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PubMed2004

Role of lipid in sulfite-dependent propofol dimerization.

BACKGROUND: During long-term intravenous infusions, sulfite in sulfite-containing propofol emulsions can cause the peroxidation of lipid and dimerization of propofol. This study evaluated the role of lipid in sulfite-dependent propofol dimerization by determining the effects of individual fatty acids in soybean oil emulsion and peroxidized lipids in a model system. METHODS: Individual fatty acids, stearic (18:0), oleic (18:1), linoleic (18:2), linolenic (18:3), and arachidonic (20:4), were added to sulfite-containing propofol emulsion and incubated for 90 min at 37 degrees C. Model systems containing soybean oil (100 microl), water (900 microl), propofol (10 mg/ml), and sulfite (0.25 mg/ml) composed of oils with different peroxide values were allowed to react for 60 min at room temperature. After the reactions, propofol dimer and propofol dimer quinone were analyzed by reversed-phase high-pressure liquid chromatography. RESULTS: Propofol did not dimerize when added to aqueous sulfite unless soybean oil was also included. The addition of the polyunsaturated fatty acids (linoleic, linolenic, arachidonic) to sulfite-containing propofol emulsion resulted in large increases of propofol dimerization compared with stearic or oleic acid. Using biphasic mixtures of soybean oil and aqueous sulfite, propofol dimerization increased with increasing peroxide content of the oil. In propofol emulsion, lipoxidase and ferrous iron in the absence of sulfite also caused the dimerization of propofol. CONCLUSIONS: These results show that lipid can play a significant role in sulfite-dependent propofol dimerization. The relation of dimerization to polyunsaturated fatty acid and soybean oil peroxide content suggests that sulfite reacts with unsaturated lipid or peroxide-modified lipid to facilitate propofol dimerization.

Dimerization↗
PubMed1992

Flavin-sensitized photochemically induced dynamic nuclear polarization detection of pyrimidine dimer radicals.

A photochemically induced dynamic nuclear polarization (photo-CIDNP) study of carboxymethyllumiflavin-sensitized splitting of pyrimidine dimers has been carried out. In aqueous solution at high pH, an emission signal (delta 3.9 ppm) was observed from the dimer C(6)- and C(6')-protons of an N(1), N(1')-trimethylene-bridged thymine dimer (1). The dimer photo-CIDNP signal was seen only above pD 11.6 and was most intense at pD 12.9. Also observed were weak enhanced absorption signals from the product of splitting, trimethylenebis(thymine) (delta 1.7 and 7.2 ppm). In contrast, cis, syn-thymine dimer (3) gave no photo-CIDNP signals from the dimer. An enhanced absorption at 1.8 ppm, however, due to the product of splitting (thymine) was observed. It was found that dimer 1 and, to a lesser extent, dimer 3 quenched flavin fluorescence. An N(3),N(3')-dimethylated derivative of 1, however, failed to quench flavin fluorescence. Comparison of the pD profile of the dimer photo-CIDNP signal to the pKa values for thymidine dimer suggested that principally the dideprotonated dimer undergoes electron abstraction by the excited flavin.

Flavins↗
PubMed1995

Photo-CIDNP study of pyrimidine dimer splitting. I: Reactions involving pyrimidine radical cation intermediates.

The light-induced splitting of pyrimidine dimers was studied using the electron acceptor anthraquinone-2-sulfonate (AQS) as a photosensitizer. To this end, photochemically induced dynamic nuclear polarization (photo-CIDNP) experiments were performed on a series of pyrimidine monomers and dimers. The CIDNP spectra demonstrate the existence of both the dimer radical cation, which is formed by electron transfer from the dimer to the photoexcited sensitizer AQS*, and its dissociation product, the monomer radical cation. In spectra of 1,1'-trimethylene bridged cis, syn pyrimidine dimers, polarization is observed that originates from a spin-sorting process in the dimer radical pair. This points to a relatively long lifetime of the dimer radical cation involved, which is presumably due to stabilization by the trimethylene bridge. Polarization originating from a dimer radical pair is detected in the spectrum of trans,anti (1,3-dimethyluracil) dimer as well. The spectra of the bridged pyrimidines also demonstrate the reversibility of the dissociation of dimer radical cation into monomer radical cation, which is concluded from the observation of polarization in the dimer as a result of spin sorting in the monomer radical pair.

