Hemostasis system indices in the course of a 105-day hermetic chamber isolation experiment

Changes in hemostasis indices in the course of a 105-day hermetic chamber isolation experiment were studied. The following indices were measured: the activated partial thromboplastin time (APTT); the prothrombin time (PT); the prothrombin index (PI); the international normalized ratio (INR); the thrombin time (TT); the concentrations of fibrinogen, soluble fibrin-monomer complexes (SFMC), D-dimer, and plasminogen (PG); and the activities of antithrombin III, protein C, and α₂-antiplasmin. Isolation was accompanied by PT prolongation (with PI decreasing and INR increasing) observed until the seventh day of the aftereffect period, as well as a decrease in the PG concentration. The changes are likely to have been related to the specific motor activity mode, the effect of compensatory physical exercise, and changes in the characteristics of protein and lipid metabolisms.     

Hemostasis system indices after short-term space flights and during 7-day “dry” immersion experiment

The present paper deals with studying the hemostasis system indices after short-term (10–11 days) space flights, as well as in the course of the experiment with a 7-day “dry” immersion. The following values were determined: activated partial thromboplastin time, prothrombin time, prothrombin index, international normalized ratio (INR), thrombin time (TT); fibrinogen, soluble fibrin-monomer complexes, D-dimer, plasminogen (PG) concentrations; activity of antithrombin III (ATIII), protein C (PC), and alpha2-antiplasmin (AP).     

Oral anticoagulant therapy and international normalized ratios in swine

While monitoring coagulation testing in Yucatan miniature swine being given oral anticoagulants, we noticed instances of high international normalized ratios (INR) without clinical complications in our animal model. All pigs (n = 17) weighed approximately 35.2 kg and were dosed daily with 2 to 3 mg of coumadin. Plasma samples were obtained and assayed for prothrombin time (PT), calculated INR, and activated partial thromboplastin time (APTT) at baseline, and after 7 and 14 days of coumadin therapy. Results of initial testing indicated high INR values after anticoagulation and short APTT values at baseline, which led us to consider the activity of vitamin K-dependent coagulation factors in the pig. This information was not available in literature concerning this strain of swine, and was surprising given that pigs are frequently used cardiac research models. Using the same plasma samples, we repeated the PT, INR, and APTT determinations using different reagents and a different analyzer. We also determined activities of coagulation factors II, VII, IX, and X. Large PT and INR differences were seen between the two instrument/reagent combinations, possibly due to the differences in the thromboplastins used and differences in the photo-optic versus manual clot-detection method of the instruments. Vitamin K-dependent factors in all pigs responded to coumadin by decreasing to < 30.0% activity, except for factor IX. The high INR values were not as pronounced when the second instrument/reagent combination was used, and the results seemed more in line with the animals' clinical condition. With this instrument/reagent combination, the pig can be considered a good model for research requiring oral anticoagulant medication.     

The influence on coagulation of transdermal estrogen hormone replacement therapy as a preoperative preparation of the tissue before vaginal hysterectomy

In 32 postmenopausal patients who underwent vaginal hysterectomy due to the presence of uterine prolapse at the Department of Uro-gynaecology and Pelvic Floor Disorders in the Clinic of Gynaecology and Obstetrics, Medical School, Skopje in the period from 1st January 2002 to 1st January 2003, and who were preoperatively treated with transdermal estradiol 50 microg/day during 14 days the following parameters of the coagulating status were estimated: prothrombin time (PT) that is expressed in: absolute value, percentage and INR; activated partial thromboplastin time (aPTT Pathrombin SL); thrombin time and platelets number before and after hormone replacement therapy. After 14-day transdermal estrogen therapy, the parameters: PT, PT%, PT INR, aPTT Pathrombin SL didn't expressed significant changes, the thrombin time expressed significant extension, and the platelets expressed a significant decrease. According to our results, the transdermal estrogens might not have any influence on the hepatic synthesis of coagulating factors till the step of prothrombin formation. They might have an essential influence on the step of prothrombin transformation into thrombin, as well as on the process of megacaryocytes segregation into platelets.     

