MK-383 (tirofiban) induces a GPIIb/IIIa receptor conformation which differs from the resting and activated receptor

The platelet integrin alphaIIb beta3 (GPIIb/IIIa) acts as a receptor for fibrinogen, playing a critical role in platelet aggregation. GPIIb/IIIa antagonists, which block the receptor-ligand interaction, have been accused of causing occasional thrombocytopenia, probably via drug-induced platelet activation or immunogenic neoepitopes. We, therefore, analyzed the effects of the GPIIb/IIIa antagonist MK-383 (tirofiban) on platelet activation and GpIIb/IIIa conformation. At a concentration of 10(-7) mol/l, MK-383 completely inhibited fibrinogen binding to in vitro stimulated platelets. Simultaneously, the GPIIb/IIIa expression density increased, similar to that on activated platelets, but no effect on P-selectin expression or the formation of platelet-leukocyte aggregates could be observed, indicating that MK-383 binding did not induce general platelet activation. The GPIIb/IIIa receptor conformation was further analyzed by fluorescence resonance energy transfer analysis between fluorochrome-labeled antibodies against different GpIIb/IIIa epitopes. As a result, MK-383 induced a receptor conformation that differed from the resting as well as the activated receptor as induced by ADP or TRAP-6. This conformational modulation of GPIIb/IIIa presents an interesting mechanism which may be linked to receptor recruitment without inducing general platelet activation.     

MK-383 (Tirofiban) induces a GPIIb/IIIa receptor conformation which differs from the resting and activated receptor

The platelet integrin α_IIb β₃ (GPIIb/IIIa) acts as a receptor for fibrinogen, playing a critical role in platelet aggregation. GPIIb/IIIa antagonists, which block the receptor-ligand interaction, have been accused of causing occasional thrombocytopenia, probably via drug-induced platelet activation or immunogenic neoepitopes. We, therefore, analyzed the effects of the GPIIb/IIIa antagonist MK-383 (tirofiban) on platelet activation and GpIIb/IIIa conformation. At a concentration of 10^-7 mol/l, MK-383 completely inhibited fibrinogen binding to in vitro stimulated platelets. Simultaneously, the GPIIb/IIIa expression density increased, similar to that on activated platelets, but no effect on P-selectin expression or the formation of platelet-leukocyte aggregates could be observed, indicating that MK-383 binding did not induce general platelet activation. The GPIIb/IIIa receptor conformation was further analyzed by fluorescence resonance energy transfer analysis between fluorochrome-labeled antibodies against different GpIIb/IIIa epitopes. As a result, MK-383 induced a receptor conformation that differed from the resting as well as the activated receptor as induced by ADP or TRAP-6. This conformational modulation of GPIIb/IIIa presents an interesting mechanism which may be linked to receptor recruitment without inducing general platelet activation.     

Association of CIB with GPIIb/IIIa during outside-in signaling is required for platelet spreading on fibrinogen

Platelet spreading on immobilized fibrinogen (Fg) involves progression through a number of morphologic stages that, although distinctive, are not well understood mechanistically. Here we demonstrate that an association between GPIIb/IIIa and calcium- and integrin-binding protein (CIB) is required for the process of platelet spreading. Upon platelet adhesion to immobilized Fg, CIB localizes to the transiently formed filopodia and then redistributes diffusely along the membrane periphery of spread platelets. Immunoprecipitation analyses indicate that CIB and glycoprotein IIb/IIIa (GPIIb/IIIa) interact with each other as platelets adhere to immobilized Fg, and together they associate with the platelet cytoskeleton. Introduction of anti-CIB antibody or GPIIb cytoplasmic peptide into platelets blocks lamellipodia but not filopodia formation. GPIIb peptide-induced inhibition of platelet spreading is recovered by the incorporation of recombinant CIB protein, suggesting that interaction between CIB and GPIIb/IIIa is required for progression from filopodial to spread morphologies. Further, anti-CIB- or GPIIb peptide-induced inhibition of platelet spreading can be overcome by the addition of exogenous adenosine diphosphate (ADP). These data suggest that formation of the CIB-GPIIb/IIIa complex may be necessary for initiation of downstream signaling events, such as ADP secretion, that lead to platelet spreading.     

