Fibrinogen angers with a new deletion (gamma GVYYQ 346-350) causes hypofibrinogenemia with hepatic storage.

INTRODUCTION: This study reports a family with chronically abnormal blood liver function tests (LFT) and congenital hypofibrinogenemia. The proposita had cirrhosis initially related to alcohol abuse and chronic viral hepatitis C (HCV), but abnormal LFT persisted even when alcohol intake was stopped and despite HCV treatment was efficient based on serum RNA negative testing. RESULTS: Needle biopsy specimens of the proposita and her brother showed eosinophilic intra-cytoplasmic inclusions that reacted strongly with fibrinogen antisera on direct immunofluorescence. Electron microscopic examination showed that the rough endoplasmic reticulum was filled with inclusions that consisted of densely packed, curved tubular structures arranged in a fingerprint-like pattern. Coagulation studies revealed low functional and antigenic fibrinogen concentrations suggestive of hypofibrinogenemia. Amplification and DNA sequencing showed a heterozygous deletion of the a7690 to g7704 nucleotides of the gamma chain gene in the 3'end of exon 8 (g 7690_7704del14; Genbank access M10014); this deletion encompassed the splicing site at position 7703 and predicts in a new putative consensus splicing sequence (AATGgtatgtt). RNA was extracted from a liver specimen from the proposita's brother. The cDNA obtained by reverse transcription polymerase chain reaction confirmed the usage of a newly generated donor site at position 7688 of the genomic sequence resulting in an in-frame heterozygous 5 amino acid deletion (GVYYQ 346-350; p.G372_Q376del) and that this mutation is responsible for a new splicing site at position 7688 of the genomic sequence. CONCLUSION: we suggest that the molecular defect in fibrinogen Angers results in an impaired assembly and causes defective secretion and hepatic storage of fibrinogen.

[Predictive value of D-dimer assay in superficial thrombophlebitis of the lower limbs]. / Valeur des D-dimères lors des thromboses veineuses superficielles des membres inférieurs.

OBJECTIVE: The negative predictive value of D-dimer (DD) assay in patients with venous thromboembolic disease is well established for deep vein thrombosis and pulmonary embolism. Little is known about the value of DD assay in patients with superficial thrombophlebitis (ST). The purpose of this study was to assess the value of DD assay in patients with ST of the lower limb. METHOD: The study group was composed of 100 consecutive patients, irrespective of age. Patients with clinical manifestations suggestive of ST of the lower limbs with positive duplex color Doppler evidence confirming the diagnosis and DD assay results (Vidas D-Dimer Exclusion) within 24 hours were included in the study. Patients with thrombosis in another site in addition to the superficial vein of the lower limb, those taking anticoagulants for more than 48 hours, and those with a condition known to potentially elevate DD levels were excluded. The volume of the thrombus was determined echographically and reported as mean diameter and length. RESULTS: Sixty-two women and 38 men were included. Mean age (+/- 5) was 58 years +/- 13.48 (range 18-90; median: 57). The ST involved the Great saphenous (n=74), the small saphenous (n=11) or another vein (n=15). Mean thrombus volume was 4453 mm(3) +/- 7101 (range 94-38484; median: 1751). Mean DD level was 829 ng/ml +/- 516.72 (range 100-2567; median: 715.5). DD assay was negative (<500 ng/ml) in 32 patients (32%) and positive in 68 (68%). For these three items, there was no significant difference between ST with and without varicose veins. DD assay was always positive (>or=500 ng/ml) in all patients aged over 70 years (n=22). In patients aged less than 70 years (n=78), DD assay was positive in 46 (59%) and negative in 32 (41%). DD level was positively correlated with thrombus volume in patients aged less than 70 years (P<0.0001). ROC analysis, sensitivity as a function of specificity by thrombus volume for the entire population, determined the usefulness of a negative DD assay. Considering the critical threshold at 5914 mm(3), sensitivity was 1.0 (95CI 0.89-1.0), with 0.29 specificity (95CI 0.19-0.42), 1.00 negative predictive value and 0.75 positive predictive value. However, the thrombus volume was less than this threshold value in three of the nine cases of ST with extension to the terminal portion of the saphenous. CONCLUSION: A positive DD assay was observed in 68% of patients with ST, with no significant difference with or without varicose veins. The test was positive in all patients aged over 70 years and in 59% of those aged under 70 years. There was a correlation between DD level and thrombus volume, yielding a threshold volume (5914 m(3)) above which all DD tests were positive. Nevertheless, this threshold volume was too great to include all ST extending to the terminal portion of the saphenous. Measurement of DD level is thus not contributive to the diagnosis of ST.

