Extracellular matrix component Receptor 

Cells use a wide spectrum of proteins and mechanisms to recognize their environment. Evidence demonstrates that extracellular matrix components receptors can be used by Mks to control the site of platelet formation and release. Whether and how extracellular matrix component receptor activity is regulated during thrombopoiesis in vivo is not known. To progress towards a better understanding of these critical mechanisms, significantly improved knowledge of the physical, cellular and biochemical interactions in the bone marrow environment is needed. Integrins are the major human receptors for cell adhesion on extracellular matrix components, however a variety of other interaction mechanisms are possible. Besides integrin alpha2beta1 and GPVI, expression and function of other collagen receptors on human megakaryocytes are unknown. Discoidin domain receptors (DDR1 and DDR2) are tyrosine-kinase collagen receptors that are stimulated by fibrillar and basement membrane collagens and mediate cell adhesion and migration in different tissues. We recently discovered that DDR1 is expressed by both human megakaryocytes and platelets. DDR1 is activated upon megakaryocyte adhesion on fibrillar type I collagen and regulates megakaryocyte Syk-mediated migration through activation of the tyrosine phosphatase SHP1. Altogether, these data point out that DDR1 may represent an important new regulator of megakaryocyte function. 

Figure 1: Human MKs express and synthesize DDR1 tyrosine kinase. A, total cellular RNA was extracted from MKs and fibroblasts (Fb) as positive control. alpha2-microglobulin was used as housekeeping gene. NTC indicates “no template” controls in the reverse transcriptase and PCR steps. RT-PCR products were loaded in duplicates for each cell type. B, MK and fibroblast lysates were subjected to Western blot analysis using an anti-DDR1 antibody. The anti-DDR1 blocking peptide (B.P.) was used to confirm the specificity of the antibody. Actin was probed to show equal loading. C, DDR1 expression was demonstrated in peripheral blood platelet lysate (Plt) by Western blot. Shown here are representative Western blots out of three independent experiments. D, MKs were cytospun on polylysine-coated glass coverslips, fixed, and stained with an anti-DDR1 antibody (red) and an anti-CD61 antibody (green). The graphs report the intensity of the fluorescence signal along the x axis for each fluorochrome on the optical section. Scale bars are 25 mm. Nuclei were counterstained with Hoechst 33288 (blue).

Figure 1: Human MKs express and synthesize DDR1 tyrosine kinase. A, total cellular RNA was extracted from MKs and fibroblasts (Fb) as positive control. alpha2-microglobulin was used as housekeeping gene. NTC indicates “no template” controls in the reverse transcriptase and PCR steps. RT-PCR products were loaded in duplicates for each cell type. B, MK and fibroblast lysates were subjected to Western blot analysis using an anti-DDR1 antibody. The anti-DDR1 blocking peptide (B.P.) was used to confirm the specificity of the antibody. Actin was probed to show equal loading. C, DDR1 expression was demonstrated in peripheral blood platelet lysate (Plt) by Western blot. Shown here are representative Western blots out of three independent experiments. D, MKs were cytospun on polylysine-coated glass coverslips, fixed, and stained with an anti-DDR1 antibody (red) and an anti-CD61 antibody (green). The graphs report the intensity of the fluorescence signal along the x axis for each fluorochrome on the optical section. Scale bars are 25 mm. Nuclei were counterstained with Hoechst 33288 (blue).