Platelet aggregation induced by type IIb platelet von Willebrand factor

Platelet lysates from five patients with a form of type IIb von Willebrand's disease (vWd), associated with spontaneous platelet aggregation and thrombocytopenia, induced platelet aggregation of normal and other vWd's platelet-rich plasma (PRP). Platelet lysate from normals, type I or type IIa vWd did not cause platelet aggregation of normal PRP. When polyclonal monospecific antibodies directed against plasma von Willebrand factor (vWf) were incubated with the type IIb platelet lysate, they inhibited the platelet aggregation. Monoclonal antibodies directed against the glycoprotein (GP) Ib binding domain of plasma vWf incubated with the type IIb platelet lysate did not inhibit the platelet aggregation. Normal platelets suspended in afibrinogenaemic plasma did not aggregate when type IIb vWd platelet lysate was added. Normal platelets incubated with monoclonal antibodies directed against the fibrinogen and vWf binding site(s) on the GPIIb/IIIa were not aggregated by the type IIb platelet lysate. Bernard-Soulier PRP aggregated when type IIb vWd platelet lysate was added, while Glanzmann's thrombasthenic platelets did not. Peptides containing the RGDS sequence or the sequence of the carboxy terminal 15 amino acids of the gamma chain of fibrinogen inhibited the type IIb vWd platelet lysate-induced platelet aggregation. These data suggest that type IIb platelet vWf can cause platelet aggregation of PRP without the addition of any agonist. This interaction is different from that observed with the plasma vWf from these patients.     

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.     

Synthetic inhibitors of platelet glycoprotein IIb/IIIa in clinical development

Activation of the platelet glycoprotein (GP IIb/IIIa) receptor on the platelet surface is the final pathway of platelet aggregation, regardless of the initiating stimulus. Inhibitors of GP IIb/IIIa receptors include monoclonal antibodies (abciximab) against this receptor and peptidic and nonpeptidic synthetic specific receptor blockers. Abciximab exchanges between and binds to platelets for as long as 2 weeks, whereas synthetic GP IIb/IIIa inhibitors inhibit ex vivo platelet aggregation for only a few hours after the end of infusion, but some have the advantage of also being orally active. In the secondary prevention of atherothrombosis, large-scale trials were successfully conducted with aspirin, dipyridamole, ticlopidine, and clopidogrel. In the first large-scale trials with GP IIb/IIIa inhibitors, abciximab was investigated. In aggregate, synthetic GP IIb/IIIa inhibitors, combined with aspirin and heparin, were shown to reduce ischemic events in patients with high- and low-risk coronary intervention, stents, unstable angina, and non-Q-wave infarction. With short-term use of synthetic GP IIb/IIIa inhibitors, there is no suppression of clinical evident restenosis 6 months after the end of treatment. With the doses currently used, bleeding occurs more often with the synthetic GP IIb/IIIa inhibitors (used for 3 days) than with abciximab (used for 12 hours), but there are no direct comparisons between these drugs.     

Platelet Inhibition in Cardiovascular Disease Management: Aspirin and Beyond

Swine platelets are very similar to those of humans and are therefore relevant to cardiovascular research. The swine coronary circulation mimics the human circulation and is large enough to obtain multiple blood samples in survival experiments. In swine regional ischemia similar to the human condition is easily obtainable, which makes the porcine model an ideal choice to study coronary artery disease. However, little is known about the similarity between swine and human platelet surface antigens. We tested the hypothesis that certain swine platelet antigens could crossreact with antihuman antibodies. Using FITC-conjugated monoclonal murine antihuman platelet antibodies, surface antigen expression was determined for human and Yorkshire swine platelets. Expression of CD9 (p24), CD42B (Ib), CD41b (IIb), CD61 (IIIa), CD41a (IIb/IIIa), CD49b (VLA-2), CD62p, (P selectin), CD31 (PECAM-1), and CD51/CD61 (vitronectin) was measured by flow cytometry. Significant crossreactivity with human platelets was observed consistently for swine platelet GP Ib and GP IIIa. Crossreactivity of the swine GPIb and GP IIIa with the human receptors is evidence of receptor similarity between human and swine platelets. The implications of significant crossreactivity of these antigens and the lack of recognition of IIb/IIIa needs to be understood in cardiovascular research. Determining commercially available antihuman GP Ib and GP IIIa, rather than GP IIb/IIIa, would contribute to better elucidation of the effects of von Willebrand factor and the booming family of platelet inhibitors in the swine model of ischemia-reperfusion.     

