The C-Type Lectin Receptor CD93 Regulates Platelet Activation and Surface Expression of the Protease Activated Receptor 4.

BACKGROUND: The C-type lectin receptor CD93 is a single pass type I transmembrane glycoprotein involved in inflammation, immunity, and angiogenesis. This study investigates the role of CD93 in platelet function. CD93 knockout (KO) mice and wild-type (WT) controls were compared in this study. METHODS: Platelet activation and aggregation were investigated by flow cytometry and light transmission aggregometry, respectively. Protein expression and phosphorylation were analyzed by immunoblotting. Subcellular localization of membrane receptors was investigated by wide-field and confocal microscopy. RESULTS: The lack of CD93 in mice was not associated to any evident bleeding defect and no alterations of platelet activation were observed upon stimulation with thromboxane A2 analogue and convulxin. Conversely, platelet aggregation induced by stimulation of the thrombin receptor PAR4 was significantly reduced in the absence of CD93. This defect was associated with a significant reduction of α-granule secretion, integrin αIIbß3 activation, and protein kinase C (PKC) stimulation. Resting WT and CD93-deficient platelets expressed comparable amounts of PAR4. However, upon stimulation with a PAR4 activating peptide, a more pronounced clearance of PAR4 from the platelet surface was observed in CD93-deficient platelets compared with WT controls. Confocal microscopy analysis revealed a massive movement of PAR4 in cytosolic compartments of activated platelets lacking CD93. Accordingly, platelet desensitization following PAR4 stimulation was more pronounced in CD93 KO platelets compared with WT controls. CONCLUSION: These results demonstrate that CD93 supports platelet activation triggered by PAR4 stimulation and is required to stabilize the expression of the thrombin receptor on the cell surface.

Blood Platelets in Infection: The Multiple Roles of the Platelet Signalling Machinery.

Platelets are classically recognized for their important role in hemostasis and thrombosis but they are also involved in many other physiological and pathophysiological processes, including infection. Platelets are among the first cells recruited to sites of inflammation and infection and they exert their antimicrobial response actively cooperating with the immune system. This review aims to summarize the current knowledge on platelet receptor interaction with different types of pathogens and the consequent modulations of innate and adaptive immune responses.

Proteomic and functional profiling of platelet-derived extracellular vesicles released under physiological or tumor-associated conditions.

During hemostasis, thrombosis, and inflammation, activated blood platelets release extracellular vesicles (PEVs) that represent biological mediators of physiological and pathological processes. We have recently demonstrated that the activation of platelets by breast cancer cells is accompanied by a massive release of PEVs, evidence that matches with the observation that breast cancer patients display increased levels of circulating PEVs. A core concept in PEVs biology is that their nature, composition and biological function are strongly influenced by the conditions that induced their release. In this study we have performed a comparative characterization of PEVs released by platelets upon activation with thrombin, a potent thrombotic stimulus, and upon exposure to the breast cancer cell line MDA-MB-231. By nanoparticle tracking analysis and tandem mass spectrometry we have characterized the two populations of PEVs, showing that the thrombotic and tumoral stimuli produced vesicles that largely differ in protein composition. The bioinformatic analysis of the proteomic data led to the identification of signaling pathways that can be differently affected by the two PEVs population in target cells. Specifically, we have demonstrated that both thrombin- and cancer-cell-induced PEVs reduce the migration and potentiate Ca2+-induced apoptosis of Jurkat cells, but only thrombin-derived PEVs also potentiate cell necrosis. Our results demonstrate that stimulation of platelets by thrombotic or tumoral stimuli induces the release of PEVs with different protein composition that, in turn, may elicit selective biological responses in target cells.

Platelet-Derived Extracellular Vesicles Stimulate Migration through Partial Remodelling of the Ca2+ Handling Machinery in MDA-MB-231 Breast Cancer Cells.

