Response of human platelets to activating monoclonal antibodies: importance of Fc gamma RII (CD32) phenotype and level of expression.

Certain monoclonal antibodies (MoAbs) specific for platelet membrane glycoproteins are known to be capable of activating platelets, and it is generally thought that platelets from normal subjects are equally susceptible to stimulation by such MoAbs. We found that platelets from 20 normal donors varied significantly in their sensitivity to three IgG1 murine MoAbs specific for membrane glycoproteins CD9, GPIV (CD36), and the GPIIb/IIIa complex (CD41), respectively. The response of platelets to these MoAbs was blocked by prior addition of MoAb IV.3 specific for the Fc gamma RII receptor, indicating that activation was Fc receptor mediated. Platelets that responded poorly to these MoAbs failed to bind the MoAb 41H.16, specific for the "responder" form of Fc gamma RII, but platelets that responded well reacted with this MoAb. The average number of Fc gamma RII receptors on platelets from "responders" and "non-responders" was approximately the same. However, the number of Fc gamma RII receptors expressed influenced sensitivity of a subgroup of "responder" platelets to the anti-CD41 MoAb. These platelets were judged on the basis of MoAb binding studies to be heterozygous for the two alleles of Fc gamma RIIA. In contrast to their varying sensitivity to IgG1 MoAbs, members of the platelet panel responded equally well to 50H.19, an IgG2a MoAb specific for CD9, and these responses could not be blocked by MoAb IV.3 in the presence of plasma. This appears to be because of dual actions of 50H.19 on platelets: one FcR-dependent and the other complement-dependent. Our findings confirm previous reports that certain IgG1 MoAbs activate platelets through binding of their Fc domains to Fc gamma RII receptors and demonstrate that this response is influenced both by Fc gamma RII phenotype and (in the case of the anti-CD41 MoAb) by the number of Fc gamma RII receptors expressed. The failure of nonresponding platelets to bind detectable amounts of MoAb 41H.16, which is thought to recognize all Fc gamma RII receptors except for one allele of the Fc gamma RIIA gene, is consistent with the possibility that Fc gamma RIIA gene products, but not Fc gamma RIIB or Fc gamma RIIC gene products, are expressed on platelets.     

Interaction of porcine von Willebrand factor with the platelet glycoproteins Ib and IIb/IIIa complex

Porcine von Willebrand factor (PvWF) induces platelet aggregation which is thought to be responsible for the thrombocytopenia that occurs in haemophilic patients treated with commercial preparations of porcine factor VIII. This study demonstrates that such aggregation can be completely inhibited by a monoclonal antibody against human platelet glycoprotein GPIb and partially inhibited by an antibody directed against platelet GPIIb/IIIa. The interaction of PvWF with GPIb is also demonstrated by the inhibitory effect of purified glycocalycin on aggregation. The binding site of PvWF to GPIb is very close to that of human vWF, since a recombinant peptide blocks the binding of both molecules to GPIb. When platelets are incubated with PvWF, the GPIIb/IIIa receptor is activated and binds fibrinogen. PvWF also binds to GPIIb/IIIa when platelets are stimulated with thrombin, suggesting that the molecule has the same RGD sequence as other adhesive proteins (human vWF, fibrinogen, fibronectin and vitronectin). These findings identify the dual mechanisms responsible for in vivo platelet aggregation induced by PvWF, i.e. binding to GPIb and activation of the GPIIb/IIIa receptor.     

Differential redistribution of platelet glycoproteins Ib and IIb-IIIa after plasmin stimulation.

