Search

Aim: To develop a fibrin-specific urokinase nanomedicine thrombolytic agent. Materials & Methods:In vitro fibrin-clot dissolution studies were utilized to develop and characterize simultaneous coupling and loading of anti-fibrin monoclonal antibody and urokinase onto perfluorocarbon nanoparticle (NP) surface. In vivo pharmacokinetics and fibrin-specific targeting of the nanolytic agent was studied in dogs. Results: Simultaneous coupling of up to 40 anti-fibrin antibodies and 400 urokinase enzymes per perfluorocarbon NP produced an effective targeted nanolytic agent with no significant surface protein–protein interference. Fibrin clot dissolution was not improved by increasing homing capacity from 10 to 40 antibodies/NP, but increasing enzymatic payload from 100 to 400/NP resulted in maximized lytic effect. Fluorescent microscopy showed that rhodamine-labeled urokinase nanoparticles densely decorated the intraluminal thrombus in canine clots in vivo analogous to the fibrin pattern, while an irrelevant-targeted agent had negligible binding. Conclusion: This agent offers a vascularly constrained, simple to administer, low-dose nanomedicine approach that may present an attractive alternative for treating acute stroke victims.

A synthetic methodology for developing a polymeric nanoparticle for targeted computed tomographic (CT) imaging is revealed in this manuscript. The work describes a new class of “soft” type, vascularly constrained, stable colloidal radio-opaque metal-entrapped polymeric nanoparticle using organically soluble radio-opaque elements encapsulated by synthetic amphiphile. This agent offers several fold CT signal enhancement in vitro and in vivo demonstrating detection sensitivity reaching to the low nanomolar particulate concentration range.

Aprotinin has been used widely in surgery as an anti-bleeding agent but is associated with a number of side effects. We report that textilinin-1, a serine protease inhibitor from Pseudonaja textilis venom with sequence relatedness to aprotinin, is a potent but reversible plasmin inhibitor and has a narrower range of protease inhibition compared to aprotinin. Like aprotinin, textilinin-1 at 5 micromol/l gave almost complete inhibition of tissue plasminogen activator-induced fibrinolysis of whole blood clots. The activated partial thromboplastin time for plasma was markedly increased by aprotinin but unaffected by textilinin-1. In a mouse tail-vein bleeding model, intravenous textilinin-1 and aprotinin caused similar decreases in blood loss but time to haemostasis in the textilinin-treated animals was significantly shorter than in aprotinin-treated mice. Based on these data, textilinin-1 merits further investigation as a therapeutic alternative to aprotinin.

The use of antiangiogenic therapy in conjunction with traditional chemotherapy is becoming increasingly in cancer management, but the optimal benefit of these targeted pharmaceuticals has been limited to a subset of the population treated. Improved imaging probes that permit sensitive detection and high-resolution characterization of tumor angiogenesis could improve patient risk-benefit stratification. The overarching objective of these experiments was to develop a dual modality alpha(nu)beta3-targeted nanoparticle molecular imaging agent that affords sensitive nuclear detection in conjunction with high-resolution MR characterization of tumor angiogenesis.In part 1, New Zealand white rabbits (n = 21) bearing 14d Vx2 tumor received either alpha(nu)beta3-targeted 99mTc nanoparticles at doses of 11, 22, or 44 MBq/kg, nontargeted 99mTc nanoparticles at 22 MBq/kg, or alpha(nu)beta3-targeted 99mTc nanoparticles (22 MBq/kg) competitively inhibited with unlabeled alpha(nu)beta3-nanoparticles. All animals were imaged dynamically over 2 hours with a planar camera using a pinhole collimator. In part 2, the effectiveness of alpha(nu)beta3-targeted 99mTc nanoparticles in the Vx2 rabbit model was demonstrated using clinical SPECT-CT imaging techniques. Next, MR functionality was incorporated into alpha(nu)beta3-targeted 99mTc nanoparticles by inclusion of lipophilic gadolinium chelates into the outer phospholipid layer, and the concept of high sensitivity - high-resolution detection and characterization of tumor angiogenesis was shown using sequential SPECT-CT and MR molecular imaging with 3D neovascular mapping.alpha(nu)beta3-Targeted 99mTc nanoparticles at 22 MBq/kg produced the highest tumor-to-muscle contrast ratio (8.56 +/- 0.13, TMR) versus the 11 MBq/kg (7.32 +/- 0.12) and 44 MBq/kg (6.55 +/- 0.07) doses, (P0.05). TMR of nontargeted particles at 22.2 MBq/kg (5.48 +/- 0.09) was less (P0.05) than the equivalent dosage of alpha(nu)beta3-targeted 99mTc nanoparticles. Competitively inhibition of 99mTc alpha(nu)beta3-integrin-targeted nanoparticles at 22.2 MBq/kg reduced (P0.05) TMR (5.31 +/- 0.06) to the nontargeted control contrast level. Multislice CT imaging could not distinguish the presence of Vx2 tumor implanted in the popliteal fossa from lymph nodes in the same fossa or in the contralateral leg. However, the use of 99mTc alpha(nu)beta3-nanoparticles with SPECT-CT produced a clear neovasculature signal from the tumor that was absent in the nonimplanted hind leg. Using alpha(nu)beta3-targeted 99mTc-gadolinium nanoparticles, the sensitive detection of the Vx2 tumor was extended to allow MR molecular imaging and 3D mapping of angiogenesis in the small tumor, revealing an asymmetrically distributed, patchy neovasculature along the periphery of the cancer.Dual modality molecular imaging with alpha(nu)beta3-targeted 99mTc-gadolinium nanoparticles can afford highly sensitive and specific localization of tumor angiogenesis, which can be further characterized with high-resolution MR neovascular mapping, which may predict responsiveness to antiangiogenic therapy.

Summary. Cardiopulmonary bypass (CPB) induces a bleeding defect which leads to enhanced blood loss. A double-blind study was carried out comparing aprotinin with placebo in patients undergoing re-operation for heart valve replacement. The results confirm that aprotinin is effective at reducing such loss. In the placebo treated group, significant increases were observed, during CPB, in the plasma concentrations of fibrinolytic activity, tissue plasminogen activator antigen. D-dimer, and β-thromboglobulin. Platelet counts fell within 5–10 min of the patients going onto CPB, but this could be accounted for by the dilutional effect of the extracorporeal circuit. Inhibition of responsiveness of platelets, as judged by aggregometry. was significant only at the end of bypass when collagen was the agonist and after protamine reversal when ristocetin was the agonist. CPB did not enhance the release, into the circulation, of glycocalicin (a proteolytic fragment of glycoprotein Ib). In the aprotinin-treated group, the formation of fibrin degradation products as measured by D-dimer was inhibited. However, aprotinin did not influence the change in platelet count, suppress β-thromboglobulin release from platelets, prevent the inhibition of platelet function or influence the concentration of plasma glycocalicin during the study period. These observations confirm that CPB leads to a fibrinolytic state and less responsive platelets. This study also indicates that aprotinin-induced reduction in blood loss is associated with inhibition of plasmin-mediated fibrin digestion and that the mechanism by which aprotinin reduces blood loss is not via protection of platelets during CPB.

