Blood vessel occlusion with erythrocyte aggregates causes burn injury progression-microvasculature dilation as a possible therapy.

Burns are dynamic injuries characterized by progressive tissue death and continuous severe pain over the course of several days. The extent of burn injury progression determines the ultimate patient outcome. Initial burns result in a central zone of necrosis surrounded by a potentially viable zone of ischemia. Several mechanisms have been proposed to explain injury progression, including oxidant and cytokine stress resulting from either ischemia/reperfusion and/or inflammation, but no proven therapy has emerged. To address the unmet need to limit burn injury progression, the root cause of this process must be delineated. For this reason, we have recently focused on post-burn blood vessel occlusion, currently ascribed to microthrombi. We have found that blood vessel occlusion is initially, mainly and persistently caused by erythrocyte aggregation. Although thermal-induced cell necrosis is the immediate cause of cell death, apoptotic cells from persistent ischemia/anoxia, admixed with inflammatory cells, form a band between viable and nonviable tissue 24 hours later. The delayed cell death by apoptosis appears to be the main attractant for inflammatory cells. Finally, we posit that fibrinogen elevation arising from inflammation provides stimulus for additional erythrocyte aggregation, further extending blood vessel occlusion. In our view this persistent occlusion with resultant prolonged tissue ischemia/anoxia, not ischemia/reperfusion, is the root cause of burn injury progression concomitant with associated severe and persistent pain. Epiviosamines, a new class of peptides, appear to selectively dilate microvasculature, and may provide therapy for burn injury progression.

The antileukemic activity of modified fibrinogen-methotrexate conjugate.

BACKGROUND: The search for new, innovative methods to treat all types of diseases, especially cancer-related ones, is a challenge taken by pharmaceutical companies and academic institutions. The use of conjugates containing widely-known and widely-used bioactive substances is one of the ways to solve this problem. Research into drug binding with macromolecular carrier systems has joined the search for new therapeutic strategies. METHODS: The main goal of this paper is the potential offered by the use of fibrinogen derivatives as an antileukemic drug carrier. Physicochemical properties of the obtained conjugate were analyzed, characterizing alterations in relation to the starting carrier and analyzing biological implications. The intraperitoneally (i.p.) inoculated P388 mouse leukemia model for in vivo studies was used. RESULTS AND CONCLUSIONS: Conjugates consisting of a fibrinogen derivative with a covalently bound anticancer drug were developed. Carrier preparation and a conjugate synthesis in aqueous solution were formulated, as well as purification of the conjugate was performed. The study showed that the survival of leukemia mice treated with FH-MTX conjugate was indeed significantly longer than survival in both untreated animals (control) and mice treated with unbound MTX. A significant increase in the antileukemic activity of MTX conjugated with hydrolysed fibrinogen was observed as compared with the unconjugated drug. Reported data suggest that hydrolysed fibrinogen can serve as a carrier molecule for the MTX drug with the aim of enhancing its antileukemic activity. GENERAL SIGNIFICANCE: Conjugates consisting of a fibrinogen derivative with a covalently bound anticancer drug seem to be a promising anticancer drug.

From artificial red blood cells, oxygen carriers, and oxygen therapeutics to artificial cells, nanomedicine, and beyond.

The first experimental artificial red blood cells have all three major functions of red blood cells (rbc). However, the first practical one is a simple polyhemoglobin (PolyHb) that only has an oxygen-carrying function. This is now in routine clinical use in South Africa and Russia. An oxygen carrier with antioxidant functions, PolyHb-catalase-superoxide dismutase, can fulfill two of the three functions of rbc. Even more complete is one with all three functions of rbc in the form of PolyHb-catalase-superoxide dismutase-carbonic anhydrase. The most advanced ones are nanodimension artificial rbc with either PEG-lipid membrane or PEG-PLA polymer membrane. Extensions into oxygen therapeutics include a PolyHb-tyrosinase that suppresses the growth of melanoma in a mice model. Another is a PolyHb-fibrinogen that is an oxygen carrier with platelet-like function. Research has now extended well beyond the original research on artificial rbc into many areas of artificial cells. These include nanoparticles, nanotubules, lipid vesicles, liposomes, polymer-tethered lipid vesicles, polymersomes, microcapsules, bioencapsulation, nanocapules, macroencapsulation, synthetic cells, and others. These are being used in nanotechnology, nanomedicine, regenerative medicine, enzyme/gene therapy, cell/stem cell therapy, biotechnology, drug delivery, hemoperfusion, nanosensers, and even by some groups in agriculture, industry, aquatic culture, nanocomputers, and nanorobotics.