BACKGROUND: Platelets can support cancer progression via the release of microparticles and microvesicles that enhance the migratory behaviour of recipient cancer cells. We recently showed that platelet-derived extracellular vesicles (PEVs) stimulate migration and invasiveness in highly metastatic MDA-MB-231 cells by stimulating the phosphorylation of p38 MAPK and the myosin light chain 2 (MLC2). Herein, we assessed whether the pro-migratory effect of PEVs involves the remodelling of the Ca2+ handling machinery, which drives MDA-MB-231 cell motility. METHODS: PEVs were isolated from human blood platelets, and Fura-2/AM Ca2+ imaging, RT-qPCR, and immunoblotting were exploited to assess their effect on intracellular Ca2+ dynamics and Ca2+-dependent migratory processes in MDA-MB-231 cells. RESULTS: Pretreating MDA-MB-231 cells with PEVs for 24 h caused an increase in Ca2+ release from the endoplasmic reticulum (ER) due to the up-regulation of SERCA2B and InsP3R1/InsP3R2 mRNAs and proteins. The consequent enhancement of ER Ca2+ depletion led to a significant increase in store-operated Ca2+ entry. The larger Ca2+ mobilization from the ER was required to potentiate serum-induced migration by recruiting p38 MAPK and MLC2. CONCLUSIONS: PEVs stimulate migration in the highly metastatic MDA-MB-231 breast cancer cell line by inducing a partial remodelling of the Ca2+ handling machinery.

The Controversial Role of LPS in Platelet Activation In Vitro.

Circulating platelets are responsible for hemostasis and thrombosis but are also primary sensors of pathogens and are involved in innate immunity, inflammation, and sepsis. Sepsis is commonly caused by an exaggerated immune response to bacterial, viral, and fungal infections, and leads to severe thrombotic complications. Among others, the endotoxin lipopolysaccharide (LPS) found in the outer membrane of Gram-negative bacteria is the most common trigger of sepsis. Since the discovery of the expression of the LPS receptor TLR4 in platelets, several studies have investigated the ability of LPS to induce platelet activation and to contribute to a prothrombotic phenotype, per se or in combination with plasma proteins and platelet agonists. This issue, however, is still controversial, as different sources, purity, and concentrations of LPS, different platelet-purification protocols, and different methods of analysis have been used in the past two decades, giving contradictory results. This review summarizes and critically analyzes past and recent publications about LPS-induced platelet activation in vitro. A methodological section illustrates the principal platelet preparation protocols and significant differences. The ability of various sources of LPS to elicit platelet activation in terms of aggregation, granule secretion, cytokine release, ROS production, and interaction with leukocytes and NET formation is discussed.

Proline-rich tyrosine kinase Pyk2 regulates deep vein thrombosis.

Deep vein thrombosis results from the cooperative action of leukocytes, platelets, and endothelial cells. The proline-rich tyrosine kinase Pyk2 regulates platelet activation and supports arterial thrombosis. In this study, we combined pharmacological and genetic approaches to unravel the role of Pyk2 in venous thrombosis. We found that mice lacking Pyk2 almost completely failed to develop deep venous thrombi upon partial ligation of the inferior vena cava. Pyk2-deficient platelets displayed impaired exposure of phosphatidylserine and tissue factor expression by endothelial cells and monocytes was completely prevented by inhibition of Pyk2. In human umbilical vein endothelial cells (HUVEC), inhibition of Pyk2 hampered IL-1b-induced expression of VCAM and P-selectin, and von Willebrand factor release. Pyk2-deficient platelets showed defective adhesion on von Willebrand factor and reduced ability to bind activated HUVEC under flow. Moreover, inhibition of Pyk2 in HUVEC strongly reduced platelet adhesion. Similarly, Pyk2-deficient neutrophils were unable to efficiently roll and adhere to immobilized endothelial cells under venous flow conditions. Moreover, platelets and neutrophils from Pyk2- knockout mice showed defective ability to form heterogeneous aggregates upon stimulation, while platelet monocyte interaction occurred normally. Consequently, platelet neutrophil aggregates, abundant in blood of wild-type mice upon inferior vena cava ligation, were virtually undetectable in Pyk2-knockout mice. Finally, we found that expression of Pyk2 was required for NETosis induced by activated platelets. Altogether our results demonstrate a critical role of Pyk2 in the regulation of the coordinated thromboinflammatory responses of endothelial cells, leukocytes and platelets leading to venous thrombosis. Pyk2 may represent a novel promising target in the treatment of deep vein thrombosis.