This study explored the use of F spectroscopy and imaging with targeted perfluorocarbon nanoparticles for the simultaneous identification of multiple bio-signatures at 1.5 T.Two nanoparticle emulsions with perfluoro-15-crown-5-ether (CE) or perfluorooctylbromide (PFOB) cores were targeted in vitro to fibrin clot phantoms (n=12) in 4 progressive ratios using biotin-avidin interactions. The CE nanoparticles incorporated gadolinium. Fluorine images were acquired using steady-state gradient-echo techniques; spectra using volume-selective and nonselective sampling.On conventional T1-weighted imaging, clots with CE nanoparticles enhanced as expected, with intensity decreasing monotonically with CE concentration. All clots were visualized using wide bandwidth fluorine imaging, while restricted bandwidth excitation permitted independent imaging of CE or PFOB nanoparticles. Furthermore, F imaging and spectroscopy allowed visual and quantitative confirmation of relative perfluorocarbon nanoparticle distributions.F MRI/S molecular imaging of perfluorocarbon nanoparticles in vitro suggests that noninvasive phenotypic characterization of pathologic bio-signatures is feasible at clinical field strengths.

This review attempts to relate subunit structures of fibrin degradation products (FnDP) made in vitro with structures found in vivo. The domainally directed fragmentation in vitro of both fibrinogen and fibrin is emphasized, all fragments being various associations of the two core fibrin fragments D and E. The digestion of fibrinogen by plasmin in vivo is rare, and the crosslinking of fibrin in vivo takes place at a very early stage in the clotting/polymerization process. The notion that as fibrin forms in vivo it orchestrates its own destruction is developed. Plasmas from patients suffering from disseminated intravascular coagulation demonstrate very large crosslinked FnDP fragments in their plasmas which seem to contain not alone fibrinopeptide A but also subunits with intact alpha chains. This is interpreted to mean that many of the large soluble fragments found in vivo, and heretofore known as FnDP, are in reality long fibrin polymers, random parts of whose structures have been converted to FnDP by lysis of the carboxy terminal regions of the alpha chains in the polymer. The ratio of intact fibrin to FnDP in these large soluble structures may be a useful clinical marker; however, such data can only be relied upon when blood samples are taken into an anticoagulant mix that contains a fibrinolytic inhibitor. Some of the biological effects of FnDP structures in vivo (fibrinogen synthesis, vasoactivity) are still quite ambiguous.

A lipid-encapsulated perfluorocarbon nanoparticle molecular imaging contrast agent that utilizes a paramagnetic chemical exchange saturation transfer (PARACEST) chelate is presented. PARACEST agents are ideally suited for molecular imaging applications because one can switch the contrast on and off at will simply by adjusting the pulse sequence parameters. This obviates the need for pre- and postinjection images to define contrast agent binding. Spectroscopy (4.7T) of PARACEST nanoparticles revealed a bound water peak at 52 ppm, in agreement with results from the water-soluble chelate. Imaging of control nanoparticles showed no appreciable contrast, while PARACEST nanoparticles produced >10% signal enhancement. PARACEST nanoparticles were targeted to clots via antifibrin antibodies and produced a contrast-to-noise ratio (CNR) of 10 at the clot surface.

The extensively studied para gene encodes a α-subunit of the voltage-activated sodium channel in Drosophila melanogaster , which is the documented target site of DDT and pyrethroid insecticides. The para ts-1 fruit fly line carries a recessive sex-linked insecticide-resistance trait ( para ts-1 allele) that has been defined on the basis of the behavioral phenotype of temperature-sensitive paralysis. We have determined that para ts-1 confers hyper-susceptibility to deltamethrin in addition to the previously annotated resistance to DDT, revealing the presence of negative cross-resistance. We investigated the potential use of negative cross-resistance shifting para ts-1 gene frequencies in D. melanogaster populations. After five generations of selection, the para ts-1 allele, respectively, became more or less frequent whether Drosophila populations were selected with DDT or deltamethrin.

Molecular imaging of microthrombus within fissures of unstable atherosclerotic plaques requires sensitive detection with a thrombus-specific agent. Effective molecular imaging has been previously demonstrated with fibrin-targeted Gd-DTPA-bis-oleate (BOA) nanoparticles. In this study, the relaxivity of an improved fibrin-targeted paramagnetic formulation, Gd-DTPA-phosphatidylethanolamine (PE), was compared with Gd-DTPA-BOA at 0.05-4.7 T. Ion- and particle-based r(1) relaxivities (1.5 T) for Gd-DTPA-PE (33.7 (s*mM)(-1) and 2.48 x 10(6) (s*mM)(-1), respectively) were about twofold higher than for Gd-DTPA-BOA, perhaps due to faster water exchange with surface gadolinium. Gd-DTPA-PE nanoparticles bound to thrombus surfaces via anti-fibrin antibodies (1H10) induced 72% +/- 5% higher change in R(1) values at 1.5 T (deltaR(1) = 0.77 +/- 0.02 1/s) relative to Gd-DTPA-BOA (deltaR(1) = 0.45 +/- 0.02 1/s). These studies demonstrate marked improvement in a fibrin-specific molecular imaging agent that might allow sensitive, early detection of vascular microthrombi, the antecedent to stroke and heart attack.

Two peptides, textilinins 1 and 2, isolated from the venom of the Australian common brown snake, Pseudonaja textilis textilis, are effective in preventing blood loss. To further investigate the potential of textilinins as anti-haemorrhagic agents, we cloned cDNAs encoding these proteins. The isolated full-length cDNA (430 bp in size) was shown to code for a 59 amino acid protein, corresponding in size to the native peptide, plus an additional 24 amino acid propeptide. Six such cDNAs were identified, differing in nucleotide sequence in the coding region but with an identical propeptide. All six sequences predicted peptides containing six conserved cysteines common to Kunitz-type serine protease inhibitors. When expressed as glutathione S-transferase (GST) fusion proteins and released by cleavage with thrombin, only those peptides corresponding to textilinin 1 and 2 were active in inhibiting plasmin with K-i values similar to those of their native counterparts and in binding to plasmin less tightly than aprotinin by two orders of magnitude. Similarly, in the mouse tail vein blood loss model only recombinant textilinin 1 and 2 were effective in reducing blood loss. These recombinant textilinins have potential as therapeutic agents for reducing blood loss in humans, obviating the need for reliance on aprotinin, a bovine product with possible risk of transmissible disease, and compromising the fibrinolytic system in a less irreversible manner.