Cations↗
PubMed2001

Role of stem B, loop B, and nucleotides next to the primer binding site and the kissing-loop domain in human immunodeficiency virus type 1 replication and genomic-RNA dimerization.

Stem-loop B is a 12-nucleotide [nt]-long completely conserved sequence postulated to form a 4-bp stem and a 4-nt internal loop under the kissing-loop hairpin (klh) (nt 248 to 270) of human immunodeficiency virus type 1 (HIV-1) genomic RNA. We investigated its role in viral replication, genomic RNA dimerization, and dimerization of partial HIV-1 RNA transcripts. The putative CUCG246-CGAG277 duplex was replaced by nine alternative complementary sequences, five likely to base pair only in short RNAs and four likely to base pair in long (approximately 500-nt) RNAs, as assessed by the algorithm mfold. Among the five former sequences, none preserved genome dimerization and all reduced viral replication by 98 to 99.9%. Among the four latter sequences, three (MB6, -9, and -10) preserved genome dimerization, one (MB7) did not significantly inhibit it, and two (MB9 and -10) preserved viral replication. We conclude that duplex formation by stem B nucleotides is necessary for viral infectivity and complete genome dimerization. Deleting the 5' or 3' side of loop B or of stem B had little impact on dimerization of partial RNA transcript and no impact on klh folding (and, for loop B mutations, on stem B folding), but each deletion inhibited genome dimerization almost as much as klh destruction. This suggests that loop B is required for complete genome dimerization and that loop B and stem B stimulate dimerization only in very long RNAs and/or in the presence of unidentified viral and cellular factors. Finally, we asked if nine deletions or nucleotide substitutions within nt 200 to 242 and/or nt 282 to 335 could influence genome dimerization. These mutations had intermediate inhibitory impacts consistent with their predicted influence on stem B, loop B, and klh formation. Two exceptions were Delta200-226 and Delta236-242 genomic RNAs, which dimerized relatively poorly despite having neutral or positive influences on stem B, loop B, and klh folding.

Animals↗
PubMed2002

Bipartite signal for genomic RNA dimerization in Moloney murine leukemia virus.

Retroviral virions each contain two identical genomic RNA strands that are stably but noncovalently joined in parallel near their 5' ends. For certain viruses, this dimerization has been shown to depend on a unique RNA stem-loop locus, called the dimer initiation site (DIS), that efficiently homodimerizes through a palindromic base sequence in its loop. Previous studies with Moloney murine leukemia virus (Mo-MuLV) identified two alternative DIS loci that can each independently support RNA dimerization in vitro but whose relative contributions are unknown. We now report that both of these loci contribute to the assembly of the Mo-MuLV dimer. Using targeted deletions, point mutagenesis, and antisense oligonucleotides, we found that each of the two stem-loops forms as predicted and contributes independently to dimerization in vitro through a mechanism involving autocomplementary interactions of its loop. Disruption of either DIS locus individually reduced both the yield and the thermal stability of the in vitro dimers, whereas disruption of both eliminated dimerization altogether. Similarly, the thermal stability of virion-derived dimers was impaired by deletion of both DIS elements, and point mutations in either element produced defects in viral replication that correlated with their effects on in vitro RNA dimerization. These findings support the view that in some retroviruses, dimer initiation and stability involve two or more closely linked DIS loci which together align the nascent dimer strands in parallel and in register.

Animals↗
PubMed2004

mRNA molecules containing murine leukemia virus packaging signals are encapsidated as dimers.

Prior work by others has shown that insertion of psi (i.e., leader) sequences from the Moloney murine leukemia virus (MLV) genome into the 3' untranslated region of a nonviral mRNA leads to the specific encapsidation of this RNA in MLV particles. We now report that these RNAs are, like genomic RNAs, encapsidated as dimers. These dimers have the same thermostability as MLV genomic RNA dimers; like them, these dimers are more stable if isolated from mature virions than from immature virions. We characterized encapsidated mRNAs containing deletions or truncations of MLV psi or with psi sequences from MLV-related acute transforming viruses. The results indicate that the dimeric linkage in genomic RNA can be completely attributed to the psi region of the genome. While this conclusion agrees with earlier electron microscopic studies on mature MLV dimers, it is the first evidence as to the site of the linkage in immature dimers for any retrovirus. Since the Psi(+) mRNA is not encapsidated as well as genomic RNA, it is only present in a minority of virions. The fact that it is nevertheless dimeric argues strongly that two of these molecules are packaged into particles together. We also found that the kissing loop is unnecessary for this coencapsidation or for the stability of mature dimers but makes a major contribution to the stability of immature dimers. Our results are consistent with the hypothesis that the packaging signal involves a dimeric structure in which the RNAs are joined by intermolecular interactions between GACG loops.

