Inhibition of fibrin(ogen) binding to stimulated platelets by a monoclonal antibody specific for a conformational determinant of GPIIIa

We have purified the integrin GPIIb:IIIa from the membrane fraction of human blood platelets by lentil lectin affinity chromatography followed by gel filtration chromatography. With purified GPIIb:IIIa as an antigen, we have produced monoclonal antibody CS-1, which immunoblotting demonstrates to be specific for native GPIIIa; disulfide bond reduction of GPIIIa resulted in loss of immunoreactivity. Radiolabelled ligand binding studies revealed that CS-1 recognized approximately 55,000 sites per platelet and bound with a K_d in the nanomolar range, independent of the state of platelet activation. Binding of CS-1 or its Fab fragment to ADP-and thrombin-stimulated gel filtered platelets caused a 2–3 fold inhibition of binding the soluble ligands fibrinogen and fibrin protofibrils. CS-1 also inhibited aggregation of ADP- and thrombin-stimulated platelets by 2- and 4-fold, respectively. Since CS-1 inhibits fibrin(ogen) interactions with GPIIb:IIIa, we propose that the conformationally dependent epitope on GPIIIa recognized by CS-1 constitutes a region of the receptor which is involved in fibrin(ogen) binding.     

Studies on the dual effects on platelets of a monoclonal antibody to CD9, and on the properties of platelet CD9.

The article describes effects on human platelets of a murine monoclonal antibody of the IgG2a subtype (clone FN99) directed against the membrane glycoprotein CD9. This antibody exerts a dual action on human platelets in plasma depending on whether the complement system can be activated or not, resulting either in membrane permeabilization or a true platelet aggregation. Secretion from the alpha-granules during permeabilisation was not observed in the sense that the granule-located protein thrombospondin was retained in the platelets, as opposed to what was seen with platelets that had undergone an antibody-induced aggregation. Only a small fraction of P-selectin was found on the surface of the permeabilised platelets. The cytoskeletal protein actin-binding protein (filamin) was profoundly degraded during membrane permeabilisation, however, and scanning electron microscopy showed platelets that were swollen with only a few pseudopodia. Preincubation of platelets with three different antibodies to CD9 showed strong inhibition of a subsequent binding of FITC-labelled Fab fragment of FN99 indicating that antibodies tend to bind in the same area of the CD9 molecule. No association of CD9 to the platelet actin-based cytoskeleton was observed. CD9 was present on the surface of microvesicles derived from calcium ionophore-treated platelets.     

An immunoradiometric assay for factor VIII related antigen (VIIIRAg) using two monoclonal antibodies-comparison with polyclonal rabbit antibodies for use in von Willebrand's disease diagnosis

Two monoclonal antibodies raised against FVIII/von Willebrand protein were used in an immunoradiometric assay (IRMA) to measure this antigen in normal plasma and plasma of patients with different forms of von Willebrand's disease. The first antibody, an IgG1, was used to coat polystyrene tubes, the second one, an IgG2a, iodinated and used in the second step. Both antibodies inhibit ristocetin induced platelet agglutination and react strongly with platelets, megakaryocytes and endothelial cells. The IRMA test using these antibodies showed greater sensitivity than that using rabbit polyclonal anti VIIIRAg antibodies. A good correlation between the two tests was nevertheless found when VIIIRAg was measured in the majority of patient's plasma. However 5 patients from 3 different families showed more antigenic material in the rabbit antibody IRMA than in the monoclonal antibody IRMA. It is suggested therefore that the monoclonal antibodies identify part of the VIIIR:Ag molecule showing structural abnormalities in these vWd patients, these structural changes remaining undetected by the polyclonal antibodies.     

Neutralization of the antiheparin activity of platelet factor 4 by a monoclonal antibody.

