Soluble Fibrin Complexes Research Articles

Hematocytological, biochemical, and hemostasis parameters' role in predicting the possibility of the various forms of the COVID‐19 course in hospitalized Ukrainian patients: A cross‐sectional study

AbstractBackground and AimsThe main objective of this study is to establish the characteristics of blood laboratory parameters in hospitalized coronavirus disease 2019 (COVID‐19) Ukrainian patients and the significance of the above‐mentioned parameters for predicting the course of the disease.MethodsHematocytological, biochemical, and hemostasis methods of research have been used. Groups of patients with different forms of the coronavirus disease course have been analyzed (lethality ‐ recovery, recovery with a severe and mild course).Results and ConclusionAge is one of the risk factors for COVID‐19 mortality. Absolute values of neutrophils, neutrophil‐lymphocyte ratio (NLR), systemic inflammation index, d‐dimer, C‐reactive protein, and soluble fibrin complex can be used by clinicians to effectively differentiate between two possible outcomes (lethality vs. recovery). A higher number of stab leukocytes, d‐NLR, and platelets concentration have been recorded in patients with severe COVID‐19 cases, compared to mild ones. The risk of adverse COVID‐19 outcome (lethality) is significantly linked with d‐dimer and NLR (odds ratio 1.42). The risk of a severe course of the disease was significantly associated with the count of leukocytes (odds ratio 4.96).

Dynamics of Blood Values in Patients with Radial Shaft Injury after Using Transosseous Osteosynthesis by external Fixation Pin Device

Transosseous osteosynthesis continues to occupy a leading position among the methods of treatment of damage to the tubular bones of the extremities. This article presents a survey of 47 patients: 22 men and 25 women aged 25 to 60 years with fractures in the middle third of the radial shaft with displacement of bone fragments. The peculiarity of the external fixation device we used is that all transosseous elements are represented by 1.5 mm pins, inserted through both cortical layers of each bone, at levels from I and VIII. The pins are inserted simultaneously through both bones, thus excluding the rotational function of the forearm.The essence of the study is a comparative analysis of the blood system parameters in patients with diaphyseal injuries of the radial bone, operated by the transosseous osteosynthesis method using the external fixation device of pin configuration. Any damage to the body, (a fracture or related surgery), is accompanied with various changes in the system of hemodynamics and metabolism.The laboratory blood tests performed in the group of patients with damage to the radial shaft treated with transosseous osteosynthesis by external fixation device of pin configuration, revealed certain regularities in changing of blood parameters: increased number of platelets, increased level of fibrinogen and level of soluble fibrin complexes, which promotes the activation of the internal mechanism of blood clotting.

Diagnosis and management of DIC complicated by hematological malignancies.

The clinical features noted in individuals with disseminated intravascular coagulation (DIC) complicated by hematological malignancies include life threatening hemorrhage that is associated with thrombocytopenia and consumptive deficiency of coagulation factors. Exacerbation of DIC after the initiation of chemotherapy is also related to fatal hemorrhage. The Japanese Society of Thrombosis and Hemostasis recently proposed provisional DIC diagnostic criteria allowing evaluation of hypercoagulable markers such as soluble fibrin and thrombin-antithrombin complex to help physicians to diagnose DIC and initiate treatment in the early phase of coagulopathy. A phase III clinical trial showed that human soluble recombinant thrombomodulin (rTM) more potently improved DIC than unfractionated heparin and was approved for treatment of DIC in 2008 in Japan. rTM exerts anti-inflammatory and cytoprotective actions and may improve clinical outcomes of DIC patients.