The subcellular localization of the platelet membrane receptors glycoproteins (GP) Ib and IIb/IIIa [corrected] has been studied within resting platelets by a combination of biochemical and cytochemical techniques. While both GPIb and GPIIb/IIIa are localized within the plasma membrane and surface-connected canalicular system (SCCS) membranes, only GPIIb/IIIa is present within the internal face of alpha-granular membranes. Previous studies demonstrated that plasmin can induce platelet stimulation and also decrease ristocetin-induced platelet aggregation; it was suggested that this was because of GPIb degradation by plasmin. In this study, the respective localizations of both GPIb and GPIIb/IIIa were visualized during in vitro plasmin stimulation of platelets. Generally, plasmin induced shape change, pseudopod formation, organelle centralization either with or without alpha-granule release depending on the conditions of stimulation. Plasmin treatment of platelets at 37 degrees C resulted in the disappearance of GPIb from the cell surface and its subsequent redistribution into the channels and vesicles of the SCCS with no significant modification of GPIIb/IIIa remaining on the plasma membrane. Within degranulated platelets, GPIIb/IIIa was expressed on the plasma membrane and within membranes of large vacuoles containing the alpha-granule proteins. GPIb was virtually absent from these structures and mainly restricted to the SCCS. Addition of cytochalasin D inhibited the migration of GPIb to the SCCS. Biochemical measurements confirmed that no important hydrolysis of GPIb had occurred because only very little amounts of glycocalicin were generated during the reaction. In conclusion, in plasmin-treated platelets GPIIb/IIIa is externalized to the plasma membrane while GPIb is internalized into the SCCS. Although previous studies have suggested that plasmin degrades GPIb, the reduction in ristocetin-induced aggregation may be explained by its apparent redistribution within the membranes of the SCCS.     

Antiplatelet antibody predicts platelet desialylation and apoptosis in immune thrombocytopenia

Immune thrombocytopenia (ITP) is a bleeding disorder caused by dysregulated B- and T- cell functions, which lead to platelet destruction. A well-recognized mechanism of ITP pathogenesis involves anti-platelet and anti-megakaryocyte antibodies recognizing membrane glycoprotein (GP) complexes, mainly GPIb/IX and GPIIb/IIIa. In addition to the current view of phagocytosis of the opsonised platelets by splenic and hepatic macrophages via their Fc γ receptors, antibody-induced platelet desialylation and apoptosis have also been reported to contribute to ITP pathogenesis. Nevertheless, the relationship between the specific thrombocytopenic mechanisms and various types of anti-platelet antibodies has not been established. In order to ascertain such association, we used sera from 61 ITP patients and assessed the capacity of anti-platelet antibodies to induce neuraminidase 1 (NEU1) surface expression, RCA-1 lectin binding and loss of mitochondrial inner membrane potential on donors’ platelets. Sera from ITP patients with detectable antibodies caused significant platelet desialylation and apoptosis. Anti-GPIIb/IIIa antibodies appeared more capable of causing NEU1 surface translocation while anti-GPIb/IX complex antibodies resulted in a higher degree of platelet apoptosis. In ITP patients with anti-GPIIb/IIIa antibodies, both desialylation and apoptosis were dependent on FcγRIIa signaling rather than platelet activation. Finally, we confirmed in a murine model of ITP that destruction of human platelets induced by anti-GPIIb/IIIa antibodies can be prevented with the NEU1 inhibitor oseltamivir. A collaborative clinical trial is warranted to investigate the utility of oseltamivir in the treatment of ITP.     

Effect of aspirin on platelet-von Willebrand factor surface expression on thrombin and ADP-stimulated platelets

Platelets contain a pool of endogenous platelet-von Willebrand factor (vWF) that becomes expressed on the platelet surface when platelets are stimulated by a variety of agonists. Maximal platelet-vWF expression occurs in concert with platelet alpha-granule secretion. Aspirin (ASA) is known to impair platelet activation and alpha-granule secretion by irreversible inhibition of platelet cyclo-oxygenase. We studied native and ASA-treated platelets for their ability to mobilize and to express platelet-vWF in response to adenosine diphosphate (ADP) or thrombin. We found that each agonist was effective in promoting increased platelet- vWF surface expression on native and ASA-treated platelets. ASA-treated platelets responded identically to native platelets to low (0.01 U/mL) and high (1.0 U/mL) concentrations of thrombin, while the ADP-induced increase in ASA-treated platelets was only 50% to 60% of that for control platelets. Measurement of secreted platelet-vWF and beta- thromboglobulin indicated that the increase seen with ADP was largely independent of alpha-granule secretion. Using monoclonal antibodies (MoAbs) against the platelet glycoproteins (GP) IIb/IIIa and Ib (MoAbs 10E5 and 6D1, respectively), we demonstrated that the ADP-induced increase in platelet-vWF expression on control platelets primarily involved the binding of secreted platelet-vWF to the platelet GPIIb/IIIa. In contrast, the increase in platelet-vWF that occurred following ADP stimulation of ASA-treated platelets was largely insensitive to GPIIb/IIIa blockade. No effect of GPIb blockade in platelet-vWf expression was noted for either control or ASA-treated platelets. When platelet shape change was prevented by the addition of cytochalasin D, ADP-induced platelet-vWf surface expression on ASA- treated platelets was reduced by more than 80%. Our data indicate that platelets in which the cyclooxygenase pathway is blocked by the action of aspirin can increase surface expression of platelet-vWf as a consequence of platelet shape change. We speculate that this process exposes platelet-vWf bound to GPIIb/IIIa, or possibly GPIb, within the surface connected canalicular system.     

