A new murine monoclonal antibody reports an activation-dependent change in the conformation and/or microenvironment of the platelet glycoprotein IIb/IIIa complex.

Considerable evidence indicates that the glycoprotein (GP) IIb/IIIa complex on human platelets functions as a receptor for fibrinogen, but little is known about the mechanism of receptor "exposure." To investigate this mechanism, our previously described murine monoclonal antibody (10E5) and a new monoclonal antibody (7E3), both of which block the binding of fibrinogen to platelets and bind to GPIIb and/or GPIIIa, were radiolabeled and their rates of binding to native and ADP-activated platelets were studied. At low concentrations, 125I-10E5 bound nearly equally rapidly to both native and activated platelets, whereas 125I-7E3 bound slowly to native platelets and much more rapidly to activated platelets. This increased rate of 7E3 binding is unlikely to be due to an increase in the number of GPIIb/IIIa sites on the surface of activated platelets because: (a) the rate of 10E5 binding was unchanged; (b) the total number of surface GPIIb/IIIa sites increased by only 2-10% with activation as judged by equilibrium binding of near-saturating concentrations of 10E5 and 7E3, and (c) there was less than 1% release of platelet factor 4 with activation, indicating minimal fusion of alpha-granule membranes (a potential source of GPIIb/IIIa) with the plasma membrane. Other activators (epinephrine, thrombin, and ionophore A 23187) also increased the rate of 7E3 binding, as did digestion of platelets with chymotrypsin. Aspirin did not affect the rate of binding of 7E3, whereas apyrase, prostaglandin E1, and dibucaine all inhibited the enhancement of the 7E3-binding rate produced by ADP. These data provide evidence for an activation-dependent change in the conformation and/or microenvironment of the GPIIb/IIIa complex, and offer a method of studying the receptor exposure mechanism that does not rely on the binding of fibrinogen itself.     

Independent modulation of von Willebrand factor and fibrinogen binding to the platelet membrane glycoprotein IIb/IIIa complex as demonstrated by monoclonal antibody.

In this study we have used two new monoclonal antibodies, designated LJP5 and LJP9, as well as a previously described one, AP2, all specific for the platelet membrane glycoprotein (GP)IIb/IIIa complex. None of them reacted with dissociated GPIIb or GPIIIa. The monovalent Fab fragment of both LJP5 and LJP9 bound to unstimulated platelets in a saturable manner, but binding was markedly decreased after platelets had been incubated at 37 degrees C in the absence of added extracellular calcium. The binding of LJP9 was not affected by AP2, but was blocked by excess LJP5. On the contrary, the binding of LJP5 was blocked in the presence of both AP2 and LJP9. Thus, these antibodies bound to distinct epitopes of GPIIb/IIIa. At saturation, the binding to unstimulated platelets was between 2.41 and 10.9 X 10(4) molecules/platelet for LJP5 and between 3.47 and 9.1 X 10(4) molecules/platelet for LJP9 (range of 11 and 10 experiments, respectively). Binding increased up to 50% after thrombin stimulation. The estimated association constant, Ka, was 2.7 X 10(7) M-1 for LJP5 and 3.85 X 10(7) M-1 for LJP9. Both LJP5 and LJP9 partially inhibited the association of 45Ca2+ with the surface of unstimulated platelets. Moreover, both antibodies blocked the binding of von Willebrand factor (vWF) to stimulated platelets, whereas only LJP9, but not LJP5, blocked fibrinogen binding. LJP9 was also a potent inhibitor of platelet aggregation, whereas LJP5 was without effect in this regard. The results of the present study demonstrate that independent modulation of vWF and fibrinogen binding to stimulated platelets can be attained with monoclonal antibodies directed against distinct epitopes of GPIIb/IIIa.     

Asialo von Willebrand factor interactions with platelets. Interdependence of glycoproteins Ib and IIb/IIIa for binding and aggregation.

