2.1. Marco teórico
2.1.5. Sistema nacional de contrataciones del estado
Weibal-Palade bodies are rod-shaped organelles of 4|im in length that contain longitudinally orientated tubular structures (Matsuda and Sugiura, 1970). Similar structures are present in regions of platelet a-granules and may constitute mature von Wülebrand factor polymers (Wagner et at., 1991).
Alpha-granules of platelets are spherical, oval and sometimes elongated structures with diameters ranging form 200nm to SOOnm. They contain adhesive proteins (such as fibrinogen, von Willebrand factor (vWf) and thrombospondin (TSP)), plasma proteins (such as IgG and albumin), cellular mitogens (such as platelet derived growth factor (PDGF) and transforming growth factor (TGF)), coagulation factors (such as factor V) and protease inhibitors (such as ot2-macroglobulin and 0 2-antiplasmin). The a-granule membrane contains the receptors glycoproteins nb/rUa (ccIIbPs) and p-selectin. Alpha- granular contents are destined for release during platelet activation at sites of vessel wall injury. They thus play an important role in homeostasis, inflammation, wound repair and in the pathogenesis of antherosclerosis (reviewed by Harison and Cramer, 1992).
Two constituents common to Weibel-Palade bodies and a-granules have been exploited to reveal the nature of protein trafficking to dense core granules and dense core granule biogenesis. These are the content protein von Willebrand factor and the integral membrane protein p-selectin:
1.5.4.1. von Willebrand factor (vWf).
von Willebrand factor (vWf) is a large adhesive glycoprotein that mediates the attachment of platelets to blood vessel basement membranes after tissue injury and contributes to normal platelet aggregate formation (Harison and Cramer, 1992; Spom et al., 1986; Spom et al., 1989 and references therein). vWf is synthesised as the precursor pro-vWf, which dimerises in the ER and becomes glycosylated and sulphated in the Golgi
apparatus. Interdimer disulfide bonds can form to produce large vWf multimers of molecular weights estimated at ten to twenty million daltons. Although covalent
multimerisation of vWf is not necessary for storage (Wagner et al., 1991), Weibel-Palade bodies contain this subclass of very large vWf multimers that are particularly biologically potent, while dimeric pro-vWf and mature vWf are secreted (Spom et al., 1986). Indeed, when expressed in AtT-20 cells, vWf-containing, rod-shaped Weibel-Palade body-like structures are induced (Wagner etal., 1991), suggesting that this multimerisation of vWf is responsible for the characteristic morphology of Weibel-Palade bodies. The pro region of the pro-vWf, on the other hand, is required for vWf storage. Efficient cleavage of this pro sequence only occurs after sorting to the Weibel-Palade body, indicating that Weibel- Palade bodies contain a protease specific for this reaction (Wagner et al., 1991).
1.5.4.2. P-selectin (PADGEM/CD62/GMP-140).
P-selectin is a 140kD glycosylated type I integral membrane protein that belongs to a family of proteins that selectively adhere to cells with a lectin-like domain and are hence called "selectins" (figure 1.10.). These animal lectins have a carbohydrate recognition domain (CRD) that consists of invariant, highly conserved amino acid residues of characteristic spacing that can discriminate between a myriad of complex carbohydrate structures (reviewed by Drickamer and Taylor, 1993). This lectin-like domain is situated at the N-terminal tip of the extracellular/lumenal domain of p-selectin and contains the critical residues Lysl 13, Tyr48 and Tyi94 (Hollenbaugh et al., 1993) thought to bind specific sialyc acid residues ("Lewis ligands") of glycoproteins exposed on cell surface of
C y t o p l a s m i c
Domain
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Domain C3b C4b R egulatory Protein* R e p e a ts T ransm em brane Domain
Figure 1.10. The predicted structure o f p-selectin.
Diagrammatic representation of predicted p-selectin folding and disulphide bridge formation within the “lectin”, “E G F ’ and repeat domains (adapted from Johnston et. al.,
Figure l . i l . Regulated cell adhesion (McEver, 1991). platelet p-solectin activation platelet Welbal-Palade body endothelial cell endothelial cell platelet monocyte neutropNI
vessel wall endothelial cell
white blood cells (Lenter et a l, 1994; Moore et a i, 1995 and references therein). Thus, when endothelial cells and platelets are stimulated with histamine or thrombin: Weibel- Palade bodies and a-granules fuse with the plasma membrane; p-selectin is transferred to the cell surface in a regulated fashion; p-selectin acts as a cell adhesion molecule for monocytes and neutrophils (Harison and Cramer, 1992; McEver, 1991; McEver era/.,
1989; figure 1.11.).
The extracellular region of p-selectin also contains an epidermal growth factor-like
domain that has a role in ligand recognition (Kansas et a/., 1994) and nine repeat domains that have similar sequences to those of proteins from the complement system (Johnston et
a/., 1989; McEver, 1991; figure 1.10). It is the cytoplasmic domain of p-selectin that is thought to be involved in the incorporation of p-selectin to secretory granules (Disdier et
a/., 1992; Green etal.y 1994; Koedam etal., 1992; sections 1.5.10. and 1.6.3.). This cytoplasmic domain is encoded by three exons that contain (figure 1.12.): An acylation site at cysteine 766 that bears palmitic and stearic acid modifications (Fujimoto and McEver, 1993); and a potential tight turn motif between residues 777 and 710 (Johnston
et al.y 1989), although this has not been shown to act as an internalisation signal (section 1.2.1.).
The cytoplasmic domain also contains phosphorylation sites on serine, threonine and tyrosine residues that become phosphorylated within fifteen to twenty seconds of thrombin stimulation of platelets. Within five minutes, the threonine and tyrosine residues become dephosphorylated, while the serine residues remain phosphorylated (Crovello et a/., 1993). In both platelets and endothelial cells, these phosphorylation events are dependent on protein kinase C (PKC) (Fujimoto and McEver, 1993). P- selectin is also phosphorylated when expressed in Chinese hamster ovary (CHO) fibroblasts (which have no regulated secretory pathway) and mutagenesis studies show
Figure 1.12. The primary sequence o f the p-selectin cytoplasmic tail.