This study was aimed to investigate the stability and in vitro function of glycosylated platelets concentrates after long-term refrigeration. The experiments were divided into 4 groups: group preserved at room temperature (RT group), group preserved at 4 degrees C (4T group), group glycosylated and preserved at 4 degrees C (U + 4 group) and group preserved at 4 degrees C and glycosylated (4 + U group). All groups followed for up to 14 days. The binding rate of RCA I lectin and expression of Plt surface markers CD62P, CD42b and Annexin V binding were determined by flow cytometry. pH and mean volume were determined by pH meter and hematotocytometer respectively. Platelet aggregation was detected by aggregometer. The results showed that during storage up to 14 days RCAI binding rate of modified groups was 5 - 6 fold of RT group. The pH of platelets suspension had no significant difference between these two groups (p > 0.05). Mean volumes of both groups (10.6 +/- 1.9 fL and 11.14 +/- 1.1 fL) were also no significant difference (p > 0.05). Furthermore, aggregation responsiveness of modified groups was better than that of RT groups (p < 0.05) although both decreased during the storage. The expression level of CD62P, CD42b and Annexin V binding during 5 days of storage had no significant difference between modified and fresh platelet groups (p > 0.05). While the expression level of CD62P and PS increased and the expression level of CD42b decreased during storage up to 14 days, there was significant difference between modified and fresh platelet groups (p < 0.01). It is concluded that the glycan modification is stable during storage up to 14 days. The glycosylated platelets retain in vitro function better than RT platelets during storage, but it shows activation to varying degrees in vitro after storage for 5 days.
Blood Platelets ; cytology ; metabolism ; Blood Preservation ; Cryopreservation ; methods ; Galactose ; pharmacology ; Glycosylation ; Humans ; Platelet Aggregation ; drug effects

Blood Platelets ; drug effects ; metabolism ; Calcium ; blood ; Deoxycholic Acid ; pharmacology ; Humans ; Quinazolines ; pharmacology ; Quinazolinones

This study was aimed to investigate the aggregation of rehydrated-lyophilized platelets. The aggregation rate of fresh and rehydrated-lyophilized platelets were measured by using thrombin, ristocetin, ADP and collagen as inductors and APACT2 aggregameter; the effects of intra- and extra-cellular trehalose on maximum aggregation rate of rehydrated-lyophilized platelets were detected by using ADP as an inductor. The results showed that the aggregation rate of fresh platelets was all about 100%, while aggregation rate of rehydrated lyophilized platelets was (70.17 +/- 7.36)%, (15.3 +/- 2.81)%, (68.67 +/- 6.86)%, (64.67 +/- 11.6)% respectively, when the concentration of thrombin, ristocetin, ADP and collagen was 1 U/ml, 1.6 mg/ml, 20 micromol/L and 2 microg/ml. The maximum aggregation rates of rehydrated-lyophilized platelets in intra- and extra-cellular trehalose, extracellular trehalose and blank control groups were (66.0 +/- 4.69)%, (25.3 +/- 2.42)% and (11.5 +/- 1.87)% (P < 0.01), meanwhile there was significant difference of rehydrated-lyophilized platelet aggregation rate between intra- and extra-cellular trehalose and extracellular trehalose groups (P < 0.01). It is concluded that the concentrations of thrombin (1 U/ml), ristocetin (1.6 mg/ml), ADP (20 micromol/L) and collagen (2 microg/ml) are optimal for platelets aggregation tests, the internal and extracellular trehalose significantly enhance the aggregation of rehydrated-lyophilized platelets.
Blood Platelets ; cytology ; drug effects ; metabolism ; Blood Preservation ; methods ; Freeze Drying ; methods ; Humans ; Platelet Aggregation ; physiology

The study was purposed to investigate whether the cyclooxygenase inhibitors from some dietary vegetables can inhibit platelet aggregation function by the arachidonic acid (AA). The vegetable juice was mixed with platelet rich plasma (PRP), and asprin was used as positive control. The maximum ratio of platelet aggregation induced by AA was measured on the aggregometer; heme and cyclooxygenase-1 (COX(1)) or cyclooxygenase-2 (COX(2)) were added to test tubes containing COX reaction buffer, the mixture was vortex-mixed and exposed to aspirin or vegetable juice, followed by addition of AA and then hydrochloric acid (1 mol/L) was added to stop the COX reaction, followed by chemical reduction with stannous chloride solution. The concentration of COX inhibitors was detected by the enzyme immunoassay kit; vegetable juice (aspirin as positive control) was mixed with whole blood, which was followed by the addition of AA, and then the reaction was stopped by adding indomethacin, centrifuged, then the supernatant was collected, and the plasma thromboxane B(2) (TXB(2)) was measured by radioimmunoassay. The results showed that spinach juice, garlic bolt juice, blanched garlic leave juice and Chinese leek juice could inhibit by 80% human platelet aggregation induced by AA. 4 kinds of vegetables were all found a certain amount of cyclooxygenase inhibitors, which COX(1) and COX(2) inhibitor concentrations of spinach were higher than that of aspirin; 4 vegetable juice could significantly reduce the human plasma concentrations of TXB(2) induced by AA (p < 0.05). It is concluded that 4 kinds of raw vegetables containing cyclooxygenase inhibitors inhibit the production of TXA(2) and thus hinder platelet aggregation. Raw spinach, garlic bolt, blanched garlic and chinese leek inhibit significantly AA-induced human platelet aggregation in vitro. 4 kinds of vegetables may have a good potential perspective of anti-platelet aggregation therapy or prevention of thrombosis.
Adult ; Arachidonic Acid ; metabolism ; Blood Platelets ; drug effects ; Cyclooxygenase Inhibitors ; pharmacology ; Female ; Humans ; Male ; Platelet Aggregation ; drug effects ; Vegetables ; chemistry

