Determinants of donor platelet variability when testing for heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia is a common immune-mediated drug reaction that can be complicated by life-threatening arterial thrombosis. The diagnosis can be confirmed by demonstrating heparin-dependent release of radiolabeled serotonin from washed normal platelets in the presence of patient serum. However, certain serum samples from these patients produce 14C-serotonin release from some but not other normal donor platelets. We investigated this problem of donor platelet variability by studying the reactivities of 10 serum samples from patients with heparin-induced thrombocytopenia with platelets from 10 normal donors (100 serum and platelet combinations). We observed a marked variability in reactivity for patient serum and platelets from normal donors; this initially appeared random. However, closer examination indicated that the reactivities varied hierarchically. Because heparin-induced thrombocytopenia is caused by binding of heparin-dependent IgG to platelet Fc receptors, we examined whether platelet Fc receptor number or function explained the variability in platelet reactivity. We observed that platelet Fc receptor function, as measured by platelet release associated with heat-aggregated IgG, was highly correlated with platelet reactivity to heparin-induced thrombocytopenia serum samples. No significant correlation, however, was found between Fc receptor number and platelet response. Reaction of murine monoclonal antibodies that activate human platelets by means of the platelet Fc receptors was not predictive of platelet reactivity to heparin-induced thrombocytopenia serum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of Platelet Membrane Receptors with von Willebrand Factor, Ristocetin, and the Fc Region of Immunoglobulin G

The agglutination of human platelets by ristocetin and von Willebrand factor was inhibited by aggregated immunoglobulin (Ig)G and by Fc fragments of IgG, but not by Fab, F(ab')(2) or pFc fragments of IgG. Because this inhibition occurred with formalin-fixed platelets as well as with normal platelets, a generalized aggregation of fluid membrane components by Fc fragments was not responsible for this inhibition of ristocetin and von Willebrand factor-induced agglutination. Reciprocal inhibition of platelet Fc receptors was produced by prior incubation of platelets with von Willebrand factor and ristocetin. Sucrose density gradient ultracentrifugation studies demonstrated that aggregated IgG did not form fluid-phase complexes with von Willebrand factor and ristocetin. Furthermore, passage of von Willebrand factor and ristocetin through a column of immobilized heat-aggregated IgG did not alter platelet agglutinating activity which indicates that aggregated IgG did not inactivate von Willebrand factor or ristocetin. Thus, it was likely that the IgG-mediated interference with platelet agglutination by ristocetin and von Willebrand factor did not occur in the fluid phase but at the platelet surface. These studies suggest that the platelet membrane Fc receptor may be either a part of, or sterically related to, the membrane glycoprotein I complex that interacts with von Willebrand factor, and that occupation of one of these surface components blocks the availability of the other.     

Release of serotonin from human platelets induced by aggregated immunoglobulins of different classes and subclasses

The ability of human myeloma proteins of different classes and subclasses and of macroglobulins (all aggregated with bis-diazotized benzidine or heat) to aggregate washed human platelets and release [(3)H]-serotonin from the platelets was investigated and compared with the activity of normal IgG and tetanus-antitetanus IgG antigen-antibody complexes. Aggregated IgG1, IgG2, IgG3, IgG4, and normal IgG complexes all aggregated platelets and caused release of serotonin to similar extents. In contrast, IgA1, IgA2, IgD, and IgE myeloma proteins as well as IgM macroglobulins were completely inactive in this respect. Approximately 50% of the actvity remained in aggregated, mildly reduced and alkylated IgG myeloma proteins and their Fc fragments, whereas aggregated F(ab')(2) fragments were completely inactive. Addition of fresh serum inhibited the release of serotonin caused by aggregated IgG1 and IgG3 proteins and normal IgG antigen-antibody complexes by about 50% but had no effect upon the release of serotonin obtained with IgG2 and IgG4 proteins. This inhibition appeared to be mediated by complement. The release of serotonin was not accompanied by liberation of the cytoplasmic enzyme lactic dehydrogenase, indicating that no significant lysis of the platelets occurred. Addition of neutrophils did not enhance the serotonin release.     

