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Biomedical subjects

C M Ward

Publications and source records attributed to C M Ward.

At least 37 records · Page 2Linked to original sources
PubMed1997

Binding of the von Willebrand factor A1 domain to histone.

Activation of the von Willebrand Factor (vWF) A1 domain is a critical factor in regulating the interaction of vWF with its platelet membrane receptor, the glycoprotein (GP) Ib-IX-V complex. This activation controls vWF-dependent platelet adhesion at high shear. The vWF-GP Ib-IX-V interaction is induced in vivo by exposure of platelet-rich plasma to high shear force, or by association of vWF with one or more unidentified components of the subendothelial matrix. In vitro, soluble vWF is activated to bind to platelets by nonphysiological modulators, such as the bacterial glycopeptide, ristocetin, or the snake venom protein, botrocetin, or by removal of negatively-charged sialic acid residues. Analysis of vWF modulators and the very marked charge asymmetry of amino acid sequences within the A1 domain has led to an electrostatic model for vWF modulation. Endothelial membrane/matrix and detergent-soluble fractions of human placenta were screened for the ability to bind vWF by electrophoresis of extracts on SDS-polyacrylamide gels, electrotransferring to nitrocellulose and probing with fluid-phase 125I-labeled vWF or a 39/34-kDa vWF fragment (Leu-480-Gly-718) that encompasses the A1 domain. In the course of these studies, it was found that both vWF and the 39/34-kDa vWF fragment bound strongly to histone. Purified soluble histone also bound vWF since, like ristocetin, it induced vWF flocculation. Histone binding to vWF did not activate or inhibit vWF binding to platelets. While the vWF-histone interaction has no conceivable physiological role, it suggests that binding to the A1 domain of vWF alone is insufficient to modulate vWF adhesive activity. This implies that specific interactions of the vWF A1 domain with either ristocetin or botrocetin are required for GP Ib-IX-V recognition to occur.

Amino Acid Sequence↗
PubMed1997

The protein-tyrosine phosphatase SHP-2 binds platelet/endothelial cell adhesion molecule-1 (PECAM-1) and forms a distinct signaling complex during platelet aggregation. Evidence for a mechanistic link between PECAM-1- and integrin-mediated cellular signaling.

Platelet/endothelial cell adhesion molecule-1 (PECAM-1) is a homophilic adhesion receptor that mediates leukocyte/endothelial cell interactions that take place during transendothelial migration. Recent reports have shown that the binding of certain anti-PECAM-1 antibodies results in up-regulation of integrin function on the surface of leukocytes and platelets, suggesting that PECAM-1 may be capable of transmitting information into the cell following its engagement. PECAM-1 isolated from resting or activated but nonaggregated platelets was phosphorylated predominantly on serine residues; however, PECAM-1 derived from activated, aggregated platelets was strongly phosphorylated on tyrosine. Synthetic tyrosine-phosphorylated peptides derived from five different regions within the cytoplasmic domain of PECAM-1 were screened for their ability to associate with cytoplasmic signaling molecules. The protein-tyrosine phosphatase SHP-2 was found to interact specifically with two different PECAM-1 phosphopeptides containing highly conserved phosphatase-binding motifs on PECAM-1 with the sequences VQpY663TEV and TVpY686SEV. More important, SHP-2 bound not only PECAM-1 phosphopeptides, but also became associated with full-length cellular PECAM-1 during the platelet aggregation process, and this interaction was mediated by the amino-terminal Src homology 2 domains of the phosphatase. Since SHP-2 normally serves as a positive regulator of signal transduction, its association with activated PECAM-1 suggests a number of potential mechanisms by which PECAM-1 engagement might be coupled to integrin activation in vascular cells.

Amino Acid Sequence↗
PubMed1996

Binding of a novel 50-kilodalton alboaggregin from Trimeresurus albolabris and related viper venom proteins to the platelet membrane glycoprotein Ib-IX-V complex. Effect on platelet aggregation and glycoprotein Ib-mediated platelet activation.

