Thomas M. Chiang

Platelet contributions to the pathogenesis of systemic sclerosis

Purpose of reviewThe 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 findingsAn extensive literature provides strong support for varying degrees of platelet activation and aggregation in different forms and stages of systemic sclerosis. A recent finding is that systemic sclerosis platelets overexpress a specific nonintegrin 65 kDa receptor for type I collagen as well as expressing enhanced phosphilidylinositol-3 kinase as an activation signature. Overexpression of a type I collagen receptor would make systemic sclerosis platelets more susceptible to binding to exposed type I collagen in the subendothelial lining of damaged blood vessels, facilitating the cycle of platelet aggregation and release of preformed bioactive molecules that include a host of inflammatory and fibrogenic, chemokines, cytokines and growth factors. This activation phenotype of systemic sclerosis platelets may be secondary to autoimmunity and driven by cytokines from autoreactive T cells. SummaryThe contributions of platelets to the pathogenesis of systemic sclerosis is likely substantial and may not be adequately represented in gene profiling of systemic sclerosis tissue due to the small amounts of RNA contained in platelets.

Binding of Chemotactic Collagen-Derived Peptides to Fibroblasts: THE RELATIONSHIP TO FIBROBLAST CHEMOTAXIS

We previously showed that collagen, alpha-chains, and collagen-derived peptide fragments induce chemotactic migration of human fibroblasts in vitro. We now describe biochemical and immunological evidence showing there are binding sites for collagen peptides on fibroblast membranes.By the use of (14)C-labeled alpha1(I) chain, binding to intact fibroblasts was demonstrated. The process was reversible, and time- and fibroblast concentration-dependent. Scatchard plot analyses of the data obtained for the binding of alpha1(I) suggested that there are congruent with 16 x 10(6) binding sites per fibroblast with an association constant of 1.1 x 10(7)/M for alpha1(I). Dissociation of the bound radioactivity and subsequent chromatographic analysis on agarose A-1.5 m revealed that the alpha1 was unaltered. The binding of (14)C-labeled alpha1 was inhibited by each of the CNBr peptides derived from alpha1 chain of chick skin collagen and CNBr peptide mixtures of various genetic types of collagen chains. Immunofluorescence studies with anti-alpha1 antibody showed that alpha1-treated fibroblasts exhibited strong immunofluorescence. The intensity of fluorescence was markedly diminished by prior absorption of the antibody with alpha1. The alpha1-treated cells stained with preimmune sera did not show significant fluorescence.Dose-response curves of fibroblast chemotaxis induced by alpha1 and the binding of alpha1 by fibroblasts correlate closely. Furthermore, the potency of alpha1-CNBr peptides as chemotactic agents correlates with their ability to inhibit the binding of labeled alpha1(I). These data suggest the hypothesis that collagenderived peptides cause fibroblast chemotactic migration by acting on fibroblast membranes.

A synthetic peptide derived from the sequence of a type I collagen receptor inhibits type I collagen-mediated platelet aggregation.

A synthetic peptide-1, an 18 amino acid residue peptide derived from a hydrophilic domain of a cloned platelet type I collagen receptor, was used to study the role of the receptor on types I and III collagen-induced platelet aggregation and the release of ATP. The peptide inhibits the type I, but not the type III, collagen-induced platelet aggregation and the release of ATP in a dose-dependent manner. The [125I]peptide-1 specifically binds to type I collagen-coated microtiter wells in a dose-dependent manner (with Kd = 10 nM). The binding of [125I]peptide-1 can be inhibited by an excess of unlabeled peptide-1 suggesting that the binding is specific. The labeled peptide-1 does not bind to type III collagen-coated microtiter wells. Results from an enzyme-linked immunosorbent assay show that the peptide reacts with the poly- and monoclonal antibodies raised against the purified platelet type I collagen receptor (Mr 65 kD). The peptide also inhibits the adhesion of platelets on type I collagen matrix and rabbit aortic segments in a dose-dependent manner. These results suggest that the reactive site of the platelet receptor for type I collagen resides in this portion of the molecule.

An enzyme hydrolyzing methylated inhibitors of nitric oxide synthase is present in circulating human red blood cells

NG,NG-dimethyl-l-arginine (asymmetric dimethylarginine or ADMA) and NG-monomethyl-l-arginine (l-NMMA) are post-translationally synthesized amino acids of nuclear proteins. Upon release during protein turnover, they are not used in protein synthesis, but are excreted or metabolized by dimethylarginine dimethylaminohydrolase (DDAH) found in many tissues. DDAH is present in monocytic and polynuclear cells of blood, but no report has appeared of its presence in red blood cells (RBCs). Because methylated arginines can inhibit nitric oxide synthase (NOS) and elevations are reported in several diseases, we explored whether RBCs express this enzyme. DDAH is present in RBCs as supported by hydrolysis of both ADMA and l-NMMA, but not symmetric dimethylarginine, and by immunoprecipitation/Western blot using a specific monoclonal antibody to human DDAH. In a pilot study of end-stage renal disease (ESRD) patients, RBC DDAH activity with ADMA as substrate correlated inversely with age (p = 0.005) and enzyme activities were higher in patients with greater diastolic blood pressure drops during hemodialysis (p = 0.02). Similar correlations were found with white cell DDAH activity. Thus, human RBCs can hydrolyze methylated arginines. These findings indicate the RBC could be used to assess the status of DDAH in various disease states.

