Von Willebrand''s disease with spontaneous platelet aggregation induced by an abnormal plasma von Willebrand factor.

We have investigated and characterized the abnormalities in four unrelated patients with von Willebrand's disease (vWd) who have (a) enhanced ristocetin-induced platelet aggregation (RIPA) at low ristocetin concentrations, (b) absence of the largest plasma von Willebrand factor (vWf) multimers, and (c) thrombocytopenia. The platelet-rich plasma of these patients aggregates spontaneously without the addition of any agonists. When isolated normal platelets are resuspended in patient plasma spontaneous aggregation occurs; however, the patients' plasmas did not induce platelet aggregation of normal washed formalinized platelets. When the patients' platelets are suspended in normal plasma, spontaneous aggregation is not observed. The spontaneous platelet aggregation (SPA) is associated with dense granule secretion as measured by ATP release and alpha granule release as measured by beta-thromboglobulin and platelet factor 4 release. The SPA is totally inhibited by 5 mM EDTA, prostaglandin I2, and dibutryl cyclic AMP, while it is only partially inhibited by 1 mM EDTA, acetylsalicylic acid, or apyrase. A monoclonal antibody directed against glycoprotein Ib (GPIb) and/or a monoclonal antibody against the glycoprotein IIb/IIIa (GPIIb/IIIa) complex totally inhibits the SPA. The vWf was isolated from the plasma of one of these patients. The purified vWf induced platelet aggregation of normal platelets resuspended in either normal or severe vWd plasma, but the vWf did not induce platelet aggregation of normal platelets resuspended in afibrinognemic plasma. Sialic acid and galactose quantification of the patient's vWf revealed approximately a 50% reduction compared with normal vWf. These studies indicate that a form of vWd exists, which is characterized by SPA that is induced by the abnormal plasma vWf. The SPA is dependent on the presence of plasma fibrinogen, and the availability of the GPIb and the GPIIb/IIIa complex. In this variant form of vWd the abnormal vWf causes enhanced RIPA, SPA, and thrombocytopenia.     

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.     

Disorders of platelet function.

Platelets provide for primary hemostasis by forming a hemostatic plug at sites of vascular damage. They also provide a surface for the assembly of the coagulation protein complexes that generate thrombin, serve as a nidus for fibrin clots, and secrete factors involved in wound repair. Normal platelet function can be divided into four phases: adhesion, aggregation, secretion, and expression of procoagulant activity. Platelet adhesion initiates plug formation as platelets adhere to the connective tissue at the edges of a wound within seconds after vascular damage. When damage occurs in regions of slow blood flow, platelets adhere to subendothelial collagen, fibronectin, and laminin. However, when damage occurs in regions of rapid flow, platelet adhesion requires the presence of subendothelial von Willebrand factor (vWf) and a specific platelet receptor, the glycoprotein Ib/IX (GPIb/IX) complex. Following initial adhesion, platelets aggregate to complete the formation of a hemostatic plug. Platelet aggregation requires active platelet metabolism, platelet stimulation by agonists such as ADP, thrombin, collagen, or epinephrine; the presence of calcium or magnesium ions and specific plasma proteins such as fibrinogen or vWf; and a platelet receptor, the glycoprotein IIb/IIIa (GPIIb/IIIa) complex. Thus, platelet stimulation results in the generation of intracellular second messengers that transmit the stimulus back to the platelet surface, exposing protein binding sites on GPIIb/IIIa. Fibrinogen (or vWf) then binds to GPIIb/IIIa and crosslinks adjacent platelets to produce platelet aggregates. Platelet stimulation also results in platelet secretion and the elaboration of platelet procoagulant activity. During secretion, substances are released to propagate the aggregation response and to promote wound healing; the expression of procoagulant activity localizes thrombin generation to the site of vascular damage. Disorders of platelet function can be divided into those of congenital and those of acquired origin. Although congenital disorders are uncommon, acquired disorders are encountered frequently in clinical practice. Congenital absence of GPIb/IX and GPIIb/IIIa results in the Bernard-Soulier syndrome (BSS) and Glanzmann thrombasthenia (GT), respectively. Each is an autosomal recessive bleeding disorder in which absence of a protein complex renders the affected platelets incapable of undergoing either vWf-mediated adhesion (BSS) or fibrinogen-mediated aggregation (GT).(ABSTRACT TRUNCATED AT 400 WORDS)     

Interaction of asialo von Willebrand factor with glycoprotein Ib induces fibrinogen binding to the glycoprotein IIb/IIIa complex and mediates platelet aggregation.

