Complement-Independent, Peroxide-Induced Antibody Lysis of Platelets in HIV-1-Related Immune Thrombocytopenia

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1 Cell, Vol. 106, , September 7, 2001, Copyright 2001 by Cell Press Complement-Independent, Peroxide-Induced Antibody Lysis of Platelets in HIV-1-Related Immune Thrombocytopenia Michael Nardi, 1 Stephen Tomlinson, 3 M. Alba Greco, 1 and Simon Karpatkin 2,4 1 Department of Pediatrics 2 Department of Medicine 3 Department of Pathology New York University School of Medicine 550 First Avenue New York, New York Summary Affinity purification of anti-platelet GPIIIa Ab from CIC s of these patients has revealed a high affinity IgG1 (Karpatkin et al., 1995) reactive against a specific sequence within the GPIIIa protein corresponding to residues (Nardi et al., 1997). The presence of anti- GPIIIa49-66 Ab correlates inversely with platelet count (r 0.71) and induces severe thrombocytopenia in mice (Nardi et al., 1997) (mouse GPIIIa is 83% homologous with human GPIIIa, and macrophages have Fc receptors for human IgG1). Murine thrombocytopenia can be prevented or reversed with GPIIIa49-66 peptide (Nardi et al., 1997), as well as anti-idiotype blocking Ab (Nardi and Karpatkin, 2000). Anti-GPIIIa49-66 Ab CICs can be removed from serum by centrifugation (Nardi et al., 1997), suggesting the presence of particulate platelet membrane fragments within the CIC. This report confirms the presence of these fragments in HIV-1-ITP serum. The CICs from HIV-1-ITP patients contain platelet membrane receptor antigen GPIIIa as well as GPIIb and GPIb, and we show that platelet fragments can be induced in vitro and in vivo with anti-gpiiia49-66 Ab. We further report that Abmediated fragmentation is complement independent and occurs via a novel mechanism involving the generation of hydrogen peroxide by stimulation of an NADPH oxidase pathway in platelets. Immunologic thrombocytopenia is seen commonly in HIV-1 infection. The pathogenesis of this problem has been unclear, but it is associated with circulating immune complexes that contain platelet membrane components and anti-platelet membrane GPIIIa49-66 IgG antibodies. These antibodies cause acute thrombocytopenia when injected into mice. We now show that purified anti-gpiiia49-66 causes platelet fragmentation, in vitro in the absence of complement, and in vivo in wild-type and C3-deficient mice. The mechanism of complement-independent platelet lysis is shown to be caused by the antibody-induced generation of H 2 0 2, as indicated by in vitro experiments with inhibitors of reactive oxygen species, and in vivo studies carried out with p47phox-deficient mice. Thus, a novel mechanism of immunologic platelet clearance is de- Results scribed in which an anti-platelet IgG causes platelet fragmentation via the induction of reactive oxygen Detection of Platelet Glycoproteins in PEG-IC s species. of HIV-1-ITP Patients Previous results have shown increased serum concen- Introduction tration of CIC in patients with HIV-1-ITP and that these CIC s contain Ab specific for GPIIIa We confirmed Immunologic thrombocytopenia is a common complica- these results in the population studied by demonstrating tion of HIV-1 infection (Jost et al., 1988; Morris et al., a 5.5-fold greater protein concentration of PEG-IC s de- 1982; Murphy et al., 1987). Kinetic studies on platelet rived from 46 HIV-1-ITP patients compared to 22 normal survival strongly suggest that early-onset HIV-1-ITP is control subjects. PEG-IC size was also measured in a secondary to increased peripheral destruction of plate- similar cohort of patients. There was a 2-fold greater size lets, whereas patients with AIDS are more likely to have in 35 HIV-1-ITP patients compared to 15 control subjects, decreased platelet production (Najean and Rain, 1994). as determined by forward light scatter (p 0.01). Patients with early-onset HIV-1-ITP have a thrombocy- In a previous report, the loss of 75% of antitopenic disorder that is indistinguishable from classic GPIIIa49-66 activity in PEG-IC following centrifugation autoimmune thrombocytopenia (ATP), seen predomi- at 100,000 g for 1 hr suggested the possible presence nantly in females (Karpatkin, 1997; Morris et al., 1982; of platelet membrane fragments in the IC s (Nardi et al., Ratnoff et al., 1983; Savona et al., 1985; Walsh et al., 1997). This was supported by immunoblot of the IC s 1984). However, HIV-1-ITP is different from classic ATP with mab s versus platelet membrane GPIIIa and GPIb, in that HIV-1-ITP patients have a male predominance which demonstrated molecular fragments of these rewith markedly elevated platelet-associated IgG, IgM, ceptors (Figure 1A). This observation was confirmed complement proteins C3 and C4, and the presence of employing an ELISA analysis of IC samples from 35 circulating serum immune complexes (CIC s) composed patients with HIV-1-ITP compared to 15 control subjects of the same (Savona et al., 1985; Walsh et al., 1984). employing a FITC-mAb against GPIIb (data not shown). Past studies have revealed that these complexes contain anti-platelet integrin GPIIIa ( 3) Ab (Karpatkin et Antibody Specific for GPIIIa49-66 Induces Platelet al., 1995), and its anti-idiotype blocking Ab (Nardi and Fragmentation In Vitro Karpatkin, 2000), as well as other Ab s and their anti- The presence of platelet membrane antigens in idiotypes (Karpatkin et al., 1988, 1992a; Yu et al., 1986). PEG-IC s of HIV-1-ITP patients raises the possibility that 4 Correspondence: simon.karpatkin@med.nyu.edu anti-gpiiia49-66 Ab may be inducing fragmentation. To investigate this possibility, we determined whether affin-

2 Cell 552 Figure 1. In Vitro Platelet Fragmentation Induced by Anti-GPIIIa49-66 Ab (A) Immunoblot of PEG-IC of control and HIV-1-ITP patients employing anti-gpiiia, anti-gpib, and control mouse monoclonal Ab, MoPC. CTL refers to control. (B) Flow cytometry of platelet in vitro particle formation. Gel-filtered platelets were labeled with anti-gpiib-fitc monoclonal Ab, washed, and then treated with various agents: buffer, control IgG, and patient anti-gpiiia49-66 for 4hrat37 C. Three panels represent: CTL, buffer alone; CTL IgG, IgG isolated from control PEG-IC; and PT IgG, IgG isolated from HIV-1-ITP patient PEG-IC. X axis refers to fluorescence, y axis to forward scatter. Numbers in left upper quadrant refer to the percentage of Ab-induced particles with decreased fluorescence attributed to platelet fragmentation. A highly-reactive anti-gpiiia49-66 IgG is shown. (C) Distribution of percentage of platelet particle formation in control versus HIV-1-ITP versus ATP patients at 1 hr incubation. Results shown for IgG isolated from PEG-IC s of 12 control, 16 HIV-1-ITP, and 5 ATP patients. Box Plot. Mean is shown by the solid black box; median by the horizontal line in the large open box; 25 th and 75 th percentiles by the lower and upper border of the large open box from which spread of the data from the position of the median can be assessed. Whiskers include 99% of a Gaussian distribution. Specificity of Anti-GPIIIa49-66 for Platelet Fragmentation Five different anti-gpiiia mab s with different specificities for GPIIIa (Liu et al., 1995), and an anti-gpib mab failed to induce platelet particle formation (Table 1). In striking contrast, an affinity-purified anti-gpiiia49-66 rabbit polyclonal Ab was effective at inducing platelet particle formation, albeit at an 8-fold higher concentra- tion than patient IgG (Figure 2). Preimmune rabbit IgG was inactive. ity-purified anti-gpiiia49-66 Ab could induce platelet fragmentation in vitro in the absence of serum or complement. Figure 1B shows the flow cytometric analysis of one such experiment in which anti-gpiib-fitclabeled platelets shifted their fluorescence intensity and distribution from the RUQ to the lower end of the LUQ, indicating platelet fragmentation and diminished fluorescence intensity, presumably via the loss of membrane GPIIb fluorescence as well as the production of smaller particles. Analysis of Platelet Fragmentation Induced by Anti-GPIIIa49-66 Optimum platelet particle formation occurred at 4 hr, with 25 g/ml anti-gpiiia49-66 Ab. Platelet particle formation by anti-gpiiia49-66 Ab was concentration de- pendent, with an optimum Ab concentration of 40 g/ml for fragmentation. Ab-induced platelet particle formation was also temperature dependent. Inactivity at 4 C permitted overnight storage of samples prior to analysis by flow cytometry, whenever necessary. Induction of Platelet Fragmentation in HIV-1-ITP versus Classic ATP Patients Platelet particle formation was compared in HIV-1-ITP patients, ATP patients, and control subjects. There was approximately 5-fold greater platelet particle formation in HIV-1-ITP patients compared to control subjects or ATP patients (Figure 1C). Exposure of Membrane Fragment Phosphatidylserine and Thrombin-Generating Capacity by Anti-GPIIIa49-66 Since platelet activation/vesicle formation is associated with inside-outside membrane exposure as reflected by phosphatidylserine exposure, attempts were made to measure this reaction via binding of the reaction prod- ucts to Annexin-V. Figure 3A demonstrates increased Annexin-V binding with time, with all of the activity in the supernatant obtained after centrifugation of the platelet/ platelet particle mixture at 15,000 g for 1 hr at room temperature, indicating that fragments contain phosphatidylserine. The exposure of phosphatidylserine on membranes generally leads to thrombin generation, since it provides a catalytic surface for binding of plasma coagulation pro- teins to the surface. We therefore analyzed Ab-induced platelet particles for their ability to generate thrombin.