Release of soluble CD40L from platelets is regulated by glycoprotein IIb/IIIa and actin polymerization

OBJECTIVES: The purpose of this study was to examine the effects of glycoprotein (GP) IIb/IIIa antagonists (abciximab, eptifibatide, and tirofiban) and other inhibitors on translocation of CD40L from intraplatelet stores to the platelet surface and on the release of soluble CD40L (sCD40L) from platelets. BACKGROUND: CD40L is a proinflammatory and prothrombotic ligand in the tumor necrosis factor family. METHODS: Platelet surface CD40L was measured by flow cytometry, and sCD40L was measured by enzyme-linked immunosorbent assay. RESULTS: Translocation of CD40L from intraplatelet stores to the platelet surface was not inhibited by GP IIb/IIIa antagonists. However, release of sCD40L from the surface of activated platelets was inhibited by GP IIb/IIIa antagonists in a dose-dependent manner, in concert with inhibition of PAC1 binding to platelets (a surrogate marker for fibrinogen binding). Release of sCD40L from activated platelets was also markedly reduced in Glanzmann platelets (deficient in GP IIb/IIIa). Ethylenediaminetetraacetic acid was an effective inhibitor of sCD40L release, but only when added before platelet activation. Both cytochalasin D (an inhibitor of actin polymerization) and GM6001 (an inhibitor of matrix metalloproteinases [MMPs]) inhibited the release of sCD40L from platelets when added before, as well as 3 min after, platelet activation. However, neither cytochalasin D nor GM6001 affected translocation of CD40L to the platelet surface. CONCLUSIONS: The GP IIb/IIIa antagonists inhibit release of sCD40L from activated platelets. Release of sCD40L from platelets is regulated, at least in part, by GP IIb/IIIa, actin polymerization, and an MMP inhibitor-sensitive pathway. In addition to their well-characterized inhibition of platelet aggregation, GP IIb/IIIa antagonists may obviate the proinflammatory and prothrombotic effects of sCD40L.     

Platelets' glycoproteins and their ligands in patients with intermittent claudication.

BACKGROUND: Platelet thrombi play critical role in pathogenesis of cardiovascular complications in atherosclerotic peripheral arterial disease (PAOD). The aim of this study was to evaluate the concentration of platelets GP IIb/IIIa, GP I b/IX and plasma levels of their ligands (fibrinogen and vWF) and their relation to other atherosclerotic risk factors in the patients with intermittent claudication secondary to PAOD. METHODS: Consecutive patients of the University Vascular Clinic were studied: 64 claudicants and 38 controls were enrolled. The concentration of platelets GPII b/IIIa and GP Ib/IX was estimated by ELISA method using monoclonal antibody against GPII b/IIIa (CD41a) and GPI b/IX (CD42a Immunotech). Plasma levels of vWF, fibrinogen, and platelets were measured by routine METHODS: RESULTS: Plasma vWF (145+/-41%), fibrinogen (3.8+/-1 g/l) and platelet concentration of GP Ib/IX (121.1+/-23.39), GPIIb/IIIa (117.9 6 +/-32.7%), as well as plasma lipids and uric acid were statistically higher in claudicants than in controls (vWF: 103+/-42%, fibrinogen: 2.9+/-0.5 g/l, GP Ib/IX: 100+/-16.9%, GP IIb/IIIa: 100+/-29.4%). We have observed statistically higher concentration of GP IIb/IIIa and GP Ib/IX in smoking patients than in non-smoking patients with PAOD and significant correlation between the concentration of GP Ib/IX and GP IIb/IIIa and plasma fibrinogen in patients with PAOD and controls. CONCLUSIONS: Our results demonstrate higher platelet concentration of GP Ib/IX,GP IIb/IIIa and elevated plasma levels of ligands for platelets receptors-fibrinogen and vWF in patients with PAOD. This prothrombotic conditions may explain increased cardiovascular morbidity and mortality in this patient's group.     

