SEARCH:   Advanced

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by McRedmond, J. P. Articles by Fitzgerald, D. J. Search for Related Content PubMed PubMed Citation Articles by McRedmond, J. P. Articles by Fitzgerald, D. J. Related Collections Hemostasis, Thrombosis, and Vascular Biology Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, Vol. 95 No. 4 (February 15), 2000: pp. 1301-1308 HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGYAU#0 Streptokinase-induced platelet activation involves antistreptokinase antibodies and cleavage of protease-activated receptor-1 James P. McRedmond, Patrick Harriott, Brian Walker, and Desmond J. Fitzgerald Centre for Cardiovascular Science, Department of Clinical Pharmacology, The Royal College of Surgeons in Ireland, Dublin, Ireland; Centre for Peptide and Protein Engineering, School of Biology and Biochemistry, Queen's University of Belfast, Northern Ireland.     Abstract Top Abstract Introduction Materials and methods Results Discussion References Streptokinase activates platelets, limiting its effectiveness as a thrombolytic agent. The role of antistreptokinase antibodies and proteases in streptokinase-induced platelet activation was investigated. Streptokinase induced localization of human IgG to the platelet surface, platelet aggregation, and thromboxane A2 production. These effects were inhibited by a monoclonal antibody to the platelet Fc receptor, IV.3. The platelet response to streptokinase was also blocked by an antibody directed against the cleavage site of the platelet thrombin receptor, protease-activated receptor-1 (PAR-1), but not by hirudin or an active site thrombin inhibitor, Ro46-6240. In plasma depleted of plasminogen, exogenous wild-type plasminogen, but not an inactive mutant protein, S741A plasminogen, supported platelet aggregation, suggesting that the protease cleaving PAR-1 was streptokinase-plasminogen. Streptokinase-plasminogen cleaved a synthetic peptide corresponding to PAR-1, resulting in generation of PAR-1 tethered ligand sequence and selectively reduced binding of a cleavage-sensitive PAR-1 antibody in intact cells. A combination of streptokinase, plasminogen, and antistreptokinase antibodies activated human erythroleukemic cells and was inhibited by pretreatment with IV.3 or pretreating the cells with the PAR-1 agonist SFLLRN, suggesting Fc receptor and PAR-1 interactions are necessary for cell activation in this system also. Streptokinase-induced platelet activation is dependent on both antistreptokinase-Fc receptor interactions and cleavage of PAR-1. (Blood. 2000;95:1301-1308) © 2000 by The American Society of Hematology.     Introduction Top Abstract Introduction Materials and methods Results Discussion References Streptokinase (Sk) is a bacterial protein that is used as a thrombolytic agent in the treatment of myocardial infarction.1,2 It is a plasminogen activator that has no intrinsic protease activity. Rather, in blood it forms an equimolar complex with the zymogen plasminogen, which is an active protease. The active complex is a plasminogen activator, converting further plasminogen to the fibrinolytic enzyme, plasmin.3 Due to its bacterial origin, Sk is antigenic. Low levels of antibodies directed against Sk are detectable in the general population, presumably due to previous streptococcal infections.4 Following administration of Sk, the level of antibodies to Sk rises dramatically in most patients, peaking at about 2 weeks and may remain elevated for up to 4 years.5,6 Anti-Sk antibodies are presumed to cause allergic reactions during Sk treatment and may neutralize the activity of Sk, necessitating the administration of a high standard dose of Sk to ensure effective thrombolysis in most patients.7 Antibodies to Sk are also capable of activating platelets8,9 and this may explain how thrombolytic therapy with Sk results in marked platelet activation in vivo, as measured by the production of the platelet-derived eicosanoid thromboxane (TX) A2.10 This platelet activation is functionally important because it delays arterial reperfusion and contributes to early reocclusion.11 Platelets are activated through a low-affinity receptor for IgG on the platelet surface (FcRII, CD 32) by immobilized immunoglobulin,12 aggregates of IgG,13 or cross-linking of the Fc receptor.14 High concentrations (5000 U/mL) of Sk and anti-Sk IgG cause platelet activation in an Fc receptor-dependent manner.15 Alternatively, proteolytic agents can initiate cellular signaling and activation by cleaving protease-activated receptors (PARs). The original protease receptor, designated PAR-1, was cloned from a megakaryocytic cell line and is present on platelets.16 It belongs to the family of cell surface receptors with 7 transmembrane domains and coupled to G-proteins. PAR-1 has an extended extracellular amino terminus, which includes a site recognized and cleaved by thrombin. This cleavage generates a new amino-terminal sequence on the receptor, which acts as a tethered ligand and activates the receptor.16 Peptides as short as 5 amino acids in length corresponding to the tethered ligand sequence (starting SFLLRN.) activate intact PAR-1 at micromolar concentrations. Other proteases, such as trypsin,16 cathepsin G,17 and granzyme A18 activate PAR-1 