Role of substrate in determining the phospholipid specificity of protein kinase C activation

The phospholipid selectivity of protein kinase C (PKC) activation was examined by using two substrates, histone and a random copolymer of lysine and serine [poly(lysine, serine)] (PLS), plus phospholipids provided as vesicles or as Triton-mixed micelle preparations. The results indicated that substrate-phospholipid interaction was an essential component of PKC activation and that many in vitro properties of PKC activation are attributable to this interaction. The substrate histone interacted with phospholipid-Triton mixed micelles containing phosphatidylserine (PS), but not with those containing phosphatidylinositol (PI) or phosphatidylglycerol (PG). In direct correlation, only PS-Triton mixed micelles were effective in supporting PKC activity. Also, the minimum PS composition (4 mol % in Triton) required to induce significant histone-PS interaction coincided with the minimum composition required for phosphorylation of histones. Moreover, the PS composition required for maximum activity varied with the histone concentration of the reaction. In contrast to histone, PLS interacted with phospholipid-Triton mixed micelles containing either PS, PI, or PG, and all these mixed micelles supported the phosphorylation of PLS. In fact, by selection of appropriate experimental conditions (e.g., concentration of substrate and phospholipid), any of the three mixed micelles could appear the most effective in supporting PKC activity. Phospholipid vesicles containing PS, PG, or PI were found to interact with both histone and PLS and to support the activity of PKC. Physical properties of the solution and conditions used for preparation of phospholipid vesicles had considerable influence on PKC activation. At high phospholipid concentrations, vesicles containing PS, PI, or PG supported the activity of PKC to essentially the same level, provided that the physical differences among the phospholipid vesicles were minimized.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comprehensive assessment of the hemostasis system in the participants of the Mars-500 experiment

Hemostasis has been studied in the course of long-term (520 days) isolation in hermetic chamber. Measured parameters included activated partial thromboplastin time (APTT), international normalized ratio (INR), thrombin time (ТT); concentrations of fibrinogen (FBG), plasminogen (PG), Willebrand factor (WF), tissue factor pathway inhibitor (TFPI), tissue plasminogen activator (TPA), and thrombomodulin (ТМ); activities of the coagulation cascade factors II, V, VII, X, VIII, IX, XI, and XII, antithrombin III (ATIII), protein С (PC), С1-inhibitor (С1), α₂-antiplasmin (АP), TPA and TFPI. The investigation revealed a diversity of changes in plasma FBG concentration, slower blood coagulation in the intrinsic pathway and in final stage, and a relative rise in the activities of ATIII and PC-inhibited factors. The remaining parameters exhibited different trends.     

重组线虫抗凝肽在毕赤酵母中的表达

目的：获得具有抗凝活性的重组线虫抗凝肽（NAP）蛋白, 为新型抗凝药物的开发奠定基础。方法：将pPIC3.5K-rNAP质粒转化毕赤酵母菌株GS115,筛选出阳性菌株后,用甲醇诱导表达。收集酵母培养上清,用SDS-PAGE和Western blot分析表达产物,通过测定PT(血浆凝血酶原时间)、INR（血浆凝血酶原国际标准化比率）和APTT（活化部分凝血活酶时间）来验证其生物学活性。结果：获得了稳定表达NAP的重组酵母菌株。重组NAP被分泌表达,其分子量比预计分子量（8.7KD）稍大,约为10KD左右,可能与糖基化有关。体外活性检测证实其有较强的抗凝活性。结论：在毕赤酵母中成功表达了具有生物学活性的重组线虫抗凝肽,可用于新型抗凝药物的开发。     

Expression of recombinant rabbit tissue factor in Pichia pastoris,and its application in a prothrombin time reagent