von Willebrand factor (vWF) is necessary for the initial attachment of platelets to exposed subendothelium, particularly under flow conditions like those prevailing in the microcirculation. Little is known about its possible participation in subsequent events leading to formation of platelet thrombi at sites of vascular injury. We addressed this question by studying the mechanisms by which desialylated vWF induces platelet aggregation in the absence of any other stimulus. Asialo vWF, unlike the native molecule, does not require ristocetin to interact with platelets. Agglutination induced by ristocetin is largely independent of active platelet metabolism and only partially reflects physiological events. We have shown here that binding of asialo vWF to platelets was accompanied by release of dense granule content and subsequent ADP-dependent fibrinogen binding to receptors on the glycoprotein (GP) IIb/IIIa complex. The initial interaction of asialo vWF with platelets was mediated by GPIb, as shown by blocking obtained with monoclonal antibody. Inhibition of this initial interaction completely abolished platelet aggregation induced by asialo vWF. The same effect was obtained with a monoclonal anti-GPIIb/IIIa antibody. This, however, did not block asialo vWF binding to platelets, but rather inhibited subsequent fibrinogen binding induced by asialo vWF. Therefore, the latter process was also essential for platelet aggregation under the conditions described. At saturation, asialo vWF induced binding of between 3.2 and 27.7 X 10(3) fibrinogen molecules/platelet, with an apparent dissociation constant between 0.28 and 1.18 X 10(-6) M. This study shows that asialo, and possibly native, vWF acts as a platelet agonist after its binding to GPIb and induces aggregation through a pathway dependent on GPIIb/IIIa-related receptors.     

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.     

Identification of the thrombin receptor on human platelets by chemical crosslinking

To identify the molecular site of thrombin binding to the platelet membrane, we covalently linked 125I-thrombin to platelets by using the bifunctional chemical cross-linking agents disuccinimidyl suberate and dithiobis(succinimidyl propionate). The proteins cross-linked to 125I-thrombin by this method were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and followed by autoradiography. Two radiolabeled thrombin complexes were identified, a major species of Mr approximately 200,000 and a minor one of Mr approximately 400,000. Hirudin prevented the formation of both complexes. The radioactivity of the approximately 200,000-Mr complex was always 7-10-fold greater than the radioactivity of the approximately 400,000-Mr complex regardless of the thrombin concentration to which the platelets were exposed (0.1-29 nM). Although 125I-thrombin complexes generated with thrombasthenic platelets (lacking glycoprotein IIb/IIIa) were indistinguishable from normal, no complexes appeared when Bernard-Soulier platelets (lacking glycoprotein Ib [GPIb]) were used. Complex formation was blocked by rabbit antiglycocalicin antiserum, but not by the monoclonal antibody 6D1, which is directed against the site on GPIb where von Willebrand factor (vWf) binds in the presence of ristocetin. Although cross-linking studies suggested that vWf might partially inhibit thrombin binding to platelets, this was not confirmed by equilibrium binding studies in the presence of vWf and ristocetin. The data suggest, therefore, that at all thrombin concentrations binding occurs at the same membrane site, despite evidence from equilibrium studies for high and low affinity classes of receptors, and that the approximately 400,000-Mr complex is simply a dimer of the approximately 200,000-Mr species. We conclude that the membrane site to which thrombin binds is the glycocalicin portion of platelet GPIb at a site remote from the point of ristocetin-dependent vWf binding.     

von Willebrand factor interaction with the glycoprotein IIb/IIa complex. Its role in platelet function as demonstrated in patients with congenital afibrinogenemia.