Pathogenesis of nasal bleeding in the patients presenting with arterial hypertension

The objective of the present work was to study the pathogenetic mechanisms underlying nasal bleeding (NB) in the patients presenting with arterial hypertension (AH). A total of 47 patients with AH suffering from NB were available for the examination of whom 28 experienced a single bleeding episode and 19 had recurrent bleeding. 11 of them were treated by endonasal surgical intervention for the achievement of hemostasis. Simultaneously, mucoperichondreal biopsies were taken from the anterior septal portions and used for histological and ultrastructural investigations. The laboratory examination of all the patients has demonstrated hyperfibrinogenemia and the enhanced level of soluble monomeric fibrin complexes in the blood (especially in the patients with recurrent nasal bleeding) despite the normal platelet levels, prothrombin and thrombin times. These findings gave reason to characterize the hemostasiological status of the patients as chronic compensated disseminated intravascular coagulation (DIC) syndrome. The histological study revealed hyaline, erythrocyte-rich and fibrin thrombi in the vessels of the microcirculatory system whereas ultrastructural studies showed desquamation of endothelial cells, massive desendothelization, and disintegration of the basal membrane with the exposure of subendothelium. It is supposed that these changes provoked the development of thrombosis and focal necrosis in nasal mucosa. The authors conclude that the cause of nasal bleeding associated with arterial hypertension is directly related to the lesions of vascular endothelial, microcirculatory disorders, and modification of the blood coagulation processes rather than to the mechanical rupture of blood vessels. These changes are believed to be responsible for the development of local intravascular coagulation.

The role of fibrin related markers in the diagnosis and prognosis of thrombosis

Fibrin related markers include several proteins and fragments developed in the conversion of fibrinogen to cross-linked fibrin and following degradation.FDP played as a more important role than conventional global coagulation test before application of D-dimer in clinical settings,moreover,combination of FDP and D-dimer improve the diagnostic of DIC greatly,and D-dimer has become an effective tool excluding venous thromboembolism besides the imaging.Whether basic or clinical research on soluble fibrin monomers and complexes were focused in an early stage,however,limited by diversity of analytes and non-standardization,no full application was come into being.It is worthwhile to note that fibrin related markers release into plasma sequentially from initialization of fibrinogen activation to fibrinolysis. Thus,the combination of these index is useful to understand patient′s condition (such as DIC),prognosis of VTE recurrence,and prophylaxes of post-operation thrombosis.(Chin J Lab Med, 2014, 37:233-235) Key words: Disseminated intravascular coagulation; Fibrin; Biological markers

Hypercoagulability in different respiratory diseases

BackgroundThe risk of venous thromboembolism (VTE) is equally high in medical patients admitted to the hospital and those treated in the surgery wards. Patients who are immobilized due to heart failure, severe respiratory disease, cerebrovascular stroke and cancer are at a high risk of venous thrombosis. Aim of the workThe aim of the study was to assess the impact of different respiratory insults on blood coagulation for early detection and prevention of thrombosis to open the way for thromboprophylaxis in such cases. Patients and methodsThe study included 25 apparently normal healthy control subjects and 141 patients with different respiratory disorders. All patients were subjected to full medical history taking, full clinical examination, and radiological evaluation. Computed tomography (CT) pulmonary angiography was done for all cases and lower limb duplex ultrasonography for all patients, at day 1 and day 5 of admission. The following coagulation markers were evaluated for all patients at day 1, 2,3,4, and 5 of admission including: Soluble fibrin complex, D dimer, thrombin antithrombin complex, antithrombin, protein C, protein S, and fibrinogen. ResultsIt was found that, the mean values of soluble fibrin complex, D dimer and thrombin antithrombin complex were higher in patients with positive duplex ultrasonograghy and positive CT angiography than patients with negative duplex ultrasonograghy and CT angiography and the difference was statistically highly significant. The higher mean values for soluble fibrin complex was on the third day of admission, while the higher mean values for D dimer, thrombin antithrombin complex and fibrinogen were on the fifth day. Whereas, there are no detectable variations in levels of antithrombin, protein C and protein S on all 5days there were highly statistically significant differences in soluble fibrin complex, D dimer and fibrinogen levels between all disease groups and the control subjects. The mean values of soluble fibrin complex, D dimer, thrombin antithrombin complex and fibrinogen were higher in severe COPD patients than in moderate COPD patients and the difference was statistically significant while there was no statistically significant difference in coagulation marker levels between moderate and severe asthmatic patients and also, between bronchogenic carcinoma and mesothelioma patients. ConclusionsEstimation of soluble fibrin complex, D dimer, thrombin antithrombin complex and fibrinogen may be useful for early identification of the prethrombotic state which may help to prevent the onset of thrombotic disorders and thereby improve the outcome of various respiratory diseases. Common respiratory disorders especially, COPD exacerbation, pneumonia, interstitial pulmonary fibrosis, and lung cancer are considered to be hypercoagulable states which carry high risks for the development of venous thromboembolism.