Tissue factor (TF), or thromboplastin, is a cell membrane-associated glycoprotein composed, in full length, of cytoplasmic, transmembrane, and extracellular domains. It functions as a cofactor in a complex with factor VII (FVII), generating activated factor VII (FVIIa) and initiating blood coagulation. The prothrombin time (PT) assay uses TF as the in vitro activator of coagulation under defined conditions, and it is primarily used to diagnose and manage the extrinsic-pathway factor defficiencies. To overcome the limitations of natural-source TF, we have expressed the mature full-length recombinant rabbit TF (rRTF) protein in Pichia pastoris. Isolation, by purification by immobilized metal-affinity chromatography, of full-length rRTF was facilitated by engineering a (His)(6) tail on its C-terminus, which maximizes the selection of rRTF with intact transmembrane and cytoplasmic domains, critical for proper activity. A PT reagent that incorporates this purified rRTF has performance characteristics similar to those of PT reagents made with natural TF as indicated in method comparison studies, and shows lot-to-lot consistency and reproducibility.     

Fibrinolytic and antithrombotic protease from Spirodela polyrhiza

A fibrinolytic protease was purified from a Chinese herb (Spirodela polyrhiza). The protease has a molecular mass of 145 kDa and 70 kDa in gel filtration and SDS-polyacrlamide gel electrophoresis (PAGE), respectively, implying it is a dimer. Its optimum pH was 4.5-5.0. The enzyme was stable below 42 degrees C and after lyophilization. The enzyme activity was inhibited significantly by leupeptin and aprotinin. The protease hydrolyzed not only fibrin but also fibrinogen, cleaving Aalpha and Bbeta without affecting the gamma chain of fibrinogen. It preferentially cleaved the peptide bond of Arg or Lys of synthetic substrates (P1 position). The enzyme had an anticoagulating activity measured with activated partial thromboplastin time (APTT), thrombin time (TT), and prothrombin time (PT) tests. It delayed APTT, TT, and PT two times at the concentration of 36, 39, and 128 nM, respectively and this was drastically reduced after heat treatment.     

玉米芯木聚糖硫酸酯抗凝血活性及其机制的研究

采用活化部分凝血活酶时间(APTT)、凝血酶时间(TT)和凝血酶原时间(PT)检测了玉米芯木聚糖硫酸酯(wisX-SB)的抗凝活性,结果表明wisX-SB能明显延长APTT和TT,而不影响PT,提示wisX-SB是通过内源性和/或共同途径发挥抗凝血作用的。采用发色底物法及纤维蛋白原转化实验分别考察了wisX-SB对凝血酶及纤维蛋白原的作用,结果提示wisX-SB的抗凝机制包括:直接抑制纤维蛋白原的转化;通过增强抗凝血酶III(AT-III)的活性,抑制凝血酶活性,从而达到抗凝目的。较低浓度时以前者为主,较高浓度下两者皆起作用。     

Multifunctional specificity of the protein C/activated protein C Gla domain

Activated protein C (APC) has potent anticoagulant and anti-inflammatory properties that are mediated in part by its interactions with its cofactor protein S and the endothelial cell protein C receptor (EPCR). The protein C/APC Gla domain is implicated in both interactions. We sought to identify how the protein C Gla domain enables specific protein-protein interactions in addition to its conserved role in phospholipid binding. The human prothrombin Gla domain, which cannot bind EPCR or support protein S cofactor activity, has 22/45 residues that are not shared with the human protein C Gla domain. We hypothesized that the unique protein C/APC Gla domain residues were responsible for mediating the specific interactions. To assess this, we generated 13 recombinant protein C/APC variants incorporating the prothrombin residue substitutions. Despite anticoagulant activity similar to wild-type APC in the absence of protein S, APC variants APC(PT33-39) (N33S/V34S/D35T/D36A/L38D/A39V) and APC(PT36/38/39) (D36A/L38D/A39V) were not stimulated by protein S, whereas APC(PT35/36) (D35T/D36A) exhibited reduced protein S sensitivity. Moreover, PC(PT8/10) (L8V/H10K) displayed negligible EPCR affinity, despite normal binding to anionic phospholipid vesicles and factor Va proteolysis in the presence and absence of protein S. A single residue variant, PC(PT8), also failed to bind EPCR. Factor VIIa, which also possesses Leu-8, bound soluble EPCR with similar affinity to wild-type protein C, collectively confirming Leu-8 as the critical residue for EPCR recognition. These results reveal the specific Gla domain residues responsible for mediating protein C/APC molecular recognition with both its cofactor and receptor and further illustrate the multifunctional potential of Gla domains.     