We have studied three afibrinogenemic patients, who had only trace amounts of plasma and platelet fibrinogen as measured by radioimmunoassay, and demonstrate here that the residual aggregation observed in their platelet-rich plasma is dependent upon von Willebrand factor (vWF) binding to the platelet membrane glycoprotein (GP)IIb/IIIa complex. The abnormality of aggregation was more pronounced when ADP, rather than thrombin, collagen, or the combination of ADP plus adrenaline was used to stimulate platelets. With all stimuli, nevertheless, the platelet response was completely inhibited by a monoclonal antibody (LJP5) that is known to block vWF, but not fibrinogen binding to GPIIb/IIIa. Addition of purified vWF to the afibrinogenemic plasma resulted in marked increase in the rate and extent of aggregation, particularly when platelets were stimulated with ADP. This response was also completely blocked by LJP5. Addition of fibrinogen, however, restored normal aggregation even in the presence of LJP5, a finding consistent with the knowledge that antibody LJP5 has no effect on platelet aggregation mediated by fibrinogen binding to GPIIb/IIIa. Two patients gave their informed consent to receiving infusion of 1-desamino-8-D-arginine vasopressin (DDAVP), a vasopressin analogue known to raise the vWF levels in plasma by two- to fourfold. The bleeding time, measured before and 45 min after infusion, shortened from greater than 24 min to 12 min and 50 s in one patient and from 16 min to 9 min and 30 s in the other. Concurrently, the rate and extent of ADP-induced platelet aggregation improved after DDAVP infusion. The pattern, however, reversed to baseline levels within 4 h. The concentration of plasma vWF increased after DDAVP infusion, but that of fibrinogen remained at trace levels. We conclude that vWF interaction with GPIIb/IIIa mediates platelet-platelet interaction and may play a role in primary hemostasis.     

特发性血小板减少性紫癜患者IgG酶切片段F(ab'')2的免疫活性及其对血小板聚集功能的影响

目的制备特发性血小板减少性紫癜（ITP）患者血浆IgG及其酶切片段，探讨其与血小板GPⅡb／Ⅲa和（或）GPⅠb／Ⅸ结合的免疫活性及其对正常人血小板聚集功能的影响。方法用改良MAIPA法和比浊法血小板聚集试验筛选出自身抗体阳性并且能抑制血小板聚集的患者，用蛋白A柱纯化其血浆IgG抗体并用胃蛋白酶制备F（ab’）2片段，改良单克隆抗体俘获血小板抗原技术（MAIPA）检测完整抗体及其酶切片段与血小板膜糖蛋白的结合活性；比浊法血小板聚集试验对比观察ITP患者血浆、纯化的IgG抗体及其酶切片段对正常人血小板聚集功能的影响。结果①68例慢性ITP患者中，34例（53．6％）血浆中抗GPⅡb／Ⅲa和（或）GPⅠb／Ⅸ自身抗体阳性，其中5例（14．7％）明显抑制了二磷酸腺苷（ADP）或瑞斯托霉素对血小板聚集的诱导作用；②用蛋白A柱结合蛋白酶酶切成功获得了纯化的IgG及F（ab’）2片段；③患者纯化的IgG及F（ab’）2片段均具有抗GPⅡb／Ⅲg或GPⅠb／Ⅸ活性，但去除IgG的血浆丧失了与GPⅡb／Ⅲa或GPⅠb／Ⅸ的结合活性；④2例患者纯化的IgG及其F（ab＇）2片段抑制ADP诱导的血小板聚集。结论F（ab’）2片段是IgG自身抗体的功能片段，它不但保留了良好的抗原结合活性并可抑制血小板聚集功能，其抑制聚集作用呈剂量依赖性。     

Aggregation of chymotrypsin-treated thrombasthenic platelets is mediated by fibrinogen binding to glycoproteins IIb and IIIa

Previous experiments demonstrated that chymotrypsin, but not adenosine diphosphate (ADP), exposed fibrinogen binding sites on platelets from patients with Glanzmann's thrombasthenia. Three of these patients have been reexamined, and previous observations were confirmed. The quantity of iodine 125-labeled glycoprotein IIb (GPIIb) and glycoprotein IIIa (GPIIIa) on the platelets of these patients was considerably less than normal but was detectable by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography. The amount of residual GPIIb and GPIIIa as measured by binding studies with radiolabeled monoclonal antibodies was between 3% and 12% of the normal value. Platelet suspensions from these patients did not aggregate with fibrinogen and did not bind 125I-fibrinogen on stimulation with ADP. However, incubation of these platelets with chymotrypsin or pronase resulted in fibrinogen binding and platelet aggregation. Monoclonal antibodies specific for the GPIIb-GPIIIa complex blocked both the fibrinogen binding and the aggregation of enzyme-treated platelets. The treatment of washed platelets of a fourth thrombasthenic patient with ADP or with chymotrypsin failed to result in fibrinogen binding and aggregation. However, the level of GPIIb and GPIIIa on these platelets as measured by a Western blot technique and by monoclonal antibody binding amounted to less than 0.35% to 0.5% of normal values. In conclusion, fibrinogen binding sites exposed on thrombasthenic platelets by chymotrypsin are derived from GPIIb-GPIIIa molecules. Aggregation of chymotrypsin-treated thrombasthenic platelets by fibrinogen appears to represent a sensitive test for detection of functionally active GPIIb-GPIIIa complex on the platelet surface.     

