Aleksandra Stojanovic

A molecular switch that controls cell spreading and retraction

Integrin-dependent cell spreading and retraction are required for cell adhesion, migration, and proliferation, and thus are important in thrombosis, wound repair, immunity, and cancer development. It remains unknown how integrin outside-in signaling induces and controls these two opposite processes. This study reveals that calpain cleavage of integrin β3 at Tyr759 switches the functional outcome of integrin signaling from cell spreading to retraction. Expression of a calpain cleavage–resistant β3 mutant in Chinese hamster ovary cells causes defective clot retraction and RhoA-mediated retraction signaling but enhances cell spreading. Conversely, a calpain-cleaved form of β3 fails to mediate cell spreading, but inhibition of the RhoA signaling pathway corrects this defect. Importantly, the calpain-cleaved β3 fails to bind c-Src, which is required for integrin-induced cell spreading, and this requirement of β3-associated c-Src results from its inhibition of RhoA-dependent contractile signals. Thus, calpain cleavage of β3 at Tyr759 relieves c-Src–mediated RhoA inhibition, activating the RhoA pathway that confines cell spreading and causes cell retraction.

A Phosphoinositide 3-Kinase-AKT-Nitric Oxide-cGMP Signaling Pathway in Stimulating Platelet Secretion and Aggregation

Phosphoinositide 3-kinase (PI3K) and Akt play important roles in platelet activation. However, the downstream mechanisms mediating their functions are unclear. We have recently shown that nitric-oxide (NO) synthase 3 and cGMP-dependent protein kinase stimulate platelet secretion and aggregation. Here we show that PI3K-mediated Akt activation plays an important role in agonist-stimulated platelet NO synthesis and cGMP elevation. Agonist-induced elevation of NO and cGMP was inhibited by Akt inhibitors and reduced in Akt-1 knock-out platelets. Akt-1 knock-out or Akt inhibitor-treated platelets showed reduced platelet secretion and aggregation in response to low concentrations of agonists, which can be reversed by low concentrations of 8-bromo-cGMP or sodium nitroprusside (an NO donor). Similarly, PI3K inhibitors diminished elevation of cGMP and inhibited platelet secretion and the second wave platelet aggregation, which was also partially reversed by 8-bromo-cGMP. These results indicate that the NO-cGMP pathway is an important downstream mechanism mediating PI3K and Akt signals leading to platelet secretion and aggregation. Conversely, the PI3K-Akt pathway is the major upstream mechanism responsible for activating the NO-cGMP pathway in platelets. Thus, this study delineates a novel platelet activation pathway involving sequential activation of PI3K, Akt, nitric-oxide synthase 3, sGC, and cGMP-dependent protein kinase.

Stimulatory roles of nitric-oxide synthase 3 and guanylyl cyclase in platelet activation

Nitric oxide (NO) stimulates soluble guanylyl cyclase and, thus, enhances cyclic guanosine monophosphate (cGMP) levels. It is a currently prevailing concept that NO inhibits platelet activation. This concept, however, does not fully explain why platelet agonists stimulate NO production. Here we show that a major platelet NO synthase (NOS) isoform, NOS3, plays a stimulatory role in platelet secretion and aggregation induced by low doses of platelet agonists. Furthermore, we show that NOS3 promotes thrombosis in vivo. The stimulatory role of NOS is mediated by soluble guanylyl cyclase and results from a cGMP-dependent stimulation of platelet granule secretion. These findings delineate a novel signaling pathway in which agonists sequentially activate NOS3, elevate cGMP, and induce platelet secretion and aggregation. Our data also suggest that NO plays a biphasic role in platelet activation, a stimulatory role at low NO concentrations and an inhibitory role at high NO concentrations.

Tyrosine Phosphorylation of the Integrin β3 Subunit Regulates β3 Cleavage by Calpain

Outside-insignalingofβ3integrinsinducesandrequiresphosphorylation at tyrosine 747 (Tyr747) and tyrosine 759 (Tyr759) of the β3 subunit, but the mechanism for this requirement is unclear. On the other hand, a key consequence of integrin signaling, cell spreading, is inhibited by calpain cleavage of β3 cytoplasmic domain. Here we show that β3 tyrosine phosphorylation inhibits calpain cleavage. Mutating both tyrosines to phenylalanine sensitizes β3 to calpain cleavage. Furthermore, phosphorylation at Tyr747 and Tyr759 of β3 in the focal adhesion sites and the leading edge of spreading platelets was differentially regulated. Selective dephosphorylation of Tyr759 is associated with calpain cleavage at Tyr759. Thus, one mechanism by which tyrosine phosphorylation promotes integrin signaling and cell spreading is its inhibition of calpain cleavage of the β3 cytoplasmic domain.

