Biochemical and Physical Properties of a Solvent-Detergent-Treated Fibrin Glue

A fibrin glue preparation has been obtained from pooled human plasma using a procedure which includes a solvent-detergent (SD) treatment to inactivate lipid-enveloped viruses. The SD treatment inactivated greater than or equal to 5.5 log10 of HIV in less than 45 min, and greater than or equal to 5 log10 and greater than or equal to 6.5 log10 of VSV and Sindbis virus, respectively, in less than 2 h. The product was found to contain high quantities of fibrinogen (116 +/- 2.49 g/l; n = 12), factor XIII (35 +/- 2.88 U/ml) and von Willebrand factor (23 +/- 1.9 U/ml ristocetin cofactor activity), and relatively low levels of fibronectin (5.9 +/- 0.51 g/l). Plasminogen, the precursor of plasmin, which may play a negative role by decreasing the resistance of the fibrin clot, was at only 0.03 g/l. Cellulose acetate electrophoresis showed 95% gamma-proteins and 5% alpha-2-beta proteins. Sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions detected three main protein bands with apparent molecular weights of 65, 56 and 47 kilodaltons, probably corresponding to the alpha, beta, and gamma fibrinogen subunits. Other characteristics of the product included (1) high clottability of fibrinogen (over 85%); (2) absence of low molecular weight fibrin degradation products; (3) rapid solubilization at room temperature (less than 10 min); (4) high tensile strength (202 +/- 27 g/cm2 after 2 h of application), and (5) high elasticity of the fibrin clot. In addition, scanning electron microscopy revealed a highly organized structure showing tridimensional arrangement of the fibrin fibers. SD treated fibrin glue should efficiently replace autologous fibrinogen or cryoprecipitate preparations for surgical application.     

Antibiotic concentrations in serum and wound fluid after local gentamicin or intravenous dicloxacillin prophylaxis in cardiac surgery

One important aim of antibiotic prophylaxis in cardiac surgery is preventing mediastinitis and thus it would appear to be relevant to study the antibiotic concentrations in pericardial/mediastinal fluid. Local administration of gentamicin in the wound before sternal closure is a novel way of antibiotic prophylaxis and could be effective against bacteria resistant to intravenous antibiotics. This study measured dicloxacillin concentrations in 101 patients in serum and wound fluid following intravenous administration of dicloxacillin. Similarly, concentrations of gentamicin in serum and wound fluid were determined in 30 patients after administration of 260 mg gentamicin in the wound at sternal closure. Median dicloxacillin concentrations in serum and wound fluid at sternal closure were 59.4 and 55.35 mg/l, respectively. Gentamicin levels in the wound were very high (median 304 mg/l), whereas serum concentrations were low (peak median 2.05 mg/l). Dicloxacillin, 1 g given intravenously, according to the clinical protocol, resulted in levels in serum and wound fluid at sternal closure likely to prevent Staphylococcus aureus infections. Locally administered gentamicin resulted in high local concentrations, potentially effective against agents normally considered resistant.     

Quantitation of the Reticuloendothelial System Clearance of Soluble Fibrin

Phagocytosis of particulate fibrin has been previously established as a function of the reticuloendothelial system (RES). More recently, reticuloendothelial cells have been shown to bind soluble fibrin/fibrinogen (f/F) complexes in vitro. To quantitate RES clearance vs microthrombus formation, varying doses (0.1-6 mg/kg) of 125I-soluble f/F was injected into rabbits. One hour later the animals were killed, at which time 48 +/- 8% of the 125I f/F had been cleared from the blood. Homogenized organ samples were separated into insoluble pellet, soluble protein bound, and free 125I. Treatment of other samples with plasmin prior to homogenization differentiated the insoluble 125I into RES cleared (intracellular-plasmin resistant) vs microthrombi (plasmin sensitive pellet 125I). 125I-f/F was chiefly found in liver and spleen. Injection of low f/F concentrations resulted in no plasmin sensitive pellet 125I. 3 mg/kg f/F caused small, variable amounts of plasmin sensitive pellet 125I, chiefly in the kidney. With 6 mg/kg, 21-50% of the insoluble 125I in all organs was plasmin sensitive, and occasional 1-2 mm thrombi were found. The data indicate complete and rapid RES clearance of small amounts of soluble f/F from the blood, without microthrombi being formed. The RES was acutely saturated at 1.5-3.0 mg f/F/kg, which is equivalent to immediate conversion to fibrin of 1-2% of the intravascular fibrinogen pool.     