Evidence that a 210,000-molecular-weight glycoprotein (GP 210) serves as a platelet Fc receptor.

We previously identified a 210,000-mol-wt platelet glycoprotein (GP 210) that is missing from Bernard-Soulier platelets, and found that an antibody against GP 210 inhibits ristocetin-induced platelet agglutination. We now show by immunoblotting that GP 210 binds heat-aggregated rabbit and human IgG, as well as keyhole limpet hemocyanin (KLH)-anti-KLH and ovalbumin (OA)-anti-OA immune complexes. Immune complex binding to GP 210 was preserved on chymotrypsin-treated platelets that lacked glycoprotein Ib (GP Ib). In contrast, ristocetin-induced platelet agglutination resulted in disappearance of immunologically detectable GP 210 and loss of immune complex binding, even though GP Ib remained intact. Purified Fc fragments inhibited binding of anti-GP 210 antibody to intact platelets and to GP 210 on immunoblots. The Fc fragments also blocked immune complex binding to GP 210. Conversely, anti-GP 210 antiserum and F(ab)2 fragments inhibited binding of fluorescein-labeled Fc fragments to intact platelets. We conclude that GP 210 functions as a platelet Fc receptor.     

Staphylococci-induced Human Platelet Injury Mediated by Protein A and Immunoglobulin G Fc Fragment Receptor

Bloodstream infections with staphylococci are accompanied by thromboembolic complications. We have studied the mechanism of the interaction of staphylococci with human blood platelets.Staphylococci that possess protein A, a bacterial receptor for the Fc fragment of immunoglobulin G (IgG), caused aggregation of human platelets in whole plasma accompanied by release of [(3)H]serotonin. These reactions were time and concentration dependent, requiring two or more staphylococci per platelet to give maximal response within 5 min. The interaction between staphylococci and platelets required the presence of cell wall-bound protein A and of IgG with an intact Fc fragment. It did not require an intact complement system. Cell wall-bound protein A (solid phase) was capable of aggregating human platelets in whole plasma. In contrast, free, solubilized protein A (fluid phase) did not cause measurable aggregation, and release of [(3)H]serotonin was reduced. An excess of free, solubilized protein A blocked aggregation of human platelets induced by staphylococci in whole plasma.The role of the Fc fragment of IgG in the staphylococci-human platelet interaction was demonstrated by an experiment in which free, isolated Fc fragment blocked aggregation of platelets in whole plasma induced by staphylococci. Furthermore, binding of (125)I-protein A to human platelets was demonstrated in the presence of complete IgG with intact Fc fragment but not in the presence of the F(ab)(2) fragment. Binding of the protein A-IgG complex to the human platelet Fc receptor was paralleled by the release of [(3)H]serotonin.These results represent a novel example of the interaction of two phylogenetically different Fc receptors, one on prokaryotic staphylococci and the other on human platelets. Their common ligand, IgG, is amplified by one Fc receptor (protein A) to react with another Fc receptor present on human platelets, which results in membrane-mediated aggregation and release reaction occurring in whole plasma. This mechanism can be of significance in the pathomechanism of thromboembolic complications at the site(s) of intravascular staphylococcal infection.     

