Microparticle generation during in vitro platelet activation by anti-CD9 murine monoclonal antibodies

THROMBOSIS RESEARCH 62; 429-439,199l 0049-3848/91 $3.00 + .OOPrinted in the USA. Copyright (c) 1991 Pergamon Press plc. All rights reserved.

MICROPARTICLE GENERATION DURING IN VITRO PLATELET ACTIVATION BY ANTI-CD9 MURINE MONOCLONAL ANTIBODIES Shosaku Nomura, Hirokazu Nagata, Masahiko Suzuki, Koji Kondo, Shigetoshi Ohga, Toshihiro Kawakatsu, Hirofumi Kido, Tsutomu Fukuroi, Kazuyuki Yamaguchi, Koji Iwata, Mutsumasa Yanabu, Tetsuji Soga, Terutoshi Kokawa, and Kojiro Yasunaga The First Department of Internal Medicine, Kansai Medical University, 1 Fumizono-cho, Moriguchi, Osaka 570, Japan

(Received 15.10.1990; accepted in revised form 16.2.1991 by Editor H. Yamazaki)

ABSTRACT We used flow cytometry and two anti-CD9 murine monoclonal antibodies (NNKYI-19, MALL13) to investigate the glycoprotein composition and the potential functions of microparticles (MP) released by platelets exposed to these antibodies in vitro. NNKYI-19 produced aggregation with characteristics similar to those noted in previous reports. The action of MALL13 on platelets in platelet-rich plasma (PRP), however, differs from that of other anti-CD9 antibodies. The normal fluctuation in the MALLlj-induced change in optical density disappeared when complement was present. MALL13-induced effect for platelet in PRP was not inhibited by preincubation with monoclonal anti-GPIIb/IIIa antibody, but was inhibited in washed platelets (WP). Furthermore, following MALL13 stimulation in PRP platelets, the amount of buffer LDH markedly increased and electron microscopy findings showed vacuoles appearing inside the platelets. These results suggest that MALL13 has at least two effects on platelets that differ for PRP platelets and WP. The number of MP released was increased by the addition of antiCD9 antibodies. MP surfaces were found to be rich in CD9 protein. MALL13 stimulation lead to a significant increase in the binding of Clq and C3 to platelets and caused the production of MP to occur more rapidly than it did the exposure of fibrinogen binding sites in the presence of complement. The analysis of the relationship of MP to anti-CD9 monoclbnal antibody may be useful in the investigation of the relationship between platelet function and coagulation regulation. INTRODUCTION When platelets are exposed to certain stimuli, activation is accompanied by the release of small membrane vesicles or microparticles (MP) from their surface (1,2). Sims et al. have reported that the C5b-9 proteins of the Key Words: anti-CD9 murine monoclonal antibody, microparticle, flow cytometry electron microscopy 429

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complement system in particular provide a unique stimulus resulting in the nonlytic activation of platelets and other blood cells (2). In human platelets, membrane deposition of complement proteins C5b-9 has been shown to induce the secretory release of both Cf.,-granules and dense granules as well as the de novo expression of membrane receptors for coagulation factor Va. The latter results in a marked increase in procoagulant activity through the production and activation of the prothrombinase enzyme complex involving factors Va and Xa (3). Furthermore, Abrams et al. have recently reported that MP can be resolved from intact platelets by fluorescence flow cytometry, allowing the membrane composition of individual particles to be analyzed using monoclonal antibodies against cell surface determinants (4). It has also been reported that anti-CD9 murine monoclonal antibodies, which react with the leukocyte differentiation antigen CD9, induce platelet aggregation and granule release (5,6,7,8). Some studies have suggested that CD9 antibody-induced platelet aggregation may be closely related to the platelet glycoprotein (GP) IIb-IIIa complex (8,9). CD9 antibodies can also elicit the aggregation of washed platelets (WP) and produce a calcium signal following a lag phase (10,ll). Although the widespread distribution of the CD9 antigen suggests that it plays an important role in general biological responses, the nature of that role has remained obscure. We recently developed two monoclonal CD9 antibodies, NNKYI-19 and MALL13, antibodies against human platelets and common ALL cells, respectively (12). In the present study, we used flow cytometry to characterize the glycoprotein composition and the potential functional significance of MP released by platelets exposed to anti-CD9 murine monoclonal antibodies in vitro.