Higher prognostic value of soluble fibrin complexes than D-dimer and fibrin degradation product for disseminated intravascular coagulation in patients with liver cirrhosis

Fibrin-related markers may help differentiate disseminated intravascular coagulation (DIC) from liver cirrhosis-associated dysfunctional coagulation. We investigated the significance of three fibrin-related markers [D-dimer, fibrin degradation product (FDP), and soluble fibrin complexes (sFC)] for the assessment of DIC status and prognosis. We classified 235 patients with suspected DIC into two groups according to their condition: the liver cirrhosis group (n = 47) and the no liver cirrhosis group (n = 188). Prothrombin time (PT), and fibrinogen, sFC, D-dimer, antithrombin, and protein C concentrations were measured and DIC scores were calculated using four parameters: platelet count, D-dimer, fibrinogen, and PT. In the liver cirrhosis group, the sFC concentration increased significantly in accordance with DIC score compared with the no liver cirrhosis group, and this increase was more prominent than D-dimer and FDP concentration increases. For the diagnosis of overt DIC in patients with liver cirrhosis, the area under the concentration curve (AUC) was larger for sFC (0.746) than for D-dimer (0.733) and FDP (0.687). Cox analysis also indicated that an elevated sFC concentration is a more significant prognostic factor of DIC than D-dimer or FDP (hazard ratio: 10.78; P = 0.036) in liver cirrhosis group; however, it was not a prognostic factor in the no liver cirrhosis group. sFC is a powerful diagnostic and prognostic marker of DIC in patient with liver cirrhosis. The use of sFC is expected to enhance the diagnosis and prognosis of DIC, particularly in patients with liver cirrhosis.

Thrombophilic Variables Do Not Increase the Generation or Procoagulant Activity of Thrombin During Cardiopulmonary Bypass

The generation of thrombin and its procoagulant activity are upregulated during cardiopulmonary bypass (CPB). Thrombophilia associates with increased basal thrombin generation and might therefore propagate thrombin generation during CPB. The objective of this study was to test whether preoperative thrombophilic variables associate with increased generation of thrombin or its procoagulant activity during and after CPB. Comprehensive thrombophilia screening was performed in patients (n = 100) before elective coronary artery bypass grafting (CABG) with CPB. Markers of thrombin generation (prothrombin fragment F1+2), its procoagulant activity (soluble fibrin complexes), and a marker of fibrin degradation (D-dimer) were measured serially at eight time points before, during, and after CABG. Abnormal thrombophilia screening was common (44%). While patients with thrombophilic variables had higher preoperative prothrombin fragment F1+2 than others (median [interquartile range] 0.55 [0.34] vs 0.45 (0.21) nmol/L, p = 0.009) they did not have higher F1+2, D-dimer, or soluble fibrin complex levels during CPB or postoperatively than patients without thrombophilic variables. Preoperative thrombophilic variables do not associate with perioperative thrombin generation or its procoagulant activity in patients undergoing CABG. Our results do not support routine thrombophilia screening before CABG.

Monitoring high-dose heparinization during cardiopulmonary bypass – A comparison between prothrombinase-induced clotting time (PiCT) and two chromogenic anti-factor Xa activity assays