The aim of this research was to study the technology and methods of loading lyoprotectant-trehalose into cytoplasm of human platelets before lyophilization, to optimize experimental conditions of loading trehalose, to investigate the changes of platelets response to agonists and activation after incubation of platelets for 4 hours at 37 degrees C in the presence of lyoprotectant-trehalose, to protract the figures of loading efficiency and intracellular trehalose concentration versus incubation time, temperature and external trehalose concentration, to optimize loading parameters. The response of platelets to different agonists--thrombin, ADP, collagen and ristocetin were measured respectively by APACT2 aggregometer before and after loading trehalose into platelets; the expressions of CD62p and PAC-1 on platelet membranes in the presence and absence of reversible platelets activation inhibitors were measured by flow cytometry respectively before and after loading trehalose into cytoplasm of platelets. The results showed that the loading efficiency was linear to incubation time (2 hours later) and incubation temperature (rang from 30 degrees C to 40 degrees C), respectively. The loading efficiency almost reached 60% when the platelets were incubated at 37 degrees C for 4 hours. The intracellular trehalose concentration was higher with the increase of the extracellular trehalose concentration (< 50 mmol/L). Compared to untreated groups, the values of MPV and aggregation to different agonists in treated groups showed no significant difference, respectively (P > 0.01). After incubation of platelets for 4 hours, the expression of CD62p increased to some extent, however, the expression of CD62p decreased again when the reversible platelets activation inhibitor PGE-1 and adenosine were added to the incubation buffer. It is concluded that 37 degrees C, 4 hours and the extracellular trehalose concentration < 50 mmol/L are the optimal conditions for loading with trehalose. The processing of loading with trehalose before platelet lyophilization has no significant effects on response of platelets to agonists and activation.
Blood Platelets ; cytology ; drug effects ; metabolism ; Blood Preservation ; methods ; Cell Survival ; Cryopreservation ; methods ; Freeze Drying ; Humans ; Trehalose ; blood ; pharmacology

OBJECTIVETo investigate the effect of benzo(a)pyrene (BaP) on platelet aggregation and expression of P-selectin.

METHODSBlood samples were collected from healthy volunteers and the platelets was washed. Platelet aggregation was monitored by aggregometer and the expression of P-seletin was detected by whole blood flow cytometry.

RESULTBaP (10 μmol/L, 1 μmol/L and 0.1 μmol/L) did not induce platelet aggregation; however, preincubation with BaP (10 μmol/L) significantly enhanced ADP-induced platelet aggregation (P < 0.01) and platelet aggregation was (80 ± 10)%, while BaP-preincubation failed to enhance platelet aggregation under collagen and thrombin stimulation. Flow cytometry showed that preincubation with BaP increased ADP-induced, but not thrombin-induced P-selectin expression (P < 0.01).

CONCLUSIONBaP can stimulate ADP-induced platelet aggregation and P-selectin expression, probably through the interaction with ADP-mediated signal pathway.

Adenosine Diphosphate ; pharmacology ; Benzo(a)pyrene ; pharmacology ; Blood Platelets ; drug effects ; metabolism ; Collagen ; pharmacology ; Humans ; P-Selectin ; blood ; drug effects ; Platelet Aggregation ; drug effects ; Thrombin ; pharmacology

This study was aimed to investigate the inhibiting effects of adenosine on platelet in vitro in order to select functional protectants for platelets before lyophilization. Platelet membrane surface CD62P expression was assayed by flow cytometer (FCM). Platelet aggregations induced by restocetin, thrombin, ADP and propyl gallate were detected by APACT-2. The results showed that platelets membrane surface CD62P expression increased significantly after pre-treating of freeze-drying. 0.75 mmol/L adenosine could inhibit CD62P expression in a dose-dependent manner. Adenosine could inhibit platelet aggregation induced by propyl gallate, but no action on restocetin. When adenosine concentration was 1.0 mmol/L or higher, the aggregation induced by thrombin was significantly restrained. When concentration of adenosine was 0.75 mmol/L, platelet activation resulted from retreating could be inhibited and platelet aggregation induced by restocetin and thrombin were not affected markedly. It is concluded that adenosine can be one of the functional protectants and activation inhibitors in vitro for platelet cryo-preservation.
Adenosine ; pharmacology ; Blood Platelets ; drug effects ; metabolism ; Blood Preservation ; Cryopreservation ; Flow Cytometry ; Humans ; P-Selectin ; analysis ; Platelet Activation ; drug effects ; Platelet Aggregation ; drug effects ; Platelet Aggregation Inhibitors ; pharmacology