Human platelet IgG Fc receptor FcγRIIA in immunity and thrombosis

Beyond their prominent role in hemostasis and thrombosis, platelets are increasingly recognized as having immunologic functions. Supporting this, human platelets express FcγRIIA (CD32a), a low‐affinity Fc receptor (FcR) for the constant region of IgG that recognizes immune complexes (ICs) and IgG‐opsonized cells with high avidity. In leukocytes, FcγRIIA engagement initiates strong effector functions that are key for immune and inflammatory responses, including cytokine release, antibody‐dependent cell‐mediated killing of pathogens, and internalization of ICs. However, the physiologic relevance of platelet‐expressed FcγRIIA has received little attention in previous reviews on FcRs. This article summarizes and discusses the available information on human platelet FcγRIIA. The importance of this receptor in heparin‐induced thrombocytopenia, a prothrombotic adverse drug effect, is well documented. However, studies demonstrating platelet activation by IgG‐opsonized bacteria point to the physiologic relevance of platelet FcγRIIA in immunity. In this context, platelet activation and secretion may facilitate both a direct antimicrobial function of platelets and crosstalk with other immune cells. Additionally, a role for platelet FcγRIIA in IgG‐independent hemostasis and physiologic thrombosis, by means of amplifying integrin α_IIbβ₃ outside‐in signaling, has also been proposed. Nonetheless, the thrombotic complications found in some infective and autoimmune diseases may result from unbalanced FcγRIIA‐mediated platelet aggregation. Moreover, FcγRIIA is not expressed in mice, and thrombocytopenia and/or thrombotic events found after drug administration can only be recapitulated by the use of human FcγRIIA‐transgenic mice. Altogether, the available data support a functional role for platelet FcγRIIA in health and disease, and emphasize the need for further investigation of this receptor.     

Human platelet Fc receptor for immunoglobulin G. Identification as a 40,000-molecular-weight membrane protein shared by monocytes

We have recently shown that human monocytes and U937 cells possess two molecular classes of Fc gamma receptor. One, a 72,000-mol-wt sialoglycoprotein, has high affinity for certain subclasses of human and murine monomeric IgG. The other is a 40,000-mol-wt protein (p40) with low affinity for monomeric IgG but with the capacity to bind IgG aggregates or IgG-coated particles. In the present study, a 40,000-mol-wt single chain protein, apparently identical to p40 from U937 cells, was isolated from surface-radioiodinated human platelets by affinity purification using a murine IgG2b monoclonal antibody to p40. This 40,000-mol-wt protein was not seen when control IgG2b or unrelated murine monoclonal antibodies were employed in place of anti-p40. The same 40,000-mol-wt protein was also recovered from an IgG-Sepharose affinity adsorbent, but not from ovalbumin-or myoglobin-Sepharose. The 72,000-mol-wt Fc gamma receptor of monocytes was not identified on platelets. Monoclonal anti-p40 and Fab fragments derived from this antibody blocked platelet aggregation by heat-aggregated human IgG, whereas a control murine IgG2b protein had little or no inhibitory effect at 500-1,000-fold higher concentrations. A murine IgG1 monoclonal antibody, reactive with an unrelated platelet-specific membrane antigen, did not inhibit platelet responses to aggregated IgG. Anti-p40 did not affect platelet aggregation by thrombin, collagen, or fibrinogen plus ADP. Although anti-p40 did not directly aggregate platelets in the concentrations employed, cross-linking with F(ab')2 goat anti-murine Ig induced apyrase-sensitive aggregation of anti-p40-treated platelets. This indicates that p40 possesses transmembrane linkage for platelet activation and secretion. These observations strongly suggest that this newly recognized 40,000-mol-wt platelet membrane protein serves as an Fc gamma receptor.     

BINDING OF SOLUBLE IMMUNE COMPLEXES TO HUMAN LYMPHOBLASTOID CELLS : II. Use of Raji Cells to Detect Circulating Immune Complexes in Animal and Human Sera