Advances in Antiplatelet Therapy in Coronary Artery Disease: Importance of the Platelet GPIIb/IIIa Receptor

It is now widely agreed that platelets are intimately involved in and contribute to the pathogenesis of acute coronary thrombosis. Aspirin, a relatively weak inhibitor of platelet activation, saves lives when administered early after acute myocardial infarction and should be routinely used as lifelong therapy in patients with coronary atherosclerosis. Ticlopidine has a mechanism of action distinct from and additive to that of aspirin; it inhibits activation of platelets mediated by the agonist, adenosine diphosphate (ADP). The reduction in subacute coronary thrombosis attained by the use of combination therapy with aspirin and ticlopidine (for 2–4 weeks) after intracoronary stenting is further evidence of the role of platelets in mediating acute arterial thrombosis. Potent platelet agonists (like thrombin) can override the effect of aspirin and ticlopidine; therefore these agents are of limited efficacy. In contrast, inhibitors of the platelet glycoprotein (GP) IIb/IIIa receptor are potentially more potent inhibitors of adhesive platelet interaction and may therefore be effective in blocking adhesive platelet interactions irrespective of the activating agonist. The GPIIb/IIIa receptor mediates the bridging of platelets (platelet aggregation) via fibrinogen, thus allowing platelet to bind other platelets at the injured vessel wall. Antagonists of this receptor are thus capable of blocking the “effector function” by acting at a step that is downstream to platelet activation. By abrogating the final common pathway of platelet aggregation, antagonists of GPIIb/IIIa also affect the most proximal step in thrombin generation (that most efficiently occurs on the membrane surface provided by platelets). Accordingly, these agents can profoundly inhibit arterial thrombosis. The clinical use of the antibody fragment directed against the GPIIb/IIIa receptor (c7E3 Fab) has truly revolutionized the practice of interventional cardiology and has the potential to effectively treat heparin-resistant intracoronary thrombosis. Synthetic antagonists of fibrinogen binding to the GPIIb/IIIa receptor (the “fibans”) are currently under initial clinical testing.     

Platelet interaction with subendothelial extracellular matrix: platelet- fibrinogen interactions are essential for platelet aggregation but not for the matrix-induced release reaction

Cultured endothelial cells produce an extracellular matrix (ECM) to which platelets adhere and spread, ultimately resulting in platelet aggregation, thromboxane B2 production, and serotonin release. We have investigated the role of fibrinogen binding to the platelet GPIIb/IIIa complex in these reactions by comparing normal platelet-rich plasma (PRP), PRP from patients with Glanzman's thrombasthenia (whose platelets lack the GPIIb/IIIa complex), PRP in the presence of a monoclonal antibody that blocks the binding of fibrinogen to the GPIIb/IIIa complex, platelets washed free of fibrinogen, and washed platelets to which fibrinogen was added. Although platelet aggregation was virtually completely inhibited in the samples in which the normal interaction between fibrinogen and GPIIb/IIIa was impaired, adhesion of platelets to the matrix, spreading, and release of [14C]-serotonin were not affected. All of the platelet preparations released significant amounts of T X B2 with time, but there was a decrease in the amount produced by both the thrombasthenic and antibody-treated platelets. We conclude that the interaction of fibrinogen with platelet GPIIb/IIIa is not required for platelet adhesion to ECM or for adhesion-induced shape change or serotonin release. On the other hand, the platelet-fibrinogen interaction may play some role in augmenting adhesion-induced T X B2 production, and it is absolutely required for adhesion-induced platelet aggregation.     

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.     

Immobilized Arg-Gly-Asp (RGD) peptides of varying lengths as structural probes of the platelet glycoprotein IIb/IIIa receptor

The interactions between ligands containing the recognition sequence arginine-glycine-aspartic acid (RGD) and integrin receptors are important in many cell-cell and cell-protein interactions. The platelet contains five integrin receptors and they contribute significantly to platelet adhesion and aggregation. To investigate the RGD binding domains on platelet integrins, we immobilized a series of RGD peptides containing variable numbers of glycine residues [(G)n-RGDF] on polyacrylonitrile beads and evaluated the ability of the beads to interact with platelets. With native platelets, virtually no interaction occurred with G1-RGDF beads, but the interactions increased as the number of glycine residues increased, plateauing with the G9- RGDF and G11-RGDF beads. ADP pretreatment enhanced the interactions with all of the beads, whereas prostaglandin E1 pretreatment eliminated the interactions with the shortest peptide beads, but only partially inhibited interactions with the longer peptide beads. Monoclonal antibodies to glycoprotein (GP) IIb/IIIa were most effective in inhibiting the interactions, but antibodies to GPIIb/IIIa with similar inhibitory effects on fibrinogen binding varied dramatically in their ability to inhibit the interaction between platelets and immobilized RGD peptides. Our data indicate that the majority of RGD binding sites on GPIIb/IIIa can be reached by peptides that extend out approximately 11 to 32 A from the surface of the bead, and these results are in accord with the dimensions of integrin receptors deduced from electron microscopy. Activation of GPIIb/IIIa facilitates the interactions, but platelet inhibition fails to eliminate the interactions with the longer peptide beads, suggesting that access to the RGD binding site on at least a fraction of the GPIIb/IIIa receptors is always possible for preferred ligands. Finally, we found that the G3-RGDF peptide beads were uniquely sensitive to the activation state of the GPIIb/IIIa receptor.     