Platelet-derived extracellular vesicles regulate cell cycle progression and cell migration in breast cancer cells.

Platelets have been extensively implicated in the progression of cancer and platelet-derived extracellular vesicles (PEVs) are gaining growing attention as potential mediators of the platelet-cancer interplay. PEVs are shed from platelet membrane in response to extracellular stimuli and carry important biological signals for intercellular communication. In this study we demonstrate that PEVs specifically bind to different breast cancer cells and elicit cell-specific functional responses. PEVs were massively internalized by the metastatic cell lines MDA-MB-231 and SKBR3 and the ductal carcinoma cell line BT474, but not by the MCF-7 cell line. In SKBR3 cells, PEVs decreased mitochondrial dehydrogenase activities and altered cell cycle progression without affecting cell viability. Conversely, PEVs potently stimulated migration and invasion of MDA-MB-231, without affecting the distribution in the different phases of the cell cycle. In all the analyzed breast cancer cells, PEVs triggered a sustained increase of intracellular Ca2+, but only in MDA-MB-231 cells, this was associated to the stimulation of selected signaling proteins implicated in migration, including p38MAPK and myosin light chain. Importantly, inhibition of myosin light chain phosphorylation by a Rho kinase inhibitor prevented PEVs-stimulated migration of MDA-MB-231 cells. Our results demonstrate that PEVs are versatile regulators of cancer cell behavior and elicit a variety of different responses depending on the specific breast cancer cell subtype.

Hydrogen Sulfide-Evoked Intracellular Ca2+ Signals in Primary Cultures of Metastatic Colorectal Cancer Cells.

Exogenous administration of hydrogen sulfide (H2S) is emerging as an alternative anticancer treatment. H2S-releasing compounds have been shown to exert a strong anticancer effect by suppressing proliferation and/or inducing apoptosis in several cancer cell types, including colorectal carcinoma (CRC). The mechanism whereby exogenous H2S affects CRC cell proliferation is yet to be clearly elucidated, but it could involve an increase in intracellular Ca2+ concentration ([Ca2+]i). Herein, we sought to assess for the first time whether (and how) sodium hydrosulfide (NaHS), one of the most widely employed H2S donors, induced intracellular Ca2+ signals in primary cultures of human metastatic CRC (mCRC) cells. We provided the evidence that NaHS induced extracellular Ca2+ entry in mCRC cells by activating the Ca2+-permeable channel Transient Receptor Potential Vanilloid 1 (TRPV1) followed by the Na+-dependent recruitment of the reverse-mode of the Na+/Ca2+ (NCX) exchanger. In agreement with these observations, TRPV1 protein was expressed and capsaicin, a selective TRPV1 agonist, induced Ca2+ influx by engaging both TRPV1 and NCX in mCRC cells. Finally, NaHS reduced mCRC cell proliferation, but did not promote apoptosis or aberrant mitochondrial depolarization. These data support the notion that exogenous administration of H2S may prevent mCRC cell proliferation through an increase in [Ca2+]i, which is triggered by TRPV1.

Fibrillar amyloid peptides promote platelet aggregation through the coordinated action of ITAM- and ROS-dependent pathways.