A role for glycoprotein IIb-IIIa complex in the binding of IgE to human platelets and platelet IgE-dependent cytotoxic functions

A possible relationship between binding sites for Immunoglobulin E (IgE) on human platelets, involved in IgE-dependent cytotoxic functions of platelets against helminth parasites, and well-characterized platelet constituents involved in haemostasis, was investigated. We first explored the interaction with IgE of platelets from patients with rare inherited deficiencies of defined platelet constituents and functions: Glanzmann's thrombasthenia, Bernard-Soulier and grey platelet syndromes. We report that only type I and II thrombasthenic platelets, which lack the membrane glycoproteins (GP) IIb and IIIa, failed to bind IgE and to exhibit IgE-dependent effector functions. Since thrombasthenic monocytes, however, showed normal interaction with IgE, this defect appeared restricted to platelets. Polyclonal and monoclonal antibodies directed against GP IIb-IIIa complex, but not monoclonal antibody directed against GP Ib, inhibited the binding of IgE to normal platelets, and their IgE-dependent cytotoxicity. Taken together, these findings indicate a relation between the GP IIb-IIIa complex and the expression of IgE binding sites and IgE-dependent effector functions in human platelets.     

Increased surface expression of the membrane glycoprotein IIb/IIIa complex induced by platelet activation. Relationship to the binding of fibrinogen and platelet aggregation

Platelet activation altered the binding of three monoclonal antibodies (monovalent Fab' fragment) directed against the glycoprotein (GP) IIb/IIIa complex. An increased binding of two- to threefold occurred after stimulation with thrombin or phorbol myristate acetate (PMA), with slight but significant increase in the dissociation constants (Kd) of two antibodies (LJ-CP8 and LJ-P9). In contrast, no statistically significant changes were observed with ADP-stimulated platelets. The increased binding of LJ-CP3, but not of the other two antibodies, to activated platelets decreased by 30% to 40% in the presence of EDTA at 22 to 25 degrees C. Platelets stimulated by thrombin or PMA bound more fibrinogen than did those stimulated by ADP, and significant differences in the extent but not in the affinity of fibrinogen binding were observed with various platelet agonists. When the pool of GP IIb/IIIa molecules exposed on the surface of unstimulated platelets was reacted with the monoclonal antibody LJ-CP3 to block ADP-induced fibrinogen binding and platelet aggregation, stimulation with thrombin or PMA still induced substantial binding of antibody and fibrinogen, and aggregation ensued. Therefore, platelets exposed to "strong" agonists exhibit an increased number of surface-oriented epitopes associated with GP IIb/IIIa. The GP IIb/IIIa molecules bearing these newly exposed epitopes are functional in that they can bind fibrinogen and mediate platelet aggregation.     

Cross-reaction of antibody against Helicobacter pylori urease B with platelet glycoprotein IIIa and its significance in the pathogenesis of immune thrombocytopenic purpura

Many clinical investigations have suggested that Helicobacter pylori (H. pylori) infection might be associated with immune thrombocytopenic purpura (ITP), but its role in the pathogenesis of ITP is unsettled. In this study, we cultured H. pylori, produced recombinant H. pylori urease (ure) B, and then prepared monoclonal antibody (MoAb) against ureB, 1F11, both 1F11 and MoAb against human platelet glycoprotein (GP) IIIa, SZ21, could bind to the band of GP IIIa of normal platelet lysate, but not to that from a patient with Glanzmann thrombasthenia (GT) whose GP IIb–IIIa complex was absent. Flow cytometry showed that normal platelets were reacted with 1F11 and SZ21, while GT platelets were not. In immuno-radiometric assay, the binding of ¹²⁵I-labeled 1F11 to GP IIIa was higher than that to GP Ib, GP IIb, GP VI, and P-selectin. 1F11 could partly compete with SZ21 in a binding to platelet surface. In addition, 1F11 inhibited platelet aggregation induced by adenosine diphosphate, but had no effect on platelet P-selectin expression or Thromboxane B₂ production of platelets. These results indicate that H. pylori ureB antibody could cross-react with human platelet GP IIIa and partly inhibit platelet aggregation. UreB may be a crucial component of H. pylori involved in the pathogenesis of a subset of ITP. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Monoclonal antibodies against porcine platelet membrane glycoproteins Ib and IIb/IIIa