Acquired thrombasthenia due to GPIIb/IIIa-specific platelet autoantibodies

An otherwise healthy woman developed a hemorrhagic diathesis with fluctuating clinical symptoms and laboratory findings, but without thrombocytopenia, over 8 years. In periods of bad clinical condition, a platelet defect, characteristic of thrombasthenia, was found. In contrast to classic thrombasthenia, electrophoresis of the patient's platelet membranes revealed normal amounts of glycoproteins IIb alpha, IIb beta, and IIIa in the normal positions. Monoclonal antibodies, specific for GPIIIa and GPIIb/IIIa, respectively, bound normally to the P1A1-positive platelets from the patient. Although no antibody and no platelet function inhibitor were evident in the autologous plasma, an IgG1 antibody that was bound to the patient's platelets and was directed against GPIIb/IIIa could be demonstrated. After elution from the patient's platelets, this antibody immunoprecipitated GPIIb (both subunits), IIIa, and a 200-kilodalton (kd) band (probably undissociated GPIIb/IIIa complex) from solubilized normal platelets, but did not react with thrombasthenic platelets. Adding the eluate from the patient's platelets to normal platelet-rich plasma immediately caused concentration-dependent inhibition of adenosine diphosphate (ADP)-induced and collagen-induced aggregation and also strong inhibition of ADP-stimulated fibrinogen binding. Because it was very unlikely from the patient's medical history that the antibody was caused by alloimmunization, the hemorrhagic diathesis must be interpreted as acquired thrombasthenia due to an anti-GPIIb/IIIa autoantibody.     

Differential inhibition of adenosine diphosphate- versus thrombin receptor-activating peptide-stimulated platelet fibrinogen binding by abciximab due to different glycoprotein IIb/IIIa activation kinetics

The exposure of internal glycoprotein (GP) IIb/IIIa receptors has been proposed to explain the incomplete inhibition of aggregation of thrombin receptor-activating peptide (TRAP)-stimulated platelets by abciximab. However, a marked and rapid externalization of GPIIb/IIIa was also observed upon stimulation with 30 microM adenosine diphosphate (ADP). ADP-induced fibrinogen binding was completely inhibited by 10 microg/mL abciximab, 30 nM tirofiban, or 3 microg/mL eptifibatide, while fibrinogen binding induced by 100 microM TRAP was inhibited only by 50%. Interestingly, striking differences in fibrinogen binding kinetics in ADP- versus TRAP-stimulated platelets were observed. ADP-induced fibrinogen binding was much slower than that of abciximab. These differences in the fibrinogen binding rate were due to differential GPIIb/IIIa activation kinetics because the actual fibrinogen binding rate (measured by adding fibrinogen after platelet activation) was similar in ADP- and TRAP-stimulated platelets. Thus, the TRAP-induced GPIIb/IIIa activation rate would allow significant amounts of fibrinogen to occupy externalized GPIIb/IIIa receptors even in the presence of the inhibitor.     

Studies of fibrinogen binding to porcine platelets by flow cytometry: a method for studies of porcine platelet activation

Platelets have a central function in haemostasis. They also participate in arterial thrombus formation in vascular disorders. Platelets have an important role in initiating and mediating ischaemia and related complications of ischemic heart disease. Several research groups are thus studying platelet activation and developing new platelet inhibitors. Platelet function is dependent upon membrane receptors and their interaction with other proteins. Binding of fibrinogen to the platelet glycoprotein (GP) IIb/IIIa receptor is a prerequisite for platelet aggregation and thrombus formation. Thus, several GPIIb/IIIa inhibitors have been developed of which abciximab is the clinically most widely used. Pigs are often used for experimental studies. We have developed a flow cytometry assay for measuring porcine platelet activation utilising an FITC-labelled chicken anti-fibrinogen antibody. ADP, ristocetin and thrombin induce fibrinogen binding to porcine platelets similarly to human platelets. Ristocetin induces platelet aggregation and microvesicle formation from porcine platelets as well as from human platelets.     