We have produced a panel of monoclonal antibodies (mAbs) against rabbit platelet factor 4 (PF4). Two of these mAbs have been characterized in this study. In particular the antibody called 10B2, which also recognizes the human molecule, is able to block PF4's ability to neutralize heparin in a modified Heparin-Factor Xa chromogenic assay. The inhibition appears to be more than 95% at 1:1 mAb/PF4 molar ratio both for purified rabbit and human PF4. Similar results were obtained using supernatants from stimulated human platelets (90% of inhibition at 1:1 mAb/PF4 molar ratio) or using Fab fragments from 10B2. Studies to determine the antigenic determinant against which 10B2 is directed, show that this is an assembled epitope which involves disulfide bonds of the PF4.     

Circulating activated platelets in myeloproliferative disorders.

Platelet activation in patients with myeloproliferative disorders is often suggested by increased platelet alpha-granule secretion and an acquired storage pool defect of dense granules. To determine whether activated platelets circulate in patients with chronic myeloproliferative disorders, we evaluated the binding of monoclonal antibodies against activation-dependent epitopes on resting platelets (P 12, CD 63, and CD 62) in 12 patients with prominent megakaryocytic proliferation (8 patients with essential thrombocythemia, 2 with chronic myeloid leukemia, and 2 patients with polycythemia rubra vera). In addition, platelet aggregation in response to collagen, adenosine diphosphate, platelet activating factor, and agglutination with ristocetin was investigated. In 3 patients there was an increased percentage of platelets binding at least 1 activation marker. In 2 other patients, a trend towards increased antibody binding was observed. Binding of the antibody to thrombospondin (P 12) was related to expression of the GMP 140 protein (CD 62, r = 0.76, p = 0.004). There was no correlation of platelet aggregation defects in vitro to increased expression of platelet activation markers or to thrombohaemorrhagic complications. However, circulating activated platelets were detected in three out of five patients with a history of bleeding or thrombotic complications. The results of this preliminary study suggest that some but not all patients with myeloproliferative disorders showed increased amounts of circulating activated platelets. The relation of bleeding and thrombotic complications to the expression of activation-dependent epitopes on platelets in myeloproliferative disorders requires further investigation.     

"A" subunit of factor XIII is present on bovine platelet membrane and mediates collagen-induced platelet activation

The Fab fragment of a polyclonal antibody against platelet factor XIII inhibited the collagen-induced platelet aggregation in a dose-dependent manner. This inhibitory effect was specific for collagen, and it had no effect on arachidonic acid-, ADP-, and serotonin-induced aggregations. This finding strengthens our notion that platelet factor XIII is involved in collagen-induced platelet aggregation. (Saito, Y., Imada, T., Takagi, J., Kikuchi, T. and Inada, Y. J. Biol. Chem. 261, 1355-1358, 1986). We have investigated membrane localization of bovine platelet factor XIII using immunological techniques. Immunofluorescent visualization revealed that the factor XIII was expressed on the surface of non-permeabilized bovine platelets, where we detected neither lactate dehydrogenase, a cytoplasmic enzyme marker, nor B subunit of factor XIII, which is present in plasma. Cell surface iodination and immunoprecipitation also confirmed that it existed on the surface of platelets.     

The effect of recombinant IgG antibodies against the leucine-33 form of the platelet beta3 integrin (HPA-1a) on platelet function

Recombinant HPA-1a antibodies with Fc, mutated to remove destructive effector functions, have been developed as a potential therapy for fetomaternal alloimmune thrombocytopenia (FMAIT), via blockade of binding of human HPA-1a polyclonal antibodies to fetal HPA-1a1b platelets. We have assessed the effect of the IgG1 HPA-1a antibody B2G1 and two mutated derivatives in various functional assays in resting and agonist-stimulated platelets of the three HPA-1 genotypes. With HPA-1a1b platelets (fetal genotype), the antibodies did not activate signalling or CD62P expression in resting platelets, did not change in vitro bleeding time (IVBT), and did not inhibit platelet adhesion to collagen in flowing blood. Adhesion of HPA-1a1b platelets to fibrinogen was reduced by 20%, and aggregation induced by ADP by 50%, but collagen-related peptide (CRP-XL)-induced aggregation was normal. With HPA-1a1a platelets, aggregation to both ADP and CRP-XL was inhibited, with total blockade of adhesion to fibrinogen and of IVBT responses. Interestingly, a monovalent antibody fragment with identical specificity had no inhibitory effect on aggregation. In static adhesion assays using human alphaIIbbeta3 or alphaVbeta3 transfectants of HPA-1a (Leu(33)) phenotype, attachment to fibrinogen of the latter but not of the former was completely blocked by the HPA-1a antibodies. These observations are best explained by antibody-mediated blockade of the RGD binding site on beta3 by a mechanism of steric hindrance. As the effect on platelet function is modest with HPA-1a1b (fetal type) platelets, the mutated HPA-1a antibodies described here could be developed further for FMAIT therapy.     