also, albeit at higher concentrations than thrombin. Several other PARs have been cloned.19-21 PAR-2 is cleaved by low concentrations of trypsin, but not by thrombin. Its physiologic role is unknown. PAR-3 is activated by thrombin, but appears to be more important in mouse than human platelets.20 Recently a fourth receptor, PAR-4, has been cloned from mouse22 and human21 tissues, and mediates thrombin-induced platelet activation in both species. It has a lower affinity for thrombin than PAR-1 or PAR-3,22 requires higher concentrations of soluble peptide ligand to induce platelet activation,23 and has been postulated to act as a "standby" protease receptor.22 Moreover, in man the platelet effects of thrombin appear to be mediated predominately by PAR-1; only at high concentrations and when PAR-1 activation has been inhibited is platelet activation by thrombin dependent on PAR-4.23 Recent evidence has shown that plasmin can cleave PAR-1 at the site resulting in receptor activation. However, cleavage at other sites in the receptor's extracellular N-terminal domain predominate, resulting in desensitization of the receptor to subsequent challenge with thrombin.24,25 We have previously shown that Sk-induced platelet activation was mediated by a protease,10 although others found immunologic mechanisms predominate.9 This study examines the mechanism of Sk-induced platelet activation, particularly, the interaction between PAR-1 cleavage and anti-Sk antibodies in inducing this response.     Materials and methods Top Abstract Introduction Materials and methods Results Discussion References Materials Sepharose 4B, Sk, adenosine diphosphate (ADP), hirudin, thrombin, human plasminogen, plasmin and 2-antiplasmin were purchased from Sigma (Poole, UK). Monoclonal antibody IV.3 was purchased from Medarex Inc (Annandale, NJ). Monoclonal antibodies SPAN 12 and WEDE 15 were purchased from Immunotech (Marseille, France). Monoclonal antibody 2/389 was a kind gift from the late Dr Stuart Stone, Department of Biochemistry, Cambridge University. Platelet preparation Blood was drawn from human volunteers, free from aspirin and other drugs likely to affect platelet function for 10 days. Blood was drawn into 10% final volume of 3.8% sodium citrate. Platelet-rich plasma (PRP) was prepared by centrifugation of whole blood at 150g for 10 minutes. Aggregation was assayed by light transmission in a final volume of 500 µL (PAP-4 aggregometer, Biodata, Horsham, PA). Inhibitory monoclonal antibodies were preincubated in PRP for 2 minutes before addition of agonists. Aliquots of PRP were frozen in liquid nitrogen for subsequent assay of TXA2 formation by enzyme-linked immunoassay for the stable metabolite, TXB2 (Assay Designs, Ann Arbor, MI). For some experiments, PRP was depleted of plasminogen by passing it over a column of lysine immobilized on Sepharose 4B. To this was added 50 µL of 8 µM native plasminogen, plasminogen inactivated by mutation of its active site (S741A plasminogen), or buffer in a final volume of 500 µL. Antibody purification Anti-Sk antibodies were purified from plasma as follows. Plasma from a normal donor was incubated overnight with 1 g Protein A-Sepharose 4B at 4°C. Sepharose beads were washed in phosphate-bufferred saline (PBS), bound antibody was eluted with glycine HCl (0.1 M, pH 3) and neutralized with Tris HCl buffer (1 M, pH 8). In some cases this IgG was concentrated to 0.1 of the original volume for use in platelet aggregation, by centrifugation through a porous membrane with a molecular weight cut-off of 30 kd (Centricon-30 concentrator, Amicon Inc., Beverly, MA). PRP was treated with ADP and Sk in the presence of buffer alone or IgG purified from different donors. To specifically purify anti-Sk antibodies, Sk was immobilized on activated CH Sepharose 4B (Pharmacia Biotech AB, Uppsala, Sweden), according to the manufacturer's instructions. IgG obtained from plasma as above was incubated overnight with Sk-Sepharose at 4°C and specific anti-Sk antibody eluted and concentrated as described above. Serum from patients recently treated with Sk for myocardial infarction contains high levels of anti-Sk. In some experiments such serum was depleted of Sk-binding antibodies by overnight incubation with Sk immobilized on Sepharose, followed by centrifugation and removal of the supernatant. As a control, aliquots of serum were incubated with bovine serum albumin (BSA) immobilized on Sepharose. Flow cytometry Platelet-rich plasma was first treated with the platelet glycoprotein IIb/IIIa antagonist integrelin to prevent aggregation and Sk and ADP added at fully activating concentrations. PRP was then fixed in an equal volume of 0.4% formaldehyde, washed twice in PBS, and incubated at 4°C for 30 minutes with the fluorescein isothiocyanate (FITC)-conjugated secondary antibody (antihuman IgG, Becton Dickinson, San Jose, CA). Platelets were then washed twice and resuspended in formaldehyde for analysis (FACScan flow cytometer, Becton Dickinson). Synthesis and cleavage of PAR1 peptides Thrombin receptor peptides LDPRSFLLRNPNDKYEPF (TR18), LDPR (TR4), and SFLLRNPNDKYEPF (TR14) were synthesized on an Applied Biosystems automated peptide synthesiser (model 432A, Norwalk, CT), using a standard solid phase Fmoc (N-[9-fluorenyl] methoxycarbonyl) procedure. All