OBJECTIVE: To investigate the effects of oridonin on platelet function and related mechanisms. METHODS: Washed platelets from healthy adults and mice were incubated with different concentrations of oridonin (2.5, 5 and 10 μmol/L) in vitro . The surface expression level of P-selectin and the activation of integrin αIIbβ3 in platelets were detected by flow cytometry, and the aggregation ability of platelets under the stimulation by various agonists was detected by light transmission aggregometry. The expression of P-AKT (Ser473) was detected by protein immunoblotting. Arterial thrombosis model was established in mice with mesenteric injury induced by ferric chloride, and tail hemorrhage model was established by cutting off the tail of mice. The effect of intraperitoneal injection of oridonin (10 mg/kg) on thrombosis and haemostasis was tested. RESULTS: Oridonin inhibited platelet P-selectin expression and integrin αIIbβ3 activation. In the presence of different stimulants, oridonin inhibited platelet aggregation in a concentration-dependent manner. The phosphorylation level of AKT Ser473 was reduced in the groups treated with different concentrations of oridonin. Oridonin significantly prolonged the time of mesenteric artery thrombosis in mice, but did not affect the tail bleeding time. CONCLUSION Oridonin inhibits platelet activation, aggregation, and thrombosis by inhibiting AKT phosphorylation, and may be used as a potential antiplatelet drug.
Diterpenes, Kaurane/pharmacology* ; Animals ; Mice ; Blood Platelets/drug effects* ; Platelet Aggregation/drug effects* ; P-Selectin/metabolism* ; Thrombosis ; Platelet Glycoprotein GPIIb-IIIa Complex/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Humans ; Phosphorylation ; Platelet Activation/drug effects*

OBJECTIVE: To examine the antiplatelet and antithrombotic activity of Rumex acetosella extract. METHODS: Standard light aggregometry was used for platelet aggregation, intracellular calcium mobilization assessed using Fura-2/AM, granule secretion (ATP release) by luminometer, and fibrinogen binding to integrin α_IIbβ₃ detected using flow cytometry. Western blotting is carried out to determine the phosphorylation of mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/Akt signaling. RESULTS: Rumex acetosella displayed the ability to inhibit platelet aggregation, calcium mobilization, granule secretion, and fibrinogen binding to integrin α_IIbβ₃. Rumex acetosella has also down-regulated MAPK and PI3K/Akt phosphorylation (all P<0.01). CONCLUSION Rumex acetosella extract exhibits antiplatelet activity via modulating GPVI signaling, and it may protect against the development of platelet-related cardiovascular diseases.
Blood Platelets/metabolism* ; Calcium/metabolism* ; Fibrinogen/metabolism* ; Mitogen-Activated Protein Kinases/metabolism* ; Phosphatidylinositol 3-Kinase/pharmacology* ; Phosphatidylinositol 3-Kinases/metabolism* ; Phosphorylation ; Plant Extracts/pharmacology* ; Platelet Aggregation ; Platelet Aggregation Inhibitors/pharmacology* ; Platelet Glycoprotein GPIIb-IIIa Complex/pharmacology* ; Proto-Oncogene Proteins c-akt/metabolism* ; Rumex/metabolism*

OBJECTIVE: To establish 293T cell lines stably expressing Calpain-cleavage related α3 cytoplasmic tail mutants, and to explore the effect of amino acid motifs in integrin β3 cytoplasmic tail on αⅡbβ3-mediated cell function. METHODS: 293T cell lines stably co-expressing human wild type integrin αⅡb and full length β3 or mutant β3, including β3-ΔNITY (β3 cytoplasmic tail NITY motif deleted), β3-Δ754 (β3 cytoplasmic tail TNITYRGT motif deleted) and β3-Δ759 (β3 cytoplasmic tail RGT motif deleted) were established. Spreading and adhesion of these stable cell lines on immobilized fibrinogen were tested. RESULTS: 293T-αⅡbβ3ΔNITY, 293T-αⅡbβ3Δ754, 293T-αⅡbβ3Δ759 and 293T-αⅡbβ3 cell lines were successfully established. Compared with the 293T cells, 293T-αⅡbβ3 cells which expressed full β3, possessed well adhesion and spread ability on immobilized fibrinogen, suggesting it can be as a surrogate for platelet. Compared with 293T-αⅡbβ3 cells, the 293T-αⅡbβ3ΔNITY cells showed a partial impairment of adhesion and spreadability on immobilized fibrinogen. while the 293T-αⅡbβ3Δ754 cells and 293T-αⅡbβ3Δ759 cells failed to adhere or spread on immobilized fibrinogen. CONCLUSION To the cell spreading function mediated by integrin β3, RGT motif is vital, while NITY can be dispensable. These established 293T cell lines stably expressing different β3 mutants provide a solid basis for a further analysis of mass spectrometry.
Amino Acid Motifs ; Animals ; CHO Cells ; Cell Adhesion ; Cricetinae ; Cricetulus ; HEK293 Cells ; Humans ; Integrin beta3 ; genetics ; metabolism ; Platelet Glycoprotein GPIIb-IIIa Complex ; genetics ; metabolism ; Signal Transduction