We prepared murine monoclonal antibodies to porcine platelet membrane glycoprotein (GP) Ib and GP IIb/IIIa for further study of the porcine hemostatic mechanism. One monoclonal antibody, designated PP3-4C, blocked Ristocetin-induced platelet agglutination and caused 80% inhibition of Ristocetin-induced 125I-von Willebrand factor (vWF) binding to porcine platelets at a concentration of greater than or equal to 12 micrograms IgG/mL. PP3-4C did not affect adenosine diphosphate (ADP)- or collagen-induced platelet aggregation. Binding of 125I-Fab fragments of PP3-4C to platelets was saturable at 3.7 x 10(4) +/- 0.8 x 10(4) molecules per platelet. Another monoclonal antibody, designated PP3-3A, blocked ADP- or collagen-induced platelet aggregation at 6 micrograms IgG/mL. At a concentration of 10 micrograms IgG/mL, PP3-3A completely inhibited binding either of 125I-fibrinogen or of 125I-vWF to ADP-stimulated platelets. PP3-3A did not affect Ristocetin-induced platelet agglutination nor 125I-vWF binding to platelets in the presence of Ristocetin. Binding of 125I-Fab' fragments of PP3-3A to platelets was saturable at 9.8 x 10(4) +/- 1.2 x 10(4) molecules per platelet. PP3-4C antibody (anti-GP Ib) did not bind to human platelets; however, PP3-3A antibody (anti-GP IIb-IIIa) had partial cross-reactivity with human platelets. Immunoaffinity chromatography of solubilized surface-radiolabeled porcine platelets and subsequent sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis demonstrated that PP3-4C recognized a GP with an apparent molecular weight of 160,000 (nonreduced), and 140,000 (reduced). PP3-3A recognized GPs with apparent molecular weights of 130,000 and 80,000 (nonreduced), and 115,000 and 95,000 (reduced). These monoclonal antibodies to porcine platelet membrane GPs, which are structural and functional analogues of human GP Ib and GP IIb/IIIa, will be useful for in vitro and in vivo studies of the mammalian hemostatic mechanism.     

Effect of monoclonal antibodies against von Willebrand factor and platelet glycoproteins IIb/IIIa on the platelet retention test

The platelet retention test provides a measure of the number of platelets retained in a column of glass beads and is one of the few in vitro platelet function tests that is abnormal in von Willebrand's disease (vWd). In a two-stage test, 1 mL of blood (designated A) was passed through the column, followed by 5 mL of isotonic saline and then 5 mL of blood (B) in which platelet retention was measured. With normal blood as A and B, retention is very high in all 5 mL of blood B. In the first stage, platelets adhere to the glass beads; this requires fibrinogen but not von Willebrand factor (vWf). The platelet-platelet adhesion in the second stage requires vWf, is dependent on release of ADP, and fails to occur if thrombasthenic platelets are tested. Retention was normal when blood from a patient with afibrinogenemia was used as blood B. We have now used monoclonal antibodies to elucidate further the mechanism of platelet retention. Five antibodies to different epitopes on vWf essentially abolished retention in the one- stage test and in the second stage of the two-stage test, but had no effect on the first stage. Thus, the entire vWf molecule must be free of antibody to function in the platelet-platelet adhesion of the second stage of this test. Binding of the antigen-antibody complex to the platelet Fc receptor was not responsible, as Fab and F(ab')2 fragments of one of the antibodies were as effective as intact antibody, and as neither heat-aggregated IgG nor a polyclonal antibody to plasma factor IX inhibited retention. F(ab')2 fragments of 6D1, an antibody to platelet GP Ib that prevents binding of vWf to platelets, also inhibited the second phase of retention. An antibody that inhibits binding of fibrinogen and vWf to GP IIb/IIIa (LJ-CP8) inhibited both the first and second stages of retention, whereas LJ-P5, an antibody that inhibits only the binding of vWf to GP IIb/IIIa, caused slight inhibition of retention when normal or afibrinogenemic blood was used as blood B and was reported to cause only partial inhibition of ADP- induced platelet aggregation in this afibrinogenemic patient. The results suggest that vWf is altered during rapid passage of blood through the glass-bead column so that it attaches to GP Ib, exposing GP IIb/IIIa, which then binds the altered vWf or fibrinogen, either of which can induce platelet aggregation (platelet-platelet adhesion) and thus retention in the column.     