Pharmacodynamics of thrombolysis with recombinant tissue-type plasminogen activator. Correlation with characteristics of and clinical outcomes in patients with acute myocardial infarction. The TAMI Study Group

Coagulation analysis was performed on blood samples from 386 patients with acute myocardial infarction drawn before, during, and after a continuous intravenous infusion of 150 mg recombinant tissue-type plasminogen activator (rt-PA) (Activase). Plasma rt-PA rose to peak levels of 2.1 +/- 3.1 micrograms/ml (mean +/- SD). Fibrinogen levels measured by coagulation rate and by sulfite precipitation decreased from baseline levels of 3.0 +/- 0.9 and 3.2 +/- 1.0 g/l, respectively, to nadir levels of 1.4 +/- 0.75 and 1.8 +/- 0.92 g/l, respectively, and were associated with peak levels in serum of fibrinogen-degradation products (FDP) of 230 +/- 470 micrograms/ml. Forty percent of patients experienced a nadir functional-fibrinogen level of less than 1.0 g/l, whereas 20% fell below 0.5 g/l. Nadir fibrinogen levels did not correlate with patency of the infarct-related coronary artery at 90 minutes or with risk of coronary vessel reocclusion within 7-10 days. However, the risk of coronary artery reocclusion was inversely related to the baseline functional fibrinogen level (p = 0.0008), with the magnitude of its drop to nadir level (p = 0.0003) as well as to peak levels of FDP (p = 0.038). Quantitative blood loss correlated with all markers for systemic fibrinogenolysis including nadir fibrinogen level (r = -0.20, p = 0.0011), percent decrease of fibrinogen (r = 0.22, p = 0.001), and peak FDP levels (r = 0.14, p = 0.020). Both patients who experienced intracranial hemorrhage presented with high baseline fibrinogen levels and experienced extensive degradation of coagulable fibrinogen. Overall, patients at greatest risk of systemic fibrinogenolysis tended to be relatively older women with lower body weight.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prothrombin fragments F1+2, thrombin-antithrombin III complexes, fibrin monomers and fibrinogen in patients with coronary atherosclerosis

We determined the plasma levels of prothrombin fragment F1+2, thrombin-antithrombin III complexes (TAT), fibrin monomers (FM), D-dimers (DD) and fibrinogen in 57 patients with angiographically verified graded coronary artery disease (CAD) free of concomitant peripheral atherosclerosis, cerebrovascular disease or diabetes mellitus and a group of 21 apparently healthy controls. Blood was collected from the antecubital vein through atraumatic venipuncture prior to the angiographic procedure. Plasma levels of hemostatic markers were related to the presence and graded severity of CAD. The levels of prothrombin fragment F1+2 (1.74+/-0.11 vs. 1.0+/-0.07 nmol/l, P<0.001), FM (41.6+/-5.5 vs. 7.42+/-3.05 nmol/l, P<0.001), TAT (15.6+/-2.7 vs. 2.96+/-0.32 microg/l, P<0.001) and fibrinogen (3.64+/-1.3 vs. 3.08+/-0.33 g/l, P<0.01) were significantly higher in patients with CAD compared to controls, while there was no difference regarding the fibrinolytic system represented by DD (441.6+/-58.9 vs. 337.4+/-42.05 microg/l, n.s.). Within the CAD group, patients with extensive coronary atherosclerosis (> or =2 vessel disease) had significantly higher values for prothrombin fragment F1+2 (1.89 vs. 1.57 nmol/l, P = 0.04), FM (50.7 vs. 29.8 nmol/l, P = 0.03), and a trend to significance was noted for fibrinogen (3.9 vs. 3.3 g/l, P = 0.07) suggesting that blood coagulability was related to the severity of the disease and that hemostatic markers of thrombin activity represent a useful tool to identify patients with a latent hypercoagulable state with a higher susceptibility to sustain coronary thrombosis.     