Monoclonal antibody (mAb) III-3b binds D-dimer with Kd = 1.4 × 10-10 M without cross-reaction with fibrin(ogen). The epitope for this mAb is in Bβ134-190, presumably in Bβ155-160. The latter site is buried in the coiled coil structure of fibrin(ogen) but it is exposed as a neoantigenic determinant in D-dimer upon plasmic lysis of fibrin. mAb III-3b may be used as a tool for immunodiagnostic quantification of D-dimer in blood plasma. © 2002 Elsevier Science Ltd. All rights reserved. Chemicals/CAS: Antibodies, Monoclonal; Epitopes; Fibrin Fibrinogen Degradation Products; fibrin fragment D; Fibrin, 9001-31-4

An experimental venous clot model using the lateral saphenous vein of sheep is described. Eight experimental Suffolk crossbred sheep were used. A mixture of human fibrinogen, in some cases labelled with (125)I, bovine thrombin and homologous whole blood was placed via a catheter into a surgically isolated segment of the lateral saphenous vein. The resulting heterologous clot was imaged daily for 6 days using venography, or monitored using an external gamma ray detector. Clots were radiographically detectable for the 6 days of the study. They were totally occlusive for a mean of 4.2 days (SD 2.2) and were occlusive in the immediate 24 hour period after surgery. The fibrin component of the clot was persistent (85 per cent of the initial fibrin[ogen] present after 6 days). Radiographically the clots were seen as filling defects within partially filled vessels, or their presence was inferred from the absence of filling. A collateral blood supply was apparent immediately on vessel occlusion. No adverse effects, evidence of infection or limb oedema were seen. The model provided a reproducible blood clot within the lateral saphenous vein of the sheep. Clot imaging using venography was effective and readily achieved. It is suggested that the model is useful when a stable, intravenous deposit of heterologous (e.g. human) fibrin is required in vivo at a site suitable for venography and radionucleid monitoring.

This study investigates reaction kinetics by slow-binding kinetics methods of both adult and fetal plasmin (Types 1 and 2) with adult and fetal alpha(2)-antiplasmin. In addition, carbohydrate sequences of Fetal and Adult Plasminogen Types 1 and 2, as well as fetal and adult alpha(2)-antiplasmin, were determined by mass spectrometric analysis. All curves of plasmin-alpha(2)-antiplasmin interaction followed the same pattern, indicating reversible slow-binding inhibition with an initial loose complex and a following tight complex. Differences between fetal and adult plasmin reactions with alpha(2)-antiplasmin were predominantly due to the initial loose complex. Values for K(i initial) in the reaction with adult alpha(2)-antiplasmin were 1.5 and 1.6 nM for Fetal Plasmin Types 1 and 2, respectively; compared to 0.3 and 0.7 nM for the corresponding adult types. Increasing concentrations of tranexamic acid resulted in a continuous increase of K(i initial) until a plateau was reached which was similar for all plasmin types. Almost identical values could be obtained when fetal alpha(2)-antiplasmin was used instead of adult alpha(2)-antiplasmin. Mass spectrometric analyses of the glycans present on plasminogen revealed a higher level of truncated N-glycans on the fetal material compared to the adult. The O-glycans of fetal and adult plasminogen were closely similar and only minor differences were observed between N-glycans of fetal and adult alpha(2)-antiplasmin. In conclusion, both fetal plasmin isoforms are less inhibited by alpha(2)-antiplasmin compared to the adult plasmin variants. These findings are important for the understanding of the physiology of the fibrinolytic system in neonates and provide further evidence that differences in glycosylation could be associated with marked effects on protein function.

During the 20th century, pesticide use has become integral for current agricultural practices, as has the challenge associated with pesticide resistance. Traditional resistance management plans have often used a “use and discard” approach, changing the chemical to target a different mode of action in the pest species once resistance becomes a problem in the field. An alternative strategy is to identify compounds that confer negative cross-resistance (NCR), where the NCR compound is more toxic to pesticide resistant insects as compared to their pesticide susceptible counterparts. Examples of NCR exist in the literature, however, a systematic approach to discover and use these compounds has been lacking in industrial agriculture. In the following chapter we explore both the limitations and the potential for use of NCR strategies in relation to resistance management.

Background Molecular imaging of thrombus within fissures of vulnerable atherosclerotic plaques requires sensitive detection of a robust thrombus-specific contrast agent. In this study, we report the development and characterization of a novel ligand-targeted paramagnetic molecular imaging agent with high avidity for fibrin and the potential to sensitively detect active vulnerable plaques. Methods and Results The nanoparticles were formulated with 2.5 to 50 mol% Gd-DTPA-BOA, which corresponds to >50 000 Gd 3+ atoms/particle. Paramagnetic nanoparticles were characterized in vitro and evaluated in vivo. In contradistinction to traditional blood-pool agents, T1 relaxation rate as a function of paramagnetic nanoparticle number was increased monotonically with Gd-DTPA concentration from 0.18 mL · s −1 · pmol −1 (10% Gd-DTPA nanoparticles) to 0.54 mL · s −1 · pmol −1 for the 40 mol% Gd-DTPA formulations. Fibrin clots targeted in vitro with paramagnetic nanoparticles presented a highly detectable, homogeneous T1-weighted contrast enhancement that improved with increasing gadolinium level (0, 2.5, and 20 mol% Gd). Higher-resolution scans and scanning electron microscopy revealed that the nanoparticles were present as a thin layer over the clot surface. In vivo contrast enhancement under open-circulation conditions was assessed in dogs. The contrast-to-noise ratio between the targeted clot (20 mol% Gd-DTPA nanoparticles) and blood was ≈118±21, and that between the targeted clot and the control clot was 131±37. Conclusions These results suggest that molecular imaging of fibrin-targeted paramagnetic nanoparticles can provide sensitive detection and localization of fibrin and may allow early, direct identification of vulnerable plaques, leading to early therapeutic decisions.