The purpose of study was to investigate the effects of L-arginine and cilostazol on platelet-activation and aggregation reserve in vitro, so as to provide proof for selecting reversible activation-inhibitors for platelets lyophilization. Activation and function of platelets were investigated by using flow cytometry with the CD62p and PAC-1 expression and re-expression after being activated by thrombin, and by means of platelet aggregation reaction to thrombin, ADP and propyl gallate, as well as coagulation activity of platelets. The results showed that expression of CD62p and PAC-1 increased after being pretreated. Both L-arginie and cilostazol could inhibit CD62p and PAC-1 expression and related with their concentrations. Cilostazol had an intensive inhibition effect on expressions of CD62p and PAC-1 induced by thrombin, and the inhibition increased when concentration augmented. L-arginine had the same effects on PAC-1, but had no effects on CD62p. L-arginine and cilostazol inhibited aggregation induced by thrombin, ADP and propyl gallate, and the inhibitions were related directly with dosage. When L-arginine concentration was higher or equal to 15 mmol/L, or cilostazol concentration was in range of 1 - 4 mmol/L, the aggregation time were prolonged so much or even no aggregation. It is concluded that when L-Arginine concentration is 5 mmol/L and 10 mmol/L, platelet activation can be inhibited, but aggregation ability and characters keep intact. Concentration at 5 mmol/L may be the best. 1 mmol/L of cilostazol can inhibit activation in vitro and retain part of platelet ability of aggregation and reexpression.
Arginine ; pharmacology ; Blood Platelets ; metabolism ; Humans ; P-Selectin ; drug effects ; metabolism ; Platelet Activation ; drug effects ; Platelet Aggregation ; drug effects ; Platelet Aggregation Inhibitors ; pharmacology ; Tetrazoles ; pharmacology

The aim of this study was to search a procedure of platelet lyophilization and find a way of long-term storage of human platelets at normal temperature with smaller size and lighter weight, to be convenient to transport at long distance thus to meet the demands in accidents and war time. Human platelets were pretreated by freezing, the first and the second desiccation, and were added with reversible activation-inhibitors of platelets, DMSO and trehalose, then were rehydrated. At the same time, the recovery rate of platelets, platelet maximal aggregation induced by thrombin, coagulation of platelets, CD62p expression and PAC-1 expression were assayed. The results indicated that the recovery rate of the platelets was 56.29%. The platelet maximal aggregation induced by thrombin had no significant difference between lyophilized platelets and the fresh platelet-rich plasma (FPRP), but the aggregation of platelets induced by ADP or propyl gallate was decreased by 49.34% and 26.25%. Coagulation of the lyophilized platelets was not significantly different from FPRP. CD62p expression of the lyophilized platelets (42.36%) was higher than that in FPRP while PAC-1 expression was 2.12%. CD62p re-expression rate induced by thrombin was 50.88% and PAC-1 re-expression was 54.55%. It is concluded that the ability of recovered lyophilized platelets added with reversible activation-inhibitors, DMSO and trehalose to aggregate and coagulate has showed no significant difference as compared with FPRP. The reversible activation-inhibitors can decrease CD62p expression of lyophilized platelets, and may enhance their survival ability and prolongate survival time. Therefore the efficiency of lyophilizing platelets can be improved based on this freeze-drying procedure.
Blood Platelets ; cytology ; drug effects ; metabolism ; Blood Preservation ; methods ; Cell Survival ; Freeze Drying ; methods ; Humans ; Trehalose ; pharmacology

OBJECTIVETo observe the effect of novel air neuromodulator H2S on platelet function of L-Arg transport for discussing H2S of effect on platelet function.

METHODSSaturate H2S solution as donate made rat rich platelet plasma and pre-incubation rat platelet with different density of H2S. To measure the velocity of L-Arg transport in platelet by radioactivity technique.

RESULTSAt different concentrations of H2S (6.25, 12.5, 25, 50, 100 micromol/L), the velocity of L-Arg transport was lower than that in control. H2S reduced rapidly the Vmax and velocity of L-Arg transport in platelet (P < 0.05) and this effect had no effect to Km.

CONCLUSIONH2S can affect platelet function by changing rapidly platelet L-Arg transport system function.

Animals ; Arginine ; metabolism ; Biological Transport ; drug effects ; Blood Platelets ; drug effects ; metabolism ; Hydrogen Sulfide ; metabolism ; pharmacology ; Male ; Rats ; Rats, Sprague-Dawley