Thrombolytic versus fibrinogenolytic activity of rt-PA and streptokinase in patients with acute myocardial infarction

In this study the concentration of plasma breakdown products of cross-linked fibrin (XDP), serum fibrinogen-fibrin degradation products (FDP), and plasma fibrinogen were measured before and at the end of the administration of single-chain recombinant tissue-type plasminogen activator (rt-PA, 100 mg IV over three hours) or streptokinase (1.5 million units over one hour), respectively, in two groups, each composed of 22 patients with acute myocardial infarction. The XDP concentration was not statistically different between the two groups at the end of thrombolytic treatment, whereas FDP and fibrinogen concentrations were significantly different (FDP: streptokinase 396 +/- 287 vs rt-PA 177 +/- 222 micrograms/mL, p less than 0.01; fibrinogen: streptokinase 71 +/- 43 vs rt-PA 181 +/- 49 mg/dL, p less than 0.001). These results indicate that the two drugs have equipotent thrombolytic activity at this administration regimen but that rt-PA causes a markedly more selective lysis of fibrin in comparison with streptokinase.     

Plasma tissue factor plus activated peripheral mononuclear cells activate factors VII and X in cardiac surgical wounds

OBJECTIVES: The purpose of this study was to test the hypothesis that activated monocytes with soluble plasma tissue factor (pTF) activate factors VII and X to generate thrombin. BACKGROUND: Despite heparin, thrombin is progressively generated during cardiac surgery with cardiopulmonary bypass (CPB), produces intravascular fibrin and fibrinolysis, and causes serious thromboembolic and nonsurgical bleeding complications. Thrombin is primarily produced in the surgical wound, but mechanisms are unclear. METHODS: In 13 patients, interactions of mononuclear cells, platelets, pTF, and pTF fractions to activate factors VII and X were evaluated in pre-bypass, perfusate, and pericardial wound blood before and during CPB. RESULTS: Monocytes are activated in wound, but not in pre-bypass or perfusate plasma (monocyte chemotactic protein-1 = 29.5 +/- 2.1 pmoles/l vs. 2.8 +/- 1.2 pmoles/l and 3.3 +/-1.4 pmoles/l, respectively). Wound pTF is substantially elevated compared to other locations (3.64 +/- 0.45 pmoles/l vs. 0.71 +/- 0.65 pmoles/l and 1.31 +/- 1.4 pmoles/l). Supernatant wound pTF contains 81.7% of TF antigen; wound microparticle pTF contains 18.3%. Wound monocytes and all C5a-stimulated monocytes (but not activated platelets) completely convert factor VII to factor VIIa with wound pTF. Activated monocytes more efficiently activate factor X with wound supernatant TF/factor VII(VIIa) complex than with wound microparticle TF/factor VII(fVIIa). The correlation coefficient (r) between wound thrombin generation (F1.2) and wound pTF concentration is 0.944 (p = 0.0004). CONCLUSIONS: During cardiac surgery with CPB, wound monocytes plus wound pTF or wound microparticle-free supernatant pTF preferentially accelerate activation of factor VII and factor X. This system represents a novel mechanism for thrombin generation via the TF coagulation pathway.     

Fibrin fragment D-dimer and fibrinogen B beta peptides in plasma as markers of clot lysis during thrombolytic therapy in acute myocardial infarction