MATERIALS AND METHODS

Monoclonal Antibody Preparation: The anti-CD9 murine monoclonal antibodies used (NNKYI-19 and MALL13) have been described previously (12). NNKYI-19 was developed by immunizing mice with human platelets and MALL13 by immunization with common ALL cells. The respective subclasses of NNKYl-19 and MALL13 are IgGl and IgG2a. The molecular weight of the platelet-membrane antigens recognized by NNKYI-19 and MALL13 was 24 kDa under nonreducing conditions. The following antibodies were used for comparison: ALB6 (anti-CDS; Cosmo Bio Co., Tokyo, Japan)(6), TP82 (anti-CDS; Nichirei Co., Tokyo, Japan)(7), NNKYl-32 (anti-GPIIb/IIIa)(l3,l4), and NNKYS-5 (anti-GPIb)(l4). Washed Human Platelets Preparation: Platelet-rich plasma (PRP) and washed platelets (WI')were obtained by methods described previously (13,14). After washing, the platelets were resuspended in Tyrode's buffer (137 mM NaCl, 12 mM NaHC03, 5.5 mM glucose, 2 mM KCl, 1 mM MgC12, and 0.3 mM Na2HP04, pH 7.4). Platelet Aggregation and ATP Release: Platelet aggregation studies on citrated PRP or WP were performed using an NKK Hematracer 1 (Niko Bioscience, Tokyo, Japan), and the maximal percent change in light transmission relative to platelet-poor plasma was recorded. ATP release from PRP or WP was measured using a Lumi-aggregometer (Payton Association Limited, Canada). Aggregation curves and ATP release were evaluated for aggregation induced by NNKYI-19 and MALL13 at 10 ug/ml. ADP (10 fl), Collagen (4 pg/ml), Thrombin (0.25 U/ml), and A23187 (2 uM) were used for comparison. The following reagents were used for aggregation inhibition testing: ethylene diamine tetraacetic acids (EDTA)

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and the peptides Arg-Gly-Asp-Ser (RGDS; Peninsula Laboratories, Belmont, CA). Paraformaldehyde-Treated Platelets Preparation: The platelets were suspended in 1% paraformaldehyde (PFA) and incubated at room temperature for 15 minutes. They were then washed twice, resuspended in stock solution (9 mM Na2EDTA, 26.4 mMNa2HP0 2H20, 140 mM NaCl, 0.1% NaN3, and 2% fetal bovine serum, pH 7.2) and store'dr at 4°C until analysis. Cytoplasmic Free Calcium Ion Concentration: Calcium levels in aequorinloated platelets (Chrono-Log Corp., Ltd., Havertown Penn., USA) were calculated using the method of Johnson et al. (15). The platelets were washed with Hepes-Tyrode's buffer containing PGE (1 pM) then incubated with aequorin (0.2 mg/ml) in the presence of EGTA (10 &) and ATP (5 mM). The aequorin-loaded platelets were washed twice at 10,OOOg over 90 seconds and resuspended in PBS with 10 mM MgC12 and 5 mM ATP. The platelets were incubated for 60 minutes at O'C, passed through a Sepharose 2B column, and resuspended in Hepes-Tyrode's buffer. Platelet aggregation and cytoplasmic free calcium ion concentration ([Ca2+]i) were measured using a Platelet Calcium Aggregometer (PICA; ChronoLog Corp., Ltd.). Lactate Dehydrogenase Release: Lactate dehydrogenase (LDH) assays were performed on sample supernates by UV methods to determine the percentage of LDH released during agonist stimulation. The total amount of platelet LDH and the the supernated LDH activity were used to calculate the percentage of LDH leakage. The total amount of LDH was measured following platelet lysis with detergent 1% Triton X-100. Flow Cytometry: 1. Monoclonal Antibody Binding Inhibition Testing. PFA-fixed platelets treated with PBS, NNKYl-19, MALL13, ALB6, TP82, NNKY l-32, or NNKY5-5 then incubated with FITC-conjugated NNKYI-19 for 30 minutes at room temperature, washed 3 times by centrifugation, and analyzed using a FACS analyzer. 2. Platelet Activation. Agonists stimulations were performed by incubating PRP or WP for 10 min at 22°C without stirring in the presence of 10 fl ADP, 5 @i A23187, 10 Pg/ml NNKYI-19, or 10 pglml MALL13. Following incubation, the samples were immediately prepared without centrifugation using PFA with EDTA-PBS(-)(final concentration of PFA: I%, pH 7.2). Activated or control platelets were incubated in saturated concentrations of one of the following fluorescein-conjugated antibodies: FITC-NNKYI-32 (IO pg/ml), FITC-NNKYS-5 (IO pg/ml), FITC-antiFibrinogen (Fbg)(S pg/ml; Binding Site Ltd., Birmingham, England), FITC-antiClq (5 pg/ml; Binding Site Ltd..),and FITC-anti-C3 (5 pg/ml; Binding Site Ltd.). Operating Conditions for The FACS Analyzer. 3. Samples were assayed on a Becton Dickinson FACS Research Analyzer. The assay specifics have been published previously (13,14). To quantify fluorescence, SIGMA was defined as: SIGMA = [Mean channel of positive region x total platelet counts of positive region] / IO3 Ten thousand events were analyzed in the one-color analysis. For the twoparameter analysis, a 64 x 64-channel histogram of log green fluorescence versus volume was produced, with 3,000 events being analyzed. The flow cytometry data were then analyzed using an NEC PC-98OlVX personal computer. Electron Microscopy: PRP was stimulated by 10 Pg/ml of MALL13 IgG. The stimulated platelets were then fixed for 30-60 minutes at 4°C in a 2% glutar-