Binding of the multimeric adhesive glycoprotein, von Willebrand Factor (vWF), to the platelet membrane glycoprotein (GP) Ib-IX-V complex mediates platelet adhesion and initiates signal transduction leading to platelet activation. Recently described viper venom proteins that bind to the GP Ib alpha-chain and inhibit vWF binding provide novel probes for studying receptor function. We have purified a 50-kDa form of alboaggregin from the white-lipped tree viper (Trimeresurus albolabris) and two 25-kDa proteins, CHH-A and CHH-B, from the timber rattlesnake (Crotalus horridus horridus) in addition to a previously described 25-kDa alboaggregin and echicetin. Complete or partial amino acid sequencing of CHH-A, CHH-B, and 50-kDa alboaggregin and cross-reactivity of these proteins with an anti-botrocetin antiserum confirmed that they were disulfide-linked heterodimers or higher multimers of the C-type lectin protein family. These proteins, together with 25-kDa alboaggregin and echicetin, specifically bound to GP Ib alpha within the N-terminal peptide domain, His-1-Glu-282, and inhibited vWF binding with comparable IC50 values (approximately 0.2 microgram/mL). However, cross-blocking studies between these structurally related proteins and anti-GP Ib alpha monoclonal antibodies demonstrated that the venom protein binding sites were not congruent. Further, the 50-kDa alboaggregin, but not the other venom proteins, potently induced platelet activation as assessed by dense granule serotonin release or elevation of cytosolic ionized calcium. Treatment of platelets with the 50-kDa alboaggregin was associated with activation of protein kinase C and tyrosine kinase(s), resulting in a platelet protein phosphorylation profile similar to that seen on shear-stress-induced vWF binding to platelets. These results suggest that the 50-kDa alboaggregin induces cytoplasmic signaling coincident with its binding to the GP Ib-IX-V complex and provides a potentially useful probe for studying the mechanism of vWF-dependent platelet activation.

Amino Acid Sequence↗
PubMed1996

Mocarhagin, a novel cobra venom metalloproteinase, cleaves the platelet von Willebrand factor receptor glycoprotein Ibalpha. Identification of the sulfated tyrosine/anionic sequence Tyr-276-Glu-282 of glycoprotein Ibalpha as a binding site for von Willebrand factor and alpha-thrombin.

Platelet adhesion to the subendothelium is the initiating event in hemostasis and thrombosis and involves the binding of von Willebrand factor (vWF) by the platelet membrane glycoprotein (GP) Ib-IX complex. The alpha-chain of GP Ib contains binding sites for both vWF and alpha-thrombin within a 45-kDa N-terminal tryptic fragment. In the present study, we have further delineated these sites using smaller proteolytic fragments and functional antibodies. Mocarhagin, a cobra venom metallaproteinase, generates the fragment His-1-Glu-282, while cathepsin G, a neutrophil granule serine protease, generates a slightly smaller fragment, His-1-Leu-275. His-1-Glu-282 was as effective as intact soluble GP Ibalpha (glycocalicin) in inhibiting botrocetin-dependent binding of vWF to washed platelets (IC50 approximately 0.3 microM) whereas His-1-Leu-275 was an order of magnitude less effective (IC50 approximately 3 microM). Residues Tyr-276-Glu-282 (YDYYPEE) are part of an anionic region homologous to thrombin-binding molecules such as hirudin. In ligand blot analysis, thrombin blotted the His-1-Glu-282 fragment, but not His-1-Leu-275. The three tyrosine residues within Tyr-276-Glu-282 meet the consensus criteria for O-sulfation. A method was developed to distinguish O-sulfated from nonsulfated tyrosine residues based on differences in the UV absorbance spectra. Residues Tyr-276-Glu-282 were isolated from glycocalicin by proteolysis with mocarhagin and cathepsin G. Ion spray mass spectrometry confirmed that Tyr-278 and Tyr-279 was only approximately 50% O-sulfated. Four anti-GP Ibalpha monoclonal antibodies (SZ2, ES85, C34 and VM16d) were found to be modulator-specific, strongly inhibiting botrocetin-dependent binding of vWF, but having less or no effect on ristocetin-dependent vWF binding. These antibodies also inhibited the binding of thrombin to fixed platelets. Immunoprecipitation with GP ibalpha fragments defined the epitopes for these antibodies as SZ2 (Tyr-276-Glu-282), ES85 (Asp-283-Arg-293), C34 (His-1-Glu-282), and VM16d (His-1-Leu-275). An antibody which inhibited ristocetin-dependent, as well as botrocetin-dependent, vWF binding but had no effect on thrombin binding (Ak2) had an epitope within His-1-Leu-275. These findings indicate that the sulfated tyrosine/anionic GP Ibalpha residues Tyr-276-Glu-282 are important for the binding of thrombin and botrocetin-dependent binding of thrombin and the botrocetin-dependent binding of vWF, but that vWF also interacts with residues within His-1-Leu-275.

Amino Acid Sequence↗
PubMed1996

Characterization of mocarhagin, a cobra venom metalloproteinase from Naja mocambique mocambique, and related proteins from other Elapidae venoms.

Mocarhagin, a cobra venom metalloproteinase from Naja mocambique mocambique, has previously been shown to cleave selectively two mucin-like substrates on platelets and neutrophils within anionic amino acid sequences containing sulfated tyrosines. We now show that purified mocarhagin has haemagglutinin activity, and a similar profile for inhibition of mocarhagin-dependent haemagglutination and proteolysis suggests that the lectin-like domain may account for its substrate specificity. In addition, immunologically and functionally related proteins were detected in other Elapidae venoms.

Animals↗
PubMed1995

Jararhagin and jaracetin: novel snake venom inhibitors of the integrin collagen receptor, alpha 2 beta 1.