The validity of markers in plasma of in vitro thrombolysis was investigated in 12 patients with extensive fibrinogen breakdown (greater than 80%, group 1) and in 12 patients with minimal breakdown (less than 20%, group 2). The patients were treated with 100 mg of recombinant tissue-type plasminogen activator (rt-PA) in the "Thrombolysis in Myocardial Infarction II" (TIMI II) trial. Cross-linked fibrin degradation product levels were measured with two variant enzyme-linked immunosorbent assays (ELISAs), both using a fibrin fragment D-dimer specific capture antibody. In one instance, a tag antibody was used that cross-reacts with fibrinogen (pan-specific tag ELISA); in the other, the tag antibody was specific for fibrin fragment D (fibrin-specific tag ELISA). Apparent concentrations of cross-linked fibrin degradation products at baseline were within normal limits with both assays in most patients. At 8 hours after rt-PA infusion, the measured cross-linked fibrin degradation products were increased about twofold to fourfold in group 2 with both assays. However, in group 1, levels were significantly higher with the pan-specific tag ELISA (5.8 +/- 4.2 micrograms/mL) compared with the fibrin-specific tag ELISA (1.5 +/- 1.3 micrograms/mL). This observation was most likely a result of detection of fibrinogen degradation products in the pan-specific ELISA. Apparent levels of fibrinopeptide B beta 1-42, a marker of fragment X formation, increased during thrombolysis from 4.2 +/- 2.8 pmol/mL to 2,000 +/- 230 pmol/mL in group 1 and from 4.1 +/- 2.1 pmol/mL to 300 +/- 43 pmol/mL in group 2, and were correlated significantly with the extent of fibrinogen breakdown (r = -0.8). Fibrinopeptide beta 15-42 levels increased from 4.3 +/- 3 pmol/mL to 70 +/- 19 pmol/mL in group 1, but did not increase in group 2. The apparent increase in group 1 could be explained by cross-reactivity of fibrinopeptide B beta 1-42 in the fibrinopeptide beta 15-42 assay. We conclude that cross-linked fibrin degradation product levels as measured with a pan-specific tag ELISA and fibrinopeptide beta 15-42 levels as measured with certain monoclonal antibody-based ELISA are influenced by the extent of fibrinogen degradation. Fibrinopeptide B beta 1-42 is a marker specific for fibrinogen breakdown. Cross-linked fibrin degradation product levels, measured with a fibrin-specific tag ELISA, appear to be markers specific for thrombolysis. Consequently, assays similar to the fibrin-specific tag ELISA may provide more accurate information when correlated with clinical endpoints.     

Fibrinogen metabolism in the lungs

The aim of this study was to evaluate the role of the pulmonary vessel endothelium in the metabolism of fibrinogen (FBG), by measuring the FBG, D-dimer, and fibrin(ogen) degradation product levels in the blood from pulmonary and radial arteries from 99 patients undergoing aortocoronary bypass. For comparison, protein C, protein S, and factor VII, were also measured. The results showed, with respect to the pulmonary arterial blood levels, significantly lower FBG levels (3.72 +/- 0.83 vs 3.66 +/- 0.81 g/L; P < .001) and higher fibrin(ogen) degradation product levels (7.36 +/- 1.53 vs 8.15 +/- 1.59 mg/L; P < .000 01) in the radial arterial blood. No difference was found for d -dimer, protein C, protein S, and factor VII. The study demonstrated that the pulmonary capillary endothelium contributes to the FBG catabolism for about a 0.02 fractional rate and support the view of an endothelial FBG catabolic pathway as the main catabolic pathway, owing to the fact that the pulmonary endothelial surface is about a 0.1 fraction of the peripheral vessel endothelial surface.     

Serum lipids, serum insulin, plasma fibrinogen and aerobic capacity in obese and non-obese Singaporean boys.

OBJECTIVES: To compare blood lipids, lipoproteins, apoproteins, fibrinogen, insulin and aerobic capacity in obese and non-obese Chinese Singaporean boys. To examine relationships between blood metabolites, body composition and aerobic capacity in these groups. DESIGN: Cross-sectional. SUBJECTS: Forty Chinese Singaporean boys aged 13-15 y. Classified as obese (n=20) or non-obese (n=20) based on adiposity (fat mass/fat free mass): >0.60=obese, <0.40=non-obese. MEASUREMENTS: Body composition (dual energy X-ray absorptiometry), waist circumference, peak oxygen consumption (VO(2) peak), serum concentrations of total cholesterol, triacylglycerol, high density lipoprotein cholesterol (HDL-C), total cholesterol/HDL-C, apoproteins AI and B, lipoprotein(a), insulin and glucose. Plasma concentration of fibrinogen. RESULTS: Obese boys had significantly (P<0.01) higher (mean+/-s.d.) concentrations of serum triacylglycerol (1.51+/-0.65 vs 1.04+/-0.34 mmol/l), serum insulin (24.1+/-11.5 vs 12.3+/-4.45 mU/l) and plasma fibrinogen (4.01+/-0.54 vs 3.35+/-0.76 g/l) than non-obese boys. Within the non-obese group plasma fibrinogen concentration was significantly related to percentage body fat (r=0.546, P<0.05). VO(2) peak relative to body mass (ml/kg/min or ml/kg(-0.67)/min) was significantly (P<0.001) lower in obese compared to non-obese boys but absolute VO(2) peak (l/min), adjusted for fat-free mass via analysis of covariance, was higher in obese than non-obese boys (P<0.01). Partial correlations revealed that none of the blood metabolites were significantly related to VO(2) peak independent of body fatness. CONCLUSIONS: Obesity was related to elevated concentrations of serum triacylglycerol, serum insulin and plasma fibrinogen in Chinese Singaporean boys. These elevated concentrations did not appear to be associated with a lower aerobic capacity (independent of body fatness) in the obese.     

