A descriptive investigation of the ultrastructure of fibrin networks in thrombo-embolic ischemic stroke

Stroke is one of the leading causes of death worldwide. Formation of a fibrin clot is controlled by a group of tightly regulated plasma proteases and cofactors and a change in the fibrin fiber formation causes an alteration in clot morphology. This plays an important role during thrombotic events. In the current study we investigated the ultrastructure of fibrin networks from fifteen ischemic stroke patients by using scanning electron microscopy. Clot morphology was investigated with and without the addition of human thrombin to the platelet rich plasma. Previously it was shown that, when studying the ultrastructure of fibrin networks, the addition of thrombin is necessary to form an expansive, fully coagulated layer of fibers. Results from the addition of thrombin to the plasma showed thick, matted fibrin fibers and a net covering some of the major fibers in stroke patients. Typical control morphology with major thick fibers and minor thin fibers could be seen in some areas in the stroke patients. In stroke patients, without the addition of thrombin, a matted fibrin network still formed, indicating that the factors responsible for the abnormal fibrin morphology are present in the circulating plasma and is the cause of the observed matted, layered morphology. This is not present in healthy individuals. From the results obtained we suggest that this changed morphology might be useful in a screening regime to identify the possibility of a stroke or even to follow the progress of stroke patients after treatment.     

Comparing techniques: the use of recalcified plasma in comparison with citrated plasma alone and in combination with thrombin in ultrastructural studies

Fibrin plays a vital role in the coagulation process and fibrin fiber morphology can be studied using ultrastructural techniques. When studying the ultrastructure of fibrin networks, thrombin may be added to the plasma, ensuing fibrin network formation. The question that arises is whether there are differences in morphology when thrombin is added to plasma, versus morphology observed when plasma from citrated or recalcified citrated whole blood, is studied. The current study therefore aimed to compare ultrastructure of platelets and fibrin networks from these three techniques. Results indicated comparable platelet ultrastructure between smears formed from the plasma of citrated blood and that of the citrated recalcified blood. This method might give us further information regarding the 'natural state' fibrin assembly and association with platelets, when studying haemostasis. However, when studying the ultrastructure of fibrin networks, the addition of thrombin is necessary to form an expansive, fully coagulated layer of fibrin fibers.     

Comparing techniques: the use of recalcified plasma in comparison with citrated plasma alone and in combination with thrombin in ultrastructural studies

Abstract

Fibrin plays a vital role in the coagulation process and fibrin fiber morphology can be studied using ultrastructural techniques. When studying the ultrastructure of fibrin networks, thrombin may be added to the plasma, ensuing fibrin network formation. The question that arises is whether there are differences in morphology when thrombin is added to plasma, versus morphology observed when plasma from citrated or recalcified citrated whole blood, is studied. The current study therefore aimed to compare ultrastructure of platelets and fibrin networks from these three techniques. Results indicated comparable platelet ultrastructure between smears formed from the plasma of citrated blood and that of the citrated recalcified blood. This method might give us further information regarding the ‘natural state’ fibrin assembly and association with platelets, when studying haemostasis. However, when studying the ultrastructure of fibrin networks, the addition of thrombin is necessary to form an expansive, fully coagulated layer of fibrin fibers.     

The changed ultrastructure of fibrin networks during use of oral contraception and hormone replacement

Contraceptives and hormone replacement have been extensively used since the late 1950s. However, adverse effects are common and include an increased risk of cardiovascular diseases, including thrombo-embolic diseases. Previous research has shown that ultrastructure of fibrin networks may provide great insight regarding the thrombotic potential of patients. The current study investigates the scanning electron microscopy (SEM) ultrastructure of fibrin networks of individuals using oral contraceptive therapy as well as individuals using hormone replacement. We compare micrographs of these two groups with micrographs of young, healthy individuals not using oral contraception. Platelet rich plasma and thrombin was used to prepare the fibrin clots. Here we show that during contraceptive and hormone replacement use, a netted fibrin layer forms. We suggest that oestradiol use causes fibrin network changes and these changes can be seen using SEM technology. These changes may provide further evidence regarding the increased occurrence of thrombotic events during contraceptive and hormone replacement therapy.     

