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• W1992296952 abstract "First of all, we would like to thank Akkerman et al. [1Akkerman J.W.N. Gerrits A.J. Ferreira I.A. Heemskerk J.W.M. Insulin inhibition of platelet‐endothelial interaction is mediated by insulin effects on endothelial cells without direct effects on platelets: a rebuttal.J Thromb Haemost. 2009; 7: 369-71Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar] for their valuable comments, as well as the JTH editors for the opportunity to respond to this letter. Clearly, research studies on insulin function/diabetes, and particularly on insulin effects on hemostasis/platelet function, are strongly affected by various experimental conditions. Therefore, we principally agree with the authors of the rebuttal letter that some of the differences between their previously published work, other papers in the field and our present results may be due to different experimental procedures and methods used. As an example, we would like to mention here that the authors of the rebuttal letter themselves could not reproduce [2Ferreira I.A. Eybrechts K.L. Mocking A.I. Kroner C. Akkerman J.W. IRS‐1 mediates inhibition of Ca2+ mobilization by insulin via the inhibitory G‐protein Gi.J Biol Chem. 2004; 279: 3254-64Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar] several effects of insulin on platelets published by other groups, that is, rise of cAMP [3Trovati M. Anfossi G. Massucco P. Mattiello L. Costamagna C. Piretto V. Mularoni E. Cavalot F. Bosia A. Ghigo D. Insulin stimulates nitric oxide synthesis in human platelets and, through nitric oxide, increases platelet concentrations of both guanosine‐3′,5′‐cyclic monophosphate and adenosine‐3′,5′‐cyclic monophosphate.Diabetes. 1997; 46: 742-9Crossref PubMed Scopus (121) Google Scholar] and activation of NO synthase [4Fleming I. Schulz C. Fichtlscherer B. Kemp B.E. Fisslthaler B. Busse R. AMP‐activated protein kinase (AMPK) regulates the insulin‐induced activation of the nitric oxide synthase in human platelets.Thromb Haemost. 2003; 90: 863-71Crossref PubMed Google Scholar]. The main conclusion of our recent work was that insulin inhibits platelet function primarily via an indirect, endothelium‐dependent mechanism, and not via a direct effect on a platelet insulin receptor (IR). However, the authors of the rebuttal letter raised a number of important questions that we wish to address here as follows. 1Questionable expression of IR in platelets. Up to now, there is no clear biochemical evidence that human platelets express IRs. The only evidence for IR in platelets dates from 1979 and is indirect, based on 125I‐radiolabeled insulin binding to isolated platelet plasma membranes [5Hajek A.S. Joist J.H. Baker R.K. Jarett L. Daughaday W.H. Demonstration and partial characterization of insulin receptors in human platelets.J Clin Invest. 1979; 63: 1060-5Crossref PubMed Scopus (50) Google Scholar]. Using IR antibodies, which clearly detect endogenous IR in endothelial cells and specifically cross‐react with IR overexpressed in endothelial cells, we observed only weak signals migrating at the apparent molecular weight of a putative IR in platelet lysates. As the antibody cross‐reacts with many other platelet proteins, with at an even higher intensity as compared to the ‘putative IR’, we concluded that such bands most likely represent the unspecific staining that is often observed in western blots. In one of the papers [2Ferreira I.A. Eybrechts K.L. Mocking A.I. Kroner C. Akkerman J.W. IRS‐1 mediates inhibition of Ca2+ mobilization by insulin via the inhibitory G‐protein Gi.J Biol Chem. 2004; 279: 3254-64Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar] cited by Akkerman et al., only anti‐IR phospho‐Tyr1158 antibody (but not anti‐total IR as an important control) was used. In one of our recent papers [6Gambaryan S. Kobsar A. Hartmann S. Birschmann I. Kuhlencordt P.J. Muller‐Esterl W. Lohmann S.M. Walter U. NO‐synthase‐/NO‐independent regulation of human and murine platelet soluble guanylyl cyclase activity.J Thromb Haemost. 2008; 6: 1376-84Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar], we demonstrated that phospho‐antibodies may produce surprising (unspecific) ‘positive’ data when used without controls for the respective total antigen/protein. In our recent experiments, we could detect phosphorylated IR of the same intensity as presented in Fig. 8D of ref. [2Ferreira I.A. Eybrechts K.L. Mocking A.I. Kroner C. Akkerman J.W. IRS‐1 mediates inhibition of Ca2+ mobilization by insulin via the inhibitory G‐protein Gi.J Biol Chem. 2004; 279: 3254-64Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar] only in ECV cells overexpressing IR, but not in platelets or even in ECV cells not overexpressing IR (Fig. 5 of [7Rauchfuss S. Geiger J. Walter U. Renne T. Gambaryan S. Insulin inhibition of platelet–endothelial interaction is mediated by insulin effects on endothelial cells without direct effects on platelets.J Thromb Haemost. 