Preparation and anticoagulant activity of N-succinyl chitosan sulfates

In order to develop a promising substitute for heparin, N-succinyl chitosan (NSC) was chemically modified by sulfating agent N(SO(3)Na)(3), which were synthesized with sodium bisulfite and sodium nitrite in aqueous solution. The N-succinyl chitosan sulfates (NSCS) products were characterized by infrared spectroscopy (FT-IR) and (13)C NMR. The degree of substitution (DS) of NSCS depended on the ratio of sulfating agent to N-succinyl chitosan, reaction temperature, reaction time and pH of sulfation agent. N-succinyl chitosan sulfates with DS of 1.97 were obtained under optimal conditions. The in vitro coagulation assay of NSCS was determined by activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) assays. The results showed that NSCS obviously prolonged APTT. The anticoagulant activity strongly depended on DS, molecular weight (M(w)) and concentration of NSCS. The anticoagulant activity of NSCS promoted with the increase of DS and concentration, and NSCS exhibited the best anticoagulant activity with the M(w) of 1.37×10(4).     

Prothrombin activation on membranes with anionic lipids containing phosphate, sulfate, and/or carboxyl groups

Factor Xa catalyzed prothrombin activation is strongly stimulated by the presence of negatively charged membranes plus calcium ions. Here we report experiments in which we determined the prothrombin-converting activity of phosphatidylcholine (PC) membranes that contain varying amounts of different anionic lipids, viz., phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylmethanol (MePA), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidyl-beta-lactate (PLac), sulfatides (SF), sodium dodecyl sulfate (SDS), and oleic acid. All anionic lipids tested were able to accelerate factor Xa catalyzed prothrombin activation, in both the absence and presence of the protein cofactor Va. This shows that the prothrombin-converting activity of negatively charged membranes is not strictly dependent on the presence of a phosphate group but that lipids which contain a carboxyl or sulfate moiety are also able to promote the formation of a functionally active prothrombinase complex. In the absence of factor Va, the prothrombin-converting activity of membranes with MePA, PG, PE, PLac, SF, or SDS was strongly inhibited at high ionic strength, while the activity of PS- and PA-containing membranes was hardly affected by ionic strength variation. This suggests that in the case of the ionic strength sensitive lipids electrostatic forces play an important role in the formation of the membrane-bound prothrombinase complex. For PS and to a lesser extent for PA we propose that the formation of a coordinated complex (chelate complex) with Ca2+ as central ion and ligands provided by the gamma-carboxyglutamic acid residues of prothrombin and factor Xa and the polar head group of phospholipids is the major driving force in protein-membrane association. Our data indicate that the anionic lipids used in this study can be useful tools for further investigation of the molecular interactions that play a role in the assembly of a membrane-bound prothrombinase complex. Membranes that were solely composed of PC can also considerably enhance prothrombin activation in the presence of factor Va. This activity of PC is only observed on membranes which are composed of PC that contains unsaturated hydrocarbon side chains. Membranes prepared from phosphocholine-containing lipids with saturated hydrocarbon side chains such as dimyristoyl-PC, dipalmitoyl-PC, distearoyl-PC, and dioctadecylglycerophosphocholine hardly accelerated prothrombin activation.(ABSTRACT TRUNCATED AT 250 WORDS)     

