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Objective To establish and assess the new method of APTT assay based on the combinations of Mg2+and Ca2+for lupus anticoagulants(LA)measurements.Methods This prospective study included 309 trisodium citrate anticoagulated plasma samples from 244 random patients and 65 patients with different autoimmune diseases(AID)to establish and assess the method of LA measurement, respectively.Final concentrations of 0,2.0, 4.0, 8.0,16.0 mmol/L Mg2+were added into 25 mmol/L Ca2+solution, and Actin reagent was used to measured plasma APTT of 94 patients.The applied concentration of Mg2+-Ca2+solution was confirmed through the special and significant alteration of APTT from LA-positive and -negative plasma observed in the presence of Mg 2+(test solution).Based on Actin reagent use,the test solution and 25 mmol/L Ca2+solution were applied to measure APTT of patients and normal individuals, respectively, and the ratio of Mg2+-Ca2+-APTT to Ca2+-APTT(Mg2+-Ca2+-APTT indices)and normalized Mg2+-Ca2+-APTT indices(NAR)were calculated, respectively.Mixed plasma NAR was measured,and CV%was calculated to evaluate the repeatability and stability of Mg 2+-Ca2+-APTT method.APTT of 150 patients was measured with the test solution and Actin reagent to calculate Mg 2+-Ca2+-APTT indices, and normalized LA ratio was determined with dRVVT method.The applicability of Mg2+-Ca2+-APTT assay was assessed through comparisons of the results from the two methods.Finally, NAR and NLR of 65 patients with AID(including 26 SLE patients)were measured with Mg2+-Ca2+-APTT assay and dRVVT method, respectively, and ROC curve was also used to assess the efficacy of the two methods for LA measurements.Results In all LA-negative plasma,APTT increased from 28.1 ±4.5 s to 61.2 ±7.9 s in normal APTT group,47.2 ±8.9 s to 97.5 ±10.3 s in increased APTT group,and 27.6 ± 5.1 s to 61.2 ±7.9 s in ACA-positive group when Mg2+increased from 0 to 8 mmol/L in Mg2+-Ca2+solution(F=34.12, 38.9 and 28.35,P<0.01).Following increased Mg2+concentration, APTT shortened from 0 to 4.0 mmol/L, but simultaneously prolonged from 4.0 to 16.0 mmol/L in LA-positive plasma with prolonged or normal APTT(F=31.55 and 39.51, P<0.01), and APTT was significantly higher in 8.0 mmol/L than that in 4.0 mmol/L(P<0.001).The test concentration of Mg 2+/Ca2+solution was 4.0 mmol/L.The within, inter-day CV% of NAR was 1.39%,2.30%, and 3.44%, respectively. According to the judging criteria of <0.966 and >1.034 of Mg2+/Ca2+indices, there was 141 patients with increased indices and NLR <1.20, and 9 patients with decreased ones and NLR≥1.20 in all 150 patients.The area under ROC curve of NAR and NLR for LA detection was 0.913(95%CI:0.848-0.978) and 0.892(95%CI:0.817-0.966), respectively, and the cut-off value was 0.87 and 1.13, respectively. The sensitivity and specificity of NAR(85% and 77%)was higher than that of NLR(81% and 74%), respectively.The accordant rate of positive,negative,and total results between NAR and NLR was 94.4%, 98.5%,and 98%,respectively.Conclusion The method of APTT assay based on Mg2+combining Ca2+for LA measurements is feasible,and can be used to detect plasma LA of patients.
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Objective To compare the changes in blood coagulation, fibrinolysis and endothelial damage in patients undergoing laparoscopic cholecystectomy with different durations of carbon dioxide pneumoperitoneum. Methods Sixty-four ASA Ⅰ orⅡpatients, aged 23-60 yr, weighing 45-82 kg, scheduled for elective laparoscopic cholecystectomy, were randomly divided into 3 groups according to the duration of pneumoperitoneum: duration of pneumoperitoneum ≤30 min group (group Ⅰ, n=21), 30 min ＜ duration of pneumoperitoneum ＜ 60 min (group Ⅱ, n=23) and duration of pneumoperitoneum≥ 60 min (group Ⅲ , n=20).The intra-abdominal pressure was maintained at 12-14 mm Hg. Venous blood samples were taken before surgery (baseline, T0 ),at the end of surgery(T1), and at 1, 2 and 3 d after surgery (T2-4) for determination of prothrombin time, activated partial thromboplastin time, concentrations of prothrombin fragment 1+2(F1+2), fibrinogen (Fib), tissue plasminogen activator and plasminogen activator inhibitor type-1 (PAI-1), and activities of antithrombin Ⅲ(AT-Ⅲ)and von Willebrand factor(vWF).Results Compared with groupⅠ , the vWF activity and PAI-1 concentration at T2 , concentrations of Fib, F1+2, PAI-1 and activity of vWF at T3 and concentrations of Fib and F1+2 at T4 were significantly increased, while the AT-IE activity at T3 was significantly decreased in group Ⅲ(P＜0.05) .Conclusion When the duration of pneumoperitoneum is short, no obvious changes in the blood coagulation, fibrinolysis and endothelial damage are observed postoperatively in patients undergoing laparoscopic cholecystectomy, and along with the prolongation of the duration of pneumoperitoneum, increased blood coagulation, reduced fibrinolysisand aggravated endothelial damage are observed postoperatively.