OBJECTIVETo study the expression of specific anti- platelet glycoprotein autoantibodies GP II b/III a, GP I b/IX and GP I a/II a in primary immune thrombocytopenia (ITP), and to evaluate the relationship between the therapeutic effect and the expression of specific anti- platelet glycoprotein antibodies GPIIb/IIIa, GPIb/IX and GPIa/IIa.

METHODSAnti-GPIIb/IIIa, GPIb/ IX and GP I a/II a antibodies were assayed by ELISA for patients with ITP. Total 442 patients in our hospital, who were retrospectively investigated from December 2010 to November 2012, were divided into newly diagnosed ITP, persistent and chronic ITP. The expression of specific anti- platelet glycoprotein antibody in each group was measured separately. The newly diagnosed ITP patients were treated with intravenous IgG (IVIG) and corticosteroids. The relationship between the expression of specific anti- platelet glycoprotein antibodies GPIIb/IIIa, GPIb/IX and GPIa/IIa and the complete response (CR) was studied.

RESULTSPositive rates of anti- platelet glycoprotein antibodies were 59.09%, 26.97% and 37.35% respectively in newly diagnosed ITP, persistent and chronic ITP, the difference was statistical significant (P<0.05). In newly diagnosed ITP, positive rate of antibody against GPIIb/IIIa was 38.64%, double positive rate of antibodies against both GP II b/III a and GP I a/II a was 15.91%, there was statistical significance (P<0.05) compared with that of persistent and chronic ITP. The complete response (CR) rate in newly diagnosed ITP patients with positive antibody against GP II b/III a was 80.39% after treatment with IVIG and corticosteroids. There was statistical significance compared with that in patients having no antibodies (P<0.05).

CONCLUSIONThe expression of antibodies against GP II b/III a and double positive for both GP II b/III a and GP I a/II a autoantibodies increased in newly diagnosed ITP patients. Patients with anti-GP II b/III a autoantibody had good response to medication with IVIG and corticosteroids.

Adolescent ; Adult ; Aged ; Autoantibodies ; metabolism ; Child ; Child, Preschool ; Female ; Humans ; Male ; Middle Aged ; Platelet Glycoprotein GPIIb-IIIa Complex ; immunology ; Platelet Glycoprotein GPIb-IX Complex ; immunology ; Platelet Membrane Glycoproteins ; immunology ; Retrospective Studies ; Thrombocytopenia ; drug therapy ; immunology ; metabolism ; Treatment Outcome ; Young Adult

OBJECTIVETo observe the effect of Tongxinluo Capsule on platelet activities and vascular endothelial functions as well as prognosis in patients with acute coronary syndrome (ACS) after percutaneous coronary intervention (PCI) at different stages.

METHODS160 patients with acute coronary syndrome were randomly assigned to Tongxinluo (TXL) group (80 patients) and the conventional treatment group (80 patients). And 50 healthy subjects were set up as the health control group. Patients' plasma platelet activating factors (CD62p, CD63), and glucose protein (GP) IIb/IIIa, and endothelium-1 (ET-1), von Willebrand factor (vWF), nitric oxide (NO) levels, and endothelium dependent flow-mediated dilatation (FMD) were detected respectively. Patients in the TXL group orally took TXLC for six months. The aforesaid indices were re-detected in all patients after two months and six months. Comparison between before and after treatment in the same group and inter-group comparison were performed in the two groups.