Fibrinogênio sérico pré-operatório como preditor de infarto do miocárdio na cirurgia de revascularização miocárdica / Preoperative serum fibrinogen as a predictor of myocardial infarction in the surgical myocardial revascularization / Preoperative serum fibrinogen as a predictor of myocardial infarction in the surgical myocardial revascularization

OBJETIVO: Determinar o valor preditivo do nível sérico de fibrinogênio pré-operatório para a ocorrência de infarto do miocárdio (IM) no período perioperatório de cirurgia de revascularização miocárdica (CRM), bem como para outros desfechos de impacto, como acidente vascular encefálico isquêmico (AVEI), tromboembolismo pulmonar (TEP) e morte, isoladamente e de maneira composta. MÉTODOS: Estudo de coorte retrospectivo com análise do banco de dados de cirurgia cardíaca do Hospital São Lucas da PUC-RS, com 1.471 pacientes consecutivos que realizaram CRM com circulação extracorpórea entre janeiro de 1998 e dezembro de 2002. RESULTADOS: IM perioperatório ocorreu em 14 por cento dos pacientes da amostra. Não foi observada associação entre o fibrinogênio pré-operatório e IM perioperatório (410,60 ± 148,83 mg/dl para o grupo em estudo x 401,57 ±135,23 mg/dl para o grupo controle - p = 0,381 - RC = 1,000 - IC95 por cento: 0,998-1,002 - p = 0,652), o desfecho combinado de IM, AVEI, TEP e morte (411,40 ± 153,52 mg/dl para o grupo com o desfecho x 400,31 ± 131,98 mg/dl para o grupo sem o desfecho - p = 0,232) e nem com cada um destes isoladamente. CONCLUSÃO: Nesta amostra, o nível sérico de fibrinogênio pré-operatório não apresentou associação com a ocorrência de IM perioperatório nas CRM, nem mesmo com outros desfechos de impacto, incluindo AVEI, TEP e morte, isoladamente ou em conjunto.

OBJECTIVE: Determine the predictive level of preoperative serum fibrinogen level for the occurrence of MI in perioperative surgical myocardial revascularization (SMR), as well as for other impacting outcomes, such as stroke, pulmonary thromboembolism (PTE), and death, separately or in combination. METHODS: A retrospective cohort study based on the heart surgery database analysis from São Lucas Hospital, at Rio Grande do Sul Catholic University with 1,471 consecutive patients submitted to extracorporeal SMR between January, 1998 and December, 2002. RESULTS: Perioperative MI occurred in 14 percent of sample patients. No association was shown between preoperative fibrinogen and perioperative MI (410.60 ± 148.83 mg/dl for the study group x 401.57 ± 135.23 mg/dl for control group - p = 0.381 - RC = 1.000 - CI95 percent: 0.998-1.002 - p = 0.652), combined outcome for MI, stroke, PTE, and death (411.40 ± 153.52 mg/dL for the group reporting outcome x 400.31 ± 131.98 mg/dL for the group with no outcome - p = 0.232) and neither separately. CONCLUSION: In that sample, preoperative serum fibrinogen level did not show any association with the occurrence of perioperative MI in SMR, neither with other impacting outcomes, stroke, PTE, and mortality, whether separately or as composite endpoints.

Antileukemic activity of glycated fibrinogen-methotrexate conjugates.

BACKGROUND: The aim of the study was to compare the antileukemic activity of methotrexate (MTX) conjugates with native and glycated fibrinogen. We expected that conjugates based on glycated fibrinogen would reveal higher antileukemic activity because of decreased plasmin digestibility and a higher retention rate of glycated fibrinogen in the body. MATERIALS AND METHODS: Fibrinogen was glycated using a high-temperature procedure at 65-85 degrees C. Glycated fibrinogens were examined with respect to their ability to clot and susceptibility to plasmin digestion. Native fibrinogen (F) and fibrinogens glycated at 65 and 73 degrees C (F65 and F73) were conjugated with MTX and tested in mice bearing P388 leukemia, at a dose of 40 mg of MTX per kg of body weight. RESULTS: Glycated fibrinogens retained their ability to clot. Compared to native fibrinogen, they were more resistant to digestion by plasmin. All tested conjugates revealed higher antitumor activity than the free drug. Increases in average lifespan over the control group were 34% for free MTX, 137% for F-MTX, 151% for F65-MTX and 91% for F73-MTX. The differences between the antitumor activities of all conjugates were not statistically significant. CONCLUSION: It seems necessary to compare the antitumor activities of MTX conjugates based on native and glycated fibrinogen in different tumor models, to demonstrate the expected differences.