3 Peroxide-Induced Ab Lysis of Platelets 553 Table 1. Specificity of Ab-Induced Platelet Particle Formation % Platelet Particles 0 Hrs 2 Hrs 4 Hrs PEG-IC IgG CTL PT MoAb Anti-GPIIIa LK CG LK7r LK3r LK MoAb Anti-GPIb Anti-Ib Ab concentration, 25 g/ml Figure 2. Comparison of Rabbit versus Patient Anti-GPIIIa Induced Platelet Particle Formation Preimmune rabbit and patient control IgG reactivity are cited under CTL. Affinity-purified rabbit anti-gpiiia49-66 IgG or isolated patient anti-gpiiia49-66 IgG was incubated with gel-filtered platelets at Figure 3B clearly demonstrates thrombin generation varying concentrations of Ab and analyzed by flow cytometry as in after 4 hr of exposure of Ab employing a chromogenic Figure 1B. assay (1 of 2 similar experiments). Platelet Fragmentation Induced by F(ab ) 2 be induced in vivo by F(ab ) 2 at 75% the efficacy of intact and Fab Fragments IgG, (10 versus 13.5% respectively, with 1% for control Platelet particle formation could be induced with F(ab ) 2 F(ab ) 2 fragments, n 6). Thus, clearance of opsonized fragments, indicating that complement was unlikely to platelets and fragments can take place in the absence be involved in this reaction. Of interest was the positive of Ab binding to Fc or C3 complement receptors on result obtained with 2-fold molar equivalent Fab frag- phagocytic cells, perhaps by other phagocytic scavenments, albeit at 60% the effective platelet particle for- ger mechanisms (Fadok et al., 1992; Tait and Smith, mation of F(ab ) 2 fragments (p 0.05, Student s t test), 1999). suggesting the possibility that dimerization of GPIIIa could play a role. Induction of Platelet Fragmentation via Anti- GPIIIa49-66 Is Mediated by the Generation Induction of Thrombocytopenia and Platelet of Peroxide and GPIIIa49-66 Specific Antibodies Fragmentation in Complement-Deficient Numerous attempts to elucidate the mechanism(s) of C3( / ) Mice Ab-induced platelet particle formation were unsuccess- The ability to generate platelet particles in vitro, in the ful. These included inhibitors of anerobic and aerobic absence of the Fc domain of anti-platelet GPIIIa49-66, glycolysis (3 mm 2-deoxyglucose, 10 mm NaA z ), microstrongly suggested that platelet particle formation was tubules (2 mm colchicine, 0.2 mm vinblastine), microfilaindependent of complement fixation. Nevertheless, ments (10 M cytochalasin D), calpain (100 M calpastacomplement deposition on cell membranes can induce tin, 5 g/ml leupeptin), apoptosis (100 M general membrane vesiculation (as well as cell lysis), and it is caspases [FK-011] and caspases 1 and 3), proteases possible that complement may play a role in platelet (5 g/ml leupeptin, 2 mm PMSF, 5 M SBTI, 5000 g/ml fragmentation in vivo. We therefore attempted to induce aprotinin, 4 mm EDTA), Ca 2 (4 mm EDTA, 100 M thrombocytopenia in C3-deficient and wild-type mice. BAPTA-AM), and various intracellular signaling kinases: Figures 4A and 4B show similar levels of thrombocyto- 2 M wortmannin (PI3Kinase), 200 M staurosporine penia induction and platelet particle formation in both (phospholipid/ca 2 -dependent protein kinase), 40 M wild-type and C3( / ) mice, indicating that C3 comple- H-7 (serine/threonine kinase), and 200 M PDC ment is not required for platelet fragmentation and (MAPKinase). However, a recent report by Lerner and thrombocytopenia in this model. coworkers (Wentworth et al., 2000) provided evidence that all Ab s are capable of inducing peroxide formation Induction of Thrombocytopenia and Platelet from an assortment of Ag s. This reaction required the Fragmentation with F(ab ) 2 Fragments generation of singlet via irradiation with UV or visible Induction of thrombocytopenia in complement-deficient light. This is followed by Ab-Ag induced reduction of mice indicated that in vivo thrombocytopenia was not to 0 2 (superoxide) with consequent generation of H 2 0 2, due to complement-mediated cell clearance, but likely which could be neutralized with catalase. We therefore to be due to clearance of opsonized platelets as well studied the effect of catalase on platelet particle formation as platelet fragmentation. The role of these 2 mechanisms induced by either human or rabbit anti-gpiiia49-66 was analyzed by measuring the contribution of and noted that it could inhibit the reaction with either F(ab ) 2 fragments versus intact IgG. Thrombocytopenia Ab, but in the absence of UV/light irradiation (Figure 5A). could be induced in Balb/c mice in the absence of the This suggested a different mechanism, namely that Fc domain of IgG but at 40% the efficiency of intact IgG could be generated by a specific cellular generating (n 6). Similarly, platelet particle formation could also system such as the NADH/NADPH oxidase system. This

