Variability in heparin sensitivity of APTT reagents

Activated partial thromboplastin time (APTT) is the most widely used coagulation test for monitoring heparin therapy. This study quantitates the differences in heparin sensitivity of seven commercially available APTT reagents, using plasma samples obtained from 20 subjects. The reagents studied were Actin, Actin FS, Automated APTT, Cephotest, Coagachek KAPTT, Platelin plus activator, and Activated Thrombofax. The relationship between plasma heparin and APTT was established for all reagents in each subject. For each reagent studied there was a marked intersubject variability in heparin sensitivity. There was also a marked difference in heparin sensitivity among the different reagents. The average plasma heparin activities required to double baseline APTT values ranged from 0.19 +/- 0.04 (mean +/- SD) unit/mL for the most sensitive reagents (Platelin and Actin FS) to 0.43 +/- 0.12 unit/mL for the least sensitive reagent (Actin). It is concluded that reagent variability may significantly contribute to overdosage and under dosage of heparin in the individual patient. These results stress that a standard APTT reagent be developed.     

Wide variability in the sensitivity of APTT reagents for monitoring of heparin dosage.

AIM: To assess the sensitivity of activated partial thromboplastin time (APTT) reagents for monitoring heparin dosage using data from the UK National External Quality Assessment Scheme (NEQAS) for blood coagulation. METHODS: Data were reviewed from four surveys using samples prepared by addition of heparin to normal plasma in vitro and from two surveys in which samples were prepared using plasma from patients receiving heparin therapy (ex vivo samples). RESULTS: For both in vitro and ex vivo samples, notable differences between APTT reagents with respect to heparin sensitivity were noted. This indicates that a uniform therapeutic range of 1.5-2.5 calculated by the APTT ratio may not be appropriate for all reagents. Reagent sensitivity in ex vivo samples was substantially different to that in in vitro samples. CONCLUSIONS: The results of this large series of laboratories clearly indicate that reagent specific therapeutic ranges may be necessary, and that samples prepared by the addition of heparin to normal plasma in vitro can be misleading and should not be used.     

A nonelutable low-molecular weight heparin stent coating for improved thromboresistance

Low-molecular weight heparin (LMWH) has been widely used as a systemic anticoagulant during percutaneous coronary intervention. In this study, LMWH was covalently immobilized to the surface of a cobalt chromium reservoir-based sirolimus-eluting stent to create a nonelutable nanoscale coating for enhanced thromboresistance. Toludine-blue stained stents revealed uniform heparin coverage on all surfaces of the stent. Scanning electron microscopy of stent strut cross-sections showed identical coating thickness on all sides; while the thickness was determined to be 320 nm by a focus-ion beam system. Secondary ion mass spectrometry showed constant concentrations of O, N, and S atoms throughout the depth of the surface, confirming the uniformity of the heparin coating. The nonelutable nature of the coating was confirmed in a modified Factor Xa inhibition assay which showed the stent had an equivalent of 3-5 heparin units/cm(2), while no elutable heparin was detected in wash solutions. The antithrombin binding capacity of the immobilized heparin was determined to be 60-80 pmol/cm(2) in an antithrombin uptake assay. The enhanced thromboresistance of the heparin coating was demonstrated in an in-vitro bovine blood flow loop which showed minimal visual thrombus accumulation and 95% reduction in platelet deposition compared to uncoated control stents. Drug-eluting stents with such nonelutable LMWH coating would represent a significant advance in the treatment of patients with complex lesions who are at increased risk of developing stent thrombosis.     

Comparison of the sensitivity of commercial APTT reagents in the detection of mild coagulopathies

This study was undertaken to evaluate the precision and sensitivity of three different commercial APTT reagents containing the activators kaolin, micronized silica, or ellagic acid. These reagents varied greatly in their ability to detect mild coagulopathies. The ellagic acid reagent was able to detect the mildest deficiencies for the most common coagulopathies. This reagent was sensitive to 50% levels of Factor VIIIC, whereas the APTT with the kaolin reagent was not prolonged until levels of 35% or less were attained. The micronized silica reagent was the least sensitive to Factor IX deficiency, detecting levels of 12% or less. Precision was similar for all reagents when tested with normal and slightly abnormal plasmas. Since methods and instrumentation vary, each laboratory should evaluate their APTT reagent to determine its precision and sensitivity.     