Two novel proteins, jararhagin and jaracetin, were purified from Bothrops jararaca viper venom. Jararhagin is a 55-kDa member of the metalloprotease-disintegrin family. Jaracetin is a 60-kDa dimer representing a differently processed form of jararhagin. Like botrocetin, a previously described viper venom protein, jararhagin and jaracetin modulated binding of von Willebrand Factor to the glycoprotein Ib-IX complex on platelets through a specific interaction with the von Willebrand Factor A1 domain. Both jararhagin and jaracetin, but not botrocetin, also blocked alpha 2 beta 1-dependent platelet adhesion to collagen, a receptor interaction mediated through a homologous A domain on the integrin alpha 2 subunit.

Animals↗
PubMed1995

The molecular mechanism of platelet adhesion.

One of the most primitive of host-defence mechanisms is haemostasis, the ability to control blood loss. In response to vascular trauma, platelets rapidly adhere to the exposed subendothelial matrix, a process that ultimately results in the sealing of the vessel by a plug of platelets stabilised by fibrin. Paradoxically, it is the same cascade of events that leads to thrombosis and vessel occlusion, resulting in heart attack and stroke. The molecular events involved in platelet adhesion have therefore been the subject of intense investigation. In all but the largest blood vessels, the initial contact adhesion of platelets is mediated by subendothelial matrix bound von Willebrand Factor (vWF) and a specific vWF receptor on platelets, the glycoprotein (GP) Ib-V-IX complex. Our understanding of this process arose from analysis of two congenital bleeding disorders, von Willebrand's disease and the Bernard-Soulier syndrome, in which vWF or the GP Ib-V-IX, respectively, are either absent or dysfunctional. This overview discusses our current molecular understanding of platelet adhesion and how engagement of vWF by the GP Ib-V-IX complex on platelets initiates the subsequent events in platelet activation leading to either haemostasis or thrombosis.

Animals↗
PubMed1992

Identification of aspartic acid 514 through glutamic acid 542 as a glycoprotein Ib-IX complex receptor recognition sequence in von Willebrand factor. Mechanism of modulation of von Willebrand factor by ristocetin and botrocetin.

As the first step in hemostasis, the binding of von Willebrand factor (vWF) to the platelet membrane glycoprotein (GP) Ib-IX complex is essential for platelet adhesion at high-shear blood flow. This interaction in vivo requires the prior binding of vWF to the subendothelial matrix, a process which exposes a normally cryptic binding site on vWF for the GP Ib-IX complex. This process can be mimicked in vitro by modulators such as ristocetin or the snake venom protein botrocetin or by desialation of vWF. We have previously localized the GP Ib binding site on vWF to a monomeric dispase fragment which extends from Leu-480/Val-481 to Gly-718 in the primary sequence of mature vWF [Andrews, R. K., Gorman, J. J., Booth, W. J., Corino, G. L., Castaldi, P. A., & Berndt, M. C. (1989) Biochemistry 28, 8326-8336]. This fragment also contains a distinct binding site for botrocetin. Analysis of synthetic peptides corresponding to hydrophilic stretches of sequence within this fragment indicated that the sequence Asp-514-Glu-542 represents a major adhesive sequence involved in receptor recognition. This peptide inhibited both the ristocetin- and botrocetin-mediated binding of vWF to either platelets or purified GP Ib-IX complex (IC50 approximately 50-200 microM) as well as the asialo-vWF- and bovine vWF-dependent agglutination of platelets. Both the N- and C-terminal halves of the peptide were inhibitory but less so than the intact peptide. This peptide also inhibited botrocetin binding to vWF, suggesting that botrocetin modulates vWF-GP Ib interaction by binding in close proximity to the vWF adhesion sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

New grafts for old? A review of alternatives to autologous skin.

Immediate resurfacing of skin defects is a challenging prospect, especially in patients with extensive full-thickness burns. Currently, split-thickness autografts offer the best form of wound coverage, but limited donor sites and their associated morbidity have prompted the search for alternatives. The application of allogeneic skin is restricted by availability and the risk of transmission of infection, whilst synthetic skin substitutes are simply expensive dressings. The problems of limited expansion may be overcome by culturing keratinocytes in vitro. Unlike autologous cells, allogeneic keratinocytes are available immediately, although they survive for less than a week when applied to full-thickness skin defects. Moreover, the absence of a dermal component in these grafts predisposes to instability and contracture. A cross-linked collagen and glycosaminoglycan dermal substitute, covered with thin split-skin grafts or cultured autologous keratinocytes, shows promise in burns patients. An alternative is a collagen matrix populated by allogeneic fibroblasts and overlaid with cultured autologous or allogeneic keratinocytes. The clinical application of cultured grafts remains imperfect but offers the prospect of immediate coverage and massive expansion.

Culture Techniques↗