Cells from a human lymphoblastoid cell line (Raji), with B-cell characteristics, and having receptors for human IgG Fc, C3b, and C3d, were used in an immunofluorescence test as in vitro detectors of immune complexes in animal and human sera. By this test, as little as 200–300 ng aggregated human gamma globulin or immune complexes per ml serum could be detected. The receptors for IgG Fc on the Raji cells were shown to be inefficient in detecting aggregated human gamma globulin and binding of aggregates to these receptors was inhibited by physiologic concentrations of 7S human IgG. Enhancement of aggregated human gamma globulin binding and binding of immune complexes formed in vitro to Raji cells was observed when the receptors for complement on these cells were used. By using the receptors for complement on Raji cells, circulating immune complexes were detected in rabbits with acute serum sickness, in mice with acute lymphocytic choriomeningitis virus infection, and in humans with immune complex type glomerulonephritis. The Raji cell test may be useful in detecting complement fixing immune complexes in different disease states, in monitoring circulating complexes in patients with immune complex diseases and in identifying the antigen(s) responsible for the induction of pathogenic immune complexes in humans and animals.     

Compromised ITAM-based platelet receptor function in a patient with immune thrombocytopenic purpura

Summary.  Background:  Receptors on platelets that contain immunoreceptor tyrosine-based activation motifs (ITAMs) include collagen receptor glycoprotein (GP) VI, and FcγRIIa, a low affinity receptor for immunoglobulin (Ig) G. Objectives:  We examined the function of GPVI and FcγRIIa in a patient diagnosed with immune thrombocytopenic purpura (ITP) who had unexplained pathological bruising despite normalization of the platelet count with treatment. Methods and Results:  Patient platelets aggregated normally in response to ADP, arachadonic acid and epinephrine, but not to GPVI agonists, collagen or collagen-related peptide, or to FcγRII-activating monoclonal antibody (mAb) 8.26, suggesting ITAM receptor dysfunction. Plasma contained an anti-GPVI antibody by MAIPA and aggregated normal platelets. Aggregating activity was partially (∼60%) blocked by FcγRIIa-blocking antibody, IV.3, and completely blocked by soluble GPVI ectodomain. Full-length GPVI on the patient platelet surface was reduced to ∼10% of normal levels, and a ∼10-kDa GPVI cytoplasmic tail remnant and cleaved FcγRIIa were detectable by western blot, indicating platelet receptor proteolysis. Plasma from the patient contained ∼150 ng mL⁻¹ soluble GPVI by ELISA (normal plasma, ∼15 ng mL⁻¹) and IgG purified from patient plasma caused FcγRIIa-mediated, EDTA-sensitive cleavage of both GPVI and FcγRIIa on normal platelets. C onclusions:  In ITP patients, platelet autoantibodies can curtail platelet receptor function. Platelet ITAM receptor dysfunction may contribute to the increased bleeding phenotype observed in some patients with ITP.     

Acquired Bernard-Soulier syndrome. Evidence for the role of a 210,000-molecular weight protein in the interaction of platelets with von Willebrand factor.

A patient with a lymphoproliferative disorder developed bleeding associated with a prolonged bleeding time and a selective defect of platelet aggregation in response to ristocetin. The patient's purified IgG was shown to inhibit aggregation of washed normal platelets by ristocetin and von Willebrand factor (F VIII:vWF). By Western blotting, it was shown that antibody bound specifically to an antigen of Mr 210,000 present on normal platelets but missing on platelets from patients with congenital Bernard-Soulier syndrome (BSS). Binding was effected by the F(ab)2 portion of the IgG, indicating the presence of an autoantibody rather than an immune complex. These results suggest that the 210,000-Mr protein is involved in the interaction of F VIII:vWF with platelets. Furthermore, we have demonstrated the apparent absence of an additional protein on congenital BSS platelets. Heat-aggregated IgG was also shown to bind to the 210,000-Mr protein, suggesting that this protein may function as an Fc receptor on platelets. The relationship of the 210,000-Mr protein to glycoprotein Ib and the precise role of this protein in the interaction of platelets with F VIII:vWF need to be characterized.     

IgG-Fc receptors and the clinical relevance of their polymorphisms.