Heat-shock protein gp96/grp94 is an essential chaperone for the platelet glycoprotein Ib-IX-V complex

The platelet glycoprotein Ib-IX-V complex (GPIb-IX-IV) is the receptor for VWF and is responsible for VWF-mediated platelet activation and aggregation. Loss of the GPIb-IX-V complex is pathogenic for Bernard-soulier Syndrome (BSS), which is characterized by macrothrombocytopenia and impaired platelet function. It remains unclear how the GPIb-IX-V complex is assembled and whether there is a role for a specific molecular chaperone in the process. In the present study, we report that the assembly of the GPIb-IX-V complex depends critically on a molecular chaperone in the endoplasmic reticulum (ER): gp96 (also known as grp94 and HSP90b1). gp96/grp94 deletion in the murine hematopoietic system results in thrombocytopenia, prolonged bleeding time, and giant platelets that are clinically indistinguishable from human BSS. Loss of gp96/grp94 in vivo and in vitro leads to the concomitant reduction in GPIb-IX complex expression due to ER-associated degradation. We further demonstrate that gp96/grp94 binds selectively to the GPIX subunit, but not to gpIbα or gpIbβ. Therefore, we identify the platelet GPIX subunit of the GPIb-IX-V complex as an obligate and novel client of gp96/grp94.

An Important Role of the Src Family Kinase Lyn in Stimulating Platelet Granule Secretion

The Src family kinases (SFKs) have been proposed to play stimulatory and inhibitory roles in platelet activation. The mechanisms for these apparently contradictory roles are unclear. Here we show that SFK, mainly Lyn, is important in stimulating a common signaling pathway leading to secretion of platelet granules. Lyn knock-out or an isoform-nonselective SFK inhibitor, PP2, inhibited platelet secretion of both dense and α granules and the secretion-dependent platelet aggregation induced by thrombin, collagen, and thromboxane A2. The inhibitory effect of Lyn knock-out on platelet aggregation was reversed by supplementing granule content ADP, indicating that the primary role of Lyn is to stimulate granule secretion. Inhibitory effect of PP2 on platelet aggregation induced by thrombin and thromboxane A2 were also reversed by supplementing ADP. Furthermore, PP2 treatment or Lyn knock-out diminished agonist-induced Akt activation and cyclic GMP production. The inhibitory effect of PP2 or Lyn knock-out on platelet response can be corrected by supplementing cyclic GMP. These data indicate that Lyn stimulates platelet secretion by activating the phosphoinositide 3-kinase-Akt-nitric oxide (NO)-cyclic GMP pathway and also provide an explanation why Lyn can both stimulate and inhibit platelet activation.

Signaling-mediated Functional Activation of Inducible Nitric-oxide Synthase and Its Role in Stimulating Platelet Activation

Nitric oxide (NO) is a short lived secondary messenger, synthesized by nitric-oxide synthases (NOS). It is believed that the activity of inducible NOS (iNOS) is regulated primarily at the transcription level by inducing expression of iNOS mRNA and protein, which then continuously produces NO, until its degradation. Platelets do not have the nuclear transcriptional regulatory mechanisms of the iNOS gene and are believed to generate NO in response to agonist stimulation via endothelial NOS (eNOS). However, here we show that agonist-induced NO production is only partially eNOS-dependent and is also mediated by iNOS. Platelet agonist-induced NO production is significantly reduced in iNOS-knockout platelets. Platelet NO production occurs within seconds after agonist addition and is not accompanied by changes in iNOS protein levels, indicating a signaling-mediated functional activation mechanism of iNOS. Importantly, iNOS knock-out and iNOS inhibitors reduce agonist-induced platelet secretion and aggregation and cGMP levels, indicating that iNOS activation is important in stimulating platelets via the newly identified NO-cGMP-dependent platelet secretion pathway. Furthermore, iNOS knock-out mice have prolonged bleeding time, suggesting that this novel mode of regulation of iNOS activity plays a physiologically relevant role in hemostasis.

Consistent Up-regulation of Stat3 Independently of Jak2 Mutations in a New Murine Model of Essential Thrombocythemia

Janus-activated kinase 2 (JAK2) mutations are common in myeloproliferative disorders; however, although they are detected in virtually all polycythemia vera patients, they are found in approximately 50% of essential thrombocythemia (ET) patients, suggesting that converging pathways/abnormalities underlie the onset of ET. Recently, the chromosomal translocation 3;21, leading to the fusion gene AML1/MDS1/EVI1 (AME), was observed in an ET patient. After we forced the expression of AME in the bone marrow (BM) of C57BL/6J mice, all the reconstituted mice died of a disease with symptoms similar to ET with a latency of 8 to 16 months. Peripheral blood smears consistently showed an elevated number of dysplastic platelets with anisocytosis, degranulation, and giant size. Although the AME-positive mice did not harbor Jak2 mutations, the BM of most of them had significantly higher levels of activated Stat3 than the controls. With combined biochemical and biological assays we found that AME binds to the Stat3 promoter leading to its up-regulation. Signal transducers and activators of transcription 3 (STAT3) analysis of a small group of ET patients shows that in about half of the patients, there is STAT3 hyperactivation independently of JAK2 mutations, suggesting that the hyperactivation of STAT3 by JAK2 mutations or promoter activation may be a critical step in development of ET.