Influence of different levels of heparin and antithrombin Ⅲ on human coagulation factor Ⅸ activity / 中国输血杂志

【Objective】 To study the effect of different concentrations of heparin, ATⅢ or a mixture of heparin and antithrombin Ⅲ (ATⅢ) (1∶1)on the activity of human coagulation factor Ⅸ (FⅨ). 【Methods】 The heparin or heparin/ATⅢ with different concentrations were added into human coagulation Ⅸ products or human prothrombin complex (PCC) to prepare heparin or heparin/ATⅢ samples, containing 0, 0.1, 0.3, 0.5, 0.8, 1, 2 and 4 IU per unit. ATⅢ with different concentrations were added into FⅨ or PCC to prepare ATⅢ samples containing ATⅢ 0, 0.1, 0.5 and 1 IU per unit. The FⅨ activity of the samples prepared was tested by one-stage coagulation method. Then corresponding amount of protamine sulfate were added to neutralize heparin or heparin/ATⅢ to detect the FⅨ activity again. Their influence of heparin, ATⅢ and heparin/ATⅢ with different concentrations on the activity of FⅨ were analyzed. 【Results】 When the content of heparin or heparin/ATⅢ was 0, 0.1, 0.3 and 0.5 IU per unit of FⅨ, the detection results of FⅨ titer in samples were consistent. When the content of heparin or heparin/ATⅢ per unit of FⅨ was 0.8, 1, 2 and 4 IU, the detection results of FⅨ titer were all lower than those of samples without heparin. When the ATⅢ content was 0, 0.1, 0.5 and 1 IU, the FⅨ titer of the samples was consistent. 【Conclution】 When the content of heparin or heparin/ATⅢ in the product is less than or equal to 0.5 IU per IU of FⅨ, the step of protamine sulfate adding could be omitted as it has little effect on FⅨ activity. When >0.5 IU per IU of FⅨ, however, protamine sulfate adding, to neutralize heparin, is necessary before FⅨ activity testing.

Qualities Study of Protamine Sulfate Raw Material from Different Species of Fish / 中国药学杂志

OBJECTIVE: To study the qualities of protamine sulfate raw material from different species of fish. METHODS: The identification and determination of protamine sulfate raw material from different fish species were conducted by HPLC, and the composition of amino acids was tested too. Heavy metals was tested by inductively coupled plasma mass spectrometry. The potency of protamine sulfate was measured by biological assay method. RESULTS: At present, the sources of protamine sulfate on the market were mainly salmon and herring. CONCLUSION: This HPLC method can be applied to effectively identify and determine protamine sulfate raw material in order to improve its quality control.

Comparison and analysis of two potency assay methods of protamine sulfate / 中国生化药物杂志

Objective To analyze and compare two potency assay methods of protamine sulfate.Methods Heparin titration method was according to foreign pharmacopoeia,while biological assay method was according to China pharmacopoeia(2015).Results The absorbance at the end-point was stable at 0.10 mg/mL and 0.15 mg/mL test solutions.The relative standard deviations(RSDs)of potency results were all less than 5%.Three test solutions and volumes of the titrant had a good linear relationship(R2 =1.000).The results of titration method were significantly related to those of biological assay(P=0.013),with similar RSD(P>0.05).However,the potency of titration method were significantly lower than those of biological assay (P=0.045).Conclusion Heparin titration method is good-using,convenient,and need to be applied widely at home.

Study on the inlfuening faltors of altivity detemination of four coagulation factors in human prothrombin complex concentrates / 中国生化药物杂志

Objective To study the inlfuence factors on detection of activity of four coagulation factors in prothrombin complex concentrates (PCC) by several factors. Methods Using Pharmacopoeia of the People’s Republic of China (2010) as reference, the activity of four coagulation factors in PCC were investigated by choosing different pre-treatments, different diluents, different salt concentration, different standard human plasma and different company reagents. Results The activity of FII, FVII, FX were decreased and FIX was increased in the condition of adding protamine sulfate, and there were no differences of four coagulation factors whether warm bath in 37 for 15 min or not. However, the differences of four coagulation factors were significant by using deficient plasma, saline, distilled water and commercial dilution buffer(P<0.05). The activity of coagulation factor II, X in 1 mol/L salt concentration of PCC were significantly lower than in 0.25 mol/L(P<0.05), while coagulation factor VII, IX were not. The activity of FII, FVII, FIX, and FX were different by using different standard human plasma to make standard curve. The activity of four coagulation factors existed significant difference(P=0.00) by using SIEMENS company reagents and domestic reagents. Conclusion Choosing different pre-treatments, different dilution buffers, salt concentration, standard human plasma and commercial kits will inlfuence the detection result of coagulation factors.