Asialo von Willebrand factor (AS-vWf) binds to and aggregates normal human platelets in the absence of ristocetin. Maximal specific binding of AS-vWf is 1-2 micrograms vWf protein/10(8) platelets. Despite the specificity of the binding, only 60% of the bound AS-vWf can be dissociated after equilibrium has been reached. We investigated the site of binding and the mechanism of aggregation of platelets by AS-vWf by (a) pre-incubating platelets with either of two monoclonal antibodies, one against glycoprotein Ib (GPIb) or a second against the glycoprotein IIb/IIIa complex (GPIIb/IIIa), and (b) varying the concentration of fibrinogen in the medium. The results of our studies indicate that AS-vWf binds initially to GPIb. This binding then results in the exposure of receptors for AS-vWf on GPIIb/IIIa. In the presence of plasma fibrinogen, both AS-vWf and fibrinogen bind to GPIIb/IIIa. In the presence of plasma fibrinogen, 50% more AS-vWf binds to the platelet, and this additional AS-vWf binds almost exclusively to GPIIb/IIIa. Despite this enhanced binding of AS-vWf in the absence of fibrinogen, platelet aggregation is much less than that which occurs in the presence of plasma fibrinogen. Comparative studies of AS-vWf binding to normal platelets and the platelets of patients with Glanzmann's thrombasthenia reveal decreased binding to the thrombasthenic platelets and a marked decrease in the extent of platelet aggregation. These studies indicate that AS-vWf binding to, and ensuing aggregation of, platelets is different from that observed with intact vWf protein when platelets are stimulated with either ristocetin or thrombin. The AS-vWf binds to GPIb which, in turn, makes additional AS-vWf receptors available on GPIIb/IIIa. If plasma fibrinogen is present, it competes with the AS-vWf for binding to GPIIb/IIIa and causes aggregation of platelets. In the presence of plasma fibrinogen, more of the AS-vWf binds to GPIIb/IIIa, but this AS-vWf is much less effective than fibrinogen in supporting platelet aggregation.     

Plasminogen interacts with human platelets through two distinct mechanisms.

Glu-plasminogen, the native form of plasminogen, interacts in a specific and saturable manner with unstimulated human platelets, and the binding is enhanced fivefold by thrombin stimulation (Miles and Plow, 1985. J. Biol. Chem. 260:4303). This study characterizes the nature of the Glu-plasminogen binding sites by analyzing platelets deficient in selected proteins and functions. Platelets from patients with afibrinogenemia, Gray platelet syndrome, and the Cam Variant of thrombasthenia, a form of thrombasthenia with near normal levels of glycoprotein IIb/IIIa (GPIIb/IIIa), showed minimal augmentation of plasminogen binding to thrombin-stimulated platelets but normal binding to unstimulated platelets. This selective deficiency indicates that two distinct mechanisms are involved in the interaction of plasminogen with platelets. These abnormal platelets share a deficiency in fibrinogen. Surface expression of platelet fibrinogen, however, was not sufficient for enhanced plasminogen binding to stimulated platelets, and experiments with alpha-thrombin and gamma-thrombin indicated that fibrin formation on the platelet surface is necessary for the augmented plasminogen binding. Unstimulated and stimulated thrombasthenic platelets deficient in GPIIb/IIIa bound markedly reduced levels of plasminogen, which suggests a role for GPIIb/IIIa in plasminogen binding to unstimulated platelets. Treatment of platelets to dissociate the heterodimeric complex of GPIIb/IIIa did not significantly perturb plasminogen binding to unstimulated platelets, but the complex may be necessary for thrombin-stimulated plasminogen binding via its interaction with platelet fibrin.     

Vitronectin binds to activated human platelets and plays a role in platelet aggregation.

Vitronectin (Vn) is a multifunctional 75-kD glycoprotein that is present in plasma and the extracellular matrix. Vn functions as a complement regulatory protein in plasma, and promotes the growth and attachment of cells in tissue culture. Recent cDNA cloning reveals that like other adhesive proteins, Vn contains the sequence Arg-Gly-Asp and binds to some members of the integrin class of adhesive membrane receptors. In liposomes, the platelet membrane glycoprotein complex IIb/IIIa binds Vn, as well as fibrinogen, von Willebrand factor, and fibronectin. We examined the binding of purified Vn to resting and stimulated human platelets. Vn bound to thrombin-stimulated platelets in a calcium-dependent, specific, and saturable manner with a Kd of 320 nM and 8,000 sites per platelet. Epinephrine or ADP stimulation led to specific binding with KdS of 93 and 116 nM, respectively. Binding was inhibited by the tetrapeptide Arg-Gly-Asp-Ser and by monoclonal and polyclonal antibodies to GPIIb/IIIa. Endogenous platelet Vn stores were identified in immunoblots of gel-filtered platelets and the surface expression of endogenous platelet Vn was thrombin inducible. Monoclonal as well as polyclonal antibodies to Vn inhibited platelet aggregation, suggesting that Vn plays a role in the formation of stable platelet aggregates.     