(Na+ + K+)-ATPase is a target for phosphoinositide 3-kinase/protein kinase B and protein kinase C pathways triggered by albumin

In recent decades, evidence has confirmed the crucial role of albumin in the progression of renal disease. However, the possible role of signaling pathways triggered by physiologic concentrations of albumin in the modulation of proximal tubule (PT) sodium reabsorption has not been considered. In the present work, we have shown that a physiologic concentration of albumin increases the expression of the α1 subunit of (Na(+) + K(+))-ATPase in LLC-PK1 cells leading to an increase in enzyme activity. This process involves the sequential activation of PI3K/protein kinase B and protein kinase C pathways promoting inhibition of protein kinase A. This integrative network is inhibited when albumin concentration is increased, similar to renal disease, leading to a decrease in the α1 subunit of (Na(+) + K(+))-ATPase expression. Together, the results indicate that variation in albumin concentration in PT cells has an important effect on PT sodium reabsorption and, consequently, on renal sodium excretion.     

Anticoagulant properties of a sulfated galactan preparation from a marine green alga, Codium cylindricum

An anticoagulant was isolated from a marine green alga, Codium cylindricum. The anticoagulant was composed mainly of galactose with a small amount of glucose, and was highly sulfated (13.1% as SO3Na). The anticoagulant properties of the purified anticoagulant were compared with that of heparin by assays of activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) using normal human plasma. The anticoagulant showed similar activities with heparin, however, weaker than heparin. On the other hand, the anticoagulant did not affect PT even at the concentration at which APTT and TT were strongly prolonged. The anticoagulant did not potentiate antithrombin III (AT III) and heparin cofactor II (HC II), thus the anticoagulant mechanism would be different from that of other anticoagulants isolated so far from the genus Codium.     

An anticoagulant proteoglycan from the marine green alga, Codium pugniformis

An anticoagulant isolated from the marine green alga Codium pugniformis was composed mainly of glucose with minor amounts of arabinose and galactose. It was highly sulfated (326 μg mg^-1 polysaccharide) and contained protein(52 μg mg^-1 polysaccharide) and was thus a proteoglycan. The anticoagulant properties of the purified proteoglycan were compared with those of heparin by studying the activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time(TT) using normal human plasma. The proteoglycan showed similar activities to heparin, but was weaker than heparin. On the other hand, the proteoglycan did not affect PT even at the concentration at which APTT and TT were prolonged. The anticoagulation mechanism of this proteoglycan was due to the direct inhibition of thrombin and the potentiation of antithrombin III. This revised version was published online in August 2006 with corrections to the Cover Date.     

Experimental study on anticoagulant and antiplatelet aggregation activity of a chemically sulfated marine polysaccharide YCP

A polysaccharide YCP was prepared from a marine filamentous fungus Keissleriella sp. YS4108, which exhibited as a molecular weight (Mw) of 2.4x10(3) kDa and its three sulfated derivatives (YCP-SL, YCP-SM and YCP-SH) were synthesized, the degree of substitution (DS) of which were determined to be 0.13, 0.99 and 1.3, with the average molecular weight 0.64x10(3), 0.57x10(3) and 0.45x10(3) kDa, respectively. Anticoagulant activity and antiplatelet aggregation activity of these sulfated derivates were evaluated by activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT) and platelet aggregation assay. The results showed that YCP sulfates significantly prolonged APTT, TT and PT. The derivates showed no effects on thrombin in the presence or in the absence of antithrombin III (AT III) or heparin cofactor II (HC II), while the derivates effectively inhibited factor Xa in the presence of AT III. At the same time, YCP-SH also possessed potent antiplatelet aggregation activity in vitro compared with aspirin. YCP sulfates specifically interfered with different stages of the coagulation cascade, and the anticoagulant activity improved with the increasing DS and decreased Mw.     

