The chaperone protein HSP47: a platelet collagen binding protein that contributes to thrombosis and hemostasis.

Essentials Heat shock protein 47 (HSP47), a collagen specific chaperone is present on the platelet surface. Collagen mediated platelet function was reduced following blockade or deletion of HSP47. GPVI receptor regulated signalling was reduced in HSP47 deficient platelets. Platelet HSP47 tethers to exposed collagen thus modulating thrombosis and hemostasis. SUMMARY: Objective Heat shock protein 47 (HSP47) is an intracellular chaperone protein that is vital for collagen biosynthesis in collagen secreting cells. This protein has also been shown to be present on the surface of platelets. Given the importance of collagen and its interactions with platelets in triggering hemostasis and thrombosis, in this study we sought to characterize the role of HSP47 in these cells. Methods and Results The deletion of HSP47 in mouse platelets or its inhibition in human platelets reduced their function in response to collagen and the GPVI agonist (CRP-XL), but responses to thrombin were unaltered. In the absence of functional HSP47, the interaction of collagen with platelets was reduced, and this was associated with reduced GPVI-collagen binding, signalling and platelet activation. Thrombus formation on collagen, under arterial flow conditions, was also decreased following the inhibition or deletion of HSP47, in the presence or absence of eptifibatide, consistent with a role for HSP47 in enhancing platelet adhesion to collagen. Platelet adhesion under flow to von Willebrand factor was unaltered following HSP47 inhibition. Laser-induced thrombosis in cremaster muscle arterioles was reduced and bleeding time was prolonged in HSP47-deficient mice or following inhibition of HSP47. Conclusions Our study demonstrates the presence of HSP47 on the platelet surface, where it interacts with collagen, stabilizes platelet adhesion and increases collagen-mediated signalling and therefore thrombus formation and hemostasis.

PPARγ agonists negatively regulate αIIbß3 integrin outside-in signaling and platelet function through up-regulation of protein kinase A activity.

Essentials peroxisome proliferator-activated receptor γ (PPARγ) agonists inhibit platelet function. PPARγ agonists negatively regulate outside-in signaling via integrin αIIbß3. PPARγ agonists disrupt the interaction of Gα13 with integrin ß3. This is attributed to an upregulation of protein kinase A activity. SUMMARY: Background Agonists for the peroxisome proliferator-activated receptor (PPARγ) have been shown to have inhibitory effects on platelet activity following stimulation by GPVI and GPCR agonists. Objectives Profound effects on thrombus formation led us to suspect a role for PPARγ agonists in the regulation of integrin αIIbß3 mediated signaling. Both GPVI and GPCR signaling pathways lead to αIIbß3 activation, and signaling through αIIbß3 plays a critical role in platelet function and normal hemostasis. Methods The effects of PPARγ agonists on the regulation of αIIbß3 outside-in signaling was determined by monitoring the ability of platelets to adhere and spread on fibrinogen and undergo clot retraction. Effects on signaling components downstream of αIIbß3 activation were also determined following adhesion to fibrinogen by Western blotting. Results Treatment of platelets with PPARγ agonists inhibited platelet adhesion and spreading on fibrinogen and diminished clot retraction. A reduction in phosphorylation of several components of αIIbß3 signaling, including the integrin ß3 subunit, Syk, PLCγ2, focal adhesion kinase (FAK) and Akt, was also observed as a result of reduced interaction of the integrin ß3 subunit with Gα13. Studies of VASP phosphorylation revealed that this was because of an increase in PKA activity following treatment with PPARγ receptor agonists. Conclusions This study provides further evidence for antiplatelet actions of PPARγ agonists, identifies a negative regulatory role for PPARγ agonists in the control of integrin αIIbß3 outside-in signaling, and provides a molecular basis by which the PPARγ agonists negatively regulate platelet activation and thrombus formation.

Platelet signaling: a complex interplay between inhibitory and activatory networks.

The role of platelets in hemostasis and thrombosis is dependent on a complex balance of activatory and inhibitory signaling pathways. Inhibitory signals released from the healthy vasculature suppress platelet activation in the absence of platelet receptor agonists. Activatory signals present at a site of injury initiate platelet activation and thrombus formation; subsequently, endogenous negative signaling regulators dampen activatory signals to control thrombus growth. Understanding the complex interplay between activatory and inhibitory signaling networks is an emerging challenge in the study of platelet biology, and necessitates a systematic approach to utilize experimental data effectively. In this review, we will explore the key points of platelet regulation and signaling that maintain platelets in a resting state, mediate activation to elicit thrombus formation, or provide negative feedback. Platelet signaling will be described in terms of key signaling molecules that are common to the pathways activated by platelet agonists and can be described as regulatory nodes for both positive and negative regulators.

Visualization and characterization of the acrosome reaction of human spermatozoa by immunolocalization with monoclonal antibody.

A monoclonal antibody generated against hamster epididymal spermatozoa and recognizing an antigen within the acrosome was used in conjunction with FITC-antimouse immunoglobulin as a marker of the human acrosome during sperm development, capacitation, and the acrosome reaction. The specificity of binding of the monoclonal antibody was assessed using immunolocalization by epi-fluorescence and electron microscopy. Immunofluorescence revealed that antibody bound over the entire anterior acrosome in hamster and human spermatozoa. Ultrastructural localization indicated that antigen was predominantly present on the inner face of the outer acrosomal membrane and within the acrosomal content. Qualitative specificity was studied using a highly purified preparation of hamster acrosomes in an enzyme-linked immunosorbent assay. Since the antibody rapidly visualized human acrosomes, it was used to detect abnormal acrosome morphology of mature spermatozoa and to mark spermatids present in the ejaculate. During incubation in capacitating medium, changes in the immunofluorescence of live or methanol fixed spermatozoa were correlated with incubation interval and the ability of spermatozoa to fuse with zona-free hamster oocytes. Spermatozoa bound to zona-free hamster oocytes displayed no fluorescence, confirming that acrosome loss occurred before spermatozoa attached to the vitellus.

Monoclonal antibody against a sperm antigen Mr 95,000 inhibits attachment of human spermatozoa to the zona pellucida.

A murine monoclonal antibody raised against hamster spermatozoa was found to cross-react with human spermatozoa. By immunofluorescence, the antigen was visualized over the equatorial segment of human sperm heads. In the presence of antibody, sperm binding to the zona pellucida of salt-stored human oocytes was significantly inhibited (P less than or equal to 0.005) compared with other antibodies or control preparations. Using SDS-PAGE of whole spermatozoa and membrane preparations followed by Western blot analysis, the antigen was identified as a determinant with a relative molecular weight of 95,000.