Differences in fibrin fiber diameters in healthy individuals and thromboembolic ischemic stroke patients

Cerebrovascular disease is one of the leading causes of death and the cause of long-term adult disability. An important characteristic of thromboembolic ischemic stroke is a prothrombotic or hypercoagulable state and altered fibrin clot structure, whereas a resistance to fibrinolysis is also present. An expansive fibrin network is created when adding thrombin, and in stroke, the network appears thickened, netted and matted, compared with that of healthy individuals. Although this is clearly visible in micrographs of patients, there is a need to quantify the changes. The current study, therefore, investigates fibrin fiber diameters in stroke patients and compares it to healthy individuals. The fiber diameters were measured in nanometres, with University of Texas Health Science Center at San Antonio (UTHSCSA) Image Tool. A total of 100 measurements were done for each of the 12 patients in the healthy control group, and the same number of measurements was done for 12 stroke patients. These measurements were statistically analysed with NCSS 2007, using a significance level of 0.05. Normality was assessed with the Shapiro-Wilk W test and the thickest and thinnest fiber of each individual in the two groups was quantified and differences between groups were assessed with the Student's t-test. Results showed that there is a statistical difference in fibrin fiber thickness during thromboembolic ischemic stroke. We conclude that the changed coagulation and hemostasis, typically associated with stroke, causes a statistically relevant change in fibrin thickness, and that this netted and matted network is more resistant to lyses.     

Hepatic ultrastructure after methotrexate therapy for rheumatoid arthritis

Twenty-six patients receiving long-term oral methotrexate (MTX) therapy for rheumatoid arthritis (24 patients) or psoriasis (2 patients) were prospectively evaluated for alterations in liver morphology by light microscopy, electron microscopy, and immunofluorescence microscopy. Although only 4 MTX-treated patients had light microscopic evidence of mild fibrosis, all had evidence of collagen deposition in the space of Disse near Ito cells and changes in hepatocyte lysosomes on electron microscopy. These findings were absent from control livers. Fibrinogen, fibronectin, and type IV collagen were identified by immunofluorescence in both MTX-treated patients and controls. We conclude that long-term MTX therapy for rheumatoid arthritis is associated with alterations in hepatic ultrastructure, including collagen deposition in the space of Disse and changes in hepatocyte lysosomes.     

Comparison of polymerization of ancrod and thrombin fibrin monomers.

The polymerization of thrombin and ancrod fibrin monomers was studied with a standardized technique that evaluated turbidity changes and protein incorporation into the clot. Ancrod fibrin monomers were found to polymerize more slowly and form less turbid clots (at identical protein concentrations). Changes in ionic strength and pH influences ancrod fibrin monomer polymerization to a greater extent than thrombin fibrin monomer polymerization. Benzyltriethylammonium chloride was shown to be a potent inhibitor of fibrin monomer polymerization, with a greater inhibitory effect on ancrod fibrin monomers than on thrombin fibrin monomers. The differences between ancrod and thrombin fibrin may play a role in the infrequent thrombotic complications reported with ancrod therapy.     

Fibrinopeptide A reactive peptides and procoagulant activity in bronchoalveolar lavage: relationship to rheumatoid interstitial lung disease

