The human proteinase-activated receptor-3 (PAR-3) gene. Identification within a Par gene cluster and characterization in vascular endothelial cells and platelets.

Proteolytically activated receptors (PARs) represent an emerging subset of seven transmembrane G protein-coupled receptors that mediate cell activation events by receptor cleavage at distinct scissile bonds located within receptor amino termini. Differential genomic blotting using a yeast artificial chromosome known to contain the PAR-1 and PAR-2 genes identified the PAR-3 gene within a PAR gene cluster spanning approximately 100 kilobases at 5q13. The PAR-3 gene is relatively small (approximately 12 kilobases); and, like the PAR-1 and PAR-2 genes, it displays a two-exon structure, with the majority of the coding sequence and the proteolytic cleavage site contained within the larger second exon. Sequence analysis of the 5'-flanking region demonstrates that the promoter is TATA-less, similar to that seen with PAR-1, with the identification of nucleic acid motifs potentially involved in transcriptional gene regulation, including AP-1, GATA, and octameric sequences. PAR-3 transcripts were apparent in human vascular endothelial cells, although at considerably lower levels than those of PAR-1 and not significantly modulated by the endothelial cell stimulus tumor necrosis factor-alpha. Likewise, although PAR-3 mRNA was evident in human platelets, receptor cell surface expression was modest (approximately 10%) compared with that of PAR-1. Thus, although PAR-3 is postulated to represent a second thrombin receptor, its modest endothelial cell and platelet expression suggest that PAR-3 activation by alpha-thrombin is less relevant for physiological responses in these mature cells. Rather, given its disparately greater expression in megakaryocytes (and megakaryocyte-like human erythroleukemia cells), a regulatory role in cellular development (by protease activation) could be postulated.

Examination of irreversible platelet-fibrinogen interactions.

The progressive stabilization of fibrinogen binding to ADP-treated platelets has been well described, but the nature of this interaction remains obscure. In the present study, irreversibly bound fibrinogen was defined as that fraction of bound iodinated fibrinogen that failed to dissociate from stimulated human gel-filtered platelets within 10 min of adding 10 mM ethylenediaminetetraacetic acid. It represented 16 +/- 11% (mean +/- SD, n = 10) of fibrinogen bound to ADP-treated platelets after 1 min and 52 +/- 11% of fibrinogen bound to these platelets after 60 min. Similar results were obtained if platelets were stimulated with purified human thrombin (0.1 U/ml) or epinephrine (10 microM). Irreversible fibrinogen binding was significantly reduced at 4 degrees C (27 +/- 9%, mean +/- SD, n = 6) if platelets were preincubated (30 min, 25 degrees C) with 30 micrograms/ml cytochalasin B or D (18 +/- 8%) or stimulated with chymotrypsin (0.5 mg/2-3 X 10(8) platelets) (31 +/- 8%). Formation of irreversible platelet-fibrinogen interactions correlated with the incorporation of actin and actin-binding protein into the Triton X-100-insoluble platelet cytoskeleton and the ability of platelets to retract fibrin clots. Irreversibly bound fibrinogen was available on platelets for digestion by 0.2 U/ml plasmin. The enzyme removed 96 +/- 6% (mean +/- SD, n = 6) of all bound fibrinogen from platelets after 30 min at 25 degrees C. This was not accompanied by significant release of [14C]serotonin or lactate dehydrogenase. Furthermore, platelets incubated with plasmin could bind fibrinogen normally after the enzyme had been neutralized with aprotinin.(ABSTRACT TRUNCATED AT 250 WORDS)