Characterization of a platelet membrane protein of low molecular weight associated with platelet activation following binding by monoclonal antibody AG-1

With the exception of the major platelet glycoproteins IIb/IIIa and Ib, which function as receptors for fibrinogen and von Willebrand factor, little is presently known regarding the possible role of other platelet surface proteins in mediating platelet aggregation. We report the production of a murine monoclonal antibody (AG-1) recognizing human platelet membrane surface protein of relatively low molecular weight (mol wt) that may be involved in this process. AG-1 added to human platelet-rich plasma induces dense granule secretion and aggregation, with lag phase and maximal extent of aggregation dependent on antibody concentration. Aggregation induced by AG-1 is inhibited by AG-1 Fab fragments, indicating that the response is not Fc receptor-mediated. Although AG-1 continues to produce platelet shape change in the presence of EDTA, aggregation is fully inhibited and appears to be mediated by fibrinogen binding to glycoproteins IIb/IIIa. AG-1 is a potent stimulus of thromboxane formation, but full inhibition of thromboxane production by 30 mumol/L indomethacin does not significantly inhibit platelet aggregation induced by 25 micrograms/mL AG-1, indicating that aggregation induced by AG-1 may proceed by way of an endoperoxide-independent pathway. Quantitation of AG-1 Fab binding to platelets reveals approximately 65,000 binding sites per platelet. When intact platelets are radioiodinated, immunoprecipitation of NP-40 lysates by AG-1 reveals an intensely labeled protein with an apparent mol wt of approximately 21,000 daltons, and several additional bands in the mol wt range of 22,000 to 28,000 daltons, all sharing the AG-1 epitope. These bands appear to be distinct from glycoprotein IX or from the beta-chains of glycoprotein Ib or IIb. Finally, studies with platelets labeled by the periodate-[3H]borohydride procedure suggest the possibility of complex formation between subpopulations of glycoprotein Ib and the low-mol-wt glycoproteins recognized by AG-1.     

Heparin-induced thrombocytopenia: further studies of the effects of heparin-dependent antibodies on platelets

Studies were performed with the sera of five patients with heparin-induced thrombocytopenia. Each patient serum was found to cause heparin-dependent aggregation of normal and Bernard-Soulier syndrome platelets but not Glanzmann's thrombasthenia or fixed normal platelets. This antibody effect was blocked by inhibition of both aerobic and anaerobic metabolism and an increase in intraplatelet c-AMP with prostaglandin E1. These findings suggest that the antibody-induced platelet aggregation was not a passive agglutination of cells but an active process requiring metabolic energy and membrane glycoproteins IIb/IIIa but not glycoprotein Ib. In addition, platelet aggregation by four patient sera was inhibited by aspirin or apyrase indicating a prostaglandin synthesis and ADP-dependent mechanism and the reaction could be suppressed by dipyridamole alone. However, platelet aggregation induced by the serum of one patient was only inhibited partially by the combination of aspirin and apyrase suggesting mediation of the antibody effect in part by a pathway independent of extracellular ADP and prostaglandin synthesis and this antibody reaction could only be completely suppressed by a combination of aspirin and dipyridamole. Heterogeneity exhibited by these antibodies may influence the choice and effectiveness of antiplatelet therapy in heparin-induced thrombocytopenia.     

