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The human GPIa/Ila complex, also known as integrin a2Bfl, serves as a major receptor for collagen in platelets and other cell types. In addition to its role in platelet adhesion to extracellular matrix, GPIa/IIa is also known to bear the clinically important Br' and Br' alloantigenic determinants, which can result in antibody-mediated platelet destruction. Immunochemical studies showed that the Br antigenic epitopes reside solely on the GP Ia subunit and do not depend on sialic acid residues. To define the polymorphism responsible for the Br alloantigen system platelet RNA PCR technique, was used to amplify GPIa mRNA transcripts. Nucleotide sequence analysis of the amplified platelet GPIa cDNA from Bra/a and Brb/b individuals revealed a single A * G polymorphism at base 1648. MnlI RFLP analysis of cDNA from serologically determined individuals confirmed that this polymorphism segregates with Br phenotype. This single base change results in a substitution of Lys (AAG) in Bra to Glu (GAG) in Brb at amino acid residue 505. In spite of the reversal in charge at this position, however, we found no difference in the ability of Bra and Br' homozygous platelets to adhere to collagens types I, III, or V, nor did antiBra or anti-Br' alloantibodies interfere with platelet adhesion to any of these fibrillar collagens. The identification of the nucleotide substitution that defines the Bra/Brb alloantigen system will now permit both pre-and postnatal diagnosis for Br phenotype. (J. Clin. Invest. 1993. 92:2427-2432

A new platelet-specific alloantigen, termed Sita, was identified in a severe case of neonatal alloimmune thrombocytopenia. The Sita alloantigen is of low frequency (1/400) in the German population. Immunochemical studies demonstrated that the Sita epitopes reside on platelet glycoprotein (GP) Ia. Nucleotide sequence analysis of GPIa cDNA derived from Sita-positive platelets showed C2531→T2531 point mutation, resulting in Thr799Met dimorphism. Analysis of genomic DNA from 22 Sita-negative normal individuals showed that the Thr799 is encoded by ACG2532 (90.9%) or ACA2532 (9.1%). To establish a DNA typing technique, we elucidated the organization of the GPIa gene adjacent to the polymorphic bases. The introns (421 bp and 1.2 kb) encompass a 142-bp exon with the 2 polymorphic bases 2531 and 2532. Polymerase chain reaction-restriction fragment length polymorphism analysis on DNA derived from 100 donors using the restriction enzyme MaeIII showed that the Met799 form of GPIa is restricted to Sita (+) phenotype. Analysis of stable Chinese hamster ovary transfectants expressing allele-specific recombinant forms of GPIa showed that anti-Sita exclusively reacted with the Glu505Met799, but not with the Glu505Thr799 and the Lys505Thr799 isoforms. In contrast, anti-Bra (HPA-5b) only recognized the Lys505Thr799 form, whereas anti-Brb(HPA-5a) reacted with both Glu505Thr799 and Glu505Met799 isoforms. These results demonstrated that the Met799 is responsible for formation of the Sita alloantigenic determinants, whereas amino acid 505 (Lys or Glu) specifically controls the expression of Bra and Brb epitopes, respectively. Platelet aggregation responses of Sita (+) individuals were diminished in response to collagen, indicating that the Thr799Met mutation affects the function of the GPIa/IIa complex.

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Elevated concentrations of anaphylatoxin are frequently found in FFP units produced by apheresis. Studies are necessary to investigate the reasons for complement activation and the possibilities of prevention during apheresis. As the concentrations of complement precursors and complement function did not change with activation in FFP, these studies should include measurement of the anaphylatoxins C3a and C5a.

Thrombopoietin (TPO) concentrations were determined in umbilical cord plasma of 121 healthy term newborns. The lower detection limit of the enzyme immunoassay employed was 32.5 pg/ml. Median cord plasma TPO concentration was 78 (interquartile range 55–107) pg/ml. 95th percentile was 255 pg/ml. In only 8% (10/121), TPO was below the detection limit compared to 81% of healthy adults (25/31). In cord blood and adult controls, there were no significant correlations of TPO with platelet count or mass.

WE in ve s tigated th e in vitro IntroductionHypox ia and re perfusion c ause compleme nt activation in animal ex perime nts and in clinical studie s. [1][2][3][4] It c ould be show n, that after c ytological damage, contac t w ith cellular compone nts such as mitochondria, or ex c ess of hydrox yl radicals, w as re spons ible for the activation of the compleme nt syste m. 5 ,6 On the othe r hand, in vitro studies show ed a compleme nt ac tivation by ac idosis or hypox ia only. [7][8][9] In a pre vious study, w e found complement ac tivatio n induc ed by lactic acid. 10 The aim of the pre sent in v itro study w as to inve stigate: (1) w he the r lac tate itself or metabolic acidosis is re sponsible for activation, and (2) w hethe r re spiratory acidosis is also able to ac tivate the complement syste m. Material and MethodsBlood sample s (10 ml) from 12 healthy volunte ers (6 male and 6 female, age : 28 -40 years) w e re c ollec te d and 50 IE Heparin w as adde d. Each sample w as divide d into e ight portions and plac ed in polypropylen tube s.To inve stigate the influe nce of re sp iratory as w ell as me tabolic changes of pH on anaphylatox in formation, the p ortions w e re e quilibrated w ith differe nt gas mix tures and supplemented w ith acids re sulting in marked changes in pH, pCO 2 and base de ficit ( 11 A 6 ml polyprop ylen tube (6 ml) w as filled 1.5 to 2.0 ml blood and the end of a polyprop yle n tube w ith an inne r diame te r of 3 mm w as place d into the blood a few millimetre s above the bottom of the tube . Gas mix tures (O 2 , N 2 , CO 2 ) c omposed from me dic al gas es by f low me te rs w e re put through the tube into the blood sample. Gas flow w as adjuste d to one bubble pe r second for 20 min. Foam caused by bubbling w as c ontinuou sly re move d by a suction cathete r place d above the blood level. After e quilibration, the blood samples w e re transferred into different tubes for further proce ssing.For metabolic ac idosis 10 m mol hydrochloric ac id (No. 4; Sigma-Aldrich, De isenhofe n, Ge rmany) or 5.5 m mol lactate (No: 5; Sigma-Aldrich, Deise nhofen, Ge rmany) w ere adde d per ml of blood. To achie ve compe nsated me tabolic ac idosis , the sample w as equilibrate d w ith a hypoc apnic gas mix ture for 20 min and the n 5.5 m mol lac tate per ml of blood w as added (No. 8).Blood gas analysis, potassium and lactate c onc entrations w ere me as ure d by a Radiome te r Copenhagen ABL 505 (Willich, Ge rmany) using he parinise d syringes after all samples w ere incubated at 37°C for 1 h. To stop c omplement activation after inc ubation, w e added 1 mg EDTA dissolve d in purified w ater to each sample. Plasma w as se parated by ce ntrifugation at 0962-9351/98/060417-04 $9.00 © 1998 Carfax Publishing Ltd 417 Short CommunicationMediators of Inflammation, 7, 417-420 (1998) 4°C at 3000 g for 10 min and froze n immediate ly at -80°C. Anaphylatox ins C3a and C5a w ere dete rmined as pre viously desc ribed.1 2 C3a enzyme immunoassay (EIA, Fa. Progen Biote chnik GmbH, Heide lberg, Ge rmany) sel...