Extravascular, primarily, alveolar fibrin deposition is commonly associated with the alveolitis of many interstitial lung diseases including the interstitial lung disease associated with rheumatoid arthritis (RA). We therefore hypothesized that coagulation pathways, which promote fibrin formation, would be activated in the alveolar lining fluids of patients with rheumatoid interstitial lung disease. To test this hypothesis, we studied the bronchoalveolar lavage (BAL) fluids from patients with rheumatoid interstitial lung disease (n = 7) and patients with RA unassociated with interstitial lung disease (n = 10) to characterize and quantitatively compare the BAL procoagulant material and levels of fibrinopeptide A (FPA), which is cleaved from fibrinogen by thrombin. FPA reactive peptide concentrations were significantly greater in rheumatoid interstitial lung disease than RA when normalized per ml of concentrated BAL fluid (p = 0.02), per mg BAL total protein (p = 0.01) or BAL albumin content (p = 0.03) and correlated with BAL antigenic neutrophil elastase concentrations (r = 0.87). Procoagulant activity was present in similar concentration of BAL of patients with RA and rheumatoid interstitial lung disease and was mainly attributable to tissue factor associated with factor VII (or VIIa). Our results demonstrate that tissue factor and factor VII are endogenous in the alveoli of subjects with RA and interstitial lung disease and could interact with distal coagulation substrates which may enter the alveoli in interstitial lung disease to locally promote fibrin deposition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presence of foam cells containing oxidised low density lipoprotein in the synovial membrane from patients with rheumatoid arthritis.

OBJECTIVE--Increased concentrations of lipid peroxidation products have been described in the serum and synovial fluid from patients with rheumatoid arthritis. A large proportion of the unsaturated lipids in human extracellular fluids is a component of low density lipoprotein (LDL). The oxidative modification of LDL, and its subsequent uptake by macrophages, has been implicated in the pathogenesis of atherosclerosis, but not of rheumatoid arthritis. This study aimed to assess whether oxidatively modified LDL was present in the rheumatoid synovium. METHODS--A polyclonal antiserum raised in rabbits against oxidised LDL (o-LDL) was used to perform an immunohistochemical study of a series of synovial biopsy specimens from patients with rheumatoid arthritis. RESULTS--Collections of positively stained macrophages, arranged in a linear fashion and with the morphological characteristics of foam cells--that is, 'fatty streaks', were identified around blood vessels within the intimal connective tissue. In addition, scattered, positively stained foam cells were present in association with deposits of fibrin. These staining patterns were absent from control synovial membranes (traumatic knee injuries). CONCLUSIONS--The findings in all rheumatoid patients studied suggest that atherosclerosis and rheumatoid arthritis have analogous pathogenetic features.     

Studies on cold insoluble globulin. I Concentrations in citrated plasma in rheumatic disorders.

Cold insoluble globulin (CIG) is a normal glycoprotein of human serum and plasma. The physiological significance of this protein is unknown, but is shows a temperature-dependent relation to fibrinogen and fibrin. It is possible that it represents a substrate for activated fibrin-stabilising factor in the polymerisation of fibrin. CIG is found on the surface of fibroblasts. In the present study CIG was estimated in citrated plasma in 115 patients with rheumatic diseases. Increased amounts were found in patients with systemic lupus erythematosus, secondary amyloidosis in classical and definite rheumatoid arthritis, and in male patients with juvenile rheumatoid arthritis.     

Incidence of hip involvement and need for total hip replacement in rheumatoid arthritis. An eight-year follow-up study

In an 8-year follow-up of a prospective study of recent arthritis, 103 patients were found to have erosive and 97% seropositive rheumatoid arthritis. A 20% incidence of hip joint involvement was observed among them. Moderate radiological changes could be seen in 10%, severe destruction (Larsen 4 or 5) in 3%, and acetabular protrusion in 1% of the patients. It was estimated that the minimum annual need of total hip replacement in rheumatoid arthritis in Finland would be 470. This is about 100 per million in the overall population. At the end of the follow-up the Larsen X-ray index was highly significantly (p less than 0.001), ESR significantly (p less than 0.005), and CRP clearly (p less than 0.03) higher in the 13 patients with radiologically confirmed hip changes than in the 90 without them. Thus their hip destruction is attributable to a more severe disease condition and not to the glucocorticoids administered. Furthermore, HLA-B27 was positive in 69% of the above-mentioned 13 patients compared with 27% of the rest, and this difference (p less than 0.005) may also explain the poor prognosis for true rheumatoid arthritis patients.     