Platelet aggregation by fibrinogen polymers crosslinked across the E domain

There is evidence that platelet interactions with artificial surfaces are mediated by plasma proteins, especially fibrinogen, adsorbed on the surfaces. Multiple site interactions between fibrinogen molecules adsorbed in high concentration and receptors in the unactivated platelet may be sufficient for platelet adhesion and subsequent activation. To examine this hypothesis, we prepared soluble polymers of fibrinogen. Polymers produced by interaction of fibrinogen with Fab'2 fragments of antibodies against fibrinogen's E (central) domain (Fg- Fab'2(E] induced, in gel-filtered platelets, aggregation and serotonin release, which were blocked by monoclonal antibodies against the GPIIb/IIIa complex, by Fab fragments against the D domain, and by metabolic inhibitors; aggregation was attenuated but not abolished by enzymatic removal of ADP (with CP/CPK) or by blockage of ADP binding sites (with FSBA), and when secretion was inhibited by aspirin. Fg- Fab'2(E) also induced a dose-dependent elevation in cytoplasmic Ca2+ (measured by Aequorin luminescence) which was attenuated by CP/CPK and by FSBA, and was eliminated by metabolic inhibitors and by anti- IIb/IIIa antibody. Fibrinogen complexes crosslinked with dimethylsuberimidate or Factor XIII neither aggregated gel-filtered platelets nor inhibited platelet aggregation by ADP and fibrinogen, probably because of inaccessibility of lysine residues in the D (terminal) domain of fibrinogen, which are thought to be required for platelet binding. Thus, soluble complexes of fibrinogen having multiple available platelet receptor recognition sites activate gel-filtered platelets and may provide a useful model for platelet-surface interactions mediated by adsorbed fibrinogen.     

Plasminogen interactions with platelets in plasma

In this report we used a fluorescent flow cytometry-based assay to examine plasminogen binding to platelets in plasma. Our data indicate that platelets activated in platelet-rich plasma (PRP) by adenosine-5'- diphosphate (ADP) or thrombin bind plasminogen to their surface. Fab fragments of the monoclonal antibody LJ-CP8 that are directed against the fibrinogen binding site on the glycoprotein (GP) IIb-IIIa complex inhibit both plasminogen and fibrinogen binding to ADP-stimulated platelets as does 5 mmol/L EDTA. Platelet aggregation and plasminogen and fibrinogen binding are also concurrently inhibited by the Gly-Arg- Asp (RGD) analogue Gly-Arg-Gly-Asp-Ser (GRGDS) when it is added to PRP before ADP stimulation. The scrambled peptide analogue SDGRG has no effect. The monoclonal antibody 6D1, directed against the von Willebrand factor binding site on GPIb, has no effect on plasminogen- platelet binding, nor does antithrombospondin antibody. epsilon- Aminocaproic acid (EACA), however, inhibits plasminogen binding to ADP- activated platelets. These data indicate that plasminogen binds to platelets activated in plasma, that binding occurs on platelet GPIIb/IIIa, and that binding may be mediated via plasminogen association with fibrinogen via lysine binding domains. Finally, we found both plasminogen and fibrinogen on resting platelets in PRP and demonstrated that they are equally displaced by EDTA, LJ-CP8, and 10E5 (an additional anti-GPIIb/IIIa monoclonal antibody). Plasminogen is also equally displaced by EACA. These data suggest that plasminogen is also bound to GPIIb/IIIa on resting platelets, possibly also via interaction with fibrinogen.     

Thrombasthenia with an abnormal platelet membrane glycoprotein IIb of different molecular weight

We describe an individual with abnormal platelet glycoprotein (GP) IIb of different molecular weight (mol wt), a defect that distinguishes this patient from previously reported thrombasthenics. The patient, a 21-year-old female, has a mild bleeding tendency; her platelets lack adenosine diphosphate (ADP) aggregation and have severely suppressed collagen aggregation but a normal response to ristocetin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of her platelets indicates that they contain two types of GPIIb molecules: one with an abnormal mol wt (122 kd, unreduced; 128 kd, reduced) and one with a normal mol wt (128 kd, unreduced; 118 kd, reduced). Relative to the amount of GPIIb in normal platelets, her platelets contain approximately 35% abnormal GPIIb and 20% normal GPIIb. Fibrinogen binding assays on the patient's platelets indicated that they contained 25% of the normal amount of fibrinogen receptors. Crossed immunoelectrophoresis of the patient's platelets demonstrated the formation of a GPIIb/IIIa complex that was mainly composed of normal mol wt GPIIb and GPIIIa. The patient's father has decreased ADP aggregability, and his platelets also contained both abnormal and normal GPIIb (about 50% of the normal level and about 50% of the normal number of fibrinogen receptors); her mother has only normal GPIIb. These results indicate that the patient has heterozygous GPIIb molecules with an abnormality of GPIIb at the molecular level. Studies on this abnormal GPIIb should provide information about the function of GPIIb and the mechanism of its biosynthesis.     