peptides were purified after synthesis using reverse-phase high-performance liquid chromatography (HPLC) and confirmed using electrospray mass spectrometry. The peptide LDPRSFLLRNPNDKYEPF corresponds to the site on PAR-1 recognized and cleaved by thrombin.26 This was incubated at 0.5 mM in PBS with thrombin (10 nM) or a complex formed by incubating 1.0 µM Sk with 0.5 µM plasminogen at room temperature for 30 minutes. Incubations with Sk-plasmin(ogen) were performed in the presence and absence of 2-antiplasmin (2.0 µM). Analysis of the resultant cleavage products was performed by HPLC on a C18 column (Supelco, Poole, UK). Peptides were detected at 215 nm with a SPD-6A UV detector and a C-R3A integrator (Shimadazu, Kyoto, Japan), using a mobile phase of 25% acetonitrile, 75% 5 mM phosphate buffer, pH 7.4, and compared to standard peptides corresponding to cleavage of the parent peptide at the expected site (TR4 and TR14). Calcium fluorimetry Human erythroleukemic (HEL) cells were incubated for 30 minutes at 37°C with 4 µM FURA-2-AM (Calbiochem-Novabiochem, Nottingham, UK), before being washed twice in Hank's balanced salt solution containing 1.25 mM Ca++ and finally resuspended in the same buffer. Intracellular Ca++ changes were detected using an LS-50 fluorimeter (Perkin Elmer, Norwalk, CT). Cells were stirred at 37°C in a 500-µL quartz cuvette. FURA-2 fluorescence was measured following excitation at wavelengths 340 and 380 nm, with emission at 510 nm. Fluorescent microscopy The HEL cells were pelleted and resuspended in plasma from an individual who exhibited platelet aggregation in the presence of Sk. Cells were treated with no agonist or Sk (1600 U/mL) for 10 minutes in the presence of 0.01% sodium azide to prevent receptor internalization. Treated cells were pelleted and resuspended in Tris-buffered saline (TBS), adhered by centrifugation onto poly-L-lysine-coated slides and fixed in methanol for 7 minutes. Cells were blocked with 2% normal goat IgG in TBS for 30 minutes, incubated with monoclonal antibodies SPAN 12 or WEDE 15 (directed against the cleavage site and hirudin-like site of PAR-1, respectively) at 10 µg/mL in TBS for 45 minutes and stained with goat antimouse IgG conjugated to an AlexaTM 488 fluorescent group (Molecular Probes, Eugene, OR) at 4 µg/mL for 10 minutes. Cells were visualized using a LSM 510 microscope (Carl Zeiss Ltd, Herts, UK) using an argon laser.     Results Top Abstract Introduction Materials and methods Results Discussion References Platelet aggregation and TXA2 production Low concentrations of ADP (0.5-2.0 µM) cause a small and reversible platelet aggregation. In the presence of Sk added to untreated PRP, this is converted to an irreversible platelet aggregation (Figure 1A). The effect can be demonstrated in PRP from some individuals at an Sk concentration of 300 to 500 U/mL, in the range of concentrations achieved in vivo following a standard dose of Sk.27 Sk-induced platelet aggregation was accompanied by increased TXA2 production, measured as its hydrolysis product TXB2, from 3.1 ± 0.8 ng/mL in stirred platelets (no agonist) to 78 ± 22.4 ng/mL in platelets treated with Sk and ADP. Neither ADP nor Sk alone, at the concentrations used, results in significant generation of TXB2 (Figure 2). View larger version (21K): [in this window] [in a new window]   Fig 1. The role of antibodies in platelet aggregation to Sk. (A) Sk (500 U/mL) combined with a subthreshold concentration of ADP (2 µM) results in irreversible platelet aggregation. This is inhibited by 5 µg/mL of the Fc receptor-blocking antibody IV.3. (B) In individuals who fail to aggregate to low concentrations of Sk and ADP, the addition of 50 µL purified anti-Sk antibodies results in an aggregation response. This is also sensitive to inhibition by IV.3. (C) Whole-plasma IgG from a responsive donor (donor 1) but not from a nonresponsive donor (donor 2) can confer sensitivity to Sk on PRP from a third donor. These results are representative of tracings from at least 5 (A and B) or 2 (C) individuals. View larger version (10K): [in this window] [in a new window]   Fig 2. Thromboxane production in plasma during Sk-induced platelet aggregation. Plasma was obtained from platelet aggregations as shown in Figure 1, and assayed for TXB2. Platelets were pretreated with buffer, 5 µg/mL of the Fc receptor-blocking antibody IV.3 or 4 µg/mL of the PAR-1-blocking antibody 2/389 as shown, and challenged with subthreshold concentrations of ADP, 500 U/mL of Sk or both, as shown. Results are mean ± SEM from 3 individuals. Involvement of the platelet Fc receptor The monoclonal antibody IV.3 blocks the binding of IgG antibodies to the Fc receptor found on platelets. Pretreatment of PRP with IV.3 abolished Sk-induced platelet aggregation (see Figure 1A) and TXB2 production (see Figure 2), but had no effect on aggregation to ADP alone. This implicates endogenous antibodies in the plasma in Sk-induced platelet activation. In individuals who did not exhibit platelet aggregation in response to low concentrations of Sk, aggregation to Sk was seen on addition of 50 µL of purified anti-Sk antibodies (Figure 1B). Moreover, a small volume of serum from a patient who previously received Sk and had a high level of anti-Sk