Heparinization requires monitoring, but optimal methods for measuring the anticoagulant effects of heparin remain to be determined. We compared prothrombinase-induced clotting time (PiCT) and two chromogenic anti-factor Xa activity (anti-Xa) assays in monitoring high-dose heparinization during cardiopulmonary by-pass (CPB). Heparin effects were serially measured with PiCT and two anti-Xa assays in 100 patients. Antithrombin and protein C activities were measured preoperatively, and antithrombin activity was measured during CPB. Activation of coagulation was assessed with measurements of prothrombin fragment F1+2, soluble fibrin complexes, and D-dimer before, during, and after CPB. During CPB mean ranges of PiCT and of anti-Xa heparin levels measured with (anti-Xa A) and without (anti-Xa B) dextran sulfate and antithrombin supplementation were 5.0-5.2, 4.7-5.0, and 4.5-4.9 IU/ml, respectively. There was poor agreement between PiCT and anti-Xa and between the two anti-Xa assays (r = 0.32-0.65 and broad limits of agreement). Patients with low preoperative antithrombin or protein C levels had lower PiCT (p = 0.028 and p = 0.01) and anti-Xa A (both p<0.001) levels during CPB than others. Patients with the lowest heparin activities during CPB (lowest deciles of PiCT and anti-Xa A) had higher subsequent F1+2 after CPB (p = 0.002 and p = 0.02), and patients with high heparin levels required fewer transfusions of packed red blood cells than others. In conclusion, in the challenging setting of CPB there is poor agreement between anti-Xa assays and PiCT. However, coagulation-based PiCT could provide an alternative to the chromogenic anti-Xa assays. Higher heparin levels during CPB were confirmed to associate with reduced transfusion requirements.

Latex Immunoturbidimetric Assay for Soluble Fibrin Complex

Soluble fibrin complex (SFC), composed of fibrin monomer and fibrinogen derivatives, is known to exist in the circulating blood in patients with thrombosis. Its detection and quantification are useful for obtaining information about the condition and degree of intravascular coagulation in early-stage thrombosis, but there is no rapid method to measure SFC in plasma for clinical use. We obtained a monoclonal antibody that specifically reacts with SFC, with desAA-fibrin as the immunogen, and developed a rapid and sensitive latex immunoturbidimetric assay (LIA) using latex-immobilized anti-SFC monoclonal antibody. The assay system was based on the increase in turbidity induced by the reaction of the latex-immobilized anti-SFC monoclonal antibody with SFC in plasma, and the assay procedure was fully automated on a Hitachi 911 analyzer. The method had an analytical range of 3-300 mg/L. Intra- and interassay precision studies indicated that this system provided reproducible data (CVs <3.0% and <2.0%, respectively). The assay detection limit was <0.5 mg/L. There was no interference from bilirubin (up to 440 mg/L), hemoglobin (up to 9.6 g/L), Intralipid (up to 10%), D-dimer (up to 200 mg/L), and rheumatoid factor (up to 470 000 IU/L). SFC concentrations in plasma from patients with thrombotic diseases [mean (SD), 48.9 (57.6) mg/L; n = 160) were significantly higher than those in plasma from healthy individuals [1.8 (2.1) mg/L; P <0.001; n = 304]. In terms of linearity, precision, and sensitivity, the LIA, performed on a Hitachi 911 automated analyzer, may be useful for measurement of SFC in plasma.

Cryptic D-Dimer Antigen: A Sensitive Marker of Factor XIIIa Activity during Intravascular Coagulation.