RESULTSCompared with the health control group, CD62p, CD63, GPIIb/IIIa, vWF, and ET-1 levels increased significantly in ACS patients after PCI (all P<0.01), NO and FMD significantly decreased (P<0.01). CD62p, CD63, GPIIb/IIIa and, vWF also increased, and FMD decreased after PCI (all P<0.05), but insignificant difference was found in ET-1 and NO (P>0.05). In the TXL group and the conventional treatment group, the levels of CD62p, CD63, GPIIb/IIIa, vWF and ET-1 decreased significantly (P<0.05, P<0.01), NO and FMD increased (P<0.05, P<0. 01) when compared with before treatment. Compared with the conventional treatment group, the decrement of CD62p, CD63, GPIIb/IIIa and vWF (P<0.05, P<0.01), and the increment of FMD and NO (both P<0.05) were more obvious in the TXL group. The aforesaid indices were more obviously different between 6-month treatment and 2-month treatment in the TXL group and the conventional treatment group (P<0.05, P<0.01). Seven patients suffered from angina, six from heart failure, three from ventricular tachycardiac (VT)/ventricular fibrillation (VF), and two died suddenly in the conventional treatment group after six months of treatment, while only one suffered from angina, one from heart failure, and none from VT/VF or died suddenly in the TXL treatment group after 6 months of treatment.

CONCLUSIONTXL could be used in the prevention and treatment of coronary thrombosis, protect the vascular endothelial functions, as well as improve the prognosis of ACS patients after PCI.

Acute Coronary Syndrome ; blood ; therapy ; Aged ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Female ; Humans ; Male ; Middle Aged ; Nitric Oxide ; blood ; Percutaneous Coronary Intervention ; Platelet Activation ; drug effects ; Platelet Glycoprotein GPIIb-IIIa Complex ; metabolism ; Prognosis ; von Willebrand Factor ; metabolism

Adolescent ; Adult ; Aged ; Case-Control Studies ; Child ; Female ; Flow Cytometry ; Humans ; Male ; Middle Aged ; Platelet Glycoprotein GPIIb-IIIa Complex ; metabolism ; Purpura, Thrombocytopenic ; diagnosis ; metabolism ; Young Adult

The efficacy of low molecular weight heparin (LMWH) with low dose unfractionated heparin (UFH) during percutaneous coronary intervention (PCI) with or without glycoprotein (Gp) IIb/IIIa inhibitor compared to UFH with or without Gp IIb/IIIa inhibitor has not been elucidated. Between October 2005 and July 2007, 2,535 patients with ST elevation acute myocardial infarction (STEMI) undergoing PCI in the Korean Acute Myocardial Infarction Registry (KAMIR) were assigned to either of two groups: a group with Gp IIb/IIIa inhibitor (n=476) or a group without Gp IIb/IIIa inhibitor (n=2,059). These groups were further subdivided according to the use of LMWH with low dose UFH (n=219) or UFH alone (n=257). The primary end points were cardiac death or myocardial infarction during the 30 days after the registration. The primary end point occurred in 4.1% (9/219) of patients managed with LMWH during PCI and Gp IIb/IIIa inhibitor and 10.8% (28/257) of patients managed with UFH and Gp IIb/IIIa inhibitor (odds ratio [OR], 0.290; 95% confidence interval [CI], 0.132-0.634; P=0.006). Thrombolysis In Myocardial Infarction (TIMI) with major bleeding was observed in LMHW and UFH with Gp IIb/IIIa inhibitor (1/219 [0.5%] vs 1/257 [0.4%], P=1.00). For patients with STEMI managed with a primary PCI and Gp IIb/IIIa inhibitor, LMWH is more beneficial than UFH.
Acute Disease ; Aged ; Drug Therapy, Combination ; Female ; Hemorrhage ; Heparin/*therapeutic use ; Heparin, Low-Molecular-Weight/*therapeutic use ; Humans ; Male ; Middle Aged ; Multivariate Analysis ; Myocardial Infarction/epidemiology/mortality/*therapy ; Myocardial Revascularization ; Odds Ratio ; Platelet Glycoprotein GPIIb-IIIa Complex/*antagonists & inhibitors/metabolism ; Prognosis ; Registries

