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.

Hemodialysis patients have plasmatic hypercoagulability and decreased fibrinolytic vulnerability: role of carbon monoxide.

Chronic hemodialysis is associated with significant thrombophilia. Of interest, hemodialysis patients have increased carboxyhemoglobin (COHb) and exhaled carbon monoxide (CO), signs of upregulated heme oxygenase (Hmox) activity. Given that CO enhances plasmatic coagulation, we determined whether patients requiring chronic hemodialysis had an increase in endogenous CO, plasmatic hypercoagulability and decreased fibrinolytic vulnerability. Carbon monoxide was determined by noninvasive pulse oximetry measurement of COHb. Blood samples were obtained just before hemodialysis. Thrombelastographic methods to assess plasma coagulation kinetics, fibrinolytic kinetics, and formation of carboxyhemefibrinogen (COHF) were used. Hemodialysis patients (n = 45) had abnormally increased COHb concentrations of 2.2 ± 1.9%, indicative of Hmox upregulation. Coagulation and fibrinolytic parameter normal values were determined with normal individual (n = 30) plasma. Thirty-seven patients of the hemodialysis cohort had COHF formation (82.2%, [67.9%-92.0%]; mean, [95% confidence interval]), and many of this group of patients had abnormally great velocity of clot growth (73.3%, [58.1%-85.4%]) and strength (75.6%, [60.5%-87.1%]). Furthermore, over half of COHF positive patients had a hypofibrinolytic state, evidenced by an abnormally prolonged time to maximum rate of lysis (53.3%, [37.9%-68.6%]) and clot lysis time (64.4%, [48.8%-78.1%]). Carbon monoxide enhanced coagulation and diminished fibrinolytic vulnerability in hemodialysis patients. Future investigation of hemodialysis, CO-related thrombophilia is warranted.

Detection of carboxyhemefibrinogen and methemefibrinogen in a patient with thrombosis of a HeartMate II ventricular assist device.

Ventricular assist device (VAD) thrombosis is a devastating, potentially fatal complication suffered by patients requiring mechanical circulatory support. We present a patient with thrombosis of a HeartMate II VAD with concurrent hemolysis and increased carbon monoxide formation. Using a specialized thrombelastographic assay, we detected marked plasmatic hypercoagulability mediated in part by the formation of carboxyhemefibrinogen.

Characterization of fibrinogen glycosylation and its importance for serum/plasma N-glycome analysis.

The majority of proteins present in human serum/plasma are glycoproteins, validating this fluid as an ideal starting material for N-glycan analysis and discovery of potential biomarkers. The glycoprotein content for both serum and plasma is very similar, except for proteins removed in the coagulation process, including fibrinogen. Our aim was to characterize fibrinogen glycosylation in order to determine its contribution to differences between serum and plasma N-glycomes. N-Glycans from human fibrinogen were released, labeled, and analyzed by HILIC-HPLC and MS. Structural characterization of fibrinogen subunits revealed that the α chain was not N-glycosylated, whereas ß and γ contained identical oligosaccharide structures, mainly biantennary digalactosylated monosialylated structures (A2G2S1) and biantennary digalactosylated disialylated structures (A2G2S2). Blood was collected from five healthy volunteers into four testing tubes: silicone-coated glass for serum and EDTA, Na-heparin, and Li-heparin glass tubes for plasma. N-Glycans were analyzed using the high-throughput HILIC-HPLC method. N-Glycan profiles from serum and plasma samples differed largely in glycans identified in fibrinogen, suggesting that this glycoprotein represents a major factor distinguishing these body fluids. This result emphasizes the important of consistent body fluid collection practices in biomarker discovery studies.

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.

Comparison of plasma and peritoneal indices of fibrinolysis between foals and adult horses with and without colic.

OBJECTIVE: To identify hemostatic imbalances indicative of an increased risk of intra-abdominal adhesion formation in foals versus adult horses. ANIMALS: Horses with colic undergoing exploratory laparotomy or abdominocentesis as part of a clinical examination (n = 16 foals ≤ 6 months of age and 19 adults ≥ 5 years of age) and horses without colic undergoing herniorrhaphy (15 foals) or euthanasia for noninflammatory and nongastrointestinal disease (10 foals and 20 adults). PROCEDURES: Paired abdominal fluid and blood samples were collected from each horse into buffered sodium citrate and centrifuged immediately after collection. Supernatants were stored at -80°C, then thawed for measurement of fibrinogen concentration, plasminogen activity, antiplasmin activity, and D-dimer concentration. Supernatant analyte concentrations or activities were compared within age group (foals with and without colic vs adults with and without colic) and within disease status (foals and adults without colic vs foals and adults with colic). RESULTS: All analyte concentrations or activities in abdominal fluid samples were significantly lower in the noncolic groups than in the colic groups, and none differed between foal and adult groups. Several plasma analyte values differed by disease status and age. CONCLUSIONS AND CLINICAL RELEVANCE: The risk of intra-abdominal adhesion formation in the foals in this study did not appear to be attributable to differences in intra-abdominal hemostasis between adult horses and foals. Strategies for initial medical and surgical management of colic in adult horses may be applicable to foals with similar disorders.

