[Major thrombopenia induced by heparin. Practical approach to cardiac surgery under extracorporeal circulation]. / Thrombopénies majeures induites par l'héparine. Attitude pratique en chirurgie cardiaque sous CEC.

Major heparin-induced thrombocytopaenia (HIT) is a condition which is feared more for its thrombotic complications than for the risk of haemorrhage. The platelet count is part of routine surveillance of patients receiving this treatment which must be withdrawn if HIT occurs. The use of heparin remains essential for cardio-pulmonary bypass surgery. There are two possible scenarios: The thrombocytopaenia occurs in the postoperative period: the standard heparin may be relayed by oral anti-vitamin K anticoagulants, platelet antiaggregant drugs or by low molecular weight heparin (LMWH). The diagnosis of HIT is made before surgery: three therapeutic attitudes are discussed with respect to the urgency of surgery: surgery is deferred for 6 to 8 weeks to allow the platelet count to return to normal and the responsible circulating antibody to disappear; the use of LMWH providing the tests of platelet aggregation are negative with this product; in addition, there are other problems specific to their use in cardiopulmonary bypass to be considered; blood exchange at the beginning of cardiopulmonary bypass to eliminate the circulating factor responsible and so allow the use of standard heparin during and after the operation: this is the only possible solution in cases with in vitro aggregant activity of LMWH.

[Hemorrhagic rectocolitis associated with idiopathic thrombopenic purpura]. / Rectocolite hémorragique associée à un purpura thrombopénique idiopathique.

This case report documents severe autoimmune thrombocytopenia in a 20 yrs. old patient with ulcerative colitis. Diagnosis of idiopathic thrombocytopenic purpura was made on the presence of bound antiplatelet antibodies and on the absence of any other disorder capable of provoking a platelet fall. Colonic lesions were moderate but were resistant to steroids and total parenteral nutrition. Thrombocytopenia resisted to steroids and vincristine and a major fall in the platelet count (less than 10,000/mm3) occurred in two instances. However, platelets rose transiently after high-dose intravenous gamma-globulins. The patient underwent splenectomy, colectomy and ileostomy because of life-threatening colonic hemorrhage. The platelet count rose to normal levels within one week after surgery. Ileorectal anastomosis was subsequently performed. One year later the patient was in good health and his platelet count had returned to normal.

[Posttransfusion purpura: identification of a new platelet antigen Leka. A case]. / Purpura post-transfusionnel: identification d'un nouvel antigène plaquettaire Leka. Une observation.

A young female patient with Gardner's syndrome developed post-transfusion purpura. Serological studies demonstrated the presence of an antibody directed not against PlA1 but against a new antigen, Leka. This is the first case of post-transfusion purpura not associated with immunization against the PlA1 antigen. The Leka antigen is present in 98% of the French population. Its absence on platelets from patients with thrombasthenia suggests that it is located on the IIb IIIa platelet glycoprotein complex.

Lek a, a new platelet antigen absent in Glanzmann's thrombasthenia.

The serum of a patient who developed a posttransfusion purpura contained antibodies directed against a previously undescribed platelet antigen Lek a. The antiplatelet activity was present in the IgG fraction and was detected by immunofluorescence, 51Cr lysis and 14C-serotonin release. The frequency of the Lek a phenotype in the French population is 98.18%. Lek a does not appear to be sex-linked and seems to be closely related to the Bak a antigen. The Lek a antigen is not expressed on thrombasthenic platelets but is found on platelets from patients with the Bernard-Soulier syndrome which suggests that this antigen is carried by platelet glycoproteins IIb and/or IIIa.

[Quantification of platelet associated IgG in thrombocytopenic purpura (authors transl)]. / Taux des immunoglobulines plaquettaires au cours des purpuras thrombopéniques.

One hundred and twenty four tests of quantification of platelet associated IgG were done in patients with thrombocytopenia. The level was increased in 80% of the patients with ITP. The platelet associated IgG level does not seem a prognosis tool but is useful to follow the evolution. Platelet associated IgG levels higher than 600 x 10(-16) g/platelet were only observed in ITP (control less than 54 x 10(-16) g/platelet). Level lower than 600 x 10(-16) g/platelet were observed in systemic lupus erythematosus, chronic lymphocytic leukemia or chronic hepatitis. In defective thrombopoïesis, the levels were always normal.

[Transfusion of platelets loaded with periwinkle alkaloids as a treatment for chronic idiopathic thrombocytopenic purpura (author's transl)]. / Traitement d'un purpura thrombopénique par la transfusion de plaquettes véhiculant de la vincristine (plaquettes "cheval de Troie").