FactorXIIIa (FXIIIa) plays an important role in regulating hemostatic and thrombotic events. FXIIIa modifies the structure of fibrin making the insoluble fibrin gel resistant to plasmin degradation. Methods to detect FXIIIa crosslinking of soluble fibrin(ogen) complexes (SFCs) are not readily available. The purpose of this study was to 1) develop a sensitive assay to detect FXIIIa crosslinked (XL)-SFCs designated: cryptic D-dimer assay, 2) determine whether soluble XL-SFCs are formed prior to formation of an insoluble clot, 3) determine whether soluble XL-SFCs circulated in human plasma and could be incorporated into a fibrin clot, 4) determine whether levels of soluble XL-SFCs are elevated in patients suspected of having intravascular coagulation. We postulated that the cryptic D-dimer antigen would be created by FXIIIa crosslinking of SFCs and could be detected after plasmin digestion. We added purified recombinant FXIIIa (119 nM) to afibrinogenemic plasma with varying concentrations of purified fibrinogen (10–200 μg/mL) and assayed for D-dimer antigen levels pre- and post- plasmin treatment. D-dimer levels were determined by a quantitative latex agglutination assay (MiniQuant® D-dimer, Ireland). The assay utilizes a monoclonal antibody specific for an epitope that is present on plasmin modified crosslinked D-dimer domain on either fibrinogen or fibrin. We found that generation of cryptic D-dimer antigen was a) dependent on FXIIIa, b) uncovered only after plasmin digestion and c) increased with escalating fibrinogen concentration. The level of cryptic D-dimer antigen increased from 0.45 μg/ml to 5.4 μg/ml in the presence of fibrinogen (10–200 μg/ml), FXIIIa and 5 mM calcium chloride. We had earlier confirmed that the cryptic D-dimer antigen could be detected in both EDTA and citrated plasmas and the exposure of this antigen was not modified by calcium present during plasmin digestion. We also determined that the cryptic D-dimer antigen was generated 150 sec before a visible clot appeared in recalcifed plasma in plastic tubes. We then investigated whether cryptic D-dimer antigen circulated in healthy individuals and if these levels changed in patients. Cryptic D-dimer antigen was detected (mean level 1 μg/ml, <1% of plasma fibrinogen level) in ten normal plasma samples. In comparison, patients with elevated D-dimer by ELISA assay showed cryptic D-dimer mean level in excess of 5 μg/ml. Cryptic D-dimer levels increased with rising native D-dimer (no plasmin treatment) levels in patients. To assess the thrombogenic potential of SFCs with cryptic D-dimer antigens, we added reptilase (5 BU/ml) to plasma and examined whether the cryptic D-dimer antigen or native D-dimer could bind to a fibrin clot. We found that more than 70% of the cryptic D-dimer antigen was clottable (n=20) as compared to only 48 % of the native D-dimer (n=20). This suggests that the majority of cryptic D-dimer antigen is present on clottable SFCs. In conclusion, we developed an assay that can detect the ability of FXIIIa to modify SFCs before the appearance of a fibrin clot and may serve as a sensitive marker of intravascular fibrin formation. The cryptic D-dimer antigen is a novel analyte that is present on clottable SFCs and its levels increased in individuals experiencing intravascular blood coagulation. In addition, a basal level of FXIIIa activity exists in human plasma and that regulating this process could have an important impact on the fate of intravascular fibrin and thrombotic events.

Use of soluble fibrin antigen instead of D-dimer as fibrin-related marker may enhance the prognostic power of the ISTH overt DIC score

The overt DIC score of the DIC subcommittee of the ISTH includes a fibrin-related marker (FRM) as indicator of intravascular fibrin formation. The type of marker to be used has not been specified, but D-dimer antigen, or fibrin degradation products are used by most investigators. Soluble fibrin complexes have been suggested as more specific indicators of acute intravascular fibrin formation. The aim of the present study was to compare the predictive value of the overt DIC score concerning clinical outcome in a surgical intensive care cohort, using either D-dimer antigen, or soluble fibrin antigen as FRM. The cutoff values for 2 and 3 score points for the FRM were assigned on the basis of the 25% and 75% quartiles of 1870 plasma samples obtained from 359 ICU patients during a period of 6 months. For 331 patients with complete diagnostic workup and day 1 blood samples, the Iatro SF as FRM component of the overt DIC score displayed the highest prognostic power concerning clinical outcome. The 28-day mortality of patients with overt DIC at day 1, using Iatro SF as FRM assay was 50.0%, whereas 28-day mortality of patients without overt DIC was 14.0% (p <0.0001). Using MDA D-dimer, and TINAquant D-dimer, 28-day mortality was between 35.5% and 39.3% in patients with overt DIC, and 15.5% to 15.6% in patients without overt DIC. Selection of the FRM as component of the DIC score has a small, but relevant impact on the prognostic performance of the overt DIC score. The present data on the distribution of values may provide a basis for the selection of appropriate cutoff points for assigning 2, and 3 points in the score.

Course of indicators of thrombin activity in the early phase of acute myocardial infarction: effect of fibrinolytic therapy and acute percutaneous coronary angioplasty