Effect of clot-detection methods and reagents on activated partial thromboplastin time (APTT). Implications in heparin monitoring by APTT

Two automatic coagulometers, ACL 810 (Instrumentation Laboratory), a laser-nephelometric centrifugal analyzer, and KoaguLab 40 A (Ortho Diagnostics), an optical automatic coagulometer, were compared with the manual tilt-tube method for the performance of activated partial thromboplastin time (APTT). Seven commercial APTT reagents were used for duplicate determinations in 30 normal controls, 26 patients with liver disease, and 33 patients on full-dose heparin treatment. Clotting times were longer with the manual method than with ACL 810 and, to a lesser extent, with KoaguLab 40 A. Average imprecision of duplicate determinations (coefficient of variation [CV]) was less with ACL 810 (less than 1.5%) than with KoaguLab 40 A (2.9%) and the manual method (2.4%). Differences in slope of the regression curves of clotting times obtained with the coagulometers over the tilt-tube method were observed with all the reagents tested (P less than 0.01). Transformation of clotting times of controls, patients with liver disease, and patients on heparin therapy to APTT ratios did not eliminate the bias resulting from the different reagents (P less than 0.001) and clot-detection methods (P less than 0.001); in controls, significant (P less than 0.001) reagent-method interaction was also observed. The in vitro heparin sensitivity differed with the APTT reagents evaluated and was influenced by the clot-detection method used. Transformation of APTT ratios of anticoagulated patients to apparent plasma heparin levels--as derived from in vitro dose-response curves--effectively eliminated the bias resulting from the different clot-detection methods but had no effect on the bias resulting from the different APTT reagents. In vitro heparin activity curves thus have little, if any, relevance for the ex vivo monitoring of heparin treatment.     

Cost effectiveness of low-molecular weight heparin versus warfarin following hip replacement surgery.

Little information is available on the efficacy of low-molecular-weight heparin (enoxaparin) versus warfarin for treatment of deep vein thrombosis and pulmonary embolism following hip replacement surgery. Still less is known of the comparative cost effectiveness of these two therapies. A retrospective study was done on 56 patients who underwent elective hip surgery at an urban medical center between 1991 and 1996. All patients received enoxaparin or warfarin for purposes of thromboprophylaxis. An analysis of medication cost, therapy, laboratory monitoring, and bleeding events of the two antithrombolytic agents was undertaken. Total savings with enoxaparin averaged $1253 per patient, or $137,886 over the study period. The incidence of deep vein thrombosis or pulmonary embolism was 0% with enoxaparin and 3% with warfarin. These data indicate that enoxaparin is a more cost-effective and efficacious regimen for thromboprophylaxis following hip replacement surgery than warfarin.     

低分子量肝素对滋养层细胞MMP-2、TIMP-2表达及侵袭力的影响

目的:探讨低分子量肝素对体外培养人早孕绒毛滋养层细胞MMP-2、TIMP-2表达及侵袭力的影响。方法:将经胰蛋白酶/DNA酶Ⅰ联合消化,通过Percoll细胞分离液纯化得到的绒毛滋养层细胞进行体外培养。采用不同浓度低分子量肝素干预培养24h后,ELISA法测定细胞上清液中MMP-2、TIMP-2的浓度;采用Tr-answell小室观察滋养层细胞的侵袭能力。结果:不同浓度的低分子量肝素(1.0×102IU/L,1.0×103IU/L,1.0×104IU/L)干预人早孕绒毛滋养层细胞后,与对照组相比,MMP-2的表达上调,滋养层细胞侵袭力增强,在1.0×103IU/L时MMP-2表达最高,滋养层细胞侵袭力最强(P0.05)。TIMP-2的表达随着低分子量肝素浓度的增加而逐渐下降,与对照组比较,在1.0×103IU/L、1.0×104IU/L组明显降低(P0.05),但1.0×103IU/L组与1.0×104IU/L组之间TIMP-2的表达无差异(P0.05)。结论:低分子量肝素可能直接通过影响绒毛滋养层细胞MMP-2、TIMP-2的表达进而影响绒毛滋养层细胞的侵袭能力。     