Platelet dysfunction during Bothrops jararaca snake envenomation in rabbits

Despite being well established that snake envenomation causes blood coagulation and fibrinolysis disturbances, scant information is available about blood platelet disorders. Herein we show that experimentally Bothrops jararaca-envenomed rabbits presented thrombocytopenia, hypofibrinogenemia, elevation of von Willebrand factor plasma levels, platelet hypoaggregation in platelet rich plasma and whole blood, normoaggregation in washed platelet suspensions, decreased platelet ATP secretion, normal plasma levels of platelet factor 4, and constant intraplatelet serotonin levels. Furthermore, by flow cytometric analyses, platelets displayed a significant decrease in the expression of a GPIIb-IIIa epitope recognized by P2 monoclonal antibody (p< 0.05) and an increased expression of a ligand-induced binding site (LIBS-1) of GPIIIa (p< 0.05), but total GPIIb-IIIa expression, evaluated with specific polyclonal antibodies, was normal. Fibrinogen and fibrin(ogen) degradation product (FfDP) expression on platelet surface showed no significant alteration. Nonetheless, significant elevations of platelet P-selectin were noticed on circulating platelets. The percentage of circulating reticulated platelets and the survival time of biotinylated platelets of envenomed rabbits were not statistically different from control animals. We suggest that thrombin engendered by procoagulating components of B. jararaca venom has an essential role in the pathogenesis of platelet and coagulation disorders in this experimental model. Increased expression of P-selectin in the experimental group demonstrates that platelets of envenomed rabbits are indeed activated in circulation, and that decreased fibrinogen or increased FfDP levels are not the primary cause of platelet dysfunction. These results imply the existence of an inhibitor in plasma that interferes with platelet aggregation in bothropic envenomation.     

Inhibition of Platelet-Mediated Clot Retraction by Integrin Antagonists

The effects of the platelet IIbβ3 integrin (GPIIb/IIIa) antagonists XV459 (non-peptide), c7E3 (Fab monoclonal antibody) and DMP728 (cyclic peptide) as well as the vβ3 integrin antagonists, LM609 (monoclonal antibody) and XT199 (non-peptide) on clotting and platelet-mediated clot retraction were examined. While 30 nM of XV459 had no significant effect on the kinetics of coagulation, platelet-mediated clot retraction was nearly fully inhibited at this concentration (Relative Retraction Rate = 0.09). XV459 resulted in a concentration related-response curve. Other experiments demonstrated that platelet aggregation was maximally inhibited at XV459 concentrations ranging from 30–50 nM. Similarly, c7E3 demonstrated comparable inhibitory efficacy in inhibiting either clot retraction or platelet aggregation. In contrast, DMP728, an equally potent anti-aggregatory agent with an IC₅₀ of 20–50 nM in inhibiting platelet aggregation induced by various agonists, was found to be a less potent inhibitor of platelet-mediated clot retraction with a half-maximal inhibition of clot retraction at 0.7 μM, and maximum effects at concentrations of 10 μM. The vβ3 integrin antagonists, LM609 or XT199 were without any significant effects on either platelet-mediated clot retraction or platelet aggregation. In conclusion, these data suggest a differential efficacy among different GPIIb/IIIa antagonists in inhibiting platelet-mediated clot retraction in spite of the equivalent anti-aggregatory potency. Additionally, the vβ3 integrin antagonists do not affect platelet-mediated clot retraction or aggregation. Further studies with the previously described IIbβ3 integrin antagonists as well as others revealed a distinct correlation between the Kd to resting and activated platelets and the efficacy in inhibiting platelet-mediated clot retraction.     