2008; 6: 856-64Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar]).2Platelet calcium experiments. In the methods section of our manuscript [7], we regrettably did not precisely indicate that for these experiments platelets were isolated in the presence of apyrase, as is routinely done in our laboratory to prevent desensitization of P2Y12 receptors. We established previously that intact P2Y12‐mediated signaling can be effectively demonstrated by strong ADP inhibition of prostaglandin I2‐induced (or prostaglandin E1‐induced) protein kinase A(PKA)‐mediated vasodilator‐stimulated phosphoprotein (VASP) phosphorylation [8Schwarz U.R. Geiger J. Walter U. Eigenthaler M. Flow cytometry analysis of intracellular VASP phosphorylation for the assessment of activating and inhibitory signal transduction pathways in human platelets – definition and detection of ticlopidine/clopidogrel effects.Thromb Haemost. 1999; 82: 1145-52Crossref PubMed Scopus (287) Google Scholar]. The results of western blot analysis of VASP phosphorylation at Ser157 (preferential PKA phosphorylation site, which reflects platelet cAMP content) in platelets as prepared and used in our experiments are shown in Fig. 1. Ten minutes of incubation of platelets with apyrase increases basal VASP phosphorylation (reflecting inhibition of P2Y12 receptor activation and an increase of cAMP), whereas stimulation with 10 μm ADP significantly decreases basal VASP phosphorylation (reflecting P2Y12 receptor activation with subsequent cAMP decrease).3Platelet hyperreactivity in type 2 diabetes mellitus patients. In our experiments, we did not study platelets from patients with type 2 diabetes mellitus. In the discussion, we only presented our hypothesis (which should be tested in the future) that platelet hyperreactivity in type 2 diabetes mellitus patients may not be associated with a direct insulin effect on platelets, but is more likely to be associated with impaired insulin effects on endothelial cells. This hypothesis is based on our own observation that insulin did not directly affect platelet adhesion to collagen‐coated plates [7Rauchfuss S. Geiger J. Walter U. Renne T. Gambaryan S. Insulin inhibition of platelet–endothelial interaction is mediated by insulin effects on endothelial cells without direct effects on platelets.J Thromb Haemost. 2008; 6: 856-64Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar]. Also, in our article [7Rauchfuss S. Geiger J. Walter U. Renne T. Gambaryan S. Insulin inhibition of platelet–endothelial interaction is mediated by insulin effects on endothelial cells without direct effects on platelets.J Thromb Haemost. 2008; 6: 856-64Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar], we did not deny the fact that platelets from type 2 diabetes mellitus patients studied in vitro may be ‘hyperreactive’. However, we did want to emphasize that, in our opinion, IR‐mediated platelet inhibition is most likely due to endothelial‐mediated rather than direct platelet‐mediated effects.4Physiologic relevance of insulin/IR signaling in platelet function. As mentioned by the authors of the letter, the postprandial glucose concentration rises to 7–8 mm in healthy individuals. However, there is no indication in the literature that a glucose concentration up to 10 mm has any effect on platelet function. In the cited paper (reference 19 of the letter) [9Sudic D. Razmara M. Forslund M. Ji Q. Hjemdahl P. Li N. High glucose levels enhance platelet activation: involvement of multiple mechanisms.Br J Haematol. 2006; 133: 315-22Crossref PubMed Scopus (76) Google Scholar], the authors described enhanced agonist‐induced platelet activation only at 30 mm glucose (which is extremely high for a healthy individual). Thus, the question of ‘whether the relative weak effect of insulin significantly contributes to the control of platelet responsiveness in the circulation’ still remains open. Direct, acute insulin effects on platelets appear to be rather weak, if present at all, and this may account for some of the observed discrepancies between the data obtained by different groups using different methods of washed platelet preparation. Also, unphysiologically high insulin levels may affect platelet function via insulin‐like growth factor receptors, which are present in platelets. There appear to still be many unresolved, important questions concerning insulin signaling in human platelets that require further study. The authors state that they have no conflict of interest." @default.
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• W1992296952 title "Insulin inhibition of platelet‐endothelial interaction is mediated by insulin effects on endothelial cells without direct effects on platelets: reply to a rebuttal" @default.
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