Characterization of a monoclonal antibody ITI-Pl 1 directed against human platelet membrane glycoprotein IIb using extracts of whole platelets and platelet surface and intracellular membranes

A monoclonal antibody (mAb) termed ITI-Pl 1 has been prepared by the hybridoma procedure. Using immuno-absorption and crossed immunoelectrophoresis of Triton X-100 extracts of untreated and EDTA-treated human platelets it was shown to be directed against the surface membrane glycoprotein IIb (GP IIb). This mAb binds to whole platelets independently of ADP-stimulation and the presence of Ca2+-ions. It saturates at around 870 ng/10(8) cells corresponding to approximately 35,800 molecules/platelet. ITI-Pl 1 did not significantly inhibit GP IIb-IIIa dependent functions such as platelet aggregation or fibrinogen binding. Immunofluorescence could be demonstrated using ITI-Pl 1 and intact normal platelets, but not with platelets from a Glanzmann's thrombasthenia patient. Crossed immuno-electrophoresis with platelet extracts from four different thrombasthenic patients gave a line precipitate in the intermediate gel with 125I-labelled ITI-Pl 1 and autoradiography indicating trace amounts of free GP IIb or the GP IIb-IIIa complex. The epitope on GP IIb detected by ITI-Pl 1 is not destroyed by neuraminidase treatment. Thus the mAb also interacts with neuraminidase-treated GP IIb-IIIa complex in highly purified platelet surface membrane fractions as well as with GP IIb-IIIa from untreated internal membranes isolated by continuous flow electrophoresis.     

Understanding the mechanism and prevention of arterial occlusive thrombus formation by anti-platelet agents

For many years, platelet aggregation, which is mediated exclusively by the binding of fibrinogen to activated glycoprotein (GP) IIb/IIIa, has been used for the screening of antiplatelet agents. However, clinical experience with anti-GP IIb/IIIa agents, which can completely inhibit platelet aggregation, has shown that these drugs are not the most ideal agents for the prevention of atherothrombosis. Recently, many investigators have reported that platelets play a major role in thrombus formation at sites exposed to blood flow, and also that there is a crucial difference between the mechanism of platelet thrombus formation under blood flow conditions in vivo and that of platelet aggregation occurring in conventional aggregometry. Indeed, multiple receptor-ligand interactions, including von Willebrand factor (VWF) binding with platelet GP Ibalpha and GP IIb/IIIa, collagen binding with collagen receptors, as well as stimulation of platelet receptors, such as adenosine 5'-diphosphate (ADP) receptors, appear to be involved in the process of in vivo arterial thrombus formation. Moreover, not only platelets, but also the coagulation cascade activated by the procoagulant activity expressed on the surface of activated platelets, are believed to play a crucial role in the formation of occlusive thrombi. These findings suggest that drugs which block events upstream of the final common pathway for platelet aggregation might be better antiplatelet agents than those that merely inhibit platelet aggregation. We may then expect new antiplatelet agents on the horizon that exert their actions against both thrombus formation under blood flow conditions and against the procoagulant activity appearing on the surface of activated platelets.     

Autoimmune thrombocytopenic purpura (AITP) and acquired thrombasthenia due to autoantibodies to GP IIb–IIIa in a patient with an unusual platelet membrane glycoprotein composition