4 Cell 554 Figure 3. Phosphatidylserine Exposure and Thrombin Generation from Platelet Particles Induced by Control and Patient Anti- GPIIIa49-66 Isolated from PEG-IC s (A) Phosphatidylserine exposure. Open bars refer to Annexin-V binding of entire platelet suspension; black bars refer to similar binding of supernatant platelet particles following centrifugation at 15,000g 1 hr, n 3, SEM is given. (B) Thrombin generation. Platelet microparticles were induced by Anti-GPIIIa49-66 Ab at 0 and 4 hrs. The 15,000 g supernatant containing platelet microparticles was added to defibrinated plasma in the presence of thrombin chomophore S2238 and CaCl 2 for 3 min and the developed color read spectrophotometrically at 410 nm. Typical of 2 different experiments. hypothesis was tested with the use of diphenyleneio- Additional studies revealed Ab concentration dependence donium (DPI), an inhibitor of NADH/NADPH oxidase, as with saturation at g/ml (n 2, data not well as other flavoprotein oxidases. Figure 5A demon- shown). strates inhibition by DPI as well as superoxide dismutase The GPIIIa49-66 specificity of this reaction was (SOD) in a similar manner as catalase. Similar results addressed by employing the experimentally raised rabbit were obtained with the human Ab (data not shown). anti-gpiiia49-66 Ab as well as 5 anti-gpiiia mab s Three other oxidase inhibitors failed to inhibit Ab-medi- with different specificity for GPIIIa. Similar fragmentation ated platelet particle formation: 20 M indomethacin and oxidation results for both time and concentraated against cyclooxygenase, 200 M allopurinol against tion-dependent variables were obtained with rabbit xanthine oxidase, and 200 M L-N-monomethylarginine anti-gpiiia49-66 and clinically derived platelet Ab. All 5 against NO synthetase (data not shown). anti-gpiiia mab s that had no effect on platelet fragmentation Further proof that platelets were generating peroxide also had no effect on oxidation of platelet DCFH. following incubation with anti-gpiiia49-66 was obtained using an intracellular fluorescent probe, DCFH, that Induction of Platelet Fragmentation by Thrombin measures peroxide oxidation. Platelets were loaded Does Not Require Reactive Oxygen Species with DCFH, treated with anti-gpiiia49-66 Ab, and perox- Thrombin is known to induce platelet fragmentation. ide generation assayed by measurement of fluorescence. Similar experiments were designed to test whether Figure 5B demonstrates increased fluorescence thrombin-induced fragmentation was also dependent (shift to the right of the x axis) and platelet particle on the generation of reactive oxygen species. In contrast formation (downward shift on the y axis) for both 1 and to the anti-gpiiia49-66 Ab, thrombin-induced platelet 4 hr of incubation with Ab. In 4 5 experiments, the increase fragmentation was not inhibited by catalase, DPI, or over baseline oxidation for 1 and 4 hr was 8.1 superoxide dismutase (Figure 5C). Similarly, the intracel- 1.3 and fold, respectively. The increase in the lular dye DCFH was not oxidized by thrombin. Mean platelet particle formation was and fluorescence for platelets alone, platelets plus 1 M fold, respectively, and blocked by catalase. There was H 2 0 2, or platelets plus 2.5 g/ml thrombin were 33 11, a lag period between particle formation and peroxide 304 2, and 23 6 respectively (n 3) following 4 hr of generation, as indicated by the greater increase in particle incubation at 37 C. Platelet fragmentation for platelets, formation between 1 and 4 hr compared to oxidation. platelets plus H 2 0 2, and platelets plus thrombin was 5%, Figure 4. Effect of Anti-GPIIIa49-66 Ab on Platelet Count and Platelet Particle Formation in Control and Complement-Deficient Mice (A) B6129 control and C3 / deficient mice were injected ip with 25 g of control (open square) or anti-gpiiia49-66 Ab and platelet count monitored at various time intervals. WT refers to wild-type; Mut to C3 / mice, n 5 for each group, SEM is given. (B) Percentage of platelet particles was monitored at 4 hr. Ctl refers to control IgG; Pt refers to anti-gpiiia

5 Peroxide-Induced Ab Lysis of Platelets 555 Figure 5. Generation of Peroxide and Effect of Peroxide Inhibitors, Catalase, Diphenyleneiodonium (DPI), and Superoxide Dismutase (SOD) with Platelet Particle Formation Induced by Anti-GPIIIa49-66 Ab or Thrombin (A) Gel-filtered platelets were preincubated with inhibitor or buffer for 15 min prior to the addition of rabbit anti-gpiiia49-66 Ab or control IgG. C refers to control nonimmune IgG (similar results with control plus highest inhibitor concentration employed), IS refers to rabbit anti-gpiiia49-66 immune serum. Bars labeled 2, 3, and 4 after bar 1 refer to doubling concentrations of inhibitor (50 g/ml catalase, 5 nm/ml DPI, and 12.5 g/ml SOD), n 4, SEM is given. (B) Generation of peroxide by anti-gpiiia49-66 at 1 hr (panels A, B, and C) and 4 hr (panels D, E, and F). Platelets were loaded with the intracellular dye, DCFH-DA, and then treated with buffer (panels A and D), control IgG (panels B and E), or human anti-gpiiia49-66 (panels C and F). (C) Effect of peroxide inhibitors on thrombin-induced platelet particle formation. Gel-filtered platelets were preincubated with inhibitors as in (A) and then treated with 1 g/ml thrombin for 4 hr. Bars refer to doubling concentrations, n 4. 14%, and 17% respectively (1 of 2 experiments with ference noted between patient F(ab ) 2 fragment in wildtype similar results). mice and its IgG preparation in p47phox( / ) mice. Further, anti-gpiiia49-66 Ab failed to produce any platelet particle formation in p47phox( / ) mice, compared Induction of Thrombocytopenia and Platelet Fragmentation in NADPH-Deficient to 26% platelet particle formation in wild-type mice (Fig- (p47phox[ / ]) Mice ure 6B). These data indicate that platelet particle for- Inhibition of platelet fragmentation by inhibitors of H mation is induced by H damage generated by 2 O 2 generation suggested that platelets contain a peroxide Ab-induced activation of the NADPH oxidase pathway generating pathway, namely the NADPH oxidase system and that platelet fragmentation contributes to platelet present in granulocytes/phagocytes (Segal et al., 2000). clearance. In vivo experiments were therefore performed in p47phox( / ) mice deficient in the p47 component of Electron Microscopy of Platelet Fragmentation the phagocytic oxidase complex necessary for H generation Induced by Anti-GPIIIa49-66 Ab via the NADPH oxidase pathway. Figure 6A dem- Figure 7 demonstrates the dramatic progressive platelet onstrates that thrombocytopenia induced in p47 phox damage induced by anti-gpiiia49-66 antibody at 1 and ( / ) mice by anti-gpiiia49-66 Ab was only 40% of 4 hr of incubation. Ab-damaged platelets develop breaks that obtained with wild-type C57/BL6 mice, with no dif- in their membrane, swelling, and release of cytoplasmic