Receptors for the Fc part of IgG form the bridge between immune complexes, antibodies and blood cells. Besides phagocytes, such as granulocytes and monocytes, also lymphocytes and platelets express members of the large family of IgG-Fc receptors or Fc gamma Rs. Three main classes of leukocyte Fc gamma Rs can be distinguished: Fc gamma RI, Fc gamma RII and Fc gamma RIII. Depending on the type of intracellular domain of the leukocyte Fc gamma Rs, interaction of a cell with an antibody-sensitized particle will induce either an activating signal (leading to phagocytosis, degranulation or toxic oxygen formation) or, in case of Fc gamma RIIb, an inhibiting signal. The inhibiting role of Fc gamma RIIb has long been thought to be only important for B-cell responses, however, recently it has been indicated that Fc gamma RIIb may have a much broader role in immune response, also regulating responses of phagocytes. In general, leukocyte Fc gamma Rs have high affinity for IgG1 and IgG3 subclasses. However, the affinity for the various IgG subclasses is influenced by receptor polymorphisms encoded by single nucleotide polymorphisms of the Fc gamma RIIa and Fc gamma RIIIa and Fc gamma RIIIb encoding genes. Because these subtle Fc gamma R changes determine the level of clearance of immune complexes, associations between Fc gamma R isoforms and disease risks have been investigated and also indicated as discussed in this review. Next to the leukocyte Fc gamma Rs, an IgG transport receptor, FcRn (neonatal Fc receptor) has been described. This receptor seems to be important for placental IgG transport and for IgG plasma level homeostasis. FcRn is expressed by endothelial cells and hepatocytes and functions in these cells as a receptor rescuing IgG from destruction, thereby increasing IgG half life. Also blood cells express FcRn and the exact role of FcRn in these cells is still to determined. Thus, although a lot is known, questions on the exact pattern of expression of the IgG receptors and their role in immune response still remain.     

The lymphocyte stimulation test: a sensitive technique for the detection of circulating platelet antibodies.

Platelet autoantibodies are unable to react in standard immunological reactions and this makes it difficult to detect their presence in the serum. The present work shows that peripheral blood lymphocytes from normal donors are stimulated in vitro by autologous platelets sensitized with sera from patients with different types of chronic thrombocytopenia. Comparison of the lymphocyte stimulation test with the platelet factor 3 (PF3) availability assay and the serotonin (5HT) release test demonstrated that the former method was the more sensitive one. Some sera from patients with SLE and thrombocytopenia also induced both a slight lymphocyte stimulation in the absence of platelets and a complement-dependent release of serotonin, probably due to the presence of immune complexes.     

Antibodies from patients with heparin-induced thrombocytopenia/thrombosis are specific for platelet factor 4 complexed with heparin or bound to endothelial cells.

Heparin-induced thrombocytopenia/thrombosis (HITP) is thought to be mediated by immunoglobulins that activate platelets in the presence of pharmacologic concentrations of heparin, but the molecular basis for this relatively common and often serious complication of heparin therapy has not been established. We found that plasma from each of 12 patients with HITP contained high titer (> or = 1:200) antibodies that reacted with immobilized complexes of heparin and platelet factor 4 (PF4), a heparin-binding protein contained in platelet alpha-granules. Recombinant human PF4 behaved similarly to PF4 isolated from platelets in this assay system. Complexes formed at an apparent heparin/PF4 molecular ratio of approximately 1:2 (fresh heparin) and approximately 1:12 (outdated heparin) were most effective in binding antibody. Immune complexes consisting of PF4, heparin, and antibody reacted with resting platelets; this interaction was inhibited by a monoclonal antibody specific for the Fc gamma RII receptor and by excess heparin. Human umbilical vein endothelial cells, known to express heparin-like glycosaminoglycan molecules on their surface, were recognized by antibody in the presence of PF4 alone; this reaction was inhibited by excess heparin, but not by anti-Fc gamma RII. Antibodies reactive with heparin/PF4 were not found in normal plasma, but IgG and IgM antibodies were detected at dilutions of 1:10 (IgG) and 1:50 (IgM) in 3 of 50 patients (6%) with other types of immune thrombocytopenia. These findings indicate that antibodies associated with HITP react with PF4 complexed with heparin in solution or with glycosaminoglycan molecules on the surface of endothelial cells and provide the basis for a new hypothesis to explain the development of thrombocytopenia with thrombosis or disseminated intravascular coagulation in patients sensitive to heparin.     