A randomized controlled trial of a low-molecular-weight heparin (enoxaparin) to prevent deep-vein thrombosis in patients undergoing elective hip surgery

There is experimental evidence that low-molecular-weight fractions of heparin are as effective as the standard form but cause less bleeding. We therefore performed a double-blind, randomized trial comparing PK10169 low-molecular-weight heparin with placebo for the prevention of venous thrombosis in patients undergoing elective hip surgery. Prophylactic treatment with a fixed dose was begun postoperatively and continued for 14 days. Fifty patients in each treatment group underwent surveillance with [125I]fibrinogen leg scanning and impedance plethysmography. In the first 24 patients, venography was performed only if either surveillance test was positive. Because the rate of venous thrombosis detected in those patients was unexpectedly low, venography was requested in the remaining 76 patients, even if the screening tests were negative. In this latter group, venous thrombosis occurred in 4 patients (10.8 percent) given PK10169 heparin and 20 patients (51.3 percent) given placebo (P = 0.0002); the corresponding rates for proximal-vein thrombosis were 5.4 percent and 23.1 percent, respectively (P = 0.029). In the entire group of 100 patients, venous thrombosis occurred in 12 percent of those given PK10169 heparin and 42 percent of those given placebo (P = 0.0007), and the corresponding rates for proximalvein thrombi were 4 percent and 20 percent, respectively (P = 0.014). The observed hemorrhagic rate was 4 percent in each treatment group. We conclude that prophylaxis with fixed-dose PK10169 heparin is effective and safe for patients undergoing elective hip replacement.     

Evidence for low-molecular weight antibodies in the serum of a urodele amphibian, Ambystoma mexicanum

Adult axolotls (A. mexicanum) were hyperimmunized with the haptenic determinant, azobenzene-arsonate (ARS). Specific antibodies were isolated from their serum by immune-affinity chromatography on immobilized ARS columns. Analysis of the specific ARS-binding molecules by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) indicated that these animals were capable of producing both high molecular-weight (presumably IgM) and low molecular-weight antibodies to the ARS hapten. The low molecular-weight anti-ARS antibody produced by one inbred colony of axolotls did not show any restricted heterogeneity, as assessed by isoelectric focusing. Our results suggest that regulatory events, not the absence of genetic information, may be responsible for the apparent lack of Ig isotype diversity in this species.     

Regulation of interleukin-6 fetoplacental levels could be involved in the protective effect of low-molecular weight heparin treatment on murine spontaneous abortion

PROBLEM: CBA/J x DBA/2 abortion rate could be the consequence of a deficient local production of T helper (Th2) cytokines, which cause fetal wastage via fgl2 prothrombinase. Heparin reduces significantly the abortion rate in mice and recurrent spontaneous abortion (RSA) patients. We proposed to determine the effect of enoxaparin on the levels of local interleukin (IL)-6 during murine pregnancy. METHOD OF STUDY: Recombinant human IL-6 (rhIL-6) or enoxaparin were inoculated in CBA/J x DBA/2 pregnant mice on days 6.5-12.5. IL-6 levels in sera as well as in culture supernatants of day 9.5 fetoplacental units of CBA/J x BALB/c control mice or CBA/J x DBA/2 abortion combination were determined by enzyme-linked immunosorbent assay (ELISA) test. RESULTS: CBA/J x DBA/2 fetoplacental units secreted significantly lower levels of IL-6 with regard to CBA/J x BALB/c normal units. rhIL-6h and enoxaparin treatments decreased the resorption rate and regulated IL-6 fetoplacental levels. CONCLUSION: This study suggests that regulation of IL-6 fetoplacental levels could be involved in heparin-mediated anticoagulation protection against abortion.     