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aldehyde in 0.1 M PBS solution, and washed in 0.1 M PBS with 6.SX sucrose. After postfixation in 1% osmium tetroxide, the platelets were dehydrated with a series of ethanols, and embedded in Spurr resion. Ultrathin sections were stained with uranyl acetate and lead citrate, then examined and photographed under a transmission electron microscope (H-600, Hitachi, Ibaragi, Japan) at an accelerating voltage of 100 kV.

[Al

I

PRP

J--+,(-----y/A t low/ml

t Ewe/ml

t l.Srp/ml

If FIG. 1

0.5rg/ml

[Cl WP

tB1 WP

10w/ml

t 1.Ovg/ml

zT
w

H.S.

t 10 w/ml

Platelet aggregation studies using MALL13.

All of the concentrations shown are the final concentrations after addition. [A] MALL13 induced a dose-dependent change in optical density following a specific lag period. [BI[C] A normal fluctuation was observed in washed platelets. This fluctuation disappeared when normal serum was added to the washed platelets. N.S.: Normal serum H.S.: Heat-treated serum; Serum was incubated at 56°C for 30 minutes.

RESULTS The change in optical density induced by MALL13 is shown in Fig. 1. When MALL13 IgG was added to PRP, increased light transmission was observed. MALL13-induced change in optical density was associated with a lag period that decreased with increasing antibody concentration. However, aggregation curve fluctuation which is normally present during aggregation was greatly reduced (Fig. IA). A normal fluctuation was observed in WP (Fig. 1B). When heattreated serum (56°C for 30 min) was added to WP, the fluctuation was still ob served, whereas when normal serum was added, the fluctuation disappeared (Fig. IC). MALL13 F(ab')* did not induce platelet aggregation, but blocked the in-

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duction of aggregation by NNKYI-19 or ALB6 (data not shown). NNKYl-19 also lost the ability to stimulate platelets when F(ab')2 or Fab' fragment was produced (data not shown).

TABLE 1

Binding inhibition testing of monoclonal antibodies %

First

Positive Fluorescence

Ligand Incubation with 73.4 2.8 7.6 5.4 5.2 68.6 65.6

PBS NNKYI-19 (CDS) MALL13 (CD91 ALB6 (CDS) TP82 (CDS) NNKYl-32 (GPIIb/IIIa) NNKY5-5 (GPIb)

f 4.8 + 0.8 f 1.8 f 2.1 +_1.9 zk6.2 + 4.5

Results are expressed as the

PBS: Phosphate buffered saline. mean f SD of three experiments.

