OBJECTIVE: To study the effect of gradient shear stress on platelet aggregation by microfluidic chip Technology. METHODS: Microfluidic chip was used to simulate 80% fixed stenotic microchannel, and the hydrodynamic behavior of the stenotic microchannel model was analyzed by the finite element analysis module of sollidwork software. Microfluidic chip was used to analyze the adhesion and aggregation behavior of platelets in patients with different diseases, and flow cytometry was used to detect expression of the platelet activation marker CD62p. Aspirin, Tirofiban and protocatechuic acid were used to treat the blood, and the adhesion and aggregation of platelets were observed by fluorescence microscope. RESULTS: The gradient fluid shear rate produced by the stenosis model of microfluidic chip could induce platelet aggregation, and the degree of platelet adhesion and aggregation increased with the increase of shear rate within a certain range of shear rate. The effect of platelet aggregation in patients with arterial thrombotic diseases were significantly higher than normal group (P<0.05), and the effect of platelet aggregation in patients with myelodysplastic disease was lower than normal group (P<0.05). CONCLUSION The microfluidic chip analysis technology can accurately analyze and evaluate the platelet adhesion and aggregation effects of various thrombotic diseases unde the environment of the shear rate, and is helpful for auxiliary diagnosis of clinical thrombotic diseases.
Humans ; Microfluidics ; Platelet Adhesiveness ; Platelet Aggregation ; Blood Platelets/metabolism* ; Platelet Aggregation Inhibitors/pharmacology* ; Platelet Activation/physiology* ; Thrombosis

OBJECTIVE: To explore the main factors of platelet spreading and provide the foundation for related research. METHODS: Platelets (2×10⁷/ml) were draw from C57BL/6J mouse and kept at 22 ℃ for 1-2 hours. Platelets (2×10⁷/ml) were were allowed to adhere and spread on the fibrinogen-coated slides, after staining F-actin in platelets, the platelets were observed with the confocal microscopy. The effects of different concentrations of fibrinogen (10 μg/ml, 30 μg/ml, 100 μg/ml) and kinds of agonists ［thrombin(0.01,0.05,0.1 U/ml), ADP（5,10,20 μmol/L）, U46619（0.125,0.25,0.5 μmol/L）］ on platelets were analyzed. The platelet spreading was successful if the spreading rate was higher after treated with agonists. RESULTS: Compared to the group which coated with 10 μg/ml and 100 μg/ml fibrinogen, the platelet density is optimal when coated with 30 μg/ml fibrinogen. In addition, under the stimulation of thrombin, ADP and U46619, the spreading rate of platelets showed a certain concentration-dependent increasing. CONCLUSION The platelet spreading is easily influenced by various factors, the platelet spreading can be induced successfully at 0.1 U/ml thrombin, 20 μmol/L ADP and 0.5 μmol/L U46619 on the slide coated with 30 μg/ml fibrinogen.
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid/pharmacology* ; Adenosine Diphosphate ; Animals ; Blood Platelets/physiology* ; Fibrinogen ; Humans ; Mice ; Mice, Inbred C57BL ; Platelet Adhesiveness/physiology* ; Thrombin/pharmacology*

Fibrinogen (Fg) in human plasma plays an important role in hemostasis, vascular repair and tissue integrity. The surface chemistry of extracellular matrix or biological materials affects the orientation and distribution of Fg, and changes the exposure of integrin binding sites, thereby affecting its adhesion function to platelets. Here, the quantity, morphology and side chain exposure of Fg adsorbed on hydrophilic, hydrophobic and avidin surfaces were measured by atomic force microscopy (AFM) and flow cytometry (FCM), then the rolling behavior of platelets on Fg was observed through a parallel plate flow chamber system. Our results show that the hydrophobic surface leads to a large amount of cross-linking and aggregation of Fg, while the hydrophilic surface reduces the adsorption and accumulation of Fg while causing the exposure and spreading of the α chain on Fg and further mediating the adhesion of platelets. Fg immobilized by avidin / biotin on hydrophilic surface can maintain the monomer state, avoid over exposure and stretching of α chain, and bind to the platelets activated by the A1 domain of von Willebrand factor instead of inactivated platelets. This study would be helpful for improving the blood compatibility of implant biomaterials and reasonable experimental design of coagulation
Adsorption ; Blood Platelets ; Fibrinogen ; Humans ; Platelet Adhesiveness ; von Willebrand Factor