蝮蛇毒蛋白C激活物对凝血系统的影响

目的研究蝮蛇毒纯化蛋白C激活物(PCA)的抗凝机制。方法测定PCA对正常人混浆KPTT、PT、TT的影响,对血液凝血活酶生成试验(BTGT)及PA二期法的影响以及对白兔的KPTT和Fgn的影响。结果PCA在最终浓度为0.025mg/L时对正常人混浆KPTT可明显延长,但PT和TT不受影响;当它的浓度增加到50mg/L,PT也可延长,但TT依然无明显变化;最终浓度为0.0125mg/L时,血液凝血活酶生成明显受抑制;当它的浓度增加到2.5mg/L,凝血酶生成显著减少,但抗凝血酶时间始终无明显变化;PCA可明显延长白兔的KPTT。结论PCA在低浓度时首先抑制凝血系统第一阶段内凝途径,在高浓度时也妨碍外凝途径或共同途径,BTGT和PA二期法比KPTT和PT敏感;PCA具有体内抗凝作用。     

Comparison of warfarin therapy clinical outcomes following implementation of an automated mobile phone-based critical laboratory value text alert system

Background

Computerized alert and reminder systems have been widely accepted and applied to various patient care settings, with increasing numbers of clinical laboratories communicating critical laboratory test values to professionals via either manual notification or automated alerting systems/computerized reminders. Warfarin, an oral anticoagulant, exhibits narrow therapeutic range between treatment response and adverse events. It requires close monitoring of prothrombin time (PT)/international normalized ratio (INR) to ensure patient safety. This study was aimed to evaluate clinical outcomes of patients on warfarin therapy following implementation of a Personal Handy-phone System-based (PHS) alert system capable of generating and delivering text messages to communicate critical PT/INR laboratory results to practitioners' mobile phones in a large tertiary teaching hospital.

Methods

A retrospective analysis was performed comparing patient clinical outcomes and physician prescribing behavior following conversion from a manual laboratory result alert system to an automated system. Clinical outcomes and practitioner responses to both alert systems were compared. Complications to warfarin therapy, warfarin utilization, and PT/INR results were evaluated for both systems, as well as clinician time to read alert messages, time to warfarin therapy modification, and monitoring frequency.

Results

No significant differences were detected in major hemorrhage and thromboembolism, warfarin prescribing patterns, PT/INR results, warfarin therapy modification, or monitoring frequency following implementation of the PHS text alert system. In both study periods, approximately 80% of critical results led to warfarin discontinuation or dose reduction. Senior physicians' follow-up response time to critical results was significantly decreased in the PHS alert study period (46.3% responded within 1 day) compared to the manual notification study period (24.7%; P = 0.015). No difference in follow-up response time was detected for junior physicians.

Conclusions

Implementation of an automated PHS-based text alert system did not adversely impact clinical or safety outcomes of patients on warfarin therapy. Approximately 80% immediate recognition of text alerts was achieved. The potential benefits of an automated PHS alert for senior physicians were demonstrated.

     

Interfacial adsorption of simple lipid mixtures combined with hydrophobic surfactant protein from pig lung.

Hydrophobic pulmonary surfactant protein enriched in SP-C has been mixed in amounts up to 10% by weight with various phospholipids. The lipids used were dipalmitoyl phosphatidylcholine (DPPC), or DPPC plus unsaturated phosphatidylglycerol (PG), or phosphatidylinositol (PI) in molar ratios of 9:1 and 7:3. The protein enhanced the rate and extent of adsorption of each lipid preparation into the air-water interface, and its respreading after compression on a surface balance. Maximum surface pressures attained on compression of monolayers of mixtures of lipids were slightly higher in the presence of protein. The effects on rate and extent of adsorption were proportional to the amount of protein present. Mixtures containing 30 mol% PG or PI adsorbed more readily into the interface than those containing 10% acidic lipid or DPPC alone. Mixtures containing 30% PI were slightly more rapidly adsorbed than those containing 30% PG. The results suggest that mixtures of DPPC with either acidic lipid in the presence of surfactant protein could be effective in artificial surfactants.