Increased thrombin-induced polymerization of fibrinogen associated with high protein carbonyl levels in plasma from patients post myocardial infarction.

Increased levels of protein carbonyls have been measured in plasma of patients following a myocardial infarction (Mocatta et al. J. Am. Coll. Cardiol.49:1993-2000; 2007). In this study, we have examined representative plasma samples from this group of patients. We show that carbonyls are formed preferentially on fibrinogen and that there is a strong correlation between fibrinogen and total plasma protein carbonyls. Functional properties of fibrinogen isolated from post myocardial plasmas were investigated by measuring thrombin-catalyzed polymerization. Fibrinogen from plasma with upper quartile protein carbonyls (mean 0.16 nmol/mg protein) polymerized approximately 1.4 times more rapidly and gave 1.4-fold higher maximum turbidity (12 per group, P<0.001) than fibrinogen from lower quartile carbonyl plasma (mean 0.007 nmol/mg), which behaved similarly to control plasma. Significant differences were also apparent when related to the carbonyl content of the fibrinogen itself. These changes in the high carbonyl plasma reflect a faster rate of lateral aggregation of small oligomers to form fibrin polymers that comprise thicker, more loosely woven fibers. In vivo this could be translated into a tendency to clot faster and form more fragile clots. High protein carbonyls in fibrinogen were not associated with an increased content of nitrotyrosine or chlorotyrosine. Nitrotyrosine levels were significantly lower in fibrinogen than total plasma protein, with no difference between patients and controls. Chlorotyrosine levels in fibrinogen (but not total protein) were significantly higher for the post myocardial patients. Our data suggest that high fibrinogen protein carbonyls in heart disease could be a prothrombotic risk factor.

Polyhemoglobin-fibrinogen: a novel oxygen carrier with platelet-like properties in a hemodiluted setting.

Polyhemoglobin (polyHb) is currently being assessed in phase III trials under various formulations. At present, none contain clotting factors or platelet substitutes to aid in hemostasis. We have prepared a novel blood substitute that is an oxygen carrier with platelet-like activity. This is formed by crosslinking fibrinogen to hemoglobin to form polyhemoglobin-fibrinogen (polyHb-Fg). This was studied and compared to polyHb for its effect on coagulation both in vitro and in vivo. In the in vitro experiments, PolyHb-Fg showed similar clotting times as whole blood, whereas polyHb showed significantly higher clotting times. This result was confirmed in in vivo experiments using an exchange transfusion rat-model. Using PolyHb, exchange transfusion of 80% or more increased the normal clotting time (1-2 mins) to > 10 mins. Partial clots formed with PolyHb did not adhere to the tubing wall. With PolyHb-Fg, a normal clotting time is maintained, even with 98% exchange transfusion.

Immobilization of aprotinin to fibrinogen as a novel method for controlling degradation of fibrin gels.

The goal of this work was to demonstrate that aprotinin conjugated to fibrinogen could (1) maintain its function and (2) control fibrin degradation. Using the chick chorioallantoic membrane (CAM) assay, we found that blood vessels did not directly invade fibrin constructs containing immobilized fibroblast growth factor-2. Because the fibrin quickly degraded within approximately 5 days, we hypothesized that controlling fibrinolysis may improve direct blood vessel invasion. Aprotinin, a protease inhibitor typically added to slow fibrinolysis, is a small protein and can diffuse out of the gel resulting in the loss of fibrinolysis protection. Therefore, using a novel synthesis strategy, aprotinin and a fluorescent reporter, Cy3, were chemically conjugated to fibrinogen. In vitro microplate absorbance assays showed that the conjugated aprotinin was able to inhibit plasmin-mediated fibrin degradation and that its activity was comparable to equimolar levels of soluble, nonconjugated aprotinin. Additionally, we found that fibrinolysis rates could be tuned by varying the level of conjugated aprotinin within the gel. The conjugated aprotinin also demonstrated functionality in vivo. In the chick CAM assay, fibrin gels containing conjugated aprotinin were approximately 5 times larger than gels containing soluble aprotinin after 4 days. Also, in support of our hypothesis, we found that immobilized aprotinin within fibrin gels demonstrated substantial blood vessel invasion.

