Junling Liu

Platelet-derived ERp57 mediates platelet incorporation into a growing thrombus by regulation of the αIIbβ3 integrin

The platelet protein disulfide isomerase called ERp57 mediates platelet aggregation, but its role in thrombus formation is unknown. To determine the specific role of platelet-derived ERp57 in hemostasis and thrombosis, we generated a megakaryocyte/platelet-specific knockout. Despite normal platelet counts and platelet glycoprotein expression, mice with ERp57-deficient platelets had prolonged tail-bleeding times and thrombus occlusion times with FeCl3-induced carotid artery injury. Using a mesenteric artery thrombosis model, we found decreased incorporation of ERp57-deficient platelets into a growing thrombus. Platelets lacking ERp57 have defective activation of the αIIbβ3 integrin and platelet aggregation. The defect in aggregation was corrected by the addition of exogenous ERp57, implicating surface ERp57 in platelet aggregation. Using mutants of ERp57, we demonstrate the second active site targets a platelet surface substrate to potentiate platelet aggregation. Binding of Alexa 488-labeled ERp57 to thrombin-activated and Mn(2+)-treated platelets lacking β3 was decreased substantially, suggesting a direct interaction of ERp57 with αIIbβ3. Surface expression of ERp57 protein and activity in human platelets increased with platelet activation, with protein expression occurring in a physiologically relevant time frame. In conclusion, platelet-derived ERp57 directly interacts with αIIbβ3 during activation of this receptor and is required for incorporation of platelets into a growing thrombus.

PTEN regulates collagen-induced platelet activation

Phosphatidylinositol 3-kinase (PI3K) has been shown to play an important role in collagen-induced platelet activation, but the role(s) of PTEN, a major regulator of the PI3K/Akt signaling pathway, has not been examined in platelets. Here, we report that Pten(-/-) mouse blood contains 25% more platelets than Pten(+/+) blood and that PTEN deficiency significantly shortened the bleeding time, increased the sensitivity of platelets to collagen-induced activation and aggregation, and enhanced phosphorylation of Akt at Ser473 in response to collagen. Furthermore, we found that PP2, and the combination of apyrase, indomethacin + 1B5, respectively, inhibited collagen-induced aggregation in both PTEN(+/+) and PTEN(-/-) platelets. In contrast, LY294002 (a PI3K inhibitor) prevented the aggregation of PTEN(+/+), but not PTEN(-/-), platelets. Therefore, PTEN apparently regulates collagen-induced platelet activation through PI3K/Akt-dependent and -independent signaling pathways.

Platelet P2Y12 Is Involved in Murine Pulmonary Metastasis

The involvement of platelets in tumor progression is well recognized. The depletion of circulating platelets or pharmacologic inhibitors of platelet activation decreases the metastatic potential of circulating tumor cells in metastasis mouse models. The platelet ADP receptor P2Y12 amplifies the initial hemostatic responses activated by a variety of platelet agonists and stabilizes platelet aggregation, playing a crucial role in granule secretion, integrin activation and thrombus formation. However, the relationship between P2Y12 and tumor progression is not clear. In our study, the Lewis Lung Carcinoma (LLC) spontaneous metastatic mouse model was used to evaluate the role of P2Y12 in metastasis. The results demonstrated that P2Y12 deficiency significantly reduced pulmonary metastasis. Further studies indicated that P2Y12 deficiency diminished the ability of LLC cells to induce platelet shape change and release of active TGFβ1 by a non-contact dependent mechanism resulting in a diminished, platelet-induced EMT-like transformation of the LLC cells, and that transformation probably is a prerequisite of LLC cell metastasis. Immunohistochemical analyses indicated an obvious P2Y12 deficiency related attenuation of recruitment of VEGFR1+ bone marrow derived cell clusters, and extracellular matrix fibronectin deposition in lungs, which presumably are required for pre-metastatic niche formation. In contrast to the LLC cells, non-epithelial melanoma B16 cells induced platelet aggregation in a cell number and P2Y12-dependent manner. Also, a platelet induced EMT-like transformation of B16 cells is dependent on P2Y12. In agreement with the LLC cell model, platelet P2Y12 deficiency also results in significantly less lung metastasis in the B16 melanoma experimental metastasis model. These results demonstrate that P2Y12 is a safe drug target for anti-thrombotic therapy, and that P2Y12 may serve as a new target for inhibition of tumor metastasis.

Paired immunoglobulin-like receptor B regulates platelet activation.

