Analysis of the glycoprotein and protein composition of Bernard-Soulier platelets by single and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

125

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 HIb/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.

Section: Resultssupporting

confidence: 88%

Plasminogen interacts with human platelets through two distinct mechanisms.

Miles¹,

Ginsberg²,

White³

et al. 1986

125

“…There is ample evidence that GPIb functions as the platelet binding site for vWF in the presence of the antibiotic ristocetin (2,(6)(7)(8) (34) have also reported studies with a different monoclonal antibody to GPIb that completely blocks ristocetin-induced binding of vWF to platelets. It is evident, however, that GPIb, or at least the epitope recognized by the antibody used in these studies and essential for ristocetin-induced binding, is not involved as part of the vWF binding site exposed on platelets stimulated by thrombin or ADP + EPI.…”

Section: Discussionmentioning

confidence: 99%

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

Ruggeri¹,

Marco²,

Gatti³

et al. 1983

443

190

A B ST R A CT The binding of '251-von Willebrand factor 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) lb had no effect on the binding of '251-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 1251-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 ristocetininduced binding of (vWF)' is a large multimeric glycoprotein that circulates in blood complexed with the Factor VIII procoagulant activity protein (1). It plays an essential role in platelet function as shown by the prolonged bleeding time in von Willebrand disease (1). Specific binding sites for vWF are induced on the platelet membrane by the antibiotic ristocetin (2), a nonphysiologic agent, as well as by thrombin (3, 4) and ADP (5). Platelet membrane glycoprotein (GP) lb is considered to function as the surface receptor for vWF (6). In the Bernard-Soulier syndrome, a congenital bleeding disorder, platelets lack GPIb (7) and the ristocetin-induced binding of vWF is decreased (8). On the other hand, in Glanzmann thrombasthenia, another congenital platelet abnormality, there is a marked decrease of the membrane GP complex Ilb/Illa but normal content of GPIb (9). The ristocetin-induced binding of vWF to thrombasthenic platelets has been reported to be normal (8). In contrast, we have recently shown that binding of vWF to thrombasthenic platelets stimulated by thrombin is severely deficient (4). Therefore, we postulated that different sites are involved in ' Abbreviations used Marguerie et al. (12). The final platelet suspensions were all in modified calcium-free Tyrode buffer, containing 137 mM NaCI, 2 mM MgCI2, 0.42 mM NaH2PO4, 11.9 mM NaHCO3, 2.9 mM KCI, 5.5 mM glucose, 10 mM Hepes, pH 7.35, and 20 mg/ml bovine serum albumin (Fraction V, CalbiochemBehring Corp., American Hoechst Corp., San Diego, CA). When experiments of serotonin release were performed, PRP was labeled before washing by incubating 20 ml with 0. (3.3,M). However, ADG-washed platelets usually gave reversible aggregation to ADP and were unresponsive to epinephrine (EPI). On the contrary, gel-filtered platelets always gave irreversible aggregation to ADP, and responded to 20 MM EPI in the presence of fibrinogen (3.3 ,M) with a typical two-wave aggregation. Contamination of plasma vWF in all washed platelet preparations was below 5 X 10' U/dl (1 dl of a normal plasma pool contains ...

“…In some experiments, von Willebrand's disease (vWd) and normal platelets frozen in dimethylsulfoxide were used (14). Platelets were isolated from blood of a patient with the Bernard-Soulier syndrome (kindly supplied by Dr. Margaret Johnson, Wilmington, DE), as previously described (15,16). Platelet concentration was determined by phase microscopy.…”

Section: Methodsmentioning

confidence: 99%

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

Christie¹,

Aster²

1982

A B S T R A C T 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 107 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 ,tM, 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 drugantibody-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 A preliminary report was presented at the VlIIth International Congress on Thrombosis and Haemostasis, Toronto, Canada, 1981. Thromb. Haemostasis. 46: 296.