Evidence for the involvement of platelet glycoproteins other than GP Ib in heparin-associated thrombocytopenia and thrombosis

Heparin-associated thrombocytopenia and thrombosis (HATT) is a potentially fatal complication of heparin therapy which is characterized by a progressive fall in the platelet count associated with arterial or venous thrombosis. The etiology is consistent with the development an antibody which, in the presence of heparin, induces intravascular platelet aggregation. Biochemical analysis has demonstrated that the platelet membrane glycoprotein (GP) Ib binds heparin. Recent studies have shown that polyclonal antisera or monoclonal antibodies to GP Ib can inhibit platelet aggregation induced by HATT plasma in the presence of heparin implicating GP Ib as the site of heparin-antibody interaction. The Bernard-Soulier syndrome (BSS) is an inherited bleeding disorder in which the platelets fail to adhere to exposed subendothelium due to a deficiency of GP Ib. We have used the platelets from a patient with documented BSS to further investigate the role of GP Ib in the heparin- dependent platelet aggregation induced by the plasma of three patients with HATT. We have shown that platelet aggregation by HATT plasma in the presence of heparin occurred as readily with BSS platelets as with normal donor platelets. These results indicate that glycoprotein Ib cannot be the only site of platelet-heparin-antibody interactions.     

Platelet collagen receptors, signaling and antagonism: emerging approaches for the prevention of intravascular thrombosis

Collagen, one of the major proteins of sub-endothelial vasculature get exposed following endothelium denudement, is a potent stimulator of platelet adhesion and aggregation. Adhesion of platelets following endothelial injury is the primary event usually associated with uncontrolled platelet activation culminating into intravascular thrombosis, thus needs to be intervened to prevent the pathology related to various peripheral, myocardial and cerebral ischemic episodes. Recent advances in the understanding of collagen mediated platelet adhesion and aggregation have led to the identification of two prominent receptors, glycoprotein Ia/IIa (GPIa/IIa or integrin α₂β₁) and glycoprotein VI (GPVI) and associated intracellular signaling, which are undoubtedly the new emerging targets for the development of more effective antithrombotic drugs. The optimism for collagen antagonism is based on results obtained so far by the use of monoclonal and polyclonal antibodies, peptide inhibitors, knockouts models and collagen-mimetics in various in vitro test systems and animal models. These findings have revealed that collagen receptor inhibition is an attractive and secure strategy for the new drug development to prevent intravascular thrombosis.     

Comparison of platelet fibrinogen receptors on intact and proteolytically-treated platelets by use of an anti-glycoprotein IIIa monoclonal antibody (MA 123)

A murine monoclonal antibody (MA 123) was selected by screening 153 supernatants of hybridoma cells secreting anti-human platelet antibodies for their ability to inhibit the fibrinogen-induced aggregation of chymotrypsin-treated platelets. MA 123 inhibited the binding of 125I-fibrinogen to ADP-stimulated intact human platelets and to platelets treated with chymotrypsin or pronase. Moreover, it inhibited the fibrinogen-induced aggregation of these platelet suspensions. The degree of inhibition was similar in each of the three types of platelets tested. The interactions of MA 123 with the 125I-labeled surface components of intact and chymotrypsin-treated platelets were studied by immunoprecipitation using Staphylococcus aureus coated with goat anti-mouse IgG, followed by SDS-polyacrylamide gel electrophoresis and autoradiography. MA 123 precipitated the glycoprotein IIb-glycoprotein IIIa (GPIIb-GPIIIa) complex from the surface of detergent solubilized intact human platelets; and it precipitated GPIIIa from the surface of chymotrypsin-treated platelets. Partially purified GPIIIa was also immunoprecipitated by MA 123. Our data suggest that the exposure of fibrinogen receptors by ADP, chymotrypsin or pronase, is associated with alterations of GPIIIa on the platelet surface.     