The effect of glycaemic control on fibrin network structure of type 2 diabetic subjects.

Diabetic subjects have been shown to have altered fibrin network structures. One possible cause may be fibrinogen glycation resulting in altered structure/function properties. We investigated the effect of glucose control on fibrinogen glycation and fibrin network structure in type 2 diabetes. Blood samples were taken from twenty uncontrolled diabetic subjects at baseline to determine the levels of fibrinogen glycation and fibrin network structures. The subjects were then treated with insulin until blood glucose control was achieved before end blood samples were taken. Twenty age- and BMI-matched non-diabetic subjects were included as a reference group. The diabetic subjects had significantly higher mean fibrinogen glycation at baseline than the non-diabetic subjects (7.84 vs. 3.89 mol glucose / mol fibrinogen; p < 0.001). This was significantly reduced during the intervention (7.84 to 5.24 mol glucose / mol fibrinogen; p < 0.0002) in the diabetic group. Both groups had high mean fibrinogen concentrations (4.25 and 4.02 g/l, diabetic and non-diabetic subjects respectively). There was no difference in fibrinogen concentration, porosity, compaction and kinetics of clot formation between the diabetic subjects and non-diabetic subjects at baseline, nor were there any changes during the intervention despite the reduced fibrinogen glycation. Fibrin network characteristics correlated well with fibrinogen but not with any markers of glycaemic control. Improved glycaemic control resulted in decreased fibrinogen glycation but not fibrinogen concentration. It seems as though porosity, compaction and kinetics of clot formation are more related to fibrinogen concentration than fibrinogen glycation in this model.

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.

Molecular cloning and comparative analysis of fibrinogen-related proteins from the soft tick Ornithodoros moubata and the hard tick Ixodes ricinus.

Among disease-vectors, the evolution of the tick innate immune system is still lagging when compared to insects. Such an investigation, which was initiated, by first cloning and sequencing lectins associated in the innate immunity of invertebrates and having fibrinogen related domains, helped in the sequencing of cDNA encoding for OMFREP from the soft tick, Ornithodoros moubata. Also obtained were Ixoderin A and Ixoderin B cDNA sequences from the hard tick Ixodes ricinus. Tissue-specific expression of OMFREP showed that it was present primarily in the hemocytes and salivary glands. Ixoderin A besides sharing a similar expression profile was also expressed in the midgut. Both showed significantly high homology to the lectin Dorin M, from O. moubata. Further, phylogenetic comparisons between these molecules of the soft and hard ticks showed their relatedness to Tachylectins 5A and 5B, involved in the innate immunity of Tachypleus tridentatus and ficolins from both vertebrates and invertebrates. Ixoderin B showing tissue-specific expression only in the salivary glands and the sequence displaying certain motif differences in homology point towards a possible function different from the other two molecules. This is the first report of lectin-like sequences, with a fibrinogen-domain, from the hard tick I. ricinus and a preliminary phylogenetic study of these tick sequences with related fibrinogen-domain containing sequences highlights a possible role for them in the innate immunity of the ticks.

In vivo magnetic resonance imaging of coronary thrombosis using a fibrin-binding molecular magnetic resonance contrast agent.

BACKGROUND: The advent of fibrin-binding molecular magnetic resonance (MR) contrast agents and advances in coronary MRI techniques offers the potential for direct imaging of coronary thrombosis. We tested the feasibility of this approach using a gadolinium (Gd)-based fibrin-binding contrast agent, EP-2104R (EPIX Medical Inc), in a swine model of coronary thrombus and in-stent thrombosis. METHODS AND RESULTS: Ex vivo and in vivo sensitivity of coronary MR thrombus imaging was tested by use of intracoronarily delivered Gd-DTPA-labeled fibrinogen thrombi (n=6). After successful demonstration, in-stent coronary thrombosis was induced by x-ray-guided placement of thrombogenic-coated, MR-lucent stents (n=5). After stent placement, 60 micromol of EP-2104R was injected via the left main coronary artery. Free-breathing, navigator-gated 3D coronary MR angiography and thrombus imaging were performed (1) before and after stent placement and (2) before and after EP-2104R. Thrombi were confirmed by x-ray angiography and autopsy. Fibrinogen thrombi: 5 of 6 intracoronarily delivered Gd-labeled fibrinogen clots (approximately 250 micromol/L Gd) were visible on MRI and subsequently confirmed by x-ray angiography. In-stent thrombi: in-stent thrombosis was observed in all stents after EP-2104R. Four of 5 thrombi were confirmed by x-ray angiography. Chemical analysis of 2 thrombi demonstrated 99 to 147 micromol/L Gd. CONCLUSIONS: We demonstrate the feasibility of MRI of coronary thrombus and in-stent thrombosis using a novel fibrin-binding molecular MR contrast agent. Potential applications include detection of coronary in-stent thrombosis or thrombus burden in patients with acute coronary syndromes.