A new congenital platelet abnormality characterized by spontaneous platelet aggregation, enhanced von Willebrand factor platelet interaction, and the presence of all von Willebrand factor multimers in plasma

A case is reported of a 49-year-old woman with a mild bleeding tendency. Her bleeding time, platelet count and size, plasma ristocetin cofactor activity, von Willebrand factor (vWF) antigen, and vWF multimeric pattern are all within normal limits. Spontaneous platelet aggregation is observed when citrated platelet-rich plasma (PRP) is stirred in an aggregometer cuvette. This aggregation is completely is only slightly diminished by an antiglycoprotein (GP) IIb/IIIa or by an anti GPIb monoclonal antibody. The patient's PRP shows increased sensitivity to ristocetin. The distinct feature of this patient, also present in two family members studied, is that platelet aggregation is initiated by purified vWF in the absence of any other agonist. The vWF- induced platelet aggregation is abolished by anti-GPIb and anti- GPIIb/IIIa monoclonal antibodies and by EDTA (5 mmol/L). Apyrase inhibits the second wave of aggregation. Patient's platelets in PRP are four to six times more reactive to asialo vWF-induced platelet aggregation than normal platelets. The amount of radiolabeled vWF bound to platelets in the presence of either low concentration of ristocetin or asialo vWF was increased 30% compared with normal. The patient's platelet GPIb was analyzed by SDS page and immunoblotting and by binding studies with anti-GPIb monoclonal antibodies showed one band with slightly increased migration pattern and a normal number of GPIb molecules. Unlike the previously reported patients with pseudo or platelet-type von Willebrand disease, this patient has normal vWF parameters.     

Influences of antiplatelet autoantibodies on platelet function in immune thrombocytopenic purpura

We investigated the characteristics of the antiplatelet autoantibodies in 60 patients with ITP. Using flow cytometry, the binding of monoclonal antibodies to the platelet glycoprotein (GP) IIb/IIIa complex and to GPIb was examined in these patients. The extent of binding was decreased in 15 patients (anti-GPIIb/IIIa in 12 patients and both anti-GPIIb/IIIa and anti-GPIb in 3 patients). Western blotting revealed that 10 of these 15 patients had either anti-GPIIb or anti-GPIIIa and 2 had anti-GPIb autoantibodies, ADP-induced aggregation of normal platelets was inhibited by autoantibodies in 12 of 60 patients, and 11 of these had anti-GPIIb/IIIa antibodies. Ristocetin-induced aggregation was inhibited in 4 of these patients, and 2 with prominent inhibition had anti-GPIb antibodies. There was a significant relationship between platelet-associated IgG value and ATP secretion. These results suggest that some antiplatelet autoantibodies can affect platelet function and thus have an influence on the pathophysiology of ITP.     

Increased platelet sensitivity to ristocetin is predicted by the binding characteristics of a GPIb/IX determinant

Platelets from patients with platelet-type von Willebrand disease (vWD) were used as immunogens for the production of murine monoclonal antibodies (MoAbs). One such MoAb, C-34, inhibited ristocetin-induced aggregation of patient or normal platelets, but not aggregation induced by other aggregating agents. As demonstrated by crossed-immunoelectrophoresis, C-34 recognized an epitope within the GPIb/IX complex. In indirect immunofluorescence studies on fresh platelets, the ratio of any of four different anti-GPIb MoAbs to one another was near unity (0.88-1.14) both for normals and for patients. In contrast, the ratio of the binding of C-34 to such a MoAb (AP-1) was 0.31 +/- 0.02 (means +/- SE) for normal platelets and significantly increased to 0.54 +/- 0.01 for patient platelets (P less than 0.001). In NP-40 lysates of 3H-labelled platelets, saturating concentrations of C-34 produced much fainter bands than did AS-2 or other anti-GPIb MoAbs in the GPIb and GPIX regions. In contrast to the other anti-GPIb MoAbs, C-34 did not bind to the purified 125I-labelled glycocalicin fragment of GPIb, to the glycocalicin derivative identified by crossed-immunoelectrophoresis, or to the amino-terminal approximately 40 kDa portion of GPIb alpha cleaved from 3H-labelled platelets by trypsin. C-34 appears to be the first MoAb that is quantitatively informative in identifying the abnormal platelets in platelet-type vWD. The observed differences between the patient and normal platelets may reflect an abnormality in the primary structure of the GPIb/IX complex. Alternatively, patient platelets may have an abnormality of other structures near this region that impose less of a steric hindrance upon binding of antibody to the C-34 epitope.     

