Effect of a hybrid peptide derived from platelet type I and type III collagen receptors on CHO-65 cells.

The function of the platelet collagen receptor, 65-kDa, specific for type I collagen (CI), in eukaryotic cells remains elusive. In order to examine the effect of posttranslational modification of the receptor, we have made a construct, which contains the 65-kDa cDNA ligated into pcDNA3 (eukaryotic expression vector) by using standard cloning methodologies. The construct was transfected into CHO (CHO-65) cells, which expressed the protein, detectable by immunoblots [using the anti-65kDa antibodies (65-Ab)], and flow cytometry confirmed expression on the cell surface.

Alteration in protein kinase B (AKT) activity in platelets from patients with systemic sclerosis.

We have previously reported that there is an increase in phosphotidylinositide-3 kinase activity secondary to increase in nitrotyrosylation in platelets from patients with SSc. Protein kinase B (Akt) is recruited to the plasma membrane by PI 3-K metabolites. Both enzymes play critical roles in signal transduction and activation of platelets.

Platelet contributions to the pathogenesis of systemic sclerosis.

Purpose Of Review: The purpose of this review is to focus attention on platelet contributions, in general, to systemic sclerosis. There have also been recent advances in characterization of the phenotype of platelets in systemic sclerosis which will be reviewed.

Recent Findings: An extensive literature provides strong support for varying degrees of platelet activation and aggregation in different forms and stages of systemic sclerosis.

A recombinant protein and a chemically synthesized peptide containing the active peptides of the platelet collagen receptors inhibit ferric chloride-induced thrombosis in a rat model.

We have previously reported that a recombinant protein (M(r) 47 kDa), which contains both active peptide of platelet receptors for types I and III collagen inhibits both types I and III collagen-induced platelet aggregation. In order to eliminate non-reactive portion of the protein, we have constructed a recombinant of rHyB (M r 6 kDa). In addition, we chemically synthesized a hybrid peptide with 30 amino acid residues (cHyB, M r 3 kDa) that contains each of the active peptide derived from platelet receptors for types I and III collagen and a linker of 12 amino acid residues.

A cell model system to study regulation of phosphotidylinositol 3-kinase and protein kinase B activity by cytokines/growth factors produced by type I collagen stimulated immune cells from patients with systemic sclerosis.

We have reported that posttranslational modification of systemic sclerosis patients' platelet phosphoinositide 1,3,4,5 kinase (PI 3-K) and protein kinase B (Akt) altered their enzymatic activities. In the present investigation, we have established a cell line model to study further the effects of posttranslational modification and modification by cytokines or growth factors of these two enzymes. Results from these studies suggest that posttranslational modification by phosphorylation of Akt and nitrotyrosylation of PI 3-K increases enzymatic activities, as was observed from SSc patients' platelets.

Peptides derived from platelet non-integrin collagen-receptors or types I and III collagen inhibit collagen-platelet interaction.

Platelet-collagen interaction plays an important role in hemostasis and pathological thrombosis. Upon an injury to the subendothelium of a blood vessel wall, platelets adhere to the denuded substrate, aggregate, and release biological substances. Many investigators have explored the use of blocking agents to interrupt the final step of binding fibrinogen on glycoprotein (GP) IIb/IIIa of activated platelets.

Type I and type III collagen-platelet interaction: inhibition by type specific receptor peptides.

We have previously cloned and characterized a platelet receptor for type III collagen (47 kDa) from a human bone marrow cDNA phage library and defined two active peptides. We also cloned and characterized a platelet receptor for type I collagen (65 kDa) and defined an active peptide. Our objective was to study whether there is type specificity of these active peptides.

Increase in platelet non-integrin type I collagen receptor in patients with systemic sclerosis.

Microvascular injury is one of the major pathogenetic processes involved in systemic sclerosis (SSc). Interaction of the platelet types I and III collagen receptors with their respective ligand in the exposed subendothelial stroma as a result of ongoing microvascular injury in SSc patients results in platelet activation and aggregation with the release of mediators, which contribute to vascular damage and inflammation. We have found that there is a twofold increase in radiolabeled type I collagen binding to washed platelets from patients with SSc compared to platelets obtained from normal volunteers.

Increase in phosphotidylinositide-3 kinase activity by nitrotyrosylation of lysates of platelets from patients with systemic sclerosis.

We have observed that the platelet non-integrin collagen receptor (65 kDa) and another protein (M(r) 185 kDa) are altered in the posttranslational modification by nitrotyrosylation in platelets from patients with systemic sclerosis (SSc). We reported the identification of nitrotyrosylated 65-kDa proteins in a previous study. In the present investigation, using Western blots, one- and two-dimensional gel electrophoreses and matrix assisted ionization/desorption-time of flight (MALDI-TOF) we have identified the 185-kDa protein as phosphoinositide kinase C2beta (PI 3-K).

A defined peptide that inhibits the formation of the glycoprotein IIb and IIIa complex.

Collagen-platelet interaction plays an important role in hemostasis and pathological thrombosis. The proposed mechanism of the interaction was the activation of platelets-->releasing of contents from granules-->aggregation. The common end point is the platelets and fibrin aggregates.

Sunscreen ingredients inhibit inducible nitric oxide synthase (iNOS): a possible biochemical explanation for the sunscreen melanoma controversy.

Sunscreen products are rated upon their ability to inhibit visible redness of the skin 24 h after measured doses of ultraviolet (UV) exposure (Sun Protection Factor, SPF). Although sunscreens prevent UV-induced redness, their ability to protect against melanoma or the development of moles is less clear. UV-induced redness occurs in part by the action of nitric oxide (NO), synthesized in the skin.

The signal transduction pathway of the nonintegrin receptor of 65 kDa is different from glycoprotein VI.

The identity and signal pathways of a platelet nonintegrin receptor for type I collagen, 65 kDa, are not established. In this investigation, we have examined whether there is a difference in the signal transduction pathways between the 65-kDa protein and glycoprotein VI (GP VI). Results from this study show that these two proteins are different based on the following facts.

Role of platelet endothelial form of nitric oxide synthase in collagen-platelet interaction: regulation by phosphorylation.

Different pathways have been reported to be involved in platelet-collagen interaction. We have reported that the platelet endothelial form of nitric oxide synthase (eNOS) and the platelet receptor for type I collagen, p65, are closely associated. But the controlling mechanism underlying the generation of nitric oxide (NO) by the eNOS has not been fully explored.

Cloning, characterization, and functional studies of a 47-kDa platelet receptor for type III collagen.

A 1.2-kb cDNA fragment encoding a platelet 47-kDa protein has been isolated from a human bone marrow cDNA library by using a degenerate oligonucleotide of the sequenced amino terminus of the purified platelet protein with a poly(dT)(12).(dG) by polymerase chain reaction.