In vitro measurement of platelet glycoprotein IIb/IIIa receptor blockade by abciximab: interindividual variation and increased platelet secretion

BACKGROUND AND OBJECTIVES: Inhibition of soluble fibrinogen binding to activated platelets represents the target of pharmacologic approach with antagonists of the glycoprotein IIb/IIIa (GPIIb/IIIa) complex. In this study we assessed the effects of abciximab, a recombinant chimeric Fab fraction of the antibody against GPIIb/IIIa, on several markers of platelet activation. DESIGN AND METHODS: The platelet surface expression of GPIIb/IIIa was measured by a flow cytometry technique using a two-color assay. GPIIb/IIIa was detected by FITC-conjugated antibodies in whole blood, either unstimulated or exposed to platelet stimuli. The following antibodies were used: CD41, which recognizes the IIb/IIIa complex both in activated and non-activated conformers, and PAC-1, which is directed toward the activated conformer of GPIIb/IIIa. In addition, the same blood sample was incubated with CD62 antibody to measure P-selectin, as a marker of a-granule degranulation. The effect of abciximab was also assessed by experiments carried out on shear stress-induced platelet aggregation, a test that appears to be a predictor of platelet hemostatic function. RESULTS: Abciximab inhibited CD41 binding to glycoprotein IIb (GPIIb) in a concentration-dependent manner and also inhibited the binding of PAC-1 to active GPIIb/IIIa. In contrast, membrane-associated P-selectin was significantly increased by the drug, which suggests that blockade of GPIIb/IIIa receptors results in an increased platelet degranulation in response to agonists. Shear stress-induced platelet aggregation was inhibited by abciximab, with a more pronounced effect on blood filtration, which represents an index of platelet aggregate formation. INTERPRETATION AND CONCLUSIONS: Our results indicate that GPIIb/IIIa blockade by abciximab is accompanied by an increase of a-granule secretion, suggesting that different mechanisms regulate these aspects of platelet activation. The described flow cytometry technique, that allows the simultaneous in vitro detection of several platelet markers, is a suitable method for assessing the effects of agents which interfere with platelet function.     

Vitronectin inhibits blood platelet aggregation

The effect of vitronectin on platelet aggregation has been investigated. Vitronectin inhibited both thrombin- and ADP-induced platelet aggregation in a dose-dependent manner. A monoclonal antibody (MoAb) to vitronectin increased thrombin-induced platelet aggregation. This effect of the MoAb was not mediated via the platelet Fc-receptor, suggesting that the antibody directly counteracted the inhibitory effect of vitronectin on platelet aggregation. Like some other adhesive proteins such as fibrinogen, fibronectin, and von Willebrand factor, vitronectin contains the amino-acid sequence Arg-Gly-Asp (RGD) which enables binding to the platelet membrane glycoprotein complex IIb/IIIa (GPIIb/IIIa). The results of this study indicate that vitronectin can modulate the function of fibrinogen on platelet aggregation by interfering with the binding of fibrinogen to GPIIb/IIIa in activated platelets.     

A Val193Met mutation in GPIIIa results in a GPIIb/IIIa receptor with a constitutively high affinity for a small ligand

We have identified a patient designated as (GTa) with Glanzmann's Thrombasthenia (GT) diagnosed on the basis of a prolonged bleeding time and failure of the patient's platelets to aggregate. The number of glycoprotein (GP)IIb/IIIa receptors on the platelet surface was 37% of normal and those receptors displayed a defect in soluble fibrinogen binding. Nevertheless, GTa platelets showed increased adhesion to solid-phase fibrinogen and binding affinity for the RGD-mimetic (3)H-SC52012, a non-peptide GPIIb/IIIa antagonist. Dithiothreitol (DTT) and ADP enhanced the affinity for [(3)H]-SC52012 in normal platelets, but had little effect in GTa platelets. These findings suggested that GTa platelets were locked in an altered affinity state. Genetic analysis showed that GTa was a compound heterozygote for the GPIIIa gene. One allele showed a deletion at the 3' end of exon 3 resulting in a premature stop codon. The second GPIIIa allele had a G to A transition at nucleotide 577, resulting in a Val193Met substitution. HEK 293T cells transfected with mutant GPIIb/IIIaV193M bound [(3)H]-SC52012 with a higher affinity than wild-type GPIIb/IIIa, and this was not increased by DTT. The mutant receptor distinguishes between platelet adhesion and aggregation, and demonstrates the phenotype that may be expected when platelet aggregation alone is inhibited.     