antibodies, resulted in Sk-induced aggregation (not shown). These responses were blocked by pretreatment with the antibody IV.3 (see Figure 1B). Incubation of PRP from a nonresponsive donor with whole plasma IgG purified from a responsive donor conferred Sk sensitivity on the PRP, whereas IgG from a nonresponsive donor did not (Figure 1C), indicating that antibodies purified in this way do not of themselves activate platelets. Platelet-rich plasma incubated with an FITC-conjugated monoclonal antibody directed against human immunoglobulin was analyzed using flow cytometry. In the presence of both ADP and Sk, there was a marked increase in human IgG associated with the platelet surface (Figure 3). Stimulation of cells by ADP or Sk alone did not enhance human IgG binding. Localization of human IgG to the surface was prevented by pretreatment with the Fc receptor-blocking antibody IV.3, showing that binding of the antibody to the platelet was specific and not due to secretion of IgG by the platelet or to nonspecific adsorption of plasma antibody (see Figure 3). No increase in antibody binding was seen in platelets treated with the PAR-1 agonist SFLLRN (data not shown), indicating that the platelet-associated IgG was not due solely to platelet activation. View larger version (19K): [in this window] [in a new window]   Fig 3. Platelet-associated human IgG during Sk-induced platelet aggregation. Following agonist treatment, platelets were incubated with an FITC-labeled antibody against human IgG and platelet-associated fluorescence measured using flow cytometry. (A) Platelets stirred in the absence of agonist. (B) Subthreshold concentration of ADP. (C) 500 U/mL Sk alone. (D) ADP in the presence of 500 U/mL Sk. (E) Treated with the Fc receptor-blocking antibody IV.3 prior to the addition of Sk and ADP. Role of PAR-1 Sk-induced platelet aggregation was prevented by pretreating the platelets with antibody 2/389 raised against a peptide containing the cleavage site of PAR-1 (Figure 4A). The antibody binds to the tethered ligand-binding domain of PAR-1 and prevents PAR-1 cleavage by thrombin (Dr S. Stone, personal communication, July 1995). This antibody also prevented platelet aggregation induced by nanomolar concentrations of thrombin (Figure 4B). Although thrombin-induced platelet aggregation could be prevented by pretreatment with the specific thrombin inhibitor hirudin, platelet aggregation to Sk was unaffected (see Figure 4A and B). Thrombin bound on the platelet surface, or newly generated by the prothrombinase complex, may not be inhibited by hirudin, due to inaccessibility of the anion-binding site. The low molecular weight thrombin inhibitor, Ro46-6240, is a potent, specific inhibitor of thrombin that binds to the active site of the enzyme and not to the anion-binding site.28 Ro46-6240, at a concentration that inhibited platelet aggregation to thrombin, but not to other platelet agonists, had no effect on Sk-induced platelet aggregation (see Figure 4A and B). These findings suggest that Sk-induced platelet aggregation is due to the action of a protease distinct from thrombin acting on PAR-1. View larger version (22K): [in this window] [in a new window]   Fig 4. The role of thrombin, PAR-1, and plasminogen in Sk-induced platelet aggregation. Aggregations to ADP and Sk (A) or 0.35 U/mL thrombin (B) were performed without inhibitors or in the presence of the PAR-1-blocking antibody 2/389 (4 µg/mL), hirudin (1 U/mL), or the thrombin active site inhibitor Ro46-6240 (Ro-46) (0.04 µg/mL). (C) Aggregation to ADP and Sk was performed in PRP depleted of plasminogen by passage over a lysine-Sepharose column, to which had been added buffer alone, wild-type plasminogen, or the inactive mutant S741A plasminogen. Each tracing is representative of 2 to 3 experiments. Role of plasminogen In PRP depleted of plasminogen by passage over a lysine-Sepharose column, Sk at 500 U/mL was unable to induce platelet aggregation. Platelet aggregation was restored by the addition of 0.8 µM native plasminogen. However, when plasminogen rendered inactive by mutation at its active site (S741A) was used at 0.8 µM, no aggregation to Sk was seen (Figure 4C). Sk-induced platelet activation, therefore, requires proteolytically active plasminogen. PAR-1 cleavage by Sk-plasminogen Incubation of a peptide corresponding to the thrombin recognition and cleavage sites of PAR-1 (LDPRSFLLRNPNDKYEPF) with thrombin, resulted in cleavage at the expected site and production of the 2 predicted fragments, LDPR and SFLLRNPNDKYEPF (Figure 5A-C). The peptide was also cleaved following incubation with plasmin (not shown) and Sk-plasminogen complex (Figure 5D). Pretreatment with 2-antiplasmin, which inhibits free plasmin, but not plasmin(ogen) complexed with Sk,29 did not prevent Sk-plasminogen cleaving TR18 (Figure 5E). View larger version (9K): [in this window] [in a new window]   Fig 5. Cleavage of PAR-1 peptide sequences. (A) The peptide LDPRSFLLRNPNDKYEPF (TR18), corresponding to the uncleaved receptor, elutes as a single peak at 18 minutes. (B) The peptide SFLLRNPNDKYEPF (TR14), corresponding to the receptor following cleavage at the site resulting in receptor activation, elutes as a single peak at 5 minutes. (C) TR18 following 15 minutes incubation with 10 nM