A diblock copolymer, poly(ethylene glycol) methacrylate-block-glycidyl methacrylate (PEGMA-GMA), was prepared on glass substrate by surface-initiated atom transfer radical polymerization (SI-ATRP), and endothelial specific peptide Arg-Glu-Asp-Val (REDV) was immobilized at the end of the PEGMA-GMA polymer brush by ring opening reaction through the rich epoxy groups in the GMA. The structure and hydrophilicity of the polymer brushes were characterized by static water contact angle, X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The results showed that the REDV modified copolymer brushes were successfully constructed on the glass substrates. The REDV peptide immobilized onto surface was quantitatively characterized by ultraviolet-visible spectroscopy (UV-VIS). The blood compatibility of the coating was characterized by recalcification time and platelet adhesion assay. The results showed that the polymer coating had good blood compatibility. The multifunctional active polymer coating with PEGMA and peptide produced an excellent prospect in surface construction with endothelial cells selectivity.
Biocompatible Materials ; Cells, Cultured ; Endothelial Cells ; Glass ; Humans ; Immobilized Proteins ; Methacrylates ; Oligopeptides ; Platelet Adhesiveness ; Polyethylene Glycols ; Polymers ; Surface Properties

OBJECTIVETo study the effect of interaction of the talin rod domain integrin binding site 2 with integrin β3 on platelet signal transduction.

METHODSA peptide that mimics the membrane proximal α helix 6 residues R724 KEFAK729 of the integrin β3 cytoplasmic tails was designed and synthesized, to which the myristoylation was covalently linked to the N-terminal of the peptide enabling membrane penetration. The effects of myr-RKEFAK peptide on the typical platelet outside-in signaling ovent (stable adhesion and spreading on immobilized fibrinogen, aggregation, fibrin clot retraction) and inside-out signaling events (soluble fibrinogen binding) were tested.

RESULTSmyr-RKEFAK peptide dose-dependently inhibited platelet stable adhesion and spreading on immobilized fibrinogen, irreversible aggregation, as well as fibrin clot retraction, but not soluble fibrinogen binding and reversible phase of platelet aggregation.

CONCLUSIONThe cell-penetrating peptide myr-RKEFAK causes an inhibitory effect on integrin β3 outside-in signaling-regulated platelets functions, but did not affect inside-out signaling-regulated platelets functions.

Blood Platelets ; Fibrinogen ; Humans ; Integrin beta3 ; Peptides ; Platelet Adhesiveness ; Platelet Aggregation ; Signal Transduction

The possibility of polyoxymethylene (POM) as heart valve leaflet material was investigated by comparing the hemocompatibility with that of 316L stainless steel and low-temperature isotropic pyrolytic carbon (LTIC). Surface hydrophobicity was characterized by water contact angle measurement. Platelet adhesion, APTT/PT/TT and hemolysis rate tests were applied for evaluating hemocompatibility. The results showed that POM was hydrophobic and had a low hemolytic rate, adhesion amount and activation degree of platelets on POM surface were less than 316L stainless steel, and was similar to LTIC. This research pointed out potential application of POM as heart valve leaflets.
Biocompatible Materials ; chemistry ; Blood Platelets ; Carbon ; chemistry ; Heart Valve Prosthesis ; Heart Valves ; Humans ; Platelet Adhesiveness ; Resins, Synthetic ; chemistry ; Stainless Steel ; chemistry

OBJECTIVETo observe the effects of multi-walled carbon nano-onions (MWCNOs) on platelet adhesion and experimental thrombosis in rats.

METHODSExperimental rats were randomly divided into sham operation group, solvent group, and MWCNO group, each including 6 ∼ 9 rats. An inverted fluorescence microscope and a flow chamber were used to observe the effects of 20 g/ml MWCNOs on platelet adhesion at shear rates of 500 s(-1) and 1000 s(-1); the experiment was repeated at least three times in each group. A rat model of carotid artery thrombosis was induced by 25% FeCl3, and the effects of 2 mg/kg MWCNOs on the blood flow and wet weight of thrombus per millimeter in the model were observed.

RESULTSWhen the shear rate was 500 s(-1), the MWCNO group showed a significantly smaller number of adhering platelets than the solvent group (58.3 ± 16.1 platelets/0.01 mm(2) vs 190.1 ± 36.0 platelets/0.01 mm(2)), but the inhibitory effect of MWCNOs on platelet adhesion disappeared as the shear rate increased to 1000 s(-1). The wet weights of thrombus per millimeter at 0 h after injection of a solvent or MWCNOs via the caudal vein were 0.83 ± 0.12 mg/mm in the solvent group and 0.97 ± 0.11 mg/mm in the MWCNO group, and the wet weights of thrombus per millimeter at 12 h after injection were 0.89 ± 0.12 mg/mm in the solvent group and 1.01 ± 0.15 mg/mm in the MWCNO group, exhibiting no significant differences between the two groups (P > 0.05). There were also no significant differences between the two groups in terms of blood flow at 0 h and 12 h after injection (P > 0.05).