Anti-biofilm and anti-adherence activity of Glm-U inhibitors.

Background: Intravascular catheters and urinary catheters are an important source of hospital-acquired infections. Many microorganisms colonize indwelling catheters, including central venous catheters (CVCs) forming biofilms and cause infections that are difficult to treat. Although various methods have been employed to reduce biofilms, enzymes involved in bacterial cell wall synthesis could provide novel targets for the development of anti-biofilm agents. N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) is an essential enzyme in aminosugars metabolism and catalyzes the formation of uridine-diphospho-N-acetylglucosamine (UDP-GlcNAc), an important precursor in the peptidoglycan and lipopolysaccharide biosynthesis of Gram-positive and Gram-negative bacteria. Previous study has been conducted on the anti-biofilm effect of GlmU inhibitors such as N-ethyl maleimide (NEM) and NEM analogs along with a cationic polypeptide protamine sulfate (PS), which enhanced its anti-biofilm activity. AIM: The present study aimed at finding the effect of sub-inhibitory concentrations of N-ethyl maleimide (NEM) and protamine sulfate (PS) on the biofilms produced by Pseudomonas aeruginosa and Enterococcus spp. isolated from cases of catheter-associated UTI as well as Klebsiella pneumoniae and Staphylococcus aureus isolated from cases of catheter-related bloodstream infections (CRBSI). Materials and Methods: In order to enhance the activity of NEM and to develop a broad-spectrum anti-microbial composition, NEM (50 μg/ml) was combined with protamine sulfate (50 μg/ml) and tested for anti-biofilm activity using a standard quantitative biofilm assay method. Results and Conclusion: It was observed that NEM had no effect on the biofilm produced by Pseudomonas aeruginosa as well as by Enterococcus spp. NEM also caused a significant decrease in biofilm production by Staphylococcus aureus while it had no effect on the biofilm produced by Klebsiella pneumoniae. There was a significant synergistic inhibitory effect on Staphylococcus aureus and Enterococcus spp., whereas Pseudomonas aeruginosa and Klebsiella pneumoniae remained unaffected. Combination of GlmU inhibitor-plus-protamine sulfate failed to significantly reduce bacterial adherence of Pseudomonas aeruginosa and Klebsiella pneumoniae to catheter and cannula pieces, respectively. We found that the GlmU inhibitor was mainly effective in preventing the adherence and biofilm formation by gram-positive organisms. The combination of NEM and protamine sulfate may, therefore, be tried as anti-infective coatings for medical devices such as catheters and cannulas, and thus help in overcoming microbial resistance in the current era of increasing device-associated hospital infections.

Combination of protamine sulfate and potassium chloride in establishing the interstitial cystitis animal model / 中华泌尿外科杂志

e combina-tion of protamine sulfate and potassium chloride to establish interstitial cystitis animal model is reliable and feasible. Researchers can choose the right time of irrigation based on the intent of the experiment.

The Effect of Calcium Chloride on Hemodynamic Responses to Protamine Sulfate in the Dog / 대한마취과학회지

BACKGROUND: Protamine sulfate (PS), used to neutralize the effect of heparin, is often associated with systemic hypotension. The present study was aimed to investigate the protective effects of CaCl2 on adverse hemodynamic reaction to PS. METHODS: Forty-six dogs of three groups were studied. Animals were randomly allocated to receive either; saline 10 ml (controls, n = 26), CaCl2 5 mg/kg (n = 10) or 10 mg/kg (n = 10), added to PS 3 mg/kg given intravenously 5 min after heparin (300 IU/kg, iv). Mean aortic pressure (MAP), mean pulmonary arterial pressure (MPAP), left ventricular end-diastolic pressure (LVEDP), LVdP/dtmax, and -LVdP/dtmin, and pulmonary artery (cardiac output) and left circumflex blood flow (LCX flow) were recorded up to 15 min after PS. RESULTS: PS caused significant reductions in MAP (-49%), cardiac index (CI, -66%), systemic vascular resistance index (SVRI, -22%), LVEDP (-67%), LVdP/dtmax (-36%), and LVdP/dtmin (-55%), but increased MPAP (39%) and PVRI (3.8 fold), which all increases and reductions peaked 1-3 min after PS injection in the control group. The addition of CaCl2 to PS significantly attenuated reductions of MAP (-35 and -26% in CaCl2 5 and 10 mg/kg treated dogs, respectively), CI (-34 and -37%), LVdP/dtmax (-14, -11% ), and -LVdP/dtmin (-34, -21%), and increases in PVRI (1.8 and 2.4 fold). However, both doses of CaCl2 increased MPAP (48, 82%), but not SVRI (-28, -21%) or LVEDP (-73, -75%). LCX flow increased similarly in all groups (75-137%). CaCl2 5 and 10 mg/kg iv increased plasma Ca2+ by 0.23 and 0.36 mM, respectively. CONCLUSIONS: These results suggest that the simultaneous administration of CaCl2 attenuates the adverse hemodynamic effects of PS, used to reverse heparin anticoagulation in the dog.

