Calcium changes in normal and thrombasthenic platelets were recorded using the PICA-apparatus. Aequorin was loaded in the presence of DMSO, EGTA and PGE1. Platelets of three patients with type I thrombasthenia stimulated with A-23,187, thrombin, PMA in the presence 1mM Ca++ and 1mM Mg++ were able to normally raise their calcium concentrations. The maximal values could be found below the normal range with collagen, ADP and PAF-acether. Calcium mobilization from internal stores in response to thrombin was normal. There were two calcium peaks in normal platelets stimulated with ADP. The second one was suppressed by omitting fibrinogen, stirring, or by adding aspirin, and was absent in thrombasthenic platelets. Thus the GP IIb-Illa complex is not a prerequisite for calcium fluxes but is involved, when weak agonists such ADP are used, through an aggregation-dependent reinforcement of platelet activation.

PL2-49 is a murine monoclonal IgG1 antibody obtained after immunization of Balb/c mice with EDTA washed platelets. Binding could be detected on Zwa(+) as well as Zwa(-) platelets, but not on type I Glanzmann's thrombasthenia platelets using an ELISA screening test. Immunoprecipitation studies showed that PL2-49 bound to glycoprotein IIb when the glycoprotein IIb/IIIa complex dissociation was performed after the monoclonal antibody binding. Experiments with a human alloantibody against Zwa antigen were run in parallel to control the complex dissociation. Ascitic fluid, as well as the purified antibody, induced activation and aggregation of washed platelets and ATP release. PL2-49-induced aggregation did not require exogenous fibrinogen and was inhibited, partially, in the presence of aspirin, apyrase, isosorbide dinitrate. Raising intra-platelet cyclic AMP with a stable PGI2 analogue, iloprost, and/or a phosphodiesterase inhibitor, RA 233, suppressed the responses to PL2-49. F(ab')2 fragments did not induce aggregation of normal platelets but inhibited the response to the whole immunoglobulin. Finally PL2-49 was shown to induce aequorin-detected elevations in intraplatelet Ca++ levels. Thus PL2-49 seems to differ from monoclonal antibodies so far described, since it binds to glycoprotein IIb in a complex-dependent manner at least under our experimental conditions for immunoprecipitation studies, and it induces platelet Ca++ mobilization and platelet aggregation after a lag-time. These reactions depend both on Fab and Fc domains of the antibody and require neither complement nor exogenous fibrinogen.

Summary. In systemic lupus erythematosus (SLE), the precise cause of the thrombocytopenia is unknown. Since platelet associated IgG is increased in many patients, it has been suggested that the destruction of platelets might be dependent on specific antibodies.In nine patients with SLE, platelet associated immunoglobulins were found together with free serum antibody which bound to platelets from all normal subjects.Using an immunoblotting technique with membrane proteins from normal platelets incubated with patient sera, target antigens were localized on a band of mol wt 108 000 in two cases (B. and N.) and on a band of mol wt 66 000 in a third (M.). When the same technique was applied to autologous platelets of patient N., autoantibody binding to the protein of mol wt 108 000 was demonstrated. The antigenic determinants were not removed from the platelets by enzyme treatment or by disulphide bond reduction, and were localized in the cytoplasmic fraction of the platelets.

Neonatal alloimmune thrombocytopenia (NAITP) is primarily due to a foeto-maternal incompatibility of a specific platelet alloantigen. When the PLA1 antigen [1] is involved, its incidence has been recently evaluated to 1/3000 births [2]. The PLA1 antigen has been located on the platelet membrane glycoprotein Ilia [3]. This glycoprotein is mainly involved in the platelet aggregation system. The Glanzmann thrombasthenia is due to a lack of this protein [4] and in this disease patients can have severe hemorrhages. In vitro it was shown that the platelets were unable to adhere after activation [5]. Von dem Borne et al. [6] have shown that the antibody anti-PLA1 interacts in the normal platelet aggregation process. So it was of interest to study the effect of the interaction of such an antibody on the membrane platelet fluidity and order. For this purpose fluorescence polarization was used. The method allows a submacroscopic level of investigation in membrane fluidity studies which is directly correlated with physicological events [7].

Utilizing an easy and safe procedure for fetal blood sampling in utero. we have studied 123 fetuses for congenital oracquire hemorrhagic disorders. Usually, the diagnosis is performed at the 18th week of gestation. To date, no fetal less or premature labor has beenattributed to these fetal samplings. Theduration of the procedure was less than 10 minutes in 90 % of the cases. Direct blood sampling with a needle guided by ultrasound is safer for fetuses and simpler for the patients than fetoscopy. Among the 1.465 samplings the mortality rateis 0.2 %. We have established the basis values for fetal hemostasis when the samplings were performed for non hematological purpose, and could determine the fetal sex which play a role in hereditary disorders. Hemophilia A and B [92 cases]. Willebrand disease, factor XIII, V and VII deficiencies were diagnosed on the existence of a specific fetal deficit. Theknowledge of the fetal primary hemostasis let us to establish the diagnosis of May Hegglin syndrome. Gray platelet syndrome. and Glanzmann's thrombasthenia. There were no diagnostic errors. This procedure offers a new possibility of easily taking iterative samples, until the end of pregnancy, which represents a particular interest in prenatal diagnosis.