BACKGROUND: Amyloid peptides Aß40 and Aß42, whose deposition in brain correlates with Alzheimer disease, are also present in platelets and have prothrombotic activities. OBJECTIVE: In this study, we analyze the ability of Aß peptides to form fibrils and to induce platelet activation and aggregation. METHODS: Aß40, Aß42, and their scrambled peptides were diluted in phosphate buffered saline and fibrillogenesis was investigated by ThioflavinT and Congo Red. Aggregation, protein phosphorylation, and reactive oxygen species (ROS) production were analyzed. RESULTS: Aß40 and Aß42, but not scrambled peptides, were able to form fibrils when diluted in phosphate buffered saline. Fibrillogenesis of Aß42 was very rapid, whereas fibril formation by Aß40 was completed only after 48 hours of incubation. Fibrillar Aß40 and Aß42 promoted dose-dependent aggregation of washed platelets in the presence of extracellular CaCl2 . Cleavage of GPIbα by mocarhagin or blockade of the ITAM-containing FcγRIIA prevented platelet aggregation induced by fibrillary Aß40 and Aß42. Fibrillar Aß peptides stimulated the phosphorylation of FcγRIIA, resulting in the downstream stimulation of PLC, protein kinase C, and phosphoinositide 3-kinases, whose activity was necessary for full aggregation of platelets. Fibrillar Aß peptides also induced ROS generation, and NOX inhibitors, as well as ROS scavengers, prevented platelet aggregation. However, Aß peptide-induced ROS production did not require binding to GPIbα or activation of FcγRIIA, but was initiated by CD36, which provided an important contribution to full platelet aggregation. CONCLUSION: These results suggest that fibrillar amyloid Aß40 and Aß42 induce platelet aggregation through the recruitment of GPIb-IX-V and CD36, which requires the convergence of ITAM- and ROS-dependent pathways.

The proline-rich tyrosine kinase Pyk2 modulates integrin-mediated neutrophil adhesion and reactive oxygen species generation.

Neutrophils are first responders in infection and inflammation. They are able to roll, adhere and transmigrate through the endothelium to reach the site of infection, where they fight pathogens through secretion of granule contents, production of reactive oxygen species, extrusion of neutrophil extracellular traps, and phagocytosis. In this study we explored the role of the non-receptor focal adhesion kinase Pyk2 in neutrophil adhesion and activation. Using a specific Pyk2 pharmacological inhibitor, PF-4594755, as well as Pyk2-deficient murine neutrophils, we found that Pyk2 is activated upon integrin αMß2-mediated neutrophil adhesion to fibrinogen. This process is triggered by Src family kinases-mediated phosphorylation and supported by Pyk2 autophosphorylation on Y402. In neutrophil adherent to fibrinogen, Pyk2 activates PI3K-dependent pathways promoting the phosphorylation of Akt and of its downstream effector GSK3. Pyk2 also dynamically regulates MAP kinases in fibrinogen-adherent neutrophils, as it stimulates p38MAPK but negatively regulates ERK1/2. Pharmacological inhibition of Pyk2 significantly prevented adhesion of human neutrophils to fibrinogen, and neutrophils from Pyk2-knockout mice showed a reduced ability to adhere compared to wildtype cells. Accordingly, neutrophil adhesion to fibrinogen was reduced upon inhibition of p38MAPK but potentiated by ERK1/2 inhibition. Neutrophil adherent to fibrinogen, but not to polylysine, were able to produce ROS upon lipopolysaccharide challenge and ROS production was completely suppressed upon inhibition of Pyk2. By contrast PMA-induced ROS production by neutrophil adherent to either fibrinogen or polylysine was independent from Pyk2. Altogether these results demonstrate that Pyk2 is an important effector in the coordinated puzzle regulating neutrophil adhesion and activation.

Stimulation of mTORC2 by integrin αIIbß3 is required for PI3Kß-dependent activation of Akt but is dispensable for platelet spreading on fibrinogen.