Animals↗
PubMed2001

Dimerization of receptor protein-tyrosine phosphatase alpha in living cells.

BACKGROUND: Dimerization is an important regulatory mechanism of single membrane-spanning receptors. For instance, activation of receptor protein-tyrosine kinases (RPTKs) involves dimerization. Structural, functional and biochemical studies suggested that the enzymatic counterparts of RPTKs, the receptor protein-tyrosine phosphatases (RPTPs), are inhibited by dimerization, but whether RPTPs actually dimerize in living cells remained to be determined. RESULTS: In order to assess RPTP dimerization, we have assayed Fluorescence Resonance Energy Transfer (FRET) between chimeric proteins of cyan- and yellow-emitting derivatives of green fluorescent protein, fused to RPTPalpha, using three different techniques: dual wavelength excitation, spectral imaging and fluorescence lifetime imaging. All three techniques suggested that FRET occurred between RPTPalpha -CFP and -YFP fusion proteins, and thus that RPTPalpha dimerized in living cells. RPTPalpha dimerization was constitutive, extensive and specific. RPTPalpha dimerization was consistent with cross-linking experiments, using a non-cell-permeable chemical cross-linker. Using a panel of deletion mutants, we found that the transmembrane domain was required and sufficient for dimerization. CONCLUSIONS: We demonstrate here that RPTPalpha dimerized constitutively in living cells, which may be mediated by the transmembrane domain, providing strong support for the model that dimerization is involved in regulation of RPTPs.

Cross-Linking Reagents↗
PubMed2005

Structural probing of a pathogenic tRNA dimer.

The A3243G mutation within the human mitochondrial (hs mt) tRNALeuUUR gene is associated with maternally inherited deafness and diabetes (MIDD) and other mitochondrial encephalopathies. One of the most pronounced structural effects of this mutation is the disruption of the native structure through stabilization of a high-affinity dimeric complex. We conducted a series of studies that address the structural properties of this tRNA dimer, and we assessed its formation under physiological conditions. Enzymatic probing was used to directly define the dimeric interface for the complex, and a discrete region of the D-stem and loop of hs mt tRNALeuUUR was identified. The dependence of dimerization on magnesium ions and temperature was also tested. The formation of the tRNA dimer is influenced by temperature, with dimerization becoming more efficient at physiological temperature. Complexation of the mutant tRNA is also affected by the amount of magnesium present, and occurs at concentrations present intracellularly. Terbium probing experiments revealed a specific metal ion-binding site localized at the site of the A3243G mutation that is unique to the dimer structure. This metal ion-binding site presents a striking parallel to dimeric complexes of viral RNAs, which use the same hexanucleotide sequence for complexation and feature a similarly positioned metal ion-binding site within the dimeric structure. Taken together, these results indicate that the unique dimeric complex formed by the hs mt tRNALeuUUR A3243G mutant exhibits interesting similarities to biological RNA dimers, and may play a role in the loss of function caused by this mutation in vivo.

Base Sequence↗
PubMed2002

The clinical usefulness of D-dimer testing in cancer patients with suspected deep venous thrombosis.

BACKGROUND: Little is known about the diagnostic value of a D-dimer test in cancer patients with clinically suspected deep venous thrombosis (DVT). OBJECTIVE: To evaluate the clinical utility of a whole blood rapid D-dimer test (SimpliRED) in cancer patients compared with noncancer patients. METHODS: In consecutive patients with suspected lower limb DVT, a D-dimer test and ultrasonogram were performed. Cancer status was recorded at presentation. If the D-dimer test and ultrasonogram results were normal, DVT was considered absent. If the D-dimer result was abnormal, ultrasonography was performed again 1 week later. Anticoagulant therapy was only instituted in those patients with an abnormal ultrasonography result. All patients were followed up for 3 months to record subsequent thromboembolic events. The accuracy of the D-dimer test was assessed, and the efficiency and safety of withholding additional ultrasonography in cancer patients with normal results on both D-dimer and ultrasonography was compared with noncancer patients. RESULTS: A total of 1739 consecutive patients were studied, 217 (12%) of whom had cancer. The negative predictive value of the D-dimer test was 97% in both cancer and noncancer patients. In 63 (29%) of all 217 cancer patients, the D-dimer and ultrasonography results were normal at referral; therefore, the diagnosis of DVT was refuted and anticoagulant treatment was withheld. In these 63 patients, one thromboembolic event occurred during follow-up (1.6%; 95% confidence interval, 0.04%-8.53%). CONCLUSIONS: The negative predictive value of a whole blood D-dimer test in cancer patients seems as high as in noncancer patients. In a substantial proportion of cancer patients, the diagnosis can likely be refuted at referral, based on normal D-dimer test and ultrasonogram results. Furthermore, it seems safe to withhold anticoagulant therapy in these patients.