Enhancement of platelet response to immune complexes and IgG aggregates by lipid A-rich bacterial lipopolysaccharides

The effect of the common lipid moiety of bacterial LPS on secretion from washed human platelets has been studied. The lipid A-rich LPS of S. minnesota R595 and a lipid A preparation both potentiated platelet serotonin secretion in response to IgG aggregates or immune complexes up to 50-fold but had little effect in the absence of IgG. Lipid A has been shown to bind immune aggregates, raising the possibility that its mechanism of action involved effective enlargement or insolubilization of the aggregates. IgG aggregates of dimer to tetramer size were shown to be platelet simuli, equivalent on a weight basis to larger soluble aggregates. The effect of both sizes of aggregates on platelets were equally enhanced by the LPS, indicating that increased size of aggregates alone could not account for the effect of LPS. Similarly, because lipid A-rich LPS enhanced platelet response to already insoluble immune complexes, its mechanism of action cannot simply be insolubilization of immune aggregates. These LPS did not enhance platelet stimulation by antiplatelet antibody, monosodium urate crystals, or thrombin and only slightly enhanced stimulation by insoluble human skin collagen. This indicates some stimulus specificity in the ability of LPS to increase platelet secretion. The enhancement of cell response to immune complexes by the common lipid region of LPS may represent a mechanism for the diverse effects of LPS in vivo and in vitro.     

Receptor for immunoglobulin Fc on pathogenic but not on nonpathogenic protozoa of the Trypanosomatidae

Members of the Trypanosomatidae were studied for their ability to acquire host IgG through a possible Fc receptor. A simple rosette test was devised in which the different species and forms of protozoa were mixed with SRBC sensitized with subagglutinating does of IgG, IgM, and F (ab') 2 anti-SRBC, and the pelleted mixture was observed for the number of clumps (rosettes) formed between the parasites and SRBC. Rosettes were formed between parasites and SRBC sensitized with IgG but not with IgM or F(ab')2, indicating the presence of a receptor for IgG Fc. The specificity of this receptor for Fc was confirmed by inhibition experiments with normal rabbit aggregated gammaglobulins or with purified normal rabbit Fc. The receptor is sensitive to treatment with trypsin but regenerates after a short period of incubation (1 h), which indicates that it is synthesized by the parasite itself. Interesting was the observation that only pathogenic members of the Trypanosomatidae formed rosettes with sensitized SRBC. In none of the nonpathogenic forms studied could we demonstrate the Fc receptor. Also important was the finding that freshly isolated blood stream forms of Trypanosoma cruzi from infected mice did not form rosettes. However, after trypsinization, these forms clearly displayed the ability to do so, possibly indicating a previous acquisition of the host IgG by the parasites in the mouse blood stream. These findings point to a possible and important means of parasite evasion of the host immune response by masking their surface with host IgG.     

Isolation and Quantitation of the Platelet Membrane Glycoprotein Deficient in Thrombasthenia Using a Monoclonal Hybridoma Antibody