Studies on the basis for the properties of fibrin produced from fibrinogen-containing gamma' chains

Human fibrinogen 1 is homodimeric with respect to its gamma chains (gammaA-gammaA'), whereas fibrinogen 2 molecules each contain one gammaA (gammaA1-411V) and one gamma' chain, which differ by containing a unique C-terminal sequence from gamma'408 to 427L that binds thrombin and factor XIII. We investigated the structural and functional features of these fibrins and made several observations. First, thrombin-treated fibrinogen 2 produced finer, more branched clot networks than did fibrin 1. These known differences in network structure were attributable to delayed release of fibrinopeptide (FP) A from fibrinogen 2 by thrombin, which in turn was likely caused by allosteric changes at the thrombin catalytic site induced by thrombin exosite 2 binding to the gamma' chains. Second, cross-linking of fibrin gamma chains was virtually the same for both types of fibrin. Third, the acceleratory effect of fibrin on thrombin-mediated XIII activation was more prominent with fibrin 1 than with fibrin 2, and this was also attributable to allosteric changes at the catalytic site induced by thrombin binding to gamma' chains. Fourth, fibrinolysis of fibrin 2 was delayed compared with fibrin 1. Altogether, differences between the structure and function of fibrins 1 and 2 are attributable to the effects of thrombin binding to gamma' chains.     

Fibrin and fibronectin in rheumatoid synovial membrane and rheumatoid synovial fluid

Normal synovial membranes and synovial membranes from patients with classic rheumatoid arthritis were investigated for the presence of fibrin and fibronectin by an indirect immunoperoxidase technique. In normal synovial membranes, fibronectin was found around the monolayer of the synovial lining cells. Staining was most intense on the surface and beneath the lining cells, but not detectable in the cytoplasm. Fibronectin was also found in the cytoplasm of the endothelial cells. No staining for fibrin was found in the normal synovial membrane. In synovial membranes from patients with rheumatoid arthritis, large amounts of fibronectin were found around the multilayer of synovial lining cells, in the cytoplasm of the endothelial cells, and in argyrophilic fiber-rich connective tissue. In superficial areas denuded of synovial lining cells, high amounts of fibronectin were found incorporated in fibrin. In some areas with noninjured synovial lining cells, fibrin was also found, but in this case no fibronectin was incorporated. No fibronectin was found in connective tissue in areas with infiltration of inflammatory cells. After treatment of normal and rheumatoid synovial membranes with hyaluronidase, fibronectin was still present around the lining cells but the staining was found to be more distinct. This study relates the presence of fibrin and fibronectin in the rheumatoid synovial membrane to the high amount of these proteins, recently described, in rheumatoid synovial fluid. It also suggests that fibronectin present in the synovial membrane is produced and secreted by the endothelial cells.     

Thrombocytosis in rheumatoid arthritis.

Of 75 patients with rheumatoid arthritis, 39 had a thrombocytosis and 36 normal platelet count. A highly significant relationship existed between the platelet count and disease severity and an inverse correlation with level of haemoglobin. An association appeared to exist between thrombocytosis and extra-articular manifestations of rheumatoid disease. By 75Selenomethionine labelling platelet and fibrinogen survival and turnover were determined. In 3 rheumatoid patients with thrombocytosis platelet survival was decreased and turnover increased. In these and a further rheumatoid patient with a normal platelet count there was reduced fibrinogen survival and increased fibrinogen turnover, and in addition excess fibrin degradation products were detected. The results suggest that thrombocytosis accompanies the more severe cases of rheumatoid disease and is due to a compensatory increase in platelet production associated with active intravascular coagulation.     