Reversible exposure of human platelet fibrinogen receptors by antiplatelet tetraspanin monoclonal antibodies via induction of a conformational change in membrane glycoprotein IIb/IIIa complex.

Antihuman platelet tetraspanin (CD9 antigen) monoclonal antibodies, HI117 and SJ9A4, can induce human platelet aggregation and secretion. As platelet aggregation is mediated by fibrinogen binding to its receptors exposed on platelet glycoprotein IIb/IIIa complex, we, therefore, investigated the induction of platelet fibrinogen receptors by HI117 and SJ9A4. It was found that HI117 and SJ9A4 induced specific fibrinogen binding to human platelets, suggesting that the two monoclonal antibodies evoked obvious exposure of fibrinogen receptors on human platelets. But in the absence of fibrinogen, the monoclonal antibody-exposed fibrinogen receptors gradually lost their capacity to bind fibrinogen and closed. Our results also showed that HI117 and SJ9A4, when activating platelets, caused a conformational change in glycoprotein IIb/IIIa complex, which must contribute to the exposure of functional fibrinogen receptors on this integrin. The effect of HI117 and SJ9A4 on glycoprotein IIb/IIIa complex seems, however, to be indirect, because the HI1117 and SJ9A4-induced fibrinogen binding was reduced by pretreatment of platelets with sphingosine, aspirin, apyrase, and/or PGI2. Taken together, we conclude that the antihuman platelet tetraspanin monoclonal antibodies, HI117 and SJ9A4, reversibly expose platelet fibrinogen receptors via inducing a conformational change in glycoprotein IIb/IlIa complex. Three signaling pathways, that is, thromboxane, secreted ADP, and cAMP pathways may be involved in this process, while protein kinase C activation seems to be the final common step of the three pathways.     

Allicin and disulfiram enhance platelet integrin αIIbβ3-fibrinogen binding

Introduction

Activation of the platelet receptor αIIbβ3 (glycoprotein IIbIIIa) involves a change in the disulfide bonds pattern in the extra-cellular domain of the receptor. The disulfide-bond reducing agent, dithiothreitol (DTT), can increase integrin activity, and point mutations of specific cysteine residues of the integrin can cause its lockage at the high affinity state. The present study is aimed to support the hypothesis that prevention of specific αIIbβ3 intra-molecular disulfide bond formation increases receptor-ligand binding activity.

Methods

Platelet aggregation was induced by collagen or ADP and epinephrine. Integrin αIIbβ3-fibrinogen binding was evaluated on prostaglandins E₁ (PGE₁)-treated washed platelets or baby hamster kidney (BHK) cells expressing human αIIbβ3. Integrin was directly activated by an anti-ligand induced binding site (LIBS) PT25-2 antibody. The effect of sulfhydryl-reactive agents, such as allicin, glutathione, dithiobis nitrobenzoic acid (DTNB) and disulfiram, was tested on αIIbβ3 activity.

Results

Allicin (40 µM) completely inhibited washed platelets agonist-induced aggregation. Both allicin and disulfiram (40 µM) inhibited αIIbβ3-fibrinogen binding and P-selectin expression in washed platelets. However, there was an increase in αIIbβ3-fibrinogen binding but not P-selectin expression in PGE₁-treated washed platelets activated by PT25-2 antibody. At a high concentration (400 µM) both inhibited αIIbβ3-fibrinogen binding. Similarly, in BHK cells expressing αIIbβ3 activated by PT25-2 antibody, allicin at a low concentration increased αIIbβ3 activity.

Conclusions

Allicin and disulfiram inhibit agonist-induced washed platelet activation probably via inhibition of platelet signaling, but enhance PT25-2 antibody-induced αIIbβ3 integrin activity most likely by preventing reformation of disulfide bridges thereby stabilizing the active conformation of the integrin.     