Adult↗
PubMed2004

Circulating D-dimer levels are better predictors of overall survival and disease progression than carcinoembryonic antigen levels in patients with metastatic colorectal carcinoma.

BACKGROUND: Fibrin formation is required for tumor angiogenesis, metastasis, and invasion. D-dimer, a fibrin degradation product, is produced when crosslinked fibrin is degraded by plasmin. The current study prospectively examined D-dimer levels in patients with metastatic colorectal carcinoma treated in a Phase II randomized trial comparing bevacizumab (Avastin, Genentech, South San Francisco, CA) plus 5-fluorouracil/leucovorin (5-FU/LV) with 5-FU/LV alone. METHODS: At least one circulating D-dimer level was evaluable in 98 of the 104 previously untreated patients with metastatic colorectal carcinoma in the current trial. Plasma D-dimer levels were determined using a quantitative immunoassay kit at enrollment, before each treatment, and at the time of trial completion or disease progression. RESULTS: At trial enrollment, 86 of 104 patients (88%) had elevated D-dimer levels (> 20 ng/mL), and 86 of 102 patients (84%) had elevated carcinoembryonic antigen (CEA) levels (> 3 ng/mL). Baseline D-dimer levels were correlated with the following baseline characteristics: CEA (Pearson coefficient, 0.31; P = 0.002), albumin levels (Pearson coefficient, -0.32; P = 0.002), tumor burden (Pearson coefficient, 0.30; P = 0.003), and number of metastatic sites (Pearson coefficient, 0.21; P = 0.04). At the time of progression, plasma D-dimer levels reached a maximum postbaseline value in 51 of 61 patients (84%), whereas the CEA level was at its maximum postbaseline value in 39 of 55 patients (71%). Baseline D-dimer levels were a strong predictor of overall survival on univariate analysis (P = 0.008) and multivariate analysis (P = 0.03). Overall, treatment with bevacizumab (5 mg/kg) and baseline D-dimer levels were the only predictors of overall survival (P < 0.05). CONCLUSIONS: The current study indicates that fibrin remodeling is an important prognostic feature in metastatic colorectal carcinoma. D-dimer levels should be incorporated into prognostic models, and D-dimer may represent a useful biomarker for patients treated with antiangiogenic agents.

Adult↗
PubMed2006

Is D-dimer a predictor of strangulated intestinal hernia?

BACKGROUND: The goal of this study was to assess the relevance of serum D-dimer measurement as a possible reliable marker for the diagnosis of strangulated intestinal hernia. METHODS: Consecutive patients admitted with nontraumatic acute abdominal gastrointestinal disorders were recruited prospectively in a tertiary referral hospital. The study was conducted in 159 patients between August 2002 and April 2004. D-dimer, lactate dehydrogenase, serum amylase, and international normalized ratio (INR) levels were tested in the emergency room prior to surgical intervention. For each patient, 15 variables, including D-dimer, were available for analysis. RESULTS: Thirty-three (20.7%) of the 159 patients had intestinal ischemia, and 28 (85%) of these 33 patients had D-dimer level > 300 ng/ml. Plasma levels of D-dimer in patients with intestinal ischemia were significantly higher than in patients without ischemia (P < 0.05). There were 29 (18.2%) patients in the hernia group with incarceration (n = 22) or strangulation requiring resection (n = 7). D-dimer levels in patients requiring intestinal resection were insignificantly higher than in patients without resection (P > 0.05). Six (85%) of the 7 hernia patients requiring resection had D-dimer levels > 360 ng/ml. The D-dimer variable correlated best with the leukocyte count in patients with hernia requiring resection. Levels of lactate dehydrogenase, serum amylase, and INR did not show any correlation with D-dimer levels. CONCLUSIONS: To help predict ischemic events, the increasing use of the D-dimer assay in clinical practice could be extended to patients presenting with intestinal emergencies. An elevated D-dimer level on admission had a high sensitivity for identifying patients with intestinal ischemia, although it had a low specificity. Whether it is predictive or preventive for resection in strangulated intestinal hernia patients still remains a question.