Identification of platelet glycoprotein IIb/IIIa as the major binding site for released platelet-von Willebrand factor [published erratum appears in Blood 1987 Jun;69(6):1789]

We studied the effects(s) of two monoclonal antibodies, 6D1 and 10E5 (directed against platelet glycoprotein Ib [GPIb] and the GPIIb/IIIa complex, respectively), and purified human plasma fibrinogen on the binding of released platelet-von Willebrand factor (vWf) to the platelet surface. Neither of the monoclonal antibodies nor fibrinogen had any effect on the amount of platelet-vWf expressed on unstimulated platelets or on the amount expressed on platelets stimulated in the absence of extracellular Ca++. However, the antibody directed against GPIIb/IIIa inhibited 72% of the thrombin-induced increase in the platelet-vWf bound to the platelet surface when platelets were stimulated in the presence of 5 mmol/L Ca++. The antibody against GPIb did not inhibit the surface expression of platelet-vWf on stimulated platelets in the presence of Ca++. Purified normal human fibrinogen inhibited the surface binding of platelet-vWf to thrombin-stimulated platelets to a degree similar to that observed with the monoclonal antibody directed against the GPIIb/IIIa complex. These data indicate that platelet-vWf released from platelets binds primarily to the GPIIb/IIIa complex at or near the plasma fibrinogen binding site.     

Spontaneous platelet aggregation during pregnancy in a patient with von Willebrand disease type IIB can be blocked by monoclonal antibodies to both platelet glycoproteins Ib and IIb/IIIa

Spontaneous platelet aggregation appeared in a patient with von Willebrand disease type IIB during the 37th week of pregnancy. This phenomenon was not associated with symptoms of thrombosis and the patient delivered by caesarean section with no complications. Her platelet-poor plasma (PPP) aggregated normal platelet-rich plasma (PRP) and washed platelets. Aggregation was inhibited by monoclonal antibodies with known specificity for the platelet receptors of von Willebrand factor (vWF), i.e. the glycoprotein Ib (GPIb) and the GPIIb/IIIa complex. A monoclonal antibody, which selectively inhibits the binding of vWF to the GPIIb/IIIa complex, did not block aggregation, suggesting that spontaneous aggregation is not dependent on the binding to GPIIb/IIIa of vWF from patient plasma. Aggregation induced by patient plasma could also be blocked either by two monoclonal antibodies raised against vWF or by a fragment derived from trypsin digestion of normal vWF which blocks the ristocetin-induced binding of normal vWF to platelets. These findings indicate that the spontaneous platelet aggregation in this patient results from the binding of her vWF to GPIb but is independent from the binding of her vWF to GPIIb/IIIa.     

Interaction of purified type IIB von Willebrand factor with the platelet membrane glycoprotein Ib induces fibrinogen binding to the glycoprotein IIb/IIIa complex and initiates aggregation.

Von Willebrand factor (vWF) was purified from the plasma of a patient with type IIB von Willebrand disease (vWF from such a patient, IIB vWF) who had a normal platelet count and showed no evidence of spontaneous platelet aggregation. Large multimers of IIB vWF were absent from purified preparations and from plasma. Ristocetin-induced platelet aggregation was enhanced by purified IIB vWF. The aggregation of washed normal platelets mixed with IIB vWF (0.4 microgram/ml) required lower amounts of ristocetin than the aggregation of normal platelets mixed with the same concentrations of normal vWF. Moreover, purified IIB vWF alone induced aggregation of platelet-rich plasma at concentrations as low as 10 micrograms of IIB vWF/ml in the absence of any other agonist. Aggregation was blocked by a monoclonal antibody against the platelet membrane glycoprotein, GPIb, as well as by an anti-GPIIb/IIIa antibody. Washed platelet suspensions were promptly aggregated by IIB vWF only when fibrinogen and CaCl2 were added to the mixture. Purified IIB vWF induces the binding of fibrinogen to platelets. Such binding was blocked by the anti-GPIb monoclonal antibody as well as by the anti-GPIIb/IIIa monoclonal antibody that inhibited aggregation. A second anti-GPIIb/IIIa antibody, which has the property of blocking vWF but not fibrinogen binding to platelets, blocked neither aggregation nor fibrinogen binding induced by IIB vWF. These studies demonstrate that platelet aggregation is triggered by the initial interaction of IIB vWF with GPIb which is followed by exposure of fibrinogen binding sites on GPIIb/IIIa. Fibrinogen binds to these sites and acts as a necessary cofactor for the aggregation response.     