Phosphatidylinositol 3 kinase (PI3K) is a major player in platelet activation and regulates thrombus formation and stabilization. The ß isoform of PI3K is implicated in integrin αIIbß3 outside-in signaling, is required for the phosphorylation of Akt, and controls efficient platelet spreading upon adhesion to fibrinogen. In this study we found that during integrin αIIbß3 outside-in signaling PI3Kß-dependent phosphorylation of Akt on Serine473 is mediated by the mammalian target of rapamycin complex 2 (mTORC2). The activity of mTORC2 is stimulated upon platelet adhesion to fibrinogen, as documented by increased autophosphorylation. However, mTORC2 activation downstream of integrin αIIbß3 is PI3Kß-independent. Inhibition of mTORC2, but not mTORC1, also prevents Akt phosphorylation of Threonine308 and affects Akt activity, resulting in the inhibition of GSK3α/ß phosphorylation. Nevertheless, mTORC2 or Akt inhibition does not alter PI3Kß-dependent platelet spreading on fibrinogen. The activation of the small GTPase Rap1b downstream of integrin αIIbß3 is regulated by PI3Kß but is not affected upon inhibition of either mTORC2 or Akt. Altogether, these results demonstrate for the first time the activation of mTORC2 and its involvement in Akt phosphorylation and stimulation during integrin αIIbß3 outside-in signaling. Moreover, the results demonstrate that the mTORC2/Akt pathway is dispensable for PI3Kß-regulated platelet spreading on fibrinogen.

Biology and Role of Extracellular Vesicles (EVs) in the Pathogenesis of Thrombosis.

Extracellular vesicles (EVs) are well-established mediators of cell-to-cell communication. EVs can be released by every cell type and they can be classified into three major groups according to their biogenesis, dimension, density, and predominant protein markers: exosomes, microvesicles, and apoptotic bodies. During their formation, EVs associate with specific cargo from their parental cell that can include RNAs, free fatty acids, surface receptors, and proteins. The biological function of EVs is to maintain cellular and tissue homeostasis by transferring critical biological cargos to distal or neighboring recipient cells. On the other hand, their role in intercellular communication may also contribute to the pathogenesis of several diseases, including thrombosis. More recently, their physiological and biochemical properties have suggested their use as a therapeutic tool in tissue regeneration as well as a novel option for drug delivery. In this review, we will summarize the impact of EVs released from blood and vascular cells in arterial and venous thrombosis, describing the mechanisms by which EVs affect thrombosis and their potential clinical applications.

Focal Adhesion Kinases in Platelet Function and Thrombosis.

The focal adhesion kinase family includes 2 homolog members, FAK and Pyk2 (proline-rich tyrosine kinase 2), primarily known for their roles in nucleated cells as regulators of cytoskeletal dynamics and cell adhesion. FAK and Pyk2 are also expressed in megakaryocytes and platelets and are activated by soluble agonists and on adhesion to the subendothelial matrix. Despite high sequence homology and similar molecular organization, FAK and Pyk2 play different roles in platelet function. Whereas FAK serves mostly as a traditional focal adhesion kinase activated downstream of integrins, Pyk2 coordinates multiple signals from different receptors. FAK, but not Pyk2, is involved in megakaryocyte maturation and platelet production. In circulating platelets, FAK is recruited by integrin αIIbß3 to regulate hemostasis, whereas it plays minimal roles in thrombosis. By contrast, Pyk2 is implicated in platelet activation and is an important regulator of thrombosis. The direct activation of Pyk2 by calcium ions provides a connection between GPCRs (G-protein coupled receptors) and Src family kinases. In this review, we provide the comprehensive overview of >20 years of investigations on the role and regulation of focal adhesion kinases in blood platelets, highlighting common and distinctive features of FAK and Pyk2 in hemostasis and thrombosis.

Amyloid precursor protein is required for in vitro platelet adhesion to amyloid peptides and potentiation of thrombus formation.

Amyloid precursor protein (APP) is the precursor of amyloid ß (Aß) peptides, whose accumulation in the brain is associated with Alzheimer's disease. APP is also expressed on the platelet surface and Aß peptides are platelet agonists. The physiological role of APP is largely unknown. In neurons, APP acts as an adhesive receptor, facilitating integrin-mediated cell adhesion, while in platelets it regulates coagulation and venous thrombosis. In this work, we analyzed platelets from APP KO mice to investigate whether membrane APP supports platelet adhesion to physiological and pathological substrates. We found that APP-null platelets adhered and spread normally on collagen, von Willebrand Factor or fibrinogen. However, adhesion on immobilized Aß peptides Aß1-40, Aß1-42 and Aß25-35 was completely abolished in platelets lacking APP. By contrast, platelet activation and aggregation induced by Aß peptides occurred normally in the absence of APP. Adhesion of APP-transfected HEK293 to Aß peptides was significantly higher than that of control cells expressing low levels of APP. Co-coating of Aß1-42 and Aß25-35 with collagen strongly potentiated platelet adhesion when whole blood from wild type mice was perfused at arterial shear rate, but had no effects with blood from APP KO mice. These results demonstrate that APP selectively mediates platelet adhesion to Aß under static condition but not platelet aggregation, and is responsible for Aß-promoted potentiation of thrombus formation under flow. Therefore, APP may facilitate an early step in thrombus formation when Aß peptides accumulate in cerebral vessel walls or atherosclerotic plaques.

