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IntroductionWe have studied the interaction of the congenitally abnormal type IIA and IIB von Willebrand factor (vWF) molecules, both lacking the larger multimeric forms, with the two vWF binding sites on platelets, the glycoprotein (GP) Ib-IX and GP (8); in the latter, vWF has increased affinity for platelets and, consequently, the larger multimers are removed from the circulation as a result ofplatelet binding (7, 9-1 1). To date, however, there is no detailed information on the modalities of interaction of IIA and IIB vWF with the corresponding binding sites on platelets. The association of vWF with both GP Ib-IX and GP IlbIla appears to be important in thrombus formation (12, 13), and knowledge ofthe molecular mechanisms responsible for it is likely to provide useful information for understanding the processes involved in normal hemostasis as well as pathological vascular occlusion. In this regard, the study of molecular variants of von Willebrand disease is of particular interest if the existence of specific molecular defects can be correlated to relevant abnormalities of interaction with platelets. To address these issues, we have purified the congenitally abnormal vWF molecule from the plasma of patients with type IIA and IIB von Willebrand disease, and evaluated in a quantitative manner the interaction with GP Ib-IX and GP Ib-IhIIa. Our findings demonstrate that IIB vWF can bind to GP Ib-IX in the absence of any mediating or activating molecule, suggesting the existence of a molecular defect resulting in increased affinity for this platelet site. Such increased affinity is also evident in the presence of ristocetin, in spite of the absence of larger multimers. In contrast, IIA vWF interacts with GP Ib-IX with extremely reduced affinity. The severity of this abnormality may reflect the existence of specific structural alterations af-

Introduction We have studied a patient with a congenital bleeding disorder and phenotypic manifestations typical of Bernard-Soulier syndrome, including giant platelets with absent ristocetin-induced von Willebrand factor binding. Two monoclonal antibodies reacting with distinct epitopes in the amino-terminal domain of the a-chain of glycoprotein (GP) Ib were used to estimate the number of GP Ib molecules on the platelet membrane. In the patient, binding of one antibody (LJ-TblO) was 50% of normal, while binding of the other (LJ-Ibl) was absent. Binding of both antibodies was reduced to -50% of normal in the mother and one sister of the propositus, and their platelets exhibited -70% of normal von Willebrand factor binding. Immunoblotting studies confirmed the presence of GP Iba, as well as GP IX, in patient platelets. Antibody LJ-Ib10, but not LJ-Ibl, could immunoprecipitate the patient's GP Iba from surfacelabeled proteins. Thus, platelets from the propositus contained a structurally and functionally altered GP Ib-IX complex lacking a specific antibody epitope and the ability to bind von Willebrand factor. In contrast, the binding of human a-thrombin to the patient's platelets was normal, and three classes of binding sites with high, intermediate, and low affinity could be detected. These studies define a distinct variant form of BernardSoulier syndrome and provide evidence, based on a naturally occurring mutant molecule, that the amino-terminal region of GP Iba contains a von Wiliebrand factor-binding domain distinct from the high affinity thrombin-binding site. Use of different monoclonal antibodies with distinct epitope specificities appears to be essential for a correct identification of variant Bernard-Soulier syndrome. (J. Clin. Invest. 1990. 86:25-31.)

The number and functional activity of membrane glycoproteins (GP) Ib and IIb/IIIa were investigated in platelets from 11 patients with myeloproliferative disorders (MPD). Three patients had essential thrombocythaemia, two had chronic myeloid leukaemia and six had polycythaemia vera. The numbers of GPIb and GPIIb/IIIa molecules were detected on the platelet surface using different 125I-labelled monoclonal antibodies. The functional properties of GPIb and GPIIb/IIIa were evaluated using purified 125I-labelled asialo von Willebrand factor (vWF) and purified 125I-labelled fibrinogen, respectively, in a binding assay. Binding of the anti-GPIIb/IIIa antibody was decreased by 40% in almost all patients studied and, when measured, it was accompanied by decreased fibrinogen binding to activated platelets. Binding of anti-GPIb antibodies to platelets was also slightly decreased or virtually the same in eight out of 11 patients. The decrease correlated with decreased binding of asialo vWF. The increased plasma glycocalicin levels, measured in four patients, depended on the high platelet count. Scatchard analysis revealed normal receptor binding affinity for all ligands tested in all but one patient. In this report we demonstrate that abnormalities in the concentrations of GPIIb/IIIa membrane proteins are commonly present in patients with MPD, while a decrease in GPIb concentration is also seen, although in fewer patients. These abnormalities are accompanied by a concurrent decrease in the respective receptor functions. These findings may explain part of the haemorrhagic tendency often encountered in MPD.

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