Anti-GPIIb/IIIa (CD41) monoclonal antibody-induced platelet activation requires Fc receptor-dependent cell-cell interaction

We studied platelet activation by UR1, a murine IgG1 anti-CD41 mAb. Like thrombin and crosslinked anti-Fc gamma RII mAb IV3, UR1 initiates prompt aggregation and Ca2+ mobilization. UR1 F(ab')2 fragments failed to activate, yet inhibited UR1 IgG-mediated activation. UR1-induced activation was blocked by anti-Fc gamma RII mAb. High viscosity (15% dextran or Ficoll), which impedes cell-cell interaction, inhibited activation by UR1. Cell-cell interaction was confirmed by cell-mixing studies. UR1 binding to platelets of one pool was blocked with UR1 F(ab')2 allowing UR1 binding only to Fc gamma RII. IV3 Fab fragments blocked ligand binding to Fc gamma RII on platelets of a second pool; thus, UR1 could bind only its epitope. UR1 initiated an immediate [Ca2+]i increase in the intermixed pools at low ionic strength. These studies indicate that UR1 IgG binds CD41 on one platelet to form immune complexes which then crosslink and stimulate Fc gamma RII on nearby platelets. Two other anti-CD41 mAb, 6C9 and C17, and two anti-CD9 mAb, AG1 and mAb7, activated platelets in a UR1-like manner. We propose that platelet Fc gamma RII crosslinking that follows the interaction of IgG-opsonized platelets may be a common mechanism by which anti-platelet antibodies activate platelets.     

Heparin-induced thrombocytopenia: mechanism of interaction of the heparin-dependent antibody with platelets

The interaction of the heparin-dependent antibody with heparin and platelets has been studied using the sera and purified IgG of four patients with heparin-induced thrombocytopenia. Both normal platelets and Bernard-Soulier syndrome (BSS) platelets which lack glycoprotein (GP) Ib. GPV and GPIX, aggregated in response to patient serum or IgG, but only in the presence of heparin. A monoclonal antibody (Mab) against platelet Fc II receptor (IV.3) strongly inhibited the heparin-dependent aggregation of both normal and BSS platelets induced by patient sera/IgG. Inhibition by the anti-GPIb Mab (AK2) was variable and occurred only with normal platelets. Anti-GPIX Mab (FMC 25) was not inhibitory with either normal or BSS platelets. Similar results were obtained using 14C-serotonin release instead of platelet aggregation as a measure of platelet activation. These findings suggest that (1) the reaction of the heparin-dependent antibody with platelets and heparin is mediated by a Fc-dependent mechanism, (2) GPIb, GPV and GPIX are not involved in this reaction, and (3) the inhibitory effect of anti-GPIb Mab on normal platelets is due to steric interference consistent with the platelet Fc receptor being in close proximity to GPIb.     

Platelet membrane abnormalities in myeloproliferative disorders: decrease in glycoproteins Ib and IIb/IIIa complex is associated with deficient receptor function