Reversibility versus persistence of GPIIb/IIIa blocker-induced conformational change of GPIIb/IIIa (alphaIIbbeta3, CD41/CD61)

Clinically used GPIIb/IIIa blockers are ligand mimetics, and thereby their binding can induce conformational changes of the platelet integrin GPIIb/IIIa. Since the reversibility of these conformational changes may be an important determinant of potential adverse effects of GPIIb/IIIa blockers, we produced a new monoclonal antibody (anti-LIBS-mAb), and by using its binding properties, we investigated the conformational changes of GPIIb/IIIa during the binding and especially the dissociation of GPIIb/IIIa blockers. Production of monoclonal antibody (mAb) clones was performed using purified GPIIb/IIIa in a high affinity conformation and using activated platelets. Clone anti-LIBS-145-mAb was chosen, since it allowed the sensitive probing of eptifibatide-induced conformational changes of GPIIb/IIIa. On resting and activated platelets and on GPIIb/IIIa-expressing Chinese hamster ovary cells, anti-LIBS-145-mAb binding returned to background binding after dissociation of eptifibatide, indicating a complete reversibility of the eptifibatide-induced conformational change. Furthermore, with the mixing of eptifibatide-preincubated and nonincubated cells, a fast reversibility could be demonstrated. However, when fibrinogen was present in a physiological concentration, the GPIIb/IIIa blocker-induced conformation was partially retained after the dissociation of eptifibatide and to the same extent binding of fibrinogen and the activation-specific mAb Pac-1 was induced. In conclusion, a fast reversibility of the conformational change of GPIIb/IIIa after dissociation of GPIIb/IIIa blockers could be demonstrated as an intrinsic property of the GPIIb/IIIa receptor. This mechanism prevents general platelet aggregation after dissociation of ligand mimetic GPIIb/IIIa blockers. Nevertheless, in the presence of fibrinogen this reversibility is not complete, which may explain some of the side effects of GPIIb/IIIa blockers, especially those of the oral GPIIb/IIIa blockers.     

Studies of activated GPIIb/IIIa receptors on the luminal surface of adherent platelets. Paradoxical loss of luminal receptors when platelets adhere to high density fibrinogen.

The accessibility of activated GPIIb/IIIa receptors on the luminal surface of platelets adherent to damaged blood vessels or atherosclerotic plaques is likely to play a crucial role in subsequent platelet recruitment. To define better the factors involved in this process, we developed a functional assay to assess the presence of activated, luminal GPIIb/IIIa receptors, based on their ability to bind erythrocytes containing a high density of covalently coupled RGD-containing peptides (thromboerythrocytes). Platelets readily adhered to wells coated with purified type I rat skin collagen and the adherent platelets bound a dense lawn of thromboerythrocytes. With fibrinogen-coated wells, platelet adhesion increased as the fibrinogen-coating concentration increased, reaching a plateau at about 11 micrograms/ml. Thromboerythrocyte binding to the platelets adherent to fibrinogen showed a paradoxical response, increasing at fibrinogen coating concentrations up to approximately 4-6 micrograms/ml and then dramatically decreasing at higher fibrinogen-coating concentrations. Scanning electron microscopy demonstrated that the morphology of platelets adherent to collagen was similar to that of platelets adherent to low density fibrinogen, with extensive filopodia formation and ruffling. In contrast, platelets adherent to high density fibrinogen showed a bland, flattened appearance. Immunogold staining of GPIIb/IIIa receptors demonstrated concentration of the receptors on the filopodia, and depletion of receptors on the flattened portion of the platelets. Thus, there is a paradoxical loss of accessible, activated GPIIb/IIIa receptors on the luminal surface of platelets adherent to high density fibrinogen. Two factors may contribute to this result: engagement of GPIIb/IIIa receptors with fibrinogen on the abluminal surface leading to the loss of luminal receptors, and loss of luminal filopodia that interact with thromboerythrocytes. These data provide insight into the differences in thrombogenicity between surfaces, and may provide a mechanism for purposefully passivating platelet-reactive artificial surfaces.     