Adult↗
PubMed2006

Estimation of the levels of D-dimer by use of an alternative method based in the reaction time of fibrinogen/fibrin degradation products assay.

BACKGROUND: D-dimer and fibrinogen/fibrin degradation products (FDP) levels are elevated in thromboembolic disorders, and its assays are used in many laboratories for the investigation of these disorders. The aim of this study was to propose a cost saving alternative method for determination of D-dimer levels based in the reaction time of FDP assay. METHODS: D-dimer and FDP immunoassays were performed in 100 blood samples from subjects by use of Liatest D-dimer and Plasma FDP. The results of D-dimer obtained by the Liatest D-dimer were compared with the results obtained by the estimation method. RESULTS: A significant association between D-dimer levels and the reaction times necessary to occur agglutination in latex agglutination test for FDP was observed. A close agreement between D-dimer results obtained by the Liatest D-dimer and by the estimation method (D-dimer = 82829 FDPt(-0.8683)) was observed. CONCLUSIONS: The estimation method described in this study could be useful for clinical laboratories because it allows estimating the D-dimer levels before of the automated systems analysis, reducing costs associated to dilutions of plasma samples and reagents.

Antifibrinolytic Agents↗
PubMed1988

Mechanism of fluorescence concentration quenching of carboxyfluorescein in liposomes: energy transfer to nonfluorescent dimers.

When 5(6)-carboxyfluorescein (6CF) is encapsulated in liposomes at 0.2 M, 97-98% of the fluorescence is quenched. We have studied the mechanism of this effect. The dye-liposome system is a special case of concentration quenching of dyes, a phenomenon recognized for 100 years. Absorption spectra of encapsulated dye show that 6CF dimerizes, and the dimer is nonfluorescent. The dimerization constant was estimated, and it was concluded that dimerization can account for only part of the quenching. In 6CF solutions, the fluorescence lifetime decreased drastically as concentration was changed over the narrow range 0.02-0.05 M, a finding which was attributed to energy transfer to dimers. Inhibition of dimerization by propylene glycol also inhibited the shortening of lifetime. Förster critical transfer distances were calculated to be 51 and 57 A for monomer-monomer and monomer-dimer transfer, respectively. Monomer-monomer transfer was demonstrated directly by steady-state or time-resolved anisotropy experiments, while transfer to dimer was modeled by using sulforhodamine B, which has a critical transfer distance like that for the dimer and also quenches 6CF emission. No direct evidence for collisional self-quenching of 6CF could be found, although a model compound, salicylate, did quench weakly. For xanthene dyes, the rate of energy transfer is much faster than that for quenching collisions, implying that collisional quenching in the usual 6CF-liposome system is insignificant. The reason why 6CF is not 100% quenched in liposomes is attributed to dye interaction with lipid as evidenced by (i) multiexponential decay of 6CF in liposomes with a long component of 3-4 ns, (ii) inhibition of dimerization in liposomes, (iii) partial protection of dye from quenching by KI, (iv) differing amounts of dimerization in liposomes made from different kinds of phospholipid, and (v) enhancement of fluorescence lifetime in the presence of Triton X-100.

Chemical Phenomena↗
PubMed1990

The use of haemoregulatory peptides (pEEDCK monomer and dimer) for reduction of cytostatic drug induced haemopoietic damage.