TABLE 2

FITC-NNKYl-19

ATP secretion and percentage of microparticles in platelets following various types of stimulation ATP released (nM)*

Microparticles (X)"

Agonist PRP none ADP (10 uM) Collagen (4 ug/ml) Thrombin (0.25 U/ml) A23187 (2 uM) NNKYI-19 (IO ug/ml) MALL13 (10 ug/ml)

0 0.7 f 1.4 f ND 3.6 f 2.2 f 7.8 f

0.2 0.3 0.4 0.3 0.4

WP

0.9 3.8 3.3 1.9 1.6

0 0 f + f f f

PRP

0.2 0.5 0.3 0.4 0.3

WP

6.8 zt1.9 8.4 f 3.6 11.2 f 3.3 9.2 f 4.0 28.6 + 7.3 24.5 + 5.6 ND 23.3 f 7.8 26.6 f 7.0 28.1 + 8.8 45.4 +- 10.3 43.3 f 9.7 78.1 f 16.2 46.1 -k9.9

*: n mo1/108 platelets WP: Washed platelets ND: not done Microparticles were defined as in the legend of Fig 2. Results are expressed as the mean f SD of three experiments.

Table 1 lists results of monoclonal antibody binding inhibition testing. The percentage of FITC-NNKYI-19 positive control samples (first ligand; PBS) was 73.4 + 4.8%. The degree of FITC-NNKYl-19 positivity after incubation with monoclonal anti-GPIIb/IIIa (NNKYI-32) or CPIb (NNKY5-5) antibodies was not significantly different from the control values. However, this value was significantly reduced in samples incubated with monoclonal anti-CD9 antibodies (NNKYl-19, MALL13, ALB6, and TP82). Aggregation of PRP or WP by NNKYI-19 and aggregation of WP by MALL13 was inhibited by preincubation with NNKYI-32 (anti-GPIIb/IIIa),whereas MALL13induced change in optical density of PRP was unaffected (data not shown). EDTA completely inhibited MALL13-induced effect for platelet, but the Fbg-

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related synthetic peptide, RGDS, only minimally inhibited MALL13-induced change in optical density of PRP (data not shown).

TABLE 3

The changes of cytosolic Ca2+ and released LDH stimulated by anti-CD9 antibodies LDH(%)

Agonist

[Ca2+]i

none ADP (IO pM) A23187 (2 PM) NNKYl-19 (IO pg/ml) MALL13 (IO pg/ml)

NT 9”:

PRP

WP

7.1 11.3 NT

NT NT 12.5

6.0

28.3

17.4

2.5

88.5

25.3(32.6)+

NT: not tested *: !4Pplus heat-treated serum Results are expressed as the mean of two experiments.

Unstained

FL1

FIG. 2

FITC-NNKYC-5

FL1

Definition of microparticles in control platelets.

The population of small-volume platelets (quad 3 + quad 4) was defined as microparticles. Both unstained and FITC-NNKY5-5 (GPIb)-stained platelets were used as unstimulated platelets.

The ATP release induced by MALL13 and NNKYl-19 is included in Table 2. In PRP, significantly more ATP release was detectable during MALL13-induced aggregation compared to that produced by NNKYI-19. However, the ATP release induced by MALL13 was decreased in WP. Table 3 documents the change of Ca2+]i and LDH release stimulated by anti-CD9 antibodies. The rise in [CaL+ ]i was very slight in MALL13-induced aggregation compared to A23187 or NNKYI-19. On the other hand, the LDH concentration of the buffer markedly increased after MALL13 stimulation in PRP.

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MICROPARTICLES

FIG. 3

AND CD9 ANTIBODY

Electron microscopy of PRP platelets stimulated with 10 pg/ml MALL13 IgG.