We evaluated assay systems for detection of in vivo thrombin activity in patients with acute myocardial infarction. The study included 31 consecutive patients with acute myocardial infarction treated either with fibrinolytic therapy (FLT), or acute PTCA. Blood samples were drawn at admission, after 30 min, 1 h, 3 h, 6 h, 12 h, 24, and 48 h. Assays related to the enzymatic action of thrombin included fibrinopeptide A ELISA, a novel latex-enhanced photometric immunoassay for soluble fibrin complexes, fibrin monomer antigen, D-dimer antigen, and soluble platelet glycoprotein V, which is released from the platelet surface by thrombin cleavage. The soluble fibrin assay displayed a high degree of correlation with fibrinopeptide A, and no correlation with D-dimer antigen, indicating that this parameter allows monitoring of in vivo thrombin activity in patients with acute myocardial infarction. Fibrin monomer antigen, and D-dimer antigen, were influenced by FLT, indicating cross-reactivity with proteolytic fragments of fibrin. No significant changes in soluble glycoprotein V were observed. The results show that the novel soluble fibrin assay appears to be a promising method for measurement of in vivo fibrin formation in patients with acute myocardial infarction, irrespective of the primary treatment decision for FLT or acute PTCA.

Analysis of fibrin formation and proteolysis during intravenous administration of ancrod

AbstractAncrod is a purified fraction of venom from the Malayan pit viper, Calloselasma rhodostoma, currently under investigation for treatment of acute ischemic stroke. Treatment with ancrod leads to fibrinogen depletion. The present study investigated the mechanisms leading to the reduction of plasma fibrinogen concentration. Twelve healthy volunteers received an intravenous infusion of 0.17 U/kg body weight of ancrod for 6 hours. Blood samples were drawn and analyzed before and at various time points until 72 hours after start of infusion. Ancrod releases fibrinopeptide A from fibrinogen, leading to the formation of desAA-fibrin monomer. In addition, a considerable proportion of desA-profibrin is formed. Production of desA-profibrin is highest at low concentrations of ancrod, whereas desA-profibrin is rapidly converted to desAA-fibrin at higher concentrations of ancrod. Both desA-profibrin and desAA-fibrin monomers form fibrin complexes. A certain proportion of complexes carries exposed fibrin polymerization sites EA, indicating that the terminal component of the protofibril is a desAA-fibrin monomer unit. Soluble fibrin complexes potentiate tissue-type plasminogen activator-induced plasminogen activation. Significant amounts of plasmin are formed when soluble fibrin in plasma reaches a threshold concentration, leading to the proteolytic degradation of fibrinogen and fibrin. In the present setting, high concentrations of soluble fibrin are detected after 1 hour of ancrod infusion, whereas a rise in fibrinogen and fibrin degradation products, and plasmin-α2–plasmin inhibitor complex levels is first detected after 2 hours of ancrod infusion. Ancrod treatment also results in the appearance of cross-inked fibrin degradation productd-dimer in plasma.

Analysis of fibrin formation and proteolysis during intravenous administration of ancrod

Ancrod is a purified fraction of venom from the Malayan pit viper, Calloselasma rhodostoma, currently under investigation for treatment of acute ischemic stroke. Treatment with ancrod leads to fibrinogen depletion. The present study investigated the mechanisms leading to the reduction of plasma fibrinogen concentration. Twelve healthy volunteers received an intravenous infusion of 0.17 U/kg body weight of ancrod for 6 hours. Blood samples were drawn and analyzed before and at various time points until 72 hours after start of infusion. Ancrod releases fibrinopeptide A from fibrinogen, leading to the formation of desAA-fibrin monomer. In addition, a considerable proportion of desA-profibrin is formed. Production of desA-profibrin is highest at low concentrations of ancrod, whereas desA-profibrin is rapidly converted to desAA-fibrin at higher concentrations of ancrod. Both desA-profibrin and desAA-fibrin monomers form fibrin complexes. A certain proportion of complexes carries exposed fibrin polymerization sites E(A), indicating that the terminal component of the protofibril is a desAA-fibrin monomer unit. Soluble fibrin complexes potentiate tissue-type plasminogen activator-induced plasminogen activation. Significant amounts of plasmin are formed when soluble fibrin in plasma reaches a threshold concentration, leading to the proteolytic degradation of fibrinogen and fibrin. In the present setting, high concentrations of soluble fibrin are detected after 1 hour of ancrod infusion, whereas a rise in fibrinogen and fibrin degradation products, and plasmin-alpha(2)-plasmin inhibitor complex levels is first detected after 2 hours of ancrod infusion. Ancrod treatment also results in the appearance of cross-inked fibrin degradation product D-dimer in plasma. (Blood. 2000;96:2793-2802)