We used the hybridoma technique to characterize further the platelet glycoprotein abnormality in Glanzmann's thrombasthenia. Spleen cells from Balb/c mice immunized with human platelets were fused to mouse myeloma cell line Sp2/0-Ag14. Hybridoma lines producing a variety of antiplatelet antibodies were isolated by hypoxanthine-aminopterin-thymidine selection and cloned, and purified monoclonal IgG from six lines was prepared. One of these lines, 8aB5-9, produced an antibody, Tab, that binds to a protein on normal but not thrombasthenic platelets. We isolated this protein from Triton X-100 solubilized normal platelet membranes by affinity chromatography on Tab-Sepharose. As determined by SDS polyacrylamide gel electrophoresis, the isolated protein is a complex of glycoproteins IIb and IIIa, because the two subunits comigrate with glycoproteins IIb and IIIa of whole platelets and show identical changes in mobility after disulfide bond reduction. We prepared (125)I-Tab to determine the number of glycoprotein IIb-IIIa complexes on normal and thrombasthenic platelets by a direct binding assay. Platelets from 17 normal donors bound 39,000+/-4,600 (SD) Tab molecules/platelet. Platelets from four patients with thrombasthenia lacked Tab binding sites (<5%). Five obligate and four presumed heterozygotes for thrombasthenia bound 24,500+/-5,800 Tab molecules/platelet. The platelet alloantigen, Pl(Al), is not that recognized by Tab, because platelets from three Pl(Al)-negative subjects bound Tab normally. Studies with the Tab antibody have (a) enabled quantitation of the number of glycoprotein IIb-IIIa complexes on normal platelet membranes, (b) demonstrated that thrombasthenic homozygotes lack and heterozygotes have a partial deficiency of this complex, and (c) made possible the isolation of this membrane protein which may be required for normal platelet aggregation and clot retraction.     

Platelet FcγRIIA HIS131ARG polymorphism and platelet function: antibodies to platelet-bound fibrinogen induce platelet activation

Summary.  The His131Arg polymorphism of platelet FcγRIIA affects the binding affinity of certain IgG subclasses. The Arg131 allele has been associated with (auto)immune thrombocytopenia and heparin-induced thrombocytopenia in some studies. Because FcγRIIA can transmit platelet activation signals, we studied platelet responsiveness from 73 healthy donors to determine if this polymorphism modulated platelet function. Platelet function was studied by agonist and shear-induced activation, and standard aggregation. FcγRIIA was genotyped by allele-specific PCR. Compared with His131, the Arg131 allele was associated with significantly greater binding of activation-dependent antibodies. This effect was most prominent for the receptor-induced binding site (RIBS) antibodies F26 ( P  < 0.0001) and RIBS1 ( P  = 0.0057), and the ligand-induced binding site antibody LIBS1 ( P  = 0.0367). Unexpectedly, Arg131-positive platelets did not show greater fibrinogen binding, platelet aggregation or shear-induced platelet activation. We considered whether enhanced Fc binding and FcγRIIA cross-linking were responsible for those discrepancies. The increased binding of the two RIBS antibodies to the Arg131 isoform was abolished by blocking FcγRIIA, and the FcγRIIA genotype effect on F26 IgG binding was lost when F26 F(ab')₂ fragments were used. Furthermore, intact F26 and RIBS1 IgG directly and specifically induced P-selectin expression, and this effect was greatest in Arg131-positive platelets. We concluded that (a) the His131Arg polymorphism of FcγRIIA does not affect intrinsic platelet reactivity; (b) RIBS antibodies are able to cross-link FcγRIIA and activate platelets, and this activation has a modest effect on Arg131 platelets; and (c) flow cytometric based platelet assays may need to compensate for this FcγRIIA His131Arg effect on platelet activation.     