We have previously shown that the stem cell inhibitory peptide pGlu-Glu-Asp-Cys-Lys (pEEDCK monomer) leads to a good tolerance of otherwise lethal multiple ara-C doses and an increased survival of ara-C + peptide treated mice. This effect was due to the prevention of drug-induced CFU-S proliferation, thus keeping stem cells in a quiescent state insensitive to ara-C. Here we show that the pEEDCK monomer also inhibits stem cell proliferation after clinically relevant (non-lethal) ara-C doses. This leads to a sustained (100%) stem cell number in the femoral bone marrow, which was greatly reduced without protective peptide treatment (27%). We have measured the kinetics of influx of CFU-S into the empty S-phase (after two consecutive ara-C injections). This influx reached peak levels of 60-70%; pEEDCK treatment reduced it to 25-30%. Due to its cysteine content the pEEDCK monomer is easily oxidized and forms a symmetric disulfide-bonded dimer (pEEDCK)2. This dimer is a potent stimulator of haemopoiesis. Various modes of protective peptide treatment (monomer and dimer) were investigated in conjunction with a standardized protocol of 2 x 300 mg/kg ara-C given 12 h apart. (a) ara-monomer-ara: The administration of pEEDCK-monomer 2 h before the second ara-C injection retarded the onset of neutropenia, shortened its duration and improved recovery after depression. The degree of short-term neutropenia was not changed. (b) ara-ara-HN2-dimer: Post chemotherapy infusion of the stimulatory (pEEDCK)2 dimer led to considerable increases of progenitor levels (6.8 CFU-GM/1000 bone marrow cells vs. 1.2 CFU-GM/1000 in normal mice) 2 days after cytostatic treatment when CFU-GM were not detectable in unprotected mice. This increase was followed by greatly elevated granulocyte counts (8000 PMN/mm3 vs. 750 PMN/mm3 in normal mice). In the dimer-treated mice, up to 75% of the peripheral leukocytes were mature PMN (normal, 10%). (c) ara-monomer-ara-dimer: ara-C and monomer treatment as above (a) followed by dimer infusion led to complete protection of haemopoiesis. Mice treated with the protective pEEDCK monomer plus stimulatory dimer did not develop the leukocyte depression seen in unprotected animals. Our results show that the haemoregulatory peptide monomer and dimer can be used to improve the haematological status of mice treated with clinically relevant doses of cytostatic drugs (anti-metabolite and alkylating, alone and in combination). The pEEDCK monomer and dimer are equally active also on human haemopoietic cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2004

D-dimer as marker for microcirculatory failure: correlation with LOD and APACHE II scores.

The relevance of plasma d-dimer levels as marker for morbidity and organ dysfunction in severely ill patients is largely unknown. In a prospective study we determined d-dimer plasma levels of 800 unselected patients at admission to our intensive care unit. In 91% of the patients' samples d-dimer levels were elevated, in some patients up to several hundredfold as compared to normal values. The highest mean d-dimer values were present in the patient group with thromboembolic diseases, and particularly in non-survivors of pulmonary embolism. In patients with circulatory impairment (r=0.794) and in patients with infections (r=0.487) a statistically significant correlation was present between d-dimer levels and the APACHE II score (P<0.001). The logistic organ dysfunction score (LOD, P<0.001) correlated with d-dimer levels only in patients with circulatory impairment (r=0.474). On the contrary, patients without circulatory impairment demonstrated no correlation of d-dimer levels to the APACHE II or LOD score. Taking all patients together, no correlations of d-dimer levels with single organ failure or with indicators of infection could be detected. In conclusion, d-dimer plasma levels strongly correlated with the severity of the disease and organ dysfunction in patients with circulatory impairment or infections suggesting that elevated d-dimer levels may reflect the extent of microcirculatory failure. Thus, a therapeutic strategy to improve the microcirculation in such patients may be monitored using d-dimer plasma levels.

Adolescent↗
PubMed1997

Discrepancy between latex and ELISA D-dimer values in sepsis may be caused by human neutrophil elastase.

We have recently shown that D-dimers are degraded by human neutrophil elastase (HNE) in vitro, causing rapid decrease in the D-dimer levels measured by a Latex test, but not with an ELISA test employing the same monoclonal antibody against D-dimer. To see if such discrepant D-dimer concentrations occurred in patients with high HNE concentration, we examined 80 plasma samples from 8 patients with sepsis with a Latex and an ELISA test and calculated the ratio between the D-dimer values obtained with the two tests. Twenty healthy pregnant and twenty pre-eclamptic patients, who are known to have raised D-dimer but low HNE concentrations, were chosen as controls. HNE levels were estimated by determining the HNE-alpha 1-proteinase inhibitor complex (HNE-A1PI) concentration. HNE-A1PI concentration was increased in sepsis patients compared with pre-eclamptic patients (p < 0.0005) and healthy pregnant women (p < 0.0005). In sepsis patients, the D-dimer results were skewed towards lower ratios between Latex and ELISA values compared to pre-eclamptic patients (p = 0.008) and healthy pregnant women (p = 0.0001). In plasma samples from patients with the largest discrepancy between Latex and ELISA D-dimer values, Western blotting with immunostaining indicated degradation of D-dimers to D-like fragments similar to those observed following degradation of cross-linked fibrin by HNE in vitro. We conclude that in sepsis patients there is a marked discrepancy between Latex and ELISA D-dimer values that may be caused by HNE. In such patients Latex D-dimer assays may cause severe underestimation of fibrinolysis.