Coagulation abnormalities in rheumatoid disease

Forty-one patients with rheumatoid arthritis, including 6 with acute vasculitis, 13 with chronic vasculitis, and 22 without vasculitis, were studied for evidence of intravascular coagulation and fibrinolysis (ICF). The mean plasma fibrinogen levels were elevated in all groups. The fibrinogen, platelet count, and fibrin split products were usually elevated in acute vasculitis. Fewer patients on corticosteroids had abnormal coagulation tests. Active plasmin was detected in 12 patients primarily with chronic vasculitis. Plasminogen activator activity was not diminished in vascular endothelium of normal appearing skin of those patients with or without vasculitis. None of the patients demonstrated decompensated intravascular coagulation and fibrinolysis. The results suggest over-compensated ICF occurring in rheumatoid arthritis, but rheumatoid patients with vasculitis cannot be clearly distinguished from those without vasculitis on the basis of the usual tests performed for coagulation and fibrinolysis abnormalities.     

Stable occurrence of knee and hip total joint replacement in Central Finland between 1986 and 2003: an indication of improved long-term outcomes of rheumatoid arthritis

BACKGROUND: Total joint replacement (TJR) surgery is an important severe long-term outcome of rheumatoid arthritis, but relatively little is known about changes of its incidence in patients with rheumatoid arthritis over the past two decades. METHODS: A population-based, retrospective, incidence case review was conducted to analyse the frequency of primary TJR surgery of the knee and hip in all patients, and specifically in patients with rheumatoid arthritis in Central Finland between 1986 and 2003. Patients with TJR surgery of the knee and hip were identified in hospital databases over the 18-year period. Age-standardised incidence rate ratios for the primary TJR of the knee and hip were calculated, stratified to sex and diagnosis, with 1986 as the reference value. RESULTS: In patients without rheumatoid arthritis the age-adjusted incidence rate ratios (with 95% CI) for TJR of the knee increased 9.8-fold from 1986 to 2003 in women and men, and for TJR of the hip 1.8-fold in women and 2-fold in men. By contrast, no meaningful change was seen over this period, in age-adjusted incidence rate ratios for TJR of the knee or hip in patients with rheumatoid arthritis, ranging from 0.7 to 1.2 in 2003 compared with 1986. CONCLUSION: The prevalence of TJR surgery has increased 2-10-fold in patients without rheumatoid arthritis patients, associated with an ageing population, but has not increased in patients with rheumatoid arthritis between 1986 and 2003. These data are consistent with emerging evidence that long-term outcomes of rheumatoid arthritis have improved substantially, even before the availability of biological agents.     

Significance of clinically latent bacterial arthritis

The classical septic bacterial arthritis is a rare event, but can be distinguished by unequivocal signs such as fibrin exudation and neutrophilic masses (pus). For a long time we have been observing an abortive form of bacterial arthritis in biopsies which subsides spontaneously without antibiotic intervention. Only very early during the course can Staphyolcoccus aureus or epidermidis be detected. Despite the brief presence of bacteria, enzymes from the neutrophils can destroy cartilage and bone. The fact that the attending physician had suspected a bacterial infection in only 7% of these patients highlights the diagnostic complexity. We have termed this form clinically latent bacterial arthritis (CLBA). Structural changes in the synovial membrane, e.g. in rheumatoid arthritis or osteoarthritis, predispose for a hematogenous seeding of endogenous staphylococci and trigger clinically unapparent, temporary infections. This clinically latent bacterial superinfection (CLSI) is also self-limiting, but the high degrading potential of the neutrophilic proteases makes CLSI a very probable contributing factor in joint destruction.     

Dynamic magnetic resonance imaging for the evaluation of synovitis in patients with rheumatoid arthritis

Objective. To investigate whether the findings of magnetic resonance imaging (MRI) reflect rheumatoid synovitis. Methods. Dynamic imaging enhanced with gadolinium—diethylenetriamine pentaacetic acid was performed on 10 affected knees of 9 patients with rheumatoid arthritis. Changes in signal intensity were correlated with pathologic findings in synovial biopsy specimens obtained during total knee arthroplasty. Results. Enhancement was greater in regions with a higher degree of fibrin exudation, cellular infiltration, villous hypertrophy, vascular proliferation, and granulation formation. Conclusion. Dynamic MRI can be used for assessing local disease activity in rheumatoid synovium.     

