Such an occurrence does not mitigate againstthe same process occurring in vivo in normal individuals,as well as in patients in whom intravascular coagulationis clincally evident. Continuous intravascular thrombinformation in normal individuals has been postulated forthree quarters of a century (39, 40), and recent evidencefor its occurrence has been provided by the demonstra-

tion of small amounts of fibrinopeptide A in the plasmasof normal individuals (41).

At this time the physiological or pathological role(s)of this phenomenon can only be postulated. The failureto demonstrate initiation of C5-9 formation by humanerythrocytes indicates that this reaction is not commonto all cell membranes. It is possible that modulation ofplatelet membrane by complement is necessary forproper platelet function. Such a role is consistent withthe markedly reduced to absent response to ristocetin re-cently observed in the platelet-rich plasma of two indi-viduals congenitally deficient in C5 (21). The receptornecessary for aggregation by these agents may not beexposed unless the platelet is acted upon by the C5-9complex.

A pathologic role for the C5-9 complex in athero-genesis has been suggested by a recent study performedwith C6-deficient and normal rabbits fed a cholesterol-rich diet (42). Though both groups of rabbits developedatheromas, the normal group had significantly more ad-vanced disease. The mechanisms by which complementmay participate in the formation of atheromas is notcertain. However, it is possible that the platelet partici-pates by forming aggregates at the sites of insipientplaques. Platelets in C6-deficient animals might be lessprone to form such aggregates because of the absence ofcomplement-dependent platelet membrane receptors.

Total hemolytic complement, as well as immunologi-cally measured C3, have been shown to be decreased inseveral clinical conditions characterized by disseminatedintravascular coagulation (43). Recent studies utilizingrabbits have shown a role for the platelet in the de-pression of total hemolytic complement and C3 that oc-curs with intravascular clotting (44). The demonstrationhere that the platelet can bind C3 and initiate formationof the C5-9 complex is consistent with these in vivoobservations. It is possible that these processes may beaccelerated secondary to platelet damage induced byintravascular clotting.

ACKNOWLEDGMENTSWe are indebted to Mrs. Janet Kimball, Ms. Judy Mein-koth, and Mr. Murray Towle for excellent technical as-sistance and to Mrs. Sharon Dinwiddie for preparing themanuscript.

This is publication no. 977 from the Scripps Clinic andResearch Foundation.

This work was supported by grants HL-15491, HL-16411,and AI-11751.

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Platelet-Initiated C5-9 Complex Formation 209

3. Gocke, D. J. 1965. In vitro damage of rabbit plateletsby an unrelated antigen-antibody reaction. II. Studiesof the plasma requirement. J. Immunol. 94: 247-252.

4. Des Prez, R. M., and R. E. Bryant. 1969. Two mecha-nisms of immunologically-induced injury to rabbit plate-lets. J. Immunol. 102: 241-252.

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