Structure and function of murine alphaIIbbeta3 (GPIIb/IIIa): studies using monoclonal antibodies and beta3-null mice,

The alphaIIbeta3 receptor (GPIIb/IIIa) is the only platelet-specific integrin receptor and the most abundant adhesion/aggregation receptor on the surface of human platelets. Since mice are increasingly being used as models of human disease, we analyzed the structure and function of murine platelet alphaIIbeta3, utilizing both beta3 integrin-deficient mice, who have a phenotype that resembles Glanzmann thrombasthenia, and our hamster monoclonal antibody (mAb) 1B5 to murine alphaIIbbeta3. By immunoblot analysis, flow cytometry, and mAb binding studies, mouse platelets express abundant amounts of alphaIIbbeta3 (60-80,000 copies/platelet). Like their human counterparts, murine alphaIIb and beta3 exhibit different electrophoretic motilities under nonreducing (aIIb 135k Da; beta3 92k Da) and reducing (aIIb 120k Da; beta3 108k Da) conditions, and the alphaIIbbeta3 complex is dissociated by EDTA at pH 8 and 37 degrees C. Murine beta3 is less susceptible to proteolysis by plasmin than is human beta3. In addition to defective platelet aggregation, mouse platelets lacking alphaIIbbeta3 and alphaVbeta3 are unable to adhere to fibrinogen and prothrombin, but retain the ability to adhere to fibronectin and collagen. Following platelet activation, beta3-null platelets express slightly less P-selectin than do wild-type mouse platelets. Moreover, beta3-null platelets have altered tyrosine phosphorylation patterns following thrombin- and collagen-induced aggregation. These results suggest fundamental similarities between human and mouse platelet activation and aggregation, but delineate subtle differences that need to be considered when comparing studies from mice and humans.     

Bilinexin, a snake C-type lectin from Agkistrodon bilineatus venom agglutinates platelets via GPIb and alpha2beta1

A new snake protein, named bilinexin, has been purified from Agkistrodon bilineatus venom by ion-exchange chromatography and gel filtration chromatography. Under non-reducing conditions it has a mass of 110 kDa protein on SDS-PAGE. On reduction, it can be separated into five subunits with masses in the range 13-25 kDa. The N-terminal sequences of these subunits are very similar to those of convulxin or the alboaggregins, identifying bilinexin as a new member of the snake C-type lectin family, unusual in having multiple subunits. Bilinexin agglutinates fixed platelets. washed platelets and platelet rich plasma (PRP) without obvious activation (shape change) as confirmed by light microscope examination. Both inhibitory and binding studies indicate that antibodies against alpha2beta1 inhibit not only platelet agglutination induced by bilinexin, but also bilinexin binding to platelets. VM16d, a monoclonal anti-GPIbalpha antibody, completely inhibits platelet agglutination induced by bilinexin, and polyclonal antibodies against GPIbalpha prevent its binding to platelets. However, neither convulxin, polyclonal anti-GPVI antibodies, nor GPIIb/IIIa inhibitors affect its binding to and agglutination of platelets. Bilinexin neither activates GPIIb/IIIa integrin on platelets nor induces tyrosine phosphorylation of platelet proteins, nor increases intracellular Ca2+ in platelets. Like alboaggregin B, bilinexin agglutinates platelets, which makes it a good tool to investigate the differences in mechanism between snake C-type lectins causing platelet agglutination and those that induce full activation.     

Preferred use of primary radiolabeled anti-platelet monoclonal antibodies. Comparison of immunoblotting methods for the analysis of functional domains on human platelets.

Several methodologies used for the identification and characterization of platelet receptors for antiplatelet monoclonal antibodies are compared. Two antiplatelet monoclonal antibodies, are investigated due to their potent effects on human platelet function. A platelet-activating monoclonal antibody, called M.Ab.F11, is able to induce platelet aggregation and granular secretion. A platelet-inhibitory monoclonal antibody, named G10, strongly blocks the platelet aggregation and granular secretion induced by M.Ab.F11, as well as by physiological agonists. In order to identify the specific antigens recognized by these monoclonal antibodies, we tested a number of immunostaining methods. Comparison of various procedures revealed that a high degree of nonspecific interactions with platelet proteins occurred when the commonly used secondary reagents, protein A and radiolabeled or enzyme-conjugated secondary antibodies interacted with the platelet proteins either in the presence or absence of primary monoclonal antibodies. On the other hand, we observed a high degree of specificity and selectivity when only radiolabeled anti-platelet monoclonal antibodies were used as single reagents. It is established that M.Ab.F11 interacts with the platelet membrane proteins of 32 and 35 Kd, and M.Ab.G10 recognizes 100 Kd protein, which corresponds to GPIIIa molecule.     