6 Cell 556 Figure 6. Effect of Anti-GPIIIa49-66 Ab on Platelet Count and Platelet Particle Formation in Control C57BL/6 and p47phox( / ) Mice Control C57BL/6 (open square, closed circle, closed square) or p47phox( / ) (open circle) mice were injected ip with 25 g of intact anti-gpiiia49-66 or 16 g of its F(ab ) 2 fragment and platelet count and percent platelet particles monitored at 2 and 4 hr. (A) Platelet count. (B) Percent platelet particles. n 6 8 for each group, SEM is given. fragments. At 4 hr, there was a 6-fold greater incidence induced platelet fragmentation by peroxide inhibitors, of fragmented platelets treated with anti-gpiiia49-66 catalase, superoxide dismutase, and DPI, as well as Ab- compared to control IgG (30% versus 5%) in 12 sections induced intracellular oxidation of DCFH in vitro, and by analyzed in each group. Conversely, there was a 5-fold the inability of anti-gpiiia49-66 to induce microparticle increase in intact undamaged platelets in control versus formation and thrombocytopenia in p47 phox ( / ) anti-gpiiia49-66 treated platelets (62% versus 13%). At mice. Specificity is shown by similar results obtained with 1 hr, platelets had cytoplasmic-like material attached to an experimentally raised rabbit Ab versus GPIIIa49-66, the external surface of their membranes (Figures 7A with lack of effect for other anti-gpiiia Ab s directed 7C). Cytoplasmic contents leaked out of the platelet against other epitopes as well as an anti-gpib Ab. through gaps in the membranes and adhered to the Membrane shedding or microparticle formation is a outer surface but the granules are preserved (Figures normal property of cells grown in culture (Armstrong et 7B and 7C). This was confirmed by measuring lactic al., 1988; Beaudoin and Grondin, 1991; Mallat and et al., dehydrogenase release into the media of platelet sus- 1999), as well as cells undergoing apoptosis (Aupeix et pensions treated with Ab. No release was noted with al., 1997; Segundo et al., 1999). Platelet microparticle control Ab at 1 and 4 hr, whereas anti-gpiiia formation is enhanced by numerous pathophysiologic treated platelets released 175 and 537 units/l, respectively, conditions such as agonist-induced platelet activation in the 10,000 g supernatant (representative of (Shcherbina and Remold-O Donnell, 1999; Sims et al., 2 experiments). Some platelets show vacuolization. At 1989; Wolf et al., 1999), complement-induced platelet 4 hr, most platelets showed signs of cellular injury (cyto- lysis (Sims et al., 1988), autoimmune thrombocytopenia plasmic denudation as well as fragmentation). Many (Jy et al., 1992b; Khan et al., 1975; Zucker-Franklin and were swollen and others showed partial or almost total Karpatkin, 1977), and other conditions (Abrams et al., disintegration of their cell membrane (Figures 7E, 7F, 1990; George et al., 1986; Holme et al., 1994, 1997; and 7H). Dense and other granules were unaffected. Hughes et al., 2000; Jansen et al., 1997; Kelton et al., Clumping of cellular debris with platelet fragments was 1996; Lee et al., 1994; Miyamoto et al., 1998; Nieuwland also seen (Figure 7H). No such changes were noted with et al., 2000; Warkentin et al., 1994). Platelet microparticles control IgG-treated platelets (Figure 7D). The supernatant induced by platelet agonists have been reported collected from the centrifuged platelet samples to contain GPIIb/GPIIIa, GPIb (Sims et al., 1988, 1989), consisted mostly of cell debris with occasional degenerating CD9 (Inngjerdingen et al., 1999), P-selectin (Abrams et platelets (data not shown). Of interest is the obser- al., 1990; Inngjerdingen et al., 1999; Sims et al., 1988), vation that a minority of platelets (perhaps young platelets and Factor V (Sims et al., 1988) and to require Ca 2 [Karpatkin, 1969]) appear resistant to this Ab (Weidmer et al., 1990), calpain (Fox et al., 1991; Weidmer damage (Figures 7G and 7I). et al., 1990; Wolf et al., 1999; Yano et al., 1993), caspase 3 (Shcherbina and Remold-O Donnell, 1999), and intact Discussion GPIIb/GPIIIa (Gemmell et al., 1993) for their formation. Whether platelet microparticles with potential bioactive These data reveal a new pathophysiologic mechanism properties contribute to the pathophysiology of disease for platelet destruction (fragmentation) by an autoanti- or are a secondary consequence has not been resolved. body specific for a platelet GPIIIa49-66 epitope. The For example, platelet microvesicles can generate throm- mechanism is complement independent and involves bin (Sims et al., 1989), bind to fibrinogen (Holme et al., peroxide damage generated by an NADPH oxidase 1998b; Sims et al., 1989), coaggregate platelets (Holme pathway in platelets. Complement independence is et al., 1998b), adhere to subendothelium (Owens et al., shown by Ab-induced microparticle formation with 1992), and stimulate monocyte-endothelial cell adhe- F(ab ) 2 fragments in vitro, and by Ab-induced thrombo- sion via upregulation of adhesion molecules for both cytopenia and microparticle formation in C3 ( / ) mice, cells (Barry et al., 1998). It has been proposed that plate- in vivo. Peroxide damage is shown by inhibition of Ab- let microvesicles exert a protective hemostatic effect