thrombin. (D) TR18 following 4 hours incubation with 0.5 µM Sk-plasminogen. (E) TR18 following 4 hours incubation with 0.5 µM Sk-plasminogen and 2 µM 2-antiplasmin. Significant peptide cleavage occurs even in the presence of 2-antiplasmin. Epitope-specific cleavage of PAR-1 on intact cells The monoclonal antibody SPAN 12 was raised against an epitope spanning the site on PAR-1 cleaved by thrombin and recognizes the intact receptor only.30 WEDE 15 binds to the hirudin-like site of PAR-1 (removed by plasmin) and recognizes intact and thrombin-cleaved receptors.30 HEL cells incubated with SPAN 12 or WEDE 15 show strong staining at the cell surface (Figure 6A and B). Cells treated with Sk in the presence of plasminogen and anti-Sk antibodies showed a substantial reduction in SPAN 12 staining without significant loss of staining of WEDE 15 (Figure 6C and D). Because plasmin cleaves PAR-1 not just at the authentic thrombin cleavage site but also at sites that would delete both the SPAN and WEDE epitopes,25 these data demonstrate that Sk-plasmin(ogen) cleaves PAR-1 primarily at the site that results in receptor activation. View larger version (52K): [in this window] [in a new window]   Fig 6. Fluorescent microscopy of PAR-1 on HEL cells. Cells were stained with the cleavage-sensitive PAR-1 antibody SPAN 12 (A and C) or the cleavage-insensitive PAR-1 antibody WEDE 15 (B and D) and treated with no agonist (A and B) or Sk (C and D) in plasma from an individual whose platelets aggregated to Sk. Intracellular calcium responses The HEL cells mimic platelets in that they express the Fc receptor and PAR-1 (J.P.M. and D.J.F., unpublished observations). HEL cells showed a marked increase in intracellular Ca++ in response to sequential addition of ADP, 1 µM plasminogen, 800 U/mL Sk, and anti-Sk antibodies purified from plasma (Figure 7A). Although some rise in Ca++ is elicited in the presence of Sk and plasminogen, this is accelerated by the addition of anti-Sk. As with the platelet activation, this response is substantially blocked by pretreatment with IV.3, implicating antibody-Fc receptor interactions in mediating this effect (Figure 7B). No response was seen to anti-Sk and Sk in the absence of plasminogen (data not shown). Moreover, desensitization of PAR-1 by prior treatment with the agonist SFLLRN substantially reduced the Ca++ response to plasminogen, Sk, and anti-Sk (Figure 7C). Thus, activation of both PAR-1 and the Fc receptor is necessary for Sk-induced cell activation. View larger version (17K): [in this window] [in a new window]   Fig 7. Activation of HEL cells by ADP, plasminogen, Sk, and anti-Sk antibodies. Cells were loaded with FURA-2-AM, and Ca++-mobilizing responses measured. Arrows indicate the addition of agonists. (A) Cells were treated with ADP (100 µM), plasminogen (2 µM), Sk (800 U/mL), and anti-Sk antibodies (30 µL). (B) Cells were treated in the presence of 5 µg/mL of the Fc receptor-blocking antibody IV.3. (C) Pretreatment with 12 µM of the PAR-1 agonist SFLLRN, rendering the cells unresponsive to further activation of PAR-1 or to the combination of ADP, plasminogen, Sk, and anti-Sk antibodies. To confirm the specificity of the antibody required to support Sk-dependent cell activation, we used serum from a patient treated with Sk for acute myocardial infarction 1 month before as a source of antibody. Plasminogen and Sk evoked a calcium response in HEL cells only after addition of 10 µL serum (Figure 8A). An equivalent volume of serum incubated overnight with Sk-coated Sepharose particles to remove Sk-binding antibodies failed to provoke a calcium response (Figure 8B). In contrast, serum incubated with BSA immobilized on Sepharose supported a calcium response (Figure 8C). View larger version (16K): [in this window] [in a new window]   Fig 8. Activation of HEL cells by plasminogen, Sk, and serum from a Sk-treated patient. Cells were loaded with FURA-2-AM, and Ca++-mobilizing responses measured. Arrows indicate the addition of agonists. (A) Cells were treated with plasminogen (1 µM), Sk (800 U/mL), and serum (10 µL). (B) The serum used was incubated overnight with Sk coupled to Sepharose beads to deplete Sk-binding antibodies. (C) The serum used was incubated overnight with BSA coupled to Sepharose beads as a control.     Discussion Top Abstract Introduction Materials and methods Results Discussion References Streptokinase is widely used in the treatment of coronary thrombosis. Despite lacking protease activity, Sk complexes with plasminogen and generates the plasminogen activator, Sk-plasmin complex. Sk induces reperfusion in the majority of patients presenting with a coronary thrombosis but patency is often delayed31 and is complicated by reocclusion in 15% to 30% of cases.32 Several clinical and experimental studies have shown evidence of platelet activation and thrombosis in patients treated with Sk, and this appears to be more marked than that seen with the nonantigenic tissue-type plasminogen activator.10,33,34 The increased platelet activity appears to be functionally important because aspirin greatly enhances the benefit seen with Sk in humans.1 The mechanism for the increased platelet activity is not understood. Although thrombin has been implicated based on measurements of fibrinopeptide A35 and