P. A. Castaldi

Bio: P. A. Castaldi is an academic researcher from University of Sydney. The author has contributed to research in topics: Platelet & Von Willebrand factor. The author has an hindex of 23, co-authored 38 publications receiving 2321 citations. Previous affiliations of P. A. Castaldi include Westmead Hospital & Royal Prince Alfred Hospital.

Characterization of GMP-140 (P-selectin) as a circulating plasma protein.

Abstract: GMP-140 is a 140-kD granule membrane protein, found in the alpha granules of platelets and the Weibel-Palade bodies of endothelial cells, that is surface expressed on cell activation and mediates neutrophil attachment. Cloning data for GMP-140 from an endothelial library predict a soluble form of the protein, the transcription message for which is also found in platelets. In this study, we report the detection by enzyme-linked immunosorbent assay of soluble GMP-140 in plasma centrifuged for 3 h at 100,000 g (to remove platelet microparticles) and confirm its identity by purification from plasma. Plasma concentrations were found to be 0.251 +/- 0.043 micrograms/ml (means +/- SD, n = 10) in normal male controls and 0.175 +/- 0.063 micrograms/ml (means +/- SD, n = 10) in normal female controls. The purified protein had an identical molecular mass (nonreduced) to platelet membrane GMP-140 (approximately 3 kD lower, reduced) and was immunoblotted by polyclonal anti-GMP-140, and the anti-GMP-140 monoclonal antibodies AK4 and AK6. Analytical gel filtration studies indicated that the plasma GMP-140 eluted as a monomer whereas detergent-free, platelet membrane GMP-140 eluted as a tetramer consistent with plasma GMP-140 lacking a transmembrane domain. Purified plasma GMP-140 bound to the same neutrophil receptor as the membrane-bound form, and when immobilized on plastic, bound neutrophils equivalently to immobilized platelet membrane GMP-140. Since it has been shown that fluid-phase GMP-140 is antiinflammatory and downregulates CD18-dependent neutrophil adhesion and respiratory burst, its presence in plasma may be of major importance in preventing the inadvertent activation of neutrophils in the circulation.

Purification and preliminary characterization of the glycoprotein Ib complex in the human platelet membrane

Abstract: Human platelet glycoprotein Ib (GP Ib) is a major integral membrane protein that has been identified as the platelet-binding site mediating the factor VIII/von Willebrand-factor-dependent adhesion of platelets to vascular subendothelium. Recent evidence suggests that GP Ib is normally complexed with another platelet membrane protein, GP IX. In this study, human platelet plasma membranes were selectively solubilized with a buffer containing 0.1% (v/v) Triton X-100. The GP Ib complex (GP Ib plus GP IX) was purified to homogeneity in ∼ 30% yield by immunoaffinity chromatography of the membrane extract using the anti-(glycoprotein Ib complex) murine monoclonal antibody, WM 23, coupled to agarose. GP Ib and GP IX were subsequently isolated as purified components by immunoaffinity chromatography of the GP Ib complex using a second anti-(glycoprotein Ib complex) monoclonal antibody, FMC 25, coupled to agarose. As assessed by dodecyl sulphate/polyacrylamide gel electrophoresis, purified GP Ib was identical to the molecule on intact platelets and had an apparent relative molecular mass of 170000 under nonreducing conditions and 135000 (α subunit) and 25000 (β subunit) under reducing conditions. GP IX had an apparent Mr of 22000 under both nonreducing and reducing conditions. Purified Gb Ib complex and GP Ib inhibited the ristocetin-mediated, human factor VIII/von Willebrand-factor-dependent and bovine factor VIII/von Willebrand-factor-dependent agglutination of washed human platelets suggesting the proteins had been isolated in functionally active form. GP Ibα had a similar amino acid composition to that previously reported for its proteolytic degradation product, glycocalicin. The amino acid compositions of GP Ibβ and GP IX were similar but showed marked differences in the levels of glutamic acid, alanine, histidine and arginine. The N-termini of GP Ibα and GP IX were blocked; GP Ibβ had the N-terminal sequence, Ile-Pro-Ala-Pro-. On crossed immunoelectrophoresis, both GP Ib and GP IX were found to occur in the same immunoprecipitin arc(s) whether the platelets had been solubilized in the absence or presence of the calcium-dependent protease inhibitor, leupeptin. Binding studies in platelet-rich plasma indicated a similar number of binding sites (x± SD) for three anti-(glycoprotein Ib complex) monoclonal antibodies: AN 51, epitope on GP Ibα (22000 ± 2700, n= 3), WM 23, epitope on GP Ibα (21000 ± 3400, n= 3), FMC 25, epitope on GP IX (20100 ± 2700, n= 3), and FMC 25 (Fab′)2 (27100 ± 800, n= 2). The combined evidence suggests that GP Ib is normally bound to GP IX and that the stoichiometry of this complex is 1:1.

Biochemistry and Genetics of von Willebrand Factor

Abstract: ▪ Abstract Von Willebrand factor (VWF) is a blood glycoprotein that is required for normal hemostasis, and deficiency of VWF, or von Willebrand disease (VWD), is the most common inherited bleeding disorder. VWF mediates the adhesion of platelets to sites of vascular damage by binding to specific platelet membrane glycoproteins and to constituents of exposed connective tissue. These activities appear to be regulated by allosteric mechanisms and possibly by hydrodynamic shear forces. VWF also is a carrier protein for blood clotting factor VIII, and this interaction is required for normal factor VIII survival in the circulation. VWF is assembled from identical ≈250 kDa subunits into disulfide-linked multimers that may be >20,000 kDa. Mutations in VWD can disrupt this complex biosynthetic process at several steps to impair the assembly, intracellular targeting, or secretion of VWF multimers. Other VWD mutations impair the survival of VWF in plasma or the function of specific ligand binding sites. This growing...

Proteinase-Activated Receptors

Abstract: Proteinase-activated receptors are a recently described, novel family of seven-transmembrane G-protein-coupled receptors. Rather then being stimulated through ligand receptor occupancy, activation is initiated by cleavage of the N terminus of the receptor by a serine protease resulting in the generation of a new tethered ligand that interacts with the receptor within extracellular loop-2. To date, four proteinase-activated receptors (PARs) have been identified, with distinct N-terminal cleavage sites and tethered ligand pharmacology. In addition to the progress in the generation of PAR-1 antagonists, we describe the role of thrombin in such processes as wound healing and the evidence implicating PAR-1 in vascular disorders and cancer. We also identify advances in the understanding of PAR-1-mediated intracellular signaling and receptor desensitization. The cellular functions, signaling events, and desensitization processes involved in PAR-2 activation are also assessed. However, other major aspects of PAR-2 are highlighted, in particular the ability of several serine protease enzymes, in addition to trypsin, to function as activators of PAR-2. The likely physiological and pathophysiological roles for PAR-2 in skin, intestine, blood vessels, and the peripheral nervous system are considered in the context of PAR-2 activation by multiple serine proteases. The recent discovery of PAR-3 and PAR-4 as additional thrombin-sensitive PARs further highlights the complexity in assessing the effects of thrombin in several different systems, an issue that remains to be fully addressed. These discoveries have also highlighted possible PAR–PAR interactions at both functional and molecular levels. The future identification of other PARs and their modes of activation are an important future direction for this expanding field of study.