In a 70 year-old woman with a chronic idiopathic thrombocytopenic purpura refractory to all the therapeutic used, the absence of antiplatelet antibodies and the action of patient mononuclear cells on normal platelet 14C serotonin release was observed. The injection of vincristine loaded platelets was followed by a remission and a normalisation of the cellular immunological tests.

Studies of platelet antibodies in patients with thrombocytopenic purpura by two different techniques.

The direct antiglobulin consumption (DAC) test, used for the detection of platelet autoantibodies, was found to be positive in 86% of a group of 150 patients with thrombocytopenic purpura. The platelet radioactive Coombs' (DRC) test was performed in 30 patients and the correlation between the two tests was statistically significant (r = 0.48, p less than 0.01). The two tests were used in an indirect mode for the detection of antiplatelet antibodies in serum. The results of the two tests were in good agreement (r = 0.06, p less than 0.001). There was not a statistically significant correlation between the level of platelet-associated IgG and the presence of platelet antibodies in the serum. These results indicate that the DAC test or DRC test can be used interchangeably for the detection of antibody in serum or of antibody bound to platelets, and that only the direct test, using the patients' platelets, is useful in the diagnosis of autoimmune thrombocytopenic purpura.

[Variant of Paris-I Lariboisière thrombasthenia, a molecular anomaly of the IIb-IIIa platelet glycoprotein complex]. / Variant de thrombasthénie Paris-I Lariboisière, anomalie moléculaire du complexe glycoprotéinique plaquettaire IIb-IIIa.

Platelet GP IIb-IIIa complex is missing or strongly reduced in thrombasthenia type I or II; in parallel the binding of fibrinogen is either nil or strongly reduced after platelet activation and these platelets do not aggregate. In the platelets of the described variant, GP IIb-IIIa level and PLA1 antigen are around 50% of the normal but the fibrinogen sites are either missing or unavailable. The hypothesis is that the patient's platelets lack the specific receptor site for fibrinogen at the GP IIb-IIIa level. However, platelet fibrinogen is normal as is the clot retraction. The study of this variant allows new concepts on platelet aggregation and clot retraction.

Immunochemical characterization of the platelet-specific alloantigen Leka: a comparative study with the PlA1 alloantigen.

We report the immunochemical characterization of a new platelet-specific alloantigen detected using an IgG antibody isolated from the serum of a patient with posttransfusion purpura (PTP). In indirect immunoprecipitation experiments, the antibody, termed anti-Leka, predominantly precipitated glycoprotein (GP) IIb from Triton X-100 lysates of normal human platelets. In an immunoblot procedure, which involved the transfer of platelet polypeptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to nitrocellulose membrane, anti-Leka bound exclusively to GP IIb. Under identical conditions, four anti-PlA1 antibodies each reacted with GP IIIa. No binding of anti-Leka IgG occurred to Leka (-) platelets or to their separated polypeptides although GP IIb was normally detected by Coomassie blue staining. After electrophoresis of reduced platelet proteins, the Leka determinant was localized to the IIb alpha chain. Thus, unlike the PlA1 antigen, the Leka determinant was not destroyed by disulfide reduction. Analysis of platelets from a patient with Glanzmann's thrombasthenia revealed little or no binding in the GP IIb position. Anti-Leka permitted the identification of 76,000 and 60,000 dalton fragments of GP IIb retained by the platelet following chymotrypsin treatment. Our results further highlight the immunogenicity of the GP IIb-IIIa complex. They also suggest that antibodies against GP IIb can cause the thrombocytopenia observed in PTP and that anti-PlA1 antibodies do not account exclusively for the pathophysiology of this immune disorder.

Determination of platelet antigens and glycoproteins in the human fetus.

The autosomal recessive transmission of Glanzmann's thrombasthenia (GT) and Bernard-Soulier syndrome (BSS), together with requests of families who already had children with these diseases, prompted us to investigate the feasibility of their antenatal diagnosis. The preliminary step leading to the early detection of GT or BSS was to characterize, in the normal human fetus, the platelet antigens and glycoproteins (GPs) and to define their normal amounts on the membrane surface. Blood samples from 32 fetuses between 18 to 26 weeks of gestation were collected by direct puncture of the umbilical vein using an ultrasound-guided needle. Polyclonal antibodies from human origin directed against PLA1, Leka antigens, and the GPIIb IIIa complex (IgGL), or murine monoclonal antibodies specific for GPIb (AN51, 6D1), GPIIIa (AP-3), or GPIIb IIIa (AP-2) were studied using platelet suspension immunofluorescence tests. The binding of each antibody was quantified using a cytofluorograph (Ortho 50H). PLA1 and Leka antigens were expressed in normal amounts on fetal platelets as early as 16 weeks of intrauterine life. The GPIIb IIIa complex quantified by polyclonal or monoclonal antibodies was in the same range in fetuses (IgGL = 427 +/- 23 AUF, AP-2 = 459.5 +/- 8.5; AP-3 = 536 +/- 14) and in adults (IgGL = 420 +/- 30; AP-2 = 498 +/- 11; AP-3 = 515 +/- 13). The platelet binding of antibodies that recognized GPIb was higher in fetuses (AN51 = 491.5 +/- 14; 6D1 = 479 +/- 15) than in adults (AN51 = 426.5 +/- 9; 6D1 = 449 +/- 8.7). These results suggest that immunological techniques can be applied as early as 18 weeks of gestation for the antenatal diagnosis of GT and BSS.