other biochemical measurements,36 neither heparin37,38 nor specific thrombin inhibitors39 influence the response to Sk. We and others have shown that Sk can induce platelet aggregation and that this was dependent on the presence of antibody.8,10 These findings were confirmed in the present study in that Sk at a concentration that is achieved in vivo induced full irreversible aggregation of platelets primed with ADP. This was accompanied by a marked increase in TXA2 formation, as also seen in vivo. Platelet aggregation was abolished by a monoclonal antibody against the platelet Fc receptor. Activation of this receptor alone by cross-linking with immune complexes can cause platelet activation and aggregation.12-14 However, Sk-induced platelet aggregation was also abolished by a monoclonal antibody directed against PAR-1. This antibody prevents cleavage of PAR-1 and thrombin-induced platelet aggregation. Cleavage of the receptor was not due to formation of thrombin because neither hirudin nor Ro46-6240, 2 highly specific thrombin inhibitors, had any effect. These studies suggest that a protease other than thrombin is responsible for Sk-mediated platelet aggregation. Aggregation depended on the presence of proteolytically active plasminogen, implicating Sk-plasmin(ogen) as the protease responsible for PAR-1 cleavage. Indeed, a PAR-1 peptide was cleaved by Sk-plasminogen at the activating site, although only slowly in solution. This cleavage was not blocked by antiplasmin, suggesting that it was largely as a consequence of the complex rather than free plasmin. Plasmin has been reported to cleave PAR-1 at the thrombin cleavage site and at other sites that delete the tethered ligand domain, preventing receptor activation.24,25 However, we showed in HEL cells that Sk-plasmin is more selective. Thus, the binding of SPAN 12, an antibody to the thrombin cleavage site, was selectively reduced by Sk-plasmin, whereas an antibody to the hirudin-like binding domain (lying in the region putatively cleaved by plasmin) was not. Although PAR-4 expression is detectable by reverse transcriptase-polymerase chain reaction in platelets23 and is a potential target for Sk-plasminogen, Sk-induced platelet activation was prevented by an antibody specific to PAR-1, so PAR-4 is unlikely to be involved. A role for PAR-1 was also seen in HEL cells activated by a combination of Sk, plasminogen, and anti-Sk antibody. These cells contain PAR-1, PAR-4 (J.P.M. and D.J.F., unpublished observations), and the Fc receptor. Pretreating the cells with the PAR-1 agonist SFLLRN to desensitize the receptor reduced the intracellular Ca++ response to Sk. Thus, 2 distinct signals are required for Sk-mediated platelet activation, the first mediated by the Fc receptor and the second by cleavage of PAR-1. Consistent with a role for Ig-Fc interactions, Sk-induced but not SFLLRN-induced platelet activation targeted IgG to the platelet surface. Moreover, the Ca++ response in HEL cells induced by Sk and plasminogen was accelerated by the addition of anti-Sk antibody and blocked by IV.3. A recent report described antibody-mediated platelet activation in heparin-induced thrombocytopenia (HIT) as dependent on ADP release from the platelet.40 In contrast, the Sk-induced platelet activation described in this report requires exogenous ADP and PAR-1 activation suggesting that signaling through the Fc receptor is not as strong as that seen in HIT. A previous study has shown also that an Sk-plasmin complex devoid of protease activity induced platelet aggregation.8 However, aggregation was induced only in the presence of high concentrations of Sk antibodies and is perhaps similar to what occurs in HIT. In our experiments, Sk-induced platelet activation was always dependent on PAR-1. One possible model is that the Sk antibody localizes the complex to the cell surface allowing a more rapid cleavage of PAR-1. Consistent with this hypothesis, the Sk-plasmin complex is unable to activate a thrombin-sensitive PAR-1 receptor expressed in COS cells, which do not express the Fc receptor (data not shown). The increased surface expression of FcRII and PAR-1 in partially activated platelets30,41 may explain why ADP sensitizes platelets to Sk. In summary, activation of platelets by Sk requires both activation of the Fc receptor and cleavage of PAR-1. It is possible that interaction with the Fc receptor provides signals essential for the activation or helps localize the Sk-plasmin complex to the cell membrane where it cleaves PAR-1. This activation of PAR-1 is not susceptible to inhibition by heparin or direct thrombin inhibitors and may explain the limited activity of such treatments during thrombolytic therapy of myocardial infarction.     Acknowledgments Integrelin was a gift of Dr David Philips, COR Therapeutics (San Francisco, CA). Recombinant plasminogen S741A was a gift of Dr Anthony Brown, British Biotech Pharmaceuticals (Oxford, England). Monoclonal antibody 2/389, directed against the PAR-1 cleavage site was a gift of Dr Stuart Stone (deceased), University of Cambridge. The thrombin inhibitor Ro46-6240 (N-[N4-[(s)-1-amidino-3-piperidinyl]-methyl]N-2(2-naphthalenesulfonyl)-L-asparaginyl]-N-cyclopropylglycine) was a gift of Dr Sebastian Roux, Hoffman-La Roche (Basel, Switzerland).     