The number and functional activity of membrane glycoproteins (GP) Ib and IIb/IIIa were investigated in platelets from 11 patients with myeloproliferative disorders (MPD). Three patients had essential thrombocythaemia, two had chronic myeloid leukaemia and six had polycythaemia vera. The numbers of GPIb and GPIIb/IIIa molecules were detected on the platelet surface using different 125I-labelled monoclonal antibodies. The functional properties of GPIb and GPIIb/IIIa were evaluated using purified 125I-labelled asialo von Willebrand factor (vWF) and purified 125I-labelled fibrinogen, respectively, in a binding assay. Binding of the anti-GPIIb/IIIa antibody was decreased by 40% in almost all patients studied and, when measured, it was accompanied by decreased fibrinogen binding to activated platelets. Binding of anti-GPIb antibodies to platelets was also slightly decreased or virtually the same in eight out of 11 patients. The decrease correlated with decreased binding of asialo vWF. The increased plasma glycocalicin levels, measured in four patients, depended on the high platelet count. Scatchard analysis revealed normal receptor binding affinity for all ligands tested in all but one patient. In this report we demonstrate that abnormalities in the concentrations of GPIIb/IIIa membrane proteins are commonly present in patients with MPD, while a decrease in GPIb concentration is also seen, although in fewer patients. These abnormalities are accompanied by a concurrent decrease in the respective receptor functions. These findings may explain part of the haemorrhagic tendency often encountered in MPD.     

Determination of platelet antigens and glycoproteins in the human fetus

The autosomal recessive transmission of Glanzmann's thrombasthenia (GT) and Bernard-Soulier syndrome (BSS), together with requests of families who already had children with these diseases, prompted us to investigate the feasibility of their antenatal diagnosis. The preliminary step leading to the early detection of GT or BSS was to characterize, in the normal human fetus, the platelet antigens and glycoproteins (GPs) and to define their normal amounts on the membrane surface. Blood samples from 32 fetuses between 18 to 26 weeks of gestation were collected by direct puncture of the umbilical vein using an ultrasound-guided needle. Polyclonal antibodies from human origin directed against PLA1, Leka antigens, and the GPIIb IIIa complex (IgGL), or murine monoclonal antibodies specific for GPIb (AN51, 6D1), GPIIIa (AP-3), or GPIIb IIIa (AP-2) were studied using platelet suspension immunofluorescence tests. The binding of each antibody was quantified using a cytofluorograph (Ortho 50H). PLA1 and Leka antigens were expressed in normal amounts on fetal platelets as early as 16 weeks of intrauterine life. The GPIIb IIIa complex quantified by polyclonal or monoclonal antibodies was in the same range in fetuses (IgGL = 427 +/- 23 AUF, AP-2 = 459.5 +/- 8.5; AP-3 = 536 +/- 14) and in adults (IgGL = 420 +/- 30; AP-2 = 498 +/- 11; AP-3 = 515 +/- 13). The platelet binding of antibodies that recognized GPIb was higher in fetuses (AN51 = 491.5 +/- 14; 6D1 = 479 +/- 15) than in adults (AN51 = 426.5 +/- 9; 6D1 = 449 +/- 8.7). These results suggest that immunological techniques can be applied as early as 18 weeks of gestation for the antenatal diagnosis of GT and BSS.     

Platelet activation mediated through membrane glycoproteins: involvement of tyrosine kinases

Fc gamma RII cross-linking and anti-CD9 mAbs included tyrosine phosphorylation of Fc gamma RII, Syk, and Lyn associated with Fc gamma RII in Fc gamma RII cross-linking but not in anti-CD9 mAb-induced platelet activation. We prepared various GST fusion proteins expressing one or two SH2 domains of Syk and evaluated the association between these GST fusion proteins with Fc gamma RII. Based on the results obtained from these experiments, we suggest that only one tyrosine residue in ITAM of Fc gamma RII is phosphorylated with anti-CD9 mAb and that both are phosphorylated with Fc gamma RII cross-linking. Platelet activation mediated by GPIb, the receptor for vWF, is also related with tyrosine phosphorylation. Botrocetin and vWF induced Syk activation. Shc was also rapidly and heavily tyrosine phosphorylated. Sre and Lyn, a 54-kDa tyrosine kinase, was associated with cytoskeletal proteins. When GPIb was immunoprecipitated with nonfunctional anti-GPIb mAbs after platelets were activated with vWF and botrocetin, an in vitro kinase assay revealed the transient association of a kinase activity with GPIb after platelet activation. Phosphoamino acid analysis of phosphorylated proteins in this assay demonstrated that only tyrosine residues but not serine or threonine were phosphorylated, suggesting that the kinase was indeed a tyrosine kinase.     

Investigation of the mechanisms of monoclonal antibody-induced platelet activation.