Inhibition of lymphocyte mitogenesis by immobilized antigen-antibody complexes

Mouse spleen cells, cultured on surfaces coated with antigen-antibody complexes, are inhibited from responding to the B-cell mitogens, lipopolysaccharide, lipid A, Pneumococcal polysaccharide SIII, and poly I:C. The response to the T-cell mitogen, concanavalin A, is also substantially inhibited by immobilized antigen-antibody complexes, but specific inhibition of the response to phytohemagglutinin is minimal. Control experiments showed that immobilized complexes prepared from IgG F(ab')2 fragments and IgA antibodies (both of which fail to bind to Fc receptors when complexed to antigen) did not show significant inhibitory activity when compared with the inhibition observed with complexes prepared from whole IgG. Suspensions of antigen-antibody complexes prepared from the same antigen and intact IgG antibody did not inhibit mitogenesis. None of the mitogens used could be demonstrated to compete with the binding of aggregated immunoglobulin to the B-cell Fc receptor. It appears that the interaction of Fc receptor-bearing lymphocytes and/or macrophages with immobilized complexes prevents lymphocyte activation by mitogens. It is suggested that the mechanism(s) involved may be relevant to antibody feedback control of the humoral immune response.     

Pneumococcus-induced serotonin release from human platelets. Identification of the participating plasma/serum factor as immunoglobulin.

Pneumococcus-induced serotonin release from human platelets is greatly facilitated by a factor present in normal human plasma and serum. We have identified this factor as immunoglobulin by: (a) removing if from plasma and serum with solid phase antiFab antibody; (b) demonstrating its absence from the serum of an individual with severe immunoglobulin deficiency; and, (c) showing its presence in IgG preparations isolated from normal individuals. Evidence suggesting that the release reaction is triggered by pneumococcal antigen-antibody complexes rather than by nonimmune interaction of immunoglobulin with pneumococcus includes: (a) the failure of isolated IgG myeloma proteins to support release; (b) a lack of correlation between IgG concentration and "releasing factor activity" in normal human sera; (c) the identification of a normal serum that supports release by types II and III pneumococci but not type VII; and, (d) the ability of most normal sera to support release by pneumococca serotypes II and VII, though these types have not shown nonimmune reactivity with the Fc portion of the IgG molecule. The ability of antibodies present in normal serum to support pneumococcus-induced serotonin release suggests that the thrombocytopenia seen in pneumococcal infection may at least in part be caused by pneumococcal antigen-antibody complexes.     

Binding of soluble immune complexes to human lymphoblastoid cells. I. Characterization of receptors for IgG Fc and complement and description of the binding mechanism

In the present work we studied the expression of membrane-bound Ig (MBIg) as well as receptors for IgG Fc and complement on nine human lymphoblastoid cell lines. When MBIg and receptors for IgG Fc were compared, four categories of cell lines could be distinguished: (a) cell lines having both MBIg and receptors for IgG Fc, (b) cell lines having MBIg but lacking receptors for IgG Fc, (c) cell lines lacking MBIg but having receptors for IgG Fc, and (d) cell lines lacking both MBIg and receptors for IgG Fc. Two types of receptors for complement could be detected on the cell lines studied, one for C3-C3b and one for C3d. When sensitized red cells carrying C3b or C3d were used for rosette tests, three categories of cell lines could be distinguished: (a) cell lines having receptors for C3b and C3d, (b) cell lines having receptors only for C3d and (c) cell lines lacking both receptors. However, when a more sensitive immunofluorescent method was used instead of the rosette technique, it was found that cell lines unable to form rosettes with EAC1423b^hu were able to bind soluble C3 or C3b which indicated the presence of these receptors on the cell surface. Inhibition experiments showed that receptors for C3-C3b and receptors for C3d are distinct and that receptors for C3-C3b and C3d are different from receptors for IgG Fc. A cell line (Raji) without MBIg but with receptors for IgG Fc, C3-C3b, and C3d was selected for use in studying the binding mechanism of soluble immune complexes to cell surface membrane. Aggregated human gamma globulin was used in place of immune complexes. Immune complexes containing complement bind to Raji cells only via receptors for complement, namely receptors for C3-C3b and C3d. Binding of immune complexes containing complement to cells is much greater than that of complexes without complement. Immune complexes bound to cells via receptors for complement can be partially released from the cell surface by addition of normal human serum as well as isolated human C3 or C3b. We postulate that such release is due to competition of immune complex bound C3b and free C3 or C3b for the receptors on Raji cells.