Integrins are allosteric cell adhesion receptors that cycle from a low to a high affinity ligand binding state, a complex process of receptor activation that is of particular importance in blood cells such as platelets or leukocytes. Here we highlight recent progress in the understanding of the molecular pathways that regulate integrin activation in platelets and leukocytes, with a special focus on the structural changes in platelet integrin αIIbβ3 brought about by key intracellular proteins, namely talin and kindlins, that are of crucial importance in the regulation of integrin function. Evidence that the small GTPase Rap1 and its guanine exchange factor CalDAG-GEF1, together with RIAM, a Rap1GTP adaptor protein, promote the interaction of talin with the integrin β subunit, has greatly contributed to fill the gap in our understanding of the signaling pathway from G-coupled agonist receptors and their phospholipase C-dependant second messengers, to integrin activation. Studies of patients with the rare blood cell disorder LAD-III have contributed to the identification of kindlins as new co-regulators of the talin-dependent integrin activation process in platelets and leukocytes, underlining the relevance for the in-depth investigation of patients with rare genetic blood cell disorders.
Amino Acid Motifs ; Amino Acid Sequence ; Animals ; Cell Adhesion ; Cytoskeleton ; metabolism ; Humans ; Intracellular Signaling Peptides and Proteins ; metabolism ; Models, Molecular ; Molecular Sequence Data ; Platelet Glycoprotein GPIIb-IIIa Complex ; metabolism ; Protein Interaction Domains and Motifs ; Recombinant Proteins ; metabolism ; Sequence Alignment ; Talin ; metabolism

This study was purposed to investigate the role of integrin beta3 cytoplasmic domain in signal transduction mediated by integrin alphaIIbbeta3 and to explore the effect of integrin beta3 on signal transduction and specificity in condition without alphaIIb subunit. The fusion protein (Tac/beta3) was stably expressed in CHO cell line expressing GPIbIX, integrin alphaIIbbeta3 (IbIX/IIbIIIa-CHO cell line) by combining extracellular and transmembrane domains (Tac) of IL-2 receptor with integrin beta3 cytoplasmic domain (beta3) for formation of fusion protein (Tac/beta3). Then a series of tests were performed, including spreading and stable adhesion of IbIX/IIbIIIa-CHO cell line in solid phase fibrinogen (Fg), fibrin clot restriction and soluble fibrinogen binding, which represent outside-in and inside-out signal transduction events. The results showed that the bidirectional signal transduction mediated by alphaIIbbeta3 in IbIX/IIbIIIa-CHO/Tac-762 cells stably expressing Tac/beta3 was seriously inhibited. It is concluded that the Tac/beta3 can play a significant role in IbIX/IIbIIIa-CHO/Tac-762 cells through a dominant negative mode, the independent presence of beta3 subunit cytoplasmic domain can regulate the bidirectional signal transduction mediated by integrin alphaIIbbeta3.
Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Platelet Glycoprotein GPIIb-IIIa Complex ; genetics ; metabolism ; Receptors, Interleukin-2 ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Signal Transduction

OBJECTIVETo explore the effect of Danhong Injection (DHI) on platelet activation and inflammatory factors in patients of acute coronary syndrome (ACS) after intervention therapy.

METHODSOne hundred ACS patients were randomly assigned to the DHI group and the control group equally. Both were treated with the conventional treatment, including aspirin, clopidogrel, beta-receptor blocker, angiotensin converting enzyme inhibitor, etc. for 2 weeks after percutaneous coronary intervention (PCI), and to the patients in the DHI group, intravenous dripping of DHI was given simultaneously. Fasting venous blood of patients were collected before PCI and on the next morning of PCI to determine the platelet activation indices, expression of CD62p and receptor complex of glucose protein (GP) II b/III a by flow cytometry; plasma fibrinogen C (FIB-C) by scattering turbidimetry, and serum high-sensitivity C-reactive protein (hs-CRP) with emulsoid immuno-enhancing turbidimetric test kit. The outcomes were compared with those determined in 40 healthy persons for control.

RESULTSSerum levels of CD62p, GP II b/III a, FIB-C and hs-CRP in ACS patients were significantly higher than those in the healthy control (all P<0.01), and those were significantly higher after PCI than before PCI (P <0.05 or P<0.01). After being treated for 2 weeks, the 4 platelet activation indices were lowered to different extent in both groups, but the lowering in the DHI group was more significant than that in the control group (P<0.05).

CONCLUSIONDHI can inhibit the platelet activation and inflammatory reaction in ACS patients after PCI.

Acute Coronary Syndrome ; metabolism ; therapy ; Aged ; Angioplasty, Balloon, Coronary ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Fibrinogen ; metabolism ; Humans ; Inflammation ; drug therapy ; metabolism ; Male ; Middle Aged ; P-Selectin ; blood ; Platelet Activation ; Platelet Glycoprotein GPIIb-IIIa Complex ; metabolism