Footnotes Submitted June 3, 1999; accepted October 5, 1999. Supported by grants from the Irish Heart Foundation, the Higher Education Authority of Ireland, and Enterprise Ireland. Reprints: Desmond J. Fitzgerald, Centre for Cardiovascular Science, Department of Clinical Pharmacology, The Royal College of Surgeons in Ireland, 123 St Stephen's Green, Dublin, 2, Ireland; e-mail: dfitzgerald{at}rcsi.ie. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.     References Top Abstract Introduction Materials and methods Results Discussion References 1. ISIS-2 (Second International Study of Infarct Survival) Collaborative Group. Randomised trial of intravenous streptokinase, oral aspirin, both, or neither among 17,187 cases of suspected acute myocardial infarction: ISIS-2. Lancet. 1988;2:349[Medline] [Order article via Infotrieve]. 2. Gruppo Italiano per lo Studio della Streptochinasi nell'Infarto Miocardico. GISSI-2: a factorial randomised trial of alteplase versus streptokinase and heparin versus no heparin among 12,490 patients with acute myocardial infarction. Lancet. 1990;336:65[Medline] [Order article via Infotrieve]. 3. Gonzalez-Gronow M, Siefring GE Jr, Castellino FJ. Mechanism of activation of human plasminogen by the activator complex, streptokinase-plasmin. J Biol Chem. 1978;253:1090[Abstract/Free Full Text]. 4. Murphy N, McRedmond J, Kane M, Fitzgerald D. Prevalence of antibodies to streptokinase in the population: determination by a highly sensitive functional assay and an ELISA. Fibrinolysis. 1996;10:10. 5. Lee HS, Cross S, Davidson R, Reid T, Jennings K. Raised levels of antistreptokinase antibody and neutralisation titres from 4 days to 54 months after administration of streptokinase or anistreplase. Eur Heart J. 1993;14:84[Abstract/Free Full Text]. 6. Patel S, Jalihal S, Dutka DP, Morris GK. Streptokinase neutralisation titres up to 866 days after intravenous streptokinase for acute myocardial infarction. Br Heart J. 1993;70:119[Abstract/Free Full Text]. 7. Verstraete M, Vermylen J, Amery A, Vermylen C. Thrombolytic therapy with streptokinase using a standard dosage scheme. BMJ. 1966;5485:454. 8. Vaughan DE, Kirshenbaum JM, Loscalzo J. Streptokinase-induced, antibody-mediated platelet aggregation: a potential cause of clot propagation in vivo. J Am Coll Cardiol. 1988;11:1343[Abstract]. 9. Vaughan DE, Van Houtte E, Declerck PJ, Collen D. Streptokinase-induced platelet aggregation. Prevalence and mechanism. Circulation. 1991;84:84[Abstract/Free Full Text]. 10. Fitzgerald DJ, Catella F, Roy L, FitzGerald GA. Marked platelet activation in vivo after intravenous streptokinase in patients with acute myocardial infarction. Circulation. 1988;77:142[Abstract/Free Full Text]. 11. Fitzgerald DJ. Platelet activation in the pathogenesis of unstable angina: importance in determining the response to plasminogen activators. Am J Cardiol. 1991;68:51B[Medline] [Order article via Infotrieve]. 12. Haimovich B, Regan C, DiFazio L, et al. The FcRII receptor triggers pp125FAK phosphorylation in platelets. J Biol Chem. 1996;271:16,332[Abstract/Free Full Text]. 13. Kang J, Cabral C, Kushner L, Salzman EW. Membrane glycoproteins and platelet cytoskeleton in immune complex-induced platelet activation. Blood. 1993;81:1505[Abstract/Free Full Text]. 14. Anderson GP, Anderson CL. Signal transduction by the platelet Fc receptor. Blood. 1990;76:1165[Abstract/Free Full Text]. 15. Lebrazi J, Helft G, Abdelouahed M, et al. Human anti-streptokinase antibodies induce platelet aggregation in an Fc receptor (CD32) dependent manner. Thromb Haemost. 1995;74:938[Medline] [Order article via Infotrieve]. 16. Vu T-KH, Hung DT, Wheaton VI, Coughlin SR. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell. 1991;64:1057[Medline] [Order article via Infotrieve]. 17. Molino M, Blanchard N, Belmonte E, et al. Proteolysis of the human platelet and endothelial cell thrombin receptor by neutrophil-derived cathepsin G. J Biol Chem. 1995;270:11,168[Abstract/Free Full Text]. 18. Suidan HS, Bouvier J, Schaerer E, Stone SR, Monard D, Tschopp J. Granzyme A released upon stimulation of cytotoxic T lymphocytes activates the thrombin receptor on neuronal cells and astrocytes. Proc Nat Acad Sci U S A. 1994;91:8112[Abstract/Free Full Text]. 19. Nystedt S, Emilsson K, Wahlestdt C, Sundelin J. Molecular cloning of a potential proteinase activated receptor. Proc Nat Acad Sci U S A. 1994;91:9208[Abstract/Free Full Text]. 20. Ishihara H, Connolly AJ, Zeng D, et al. Protease-activated receptor 3 is a second thrombin receptor in humans. Nature. 1997;386:502[Medline] [Order article via Infotrieve]. 21. Xu W-F, Andersen H, Whitmore TE, et al. Cloning and characterization of human protease-activated receptor 4. Proc Nat Acad Sci U S A. 1998;95:6642[Abstract/Free Full Text]. 22. Kahn ML, Zheng Y-W, Huang W, et al. A dual thrombin receptor system for platelet activation. Nature. 1998;394:690[Medline] [Order article via Infotrieve]. 23. Kahn ML, Nakanishi-Matsui M, Shapiro MJ, Ishihara H, Coughlin SR. Protease-activated receptors 1 and 4 mediate activation of human platelets by thrombin. J Clin Invest. 1999;103:879[Medline] [Order article via Infotrieve]. 24. Kimura M, Andersen TT, Fenton JWI, Bahou WF, Aviv A. Plasmin-platelet interaction involves cleavage of functional thrombin receptor. Am J Physiol. 