Cytotoxic and antitumor effect of fibrinogen-methotrexate conjugate.

In this paper we describe the chemical procedure of fibrinogen-methotrexate (F-MTX) conjugate preparation and its in vitro and in vivo antitumor activity. F-MTX conjugates were synthesized in reaction of fibrinogen with MTX N-hydroxysuccynimide ester. The conjugates were not cross-linked and were soluble in water. The results of the in vitro and in vivo studies have shown: (1) a lower in vitro cytotoxicity of the F-MTX conjugate as compared with MTX alone; (2) a significantly higher in vivo antitumor activity of the F-MTX conjugate in mice with P388 leukemia as compared with MTX alone; (3) a significantly increased in vivo lethal toxicity of F-MTX as compared with MTX. The results suggest the therapeutic utility of the fibrinogen-methotrexate conjugate and the usefulness of fibrinogen as a chemotherapeutic drug carrier. However, a new effort in the preparation of F-MTX conjugate should be made to decrease its in vivo toxicity.

Catabolism of unglycated and naturally glycated forms of rabbit fibrinogen: their interaction with the healthy and deendothelialized aorta wall in normal and diabetic rabbits.

The incidence of nonenzymatic glycation of proteins within the hyperglycemic environment of diabetic plasma is increased compared with that in normal (i.e., nondiabetic) plasma. Whether glycation in vivo alters the behavior of proteins within the circulation is not well understood. Glycated fibrinogen, although not detected in normal rabbit plasma, was isolated from diabetic rabbit plasmas (glucose concentration 12 to 39 mmol/L) and separated from unglycated fibrinogen by boronate chromatography. The yield of glycated fibrinogen, which amounted to 3% to 6% of total fibrinogen, correlated with the content of plasma glucose. Glycated and unglycated fibrinogens facilitated aggregation of normal or diabetic platelets to a similar extent after adding adenosine-5'-diphosphate. Normal platelets stimulated by adenosine-5'-diphosphate bound more iodine 131-glycated fibrinogen than iodine 125-unglycated fibrinogen (p < 0.05), whereas the quantities of glycated and unglycated fibrinogens bound by diabetic platelets were not significantly different. When coinjected intravenously into normal or diabetic rabbits, 131I-glycated fibrinogen was cleared from the circulation faster than 125I-unglycated fibrinogen although the catabolic rates, measured as half-life, were not significantly different. At equilibrium, glycated fibrinogen was distributed significantly more in the extravascular and less in the vascular compartments of the normal and diabetic rabbit compared with the unglycated type. After balloon injury to the aorta in vivo, the unglycated/glycated ratio of radiolabeled fibrinogens associated with the platelet monolayer was 0.94, whereas for the damaged subendothelium the ratio was 1.20 (p < 0.005). We conclude that glycation in vivo changes several metabolic characteristics of fibrinogen in the normal and diabetic rabbit.

Immunoelectrophoretic and immunohistochemical characterizations of fibrinogen derivatives in atherosclerotic aortic intimas and vascular prosthesis pseudo-intimas.

Cadaveric aortic intimas with uncomplicated atherosclerosis were examined to determine the distribution and polypeptide chain composition of fibrinogen-related protein. Immunohistochemical staining showed deposits rich in fibrinopeptides A and B. The deposits were usually disseminated throughout intimas of moderate thickness < 0.7 mm, but were distributed focally in elongate patches bounded both lumenally and medially by deposit-free tissue in thick atheromas. Saline extracts generally showed undegraded monomers and dimers by electrophoresis. The residual protein contained A alpha and gamma-chains that were cross-linked predominantly (>80%) into unresolved high M(r) (>200 kd) derivatives, whereas B beta-chains were left non-cross-linked, as occurs in late stages of cross-linking by transglutaminases. The resolved components had electrophoretic mobilities corresponding to characteristic products of both factor XIIIa and tissue-transglutaminase. A greater incorporation of alpha- rather than gamma-chains into cross-linked products implicated tissue-transglutaminase as contributing heavily. By contrast, vascular graft pseudo-intimas and a cadaveric clot were rich in degraded fibrin devoid of fibrinopeptide A, and cross-linked in patterns typical of XIIIa with gamma 2 dimers constituting the principal product. The findings indicate that the fibrinogen in the aortic intima is comparatively well protected from thrombin and plasmin, and that much of it is deposited through direct cross-linking by tissue-transglutaminase without being converted to fibrin.

Peptides and monoclonal antibodies which bind to platelet glycoproteins IIb and/or IIIa inhibit clot retraction.