The effect of fibrinogen concentrate administration on coagulation abnormalities in a rat sepsis model.

Disseminated intravascular coagulation is a disorder that affects the function of the clotting system and is frequently associated with sepsis or septic shock. One of its leading symptoms is the decrease in circulating fibrinogen. We investigated the effect of fibrinogen concentrate (Haemocomplettan P) on fibrinogen plasma levels, coagulation parameters and mortality in a rat model of sepsis-induced disseminated intravascular coagulation. The disseminated intravascular coagulation was characterized by elevated thrombin-antithrombin complex and a sharp drop in circulating fibrinogen. Coagulation abnormalities were evaluated by thrombelastography. Plasma fibrinogen levels decreased from 2.06 +/- 0.2 to 0.16 +/- 0.1 g/l following administration of bacterial lipopolysaccharide. Thrombelastographic measurements revealed a concurrent decrease in maximum amplitude and an increase in reaction time. Treatment with fibrinogen concentrate (Haemocomplettan P, 25-200 mg/kg body weight intravenously) resulted in a statistically significant dose-dependent increase in fibrinogen plasma levels and amelioration of the measured coagulation abnormalities. Fibrinogen plasma concentrations were restored to normal levels when 200 mg/kg body weight fibrinogen concentrate was administered. A significant decrease in sepsis-induced mortality was observed in animals treated with Haemocomplettan P.     

Fibrinogen (Fb) concentrations in patients with ischaemic heart disease undergoing coronary angiography

Increasing evidence suggests the role of hemostatic risk factors in the development of ischemic heart disease (IHD). A raised plasma fibrinogen has been related to increased risk of IHD. The aim of the study was to determine the relationship between plasma fibrinogen and the coronary vessels state based on the coronary angiogram. 119 patients undergoing coronary angiography were classified into 5 groups according the severity of IHD: Group 0 without significant atherosclerotic lesions (control group), Group 1 with single vessel disease, Groups 2, 3 with multivessel disease (two and three affected arteries, respectively) and Group 4 with positive history of myocardial infarction. A statistically nonsignificant rise in fibrinogen levels in Groups 1, 2, 3 (3.9 +/- 0.8 g/l, 4.0 +/- 0.9 g/l, 4.1 +/- 0.9 g/l, respectively) as compared to control Group 0 (3.7 +/- 0.7 g/l) was found. In Group 4 plasma fibrinogen was significantly lower (2.8 +/- 0.6 g/l) comparing to Group 0 (p < 0.05). In addition plasma fibrinogen was positively correlated with blood pressure. These results supports the role of raised plasma fibrinogen in the pathogenesis and development of IHD.     

Intraperitoneal heparin in peritoneal dialysis and its effect on fibrinopeptide A in plasma and dialysate

In 6 patients on continuous ambulatory peritoneal dialysis we investigated the inhibition of intraperitoneal fibrin formation by heparin. A continuous addition of 500 U of heparin per liter dialysate was used for 52 h. In plasma no heparin activity could be detected, even 52 h after intraperitoneal administration of heparin. The fibrin formation was determined by fibrinopeptide A, a thrombin-induced split product of fibrinogen. In patients under regular continuous ambulatory peritoneal dialysis we determined the fibrinopeptide A concentrations in plasma. The values were comparable with the fibrinopeptide A concentrations measured in disseminated intravascular coagulopathy. They decreased during intraperitoneal administration of heparin from 63.2 +/- 11.8 to 4.9 +/- 1.7 ng/ml. The fibrinopeptide A concentration in the 4-hour intraperitoneal dialysate (155.8 +/- 15.7 ng/ml) decreased after heparin administration to 8.5 +/- 2.0 ng/ml and was always higher than in plasma. We conclude that 500 U heparin per liter dialysate prevents the intraperitoneal fibrin formation. The low antithrombin III concentration (0.44 +/- 0.13 mg/dl) in protein-poor dialysate seems to be sufficient to inhibit the thrombin activity after acceleration by heparin.     

