Plasma and cellular fibronectin: distinct and independent functions during tissue repair

Fibronectin (FN) is a ubiquitous extracellular matrix (ECM) glycoprotein that plays vital roles during tissue repair. The plasma form of FN circulates in the blood, and upon tissue injury, is incorporated into fibrin clots to exert effects on platelet function and to mediate hemostasis. Cellular FN is then synthesized and assembled by cells as they migrate into the clot to reconstitute damaged tissue. The assembly of FN into a complex three-dimensional matrix during physiological repair plays a key role not only as a structural scaffold, but also as a regulator of cell function during this stage of tissue repair. FN fibrillogenesis is a complex, stepwise process that is strictly regulated by a multitude of factors. During fibrosis, there is excessive deposition of ECM, of which FN is one of the major components. Aberrant FN-matrix assembly is a major contributing factor to the switch from normal tissue repair to misregulated fibrosis. Understanding the mechanisms involved in FN assembly and how these interplay with cellular, fibrotic and immune responses may reveal targets for the future development of therapies to regulate aberrant tissue-repair processes

Immobilization of nonactivated unfixed platelets for real-time single-cell analysis

Existing methods for measuring the response of individual platelets to stimulation are limited. They either measure each platelet at one discrete time-point (flow cytometry) or rely on adhesive ligands to immobilize platelets that concomitantly generate activation signals (microscopy). Such methods of immobilization make it impossible to assess resting platelets, the changes that occur as platelets transition from resting to active states, or the signals generated by soluble agonists, such as ADP and thrombin, or by mechanical stimulus, independently from those generated by the adhesive ligand. Here we describe a microscopy method that allows the immobilization of platelets to a glass cover slip without triggering platelet activation. This method makes use of specific antibodies that bind platelet PECAM-1 without activating it. Platelets can therefore be immobilized to PECAM-1 antibody coated biochips without causing activation and perfused with agonists or inhibitors. Using this method, platelets can be stimulated by an array of soluble agonists at any concentration or combination, in the presence or absence of inhibitors or shear forces. This chapter describes in detail this PECAM-1 mediated immobilized platelet method and its use for measuring changes in Ca2+ signaling in individual platelets under a number of different conditions. While we focus on the measurement of Ca2+ dynamics in this chapter, it is important to consider that the basic method we describe will easily lend its self to other measures of platelet activation (integrin activation, shape change, actin dynamics, degranulation), and may, therefore, be used to measure almost any facet of platelet activation

Sc65-Null Mice Provide Evidence for a Novel Endoplasmic Reticulum Complex Regulating Collagen Lysyl Hydroxylation

Collagen is a major component of the extracellular matrix and its integrity is essential for connective tissue and organ function. The importance of proteins involved in intracellular collagen post-translational modification, folding and transport was recently highlighted from studies on recessive forms of osteogenesis imperfecta (OI). Here we describe the critical role of SC65 (Synaptonemal Complex 65, P3H4), a leprecan-family member, as part of an endoplasmic reticulum (ER) complex with prolyl 3-hydroxylase 3. This complex affects the activity of lysyl-hydroxylase 1 potentially through interactions with the enzyme and/or cyclophilin B. Loss of Sc65 in the mouse results in instability of this complex, altered collagen lysine hydroxylation and cross-linking leading to connective tissue defects that include low bone mass and skin fragility. This is the first indication of a prolyl-hydroxylase complex in the ER controlling lysyl-hydroxylase activity during collagen synthesis