Activation of guinea pig platelets by a monoclonal antibody

We herein describe the characterization of a monoclonal anti-guinea pig platelet antibody, termed GT2-12, which causes aggregation, ATP and [³h]serotonin release, and platelet protein phosphorylation. GT212 can bind to platelets and megakaryocytes in guinea pig bone marrow cells. A protein of 34,000 daltons was detected by immunoprecipitation of platelet lysates with GT2-12. Incubation of ³²P-labeled guinea pig platelets with GT2-12 resulted in rapid phosphorylation of proteins of 40,000 and 20,000 daltons. GT2-12-induced aggregation and protein phosphorylation of platelets were inhibited by diltiazem, TMB-8 and dibutyryl cAMP and partially inhibited by indometacin. The F(ab′)₂ fragment of GT2-12 IgG also induced platelet aggregation, indicating that activation is not mediated by Fc receptors. This type of antibody should be useful for studying platelet function in the guinea pig model.     

A monoclonal antibody to the human platelet glycoprotein IIb/IIIa complex induces platelet activation

The platelet glycoprotein (GP) IIb/IIIa complex functions as the receptor for fibrinogen on activated platelets. The effects of two anti-GPIIb/IIIa monoclonal antibodies on platelet function were studied. These antibodies, 6C9 and C17, recognized different epitopes, which were exclusively present on the undissociated GPIIb/IIIa complex. Whereas C17 inhibited the binding of fibrinogen to platelets and platelet aggregation induced by adenosine diphosphate (ADP) or collagen, 6C9 caused irreversible aggregation of platelets, both in the presence and absence of extracellular fibrinogen. When incubated with unstirred (non-aggregating) platelets, 6C9 induced release of alpha and dense granule-constituents as well as binding of 125I-fibrinogen to platelets. The latter was evidently mediated in part by platelet-derived ADP, since it was inhibited to a large extent by apyrase, the ADP-hydrolyzing enzyme. F(ab')2 fragments of 6C9 did not induce platelet-release reactions but caused (slow) aggregation of platelets in the presence of extracellular fibrinogen. These results indicate that binding of an antibody to a specific site on the platelet GPIIb/IIIa complex may cause fibrinogen-mediated aggregation. The Fc part of the platelet-bound antibody appears to be involved in the induction of platelet release.     

The high molecular mass, glycoprotein Ib-binding protein flavocetin-A induces only small platelet aggregates in vitro

The direct effects of snake venom glycoprotein (GP) Ib-binding proteins on platelet receptors during the formation of platelet aggregates were determined by a particle counting method using light scattering. Flavocetin-A induces small platelet aggregates, but not medium or large ones. However, neither jararaca GPIb-BP nor tokaracetin induce platelet aggregation. The flavocetin-A dose-response curve for formation of small aggregates is bell-shaped, with maximal effect at 1 to 2 microg/mL. The formation of small aggregates was not observed when fixed human platelets were used. Jararaca GPIb-BP, the anti-GPIb monoclonal antibody GUR83-35, prostaglandin I2, and ethylene diamine-N,N-dimethylformamide all inhibited flavocetin-A-induced small aggregate formation, but acetylsalicylic acid did not. Furthermore, anti-GPIIb/IIIa monoclonal antibodies, Abciximab, and YM337 significantly but partially inhibited aggregate formation, but the anti-von Willebrand factor monoclonal antibody NMC-4 had no effect. The formation of small aggregates required extracellular calcium, but flavocetin-A did not elevate cytosolic calcium. These results suggest that flavocetin-A binds to intact platelets, initiating platelet responses and inducing platelet aggregate formation by cross-linking platelets. Consequently, flavocetin-A may be a useful tool to study the mechanism of GPIb-mediated platelet activation and the structure-function relationships of GPIb.