P-selectin is an integral membrane glycoprotein stored in the secretory granules of platelets and endothelial cells. To determine whether soluble P-selectin may be present in the circulation of healthy humans, we used a sandwich immunoassay to assess citrated plasma from 50 subjects. P-selectin was present in concentrations ranging from 19 to 521 ng/ml (mean +/- SD = 121 +/- 84 ng/ml). The apparent molecular weight of P-selectin immunoisolated from platelet-poor plasma was similar to that of the detergent-soluble form isolated from platelet membrane. Plasma levels of P-selectin were unaffected by the following procedures: (1) drawing of blood in the presence of protease inhibitors; (2) stimulation of platelet-rich plasma with aggregating agents; (3) ultracentrifugation at 100,000 g for 120 min at 4 degrees C or filtration through a 0.22 micron membrane; or (4) preincubation of platelet-poor plasma with immobilized anti-platelet glycoprotein Ib monoclonal antibodies. It appeared that plasma P-selectin did not result from the in vitro activation of platelets, nor was it derived from platelet microparticles. We also found that plasma P-selectin levels were significantly elevated in patients with thrombotic thrombocytopenic purpura (12 patients, 332 +/- 184 ng/ml, P < 0.001) and haemolytic uraemic syndrome (17 patients, 297 +/- 191 ng/ml, P < 0.0001), as compared to the normal levels. Thus, these data should facilitate the study of the pathophysiological significance of circulating P-selectin.

Chemical or enzymic reduction/oxidation of integrin cysteine residues (e.g. by reducing agents and protein disulphide isomerase) may be a mechanism for regulating integrin function. It has also been proposed that unique cysteine residues in the integrin beta3 subunit are involved in the regulation of alphaIIbbeta3. In the present study, we studied systematically the role of disulphide bonds in beta3 on the ligand-binding function of alphaIIbbeta3 by mutating individual cysteine residues of beta3 to serine. We found that the disulphide bonds that are critical for alphaIIbbeta3 regulation are clustered within the EGF (epidermal growth factor) domains. Interestingly, disrupting only a single disulphide bond in the EGF domains was enough to activate alphaIIbbeta3 fully. In contrast, only two (of 13) disulphide bonds tested outside the EGF domains activated alphaIIbbeta3. These results suggest that the disulphide bonds in the EGF domains should be intact to keep alphaIIbbeta3 in an inactive state, and that there is no unique cysteine residue in the EGF domain critical for regulating the receptor. The cysteine residues in the EGF domains are potential targets for chemical or enzymic reduction.

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Summary.We analysed the molecular basis of Glanzmann thrombasthenia (GT) in four Japanese patients with type I or type II disease. Polymerase chain reaction (PCR) and subsequent direct sequencing of platelet RNA and genomic DNA revealed three single nucleotide substitutions of the aIIb gene, which were confirmed by allele-specific PCR or restriction analysis. One patient with type I GT had a T to C base substitution in exon 11 resulting in a Phe (TTT)-289 to Ser (TCT) mutation (F289S) of the subunit. Another type I patient had a G to A base substitution in exon 12 resulting in a Glu (GAA)-324 to Lys (AAA) mutation (E324K). Interestingly, two unrelated patients with type II GT shared an A to C base substitution in exon 23, a region previously not associated with GT, resulting in a Gln (CAA)-747 to Pro (CCA) mutation (Q747P). To analyse the effects of these mutations on aIIbb3 surface expression, the wild-type aIIb cDNA or mutant aIIb cDNAs were transfected into Chinese hamster ovary (CHO) cells together with a wild-type b3 cDNA. Flow cytometric analysis using an anti-aIIbb3 complex antibody revealed that 50·6% of CHO cells with wildtype aIIbb3 expressed complexes, whereas only 1·6%, 7·7% and 31·3% of cells, with aIIb(F289S)b3, aIIb(E324K)b3 and aIIb(Q747P)b3 expressed complexes, respectively. Our data indicate that these three novel point mutations in the aIIb subunit may hamper surface expression of the aIIbb3 complex, thus resulting in the quantitative GT phenotypes of platelets from these patients.