Further characterization of a low-molecular weight allergen fragment isolated from the green pea.

A low molecular weight allergen fragment present in the pea dialysate fraction was purified by ion-exchange chromatography and gel filtration. The highly purified allergen fragment inhibits both antigen-indiced passive cutaneous anaphlaxix reactions in guinea-pigs sensitized with rabbit anti-pea extract sera and Prausnitz-Küstner reactions in non-allergic volunteers sensitized with the sera of patients sensitive to green peas. Preliminary analysis of the purified allergen fragment indicates that it is a glycoprotein with a molecular weight of 1800 +/- 250.     

Passage of commercial heparin and a low molecular weight fragment of heparin, CY 222, across the placenta of pregnant rabbits

Because of a further clinical use of low-molecular weight heparin fraction in prevention or treatment of venous thrombo-embolism, it was necessary to establish whether they cross the placenta. The studies were performed in pregnant rabbits. High doses of commercial unfractionated heparin (1 000 and 1 600 anti-Xa units per kg of body-weight) and high doses of a low molecular weight heparin fraction (1 000; 8 000 and 16 000 anti-Xa units per kg of body weight) were injected to animals at the end of gestation (term: 30 days). The placenta crossing was studied by drawing blood samples from the mother and foetus for assays of heparin blood level. There is no detectable levels of heparin in the foetus at any time after injection of commercial heparin at the dose of 1 000 anti-Xa units per kg; meanwhile heparin blood level is very low at the dose of 16 000 anti-Xa units/kg. The low molecular weight heparin fraction do not cross the placenta at the dose of 1 000 anti-Xa units/kg. However for higher doses (8 000 and 16 000 anti-Xa/kg) this heparin fraction gives a foetal heparin blood level upper than the one given by commercial heparin.     

The sensitivity of different coagulation reagents to the presence of lupus anticoagulants

The sensitivity and responsiveness of seven activated partial thromboplastin time reagents to the presence of lupus anticoagulants (LAs) was evaluated with a panel of 50 well-characterized LA plasmas. The results document that some reagents are clearly less responsive and sensitive to LAs; however, there is considerable variability between individual LA samples in these features. In addition, with 16% of these samples, immediate mixing studies showed correction of the activated partial thromboplastin time to the normal range with a 1:1 mixture of normal and LA plasma and 8% to 10% showed either a normal platelet neutralization procedure or a normal tissue thromboplastin inhibition procedure. Together, these findings provide further evidence of the laboratory heterogeneity of these inhibitors. The effect of this variability on the diagnosis of these inhibitors is discussed.     

Investigation of the effects of heparin and low molecular weight heparin on E-cadherin and laminin expression in rat pregnancy by immunohistochemistry

BACKGROUND: Heparin and low molecular weight heparin (LMWH) are used widely to improve the pregnancy outcome in women with thrombophilia, miscarriage, recurrent miscarriage and fetal death. This study was designed to investigate the effects of heparin and LMWHs, enoxaparin and tinzaparin, on E-cadherin and laminin expression in placental and decidual tissues in rat pregnancy. METHODS: Wistar albino female rats (n = 48) were randomly assigned to four study groups (normal saline, heparin, enoxaparin and tinzaparin) in the preconceptional period. Tissue sections of placenta and decidua were immunohistochemically examined for the expression of E-cadherin and laminin. RESULTS: E-cadherin placental staining score of heparin group was significantly lower and E-cadherin decidual staining score of heparin and enoxaparin groups were significantly lower than control group. There were no significant differences in placental and decidual laminin staining scores among the study groups. CONCLUSIONS: Heparin and enoxaparin can reduce E-cadherin expression but not laminin expression in rat pregnancy. They might modulate trophoblast invasion. We suggest that this is the possible underlying mechanism involving in improvement of trophoblast invasion by the use of heparin and LMWH in patients with the history of miscarriage.     