Molecular mechanisms of platelet activation and aggregation induced by breast cancer cells.

Tumor cell-induced platelet aggregation represents a critical process both for successful metastatic spread of the tumor and for the development of thrombotic complications in cancer patients. To get further insights into this process, we investigated and compared the molecular mechanisms of platelet aggregation induced by two different breast cancer cell lines (MDA-MB-231 and MCF7) and a colorectal cancer cell line (Caco-2). All the three types of cancer cells were able to induce comparable platelet aggregation, which, however, was observed exclusively in the presence of CaCl2 and autologous plasma. Aggregation was supported both by fibrinogen binding to integrin αIIbß3 as well as by fibrin formation, and was completely prevented by the serine protease inhibitor PPACK. Platelet aggregation was preceded by generation of low amounts of thrombin, possibly through tumor cells-expressed tissue factor, and was supported by platelet activation, as revealed by stimulation of phospholipase C, intracellular Ca2+ increase and activation of Rap1b GTPase. Pharmacological inhibition of phospholipase C, but not of phosphatidylinositol 3-kinase or Src family kinases prevented tumor cell-induced platelet aggregation. Tumor cells also induced dense granule secretion, and the stimulation of the P2Y12 receptor by released ADP was found to be necessary for complete platelet aggregation. By contrast, prevention of thromboxane A2 synthesis by aspirin did not alter the ability of all the cancer cell lines analyzed to induce platelet aggregation. These results indicate that tumor cell-induced platelet aggregation is not related to the type of the cancer cells or to their metastatic potential, and is triggered by platelet activation and secretion driven by the generation of small amount of thrombin from plasma and supported by the positive feedback signaling through secreted ADP.

Platelet amyloid precursor protein is a modulator of venous thromboembolism in mice.

The amyloid precursor protein (APP), primarily known as the precursor of amyloid peptides that accumulate in the brain of patients with Alzheimer disease, is abundant in platelets, but its physiological function remains unknown. In this study, we investigated the role of APP in hemostasis and thrombosis, using APP knockout (KO) mice. Ex vivo aggregation, secretion, and integrin αIIbß3 inside-out activation induced by several agonists were normal in APP-deficient platelets, but the number of circulating platelets was reduced by about 20%, and their size was slightly increased. Tail bleeding time was normal, and in vivo, the absence of APP did not alter thrombus formation in the femoral artery. In contrast, in a model of vein thrombosis induced by flow restriction in the inferior vena cava, APP-KO mice, as well as chimeric mice with selective deficiency of APP in blood cells, developed much larger thrombi than control animals, and were more sensitive to embolization. Consistent with this, in a pulmonary thromboembolism model, larger vessels were occluded. APP-KO mice displayed a shorter APTT, but not PT, when measured in the presence of platelets. Moreover, the activity of factor XIa (FXIa), but not FXIIa, was higher in APP-KO mice compared with controls. APP-KO mice presented a higher number of circulating platelet-leukocyte aggregates, and neutrophils displayed a greater tendency to protrude extracellular traps, which were more strongly incorporated into venous thrombi. These results indicate that platelet APP limits venous thromboembolism through a negative regulation of both fibrin formation and neutrophil function.

Release of Prometastatic Platelet-Derived Microparticles Induced by Breast Cancer Cells: A Novel Positive Feedback Mechanism for Metastasis.