Adult↗
PubMed1996

Significance of D-dimer concentrations during and after cardiopulmonary bypass.

OBJECTIVE: To determine whether D-dimer fragments predictably increase during cardiopulmonary bypass (CPB), and if so, whether increases correlate with postoperative blood loss or predict postoperative coagulopathy. DESIGN: Prospective observational study of 65 consecutive patients undergoing first-time coronary artery bypass graft (CABG) or first-time valve replacement. SETTING: Single center University teaching hospital. PARTICIPANTS: Male and female patients between the ages of 30 and 90 years undergoing first-time CABG or valve replacement surgery using CPB. Patients were excluded from study for prolonged preoperative bleeding time, preoperative warfarin therapy, perioperative intra-aortic balloon pump support, thrombolytic therapy in the week preceding operation, reoperation, and emergency operation. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Blood sampling for platelet count, prothrombin time, partial thromboplastin time, thrombin time, fibrinogen, activated coagulation time (ACT) and D-dimer concentrations was obtained at four times during each case; (1) preoperatively, after insertion of the internal jugular introducer, before insertion of pulmonary artery catheter; (2) during CPB at 28 degrees C, immediately before rewarming; (3) after heparin neutralization (20 minutes after initial protamine dose); (4) 12 to 24 hours postoperatively. Blood loss in the intensive care unit was calculated by measuring total mediastinal drainage output at 1 and 4 hours after arrival from the operating room. An initial decrease in fibrinogen was noted during bypass, but no increase in D-dimer was identified. A few patients developed a modest increase in D-dimer after heparin neutralization, but none greater than 2.0 ug/mL. Postoperatively, fibrinogen concentration increased toward baseline levels. However, this is when six patients developed significant (> 2.0 ug/mL) D-dimer formation. Results suggest appropriate physiologic response-normalization of fibrinogen with new synthesis and remodeling of clot in the operative site causing D-dimer formation. Patients with highest D-dimer levels at 12 to 24 hours postoperatively had the highest blood loss at 4 hours postoperatively, suggesting that early postoperative excess bleeding predisposed to increased clot formation and subsequent clot remodeling causing elevated D-dimer concentrations. CONCLUSIONS: D-dimer concentration is not usually elevated in patients undergoing CPB when adequately anticoagulated as monitored using the ACT. When mild elevation of D-dimer occurs, it is most often after heparin neutralization and/or in the postoperative period and is not predictive of increased postoperative blood loss. Elevations of D-dimer concentrations in the postoperative period without corresponding decreases in fibrinogen concentrations may occur and do not signify coagulopathy.

Adult↗
PubMed1992

Apple four in human blood coagulation factor XI mediates dimer formation.

Human blood coagulation factor XI is a dimer composed of two identical subunits. Each subunit contains four apple domains as tandem repeats followed by a serine protease region. A disulfide bridge between Cys321 of each fourth apple domain links the subunits together. The role of Cys321 in the dimerization of factor XI was examined by mutagenesis followed by expression of its cDNA in baby hamster kidney cells. The recombinant proteins were then purified from the tissue culture medium and shown to have full biological activity. Normal recombinant factor XI was secreted as a dimer as determined by SDS-PAGE, while recombinant factor XI-Cys321 Ser migrated as a monomer under these conditions. Gel filtration studies, however, revealed that each protein existed as a dimer under native conditions, indicating that the disulfide bond between Cys321 of each factor XI monomer was not necessary for dimer formation. The fourth apple domain (apple4) of factor XI was then introduced into tissue plasminogen activator (tPA) to investigate its role in the dimerization of other polypeptide chains. The fusion protein, containing apple4 (apple4-tPA), formed dimers as detected by SDS-PAGE and gel filtration. Furthermore, dimerization was specific to apple4, while apple3 had no effect on dimerization. These data further indicated that the apple4 domain of factor XI mediates dimerization of the two subunits and the interchain disulfide bond involving Cys321 was not essential for dimer formation.

Amino Acid Sequence↗