Our study investigates the effect of fetal and adult soluble fibrin (SF), fetal and adult fibrinogen Aalpha- and gamma-chains, as well as adult CNBr-fibrinogen fragments on tissue-type plasminogen activator (t-PA)-catalyzed plasminogen activation of both fetal and adult Glu-plasminogen types 1 and 2. In addition, we determined carbohydrate sequences of fetal and adult Bbeta- and gamma-chains by mass spectrometric analysis. In the absence of an effector, no substantial differences in the rate of plasmin formation could be seen between the fetal and adult plasminogen types. In the presence of an effector, both fetal Glu-plasminogen types revealed lower values for k(cat app) than the respective adult types. No differences could be seen in the values for K(m app). The resulting differences in catalytic efficiencies between the fetal and adult plasminogen types were much less than previously reported. No differences could be seen between fetal and adult effectors in stimulating t-PA-catalyzed plasminogen activation. Detailed analyses of the activation kinetics revealed a longer initial phase of slow plasmin formation of both fetal Glu-plasminogen types compared to their respective adult types, indicating a slower plasmin-induced modification of CNBr-fibrinogen fragments or SF by fetal plasmin. Mass spectrometric analysis of the N-glycans present on adult and fetal Bbeta- and gamma-fibrinogen chains showed the presence of a major monosialylated biantennary structure with lesser amounts of the disialylated form. In contrast to previous data, we conclude that catalytic efficiency of t-PA-catalyzed plasminogen activation in neonates is only slightly lower than in adults.

This study deals with plasminogen activation kinetics of fetal and adult Glu-plasminogen types 1 and 2 as well as fetal and adult Lys-plasminogen by urokinase in the presence and absence of the lysine analogues epsilon-amino-n-caproic acid (EACA) and tranexamic acid. In addition, activation kinetics of single-chain urokinase-plasminogen activator (scu-PA) by adult and fetal plasmin types were investigated in the absence and presence of soluble fibrin. All Lys-plasminogen isoforms were more readily activated by urokinase than their corresponding Glu-plasminogen types. No substantial differences of the catalytic constants of urokinase-catalyzed plasminogen activation could be obtained when all fetal plasminogen types were compared to the respective adult types. In the case of all Glu-plasminogen isoforms, EACA as well as tranexamic acid first stimulated the activation process and, at higher concentrations, showed inhibitory properties. Again, the relative ability of all fetal plasminogen types to interact with lysine analogues revealed no differences compared to the respective adult glycoforms. In the absence of soluble fibrin, the catalytic efficiency of scu-PA activation by plasmin was significantly lower for both fetal plasmin isoforms. However, there were no differences in catalytic efficiency between fetal and adult plasmin types in the presence of 4 microM soluble fibrin. In conclusion, no substantial differences exist in urokinase-catalyzed plasminogen activation between neonates and adults, which is in contrast to reported data on plasminogen activation by tissue-type plasminogen activator. In the absence of soluble fibrin, scu-PA activation by fetal plasmin is markedly slower than by adult plasmin. However, this is compensated when fibrin is added at a concentration that is close to the physiological fibrinogen concentration in plasma. It can be summarized that the differences in carbohydrate structures of fetal and adult plasminogen are not associated with major differences in the global function of this part of fibrinolysis, despite functional alterations of scu-PA activation.

SummaryAntifibrin monoclonal antibodies show potential as clot targeting agents for diagnosis and possibly therapy in thrombotic disease. To be effective the antibody must bind to the fibrin component of the clot. The ability of two antifibrin mabs (NIB 1H10 and NIB 12B3) to penetrate occlusive clots in vivo was investigated Both mabs react with human fibrin but not with human fibrinogen nor with the fibrin or fibrinogen from the species used in this study.Two heterologous animal (sheep and rabbit) thrombus models were used. Clots in both cases were made within isolated vein segments using a mixture of human and native fibrinogen. The clots in sheep veins were observed radiographically and found to be occlusive for a mean of 4.2 ± 2.2 days and thereafter appeared only partially occlusive. When targeted in their occlusive phase 131I labelled mab accumulated in the clot reaching a maximum ratio of 1.82 ± 0.42 when compared to counts in homologous sheep clots in the contralateral limb. It was confirmed in the rabbit jugular vein model that total occlusivity did not prevent antibody accumulation in the heterologous clot by injecting the fibrin specific mab IH10 and examining the clot excised after 1, 6 and 24 h using immunofluorescence. In a further series of similar experiments 125I labelled mab 1H10 was used and detected using autoradiography. Both sets of experiments indicated that penetration of occlusive clots by the antibody occurred and that considerable accumulation was present at 6 and 24 h.The results indicate that a circulating antibody can readily gain access to experimentally produced clots in occluded veins.

In this study, the sensitivity of a novel fibrin-targeted contrast agent for fibrin detection was defined in vitro on human thrombus. The contrast agent was a lipid-encapsulated perfluorocarbon nanoparticle with numerous Gd-DTPA complexes incorporated into the outer surface. After binding to fibrin clots, scanning electron microscopy of treated clots revealed dense accumulation of nanoparticles on the clot surfaces. Fibrin clots with sizes ranging from 0.5‐7.0 mm were imaged at 4.7 T with or without treatment with the targeted contrast agent. Regardless of sizes, untreated clots were not detectable by T1-weighted MRI, while targeted contrast agent dramatically improved the detectability of all clots. Decreases in T1 and T2 relaxation times (20 ‐ 40%) were measured relative to the surrounding media and the control clots. These results suggest the potential for sensitive and specific detection of microthrombi that form on the intimal surfaces of unstable atherosclerotic plaque. Magn Reson Med 44:867‐ 872, 2000. © 2000 Wiley-Liss, Inc.

Fibrin is found in most solid tumours, and there is speculation regarding its role in tumour invasion and metastasis. An assay to quantitate fibrin levels in tissues would be a useful preliminary tool in assessing the above. Such an assay to quantitatively detect levels of fibrin in various types of canine tumour was developed. This procedure involved an ELISA using a monoclonal antibody (MAb 1H10) for canine fibrin as a capture antibody and a polyclonal antibody to human fibrinogen conjugated to horseradish peroxidase as the detection antibody. The ELISA is calibrated against known concentrations of freeze-fractured fibrin derived from clotted dog plasma. The assay takes 3.5 hours, and concentrations as low as 0.1 μg fibrin per milliliter of solubilised tumour can be readily detected. ELISA dilution curves for fibrin from various types of canine tumour were found to be parallel to the standard canine fibrin calibration curve. The intra-assay and interassay variabilities of the assay gave coefficients of variation in the range of 2.4–4.5% and 7.2–7.8%, respectively, for the calibrator standard, in a concentration range of 0.1–10 μg/ml. Using this assay, we reported the levels of fibrin in three different types of malignant canine tissue.