The Prolonged Thrombin Time of Nephrotic Syndrome

Little information is available that documents the incidence and possible etiology of a prolonged thrombin time (TT) found in children with nephrotic syndrome. We speculated that this prolonged TT might best be explained by an altered fibrinogen similar to that found in the newborn. We describe 20 children with nephrosis, an unexplained prolonged TT and reptilase time (RT), and an elevated fibrinogen level. Thrombin and/or plasmin effect on plasma fibrinogen was studied by analyzing for soluble monomer, D-dimer, fibrin degradation products, B beta 1-42 peptide, and by polyacrylamide gel electrophoresis (PAGE) and agarose electrophoresis. Structural changes in the fibrinogen molecule were investigated using two-dimensional gel (2D gel) electrophoresis. Fibrinogen was purified via glycine precipitation, and the sialic acid (SA) content was determined. No evidence for in vivo thrombin and/or plasmin effect on fibrinogen could be detected in 13 of 20 children tested. Additionally, no fibrin/fibrinogen degradation products or soluble fibrin complexes were detected using PAGE or agarose electrophoresis in this group of patients. The B beta isoforms of nephrosis fibrinogen were similar in isoelectric point on 2D gel electrophoresis to those of fetal fibrinogen and demonstrated a greater electronegative shift when compared with normal adult fibrinogen. Also, the SA content of nephrosis and fetal fibrinogen were greater than that measured in adult fibrinogen. Both nephrosis and fetal fibrinogen were more resistant to neuraminidase treatment than was normal adult fibrinogen. These data support the notion that an altered fibrinogen exists in some children with nephrotic syndrome characterized by an increased TT and RT, elevated fibrinogen, and both an increased negative charge and SA content.

Comparison of Immunological and Functional Assays for Measurement of Soluble Fibrin

Various assays have been developed for quantitation of soluble fibrin or fibrin monomer in clinical plasma samples, since this parameter directly reflects in vivo thrombin action on fibrinogen. Using plasma samples from healthy blood donors, patients with cerebral ischemic insult, patients with septicemia, and patients with venous thrombosis, we compared two immunologic tests using monoclonal antibodies against fibrin-specific neo-epitopes, and two functional tests based on the cofactor activity of soluble fibrin complexes in tPA-induced plasminogen activation. Test A (Enzymun-Test FM) showed the best discriminating power among normal range and pathological samples. Test B (Fibrinostika soluble fibrin) clearly separated normal range from pathological samples, but failed to discriminate among samples from patients with low grade coagulation activation in septicemia, and massive activation in venous thrombosis. Functional test C (Fibrin monomer test Behring) displayed good discriminating power between normal and pathological range samples, and correlated with test A (r = 0.61), whereas assay D (Coa-Set Fibrin monomer) showed little discriminating power at values below 10 micrograms/ml and little correlation with other assays. Standardization of assays will require further characterization of analytes detected.

Clinical efficacy of a highly purified SD‐treated factor IX concentrate prepared by conventional chromatography

Summary. A highly purified (HP) FIX concentrate was used in 12 patients with haemophilia B in order to prevent bleeding following surgery. The HP FIX concentrate (CRTS, Lille, France) was prepared by ion‐exchange chromatograpy and was SD treated. The patients underwent orthopaedic surgery (3), neurosurgery (3) or multiple dental extraction (6). They were treated for 2–18 days and received 8500 to 102000 FIX units. The mean FIX:C recovery was 0·92 ± 0·20%/U/kg. Satisfactory haemostasis was provided in all patients. None of them experienced thrombotic manifestations. There was no change in FDP or soluble fibrin complexes. Prothrombin fragments one plus two (F1 + 2), thrombin‐antithrombin III complexes (TAT) and cross‐linked fibrin degradation products (D‐dimer) remained in the normal range in one patient treated for 2·5 days for dental surgery. In contrast, elevated levels of F1+2, TAT and D‐dimer were observed in one patient undergoing a bilateral hip arthroplasty treated for 16 consecutive days with the same lot of HP FIX concentrate as the previous patient. There was no seroconversion for HIV, HBV and HCV. These results indicate that the HP FIX concentrate provides satisfactory haemostasis in surgery while avoiding excessive thrombogenic risks. However, some clinical circumstances, such as orthopaedic surgery, are known to be associated with especially high thrombotic risks that must be taken into account in case of FIX replacement.