Studies on the ultrastructure of fibrin lacking fibrinopeptide B (beta- fibrin)

Release of fibrinopeptide B from fibrinogen by copperhead venom procoagulant enzyme results in a form of fibrin (beta-fibrin) with weaker self-aggregation characteristics than the normal product (alpha beta-fibrin) produced by release of fibrinopeptides A (FPA) and B (FPB) by thrombin. We investigated the ultrastructure of these two types of fibrin as well as that of beta-fibrin prepared from fibrinogen Metz (A alpha 16 Arg----Cys), a homozygous dysfibrinogenemic mutant that does not release FPA. At 14 degrees C and physiologic solvent conditions (0.15 mol/L of NaCl, 0.015 mol/L of Tris buffer pH 7.4), the turbidity (350 nm) of rapidly polymerizing alpha beta-fibrin (thrombin 1 to 2 U/mL) plateaued in less than 6 min and formed a "coarse" matrix consisting of anastomosing fiber bundles (mean diameter 92 nm). More slowly polymerizing alpha beta-fibrin (thrombin 0.01 and 0.001 U/mL) surpassed this turbidity after greater than or equal to 60 minutes and concomitantly developed a network of thicker fiber bundles (mean diameters 118 and 186 nm, respectively). Such matrices also contained networks of highly branched, twisting, "fine" fibrils (fiber diameters 7 to 30 nm) that are usually characteristic of matrices formed at high ionic strength and pH. Slowly polymerizing beta-fibrin, like slowly polymerizing alpha beta-fibrin, displayed considerable quantities of fine matrix in addition to an underlying thick cable network (mean fiber diameter 135 nm), whereas rapidly polymerizing beta-fibrin monomer was comprised almost exclusively of wide, poorly anastomosed, striated cables (mean diameter 212 nm). Metz beta-fibrin clots were more fragile than those of normal beta-fibrin and were comprised almost entirely of a fine network. Metz fibrin could be induced, however, to form thick fiber bundles (mean diameter 76 nm) in the presence of albumin at a concentration (500 mumol/L) in the physiologic range and resembled a Metz plasma fibrin clot in that regard. The diminished capacity of Metz beta-fibrin to form thick fiber bundles may be due to impaired use or occupancy of a polymerization site exposed by FPB release. Our results indicate that twisting fibrils are an inherent structural feature of all forms of assembling fibrin, and suggest that mature beta-fibrin or alpha beta-fibrin clots develop from networks of thin fibrils that have the ability to coalesce to form thicker fiber bundles.     

Prevalence and clinical significance of antibodies to citrullinated fibrinogen (ACF) in Chinese patients with rheumatoid arthritis

It has been reported that citrullinated fibrin(ogen) deposits in the inflamed joints played an important role in the pathogenesis of rheumatoid arthritis (RA). Although antibodies to citrullinated fibrinogen (ACF) have been detected in the sera of RA patients, the associations between ACF and RA remain unclear. In this study, human fibrinogen was citrullinated by peptidylarginine deiminase in vitro, and the ACF were detected by an enzyme-linked immunosorbent assay in rheumatic patients, including 183 RA, 121 systemic lupus erythematosus, 48 osteoarthritis, and 108 healthy controls. The prevalence of ACF was determined, and the associations between ACF and RA were evaluated. It was shown that the sensitivity and specificity of ACF in RA were 67.21 and 84.84%, respectively. There were significant correlations between ACF and erythrocyte sedimentation rate, anti-cyclic citrullinated peptide antibody, and anti-keratin antibodies (AKA). In radiographic progression, the RA patients with ACF had higher scores than those without ACF according to the Sharp–van der Heijde method. In addition, ACF was often positive in the RA patients who were IgM rheumatoid factor negative or AKA negative or anti-perinuclear factor negative. The results indicate that ACF assay is helpful for the diagnosis of RA.