The rate of clot retraction in platelet-rich plasma was decreased by synthetic peptide analogues of fibrinogen and monoclonal antibodies each of which bind to the platelet plasma membrane glycoproteins IIb and/or IIIa. These and related data demonstrate that intact complexes of the glycoproteins IIb and IIIa are required for platelet-mediated clot retraction.

The effect of fibrinogen-methotrexate derivatives on HeLa cell growth.

Proteolytic cleavage of bovine fibrinogen with covalently bound methotrexate (MTX) was studied using four different proteolytic enzymes--trypsin, chymotrypsin, pepsin, and cathepsin D and the interaction of the modified fibrinogen (or fibrin) with HeLa cells was investigated. The presence of fibrin-MTX derivative did not induce any significant morphological alternations of cells. The fibrin-MTX derivative in the gel form was solubilized easily by the action of all proteinases investigated, hydrolysis of highly crosslinked denatured fibrin-MTX in suspension proceeded slower. The solubilized fibrin-MTX degradation products had a strong inhibiting effect on the growth of HeLa cells cultured in monolayer indicating the liberation of chemotherapeutically active MTX from its fibrin derivative.

Glycosylated A alpha chains in chicken fibrinogen.

Fibrinogen immunoprecipitated from cultured chick embryo hepatocytes was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and compared with fibrinogen from chicken plasma. The character and relatedness of the constituent polypeptide bands were established on the basis of enzymatic treatment, peptide analysis, metabolic labeling with [14C]glucosamine, and inhibition of glycosylation with tunicamycin. Hepatocyte-derived fibrinogen resolved into five polypeptides that, in order of decreasing apparent molecular weight, were identified as glycosylated A alpha, non-glycosylated A alpha, B beta, gamma', and gamma. Fibrinogen immunoprecipitated directly from chicken plasma yielded an identical profile except for an additional smaller A alpha chain. This small A alpha chain appears to be the product of partial proteolysis in the circulation and was the only A alpha band found in purified plasma fibrinogen (fraction I-2). The observation of glycosylated A alpha chains is novel. The gamma/gamma' chain heterogeneity appears to be due to an amino acid extension similar to that observed in mammalian fibrinogens. Fibrinogen from cells exposed to fetal bovine serum, a potent stimulator of fibrinogen production, was enriched in glycosylated A alpha chains, which constituted approximately one-third of the A alpha chain population. Serum did not affect the gamma/gamma' chain distribution.

Binding of fibrinogen to ADP-treated platelets. Comparison of plasma fibrinogen fractions and early plasmic fibrinogen derivatives.

Fibrinogen supports platelet aggregation by binding to specific receptors. The importance of the fibrinogen A alpha chain in this hemostatic function is controversial. We found that fibrinogen derivatives, isolated from plasma or obtained after limited plasmin digestion, that lacked approximately 13,000 to 46,000 MW peptides from the carboxyterminal of their A alpha chains (I-6, I-9, I-9D88) displayed undiminished capacity to support ADP-induced platelet aggregation and to bind to gel-filtered platelets. Analysis of their binding disclosed upwardly concave Scatchard plots that could be resolved into high- and low-affinity binding components similar to those of intact fibrinogen. The dissociation constant for high-affinity binding of fractions I-6 and I-9, however, was slightly higher than that of intact fibrinogen, correlating with the slight decrease in the rate of platelet aggregation observed using these fractions. Low-affinity binding was unchanged. In contrast, fibrinogen derivative I-9D88, lacking as much as 2/3 from the carboxyterminal side of both A alpha chains, was indistinguishable from intact fibrinogen in its ability to bind to platelets and support aggregation. This suggested that the small differences in binding affinities noted with fractions I-6 and I-9 were most likely due to changes in molecular conformation rather than to losses of specific peptides. A more degraded derivative (I-9D50) lacking even larger A alpha segments (MW 46,000 to 48,000), as well as aminoterminal segments (B beta 1-56) from the B beta chains, possessed only 70% to 75% of the platelet aggregating activity of intact fibrinogen. Its binding to ADP-treated platelets was quantitatively similar to that of intact fibrinogen but its Scatchard plot was linear, with loss of low-affinity binding. These data indicate that (1) fibrinogen binding to platelet receptors does not require the carboxyterminal 2/3 of the A alpha chain and (2) low-affinity platelet fibrinogen interactions as revealed by Scatchard analysis reflect fibrinogen binding to platelets via an aminoterminal segment of the A alpha and/or B beta chains, the loss of which results in a slight but significant decrease in platelet aggregation support.