Fresh, liquid-preserved, and cryopreserved platelets: adhesive surface receptors and membrane procoagulant activity

BACKGROUND: A study in humans showed that the transfusion of previously frozen human platelets after cardiopulmonary bypass, despite decreased survival, resulted in better hemostatic function than that of liquid-preserved platelets stored at 22 degrees C for 3 to 4 days. STUDY DESIGN AND METHODS: In this study, fresh, 3- to 4-day-old liquid-preserved, and cryopreserved human platelets were studied by the use of monoclonal antibodies directed against p-selectin, glycoprotein (GP)Ib, activated GPIIb/IIIa, and coagulation factor V in a three-color flow cytometric method. RESULTS: The fresh and liquid-preserved platelets had normal surface levels of GPIb, while the cryopreserved platelets were composed of distinct subpopulations of GPIb-normal and GPIb-reduced platelets. On the basis of the binding of factor V, both subpopulations of cryopreserved platelets exhibited greater surface binding of factor V than did fresh and liquid-preserved platelets. Activated GPIIb/IIIa was elevated on GPIb-normal platelets, but not on GPIb-reduced platelets. Baboon platelets frozen by a procedure identical to that used to freeze human platelets also had GPIb-normal and GPIb-reduced subpopulations after the freezing-thawing-washing procedure. Autologous cryopreserved baboon platelets labeled with biotin-X-N-hydroxysuccinimide showed a rapid removal of GPIb-reduced platelets during the 5-minute postinfusion period, whereas GPIb-normal platelets had an in vivo recovery of 48 percent and a lifespan of slightly less than 6 days. CONCLUSIONS: Improved in vivo function of cryopreserved platelets may be related to the rapid hemostatic effect of the GPIb-reduced subpopulation secondary to increased binding of factor V and expression of p-selectin.     

Blood group A antigen expression in platelets is prominently associated with glycoprotein Ib and IIb. Evidence for an A1/A2 difference

Blood group ABH antigens are associated with platelets as intrinsic determinants and extrinsically adsorbed antigens, and exist both on glycosphingolipids and on glycoproteins (GPs). We now provide direct evidence that the blood group ABH antigens are prominently associated with platelet GPIb and GPIIb. By immunoprecipitation, a murine monoclonal anti-A antibody precipitated surface-biotin-labelled blood group A₁ platelet membrane proteins with electrophoretic characteristics identical to those of GPIb/IX and GPIIb/IIIa. By immunoblotting of SDS-PAGE separated blood group A₁ platelet proteins the monoclonal anti-A antibody bound to proteins with electrophoretic characteristics identical to those of GPIb and GPIIb. When immunoaffinity purified GPIb/IX and GPIIb/IIIa, derived from blood group O, A₁ and A₂ platelets, were employed for immunoblotting, GPIb and GPIIb only from A₁ platelets bound the monoclonal anti-A antibody. By ELISA, wherein monoclonal antibodies specific for GPIb (AP1) and the GPIIb/IIIa complex (AP2) were used to capture and hold antigens from platelet lysate, human anti-A antibodies reacted with these proteins derived from blood group A₁ platelets; proteins from blood group A₂, O and B platelets showed no reactivity. These results indicate that blood group A antigen is associated with GPIb and GPIIb derived from blood group A₁ but not A₂ platelets.     