Murine paired immunoglobulin-like receptors B (PIRB), as the ortholog of human leukocyte immunoglobulin-like receptor B2 (LILRB2), is involved in a variety of biological functions. Here, we found that PIRB and LILRB2 were expressed in mouse and human platelets, respectively. PIRB intracellular domain deletion (PIRB-TM) mice had thrombocythemia and significantly higher proportions of megakaryocytes in bone marrow. Agonist-induced aggregation and spreading on immobilized fibrinogen were facilitated in PIRB-TM platelets. The rate of clot retraction in platelet-rich plasma containing PIRB-TM platelets was also increased. Characterization of signaling confirmed that PIRB associated with phosphatases Shp1/2 in platelets. The phosphorylation of Shp1/2 was significantly downregulated in PIRB-TM platelets stimulated with collagen-related peptide (CRP) or on spreading. The results further revealed that the phosphorylation levels of the linker for activation of T cells, SH2 domain-containing leukocyte protein of 76kDa, and phospholipase C were enhanced in PIRB-TM platelets stimulated with CRP. The phosphorylation levels of FAK Y397 and integrin β3 Y759 were also enhanced in PIRB-TM platelet spread on fibrinogen. The PIRB/LILRB2 ligand angiopoietin-like-protein 2 (ANGPTL2) was expressed and stored in platelet α-granules. ANGPTL2 inhibited agonist-induced platelet aggregation and spreading on fibrinogen. The data presented here reveal that PIRB and its ligand ANGPTL2 possess an antithrombotic function by suppressing collagen receptor glycoprotein VI and integrin αIIbβ3-mediated signaling.

The Role of Rac1 in Glycoprotein Ib-IX–Mediated Signal Transduction and Integrin Activation

Objective—The platelet receptor for von Willebrand factor, the glycoprotein Ib-IX (GPIb-IX) complex, mediates platelet adhesion at sites of vascular injury and transmits signals leading to platelet activation. von Willebrand factor/GPIb-IX interaction sequentially activates the Src family kinase Lyn (SFK), phosphoinositide 3-kinase (PI3K), and Akt, leading to activation of integrin &agr;IIb&bgr;3 and integrin-dependent stable platelet adhesion and aggregation. It remains unclear how Lyn activates the PI3K/Akt pathway after ligand binding to GPIb-IX. Methods and Results—Using platelet-specific Rac1−/− mice and the Rac1 inhibitor NSC23766, we examined the role of Rac1 in GPIb-IX–dependent platelet activation. Rac1−/− mouse platelets and NSC23766-treated human platelets were defective in GPIb-dependent stable adhesion to von Willebrand factor under shear stress, integrin activation, thromboxane A2 synthesis, and platelet aggregation. Interestingly, GPIb-induced activation of Rac1 and the guanine nucleotide exchange factor for Rac1, Vav, was abolished in both Lyn−/− and SFK inhibitor-treated platelets but was unaffected by the PI3K inhibitor LY294002, indicating that Lyn mediates activation of Vav and Rac1 independently of PI3K. Furthermore, GPIb-induced activation of Akt was abolished in Rac1-deficient platelets, suggesting that Rac1 is upstream of the PI3K/Akt pathway. Conclusion—A Lyn-Vav-Rac1-PI3K-Akt pathway mediates von Willebrand factor–induced activation of integrin &agr;IIb&bgr;3 to promote GPIb-IX–dependent platelet activation.

Visualizing the von Willebrand factor/glycoprotein Ib-IX axis with a platelet-type von Willebrand disease mutation

Platelet-type von Willebrand disease (PT-VWD) is a bleeding disorder of the platelet glycoprotein Ib-IX/von Willebrand factor (VWF) axis caused by mutations in the glycoprotein Ib-IX receptor that lead to an increased affinity with VWF. In this report, platelets from a mouse expressing a mutation associated with PT-VWD have been visualized using state-of-the art image collection and processing. Confocal analysis revealed that VWF bound to the surface of single platelets and bridging micro-aggregates of platelets. Surface-bound VWF appears as a large, linear structure on the surface of 50% of the PT-VWD platelets. In vivo thrombus formation after chemical injury to the carotid artery revealed a severe impairment to occlusion as a consequence of the PT-VWD mutation. In vitro stimulation of PT-VWD platelets with adenosine diphosphate or thrombin demonstrates a significant block in their ability to bind fibrinogen. The impairment of in vivo thrombus formation and in vitro fibrinogen binding are more significant than might be expected from the observed platelet binding to VWF polymers over a small portion of the plasma membrane. Visualization of the receptor/ligand interaction and characterization of a severe antithrombotic phenotype provide a new understanding on the molecular basis of bleeding associated with the PT-VWD phenotype.

Agonist-induced platelet procoagulant activity requires shear and a Rac1-dependent signaling mechanism

Activated platelets facilitate blood coagulation by exposing phosphatidylserine (PS) and releasing microvesicles (MVs). However, the potent physiological agonists thrombin and collagen poorly induce PS exposure when a single agonist is used. To obtain a greater procoagulant response, thrombin is commonly used in combination with glycoprotein VI agonists. However, even under these conditions, only a percentage of platelets express procoagulant activity. To date, it remains unclear why platelets poorly expose PS even when stimulated with multiple agonists and what the signaling pathways are of soluble agonist-induced platelet procoagulant activity. Here we show that physiological levels of shear present in blood significantly enhance agonist-induced platelet PS exposure and MV release, enabling low doses of a single agonist to induce full-scale platelet procoagulant activity. PS exposed on the platelet surface was immediately released as MVs, revealing a tight coupling between the 2 processes under shear. Using platelet-specific Rac1(-/-) mice, we discovered that Rac1 plays a common role in mediating the low-dose agonist-induced procoagulant response independent of platelet aggregation, secretion, and the apoptosis pathway. Platelet-specific Rac1 function was not only important for coagulation in vitro but also for fibrin accumulation in vivo following laser-induced arteriolar injury.