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Objective To observe the effect of duration of carbon dioxide pneumoperitoneum on coagulation, fibrinolysis and endothelial activation in elderly patients undergoing laparoscopic cholecystectomy (LC). Methods The 45 elderly patients with cholelithiasis scheduled for LC, aged over 60 yeas, were placed in different groups respectively after surgery according to the duration of pneumoperitoneum. The duration of pneumoperitoneum was ≤60 minutes in group A (n=21),and more than 60 minutes in group B (n=24). Venous blood samples were taken on admission (baseline), at the end of surgery, the 1st, 2nd and 3rd day after surgery for determination of prothrombin time (PT), activated partial thromboplastin time (APTT), prothrombin fragment F1+2 (F1+2), antithrombin 3 (AT-Ⅲ activity), fibrinogen (Fib), tissue plasminogen activator (t-PA), plasminogen activator inhibitor type-1 (PAI-1), D-dimer (D-D), von Willebrand factor (vWF activity). Results Concerning the coagulation activation, at the 3rd postoperative day, the level of F1+2 was significantly higher in group B than in group A [(1.60±0.26) μg/L vs. (1.32±0.24) μg/L, P＜0.05]; AT-III was significantly higher in group B than in group A [(84.82%±20.21%) vs. (97.49%±16.87%), P＜0.05]. At the 2nd and 3rd postoperative day, the levels of Fib were significantly higher in group B than in group A [(3.87±0.62) g/L vs. (3.42±0.72) g/L, (3.98±0.77) g/L vs. (3.42±0.63) g/L, respectively, P＜0.05]. Concerning fibrinolysis, But at the 2nd and 3rd postoperative day, the level of PAI-1 was significantly higher in group B than in group A [(33.93±10.42) μg/L vs. (26.69±9.49) μg/L, (32.90±11.25) μg/L vs. (26.31±7.06) μg/L respectively, P＜0.05]. Concerning endothelial activation, at the 2nd and 3rd postoperative day, vWF was significantly higher in group B than in group A [(174.53%±44.03%) vs. (134.37%±37.74%), (176.31%±47.6%) vs. (131.21%±36.34%), respectively, P＜0.05]. Conclusions Marked activations of coagulation-fibrinolysis and endothelial activation are observed postoperatively in elderly patients undergoing laparoscopic cholecystectomy. Along with prolonged duration of pneumoperitoneum, more pronounced alterations of increased coagulation, reduced fibrinolysis and endothelial activation are observed, which could constitute an imbalanced situation of coagulation-fibrinolysis and increases the risk of venous thrombosis.
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Objective To observe the in vitro inhibitory effects of brucea javanica oil on human washed platelet and explore the possible mechanism.Methods Human washed platelets were mixed withdifferent concentration of brucea javanica oil which were divided into four groups[untreated control group,negative control group,9.0% of brucea javanica oil group,and 22.5%of brueea javanica oil group].The maximal ratio of platelet aggregation induced by adenosine liphosphate(ADP),arachidonic acid(AA),collagen,and thrombin,respectively,was measured with platelet aggregation analyzer.The expressions of fibrinogen receptor(FIB-R)and P-selectin(CD_(62p))on external membrane of activated platelet were determinated with flow cytometry.The F-actin of cytoskeletal structure in activated platelet was detected by SDS-PAGE.Results At 9.0% of brucea javanica oil,the maximal ratio of platelet aggregation[(57.7±4.0)%,(62.2±3.9)%,(66.9±5.0)%and(71.8±5.1)%]induced by ADP,AA,collagen,and thrombin,was significantly lower than that[(75.3±4.1)%,(79.3±4.8)%,(80.6±5.4)%,(84.1±6.2)%]at negative control(0% of brucea javanica oil)(P<0.01),but makedly higher than that[(39.2±3.5)%,(45.8±3.4)%,(51.2±3.9)%and(56.7±4.8%)]at 22.5%,respectively(P<0.01).The inhibitory rate of platelet aggregation(47.9%,42.2%,36.5%and 32.6%)at 22.5%of brucea javanica oil was notably higher than that(23.4%,21.6%,17.0%and 14.6%)at 9.0%,respectively(P<0.001).There was a negative correlation between brucea javanica oil concentration and the aggregation ratio of platelet stimulated by the four agonists,respectively(r=-0.952,-0.961,-0.970,-0.975,P<0.001).At 9.0% of brucea javanica oil,the expression levels of FIB-R[(64.7±4.0)%]and CD_(62p) [(3.91±0.21)%] of platelet activated by ADP were significantly lower than that[(85.5±4.6)%and (5.05±0.27)%]at negative control,but remarkably higher than that[(36.2±3.9)%and(2.34±0.15)%]at 22.5%,respectively(P<0.01).There was a much higher inhibitory rate of platelet aggregation(57.7%)at 22.5%than that(24.3%)at 9.0%(P<0.01).The ratios(1.68±0.10 and 1.77±0.12)of F-actin photodensity at 22.5%and 9.0%to that in blank control were significantly lower than that(2.22±0.15)at negative control(P<0.01)but there was no statistical difference between the ratios in the group of 9.0%and 22.5%brucea javanica(P>0.05).Conclusions brucea javanica oil has special inhibitory effect on activated platelet and thrombosis in a dose-dependent manner.The mechanism is to inhibit the expression of fibrinogen receptor on external membrane of activated platelet,which is also related to the inhibition of F-actin and secretion of platelet.