The subject (E.B.) is a 63-year-old woman with autoimmune thrombocytopenic purpura (AITP) who was first examined some 6 years ago with symptoms of epistaxis and gum bleeding, severe thrombocytopenia, and large platelets. Her serum tested positively with control platelets in the MAIPA assay performed using monoclonal antibodies (MoAb) to glycoprotein (GP) IIIa (XIIF9, Y2/51), yet was negative in the presence of MoAbs to GP IIb (SZ 22) or to the GP IIb-IIIa complex (AP2, P2). The patient's platelets failed to aggregate with all agonists tested except for ristocetin. IgG isolated from the patient's serum inhibited ADP-induced aggregation of control platelets. Unexpectedly, flow cytometry showed an altered expression of membrane glycoproteins on the patient's platelets. Levels of GP Ib-IX were much higher than previously located by us in platelets. In contrast, the expression of GP IIb-IIIa was about half that seen with control subjects. When Western blotting was performed, a striking finding was a strong band of 250 kDa recognized by a series of MoAbs to GP Ibα in addition to the band in the normal position of GP Ibα. Finally, ADP-stimulated (E.B.) platelets failed to express activation-dependent epitopes on GP IIb-IIIa as recognized by PAC-1, AP6, or F26 and additionally gave a reduced P-selectin expression after thrombin addition. In conclusion, we present a novel patient with a severely perturbed platelet function where an altered membrane GP profile is associated with the presence of an autoantibody recognizing a complex-dependent determinant on GP IIb–IIIa and inhibitory of platelet aggregation. Am. J. Hematol. 57:164–175, 1998. © 1998 Wiley-Liss, Inc.     

Planned versus provisional use of glycoprotein IIb/IIIa inhibitors in smokers undergoing percutaneous coronary intervention

Postmortem and angiographic studies have demonstrated that thrombosis is the primary cause of coronary artery occlusion in smokers. Further, smokers have high levels of fibrinogen, increased platelet aggregation, and more platelet-dependent thrombin generation than do nonsmokers, suggesting that glycoprotein (GP) IIb/IIIa inhibitor use during percutaneous coronary intervention (PCI) may be especially useful among smokers. We evaluated a subpopulation of active smokers in the REPLACE-2 trial to assess the effect of treating smokers with bivalirudin and provisional GP IIb/IIIa blockade compared with heparin and planned GP IIb/IIIa blockade. The REPLACE-2 trial enrolled 1,558 smokers and 4,305 nonsmokers. Smokers who were treated with bivalirudin had an absolute 3.2% increase in the composite end point of death and myocardial infarction at 48 hours compared with smokers who were treated with heparin and GP IIb/IIIa inhibitors (7.7% vs 4.5%, p=0.008, interaction p=0.016). This difference was ameliorated when GP IIb/IIIa inhibitors were used consistently in a previous trial that compared bivalirudin with heparin during PCI (4.6% vs 6.7%, p=0.322). In conclusion, these results suggest that smokers may derive particular benefit with GP IIb/IIIa inhibitors for decreasing myocardial infarction and death after PCI. These findings require further validation from other large, randomized trials.     

Binding of von Willebrand factor to glycoproteins Ib and IIb/IIIa complex: affinity is related to multimeric size

We have separated von Willebrand factor (vWF) multimers of different size into several fractions which were characterized by SDS-agarose gel electrophoresis and by measuring the ratio between ristocetin cofactor activity (Ricof) and von Willebrand antigen (vWF:Ag) content. The pooled fractions contained vWF with multimeric structures and Ricof similar to those in plasma. The pool was labelled with 125I and used for inhibition binding studies with individual fractions to calculate the dissociation constants (Kd values expressed in mol/l) of the individual fractions for ristocetin-dependent binding to GP Ib and thrombin-induced binding to GP IIb/IIIa. Direct binding studies of the 125I-vWF pool gave mean Kd values of 2.02 +/- 0.05 x 10(-8) for GP Ib and 1.15 +/- 0.02 x 10(-8) for the GP IIb/IIIa complex. Inhibition binding studies gave Kd mean values one third to one tenth as high for larger multimers and 3-10 times higher for smaller multimers, for both GP Ib and IIb/IIIa complex. Similar results were observed when binding studies were carried out in the presence of platelets from a patient with afibrinogenaemia. These data on binding correlated very well with ristocetin- and thrombin-induced aggregation of afibrinogenaemic platelets, since equal concentrations of the higher molecular weight forms gave significantly higher aggregation rates. Based on these results, we conclude that the affinity of the vWF molecule for its two platelet receptors is greater for the largest multimers.     