Stimulated platelets were fixed at 45 seconds (A) and 3 minutes (B) after the addition of MALL13. Arrows show the following: A (microparticles), B (vacuole)

a

GPllbAlla

fl

GPIb

?? CD9

fl

Fbg

m

Clq

Platelets

FIG. 4 Platelet and microparticle surface changes following activation by specific agonists.

Microparticles

AOP

NNKYl-19 MALL1 A231 87

0 AOP 3

NNKYl-19 MALL13 A23187

The monoclonal antibody, fibrinogen, and complement changes are expressed as the SIGMA RATIO (ratio to SIGMA of unstimulated platelets). The surfaces of microparticles exposed to antiCD9 antibody were rich in CD9 protein. Although the binding of fibrinogen to platelets stimulated by MALL13 changed little, Clq and C3 binding increased significantly.

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As noted in Fig. 2, the population of small volume platelets (quads 3 + 4) was defined as MP. Electron microscopy revealed that significant morphological changes occurred in platelets stimulated by MALL13 IgG (Fig. 3). Small vesicular structures (MP) accompanying the platelet aggregations were already observed at 45 seconds following the addition of MALL13 (Fig. 3A). However, platelet aggregates were rarely seen at 3 minutes following the addition of MALL13 (Fig. 3B), by which time vacuoles had appeared inside the platelets and MP increased still more (Fig. 3B). Table 3 shows the percentage of MP produced during platelet activation by specific agonists. Some increase in MP number occurred with NNKYI-19 and a significant increase was observed with MALL13. Platelet activation by A23187 also caused an increase in the number of MP, but the increase was smaller than that observed with MALL13. No significant increase in MP was observed when platelets were activated by ADP. Fig. 4 illustrates certain surface events occurring on platelets and MP following activation with specific agonists. Platelets and MP stimulated by NNKYI-19 and MALL13 were found to express GPIb and GPIIb/IIIa on their surface membranes similar to platelets and MP activated by other agonists. However, the surface CD9 protein expression of MP was increased by anti-CD9 antibody stimulation, especially MALL13. Fbg binding to platelets stimulated by MALL13 changed little, yet Fbg binding to ADP-, A23187-, or NNKYI-19-stimulated platelets increased significantly. The binding of Clq and C3 to platelets stimulated by MALL13 increased significantly. Clq and C3 binding to MP evidenced little changed at stimulation with specific agonists.

DISCUSSION Although our monoclonal antibodies, NNKYI-19 and MALL13, recognized the CD9 antigen, each antibody elicited different platelet effects. Results of flow cytometric analysis suggest that the binding sites recognized by the four anti-CD9 antibodies (NNKYI-19, MALL13, ALB6, and TP82) are located near each other. NNKYI-19 produced aggregation with characteristics similar to those produced by anti-CD9 antibody, as mentioned in previous reports (5,6,7,8,9). This aggregation did not vary significantly between PRP platelets and WP. However, the action of MALL13 on platelets varied between PRP and WP. With WP, there was no significant difference in the aggregation induced by MALL13 and NNKYI-19. When F(ab')2 or Fab' fragments were produced, both MALL13 and NNKYl-19 lost the ability to stimulate platelets. This suggests that the Fc portion is important in platelet activation by these antibodies. Recently, Worthington et al. has reported that a proximity of the CD9 antigen to the Fc 8 II receptor on the platelet surface allows the CD9 monoclonal antibody Fc region to interact with the platelet Fc receptor when the antibody is the Although our results concur with this concept, antigen-binding domain (I6). we did not perform specific studies using monoclonal anti-FcrII receptor antibody and therefore can make no conclusion. The action of MALL13 on platelets in PRP obviously differs from that of other anti-CD9 antibodies. The characteristic difference in the MALL13induced aggregation curve for PRP is the disappearnce of fluctuation, suggesting the possibility that this curve represents lysis rather than aggregation. When normal serum was added to WP, the pattern of fluctuation disappeared. But, when heat-treated serum (56"~ for 30 min) was added to WP, the fluctuation was observed (Fig. 1). This result suggests that complement participates in the disappearance of this fluctuation and that this is a result of lysis. The marked increase in buffer LDH are observed following MALL13 stimulation of