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Objective To observe the inhibition of amikacin in vitro on platelet aggregation and blood coagulation tests, and to study its effects on hemostasis and the related mechanisms.Methods Plateletrich plasma and platelet-poor plasma from donors were mixed with different concentration of amikacin, which was divided into four groups:0 mg/L, 30 mg/L, 91 mg/L and 910 mg/L group.The maximial ratio of platelet aggregation induced by ADP were measured with Platelet Aggregation Analyzer.The expression levels of P-selectin, GP Ⅱ b/Ⅲ a and Fg-R were determined with Flow Cytometer.The PT, APTT, TT and Fg of platelet-poor plasma were detected with Blood Coagulation Analyzer. The four concentration of amikacin mentioned above and two anticoagulants (62.5 U/ml of sodium heparin and 109 mmol/L of sodium citrate)were interacted with fresh whole blood, in which the blood CT and plasma Ca2+ were detected. Blood samples were collected from 10 patients with acute lower respiratory tract infection before and 30 minutes after routine amikcin treatment respectively.The maximial ratio of platelet aggregation, the expression levels of P-selectin, GPⅡ b/Ⅲa and Fg-R induced by ADP were measured; while PT, APTT, CT and plasma Ca2+ were determined.Results At 30 mg/L of amikacin group, the maximal ratios of platelet aggregation (65.8±3.9)%, the expression levels of P-selectin (9.2 ± 1.0)% and Fg-R (12.6 ± 1.7)% were statistically lower than those [(88.0 ±4.6%, (16.1 ± 1.3)% and (31.0 ±2.5)%]at 0 mg/L of amikacin group ( t = 9.442,8.432,9.993,P ＜ 0.01 ).At 30 mg/L of amikacin group, the APTT (80.5 ±6.8) s and CT ( 857 ± 66) s were significantly higher than those [(33.0 ± 3.6) s and (447 ± 35 ) s] at 0 mg/L of amikacin group ( t = 11.312, 13.211, P ＜ 0.01 ). There was a negative correlation between amikacin concentration and maximial ratio of platelet aggregation ( r = - 0.832, P ＜ 0.05 ), but a positive correlation between amikacin concentration and inhibitory rates of platelet aggregation ( r = 0.939, P ＜0.05) was observed, as well as APTT (r ＞0.870, P＜0.05).At 30 mg/L, 91 mg/L, and 910 mg/L of amikacin groups, the P-selectin and Fg-R expression were remarkably inhibited with a dose-dependent manner, the CT was notably enhanced [Fwithin subjects =21.44, 26.24, ( ＞29.81 ), P ＜0.01].At 0 mg/L,30 mg/L, 91 mg/L and 910 mg/L of amikacin groups, the PT values were ( 14.7 ± 1.9) s, ( 15.2 ± 1.7) s,(15.6±1.5) s and (22.1 ±2.1) s, respectively (F=8.21,P＜0.05), but there was no markeddifference for the levels of GP Ⅱ b/Ⅲ a, TT, Fg and plasma Ca2+ among the four groups ( P ＞ 0.05 ).After 30 minutes of amikacin treatment, the maximial ratio of platelet aggregation (51.6 ± 10.1)%, the expression levels of P-selectin (6.8 ± 1.8) % and Fg-R ( 20.1 ± 5.8 ) % were significantly lower than those [(66.8 ± 11.4)%, ( 10.9 ±3.1 )% and (28.5 ±7.4)%] before amikacin treatment, but APTT (49.8 ±5.9) s and CT (660 ±59) s were remarkably higher than those [(26.9 ±3.8) s and (410 ±45) s] before amikacin treatment, respectively ( t = 5.456,8.875,7.423,10.012,11.322, P ＜ 0.01 ), while the GP Ⅱ b/Ⅲ a expression, PT and Ca2+ concentration had no significant changes ( P ＞ 0.05).Conclusions There are inhibitory effects of amikacin on platelet aggregation mainly through the inhibition of both fibrinogen receptor activation and secretion reaction of activated platelet. Amikacin may also inhibit pathway of coagulation system factor to prevent blood coagulation.Therefore, risk of hemorrhage may be investigated in the patients with amikacin for anti-infection treatment.