血小板活化过程中膜糖蛋白Ⅱb/Ⅲa构象变化的研究

目的　对血小板活化过程中膜糖蛋白（ Ｇ Ｐ） Ⅱｂ／ Ⅲａ 构象变化进行初步探讨。方法　分别用供体荧光（ Ｆ Ｉ Ｔ Ｃ） 与受体荧光（ Ｔ Ｒ） 标记识别 Ｇ ＰⅡｂ／ Ⅲａ 上不同抗原决定簇的单抗。用流式细胞仪检测活化血小板在５３０ ｎｍ 处的荧光强度值,并计算荧光供受体间的荧光共振能量转移值（ Ｆ Ｒ Ｅ Ｔ Ｖ） 。结果　不论何种单抗作为荧光供体,静息态血小板均可被测得一低而稳定的 Ｆ Ｒ Ｅ Ｔ Ｖ（ 平均５ ．５ ％ ） 。血小板被激活时 Ｆ Ｒ Ｅ Ｔ Ｖ 会有显著升高,表明 Ｇ ＰⅡｂ／ Ⅲａ 内的亚单位间发生了位置或（ 和） 方向上的变化,该变化也可因胞外钙离子的清除而发生,但不依赖于纤维蛋白原与其受体的结合。结论　血小板活化时 Ｆ Ｒ Ｅ Ｔ Ｖ 的升高可定性反映出荧光标记单抗所结合的 Ｇ ＰⅡｂ／ Ⅲａ 内部亚单位间所发生的重新排列,这种构象的改变可最终导致纤维蛋白原受体的表达。     

Platelet structure and function: immunocytochemical localizations of membrane glycoprotein and alpha-granule protein]

The platelet membrane glycoproteins (GPs) are receptors or binding sites for adhesive proteins. GPIb and GPIIb/IIIa complex are major glycoproteins and have important roles, functionally. GPIb plays an essential role in primary hemostasis as receptor for the von Willebrand factor. The GPIIb/IIIa complex acts as the binding site for adhesive proteins on activated platelets and, as such, is essential for platelet aggregation. On the other hand, four adhesive proteins (fibrinogen, fibronectin, thrombospondin and von Willebrand factor), which are present not only in plasma but also in alpha-granules, mediate or modulate the platelet adhesive response. The interaction between these adhesive proteins and platelet membrane GPs are essential for platelet adhesion and aggregation. The present report will focus on the localization of GPIb and GPIIb/IIIa on the platelet surface and that of adhesive proteins in alpha-granules in both resting and activated human platelets.     

Drug-Antibody-Platelet Interaction in Quinine- and Quinidine-induced Thrombocytopenia

Binding of quinine- and quinidine-dependent antibodies to platelets was studied using an electroimmunoassay to measure platelet-bound IgG. Antibodies from four patients with drug-induced thrombocytopenia differed significantly in their interaction with platelets: association constants for binding to platelets at high drug concentrations ranged from 0.29 to 2.6 x 10(7) M(-1), the maximum number of antibody molecules bound ranged from 36,000 to 161,000/platelet, the amount of drug necessary to achieve half-maximum binding of antibodies to platelets ranged from 2 to 60 muM, and only one of the antibodies cross-reacted with the stereoisomer of the drug to which the patient was sensitized. Binding of the antibodies to platelets was enhanced at the highest achievable molar ratio of drug:antibody, 10,000:1, rather than being inhibited, as would be expected in a conventional, hapten-dependent reaction. The drug-antibody-platelet reaction was unaffected by Factor VIII/von Willebrand protein, nonspecifically aggregated IgG, or heat-labile complement components. After pretreatment with tritiated quinine, platelets retained several hundred thousand molecules of drug each, but failed to bind detectable amounts of antibody. However, platelets treated simultaneously with quinine-dependent antibody and tritiated quinine retained significantly more drug after repeated washes than platelets treated with drug and normal serum.These findings support the proposition that in quinine- and quinidine-induced thrombocytopenia, drug and antibody combine first in the soluble phase to form a complex, which then binds with high affinity to a receptor on the platelet surface (innocent bystander reaction), and demonstrate that these antibodies are heterogeneous in respect to the amount of drug required to promote their binding to platelets, the number of platelet receptors they recognize, and their binding affinities.     