Role of platelet membrane glycoproteins Ib/IX and IIb/IIIa, and of platelet alpha-granule proteins in platelet aggregation induced by human osteosarcoma cells.

We have previously shown that the platelet-aggregating activity of human MG-63 and HOS osteosarcoma cells depends at least in part upon tumor cell surface-associated thrombospondin, and suggested that platelet-osteosarcoma cell interactions could occur through interactions with specific platelet membrane receptors. In this study, the platelet-aggregating activity of MG-63 and HOS cells was studied by using a variety of platelet disorders. Both osteosarcoma cell lines induced a biphasic platelet aggregation response when added to normal platelet-rich plasma, while the second phase of aggregation was absent when added to gray platelets (deficiency in alpha-granule proteins) and to aspirin-treated platelets. Platelets from two unrelated patients with type I Glanzmann's thrombasthenia (deficiency in glycoprotein (GP) GPIIb/IIIa) did not aggregate at all with osteosarcoma cells. Using giant platelets from three patients with Bernard-Soulier syndrome (deficiency in GPIb/IX), the aggregation response induced by MG-63 and HOS cells was monophasic and reversible when compared to normal-sized platelets and to giant platelets from a patient with May-Hegglin anomaly (no membrane GP defect). Because GPIb serves as a receptor for von Willebrand factor during hemostasis, aggregation experiments were also conducted with the platelet-rich plasma of two patients with a low plasma von Willebrand factor concentration (type I von Willebrand's disease) before and after the infusion of deamino-D-arginine vasopressin. MG-63 and HOS cells induced biphasic platelet aggregation both before and after deamino-D-arginine vasopressin treatment, while the ristocetin-dependent binding of von Willebrand factor to platelets only occurred after deamino-D-arginine vasopressin treatment. Preincubation of normal platelet-rich plasma with monoclonal antibody SZ-2 directed against the von Willebrand binding domain of GPIb did not inhibit the platelet-aggregation activity of osteosarcoma cells, whereas anti-GPIb antibody SZ-2 did inhibit ristocetin-induced platelet agglutination. In addition, anti-GPIX antibodies did not affect platelet-osteosarcoma cell interactions. In conclusion, our data demonstrate that the first phase of the platelet-aggregating activity of human osteosarcoma cells is initiated by the interaction of these tumor cells with platelet membrane GPIIb/IIIa, whereas the second phase, even if plasma von Willebrand factor is deficient, involves platelet membrane GPIb and the participation of platelet alpha-granule proteins in membrane-mediated events, making aggregation irreversible.

Thermal transitions of red blood cell deformability. Correlation with membrane rheological properties.

Red blood cell deformability has been studied by the initial filtration flow rate as a function of temperature. The well-known transition at 49-50 degrees C (probably due to spectrin denaturation) is shown. Another transition is demonstrated around 18 degrees C (the cell becomes stiffer below this temperature range). The erythrocyte membranes prepared by a mild dialysis technique have the same deformability as intact erythrocytes at room temperature; they also show the same low-temperature transition. No such transition has been found for hemoglobin solutions of viscosity 30 g X dl-1. It is interesting to compare these results with those obtained by other methods which measure the properties of natural or artificial lipid membranes and which also demonstrate a thermal transition at 15-20 degrees C. Therefore, the deformability of intact normal erythrocytes seems to depend mainly on the rheological properties of the membrane.

Two novel fibrinogen variants found in patients with pulmonary embolism and their families.

BACKGROUND: The occurrence of dysfibrinogen is quite rare in comparison with other hemostatic defects, specially in cases of venous thrombosis. OBJECTIVES: Fibrinogen is known to have multiple functions, which are not evaluated by simple coagulation testing. We have used gel electrophoresis to search for new mutations. PATIENTS AND METHODS: Specimens of purified fibrinogen from 217 consecutive patients with familial or recurrent or early thrombosis and from 490 control subjects were evaluated by electrophoresis. Plasma fibrinogen levels and coagulation-dependent tests (electromechanical and optical coagulometric determinations, immunological measurement, thrombin and Reptilase(R) times) were normal. RESULTS: Two novel familial variants were detected. For a 42-year-old patient, an in-frame 117 base pair insertion in the Aalpha-chain gene caused a 5-kDa mobility shift of the Aalpha chain. This corresponds to a 39 amino acid duplication in the connector domain (fibrinogen Champagne au Mont d'Or). This pattern was also found in the patient's mother and child. A second 31-year-old patient presented an extra band under non-reducing conditions, 30 kDa larger than HMW fibrinogen and reacting with antifibrinogen antibodies (fibrinogen Lozanne). A heterozygous 5909A-->G mutation was found on the Bbeta-chain gene leading to heterozygous Bbeta Tyr236--> stop codon. The predicted truncated Bbeta chain could participate in chain assembly. Two family members were also affected, one of whom had suffered early venous thrombosis. CONCLUSIONS: Electrophoretic testing of apparently normal fibrinogens can reveal new variants which may be clinically relevant.