A myeloma paraprotein with specificity for platelet glycoprotein IIIa in a patient with a fatal bleeding disorder.

Impaired platelet aggregation, normal shape change, and agglutination and normal ATP secretion and thromboxane synthesis in response to high concentrations of thrombin or arachidonic acid were found in a patient with multiple myeloma and hemorrhagic tendency. The purified IgG1 kappa or its F(ab1)2 fragments induced similar changes when added in vitro to platelet-rich plasma from normal subjects. In addition, the paraprotein inhibited adhesion to glass microbeads, fibrin clot retraction, and binding of radiolabeled fibrinogen or von Willebrand factor to platelets exposed to thrombin or arachidonic acid without affecting intraplatelet levels of cAMP. The radiolabeled para-protein bound to an average of 35,000 sites on normal platelets but it bound to less than 2,000 sites on the platelets from a patient with Glanzmann's thrombasthenia. Immunoprecipitation studies showed that the platelet antigen identified by the paraprotein was the glycoprotein IIIa. Furthermore, binding of radiolabeled prostaglandin E1 (PGE1) to resting platelets as well as binding of von Willebrand factor to platelets stimulated with ristocetin were entirely normal in the presence of patient's inhibitor. These studies indicate that bleeding occurring in dysproteinemia may be the result of a specific interaction of monoclonal paraproteins with platelets. In addition, our data support the concept that the interaction of fibrinogen and/or von Willebrand factor with the platelet glycoprotein IIb-IIIa complex is essential for effective hemostasis.     

A monoclonal antibody recognizes a von Willebrand factor domain within the amino-terminal portion of the subunit that modulates the function of the glycoprotein IB- and IIB/IIIA-binding domains.

We developed a monoclonal antibody, 1C1E7, against vWf that increases ristocetin-induced platelet aggregation in a dose-dependent manner and lowers the threshold concentration of ristocetin needed to obtain a full aggregatory response. The platelet aggregatory effect of asialo vWf (ASvWf) also is enhanced by 1C1E7, in the presence or absence of glycoprotein (GP) IIb/IIIa receptor antagonism. In the presence of ristocetin, both intact 1C1E7 and its Fab fragments enhance specific binding of 125I-vWf to platelets. With 1C1E7, the intermediate and higher molecular weight multimers of vWf are preferentially bound to both GP Ib and GP IIb/IIIa. Thrombin-induced 125I-vWf binding to GP IIb/IIIa also is increased by 1C1E7. Maximal binding of 1C1E7 to vWf corresponds to 0.97 mol/mol vWf monomer with a Kd of 4.7 x 10(-10) M. 1C1E7 reacts with a 34/36-kD tryptic fragment (III-T4) and a 34-kD plasmic fragment (P34), which localizes the epitope between amino acid residues 1 and 272; this was confirmed by NH2-terminal amino acid sequencing. Finally, platelet aggregation by ASvWf was associated with a sharp rise in intracellular Ca2+ only in the presence of 1C1E7. An antibody-mediated conformational change of vWf may result in an improved presentation of the GP Ib- and GP IIb/IIIa-binding domains of mainly the larger multimers; the increased density of vWf on the platelet surface leads to platelet activation. The antibody may thus recognize a domain of relevance for vWf physiology.     

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.     

Anti-platelet antibody and platelet function]

The effect of anti-platelet antibodies, including murine monoclonal antibodies, autoantibodies and alloantibodies, on platelet function was analyzed. The target antigen of these antiplatelet antibodies, investigated in the present study, was a glycoprotein IIb/IIIa, which is a receptor of fibrinogen and plays an important role in platelet aggregation. Some of these antibodies inhibited agonist-induced platelet aggregation. The target antigen of one murine monoclonal antibodies, designated OP-G2, was a glycoprotein IIb/IIIa and interestingly, this antibody induced platelet aggregation, which required divalent cation and fibrinogen. We compared the epitope of these antibodies by inhibition assay and found the epitope of these antibodies to be very close. The binding of OP-G2 to the platelets required Ca2+. These data suggest that OP-G2 recognizes an epitope at or in very close proximity to the fibrinogen binding site of GPIIb/IIIa, as compared with other antibodies.     

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.     

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.     