Integrin αIIb-Mediated PI3K/Akt Activation in Platelets

Integrin αIIbβ3 mediated bidirectional signaling plays a critical role in thrombosis and haemostasis. Signaling mediated by the β3 subunit has been extensively studied, but αIIb mediated signaling has not been characterized. Previously, we reported that platelet granule secretion and TxA2 production induced by αIIb mediated outside-in signaling is negatively regulated by the β3 cytoplasmic domain residues R724KEFAKFEEER734. In this study, we identified part of the signaling pathway utilized by αIIb mediated outside-in signaling. Platelets from humans and gene deficient mice, and genetically modified CHO cells as well as a variety of kinase inhibitors were used for this work. We found that aggregation of TxA2 production and granule secretion by β3Δ724 human platelets initiated by αIIb mediated outside-in signaling was inhibited by the Src family kinase inhibitor PP2 and the PI3K inhibitor wortmannin, respectively, but not by the MAPK inhibitor U0126. Also, PP2 and wortmannin, and the palmitoylated β3 peptide R724KEFAKFEEER734, each inhibited the phosphorylation of Akt residue Ser473 and prevented TxA2 production and storage granule secretion. Similarly, Akt phosphorylation in mouse platelets stimulated by the PAR4 agonist peptide AYPGKF was αIIbβ3-dependent, and blocked by PP2, wortmannin and the palmitoylated peptide p-RKEFAKFEEER. Akt was also phosphorylated in response to mAb D3 plus Fg treatment of CHO cells in suspension expressing αIIbβ3-Δ724 or αIIbβ3E724AERKFERKFE734, but not in cells expressing wild type αIIbβ3. In summary, SFK(s) and PI3K/Akt signaling is utilized by αIIb-mediated outside-in signaling to activate platelets even in the absence of all but 8 membrane proximal residues of the β3 cytoplasmic domain. Our results provide new insight into the signaling pathway used by αIIb-mediated outside-in signaling in platelets.

BF0801, a novel adenine derivative, inhibits platelet activation via phosphodiesterase inhibition and P2Y12 antagonism

Though antiplatelet drugs are proven beneficial to patients with coronary heart disease and stroke, more effective and safer antiplatelet drugs are still needed. In this study we report the antiplatelet effects and mechanism of BF0801, a novel adenine derivative. BF0801 dramatically inhibited platelet aggregation and ATP release induced by ADP, 2MeSADP, AYPGKF, SFLLRN or convulxin without affecting shape change in vitro . It also potentiated the inhibitory effects of adenosine-based P2Y12 antagonist AR-C69931MX or phosphodiesterase (PDE) inhibitor IBMX on platelet aggregation. The cAMP levels in both resting and forskolin-stimulated platelets were increased by BF0801 suggesting its PDE inhibitor activity, which is further confirmed by the concentration-dependent suppression of BF0801 on the native and recombinant PDE. Similar to AR-C69931MX, BF0801 drastically inhibited 2MeSADP- induced adenylyl cyclase inhibition in platelets indicating its P2Y12 antagonism activity, which is substantiated by the inhibition of BF0801 on the interaction between ADP and P2Y12 receptor expressed in CHO-K1 cells measured by atomic force microscopy. Moreover, we confirmed the antiplatelet effects of BF0801 using platelets from rats intravenously given BF0801. In summary, for the first time we developed a novel adenine derivative bearing dual activities of PDE inhibition and P2Y12 antagonism, which may have therapeutic advantage as a potential antithrombotic drug.

Sorting protein VPS33B regulates exosomal autocrine signaling to mediate hematopoiesis and leukemogenesis

Certain secretory proteins are known to be critical for maintaining the stemness of stem cells through autocrine signaling. However, the processes underlying the biogenesis, maturation, and secretion of these proteins remain largely unknown. Here we demonstrate that many secretory proteins produced by hematopoietic stem cells (HSCs) undergo exosomal maturation and release that is controlled by vacuolar protein sorting protein 33b (VPS33B). Deletion of VPS33B in either mouse or human HSCs resulted in impaired exosome maturation and secretion as well as loss of stemness. Additionally, VPS33B deficiency led to a dramatic delay in leukemogenesis. Exosomes purified from either conditioned medium or human plasma could partially rescue the defects of HSCs and leukemia-initiating cells (LICs). VPS33B co-existed in exosomes with GDI2, VPS16B, FLOT1, and other known exosome markers. Mechanistically, VPS33B interacted with the GDI2/RAB11A/RAB27A pathway to regulate the trafficking of secretory proteins as exosomes. These findings reveal an essential role for VPS33B in exosome pathways in HSCs and LICs. Moreover, they shed light on the understanding of vesicle trafficking in other stem cells and on the development of improved strategies for cancer treatment.