Targeted ultrasonic contrast systems are designed to enhance the reflectivity of selected tissues in vivo [Lanza et al., Circulation 94, 3334 (1996)]. In particular, these agents hold promise for the minimally invasive diagnosis and treatment of a wide array of pathologies, most notably tumors, thromboses, and inflamed tissues. In the present study, acoustic microscopy was used to assess the efficacy of a novel, perfluorocarbon based contrast agent to enhance the inherent acoustic reflectivity of biological and synthetic substrates. Data from these experiments were used to postulate a simple model describing the observed enhancements. Frequency averaged reflectivity (30-55 MHz) was shown to increase 7.0 +/- 1.1 dB for nitrocellulose membranes with targeted contrast. Enhancements of 36.0 +/- 2.3 dB and 8.5 +/- 0.9 dB for plasma and whole blood clots, respectively, were measured between 20 and 35 MHz. A proposed acoustic transmission line model predicted the targeted contrast system would increase the acoustic reflectivity of the nitrocellulose membrane, whole blood clot, and fibrin plasma clot by 2.6, 8.0, and 31.8 dB, respectively. These predictions were in reasonable agreement with the experimental results of this paper. In conclusion, acoustic microscopy provides a rapid and sensitive approach for in vitro chracterization, development, and testing of mathematical models of targeted contrast systems. Given the current demand for targeted contrast systems for medical diagnostic and therapeutic use, the use of acoustic microscopy may provide a useful tool in the development of these agents.

Monocyte infiltration into forming thrombus has been demonstrated in experimental models of venous thrombosis developed in our laboratories. These cells produce and release plasminogen activators as the thrombus organises and resolves. Monocytes are also capable of assembling and releasing procoagulant factors and the evidence for their importance in thrombogenesis is reviewed. The ability of monocytes to maintain this fibrinolytic balance suggests that they may have a role in both thrombosis and thrombus resolution. Control of the mechanisms which regulate these activities may therefore be important in preventing thrombus formation or stimulating its resolution.

SummarySince the finding that plasminogen activator inhibitor-1 (PAI-1) may influence the initiation and progression of acute myocardial infarction, the assay of PAI-1 in plasma using a variety of commercial kits has become commonplace. The need for a standard to define the activity of PAI-1 prompted an international collaborative study (ICS) to calibrate the functional potency of a lyophilised plasma PAI-1 preparation (92/654). Since PAI-1 inhibits the 2 major plasminogen activators, tissue-type plasminogen activator (t-PA) and urinary-type plasminogen activator (u-PA) in an equimolar manner it was important to establish the potency of the PAI-1 inhibitor in terms of both t-PA and u-PA neutralisation. While the ICS indicated a wide spread of data between the laboratories the mean value of 27.5 t-PA neutralisation units and 7.0 u-PA neutralisation units was confirmed by numerous assays at NIBSC using a tedious but technically reliable titration assay procedure. The plasma PAI-1 proposed standard (92/654) was stable at 20° C for 20 months.The Fibrinolysis Subcommittee of the Scientific and Standardization Committee (SSC) of the International Society on Thrombosis and Haemostasis (ISTH), (meeting in Leuven, Belgium in September 1994) has recommended that the plasma PAI-1 (92/654) should be accepted as the International Standard for PAI-1 and should define a unitage in terms of both t-PA and u-PA neutralisation. Subsequently the Expert Committee on Biological Standardization of the World Health Organization (ECBS-WHO) meeting in Geneva, Switzerland in October 1995 approved plasma PAI-1 (92/654) as the International Standard.

SummaryTwo anti-fibrin monoclonal antibodies, MAbs 1H10 and 5F3, raised to human freeze-fractured fibrin and thrombin-treated N-terminal disulphide knot (T-NDSK), respectively, were compared for epitope binding to various domains of the fibrinogen/fibrin moiety. Using plasmin-mediated fibrinogen digests, immunoblots showed that both MAbs crossreacted strongly with fragments X and Y, weakly with fragment-E and not at all with fragment D. Purified fragments D and E used in an ELISA confirmed that MAbs 1H10 and 5F3 cross-reacted in a dose-response fashion with the isolated fragment-E, while there was no reaction with fragment-D. The two MAbs were similarly shown to react with fibrin-derived fragment-E. Surface Plasmon Resonance (SPR) technology, employed to further evaluate the epitopes in fibrin, showed that MAb 1 HI0 had a higher affinity for fragment-E (KD = 8.04 × 10-9 M) than MAb 5F3 (KD = 1.13 × 10-8 M). Individual association and dissociation rate constants of 7.97 × 105 M-1S-1 and 3.97 × 10-3S-1, respectively, for MAb 1H10, and 5.16 × 105 M-1s-1 and 3.62 × 10-3s-1, respectively, for MAb 5F3 were also obtained. A SPR inhibition assay confirmed that MAb 1H10 had a greater affinity for fragment-E than MAb 5F3. However individual isolated polypeptide chains of fibrinogen fragment E (E-Aα, E-Bβ, E-γ) showed no reaction with the two antibodies in ELISA, immunoblot or SPR analysis procedures. Furthermore, SPR pair-wise epitope mapping analysis revealed that MAbs 1H10 and 5F3 have in fact distinct epitopes on fragment-E. These distinct epitopes appeared to be a conformational amalgam of linear sequences in two or three of the polypeptide chains of fragment-E, or distinct conformational epitopes on one of the three subunit chains alone.

The purpose of the study was to determine the effects of early plasmin-mediated digests of rat fibrinogen on the vascular tone of rat pulmonary artery in order to compare with reported vasoactive effects of high levels of isolated human peptides in various rat vascular beds. Isometric tension was monitored in isolated rings of rat pulmonary artery precontracted with phenylephrine (4×10 −8 mol). Fibrinogen degradation products (FgDPs) were produced by digestion of rat fibrinogen with plasmin to 1, 2, 3 and 60 minutes whereupon the digestion was stopped by addition of Trasylol. These FgDPs were added to the tissue bath to achieve final concentrations of 6.7, 13.3, 26.7 and 53.3 μg/mL i.e. values similar to those found in vivo during thrombolytic therapy for myocardial infarction. Results were expressed as a percentage of a maximal contraction elicited in each ring in response to phenylephrine (10 −5 M). The early FgDP fractions, at these pathophysiological concentrations, did not induce significant change in isometric tension in the isolated pulmonary arterial rings (P > 0.05, ANOVA).