7 Peroxide-Induced Ab Lysis of Platelets 557 Figure 7. Electron Microscopy of Damaged Platelets Ttreated with Anti-GPIIIa49-66 Ab (A) Patient sample at 1 hr showing platelets with a fuzzy material attached to the outer surface of the cell membranes (dotted arrows). Gaps are noted in the cell membranes with leakage of cytoplasmic content (arrows). These areas are demonstrated at higher magnification in (B) and (C) (E) Patient sample at 4 hr showing degenerating platelets and disintegration of the cell membrane (arrow). Swollen platelet (F), platelet fragments (H), and occasional normal platelets (G and I) are also seen. None of these changes were present in IgG controls at 4 hr (D). Original magnifications: (A), (D I): 4,000. (B) 50,000. (C) 40,000. in ATP patients (Jy et al., 1992a), may influence the A recent morphologic analysis of microparticles induced development of atherosclerosis (Mallat and et al., 1999; by heparin-dependent Abs revealed the elaboradevelopment Nomura et al., 1995), and may contribute to the develop- tion of membrane-bound vesicles released from swellings ment of thrombosis in heparin-induced thrombocytopenic on the platelet body or from pseudopods of purpura (HITP) (Warkentin et al., 1994). Patients with activated platelets (Hughes et al., 2000). Similar particles HIV-1 infection have a higher incidence of platelet microparticles can be produced by thrombin (Shcherbina and Remoldthrombotic (Holme et al., 1998a) and a higher incidence of O Donnell, 1999; Sims et al., 1989; Wolf et al., 1999). thrombocytopenic purpura (TTP) (Leaf et al., However, the platelet microparticles produced by anti- 1988). GPIIIa49-66 are different in that they are induced by

8 Cell 558 membrane damage secondary to peroxide generation al., 1992b). Fab fragments were prepared by papain digestion of rather than platelet activation. Nevertheless, they are IgG as described (Karpatkin et al., 1992b) and verified by SDS- similar with respect to phosphatidylserine exposure and PAGE. ability to induce thrombin generation. The difference Immune Complexes is supported by biochemical as well as morphologic Circulating immune complexes (CIC s) were isolated from serum evidence: (1) microparticle formation could not be inhib- by polyethylene glycol precipitation (PEG-IC) (Nardi and Karpatkin, ited by two anti-caspase inhibitors (FK-011, DEVD-fmk), 2000; Walsh et al., 1984). Precipitates were dissolved in one fifth two calpain inhibitors (calpastatin, leupeptin), or extracellular their serum volume in 0.01 M PBS (ph 7.4). and intracellular calcium chelators (EDTA and Immunoblot BAPTA-AM, respectively), which inhibit platelet micro- Twenty-five micrograms of PEG-IC were run on 10% SDS-PAGE, particle formation induced by platelet agonists (Fox et transferred to nitrocellulose paper, and immunoblotted with mouse al., 1991; Gemmell et al., 1993; Shcherbina and Remold- monoclonal Ab s against GPIIIa (LK7r), GPIb (1b10), and an irrele- O Donnell, 1999; Weidmer et al., 1990; Wolf et al., 1999; vant Ab (MOPC) for 2 hr, followed by washing and blocking with Yano et al., 1993); (2) thrombin-induced platelet forma- 5% BLOTTO. Ab binding was detected with a sheep anti-mouse tion did not require the generation of reactive oxygen IgG coupled to horesradish peroxidase (F(ab ) 2 specific) for 2 hr, followed by washing as above, and detection with luminol (NEN, species and was not inhibited by catalase, DPI, or super- Boston, MA). oxide dismutase; and (3) EM studies reveal cell swelling, membrane disruption with release of cellular contents, Isolation of IgG and IgM from Immune Complexes and apparent cellular debris, as well as isolated vesicles. IgG and IgM were isolated and purified as described (Karpatkin et The ability of an Ab to induce platelet fragmentation by al., 1995). In brief, polyethylene glycol (PEG)-ICs were applied to a reactivity with a specific epitope on platelet membrane protein A affinity column (Sigma-Aldrich). The bound complex was GPIIIa via elaboration of platelet generated peroxide washed with PBS and eluted with 0.1 M glycine buffer (ph 2.5). The eluted material was applied to an acidified sephadex G-200 gel is unique. The sequence specifity of anti-gpiiia Ab in filtration column (Amersham Pharmacia Biotech) preequilibrated inducing platelet fragmentation by the peroxide-depen- with the same elution buffer. Effluents of the IgG peak were isolated, dent mechanism is supported by our finding that 5 other neutralized, dialyzed against PBS, and applied to a rabbit anti-igm anti-gpiiia mab s against at least 4 different regions of affinity column (ICN Pharmaceuticals, Inc.) prepared from Affi-Gel GPIIIa (Liu et al., 1995), as well as a mab against GPIb, 10 (BioRad). The flow-through material was free of contaminating failed to induce platelet fragmentation. Sequence speciisolated, IgM by immunoblot and ELISA. Effluents of the IgM peak were neutralized, dialyzed against PBS, and applied to an anti- ficity was confirmed using an anti-peptide antibody Fc receptor affinity column to remove rheumatoid factor. The flowagainst GPIIIa49-66, which gave a similar platelet frag- through IgM was devoid of rheumatoid factor, as determined by mentation histogram to patient IgG, albeit at 8-fold inability to bind to a second Fc column. Fc fragments were prepared higher concentration with similar peroxide-dependent by papain digestion (Karpatkin et al., 1992b) and affinity purified on