Relation between diabetes compensation, albuminuria and biochemical parameters and the results of stress myocardial SPECT in asymptomatic type 2 diabetics

The patients with diabetes mellitus and another risk factors have significantly higher risk to suffer from ischemic heart disease. Myocardial stress SPECT represents the examination which correlates very well with the results of selective coronary angiography even in asymptomatic diabetic patients. The aim of our study was to evaluate the relation of SPECT result to diabetes compensation, presence of micro/macroalbuminuria, blood level of fibrinogen, CRP, homocysteine and uric acid. Out of 126 diabetic 2. type abnormal SPECT has been found in 33 (26%). Fasting blood sugar (9.3 +/- 1.4 mmol/l in patients with abnormal SPECT vs. 9.7 +/- 1.9 mmol/l in the other diabetics, n.s.) and HbA1c (7.5 +/- 1.3% vs. 7.5 +/- 1.3%, n.s.) are not significantly different in the patients with abnormal SPECT to the other diabetics without this finding. Micro/macroalbuminuria was significantly more frequently seen in patients with abnormal SPECT (60% of patients with abnormal SPECT and 29% in the rest of diabetics, p = 0.01). Fibrinogen was significantly more elevated in diabetics with abnormal SPECT (3.76 +/- 0.5 g/l in the group with abnormal SPECT vs. 3.23 +/- 0.43 g/l, p = 0.0003). In the diabetics with abnormal SPECT we have found significantly higher CRP (3.84 +/- 1.51 mg/l vs. 2.79 +/- 1.13 mg/l, p = 0.024) and homocysteine (13.78 +/- 3.26 micromol/l vs. 10.98 +/- 2.33 micromol/l, p = 0.006). Uric acid level was not significantly different in the group of diabetics with abnormal SPECT to the rest of the patients (361 +/- 64 micromol/l in abnormal SPECT vs. 353 +/- 51 micromol/l, n.s.). When we analyse our results we have found that abnormal SPECT is rarely discovered in the asymptomatic 2nd type diabetics with the combination of negative micro/macroalbuminuria and fibrinogen level below 3.5 g/l.     

The fibrinogen functional turbidimetric assay.

Hitherto, clinical fibrinogen methods were based on coagulation seconds, with assay conditions not similar to a plasma milieu. The fibrinogen functional turbidimetric assay included 50 microL citrated plasma + 100 microL 300 mIU/mL thrombin, 400 microg/mL polybrene, and 6% albumin-phosphate-buffered saline; an increase in absorbance at 405 nm/5 min at room temperature (or 2 minutes at 37 degrees C) was observed. In all, 6% albumin in the fibrinogen functional turbidimetric assay reagent abolishes falsely elevated fibrinogen to fibrin turbidity in hypoproteinemic plasma samples. This assay can detect fibrinogen activity of 250% to 300% of normal, the lower detection limit being 7% of normal (0.2 g/L). The normal range of this assay is 100% +/- 20% (mean value +/- 1 SD; coefficient of variations <4%). This assay imitates fibrinogen to fibrin conversion in clotting blood plasma; it is independent of plasmatic albumin or heparin and can be performed everywhere. This assay has a diagnostic value in pathology-disseminated intravascular coagulation and in assessing risk for atherothrombosis.     