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PRP platelets and also supports the possibility that lysis occurs in this reaction. The complement component which plays a large part in lysis is the membrane attack complex (MAC; C5b-9). The mechanisms of MALL13-induced platelet activation of PRP platelets are similar to that of platelet activation by C5bFirst, platelet activation by C5b-9 protein can occur without functional 9. expression of the Fbg receptor on the platelet membrane GPIIb/IIIa complex (17). MALL13-induced platelet activation was found to be minimally affected by monoclonal anti-GPIIb/IIIa antibody or by the Fbg-related peptides RGDS. Furthermore in electron microscopy study, aggregates were observed, via electron microscopy, at an early stage (45 seconds) after the addition of MALL13, but not at a later stage (3 minutes). These results suggest that the momentary, reversible aggregation appeared partially at the early stage, and that the functional and irreversible expression of Fbg recep or did not occur. Second, C5b-9 causes a rapid influx of extracellular2$a$t into the plateIn this (3). let cytosol, which is inhibited at a low extracellular Ca study, MALLl3-induced aggrega$$on was also compl$$ely inhibited following EDTA dependence similar to that chelation of extracellular Ca , indicating a Ca in platelet activation by C5b-9. Third, the occurrence of complement on the surface of platelets stimulated by MALL13 increased. The binding of Clq and C3 to platelets following MALL13 stimulation increased significantly. This result suggests that in this setting the complement system act proceeds via the classical pathway on the surface membrane of platelets stimulated by MALL13. Thus, MALL13 appears to have at least two effects on platelets; signal transtuction related to CD9 antigen, and complement activation on platelet surfaces. It is unclear wheather the relationship between complement reaction and CD9 antigen activity is significant. Recently, Carrol et al. have reported that Clq-complement initiates lysis of human platelets by IgG subclass murine monoclonal antibodies to the CD9 antigen (18). Our findings are similar. C5b-9 has been reported as a cause of MP generation (2,3). We found that MALL13 also caused significant MP generation in PRP. This MP mainly appear to relate the complement system. An increase in MP generation was observed not only with MALL13 in WI',but also with NNKYI-19 in PRP. These results suggest that, in addition to the complement system, CD9 antigen affects the formation of MP. Interestingly, the distribution of GPIIb/IIIa or GPIb on MP derived from MALL13 stimulation was within the normal range, but that of CD9 protein was not. Previous studies have shown that the occurrence of membrane binding sites for coagulation factor Va leads to the assemblly of the prothrombinase enzyme complex (3). A close correlation was found between this platelet procoagulant activity and phosphatidylserine occurrence on the outer surface of the plasma membrane (19). This increased exposure of phosphatidylserine is thought to be due to a partial loss of the non-random distribution of phospholipids that occurs with platelet activation (19). Moreover it has been demonstrated that the increased exposure of phosphatidylserine coincides closely with an increase in the breakdown of sreeeralcytoskeltal proteins which is mediated by calpain, an endogenous Ca -dependent protease (20). MI'appear to be released from activated platelets as part of a defense reaction against complement-mediated lysis (2,3,17). This may occur as a result of the destruction of attachments between the cytoskelton and plasma membrane glycoproteins in which calpain is active. Our finding that CD9 antigen is abundant in the MP pool is of interst as monoclonal antibodies to the CD9 antigen are powerful platelet agonists and the CD9 antigen is widely distributed in a variety of cells, including endothelial cell. Such information is clinically relevant as Komada et al. (21) have reported that the shedding of CD9 antigen into cerebrospinal fluid by acute lymphoblastic leukemia cells may enable the clinical monitoring of leukemia cell burden in the central

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nervous

system. To determine the clinical significance of CD9 antigen-enriched MP, further detailed studies are necessary. In summary, two anti-CD9 murine monoclonal antibodies, NNKYI-19 and MALL13, appear to activate platelets and to generate MP by somewhat differeht mechanisms. The analysis of the relationship of MP to anti-CD9 monoclonal antibody may be useful in the investigation of the relationship between platelet function and the coagulation regulation.

ACKNOWLEDGMENT This work was partly supported by a Research Grant for Intractable Diseases from the Ministry of Health and Welfare of Japan.

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