Anti-fibrinogen antibody mediates fibrinogen binding to platelet membrane glycoprotein IIb-IIIa

Summary The binding of fibrinogen to platelets requires the agonist activation of platelet membrane glycoprotein IIb/IIIa. We have now found an anti-fibrinogen polyclonal antibody (YCU-R3) that increases the fibrinogen affinity of GPIIb/IIIa-binding function (activation) and subsequent platelet aggregation. The addition of intact IgG, F(ab)₂ fragments or Fab fragments induced platelet aggregation. The antibody-mediated fibrinogen binding was specific and saturable. This binding was inhibited by native fibrinogen, the RGDS peptide, the peptide of the C-terminus γ chain of fibrinogen (γ397–411), and the anti-GPIIb/IIIa monoclonal antibody (LJ-CP8). The antibody-dependent fibrinogen binding was similar to that induced by ADP. Moreover, after pretreatment with the anti-fibrinogen antibody and fibrinogen, formalin-fixed platelets bound to fibrinogen saturably. These results suggest that this anti-fibrinogen antibody may function as partial agonist.     

Structural changes in platelet glycoprotein IIb/IIIa by plasmin: determinants and functional consequences

Plasmin exposure modulates platelet aggregation responses, but a direct effect of plasmin on the platelet fibrinogen receptor, glycoprotein IIb/IIIa (GPIIb/IIIa), has never been conclusively shown in a plasma milieu. To examine this issue, we incubated platelets in platelet-rich plasma with plasmin and measured the effect of this treatment on platelet aggregation, fibrinogen binding, and the structural integrity of GPIIb/IIIa. Plasmin treatment reduced maximal reversible fibrinogen binding in a dose-dependent fashion, and this reduction in binding was accompanied by a correlative reduction in the maximal rate of aggregation. Immunoblots performed with polyclonal antibodies against GPIIb/IIIa showed that GPIIIa had been cleaved by plasmin, but this cleavage was detected only after subsequent degradation of the solubilized GPIIb/IIIa with Staphylococcus aureus V8 (Glu-C) endoprotease. Peptide sequence analysis showed that cleavage occurred at the lys444-pro445 bond in the first cysteine-rich repeat domain of GPIIIa a unique proteolytic event observed only in the presence of plasma fibrinogen. These observations suggest that plasmin modifies GPIIIa by a unique proteolytic event in plasma that is dependent on fibrinogen binding and, consequently, is accompanied by significant reductions in fibrinogen binding and aggregation response.     

Is platelet adhesion assay able to quantify drug-induced platelet dysfunction?

The platelet adhesion assay (PADA) is an innovative method for the detection of both normal, pathologically increased or decreased platelet adhesiveness. Adhesion is the first important phase of platelet activation, followed by shape change and aggregation. Adhesion is triggered by glycoproteins (GP) on the platelet surface, mainly by GPIIb/IIIa, and to a lesser extent also by GPIb/V/IX. Since fibrinogen serves as adhesive protein for GPIIb/IIIa receptors, and since the PADA uses polymer particles that become coated with fibrinogen, the PADA is able to monitor GPIIb/IIIa receptor antagonists and to detect overdosing, potentially leading to bleeding complications. Ex vivo, citrated whole blood from healthy volunteers and patients was spiked with increasing GPIIb/IIIa inhibitor concentrations and PADA was measured. Comparing these results with GPIIb/IIIa receptor occupancy, determined by FACS, a basic consistency of the data was shown. Via intracellular signaling, the adenosine diphosphate (ADP) receptor mechanism is closely involved in the activation of GPIIb/IIIa receptors so that also ADP receptor antagonists of the thienopyridine type, especially clopidogrel, can be quantitatively determined by the PADA. In patients under clopidogrel therapy, the therapeutic effect was monitored and also individual dose adjustments were realized. Furthermore, patients having partial or full clopidogrel resistance were identified. Overdoses can be detected as well.     

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.