Composition of fibrin glues significantly influences axial vascularization and degradation in isolation chamber model

In this study, different fibrin sealants with varying concentrations of the fibrin components were evaluated in terms of matrix degradation and vascularization in the arteriovenous loop (AVL) model of the rat. An AVL was placed in a Teflon isolation chamber filled with 500 μl fibrin gel. The matrix was composed of commercially available fibrin gels, namely Beriplast (Behring GmbH, Marburg, Germany) (group A), Evicel (Omrix Biopharmaceuticals S.A., Somerville, New Jersey, USA) (group B), Tisseel VH S/D (Baxter, Vienna, Austria) with a thrombin concentration of 4?IU/ml and a fibrinogen concentration of 80 mg/ml [Tisseel S F80 (Baxter), group C] and with an fibrinogen concentration of 20 mg/ml [Tisseel S F20 (Baxter), group D]. After 2 and 4 weeks, five constructs per group and time point were investigated using micro-computed tomography, and histological and morphometrical analysis techniques. The aprotinin, factor XIII and thrombin concentration did not affect the degree of clot degradation. An inverse relationship was found between fibrin matrix degradation and sprouting of blood vessels. By reducing the fibrinogen concentration in group D, a significantly decreased construct weight and an increased generation of vascularized connective tissue were detected. There was an inverse relationship between matrix degradation and vascularization detectable. Fibrinogen as the major matrix component showed a significant impact on the matrix properties. Alteration of fibrin gel properties might optimize formation of blood vessels.     

Improved glycaemia in type 1 diabetes results in decreased levels of soluble adhesion molecules with no change in serum adiponectin or most acute phase proteins.

AIMS: To investigate how reduction of hyperglycaemia affects acute phase serum proteins in poorly controlled type 1 diabetic patients. METHODS: 24 patients (age 31.7 +/- 2.0 years, HbA1c 9.3 +/- 0.2%, BMI 24.2 +/- 0.7 kg/m2, diabetes duration 15.3 +/- 1.7 years) participated in the study. The treatment was optimised for 16 weeks. Blood samples were taken at baseline and at the end of the study. 16 healthy age- and BMI-matched subjects were chosen for a control group. RESULTS: At baseline, the patients had higher C-reactive protein (CRP) (1.09, median [range 0.24-18.82] mg/l vs. 0.66 [0.18-2.46] mg/l, p < 0.02), mean adiponectin (16.06 +/- 1.31 vs. 8.85 +/- 0.93 mg/l, p < 0.001), ceruloplasmin (306 +/- 16.1 vs. 205.4 +/- 5.5 mg/l, p < 0.001), fibrinogen (3.41 +/- 0.26 vs. 2.38 +/- 0.07 g/l, p < 0.001), soluble intercellular adhesion molecule-1 (sICAM-1) (255.4 +/- 10.3 vs. 194 +/- 10.6 microg/l, p < 0.001), soluble vascular adhesion molecule-1 (sVCAM-1) (533.4 +/- 21.8 vs. 422.9 +/- 20.7 microg/l, p < 0.01) and interleukin-6 (2.89 +/- 0.49 vs. 1.35 +/- 0.30 ng/l, p < 0.01) concentrations than the controls. During intensified treatment, HbA1c decreased (to 8.5 +/- 0.2%, p < 0.001). This resulted in reduced sE-selectin (from 44.6 +/- 2.6 to 38.8 +/- 2.6 microg/l, p < 0.01), alpha-1-acid-glycoprotein (A1GP) (from 622.9 +/- 47.9 to 525.7 +/- 27.9 mg/l, p < 0.01), sICAM-1 (from 255.4 +/- 10.3 to 240.8 +/- 9.1 microg/l, p < 0.05) and IL-6 (from 2.9 +/- 0.5 to 2.1 +/- 0.4 ng/l, p < 0.01). Serum amyloid A (SAA) and CRP did not change 12.00 (0.7-222.0) vs. 12.00 (1.6-277.0) mg/l for SAA and 1.09 (0.24-18.82) vs. 1.09 (0.18-23.08) mg/l for CRP, baseline vs. treatment, respectively. CONCLUSIONS: Poorly controlled type 1 diabetic patients have increased values of adiponectin, CRP, ceruloplasmin, fibrinogen, sICAM-1, sVCAM-1 and IL-6. Reduction of hyperglycaemia results in decreased sE-selectin, A1GP, sICAM-1 and IL6, while other inflammatory factors including CRP, SAA and adiponectin are not affected.     