Identification of the platelet alloantigen (PlA1) in circulating immune complexes of normal human sera.

An analysis of cell membrane material present in circulating immune complexes isolated from normal human sera by precipitation with polyethylene glycol (PEG) has been performed with the platelet alloantigen PlA1 and rhesus antigen used as markers. PEG precipitates obtained from the sera of subjects of known PlA1 and rhesus phenotype were resuspended in buffer and analyzed as representative of circulating immune complexes (CIC). The consumption of anti-PlA1 serum by CIC was determined by immunofluorescence and by inhibition of sodium 51chromate release from PlA1-positive target platelets. With these two techniques, PlA1 alloantigen activity was detected in CIC. This finding suggests that at least some of the cell membrane material present in CIC is derived from platelets.

A serologic study of red cells and sera from 18 Rh:32,-46 (RN/RN) persons.

The red cells (RBCs) and sera from 18 RN/RN persons were studied. The study confirmed the Rh type D+C+E-c-e+Cw-, which is characterized by an increased expression of the D antigen; a markedly decreased expression of the C and e antigens; the presence of a low-incidence antigen, Rh32; and the absence of a high-incidence antigen, Rh46, which is associated with an epitope recognized by a murine monoclonal antibody (MR432). Other Rh antigens of low and high incidence were investigated, and the presence of Rh17 and Rh44 on the RBCs was confirmed. Three persons exposed to Rh:46 cells by pregnancy or transfusion (or both) had anti-Rh46. This antibody gave positive reactions with all RBCs of common and rare Rh phenotype except Rhnull, and those of D--, D.., DCw-, and RN homozygotes. This antibody is considered to be of clinical significance in case of transfusion or pregnancy.

Simultaneous isolation of two platelet membrane fractions: biochemical, immunological and functional characterization.

Simultaneous isolation of two platelet membrane subfractions was achieved by centrifugation on 40% sucrose from a 100.000 g crude membrane fraction. Characterization of both types of membranes was carried out by different biochemical and immunological markers. Using a surface label, 3H Concanavalin A (3HCon A), a marker enzyme, phosphodiesterase, and lipid analysis, one of the fraction has been identified as external or plasma membranes, the other consists of intracellular membranes. Further two specific antibodies directed against external membrane antigens (LeKa and IgG L) react almost exclusively with the external membranes. Finally both kinds of membranes were able to uptake calcium but the affinity for this cation was higher for the internal than for the external membranes. This suggests that both membranes are implicated in the regulation of the cytoplasmic calcium concentration and that the internal membranes (dense tubular system) play the major part in this regulation.

A role for glycoprotein IIb-IIIa complex in the binding of IgE to human platelets and platelet IgE-dependent cytotoxic functions.

A possible relationship between binding sites for Immunoglobulin E (IgE) on human platelets, involved in IgE-dependent cytotoxic functions of platelets against helminth parasites, and well-characterized platelet constituents involved in haemostasis, was investigated. We first explored the interaction with IgE of platelets from patients with rare inherited deficiencies of defined platelet constituents and functions: Glanzmann's thrombasthenia, Bernard-Soulier and grey platelet syndromes. We report that only type I and II thrombasthenic platelets, which lack the membrane glycoproteins (GP) IIb and IIIa, failed to bind IgE and to exhibit IgE-dependent effector functions. Since thrombasthenic monocytes, however, showed normal interaction with IgE, this defect appeared restricted to platelets. Polyclonal and monoclonal antibodies directed against GP IIb-IIIa complex, but not monoclonal antibody directed against GP Ib, inhibited the binding of IgE to normal platelets, and their IgE-dependent cytotoxicity. Taken together, these findings indicate a relation between the GP IIb-IIIa complex and the expression of IgE binding sites and IgE-dependent effector functions in human platelets.

Prolonged remission in Raynaud's phenomenon after prostacyclin infusion.