Circulating platelets and platelet-derived microparticles are regulators of cancer metastasis. In this study, we show that breast cancer cells induce platelet aggregation and lead to the release of platelet-derived microparticles. Although able to cause comparable aggregation, the highly aggressive MDA-MB-231 cells were more potent than the poorly aggressive MCF7 cells in inducing platelet-derived microparticles release, which was comparable to that promoted by thrombin. MDA-MB-231 cells were able to bind and internalize both MCF7- and MDA-MB-231-induced platelet-derived microparticles with comparable efficiency. By contrast, MCF7 cells did not interact with either type of platelet-derived microparticles. Upon internalization, only platelet-derived microparticles released by platelet stimulation with MDA-MB-231 cells, but not those released upon stimulation with MCF7 cells, caused activation of MDA-MB-231 cells and promoted the phosphorylation of selected signaling proteins, including p38MAPK and myosin light chain. Accordingly, MDA-MB-231-induced, but not MCF7-induced, platelet-derived microparticles dose-dependently stimulated migration and invasion of targeted MDA-MB-231 cells. These results identify a novel paracrine positive feedback mechanism initiated by aggressive breast cancer cell types to potentiate their invasive phenotype through the release of platelet-derived microparticles.

Novel pharmacological inhibitors demonstrate the role of the tyrosine kinase Pyk2 in adhesion and aggregation of human platelets.

Pyk2 is a Ca2+-regulated kinase predominantly expressed in neuronal and in haematopoietic cells. Previous studies on Pyk2-null mice have demonstrated that Pyk2 plays a crucial role in platelet activation and thrombus formation, thus representing a possible target for antithrombotic therapy. Very limited information is available about the role of Pyk2 in human platelets, mainly because of the lack of specific pharmacological inhibitors. In this work, we have tested two novel Pyk2 inhibitors, PF-4594755 and PF-4520440, to validate their specificity and to investigate their ability to modulate platelet activation. Both molecules were able to efficiently block Pyk2 activity in human and mouse platelets stimulated with thrombin or with the Ca2+-ionophore. In wild-type murine platelets, PF-4594755 and PF-4520440 reduced thrombin-induced aggregation to the level observed in Pyk2 knockout platelets, but did not affect aggregation induced by GPVI stimulation. Importantly, neither compounds affected the residual thrombin-induced aggregation of Pyk2-null platelets, thus excluding possible off-target effects. In human platelets, PF-4594755 and PF-4520440 significantly reduced aggregation stimulated by thrombin, but not by the GPVI agonist convulxin. Both inhibitors reduced platelet adhesion on fibrinogen and prevented Akt phosphorylation in adherent cells, indicating that Pyk2 regulates PI3K and cell spreading downstream of integrins in human platelets. Finally, the Pyk2 inhibitors significantly inhibited thrombus formation upon blood perfusion on immobilized collagen under arterial flow rate. These results demonstrate that PF-4594755 and PF-4520440 are specific inhibitors of Pyk2 in intact platelets and allowed to reliably document that this kinase plays a relevant role in human platelet activation.

Pull-down assay for analysis of integrin-mediated activation of Rap proteins in adherent platelets.

Rap1 GTPases operate as molecular switches by cycling between a GDP-bound inactive state and a GTP-bound active state and regulate several cellular pathways in response to different stimuli. Circulating blood platelets express high levels of Rap1 proteins, mainly Rap1b, which plays a critical role in platelet adhesion and activation. Rap1 is a key element in the inside-out signaling pathway leading to the conversion of integrins into the high-affinity state for their ligands. In platelets, Rap1b regulates inside-out activation of both integrin αIIbß3 and α2ß1. In addition, Rap1b is also involved in integrin outside-in signaling. Integrin-mediated platelet adhesion leads to accumulation of GTP-bound Rap1b, which promotes integrin-mediated processes such as spreading and clot retraction. Rap1b is thus a bidirectional regulator of platelet integrin function. Here we describe a method to analyze Rap1b activation induced by platelet adhesion via integrin α2ß1.