The Effect of Inhaled Nitric Oxide on Protamine Sulfate-Induced Hypotension in Dogs / 대한마취과학회지

BACKGROUND: Protamine sulfate has been found to produce systemic hypotension secondary to acute pulmonary vasoconstriction and subsequent right heart failure in pigs and sheep. Nitric oxide (NO) is a potent pulmonary vasodilator. The present study was aimed to determine whether NO inhalation prevents hypotension in an open-chest canine model. METHODS: With IRB approval, 29 mongrel dogs were acutely instrumented during 1.5% enflurane anesthesia. All animals then received protamine 3 mg/kg over 30 s given through right atrium, 5 min after heparin (300 IU/kg, iv). NO inhalation was done for 20 min beginning 10 min before protamine infusion (NO group, n = 10). Control group was without NO inhalation (n = 19), which was retrospectively divided into two groups according to the magnitude of pulmonary arterial pressure (PAP) increase: normal PAP group (increase in PAP less than 6 mmHg, n = 9) and pulmonary hypertensive group (increase in PAP more than 6 mmHg, n = 10). Mean arterial pressure (MAP), PAP, left ventricular end-diastolic pressure (LVEDP), heart rate (HR), and cardiac output and left circumflex (LCX) coronary flow via a Doppler flowmeter were continuously monitored. Calculated parameters included cardiac index (CI), and systemic and pulmonary vascular resistance indices (SVRI and PVRI). RESULTS: Protamine increased PAP (66 vs 7%) and PVRI (5.1- vs 3.0- fold) more pronouncedly in pulmonary hypertensive than in normal group. However, protamine caused similar reductions of MAP (-40 vs -46%), CI (-60 vs -59%), and LVEDP (-47 vs -53%) in pulmonary hypertensive and normal groups. SVRI showed a biphasic response in both groups, an initial decrease (-15 vs -14%), followed by an increase (48 vs 25%, P<0.05). Continuous inhalation of NO at 80 ppm did not affect the protamine-induced reductions in MAP (-40%), CI (-55%), and LVEDP (-46%) and increases in PAP (45%) and PVRI (4.1-fold). LCX flow increased immediately after the protamine treatment in all groups to a similar magnitude (83-130%), indicating a rapid release of potent vasodilators. CONCLUSIONS: Protamine produces profound hypotension, which may not be causally related to an acute pulmonary vasoconstriction in the dog. It is unlikely that exogenous NO treatment affects hypotension.

Effect of Calcium Chloride on Protamine Sulfate-Induced Systemic Hypotension in Adult Open-Heart Patients / 대한마취과학회지

BACKGROUND: Protamine sulfate (PS), used to neutralize the anticoagulant effect of heparin, is often associated with systemic hypotension. The present study was aimed to investigate the protective effects of calcium chloride (CaCl2) on adverse hemodynamic reactions to PS in patients undergoing open heart surgery. METHODS: After IRB approval, sixty-one patients undergoing open heart surgery were allocated randomly to receive either saline 10 ml (control group, n = 26), CaCl2 5 mg/kg (CaCl2 5 group, n = 18) or 10 mg/kg (CaCl2 10 group, n = 18), added to PS 3 mg/kg given over 3 min through the right atrium to reverse heparinization. Hemodynamic measurements, including systolic (SAP), diastolic (DAP), and mean arterial pressure (MAP), heart rate, and central venous pressure (CVP) were continuously recorded in a baseline condition and up to 10 minutes after PS infusion. Plasma Ca2+ level and arterial blood gas tension were also measured. RESULTS: PS caused immediate and significant decreases in SAP, DAP, and MAP in all three groups. However, the degree of hypotension was significantly less in the CaCl2 10 group than in the CaCl2 5 and control groups. Heart rate and CVP remained unchanged throughout the study in all groups. Intravenous CaCl2 5 and 10 mg/kg IV caused an increase in plasma Ca2 by 0.13 +/- 0.08 mM and 0.45 0.08 mM at 2 min after its injection, respectively. Arterial oxygen tension did not change significantly throughout the study in any group. CONCLUSIONS: These results suggest that simultaneous administration of 10 mg/kg CaCl2 attenuates the hypotensive effect of PS used to reverse heparin anticoagulation in patients undergoing open heart surgery. However, hypotensive reactions may still occur.