Current issues relating to the transfusion of stored red blood cells.

The development of blood storage systems allowed donation and transfusion to be separated in time and space. This separation has permitted the regionalization of donor services with subsequent economies of scale and improvements in the quality and availability of blood products. However, the availability of storage raises the question of how long blood products can and should be stored and how long they are safe and effective. The efficacy of red blood cells was originally measured as the increment in haematocrit and safety began with typing and the effort to reduce the risk of bacterial contamination. Appreciation of a growing list of storage lesions of red blood cells has developed with our increasing understanding of red blood cell physiology and our experience with red blood cell transfusion. However, other than frank haemolysis, rare episodes of bacterial contamination and overgrowth, the reduction of oxygen-carrying capacity associated with the failure of some transfused cells to circulate, and the toxicity of lysophospholipids released from membrane breakdown, storage-induced lesions have not had obvious correlations with safety or efficacy. The safety of red blood cell storage has also been approached in retrospective epidemiologic studies of transfused patients, but the results are frequently biased by the fact that sicker patients are transfused more often and blood banks do not issue blood products in a random order. Several large prospective studies of the safety of stored red blood cells are planned.

Structure of platelet glycoprotein IIIa. A common subunit for two different membrane receptors.

The platelet membrane glycoprotein IIb/IIIa complex is a member of a family of alpha/beta heterodimers that function as receptors for adhesive proteins. In this report we describe the structure of the human beta subunit GPIIIa deduced from an analysis of 4.0 kb of overlapping cDNA sequences isolated from a human erythroleukemia (HEL) cell cDNA expression library. A continuous open reading frame encoding all 788 amino acids for GPIIIa was present. The deduced amino acid sequence included a 26-residue amino-terminal signal peptide, a 29-residue transmembrane domain near the carboxy terminus, and four tandemly repeated cysteine-rich domains of 33-38 residues. An exact correspondence of 128 amino acids from seven human platelet GPIIIa fragments with HEL GPIIIa indicates that HEL and platelet GPIIIa are the same gene product. The HEL GPIIIa sequence was compared with the sequences of the beta subunit for the human LFA-1/Mac-1/p150.95 complex and human endothelial cell GPIIIa, revealing a 38% similarity with the former and virtual identity with the latter. Northern blot analysis using RNA from both HEL and endothelial cells revealed two GPIIIa transcripts of 5.9 and 4.1 kb. However, HEL RNA, but not endothelial cell RNA, contained a transcript for GPIIb. This indicates that the GPIIIa-containing heterodimers in platelets and endothelial cells are not identical structures, but are members of a subfamily within the human family of adhesion protein receptors sharing an identical beta subunit.

The immediate-early gene product MAD-3/EDG-3/IkappaB alpha is an endogenous modulator of fibroblast growth factor-1 (FGF-1) dependent human endothelial cell growth.

The tumor promoter phorbol 12-myristic 13-acetate inhibits the growth of human endothelial cells and induces the formation of capillary-like, tubular structures. We report the novel growth regulatory function of the immediate-early gene, edg-3, which is identical to the IkappaB alpha/MAD-3 gene. We employed phosphothioate oligonucleotides (PTO) directed against the translation initiation site of IkappaB alpha to inhibit its expression. The antisense IkappaB alpha PTO-treated cells exhibited an exaggerated growth response to fibroblast growth factor-1 (FGF-1). In contrast, IL-1-induced growth arrest response was not modulated. These data suggest that the early response gene IkappaB alpha is an endogenous regulator of endothelial cell growth in vitro.

Models of in vitro angiogenesis: endothelial cell differentiation on fibrin but not matrigel is transcriptionally dependent.

Endothelial cells can be induced to form a branching network of tubular structures using a variety of cell culture conditions. We have examined this differentiation process using several sets of conditions: plating human umbilical vein endothelial cells (HUVEC) on Matrigel, adding collagen to the apical surface of HUVEC grown on fibronectin, and plating HUVEC on fibrin in the presence of FGF-1. We determined that although the first two conditions produce dramatic morphologic changes in the HUVEC population, gene transcription and translation are not required for the regulation of the process. Rather, post-translational events are involved since the Matrigel-dependent process could be inhibited by the addition of nocodazole, suramin or H7, a protein kinase inhibitor. In contrast, the fibrin matrix-dependent differentiation pathway involved transcriptional and translational events since the addition of either actinomycin D or cycloheximide inhibited this pathway. A modified differential display of RNA extracted from HUVEC after 0, 2, 5, and 24 hours on fibrin revealed expression of a novel cDNA.

The genomic organization of platelet glycoprotein IIIa.

The platelet membrane glycoprotein (GP) IIb/IIIa complex, a member of the integrin family of adhesive receptors involved in cell-cell and cell-matrix interactions, contains binding sites for fibrinogen, von Willebrand factor, fibronectin, and vitronectin. Absence or defects of this receptor result in the platelet bleeding disorder Glanzmann's thrombasthenia. In this report, we describe the isolation of genomic DNA coding for the entire mature GPIIIa protein. Mature GPIIIa is encoded by 14 exons which range in length from 90 to 3618 base pairs, which are contained within an approximately 46-kilobase (kb) stretch of genomic DNA on chromosome 17. All of the exon/intron junctions were found to conform to the consensus splice donor and acceptor sequences. The coding region of the GPIIIa gene is identical with the previously described cDNA sequence except for three silent substitutions. One substitution creates a TaqI site which may be the site of a known GPIIIa polymorphism. A second substitution eliminates a SmaI site. Aside from the start of the first exon described, which begins at the second base of the first codon of the mature protein, there is no correlation between the organization of the exons in this gene and proposed functional domains of the protein based on analysis of the primary amino acid sequence. The less frequently used polyadenylation signal AAATTAAA was present at the 3'-end of the major RNA transcript. Recently, an alternatively processed GPIIIa transcript has been described. We demonstrate that this transcript results from nonsplicing of the final intron. The description of the GPIIIa gene organization should be of importance in understanding the evolution of the integrin family of receptors and should be useful in the molecular biology analysis of thrombasthenic patients who have a defect in the GPIIIa gene.

An antisense oligonucleotide to the notch ligand jagged enhances fibroblast growth factor-induced angiogenesis in vitro.

Angiogenesis, or the formation of new blood vessels, plays a central role in a number of physiologic and pathologic conditions, including wound healing, diabetic retinopathy, and solid tumor growth, and endothelial cells can be induced to mimic this process in vitro. Using a modification of the differential display method (Zimrin, A. B., Villeponteau, B., and Maciag, T. (1995) Biochem. Biophys. Res. Commun. 213, 630-638), we isolated the human homolog of the Jagged ligand for the Notch receptor from human endothelial cells exposed to fibrin and demonstrate that the Jagged transcript, but not the Notch 1 or Notch 2 transcripts, are up-regulated by fibrin. Interestingly, the addition of an antisense Jagged oligomer to bovine microvascular endothelial cells grown on a collagen gel resulted in a marked increase in invasion and tube formation in the underlying gel in response to fibroblast growth factor. In contrast, no effect was observed on vascular endothelial growth factor-induced angiogenesis under identical conditions. These data suggest that Jagged-Notch signaling is able to regulate fibroblast growth factor-induced endothelial cell migration in vitro, an early event during angiogenesis in vivo.