Liposome transduction into cells enhanced by haptotactic peptides (Haptides) homologous to fibrinogen C-termini.

Haptides are 19-21mer cell-binding peptides equivalent to sequences on the C-termini of fibrinogen beta chain (Cbeta), gamma chain (preCgamma) and the extended alphaE chain of fibrinogen (CalphaE). In solution, Haptides accumulated in cells by non-saturable kinetics [Exp. Cell Res. 287 (2003) 116]. This study describes Haptide interactions with liposomes and Haptide-mediated liposome uptake by cells. Haptides became incorporated into negatively charged liposomes, changing their zeta potential. Atomic force microscopy and particle sizing by light scattering showed that the liposomes dissolved Haptide nanoparticles and absorbed them from solution. Pre-mixing fluorescent rhodamine-containing liposomes or "stealth" doxorubicin (DOX)-containing liposomes (Doxil) with Cbeta, preCgamma or to a lesser degree CalphaE, significantly enhanced their uptake by fibroblasts and endothelial cells. Confocal microscopy showed Haptide-induced liposome uptake saturated above approximately 40 microM Haptide. Cytotoxicity tests with lower concentrations of Doxil liposomes indicated that premixing with approximately 40 microM Cbeta or preCgamma increased their toxicity by one order of magnitude. It was evident that the liposomes complexed with an amphiphilic Haptide are transduced through cell membranes, probably by a non-receptor-mediated process. These results suggest that Cbeta or pre-Cgamma could be employed to augment the cellular uptake of drugs in liposomal formulations.

The effect of fucoidan, heparin and cyanogen bromide-fibrinogen on the activation of human glutamic-plasminogen by tissue plasminogen activator.

Earlier studies on the stimulatory effect of fucoidan, heparin, and cyanogen bromide (CNBr)-fibrinogen digest on the in-vitro activation of glutamic type plasminogen by tissue plasminogen activator, which were performed using subphysiologic ionic strengths of buffers, gave inconsistent results because of the variation in the ionic strengths of the buffers used. Studies were therefore conducted on the effect of these cofactors using 0.05 mol/l Tris buffer containing a physiologic concentration of sodium chloride. The double reciprocal plots of the activation of glutamic type plasminogen by tissue plasminogen activator in the presence of fucoidan and 6-aminohexanoic acid (6-AH) or heparin and 6-AH showed a four- to six-fold increase in K(cat), while the K(m) remained unchanged. On the other hand, there was greater than six-fold lowering of K(m) from 0.213 to 0.035 micromol/l in the presence of CNBr-fibrinogen, while K(cat) was only slightly increased. The ratios of the initial rate of plasmin generation in the presence or absence of the cofactors were plotted against the inverse of the volume fraction of glutamic type plasminogen or of tissue plasminogen activator after serial dilution. The results suggested that the enhancements by fucoidan and 6-AH or CNBr-fibrinogen were due to their interactions directed towards glutamic type plasminogen, while for heparin and 6-AH, the interaction was directed towards tissue plasminogen activator. Circular dichroism studies in the near ultraviolet range (250-308 nm) showed that 6-AH enhanced the circular dichroism spectra of glutamic type plasminogen around certain chromophores, while fucoidan and heparin had no effect, suggesting that the enhancement by the cofactors may be related to the favorable conformational changes of glutamic type plasminogen by 6-AH.

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.

[Combined thrombolysis by pharmacokinetically different plasminogen activators]. / Sochetannyi trombolizis farmakokineticheski razlichnymi aktivatorami plazminogena.

Prospects for the search for thrombolytic compositions on the basis of short-term and long-term acting plasminogen activators were shown. These will be useful as potential ambulance remedies for effective prehospital treatment. Combined proteolysis by plasminogen activators with complementary action mechanisms and significantly different pharmacokinetic behavior was suggested for this purpose.

New strategy of thrombolysis. Conjunctive effect of plasminogen activators with different pharmacokinetic profile.

Combined actions of native and prolonged thrombolytics allow the use of lower doses and simplified schemes of administration, thus yielding significant results in experimental therapy regarding the efficacy and safety of thrombolysis. Development of prolonged forms of plasminogen activators and testing their effect in combination with the thrombolysis trigger are well founded and of current interest. Thrombolytic compositions on the basis of short- and long-term-acting plasminogen activators appear to be promising and potentially eligible for bolus administration.