Inhibition of von Willebrand factor-induced platelet agglutination by ADP does not result from reduced binding of total von Willebrand factor or its larger multimers

Earlier experiments showed that platelet agglutination induced by von Willebrand factor (vWf) plus ristocetin was greatly diminished if adenosine diphosphate (ADP) was added first in the presence of ethylenediaminetetraacetic acid (to prevent aggregation). Platelets treated with ADP and then fixed also agglutinated less than control fixed platelets. The studies reported here demonstrate that ADP did not decrease ristocetin-induced binding of vWf whether binding was measured on suspended platelets with iodine 125-labeled vWf or on suspended or agglutinated platelets with the use of any of three 125I-labeled monoclonal antibodies that bind to vWf but that do not interfere with ristocetin-induced agglutination. Equal amounts of vWf were eluted from ristocetin/vWf-treated platelets when they were resuspended without ristocetin, whether or not the platelets had been exposed to ADP, and the vWf recovered in either case was composed only of large multimers. No evidence for an agglutination site other than glycoprotein Ib could be demonstrated by measuring agglutination of a mixture of platelets fixed after inhibition with antibody against glycoprotein Ib and platelets fixed after inhibition with ADP. We conclude that inhibition of agglutination by ADP must involve the way in which vWf is bound, because it does not result from a decreased amount or from a difference in multimer size of bound vWf.     

GpIIb/IIIa is the main receptor for initial platelet adhesion to glass and titanium surfaces in contact with whole blood

Platelets are the first cells to adhere to a surface in contact with blood and are capable of mediating several different responses after contact with different protein-coated surfaces. They are the main source of growth factors such as platelet-derived growth factor and are therefore important in the healing process. In this study, initial platelet adhesion to and spread on hydrophilic and hydrophobic (methylized) glass and titanium with similar wettability were investigated. Whole coagulating blood was used to simulate the in vivo situation shortly after implantation, in which bleeding precedes inflammation and wound healing. Several different antibodies directed against platelet integrins and receptors (CD9, FcgammaRII, GPIIb/IIIa, vitronectin receptor, GPIb/V/IX) were used in an attempt to block platelet adhesion to the surfaces. Immunofluorescence results show that initial platelet adhesion to all the surfaces we investigated can be almost completely inhibited (approximately 95%) by clone M148, an antibody against the GPIIb/IIIa complex (integrin alpha(IIb)beta(3); CD41/CD61), but not with other antibodies to the separate parts of the integrin. Antibodies known to inhibit fibrinogen binding to GPIIb/IIIa after adenosine diphosphate- and collagen- induced aggregation had very little effect on initial platelet adhesion. None of the other integrins were found to have such an effect on initial platelet adhesion. Antibody clone M148 was furthermore found to inhibit platelet spreading. This study shows that regardless of wettability and the biomaterial used, initial adhesion of platelets appears to be mediated by GPIIb/IIIa binding to surface adsorbed fibrinogen.     

A murine monoclonal antibody that completely blocks the binding of fibrinogen to platelets produces a thrombasthenic-like state in normal platelets and binds to glycoproteins IIb and/or IIIa.

To define better the role of the fibrinogen receptor in platelet physiology and to characterize it biochemically, a murine monoclonal antibody that completely blocks the binding of fibrinogen to the platelet surface was produced by the hybridoma technique with the aid of a functional screening assay. Purified F(ab')2 fragments and/or intact antibody completely blocked aggregation induced by ADP, thrombin, or epinephrine and the binding of radiolabeled fibrinogen to platelets induced by ADP. The antibody did not block agglutination of formaldehyde-fixed platelets by ristocetin or shape change induced by either ADP or thrombin. ADP- and epinephrine-induced release of ATP was completely inhibited by the antibody, but inhibition of release induced by collagen and thrombin was dose dependent and partial. The antibody also dramatically inhibited platelet retention in glass-bead columns, platelet adhesion to glass, and clot retraction. Thus, the antibody induced a thrombasthenic-like state. Immunofluorescent studies confirmed the specificity of the antibody for normal platelets and megakaryocytes and suggested that there is a marked decrease in detectable antigen in thrombasthenic platelets. Radiolabeled antibody bound to an average of approximately 40,000 sites on normal platelets but it bound to less than 2,000 sites on the platelets of a patient with thrombasthenia. The antibody immunoprecipitated both glycoproteins IIb and IIIa, and both glycoproteins bound to an affinity column of the antibody. These studies indicate that there is probably a single anatomic site that is crucial to the binding of all fibrinogen molecules and that this site is most likely on the glycoprotein IIb/IIIa complex. It also suggests that the thrombasthenic phenotype can be completely accounted for on the basis of the inhibition of fibrinogen binding to platelets.     

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.