A comparison of seven methods to analyze heparin in biomaterials: quantification, location, and anticoagulant activity

Glycosaminoglycans, like heparin, are frequently incorporated in biomaterials because of their capacity to bind and store growth factors and because of their hydrating properties. Heparin is also often used in biomaterials for its anticoagulant activity. Analysis of biomaterial-bound heparin is challenging because most assays are based on heparin in solution. In this study, seven different methods were probed to analyze heparin covalently attached to collagen scaffolds. For each method, the basic mechanism and the advantages and disadvantages are given. An analysis by the factor Xa assay and the Farndale assay clearly indicated that the amount of immobilized heparin cannot be determined correctly when the scaffold is intact. Scaffolds had to be proteolytically digested or acid treated to obtain reliable measurements. Methods used to quantify the amount of bound heparin included a hexosamine assay, an uronic acid assay, a Farndale assay, agarose gel electrophoresis, and immuno-dot blot analysis. Location and semiquantification of heparin were accomplished by immunofluorescence. Although all assays had their advantages and disadvantages, the hexosamine assay turned out to be the most robust and is recommended as the preferred assay to quantify the amount of heparin bound to scaffolds. It is applicable to all scaffolds that are acid hydrolyzable. This study may allow researchers in the field to select the most appropriate method to analyze glycosaminoglycans in biomaterials.     

Low molecular weight protamine: a potent but nontoxic antagonist to heparin/low molecular weight protamine

To avoid bleeding complications, protamine is routinely used after cardiovascular surgery to neutralize the anticoagulant function of heparin. However, its clinical use is associated with adverse and sometimes fatal reactions. Based on literature review of the mechanism of heparin neutralization and protamine induced immunologic toxicity, we propose the following hypothesis: If a chain shortened low molecular weight protamine (LMWP) containing the heparin neutralizing domain could be derived from native protamine, it could be a potent and yet nontoxic heparin antagonist. In this study, we present results to validate this hypothesis. LMWP fragments containing an intact arginine sequence and an average molecular weight of approximately 1,100 daltons were successfully prepared by enzymatic digestion of protamine with thermolysin. In vitro studies show that such LMWP fragments completely neutralized the anticoagulant functions of heparin and LMWH, based on the anti-Xa chromogenic and aPTT clotting time assays. In vivo results reveal that although injection of protamine to mice led to obvious production of anti-protamine antibodies, injection of LMWP did not elicit any detectable immunogenic responses. In addition, these LMWP fragments exhibited a markedly reduced antigenicity and cross-reactivity toward the mice anti-protamine antibodies.     

Effect of unfractionated heparin and a low molecular weight heparin fragment on the DNA synthesis of human thymocytes

The effect of unfractionated heparin and a low molecular weight heparin fragment was tested on the DNA synthesis of human thymocytes cultured for 5 and 48 h. At 5 h, in the absence of serum, there was a stimulation with the highest concentration of unfractionated heparin, 11.1 mg/ml. At 48 h, there was an inhibition with the highest concentration of both the unfractionated heparin and the heparin fragment. In the presence of serum unfractionated heparin in the highest concentration gave an inhibitory effect at 48 h. With 0.01-1.0 mg/ml of unfractionated heparin, a stimulating effect was obtained instead.     

Stimulating effect of unfractionated heparin and a low molecular weight heparin fragment on the DNA synthesis response of human thymocytes to mercuric chloride

The effect of unfractionated heparin and a low molecular weight heparin fragment was tested on the DNA synthesis response of human thymocytes to mercuric chloride. In a concentration of 0.01-0.11 mg/ml, unfractionated heparin somewhat stimulated this response, while the heparin fragment at 11.1 mg/ml gave an evident stimulation of the response and a lower stimulation degree at 1.1 mg/ml.