We infused prostacyclin (PGI2) (7.5 ng/kg/min) during 5 h, three times at weekly intervals in 8 patients with Raynaud's phenomenon (RP). In 4 patients, improvement was long-term, more than 90 days after the last infusion (good responders); in 3 patients, improvement was mild, less than 15 days, and in one patient no improvement was observed (poor responders). Clinical response was always accompanied by improvement, although less prolonged, of capillary appearance and/or function, as judged by microscopy and/or hemodynamic tests (pulse volume index; radial artery blood flow). Lastly, increased catabolism of PGI2 seemed to be excluded in poor responders, since no statistical difference in PGI2 metabolism could be observed between the two groups.

[Anti-JKa specificity : a rare variety of auto-immune hemolytic anemia (report of a case associated with myxoedema (author's transl)]. / Une variété rare d'anémie hémolytique auto-immune : la spécificité anti-JKa.

The authors report the case of a patient with autoimmune hemolytic anemia, due to anti-KJa antibody, and associated with primary hypothyroidism. They discuss the possible relationship between these two conditions and emphasize the particular hematological pattern related to myxoedema.

Elevated angiogenin levels in synovial fluid from patients with inflammatory arthritis and secretion of angiogenin by cultured synovial fibroblasts.

Angiogenesis is a key process in the pathogenesis of inflammatory arthritis. Angiogenin is one of the most potent inducers of neovascularization in experimental models in vivo. To look for evidence that angiogenin is involved in inflammatory joint disease, we examined plasma and synovial fluid (SF) samples from rheumatology patients and synovial fibroblast cell culture supernatants. Angiogenin levels were determined by radioimmunoassay and ELISA. Plasma angiogenin concentrations ranged from 96 to 478 ng/ml, with no significant difference between patients and normal controls. In SF, angiogenin concentrations were significantly higher in patients with acute or chronic synovitis (rheumatoid arthritis (RA): median, 104 ng/ml; range 13-748, n = 14; crystal-induced arthritis (CIA): median, 149 ng/ml; range, 37-616, n = 14, and other chronic inflammatory arthritis: median, 42 ng/ml; range, 15-205; n = 9) than in the 18 patients with osteoarthritis (OA) (median, 20 ng/ml; range 8-116) (P < 0.0001, anova). Angiogenin levels in SF from RA patients in remission with secondary OA were similar to those achieved in primary OA, and decreased in parallel with the resolution of acute gout. Angiogenin protein was released by cultured synovial fibroblasts from OA and RA patients, and reached 1.18 ng/106 cells/day. These data suggest that angiogenin may mediate local inflammation in arthritis via effects on angiogenesis and leucocyte regulation.

Blood monocyte activation in rheumatoid arthritis: increased monocyte adhesiveness, integrin expression, and cytokine release.

Infiltration of the synovium by mononuclear cells, namely lymphocytes and monocytes, is one of the main features of rheumatoid arthritis (RA) and is considered to be responsible for the development of the disease. In this study in 31 consecutive patients with RA, we investigated whether peripheral blood monocytes exhibited markers of cellular activation related to cell migration. Using flow cytometry with the respective specific antibodies, we studied the expression of integrins CD11a, CD11b, CD11c, CD49d (VLA-4), and CD49e (VLA-5) on monocytes from patients with RA and from normal (N) subjects. IL-1 beta, IL-6, and tumour necrosis factor-alpha (TNF-alpha) production by cultured monocytes was measured by immunoassay. Adhesiveness of monocytes was studied on various surfaces (plastic, human fibronectin, gelatin-coated plasma, subendothelial matrix) and on cultured endothelial cells under basal conditions or after stimulation by IL-1 beta. An increased number of CD14+ monocytes (Mo) from RA patients expressed the CD11b molecule (RA Mo = 90.3%, N Mo = 83.4%, P < 0.005). The expression of CD11b on CD14+ monocytes was significantly increased in RA patients (median fluorescence intensity (FI): RA Mo = 145 (range 80-466) units; normal Mo = 95 (range 24-164) units; P < 0.003). Production of extracellular IL-1 beta and IL-6 by RA monocytes was significantly enhanced compared with monocytes from normal subjects (IL-1 beta: RA = 2.65 +/- 0.91 ng/ml versus N = 1.35 +/- 0.85 pg/ml, P < 0.05; IL-6: RA = 4.83 +/- 0.90 ng/ml versus N = 2.40 +/- 0.95 ng/ml, P < 0.05). Compared with normal monocytes, RA monocytes exhibited increased adhesion to the various surfaces studied (plastic, P < 0.01; fibronectin, P < 0.01; and gelatin-coated normal or RA plasma, P < 0.01) as well as to unstimulated (P < 0.01) and IL-1 beta-stimulated endothelial cells (IL-1 beta for 4 h, P < 0.05; IL-1 beta for 24h, P < 0.05). In our study, blood monocytes from RA patients exhibited features of activation related to cell adhesion.