1996;271:C54[Abstract/Free Full Text]. 25. Kuliopulos A, Covic L, Seeley S, Sheridan P, Helin J, Costello C. Plasmin desensitization of the PAR-1 thrombin receptor: kinetics, sites of truncation, and implications for thrombolytic therapy. Biochemistry. 1999;38:4572[Medline] [Order article via Infotrieve]. 26. Mathews II, Padmanabhan KP, Ganesh V, et al. Crystallographic structures of thrombin complexed with thrombin receptor peptides: existence of expected and novel binding modes. Biochemistry. 1994;33:3266[Medline] [Order article via Infotrieve]. 27. Gemmill JD, Hogg KJ, Burns JM, et al. A comparison of the pharmacokinetic properties of streptokinase and anistreplase in acute myocardial infarction. Br J Clin Pharmacol. 1991;31:143[Medline] [Order article via Infotrieve]. 28. Gast A, Tschopp TB, Schmid G, Hilpert K, Ackermann J. Inhibition of clot-bound and free (fluid-phase thrombin) by a novel synthetic thrombin inhibitor (Ro 46-6240), recombinant hirudin and heparin in human plasma. Blood Coagul Fibrinolysis. 1994;5:879[Medline] [Order article via Infotrieve]. 29. Cederholm-Williams SA, De Cock F, Lijnen HR, Collen D. Kinetics of the reactions between streptokinase, plasmin and 2-antiplasmin. Eur J Biochem. 1979;100:125[Medline] [Order article via Infotrieve]. 30. Molino M, Bainton DF, Hoxie JA, Coughlin SR, Brass LF. Thrombin receptors on human platelets. Initial localization and subsequent redistribution during platelet activation. J Biol Chem. 1997;272:6011[Abstract/Free Full Text]. 31. The GUSTO Angiographic Investigators. The effects of tissue plasminogen activator, streptokinase, or both on coronary-artery patency, ventricular function, and survival after acute myocardial infarction. N Engl J Med. 1993;329:1615[Abstract/Free Full Text]. 32. Chesebro JH, Knatterud G, Roberts R, et al. Thrombolysis in Myocardial Infarction (TIMI) trial, phase I: a comparison between intravenous tissue plasminogen activator and intravenous streptokinase. Circulation. 1987;76:142[Abstract/Free Full Text]. 33. Kerins DM, Roy L, FitzGerald GA, Fitzgerald DJ. Platelet and vascular function during coronary thrombolysis with tissue-type plasminogen activator. Circulation. 1989;80:1718[Abstract/Free Full Text]. 34. Parise P, Hauert J, Iorio A, Callegari P, Agnelli G. Streptokinase and rt-PA activate platelets by a different way: implications on the rethrombosis rate after their administration in myocardial infarction. J Lab Clin Med. 1995;125:212[Medline] [Order article via Infotrieve]. 35. Eisenberg PR, Sherman LA, Jaffe AS. Paradoxic elevation of fibrinopeptide A after streptokinase: evidence for continued thrombosis despite intense fibrinolysis. J Am Coll Cardiol. 1987;10:527[Abstract]. 36. Seitz R, Blanke H, Pratorius G, Strauer BE, Egbring R. Increased thrombin activity during thrombolysis. Thromb Haemost. 1988;59:541[Medline] [Order article via Infotrieve]. 37. Galvani M, Abendschein DR, Ferrini D, Ottani F, Rusticali F, Eisenberg PR. Failure of fixed dose intravenous heparin to suppress increases in thrombin activity after coronary thrombolysis with streptokinase. J Am Coll Cardiol. 1994;24:1445[Abstract]. 38. Ridker PM, Hebert PR, Fuster V, Hennekens CH. Are both aspirin and heparin justified as adjuncts to thrombolytic therapy for acute myocardial infarction? Lancet 1993;341:1574[Medline] [Order article via Infotrieve]. 39. Antman EM. Hirudin in acute myocardial infarction: thrombolysis and thrombin inhibition in myocardial infarction (TIMI) 9B trial. Circulation. 1996;94:911[Abstract/Free Full Text]. 40. Polgar J, Eichler P, Greinacher A, Clemetson KJ. Adenosine diphosphate (ADP) and ADP receptor play a major role in platelet activation/aggregation induced by sera from heparin-induced thrombocytopenia patients. Blood. 1998;91:549[Abstract/Free Full Text]. 41. McCrae KR, Shattil SJ, Cines DB. Platelet activation induces increased Fc gamma receptor expression. J Immunol. 1990;144:3920[Abstract]. © 2000 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: M. Steinhoff, J. Buddenkotte, V. Shpacovitch, A. Rattenholl, C. Moormann, N. Vergnolle, T. A. Luger, and M. D. Hollenberg Proteinase-Activated Receptors: Transducers of Proteinase-Mediated Signaling in Inflammation and Immune Response Endocr. Rev., February 1, 2005; 26(1): 1 - 43. [Abstract] [Full Text] [PDF] M. D. Hollenberg and S. J. Compton International Union of Pharmacology. XXVIII. Proteinase-Activated Receptors Pharmacol. Rev., June 1, 2002; 54(2): 203 - 217. [Abstract] [Full Text] [PDF] A. Maree and D. Fitzgerald PAR2 Is Partout and Now in the Heart Circ. Res., March 8, 2002; 90(4): 366 - 368. [Full Text] [PDF] J. P. McRedmond, D. J. Fitzgerald ;, R. N. Pike, A. Lourbakos, J. Travis, and J. Potempa A growing set of platelet-activating bacterial proteins Blood, January 1, 2002; 99(1): 387 - 388. [Full Text] [PDF] This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by McRedmond, J. P. Articles by Fitzgerald, D. J. Search for Related Content PubMed PubMed Citation Articles by McRedmond, J. P. Articles by Fitzgerald, D. J. Related Collections Hemostasis, Thrombosis, and Vascular Biology Social Bookmarking                   What's this?

	Copyright © 2000 by American Society of Hematology         Online ISSN: 1528-0020