SummaryFour monoclonal antibodies have previously been raised in our laboratory for possible use in thrombus imaging and the targeting of thrombolytic agents. These antibodies were raised to various human fibrin-related immunogens and each antibody had been selected for its specificity towards fibrin and not fibrinogen. To study further these antibodies in animal circulation models both in vivo and in vitro, their selectivity towards human fibrin as opposed to other animal fibrins was examined. In this study dissociation constants for each antibody with each of six species fibrins (human, baboon, pig, dog, sheep, and rabbit) were estimated using both fibrin clots and monolayers. Some limited data were also obtained with Sepharose-fibrin. Of the antibodies two (denoted 12B3B10 and 12B3A11) are seen to bind almost exclusively to human fibrin with dissociation constants of about 8 × 10−10 M using fibrin clots and monolayers. These same two mabs bound to baboon fibrin with a dissociation constant of 2 × 10−9 M, while neither displayed significant levels of binding to the fibrins from dog, pig, sheep and rabbit. The other two antibodies investigated (1H10 and 5F3) were found to bind well to fibrins of human, baboon, pig and dog, with dissociation constants in the range of 1.4-4.2 × 10−9 M. However neither 1H10 nor 5F3 displayed significant recognition of sheep and rabbit fibrins. Both 1H10 and 5F3 were also found by means of competitive ELISA’s to retain their selectivity to baboon, dog and pig fibrins in the presence of their respective fibrinogens.

Purpose: The aim of this study was to measure the distribution of endogenous plasminogen activators during thrombolysis with an endothelial-conserving model of laminated thrombosis. Methods: Thrombi were raised in the inferior vena cava of rats with thrombin and flow reduction. The thrombi, adjacent vein wall, and distant veins (the superior vena cava) were removed at intervals from 1 hour to 21 days from formation and then cryohomogenized and assayed with specific bioimmunoassays for tissue-type (t-PA) and urokinase-type plasminogen activators (u-PA). Results: The measured t-PA activity of the vein wall around the thrombus was reduced compared with the control inferior vena cava at 4 days. Both the u-PA and t-PA content of the thrombus increased progressively during thrombolysis. The t-PA activity increased significantly in the distant vein walls in the animals with thrombi. Immunocytochemistry and in situ hybridization localized the t-PA to a mononuclear cell infiltrate and showed up-regulation of mRNA for rat t-PA in these monocytes. Conclusions: The local plasminogen activator response was predominantly within the thrombus itself. Increased t-PA activity was additionally found in distant veins but was reduced in the vessel wall adjacent to the thrombus. This is the first report to show that u-PA activity is increased within organizing thrombus in vivo and that most of the t-PA activity is localized to a monocyte infiltrate. (J VASC SURG 1995;22:573-9.)

Summary An in vitro circulation model comprising immobilised human fibrin clots in polyvinyl tubing connected to a peristaltic pump is described in detail. The binding to human fibrin of an 125 I-labelled anti-human fibrin monoclonal antibody (mab) 12B3.B10 was studied in sheep plasma. It was found that the accretion of sheep fibrin onto the human fibrin clot did not significantly alter the extent of mab binding to human fibrin in the model. Binding to fibrin in the presence of circulating buffer and human plasma was also studied. It was found that mab binding to human fibrin was significantly less in the presence of circulating human plasma than in buffer or in sheep plasma. In an enzyme linked immunosorbent assay, the binding profiles of mab 12B3.B10 to human fibrin were similar in freshly prepared fibrinogen solutions and buffer, but differed, showing displacement in a fibrinogen solutuon that had been passed through the circulation system. Similar displacement occurred in another freshly prepared fibrinogen solution from a different commercial source. Further, fibrinogen alteration was detected after the addition of the thrombin inhibitor, hirudin, to the circulation fluid, and also despite the omission of the clot from the system. It was concluded that a combination of the action of the peristaltic pump and contact with the polyvinyl tubing caused alteration of the human fibrinogen so that it could displace mab 12B3.B10 binding to the fibrin.

In order to study the epitopes on fibrin to which monoclonal antibodies are directed, we required pure individual polypeptide chains of human fibrinogen in milligram quantities. High purity chains of human fibrinogen were rapidly obtained, in under 3 minutes, by the novel procedure of reversed-phase perfusion chromatography and these chains were subjected to immunological characterisation using monoclonal antibodies specific to the individual chains. Cross-reactivity against these antibodies in both immunoblotting and enzyme linked immunospecific assay (ELISA) procedures showed that these isolated fibrinogen chains were of high purity and retained high immunoreactivity. These chains were employed to initiate studies to define the epitopes in fibrin to which four fibrin specific monoclonal antibodies, B10, A11, 5F3 and 1H10 are targeted. Two of these antibodies, B10 and A11, were shown to be directed to a linear sequence on the A alpha chain, although the binding profiles for the two antibodies suggested that different epitopes may be involved for each of these two antibodies. MAbs, 1H10 and 5F3, however, did not bind to any of the three fibrinogen chains, suggesting that conformational epitopes in fibrin are likely to be involved in the binding of these two antibodies to fibrin.

Summary From previous studies, it is generally accepted that fibrin potentiates the tissue plasminogen activator (t-PA) mediated activation of plasminogen (PLgN) by substantially lowering the K m of the reaction. However, in the majority of these studies, kinetic constants for PLgN activation by t-PA were derived from experiments where dissimilar PLgN concentration ranges were employed in enzyme assays with and without fibrin. From this study, and employing a similar PLgN substrate range (0.07–1.0μM) in reactions with and without fibrin I (des-AA-), we show that by Michaelis-Menten kinetics, there is little alteration in the K m of the above reaction with fibrin addition, while k cat increases up to 10-fold. In the absence of fibrin I, recombinant two chain t-PA (rTCt-PA) yielded the following kinetic parameters: k cat 0.065s −1 and 0.30s −1 , for Glu- and Lys-PLgN respectively, K m 0.45 μM for both. In the presence of fibrin I between 0.05 and 0.3μM, and with rTCt-PA as the enzyme, the activation of Glu- and Lys-PLgN obeyed Michaelis-Menten kinetics over the substrate range 0.07 to 0.7 μM. For PLgN activation at infinite fibrin concentration, values for k cat were estimated as 0.60s −1 and 1.25s −1 , values for K m as 0.31 and 0.16 μM, and values for K F (the dissociation constant for the fibrin-rTCt-PA complex) as 0.075 and 0.070 μM, for Glu- and Lys-PLgN respectively. To determine whether the apparent Michaelis-Menten parameters for t-PA activation of PLgN are dependent on the employed substrate concentration range, the kinetics of this reaction were reassessed over a broad PLgN range (0.002-30μM). For rTCt-PA activation of Lys-PLgN, reaction kinetics were found to be non-standard, and consistent with cooperativity. The reaction yielded two sets of parameters by Michaelis-Menten kinetics; k cat values were 0.26 and 0.89s −1 , and K m values were 0.30 and 2.7 μM. Taken as a whole, data presented here suggest that in the majority of previous studies, the kinetic constants estimated for reactions in the absence and presence of fibrin might be inappropriate for comparison, owing to the possibility of t-PA exhibiting non-standard Michaelis-Menten kinetics.

The in vivo formation of fibrin and its subsequent secondary fibrinolytic digestion yields a variety of crosslinked fibrin degradation products (XL/FnDP). One of these is known as D-dimer and a variety of ELISA-type commercial kits using monoclonal antibodies to D-dimer have been developed. We have investigated the possibility of establishing a standard such that these various kits might indicate the same levels of D-dimer in the same samples. We have concluded that because it seems that each individual monoclonal antibody to D-dimer targets a unique and distinct epitope in the FnDP fraction the notion of introducing a standard D-dimer which will respond uniformly to each D-dimer monoclonal antibody is not feasible. Using various monoclonal and polyclonal antibodies in conjunction with gel exclusion chromatography we have examined human plasma derived from patients with disseminated intravascular coagulation (DIC) which contained high levels of fibrin degradation products (FnDP). The data suggested that the FnDP fraction in plasma contained mostly high molecular weight (> 2 x 10(6) daltons) crosslinked fragments which contain high levels of fibrinopeptide A. Many of these crosslinked fragments crossreact with antibodies to D-dimer because they each contain D-dimer as a structural component. On the basis of this data, a novel sequence of events is proposed which may occur during the aggregation of fibrin in vivo.

SummaryThe current International Standard (IS) for α-thrombin was established in 1991. It was related in unitage with the 1st IS for Thrombin established in 1975 and contains 100 IU of thrombin activity. The National Institute of Health (NIH) standard (Lot J) is in common use for the calibration of commercial thrombin reagents and this study reports on a comparison of the thrombin units defined by these 2 standards. This study has indicated that one “NIH” unit is equivalent to 1.1 to 1.3 IU of thrombin, depending on the influence of PEG in the assay. A unitage ratio figure of 1.15 was recommended following analysis of data obtained in the presence and absence of PEG in the comparative assays. This was confirmed by amidolytic assay data.

A 7kDa protein component, isolated from the venom of the Australian brown snake, Pseudonaja textilis textilis, was found to be an inhibitor of the serine protease plasmin. Its mode of action, inhibitory efficiency and specificity were determined, and compared with those displayed by aprotinin (a Kunitz inhibitor). Using aprotinin as a model small protein protease inhibitor, and with plasmin as the enzyme, the observed slow-onset inhibition was consistent with the two-stage reversible mechanism, E+IEIEI′. Formation of the initial complex (EI) was fast, but binding was relatively loose with an initial K=3.78 nM, while transition from EI to EI′, was slow, and binding was tight, with a final K′=53.2 pM. With snake inhibitor, and in contrast to the above standard mechanism, the observed inhibition was consistent with a competitive, single-stage reversible mechanism, prior to cleavage of the inhibitor to an inactive product. Plasmin and trypsin bound the snake inhibitor via this mechanism, with K values of 0.15 μM and 0.30 μM, respectively. Snake protein concentrations up to 1.0 μM failed to inhibit a number of serine proteases, including recombinant two-chain tissue plasminogen activator, high molecular weight urokinase (55 kDa), α thrombin, elastase and α chymotrypsin. Results demonstrate that the small protein protease inhibitor from the Australian brown snake does not act via the standard slow, tight-binding mechanism common to other small protein serine protease inhibitors.

Summary Mathematical modeling studies have suggested that nonspherical, disc-shaped nanoparticles may have optimal intravascular flow and homing characteristics. In this study, we report the development of a fibrin-specific high-relaxivity bialy-shaped polymeric nanoparticle using porphyrin-chelated manganese. We anticipate that this agent would be highly effective for molecular imaging of microthrombi in ruptured atherosclerotic plaques. Background Detection of microthrombi within fissures of vulnerable atherosclerotic plaques requires a sensitive molecular imaging contrast agent. Moreover, recent reports based on mathematical modeling suggest that nonspherical, disc-shaped nanoparticles could have improved intravascular flow characteristics, which may improve liganddirected targeting. In light of the concern surrounding the use of gadolinium in patients with severe renal disease, the goal of this research was to develop a nonspherical fibrin-targeted manganese-based molecular imaging agent.

Summary Molecular imaging of fibrin offers a sensitive way to detect ruptured atherosclerotic plaque with MRI. To date we and others have heavily explored the use of gadolinium and manganese as paramagnetic metals to provide T1 contrast. In this project, we developed the first bivalent copper nanocolloids for MR molecular imaging of thrombus. Background Robust detection of fibrin expressed in the microfissues of ruptured plaque of the carotid artery offers an opportunity to intercede prophylatically in patients at high risk for stroke. Given the abundance of fibrin in microthrombus, we have focused on developing gadoliniumfree nanomedicine strategies for paramagnetic imaging of clot, respecting recent concerns regarding the pathological link between the lanthanide and Nephrogenic Systemic Fibrosis in patients with severe renal disease. The objective of this research was to develop and characterize the first molecular imaging (MI) agent for fibrin using copper-based nanocolloids (NanoQ). Methods Nanocolloids incorporating copper (II) complexes were synthesized (Dav=217 nm, ζ=-13mV) and characterized for MI of thrombus. MR properties of NanoQ in suspension were defined at 3.0 T at 25°C. Single slice inversion recovery and multi-echo spin echo sequences were used to calculate the ionic (per metal) and particulate (per particle) relaxivities from serial dilutions. T1weighted gradient echo imaging of fibrin clots with NanoQ or a control (n=3/group) using fibrin-specific antibodies (NIB5F3) were acquired. In vivo pharmacokinetics and 24 hour biodistribution, and bioelimination of NanoQ were evaluated in rodents. Results The particulate relaxivity of the NanoQ was high, r1=66,000±2200 (s●mmol [NanoQ])-1, while the ionic r1 relaxivitiy (4.3±0.1 (s●mmol [Cu])-1) was similar to Gd-DTPA. The particulate r2 relaxivities were r2=135,000±2900 and ionic r2 relaxivities of 10.4±0.34 (s●mmol [Cu])-1, respectively. NanoQ targeted to fibrin clot phantoms provided strong improvement in contrast-to-noise ratio (CNR) (40x p 0.99). ICP analysis of tissue copper 24 hours post injection revealed that approximately 90% of the metal was already eliminated from the animal. Conclusions

In order to study the epitopes in fibrin towards which monoclonal antibodies are directed we needed the pure individual polypeptide chains of human fibrinogen in reasonable quantity. We report here a simplified, rapid method of separation of high-purity human fibrinogen chains. Following reduction and S-carboxymethylation of human fibrinogen, the sample was injected directly onto a column of the polymeric reversed-phase perfusion packing POROS 20-R2, and the chains were completely resolved in less than 3 min at a flow-rate of 10 ml/min. The capacity was equivalent to that of a similar sized conventional silica-based column. However the throughput was approximately five to ten times as high. The column was durable and robust in day-to-day use.