Response of neonatal platelets to nitric oxide in vitro

OBJECTIVES: Several studies have demonstrated altered platelet function during nitric oxide inhalation (iNO) in adults and neonates. In vitro NO inhibits activation of fibrinogen receptor glycoprotein (GP) IIb/IIIa in a dose-dependent manner. In neonates GPIIb/IIIa response to stimulation is physiologically attenuated during the first days after birth in comparison to adults; the effects of NO on GPIIb/IIIa in neonates, however, are less established. We investigated the response of platelets from neonates, their mothers, and nonpregnant controls to the NO donor SIN-1 in vitro. DESIGN: Umbilical cord and venous (mother, controls) platelet-rich plasma was stimulated in vitro with 10 microM ADP or 0.05 U/ml thrombin in the presence or absence of 10 microM SIN-1. GPIIb/IIIa activation was determined by two-color flow cytometry. SETTING: Delivery department of an university hospital. PATIENTS AND PARTICIPANTS: Ten healthy term neonates, their mothers and nonpregnant controls. MEASUREMENTS AND RESULTS: NO significantly reduced GPIIb/IIIa activation in thrombin- and ADP-stimulated platelets in all groups (p < 0.001). Neonatal platelets were significantly hyporeactive to stimulation (p < 0.05), but the relative response to SIN-1 was similar in all three groups (70 +/- 5 %). CONCLUSIONS: The relative amount of NO-induced inhibition of GPIIb/ IIIa activation in neonates is thus similar to that of adults. However, due to the intrinsic hyporesponsiveness of neonatal platelets and NO-synergistic pharmacodynamic profiles of other drugs (e.g., prostacyclin), possible adverse effects of iNO must be considered.     

Platelet hyperprocoagulant activity in Type 2 diabetes mellitus: attenuation by glycoprotein IIb/IIIa inhibition

Summary. Background: Platelets are hyperactive in Type 2 diabetes mellitus (T2DM), and antiplatelet treatment with glycoprotein (GP) IIb/IIIa inhibitors provides better thrombotic protection in DM than in non‐diabetic subjects. Objective: We hypothesized that diabetic platelets are hyperprocoagulant, and that this hyperactivity can be inhibited by GPIIb/IIIa blockade. Methods: Patients with T2DM and gender/age/body mass index‐matched non‐diabetic controls were recruited (n = 12 for both) to study the effect of GPIIb/IIIa blockade on platelet procoagulant activity. Platelet phosphotidylserine (PS), factor (F) Va expression, and platelet‐derived microparticle (PDMP) generation were measured by whole blood flow cytometry. Platelet‐dependent thrombin generation and plasma clotting time were monitored in recalcified platelet‐rich plasma. Results: Compared to controls, basal platelet activation was similar, while thrombin receptor activating peptide stimulated activation was enhanced in patients with T2DM. Diabetic platelets also displayed more profound elevations of platelet PS exposure, FVa binding, and PDMP generation upon stimulation. These alterations resulted in a hyperprocoagulant state, as evidenced by a marked increase in the platelet procoagulant index, enhanced thrombin generation, and a shortened plasma clotting time. GPIIb/IIIa blockade by c7E3 or SR121566 decreased platelet PS exposure and FVa binding, and diminished platelet procoagulant activity in patients with T2DM. Conclusions: Platelets have increased procoagulant activity in patients with T2DM. The hyperprocoagulant activity is counteracted by GPIIb/IIIa blockade.     

Development of fibrinogen γ-chain peptide-coated, adenosine diphosphate-encapsulated liposomes as a synthetic platelet substitute

Summary.  Background : The dodecapeptide HHLGGAKQAGDV (H12), corresponding to the fibrinogen γ-chain carboxy-terminal sequence (γ400-411), is a specific binding site of the ligand for platelet GPIIb/IIIa complex. We have evaluated H12-coated nanoparticles (polymerized albumin or liposome) as platelet function-supporting synthetic products. Objectives : To strengthen the hemostatic ability of H12-coated particles as a platelet substitute, we exploited installation of a drug delivery function by encapsulating adenosine diphosphate (ADP) into liposomes [H12-(ADP)-liposomes]. Methods and results : Via selective interaction with activated platelets through GPIIb/IIIa, H12-(ADP)-liposomes were capable of augmenting agonist-induced platelet aggregation by releasing ADP in an aggregation-dependent manner. When intravenously injected into rats, liposomes were readily targeted to sites of vascular injury as analyzed on computed tomography. In fact, comparable to fresh platelets, liposomes exhibited considerable hemostatic ability for correcting prolonged bleeding time in a busulphan-induced thrombocytopenic rabbit model. In addition, the liposomes showed no activating or aggregating effects on circulating platelets in normal rabbits. Conclusion : H12-(ADP)-liposome may thus offer a promising platelet substitute, being made with only synthetic materials and exerting hemostatic functions in vivo via reinforcement of primary thrombus formation by residual platelets in thrombocytopenia at sites of vascular injury, but not in circulation.     