Aggregation to thrombin and collagen of platelets from a Glanzmann thrombasthenic patient lacking glycoproteins IIb and IIIa.

The aim of this study was to investigate the platelets of a Glanzmann thrombasthenic patient, which in citrated PRP failed to respond to various agonists, but aggregated and secreted to high concentrations of thrombin (0.36, 0.72 and 1 U/ml) and collagen (4, 10 and 20 micrograms/ml) when washed and resuspended in a Tyrode-albumin solution (containing 2 mM Ca2+). Aggregation of the patient platelets was not affected by anti-IIb/IIIa monoclonal antibody (P18) which strongly inhibits thrombin or collagen induced aggregation of normal platelets. Washed platelets of this patient did not aggregate to ADP (10-100 microM) in the presence of added fibrinogen (2 mg/ml) nor bind 125I-labelled fibrinogen (40 to 320 micrograms/ml) when thrombin-stimulated. Different anti-IIb/IIIa monoclonal antibodies (P2, P18) when used in binding or crossed immunoelectrophoretic studies showed a complete absence of the IIb-IIIa glycoprotein complex on the patient platelets. Moreover, glycoproteins IIb or IIIa were absent on silver-stained two-dimensional (non-reduced/reduced) polyacrylamide gel separations of the patient platelets and were not detected by Western blots used in combination with anti-PLA1 (antigen present on IIIa), anti-Leka (antigen present on IIb). This study shows that platelets lacking glycoproteins IIb or IIIa can aggregate in response to high concentrations of collagen or thrombin when resuspended in the presence of physiological concentrations of calcium. Results obtained in this study could indicate the existence of other mechanisms (other than the IIb-IIIa glycoprotein complex) involving glycolipids, heparans, proteoglycans, and/or unknown membrane glycoproteins to mediate platelet aggregation of stimulated thrombasthenic platelets.

A database for human fibrinogen variants.

Identifying and studying abnormal human fibrinogens is a source of much information, and helps in taking care of the affected patients. To permit exhaustive numbering and easy updates, an extensive register has been compiled and made available on the internet. Known molecular abnormalities are mentioned with the essential clinical features.

Cardiac surgery with cardiopulmonary bypass in patients with type II heparin-induced thrombocytopenia.

BACKGROUND: The use of cardiopulmonary bypass (CPB) in patients with a history of type II heparin-induced thrombocytopenia (HIT) may be associated with complications related to their anticoagulation management. METHODS: Between January 1997 and December 1999, among 4,850 adults patients who underwent cardiac surgery in our institution, 10 patients presented with preoperative type II HIT. In 4 patients, anticoagulation during CPB was achieved with danaparoid sodium. In 6 other patients, heparin sodium was used after pretreatment with epoprostenol sodium. RESULTS: No significant change in platelet count occurred in any patient. No intraoperative thrombotic complication was encountered. Total postoperative chest drainage ranged from 250 to 1,100 ml in patients pretreated with epoprostenol and 1,700 to 2,470 ml in patients who received danaparoid sodium during CPB (p < 0.05, Mann-Whitney U test). CONCLUSIONS: During CPB, inhibition of platelet aggregation by prostacyclin may be a safe anticoagulation approach in patients with type II HIT.

Increased plasma free gamma carboxyglutamic acid levels during deep vein thrombosis and intravascular disseminated coagulation.

Gammacarboxyglutamic acid (gla) is a non essential amino acid synthesized in presence of vitamin K, predominantly found in coagulation and bone proteins. In 14 cases of deep vein thrombosis and in 11 cases of disseminated intravascular coagulation, compared to 19 normal subjects and 9 patients hospitalized for leg pain, free plasma gla levels were found significantly elevated (respectively 372 +/- 244 and 559 +/- 361 versus 146 +/- 34 and 120 +/- 40 pmol/mL). In six paired plasma and serum, gla levels were similar. These results suggest an involvement of blood coagulation in gla generation with need of a catabolism of the activated factors. A significant decrease was noticed during vitamin K antagonist therapy and liver disease, both instances in which the synthesis of gla containing coagulation factors is affected. During hepatocellular carcinoma with elevated desgamma carboxyprothrombin, gla was found normal, denying an global impairement of the vitamin K metabolism.