CONCLUSIONMWCNOs have inhibitory effect on platelet adhesion in vitro, but the injection of MWCNOs via the caudal vein has no effects on the blood flow and wet weight of thrombus per millimeter in experimental thrombosis in rats.

Animals ; Blood Platelets ; drug effects ; Male ; Nanotubes, Carbon ; adverse effects ; Platelet Adhesiveness ; drug effects ; Rats ; Rats, Sprague-Dawley ; Thrombosis ; chemically induced ; pathology

This study was purposed to investigate the role of monocrotaline-inducing mouse liver sinusoid endothelial cell (SEC) injury in hepatic veno-occlusive disease. BALB/c mice were randomly divided into 2 groups: control group and monocrotaline group, mice were orally administrated with normal saline or monocrotaline with concentration of 200 mg/kg at days 0, 1, 2, respectively. At days 3, 4, 6, 8 and 10 after oral administration with normal saline or monocrotaline, the liver function (ALT, TBIL, AKP) and liver index were examined, and the percentage of activated platelets were detected by flow cytometry. The SEC, vascular endothelial cells and hepatic fibrosis were observed by staining with hematoxylin-eosin and Masson. Transmission electron microscopy was used to observe sinusoidal endothelial cell damage and platelet adhesion. The results showed that compared with control group, mice in monocrotaline group were characterized by severe damage of SEC, numbers of platelet aggregation and adhesion, central number and sinusoidal fibrosis. The percentage of activated platelets and liver index increased (P < 0.05). The characterization of portal hypertension was presented later, such as dysfunction of liver and ascites. It is concluded that SEC injury induced by monocrotaline may be the first step of hepatic veno-occlusive disease, and this kind of SEC injury is self-limiting, but fibrosis is always observed.
Animals ; Endothelial Cells ; pathology ; Endothelium ; cytology ; Hepatic Veins ; cytology ; pathology ; Hepatic Veno-Occlusive Disease ; chemically induced ; pathology ; Liver Cirrhosis ; chemically induced ; pathology ; Male ; Mice ; Mice, Inbred BALB C ; Monocrotaline ; adverse effects ; Platelet Adhesiveness

OBJECTIVETo explore the role of the amino acids between 551 and 565 in the cytoplasmic domain of glycoprotein (GP) I b alpha in the VWF binding to GP I b alpha.

METHODSThe VWF binding to GP I b alpha induced by ristocetin was analyzed by flow cytometry, in three GP I b-IX-expressing Chinese hamster ovary (CHO) cell lines 1b9, delta 565 and delta 551, adhesion of above cells on VWF by flow chamber analysis at shear rate of 200 s(-1). The spread of GP I b-IX-expressing cells were stimulated with botrocetin on VWF-coated coverslips by confocal microscope.

RESULTSThe VWF binding to GP I b alpha was higher in delta 565 cells stimulated by ristocetin than in delta 551 or 1b9 cells. The number of delta 565 cells adhered on the VWF-coated-chamber was more than that of controls at shear rate of 200 s(-1). Moreover, the surface spreading areas of delta 565 cells were greater than that of the controls on VWF-coated coverslips.

CONCLUSIONSThe amino acids between 551 and 565 in the cytoplasmic domain of GP I b alpha regulates the VWF binding to GP I b alpha.

Amino Acid Sequence ; Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Female ; Platelet Adhesiveness ; Platelet Glycoprotein GPIb-IX Complex ; genetics ; metabolism ; von Willebrand Factor ; metabolism

We coated a thin TiO2 film on the surface of Ni-Ti shape memory alloy by activated sputter method in the present work. The blood platelet adherence and antithrombogenicity of the TiO2-coated Ni-Ti alloy were evaluated. The results showed that the platelets on the TiO2-coated Ni-Ti alloy were fewer than those on 316L stainless steel, and no agglomeration or distortion for the platelets on the coated alloy was found, which means less probability of blood coagulation for the alloy. The coagulation time on the coated Ni-Ti shape memory alloy was longer than that on the 316L. Compared with that on the 316L stainless steel, the TiO2 coated Ni-Ti shape memory alloy showed better blood compatibility, indicating that the Ni-Ti alloy with TiO2 coating is a kind of ideal biomedical materials with high clinical value.
Alloys ; chemistry ; Blood Coagulation ; Coated Materials, Biocompatible ; chemistry ; Humans ; Materials Testing ; Nickel ; chemistry ; Platelet Adhesiveness ; Stents ; Surface Properties ; Titanium ; chemistry