Type IIB von Willebrand factor with normal sialic acid content induces platelet aggregation in the absence of ristocetin. Role of platelet activation, fibrinogen, and two distinct membrane receptors.

Three preparations of purified von Willebrand factor (vWF), obtained from unrelated patients affected by type IIB von Willebrand disease, were found to have normal sialic acid content (between 129 and 170 nmol/mg of vWF, as compared with 158 +/- 17 nmol/mg in four normal preparations) and to induce platelet aggregation in the presence of physiologic levels of divalent cations and without addition of ristocetin. A monoclonal antibody that blocks the vWF binding domain of the platelet glycoprotein (GP)Ib caused complete inhibition of IIB vWF-induced aggregation. In contrast, a monoclonal antibody that blocks the receptor for adhesive proteins on the platelet GPIIb/IIIa complex failed to inhibit the initial response of platelets to high concentrations of IIB vWF. Moreover, IIB vWF caused agglutination of formalin-fixed platelets that was blocked only by the anti-GPIb antibody, suggesting that the binding of vWF to GPIb, even in the absence of ristocetin, results in platelet-platelet interaction that is followed by exposure of the GPIIb/IIIa receptors for adhesive proteins. Endogenous ADP, normally active platelet metabolism and fibrinogen binding to GPIIb/IIIa were necessary for maximal and irreversible platelet aggregation. In the absence of fibrinogen, however, aggregation was mediated by vWF binding to GPIIb/IIIa. A 52/48-kD tryptic fragment containing the GPIb binding domain of normal vWF completely blocked the aggregation induced by all three IIB vWF preparations. The present study defines in detail the mechanisms involved in IIB vWF-induced platelet aggregation. Moreover, it establishes that the GPIb binding domain of normal and IIB vWF are closely related and that desialylation is not required for the direct interaction of IIB vWF with GPIb.     

On the association of the platelet-specific alloantigen, Pena, with glycoprotein IIIa. Evidence for heterogeneity of glycoprotein IIIa.

Neonatal alloimmune thrombocytopenic purpura associated with a new platelet-specific alloantigen Pena has been reported. We now provide direct evidence that the Pena determinant is associated with glycoprotein (GP) IIIa, but that it is distinct from epitopes that define the PlA system. By ELISA wherein monoclonal antibodies specific for GPIIb (Tab) and specific for GPIIIa (AP3) were used to capture and hold antigens from a platelet lysate prepared under conditions that generate free GPIIb and GPIIIa, anti-Pena reacted with GPIIIa held by AP3 but not with GPIIb held by Tab. In an alternative ELISA where purified GPIIIa from both PlA1-positive and PlA1-negative platelets were used individually as antigen, anti-Pena reacted with both allelic forms of GPIIIa. By radioimmuno-precipitation, anti-Pena precipitated a single surface-labeled membrane protein with electrophoretic characteristics in sodium dodecyl sulfate-polyacrylamide gels, under nonreduced or reduced conditions, identical to those of GPIIIa. By fluorocytometry, platelets from several donors, regardless of PlA phenotype, bound an amount of anti-Pena roughly equivalent to one-half that amount of anti-PlA1 bound by PlA1 homozygous (A1/A1) platelets and roughly equal to that amount of anti-PlA1 bound by PlA1 heterozygous (A1/A2) platelets. Using platelets from donors typed homozygous for PlA1 and Pena in a quantitative indirect binding assay, 14-24,000 molecules of anti-Pena and 41-51,000 molecules of anti-PlA1 were bound per platelet at saturation. Anti-Pena completely inhibited ADP-induced aggregation of Pena-positive platelets, regardless of PlA phenotype. These results indicate that the Pena determinant is associated with GPIIIa but distinct from PlA.     

Inhibition of platelet-mediated,tissue factor-induced thrombin generation by the mouse/human chimeric 7E3 antibody. Potential implications for the effect of c7E3 Fab treatment on acute thrombosis and "clinical restenosis".