Determination of the topology of factor XIIIa-induced fibrin gamma-chain cross-links by electron microscopy of ligated fragments.

After fibrin polymerizes to form a clot, the transglutaminase Factor XIIIa cross-links the gamma and alpha chains to stabilize the clot. There has been conflicting evidence on whether the gamma chain isopeptide bonds occur between molecules that are interacting in a longitudinal (end-to-end) manner or transverse (half-staggered) manner between the two strands of the protofibril. Since the topology of the cross-links has important consequences for fibrin structure, as well as for its stability and susceptibility to and pattern of fibrinolysis, cross-linked fibrin fragments were examined by electron microscopy to distinguish between these two possibilities for the arrangement of the ligated molecules. Cross-linked fibrin clots were produced by prolonged incubation of fibrinogen with thrombin and Factor XIII, and then digested with plasmin. The resulting soluble cross-linked fibrin complexes were rotary-shadowed with tungsten and examined by electron microscopy, revealing protofibril-like structures consisting of clusters of globular domains with a repeat of 22.5 nm. Longer plasmin digestion times yielded increasingly shorter structures. Rotary-shadowed cross-linked fibrin fragments, produced by dilution of the complexes into 0.125% acetic acid at pH 3.5 to dissociate all non-covalently linked fragments, showed uniformly single-stranded structures with a characteristic spacing of nodules, consistent with longitudinal cross-linking. Long, thin strands were seen at short digestion times, while shorter strands appeared with longer digestion. The smallest structures observed included two nodules together, and two such nodules with another nodule at a short distance from one or both ends, compatible with fragments DD, DY, and YY. Longer strands had the appearance of fibrin molecules that were linked end-to-end, usually with a fragment D or Y at each end. In conclusion, these results are consistent with previously proposed structures of these derivatives and clearly demonstrate that the interactions between cross-linked gamma chains are longitudinal (end-to-end) and not transverse.

Effects of Poloxamer 188 on the Assembly, Structure and Dissolution of Fibrin Clots

Poloxamer 188, N.F. (RheothRx), a nonionic block copolymer composed of 2 hydrophilic polyoxyethylene chains connected by a hydrophobic polyoxypropylene chain, normalizes the viscosity of whole blood high in soluble fibrin(ogen) complexes. Normalization may be via a poloxamer 188-induced decrease in fibrin(ogen)-red cell interaction. Our study examined the influences of poloxamer 188 on fibrin assembly and structure. Studies were performed in both purified and plasma systems with a poloxamer 188 concentration range of 0.1-20 mg/ml and specific clotting conditions (fibrinogen 1 mg/ml, thrombin 1 NIH u/ml, pH 7.4 [Tris 0.05 M], ionic strength 0.15 and calcium 5 mM). Fibrin assembly was accelerated in the presence of poloxamer 188. As poloxamer 188 concentration was increased from 0 to 8 mg/ml in plasma: a) the lag phase prior to initial turbidity rise decreased from 25 to less than 5 s; b) the final gel optical density (OD) increased from 0.65 to 1.28 and c) fiber size (mass/length ratio [mu]) increased from 4.3 to 12.6 x 10(13) daltons/cm. Similar results were seen in the purified system with a poloxamer 188 concentration range of 0-8 mg/ml. OD increased from 0.26 to 0.51, and mu increased from 2.3 to 5.3 x 10(13) daltons/cm. Above 8 mg/ml, precipitation of fibrinogen was noted in this system. Since large fibrin fibers tend to be degraded more rapidly, possible poloxamer mediated enhancement of r-tPA-mediated clot lysis was investigated. With r-tPA (70 lu/ml) present at the time of clotting, clot lysis in the presence of ploxamer 188 (8 mg/ml) was 50% complete at 1,600 s compared to 2,540 s for the control. Thus, poloxamer 188-induced alterations in fibrin structure and fibrin-cell interactions may explain some of this agent's interesting hemorrheologic and antithrombotic properties.