Inhibition of thrombus formation by activated recombinant protein C in a primate model of arterial thrombosis

Activated protein C (APC) is an antithrombotic enzyme. The therapeutic potential of infused human recombinant APC (rAPC) was studied in a primate model of platelet-dependent thrombosis. Eight baboons with chronic femoral arteriovenous shunts received rAPC infusions for 1 hour. The shunts were extended with 5-cm long, 4-mm-i.d. thrombogenic Dacron graft segments for the time of infusion. The plasma level of the enzyme, the blood flow in the shunt, and the deposition of indium-111-labeled platelets and iodine-125 fibrinogen on the graft were measured. The influence of rAPC infused at doses of 0.25 and 1.0 mg/kg-hr was compared with the effects of control infusions of saline. Five of eight control grafts occluded within 60 minutes, whereas there was no change in the blood flow during rAPC infusion. Deposition of platelets was inhibited by 13 +/- 10% and by 42 +/- 13% (mean +/- SEM) after 30 minutes of infusion at the two doses, which gave rise to circulating rAPC plasma concentrations of 0.4 and 1.9 mg/l, respectively. Both doses significantly inhibited fibrin deposition in the graft. Circulating plasma markers of thrombus formation and of fibrinolysis did not increase significantly during rAPC infusion; measurements of bleeding time were also within normal limits. Thus, rAPC, like human plasma-derived APC, inhibited thrombus formation without impairing primary hemostasis.     

Studies on the functionality of fibrinogen during rt-PA therapy: results of three different methods of fibrinogen determination.

The conversion of soluble fibrinogen to insoluble fibrin is a crucial event in the process of haemostasis. In the present study three different methods were used for the determination of plasma fibrinogen levels in 12 patients with acute myocardial infarction during rt-PA therapy. Two methods were based on fibrin formation and thus measured 'functional' fibrinogen: a clotting rate assay according to the Clauss method and a photometric polymerization rate assay. Thirdly, fibrinogen antigen was measured with a new enzyme immunoassay (EIA) for immunologically intact fibrinogen. The levels of functional and intact fibrinogen were strikingly different: 4.1 g/l (Clauss) and 3.6 g/l (photometric) vs 2.6 g/l (EIA) at baseline, 2.2 g/l (54% of preinfusion value) and 2.1 g/l (59%) vs 2.1 g/l (81%) at 90 min, 5.7 g/l (146%) and 4.5 g/l (130%) vs 2.7 g/l (106%) at 72 h. The rebound phenomenon exhibited by functional fibrinogen (130-150%) was not observed for levels of intact fibrinogen. A ratio was calculated of functional (Clauss method) to intact fibrinogen (EIA) for each individual patient and each time point. The mean ratio (n = 12 patients) was 1.6 at baseline, 1.0 at 90 min, and increased markedly between 8 and 24 h to a maximum of 2.1 (P less than 0.01). Our data indicate that the 'functionality', i.e. the clotting rate per unit concentration of circulating fibrinogen, changed during acute myocardial infarction and subsequent thrombolytic therapy. This is in keeping with data from literature that the relative amount of fast clotting high molecular weight (HMW) fibrinogen of total fibrinogen was increased in the initial phase of acute myocardial infarction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tissue factor antigen and activity in serum of postoperatively shed blood used for autologous transfusion.

Orthopaedic surgery involves extensive dissection of connective and richly vascularised tissues rich in tissue factor (TF). It was, therefore, of interest to quantify the amount of TF antigen and activity in postoperatively drained, unwashed wound blood collected for the purpose of autologous transfusion. In nine young patients subjected to surgery for idiopathic thoracic scoliosis, samples were drawn postoperatively from collected shed blood, a pulmonary artery catheter and a radial arterial cannula prior to, during and after reinfusion of shed blood (10 ml/kg body weight), and analysed for TF antigen and activity. Preoperative arterial control samples contained 128 pg/ml TF antigen compared with 40 pg/ml postoperatively. During reinfusion of drained blood, arterial TF concentration rose to 96 pg/ml and dropped to 64 pg/ml after infusion. Arterial and mixed venous blood did not differ significantly in TF levels. Serum from drained blood contained high concentrations of TF antigen (773 pg/ml), but no TF activity was detected. It is concluded that the high concentrations of TF antigen in serum from postoperatively drained blood collected for autologous transfusion are devoid of procoagulant activity. The TF antigen in plasma of drained blood is suggested to be a soluble, proteolysed TF-apoprotein or a TF complex inactivated by the TF pathway inhibitor (TFPI).