The murine/human chimeric monoclonal antibody fragment (c7E3 Fab) blocks GPIIb/IIIa and alpha v beta 3 receptors, inhibits platelet aggregation, and decreases the frequency of ischemic events after coronary artery angioplasty in patients at high risk of suffering such events. Although inhibition of platelet aggregation is likely to be the major mechanism of c7E3 Fab''s effects, since activated platelets facilitate thrombin generation, it is possible that c7E3 Fab also decreases thrombin generation. To test this hypothesis, the effects of c7E3 Fab and other antiplatelet agents were tested in a thrombin generation assay triggered by tissue factor. c7E3 Fab produced dose-dependent inhibition of thrombin generation, reaching a plateau of 45-50% inhibition at concentrations > or = 15 micrograms/ml. It also inhibited thrombin-antithrombin complex formation, prothrombin fragment F1-2 generation, platelet-derived growth factor and platelet factor 4 release, incorporation of thrombin into clots, and microparticle formation. Antibody 6D1, which blocks platelet GPIb binding of von Willebrand factor, had no effect on thrombin generation, whereas antibody 10E5, which blocks GPIIb/IIIa but not alpha v beta 3 receptors decreased thrombin generation by approximately 25%. Combining antibody LM609, which blocks alpha v beta 3 receptors, with 10E5 increased the inhibition of thrombin generation to approximately 32-41%. The platelets from three patients with Glanzmann thrombasthenia, who lacked GPIIb/IIIa receptors but had normal or increased alpha v beta 3 receptors, supported approximately 21% less thrombin generation than normal platelets. We conclude that thrombin generation initiated by tissue factor in the presence of platelets is significantly inhibited by c7E3 Fab, most likely in part through both GPIIb/IIIa and alpha v beta 3 blockade, and that this effect may contribute to its antithrombotic properties.     

Thrombin binding to thrombasthenic platelets

Platelets from two patients with Glanzmann's thrombasthenia showed decreased iodination of surface glycoproteins GPIIb and GPIII. Despite these changes, the binding of [125I] alpha-thrombin to the thrombasthenic platelets was normal. Binding was linear up to a thrombin concentration of 0.1 to 0.2 U/ml, at which point a change in the slope of the binding curve was observed. At lower concentrations of thrombin, 1,000 to 2,000 molecules of thrombin were bound per platelet, with an apparent Kdiss of 0.1 to 0.3 U/ml. With high concentrations of thrombin, thrombasthenic platelets bound 30,000 to 65,000 molecules of thrombin per platelet at saturation, with an apparent Kdiss of 5 to 10 U/ml. The release of [14C]serotonin by thrombasthenic platelets as a function of thrombin concentration was also similar to release by normal platelets. These studies indicate that the receptor(s) for thrombin on the plasma membrane of platelets from patients with Glanzmann's disease are intact and that membrane glycoproteins GPIIb and GPIII play little or no role in either the initial binding of thrombin to platelets or the transmission of this surface stimulus to release-inducing mechanisms.     

Platelets have more than one binding site for von Willebrand factor.

The binding of 125I-von Willebrand factor (125I-vWF) to platelets stimulated by thrombin, ADP, and a combination of ADP + epinephrine (EPI) is specific, saturable, and reversible. Active platelet metabolism and divalent cations are required for binding induced by these stimuli, but not by ristocetin, suggesting the existence of different mechanisms involved in the vWF-platelet interaction. A monoclonal antibody directed against an epitope of membrane glycoprotein (GP) Ib had no effect on the binding of 125I-vWF to normal platelets stimulated by thrombin or a combination of ADP + EPI, but completely blocked ristocetin-induced binding. Binding induced by thrombin to GPIb-blocked platelets was specific. Moreover, thrombin-induced binding of 125I-vWF was increased, rather than decreased, in two patients with the Bernard-Soulier syndrome whose platelets lacked GPIb. Conversely, monoclonal antibodies directed against the GPIIb/IIIa complex had no effect on ristocetin-induced binding of 125I-v-WF to normal platelets, but blocked thrombin- and ADP + EPI-induced binding. To exclude effects mediated by the platelet Fc receptor, a monoclonal IgG directed against an epitope present on human B cells and monocytes, but not expressed on resting or stimulated platelets, was used. It did not affect 125I-vWF binding induced by any of the stimuli. These studies show that platelets have more than one binding site for vWF, and that they may be exposed by different stimuli.