Antiplatelet antibodies can activate platelets causing platelet aggregation and the release reaction. However, the pathway of activation by these antibodies is unknown and several potential mechanisms are possible. In this report, we describe studies investigating potential pathways of platelet activation by IgG antibodies. We tested 16 different IgG monoclonal antibodies (MoAbs) against a variety of platelet surface components and found that six antibodies were capable of causing platelet aggregation and release. These included MoAbs against glycoprotein (GP) IIb/IIIa, CD9, GPIV, and two other not well-characterized platelet components. There was no relationship between the number of platelet binding sites and the ability of an MoAb to activate the platelets. By adding intact and F(ab')2 preparations of the MoAb to control or Fc receptor-blocked platelets, we found that in all instances the MoAbs initiated platelet activation via interacting with the platelet Fc receptors. Clustering of the platelet protein components using a secondary antibody did not cause activation. Studies into the pathway of Fc-dependent activation demonstrated that the MoAbs were capable of activating platelets by occupying Fc receptors on adjacent platelets (interplatelet activation), as well as on the same platelet (intraplatelet activation).     

Idiopathic autoimmune thrombocytopenia: evidence for redistribution of platelet antibodies into the circulation after immunoadsorption treatment

Platelet antibodies are detectable in only about 50% of patients with chronic autoimmune thrombocytopenia (AITP). We determined platelet antibodies against GPIa/IIa, GPIb/IX, GPIIb/IIIa, and GPV and reticulated platelets in three female patients with AITP, before and after immunoadsorption treatment. None of the three patients' sera contained platelet antibodies prior to treatment. Thereafter, anti-GPIIb/IIIa, anti-GPIb/IX (n = 3), and anti-GPV (n = 1) were detectable in the patients' sera. These antibody specificities were also found in the eluates from the immunoadsorption columns. Only one patient had elevated levels of reticulated platelets. Immunoadsorption treatment did not induce a sustained increase of platelet counts in any patient. Immunoadsorption treatment in AITP can induce redistribution of antibodies into the circulation.     

On the role of von Willebrand factor in promoting platelet adhesion to fibrin in flowing blood

Platelet adhesion to fibrin at high shear rates depends on both the glycoprotein (GP) IIb:IIIa complex and a secondary interaction between GPIb and von Willebrand factor (vWF). This alternative link between platelets and vWF in promoting platelet adhesion to fibrin has been examined in flowing whole blood with a rectangular perfusion chamber. Optimal adhesion required both platelets and vWF, as shown by the following observations. No binding of vWF could be detected when plasma was perfused over a fibrin surface or when coated fibrinogen was incubated with control plasma in an enzyme-linked immunosorbent assay. However, when platelets were present during perfusion, interactions between vWF and fibrin could be visualized with immunoelectron microscopy. Exposure of fibrin surfaces to normal plasma before perfusion with severe von Willebrand's disease blood did not compensate for the presence of plasma vWF necessary for adhesion. vWF mutants in which the GPIIb:IIIa binding site was mutated or the GPIb binding site was deleted showed that vWF only interacts with GPIb on platelets in supporting adhesion to fibrin and not with GPIIb:IIIa. Complementary results were obtained with specific monoclonal antibodies against vWF. Thus, vWF must first bind to platelets before it can interact with fibrin and promote platelet adhesion. Furthermore, only GPIb, but not GPIIb:IIIa is directly involved in this interaction of vWF with platelets.     

A quantitative study of human platelet membrane glycoproteins using immunocolloidal gold under the electron microscope

Two human platelet membrane glucoproteins (GP) and the quantitative changes of them induced by high shear stress (HSS) were studied using SZ-2 and SZ-21 monoclonal antibodies against GPIb and GPIIb/IIIa respectively by means of immunocolloidal gold technique with electron microscopy. Exposure to HSS (50 dynes/cm2, 5 min; 100 dynes/cm2, 3 min) resulted in the transformation of platelets from native smooth discs to activated spiny spheres followed by the centralization of organelles and lysis or aggregation of platelets. The results showed that resting platelets with few gold probes were turned into activated platelets by HSS with increased gold probes which represent the quantity of GPIb or GPIIb/IIIa. HSS also caused a specific platelet reaction--lysis. In the process of platelet lysis, platelet GPIIb/IIIa were increasingly condensed. On the contrary, GPIb reduced gradually. It suggests that the increase of GPIIb/IIIa under HSS may be one of the causes inducing platelet adhesion, release and aggregation at the opening of branched vessels, curved arteries, stenosed or partially occluded arteries due to local spasm.