Aspirin, indomethacin and dazoxiben do not affect the fibrinolytic activation induced by venous occlusion.

A cause-effect relation between the synthesis and release of prostaglandins and fibrinolytic activation has been suggested. We have reinvestigated this relation in a double-blind, placebo-controlled, cross-over study with cyclooxygenase inhibitors (aspirin and indomethacin) and a thromboxane synthase inhibitor (dazoxiben) in nine healthy volunteers. Euglobulin fibrinolytic activity (EFA) and tissue-type plasminogen activator antigen level (t-PA:Ag) were studied before and after 10 min of venous occlusion. Despite effective suppression of prostaglandin synthesis by aspirin and indomethacin and enhanced prostacyclin formation by dazoxiben, baseline EFA and t-PA:Ag levels did not significantly change within 2 hours after ingestion of the different drugs. The release of t-PA by venous occlusion was not altered by any of the drugs. Thus, our study does not support the hypothesis that prostaglandins play a significant role in the modulation of the synthesis or release of t-PA.

Functions and tocopherol content of blood platelets from elderly people after low intake of purified eicosapentaenoic acid.

Elderly people present an increased incidence of atherosclerosis and vascular cerebral damages, associated with blood platelet hyperactivity and a stimulation of arachidonic acid metabolism in vivo. The effects of a low intake of purified eicosapentaenoic acid (EPA) on platelet hyperactivity in old human subjects has been investigated. In a randomized, double blind study, 8 people took during 2 months a daily intake of 100 mg of eicosapentaenoic acid (EPA) given as a triglyceride (1,3-dioctanoyl,2-eicosapentaenoyl-glycerol), and 8 other subjects ingested a placebo. A slight, but significant reduction of platelet-rich plasma aggregation in response to epinephrine and arachidonic acid occurred after EPA intake, as well as a decreased aggregation of washed platelets induced by thrombin, although collagen- and U-46619-induced aggregations were not significantly modified. EPA intake failed to affect arachidonic acid metabolism in thrombin-stimulated platelets or in clotted venous blood. The urinary excretion of thromboxane, 6-keto-PGF1 alpha and their 2,3-dinor-metabolites was also not modified. Similarly, no change in the plasma and platelet lipid fatty acid compositions could be observed. Platelet, but not plasma, alpha- and gamma-tocopherol were enhanced by EPA intake. An increase of platelet vitamin E has been associated with a decrease of aggregation, especially in vitamin E-deficient subjects, like elderly people. Therefore, low intake of EPA might have contributed to inhibit platelet aggregation by increasing cellular vitamin E.

Conductimetric study of the binding of Mn2+ to bovine pancreatic deoxyribonuclease.

The binding of Mn2+ on bovine pancreatic deoxyribonuclease has been studied by a conductimetric method. At low ionic strengths, a high-affinity single binding site is demonstrated. The association constant value (K = 1.2 x 10(5) M-1 at pH 8) is high enough to conclude that, in standard experimental conditions for DNA hydrolysis, the reacting species is the DNAase-cation complex. Competitive binding studies in presence of Mg++ and Ca++ show that these cations do not bind on the Mn++ site.

Human plasma fibrinogen measurement derived from activated partial thromboplastin time clot formation.

Prothrombin time-derived measurement of fibrinogen (PTd) has already been described. Activated partial thromboplastin time-derived measurement of fibrinogen (aPTTd) has not yet been clearly defined. Using an MDA II coagulometer (Organon Teknika, Durham, North Carolina, USA), we have therefore compared fibrinogen levels determined with Clauss, PTd, and aPTTd assays and an enzyme immunoassay (EIA) in 172 samples. Of these, 47 were from pre-operative controls, 18 from patients with liver disease, 28 from patients with hyperfibrinogenaemia, 33 from patients treated with vitamin K antagonists, 22 from patients treated with unfractionated heparin and 24 from haemophilic patients. Within the normal range, interassay and intra-assay variations were comparable. For control samples, PTd, aPTTd and Clauss assays were well correlated, without any systematic error. EIA was also correlated but values were slightly higher (mean of difference = 0.24). Pathological samples showed an overestimation of fibrinogen when using PTd measurements in patients treated with vitamin K antagonists, as well as when using aPTTd measurements in patients presenting with factor VIII and factor IX deficiencies. These results indicate that, despite expected financial savings, aPTTd fibrinogen measurements should not be used without restriction. PTd and aPTTd fibrinogen determinations are provided without any additional cost. Their comparison with Clauss fibrinogen results may constitute a validation tool or have additional diagnostic utility (e.g. identifying polymerization abnormalities in case of dissimilar results).