requirement. These observations suggest the possibility a staphylococcal protein A column; the acid eluate was verified by of a conformational change induced at a specific region SDS-PAGE and was coupled to Affi-Gel 10. of GPIIIa which is capable of activating a peroxide-gen- Affinity Purification of Anti-Platelet IgG erating pathway in platelets. Antiplatelet IgG was affinity purified with 10 8 platelets fixed with Our observations on platelet destruction and micro- 2% paraformaldehyde for 2 hr at room temperature, followed by particle formation with IgG as well as F(ab ) 2 fragments, overnight gentle rocking at 4 C, then acid elution and neutralization, in both wild-type and C3( / ) mice, as well as abrogaby as described (Karpatkin et al., 1995). The IgG subclass, determined tion of this effect in p47 phox( / ) mice, strongly indiand radial immunodiffusion (The Binding Site), was IgG1 with both k cate that platelet destruction can be via a platelet fragmentation l light chains. mechanism induced by peroxide generation Affinity Purification of Anti-Platelet GPIIIa49-66 with clearance by other than classic Fc or complement Peptide GPIIIa49-66, CAPESIEFPVSEARVLED (synthesized by Qualreceptors. ity Controlled Biochemicals, Hopkinton, MA), was coupled to an affinity column with the heterobifunctional cross-linker sulfo-succin- Experimental Procedures imidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate as recommended by the manufacturer (Pierce Chemical Co.; cross-links the Human Population resin with NH 2 -terminal cysteine of the peptide), and was incubated Patient sera were obtained from 46 early-onset HIV-1-infected with 0.4 ml of platelet affinity-purified IgG overnight at 4 C. The thrombocytopenic patients without AIDS, 12 control subjects column was then washed, eluted at ph 2.5, and neutralized as de- (healthy laboratory personnel), and 5 classic ATP patients. scribed (Karpatkin et al., 1995). The affinity-purified anti-platelet GPIIIa49-66 was nonreactive in buffer alone by flow cytometry, demonstrating Mice absence of detectable microparticles or human platelet Female BALB/c, B6129, and C57BL/6 mice were obtained from Ta- debris. conic Farms. C3( / ) X B6129 background were mice generated by Dr. Harvey Colton, Northwestern University Medical School, Chi- Induction of Platelet Particles cago, IL (Circolo et al., 1999) and kindly supplied by Drs. Susan Gel-filtered normal platelets were prepared from platelet-rich Boackle and Michael Holers at the University of Colorado Health plasma obtained from blood collected in 0.38% sodium citrate utiliz- Science Center. NADPH-deficient mice (p47phox (phagocyte ing a sepharose 2B column preincubated with Tyrode s buffer oxidase)( / ) X C57BL/6 were kindly provided by Dr. Harry L. Ma- (ph 7.4) gel-filtered platelets/ml were either unlabeled or lech, NIAID, Bethesda, MD (Jackson et al., 1995). labeled with an anti-gpiib-fitc monoclonal Ab (mab) (3B2) (Varon and Karpatkin, 1983) or an anti-gpiiia-fitc mab (Ancell, Bayport, F(ab ) 2 and Fab MN), 10 g/ml for 30 min at 4 C, centrifuged at 1000 g 6 min at F(ab ) 2 fragments were prepared from purified IgG by pepsin diges- room temperature, and resuspended in Tyrodes buffer. Ten microliters tion as described (Karpatkin et al., 1992b), and were shown to be of unlabeled or FITC-labeled platelets (10 7 /ml) were then incution free of Fc fragments by SDS-PAGE as well as ELISA (Karpatkin et bated with 15 l of thrombin (final concentration, g/ml) or

9 Peroxide-Induced Ab Lysis of Platelets 559 affinity-purified anti-gpiiia49-66 (5 40 g/ml), and 75 l Tyrodes buffer for 0 4 hr at 37 C and then stored in an ice bucket prior to measurement of percentage of platelet particles by flow cytometry. Further particle formation is arrested at 0 C (see below). For thrombin experiments, CaCl 2 (0.9 mm) and MgCl 2 (0.5 mm) were added to the Tyrodes buffer. For mouse in vivo studies, blood was collected from the orbital sinus with Unopettes (Becton-Dickinson) or by cardiac puncture into a heparinized syringe after anesthesizing mice with metafane (Schering-Plough Animal Health, Union, NJ). Platelet-rich plasma was prepared and incubated with mab anti-mouse CD41-FITC (Integrin IIb chain, Pharmingen, San Deigo, CA) for 30 min at 4 C and then assayed directly by flow cytometry. Supported by NIH grants HL and DA (S.K.), The Doro- thy and Seymour Weinstein Platelet Research Fund (S.K.), NIH grant A134451, and a grant from the American Heart Association (S.T.). Dr. Herman Yee and Ms. Judith Grecay assisted in the presentation of the electromicroscopy data. Drs. Jayne Raper and Molina Maria del Pilar provided helpful advice with the DCFH-DA intracellular oxidation studies. Dr. John Hirst provided invaluable assistance with the flow cytometry analysis. Assay of Platelet Particle Formation Both fluorescent-labeled or unlabeled percentage of platelet particles were measured by flow cytometry, employing a FACScan (Becton-Dickinson, Montain View, CA). Similar results were obtained by both methods. Debris and dead cells were excluded using scatter gates. Only cells with low orthogonal light scattering were included in the sorting gates. Gates were adjusted for control platelets by exclusion of other blood cells. Fluorescent-labeled intact platelets were monitored in the right upper quadrant (RUQ) with the y axis measuring forward-scatter and the x axis measuring fluorescence. For nonfluorescent cells, the y axis measured forward scatter and the x axis measured side scatter. A shift in fluorescent or nonfluorescent particles from RUQ to LUQ and LLQ reflected percentage of platelet particle induction of 10,000 counted platelets/particles. logicals, Inc (Reamstown, PA) employing KLH-conjugated GPIIIa49-66 with 4 booster injections days post primary injection of 500 g. Electron Microscopy Platelets were suspended in an equal volume of 2% Bacto-Agar and fixed in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer. Samples were washed twice in buffer, post-fixed with 1.5% osmium tetroxide and rewashed 2 times with buffer. Samples were then dehydrated and embedded in Eponate-12 resin. Thin sections were cut in a Reichert Ultracut 5 ultramicrotome, counterstained with uranyl acetate and lead citrate, and anlyzed using a Zeiss EM-10 electon microscope. Materials All reagents were obtained from Sigma (St. Louis, MO) unless otherwise designated. PDC (MAPKinase inhibitor) was obtained from Research Biochemicals Inc., Natick, MA. Anti-caspases 1 and 3 and BAPTA-AM were obtained from Molecular Probes, Eugene, OR. MAb s against platelet GPIIIa (LK6-55, LK7r, LK3r, LK4-r5, and CG4, and GPIIb (3B2) were produced in our laboratory (Liu et al., 1995; Varon and Karpatkin, 1983). MAb against GPIb (1b10) was a gift from Dr. Zaverio Ruggeri, Scripps Research Institute, La Jolla, CA. Thrombin substrate S2238 was obtained from DiaPharma Group. Acknowledgments ELISA Assays CIC GPIIb and phosphatidylserine were measured by ELISA. GPIIb was measured by incubating 25 g of PEG-IC with 10 g/ml mab 3B2-FITC in 0.1 M final volume for 30 min at 4 C, and then assayed by flow cytometry. Phosphatidylserine was measured by solid phase assay, employing streptavidin-labeled plastic microtiter plates (Boehringer- Received March 19, 2001; revised July 23, Mannheim, Indianapolis, IN), preincubated with Annexin V-Biotin (Sigma), blocked and washed with TBS (50 mm Tris HCl, 100 mm NaCl) and 1% BSA CaCl 2 (1 mm). One hundred microliters of References platelet preparation (recalcified to 10 mm Ca 2 ) was then added to Abrams, C.S., Ellison, N., Budzynski, A.Z., and Shattil, S.J. (1990). the prepared microtiter plate for 2 hr before and after centrigugation Direct detection of activated platelets and platelet-derived microat 15,000 g for 1 hr. Plates were washed in the same buffer. particles in humans. Blood 75, Annexin V binding to platelets was assayed with a polyclonal anti- GPIIIa (PLA1) Ab for 2 hr at room temperature, which was washed Armstrong, M.J., Storch, J., and Dainiak, N. (1988). Stimulating disand then incubated with a goat anti-human IgG-conjugated alkaline tinct plasma membrane regions gives rise to extracellular membrane phosphatase, washed, and developed with Sigma 104 developing vesicles in normal and transformed lymphocytes. Biochim. Biophys. reagent. Acta 946, Aupeix, K., Hugel, B., Martin, T., Bischoff, P., Lill, H., Pasquali, Thrombin Generation Assay J.-L., and Freyssinet, J.-M. (1997). The significance of shed mem- Thrombin generation was assayed with the thrombin substrate chroin brane particles during programmed cell death in vitro, and in vivo, mophore S2238 (DiaPharma Group Inc., West Chester, OH) by a HIV-1 infection. J. Clin. Invest. 99, modification of the described assay (Nieuwland et al., 2000). Citrated Barry, O.P., Pratico, D., Savani, R.C., and FitzGerald, A. (1998). Modplasma was defibrinated with reptilase (Sigma), 20 l/ml for 10 min ulation of monocyte-endothelial cell interactions by platelet micro- at 37 C and 10 min in melting ice. Fibrin was removed by centrifugation particles. J. Clin. Invest. 102, at 15,000 gfor1hrat25 C. The defibrinated plasma (50 l) Bass, D.A., Parce, J.W., Dechatelet, L.R., Szejda, P., Seeds, M.C., was then incubated with 35 l of platelet/platelet particle suspension and Thomas, M. (1983). Flow cytometic studies of oxidative product and 15 l of 17 mm CaCl 2 for 4 min at 37 C, followed by the addition formation by neutrophils: a graded response to membrane stimulaof 100 l of S2238 (4 mm in TBS-20 mm EDTA) for 3 min. The tion. J. Immunol. 130, reaction was stopped with 200 l of 1 M citric acid and the color Beaudoin, A.R., and Grondin, G. (1991). Shedding of vesicular matechange measured spectrophotometrically at 410 nm. rial from the cell surface of eukaryocytic cells: different cellular phenomena. Biochim. Biophys. Acta 1071, Peroxide Generation Assay Gel-filtered platelets were preloaded with 10 M 2 7 dichlorodihy- Circolo, A., Garnier, G., Fukuda, W., Wang, X., Hidvegi, T., Szalai, drofluorescein diacetate (DCFH-DA, Molecular Probes, Eugene, OR) A.J., Briles, D.E., Volanakis, J.E., Wetsel, R.A., and Colten, H.R. for 30 min at 37 C. DCFH-DA is a nonfluorescent lipid soluble probe (1999). Genetic disruption of the murine complement C3 promoter which diffuses across the plasma membrane and is converted intramrna. Immuno. Pharma. 42, region generates deficient mice with extrahepatic expression of c3 cellularly to a cell impermeant (DCFH) by intracellular esterases. The probe can then be oxidized by H to the fluorescent species Fadok, V.A., Savill, J.S., Haslett, C., Bratton, D.L., Doherty, D.E., (Bass et al., 1983). Campbell, A., and Henson, P.M. (1992). Different populations of macrophages use either the vitronectin receptor or the phosphatidylserine Preparation of Rabbit Anti-GPIIIa receptor to recognize and remove apoptotic cells. J. Immu- GPIIIa49-66 was synthesized by Quality Control Biochemicals (Hopkinton, nol. 149, MA). Antibody was prepared commercially by Cocalico Bio- Fox, J.E.B., Austin, C.D., Reynolds, C.C., and Steffen, P.K. (1991).

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