Collagen-platelet interaction: Separate receptor sites for types I and III collagen

We have isolated a platelet membrane protein of M(r) 47 kDa which is responsible for the interaction of platelets with type III collagen. The 47 kDa protein was purified to apparent homogeneity by type III collagen-Sepharose 2B column chromatography and preparative slab gel electrophoreses. The 47 kDa protein blocked the adhesion of platelets to type III but not to type I collagen. Polyclonal antibodies were obtained from rabbits immunized with the purified 47 kDa protein emulsified in complete Freunds adjuvant. The polyclonal antibodies inhibited the type III collagen but not type I collagen-induced platelet aggregation. The inhibitory effect of the antibodies on type III collagen-induced platelet aggregation was dose-dependent. Cross-inhibition on platelet aggregation studies showed that type I collagen receptor antibodies (M(r) 65 kDa) did not inhibit type III collagen-induced platelet aggregation and type III collagen receptor antibodies did not inhibit type I collagen induced platelet aggregation. These results suggest that type I and type III collagens interact with platelets at separate sites.

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. A computer search revealed that the cDNA represents the coding sequence of a protein with a fragmentary homology to several proteins. Using a prokaryotic expression system, pBad TOPO-47 cDNA, a 47-kDa recombinant protein was obtained and purified to apparent homogeneity by nickel-nitrilotriacetic acid resin and collagen affinity column. The recombinant protein binds to type III but not type I collagen-Sepharose 2B affinity columns. Anti-47-kDa but not anti-65-kDa antibody inhibits the binding of the recombinant protein to the type III collagen-coated micro titer wells in a dose-dependent manner. Like the receptor protein purified from platelet membranes, the recombinant protein inhibits type III collagen-induced platelet aggregation also in a dose-dependent manner. We have defined two active peptides from the cloned deduced amino acid sequence. Both peptides inhibit type III but not type I collagen-induced platelet aggregation in a dose-dependent fashion. These results suggest that the active binding site of the platelet receptor to type III collagen resides in these portions of the protein.

Activation of phospholipase D in human platelets by collagen and thrombin and its relationship to platelet aggregation

Stimulation of phospholipase D after activation of cell surface receptors has been reported in many cell types. We have investigated the mechanism of activation of this enzyme by collagen in the human platelet by assaying the release of [3H]methylcholine from [3H]methylphosphatidylcholine. Results from these studies suggest that phospholipase D activity is regulated by reversible phosphorylation. Phospholipase D activity was stimulated when platelet-rich plasma was preincubated with collagen and was not inhibited by aspirin. Among various aggregating agents tested, collagen and thrombin but not ADP activated phospholipase D activity (2- to 3-fold). The addition of sphingosine inhibited phospholipase D activity. Preincubation of platelet-rich plasma with sphingosine inhibited collagen- and thrombin-induced platelet aggregation and the release of ATP. The inhibitory effect of sphingosine on collagen- and thrombin- induced platelet aggregation and release of ATP was dose-dependent. The functional significance of phospholipase D activation was also tested by examining the effect of the product, phosphatidic acid, on collagen-induced platelet aggregation and release of ATP. Platelet shape change and the reversibility of platelet aggregation resulted by the addition of phosphatidic acid to platelet-rich plasma. Furthermore, the simultaneous addition of phosphatidic acid and collagen shortened the latency period but had no effect on platelet aggregation. Two platelet proteins (47 kDa and 22 kDa) increased in phosphorylation after the addition of 1 microM phosphatidic acid which did not cause platelet aggregation. These results suggest that collagen stimulates phospholipase D activity which plays a secondary role in platelet aggregation and the release reaction.

Upregulation of nitric oxide synthase in cultured human keratinocytes after ultraviolet B and bradykinin.

Kang‐Rotondo CH, Major S, Chiang TM, Myers LK, Kang ES. Upregulation of nitric oxide synthase in cultured human keratinocytes after ultraviolet B and bradykinin. Photodermatol Photoimmunol Photomed 1996: 12: 57–65.

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. In this investigation, Western blot analyses of time course samples with anti-phosphorylated tyrosine, and anti-serine/threonine showed a marked increase in serine/threonine phosphorylation of eNOS during type I collagen-induced platelet aggregation. Meanwhile, the eNOS activity measured by the conversion of [3H]-arginine to [3H]-citrulline is significantly decreased. Correlation of type I collagen-induced platelet aggregation and the activity of eNOS in the presence of the serine/threonine phosphatase inhibitor, okadiac acid and the tyrosine phosphatase inhibitor, vanadate were performed with PRP. Results show the decrease in eNOS activity by adding okadiac acid correlated with the inhibitory effect on platelet aggregation in a dose-dependent manner. On the other hand, vanadate significantly inhibits platelet aggregation and also inhibits eNOS activity when the concentration of vanadate is greater than 2 mM. These results suggest that phosphorylation of serine/threonine and tyrosine residues control the activity of eNOS through different mechanisms to affect collagen-induced platelet aggregation.

Okadaic acid and vanadate inhibit collagen-induced platelet aggregation; the functional relation of phosphatases on platelet aggregation

The different specific inhibitors for phosphoserine/threonine and phosphotyrosine protein phosphatases were used to study the role of these protein phosphatases in collagen-platelet interaction. The collagen-induced platelet aggregation and the release reaction as measured ATP release were inhibited in a dose-dependent fashion by the addition of okadaic acid, a specific inhibitor of phosphoserine/threonine protein phosphatase 1 and 2A. The inhibition was also observed by the addition of a phosphotyrosine protein phosphatase inhibitor, vanadate. Suboptimal concentrations of both inhibitors together also inhibited collagen-induced platelet aggregation and release reaction in a concentration-dependent fashion. These results suggest that collagen-platelet interaction is modulated by both protein phosphatases.