Erythrocyte filtrability measurement by the initial flow rate method.

A new filtration technique, based on the initial filtration rate of a diluted RBC suspension through 5 mu Nucleopore filter is described. As only a few hundreds RBCs traverse each pore and as the measurement are made in a few seconds, the method is by large insensitive to filter plugging and to sedimentation effects. The results are given as a filtration index IF which is, as a first order approximation, independent of the filter conductance and of the suspending medium viscosity. The filtration times are measured electronically. The filters are re-used many times. The influence on the results reproducibility of RBC washing, of the anticoagulant, of the blood sample and the suspension storage times are considered. With our technical procedure, the relative incertitude on the measurement of I.F. is about +/- 10%. The filtration index is shown to be an intrinsic RBC filterability property.

Determination of erythrocytes transit times through a 5 mu "nuclepore" filter.

Filtration experiments through 5 or 3 mu Nuclepore membranes are often performed in order to assess the so-called erythrocyte deformability. The relation between this parameter and the RBC filterability is not straightforward. A simple theoretical treatment relating filtration index (as determined by the initial flow rate method) to the average RBC flow resistance through an average pore is presented. In order to deduce the average RBC transit time through the membrane from the initial flow rate data, the suspension hematocrit change after filtration has been determined. The calculated average transit time is comparable to experimental values, as determined by KIESEWETTER et al. with the single pore technique.

Indices of filterability of red blood cell suspensions.

A number of different experimental techniques have been devised in recent years to use microsieving as a test of the filterability of suspensions of red blood cells. Various indices have been proposed to express the results of these tests. In the present paper a correlation is made of the intrinsic increase in resistance at the level of a single pore in the filter to the macroscopically observed pressure and flow through the entire filter. Further it is shown how a number of different tests may be used to derive the same index. The results apply only to situations in which there is no plugging of pores.

Physico-chemical factors of erythrocyte deformability.

The aim of this study is to assess the role of different physico-chemical factors on the deformability measurements by using the initial filtration flow rate method, and to differentiate between the membrane or internal origin of some rigidity changes. The deformability is maximum for the physiological pH value and it decreases sharply for hypotonic and hypertonic buffer. For normal RBC, the deformability is independent of the pO2 level and a small decrease is observed for increasing pCO2 values (with constant pH). A theoretical model of filtration for the "Hemorheometre" will be also developed.

[Diagnosis of venous thrombosis and/or pulmonary embolism by determination of d-dimers using ELISA. Review based on a study of 80 consecutive patients hospitalized in an emergency unit]. / Le diagnostic de la thrombose veineuse et/ou de l'embolie pulmonaire par le dosage des D-Dimères ELISA. Mise au point à partir d'une étude portant sur 80 patients consécutifs hospitalisés dans un service d'urgence médicale.

The sensitivity and specificity of an ELISA method (Fibrinostika Fbdp Organon Teknika) for assay of D-dimers in the diagnosis of deep vein thrombosis and/or pulmonary embolism was studied in 80 consecutive patients seen at an emergency unit. Fifty-six of the patients presented clinical signs of deep vein thrombosis. Diagnosis was confirmed in 26 of the 56 patients with a D-dimer level above 370 ng/ml (sensitivity 92.3%) and 370 ng/ml for 13 of 30 patients with a negative venous ultrasound Doppler examination (specificity 43.3%). The positive predictive value was 58.5% and the negative predictive value was 87%. There was a significant difference in the level of D-dimers between distal and proximal deep vein thrombosis. In 40 cases with suspected pulmonary embolis, either alone or with suspected deep vein thrombosis, diagnosis was made in only 4 of 9 with a highly or intermediately probable ventilation/perfusion scan. D-dimer level was always above 3,000 ng/ml. Coupling the ELISA dimer test with noninvasive explorations improves negative predictive value but can also avoid invasive explorations (venography, pulmonary angiography) in certain patients. A D-dimer test as sensitive as the ELISA test and as rapid as the latex test remains to be described.

[Conductometric microdosage of blood and urine urea by use of a semi-automatic analyser]. / Microdosage conductimétrique de l'urée sanguine et urinarie à l'aide d'un analyseur semi-automatique.

The authors present the principle of a new method of urea estimation based on conductimetry. Its results are compared with those given by the colorimetric method using diacetylmonoxime. Correlation of the results was satisfactory. With the apparatus studied, it was possible to estimate very simply plasma and urinary urea with low volume samples 10 microliter). The titration took 30 seconds.