Autoantibody-mediated complement activation on platelets is a common finding in patients with immune thrombocytopenic purpura (ITP)

Background: It is commonly accepted that antibody‐mediated removal of platelets represents a major mechanism of platelet destruction in immune thrombocytopenic purpura (ITP). Although complement activation may participate in platelet clearance, frequency and specificity of complement activation have not yet been studied systematically in ITP. Patients and methods: We examined blood samples from 240 patients with ITP. Samples were assessed for the presence of free and bound platelet autoantibodies by a standard glycoprotein‐specific assay (monoclonal antibody‐specific immobilization of platelet antigens). The ability of all sera to fix complement to a panel of human platelets was investigated in a complement fixation (CF) assay. Fixation of C1q to isolated GP IIb/IIIa was assessed by flow cytometry. Results: Glycoprotein‐specific autoantibodies were detected as platelet‐bound antibodies in 129 (54%) and as additional free antibodies in 26 (11%) and were undetectable in 111 (46%) patients. Assessing these subgroups for CF, 103 (65%), 21 (81%), and 33 (30%) sera gave positive results. If GP IIb/IIIa was absent from the test platelets, 81 (67%) lost their ability to fix complement; if GP Ib/IX was absent, 37 (30%) lost their ability to fix complement. C1q fixation to immunobeads coated with GP IIb/IIIa was observed in 50% of sera containing anti‐GP IIb/IIIa antibodies. Conclusions: In a significant number of patients with chronic ITP, platelet autoantibodies are capable of activating the classical complement pathway. CF is even present in ITP sera without detectable autoantibodies, indicating that current techniques for autoantibody detection may be insufficient. The major targets for complement‐fixing autoantibodies in ITP are GP IIb/IIIa and GP Ib/IX.     

Association of autoantibodies against platelet glycoproteins Ib/IX and IIb/IIIa, and platelet-reactive anti-HIV antibodies in thrombocytopenic narcotic addicts

The levels of platelet-associated Igs (PAIgs) and plasma circulating antiplatelet antibodies were evaluated by a flow cytometric immunofluorescence assay (FCIFA), an enzyme-linked immunoassay (ELISA), and a platelet radioactive antiglobulin test (PRAT), in a group of 45 human immunodeficiency virus (HIV)-infected intravenous drug users (IVDUs), with or without thrombocytopenia (TCP). Direct tests demonstrated an increased amount of PAIgs in 40% of the patients, irrespective of their platelet count. These PAIgs were mainly of IgG class and could not be eluted with ether. Plasma IgG with antiplatelet activity was found in 70% of the thrombocytopenic individuals, whereas it was detected in only one patient without TCP. The relative frequencies of antibodies against the platelet glycoproteins (GPs) Ib/IX and IIb/ IIIa were assessed in plasma from all patients by means of the monoclonal antibody-specific immobilization of platelet antigens assay (MAIPA). Plasmas from all non-thrombocytopenic patients were negative when tested by indirect MAIPA. In contrast, 10/23 plasmas from thrombocytopenic patients reacted with either GP IIb/IIIa, GP Ib/IX, or both GPs. Finally, aiming to investigate whether HIV antibodies from these patients are reactive with normal platelets, we performed absorption-elution experiments, followed by evaluation of HIV antibodies in the indirect eluates by ELISA and Western blot. Interestingly, we detected anti-HIV antibodies that bind to normal platelet antigens in 50% of the ether eluates prepared from control platelets sensitized with plasma from patients with TCP, but in only 5% of eluates obtained from platelets sensitized with plasma from non-thrombocytopenic patients. The present study provides direct evidence that specific autoantibodies against platelet membrane GPs Ib/IX and IIb/IIIa are common in HIV positive thrombocytopenic individuals. The finding in these patients of HIV antibodies cross-reactive with normal platelets, suggests that mimicry of human antigens by HIV could play a key role in the pathophysiology of the HIV-related TCP.