A new site-targeted molecular imaging contrast agent based on a nanocolloidal suspension of lipid-encapsulated, organically soluble divalent copper has been developed. Concentrating a high payload of divalent copper ions per nanoparticle, this agent provides a high per-particle r1 relaxivity, allowing sensitive detection in T1-weighted magnetic resonance imaging when targeted to fibrin clots in vitro. The particle also exhibits a defined clearance and safety profile in vivo.

BACKGROUND Targeting of plasminogen activators to the thrombus by means of fibrin-specific monoclonal antibodies may enhance their thrombolytic potency. The kinetics of clot binding of two human fibrin-specific monoclonal antibodies (MA-12B3 and MA-15C5) and of clot lysis with their chemical 1:1 stoichiometric complexes with recombinant single-chain urokinase-type plasminogen activator (rscu-PA) (rscu-PA/MA-12B3 and rscu-PA/MA-15C5) were determined in hamsters and rabbits. Thrombolytic potencies, maximal rates of clot lysis, and the duration of the lag phases before clot lysis of the antibody/rscu-PA conjugates were compared with those of rscu-PA and tissue-type plasminogen activator (rt-PA). METHODS AND RESULTS Bolus injection of 7.5 micrograms of 125I-labeled antibody in rabbits with an extracorporeal arteriovenous loop containing a 0.3-mL human plasma clot produced clot-to-blood ratios of 6.6 +/- 1.0 (mean +/- SEM) for MA-12B3 and 1.1 +/- 0.15 for MA-15C5 (p < 0.001 versus MA-12B3) within 6 hours. Progressive digestion of the clot did not alter the binding of MA-12B3 but resulted in as much as a 10-fold increase of the binding of MA-15C5. The conjugates infused intravenously over 90 minutes in hamsters with a human plasma clot in the pulmonary artery produced dose-related in vivo clot lysis. Thrombolytic potencies (maximal slope of the percent lysis versus dose in milligrams of u-PA equivalent per kilogram body weight) were 2,500 +/- 440 for rscu-PA/MA-12B3, 3,600 +/- 640 for rscu-PA/MA-15C5 (p = NS vs. rscu-PA/MA-12B3), 60 +/- 8 for rscu-PA (p < 0.001 versus both conjugates), and 380 +/- 66 for rt-PA (p < 0.001 versus both conjugates). The plasma clearances of the conjugates were fourfold to sixfold slower than those of rscu-PA and rt-PA. Maximal rates of clot lysis, determined by continuous external radioisotope scanning over the thorax, were 0.90 +/- 0.13%, 0.91 +/- 0.17%, 0.84 +/- 0.12%, and 1.1 +/- 0.16% lysis per minute for rscu-PA/MA-12B3, rscu-PA/MA-15C5, rscu-PA, and rt-PA, respectively; these maximal rates were obtained with 0.016, 0.016, 1.0, and 0.25 mg/kg, respectively, and were associated with minimal lag phases of 18 +/- 3.2, 28 +/- 4.9, 34 +/- 3.7, and 25 +/- 3.9 minutes, respectively. CONCLUSIONS The thrombolytic potency of the rscu-PA/antifibrin conjugates is determined by their clearance, as well as by rate and extent of initial binding to clots and by changes in binding during clot lysis. Clot targeting of rscu-PA with fibrin-specific antibodies increases its thrombolytic potency but does not alter the maximal rate or the minimal lag phase of clot lysis. These parameters appear to be independent of the nature of the plasminogen activator and of targeting.

Ancrod is a purified coagulant venom which renders blood incoagulable by cleaving fibrinopeptide A (FPA) from fibrinogen, but the mechanism involved in the clearance of fibrin from the circulation is unknown. To investigate the fibrinolytic response to ancrod, and to increase understanding of clearance mechanisms, six patients with peripheral vascular disease causing claudication were infused with ancrod at 2 u/kg over 6 h followed by 2 u/kg at 12 h intervals for 38 h. Venous blood samples were taken at time 0, 3, 6, 25 and 49 h for assay of fibrinogen (Fbg), fibrinopeptide A (FPA), total fibrin(ogen) degradation products (TDP), fibrin degradation products (FbDP), fibrinogen degradation products (FgDP), cross-linked fibrin degradation products (XL-FDP), tissue plasminogen activator (tPA), urinary type plasminogen activator (u-PA), plasminogen, alpha 2 antiplasmin (alpha 2 AP) and plasminogen activator inhibitor-1 (PAI-1). Fibrinogen (median and range) was 2.3 (1.4-3.90) g/l at time 0 and thereafter was undetectable. FPA rose from 2.5 (1.8-3.6) to 600 and 188 pmol/l at 3 h and 6 h and remained elevated. TDP, FbDP and FgDP increased greatly following ancrod while there was no evidence of XL-FDP. The surprising increase in FgDP during defibrination suggests either that fibrinogen is digested following its incorporation into circulating fibrin protofibrils or that some of the fibrin subunits in the photofibril retain one of the two fibrinopeptide A's. tPA and uPA remained unchanged. Plasminogen fell from 125 (100-155)% to 79 (40-118)% at 49 h and alpha 2 AP fell from 91 (75-107)% to 24 (10-35)% at 49 h. The level of PAI-1 was depressed during defibrination, with the exception of the 6 h data. The results demonstrate that ancrod removes FPA from fibrinogen to produce non-cross-linked (soluble) fibrin. This is cleared from the circulation without evidence of an increase in the circulating activities of the plasminogen activators, tPA or UK, but with evidence of plasminogen activation and consumption.

SummaryAn international collaborative study has been carried out to investigate the reproducibility of hirudin assays in 13 laboratories using four recombinant hirudins and one natural, sulphated product. A simple assay procedure was proposed involving the titration of α-thrombin with inhibitor and measurement of residual activity using a chromogenic substrate. A standard α-thrombin preparation was supplied to ensure that this reagent was of uniform quality throughout the study. The method appeared to present no difficulties and laboratories reported similar potencies for the 5 hirudin samples, in line with expected values. This gave 200–222 Thrombin Inhibitory Units/ampoule (TIU/ampoule) of lyophilised hirudin, with geometric coefficient of variation (gcv) values ranging from 10.15–15.97%. This corresponds to specific activities of approximately 14,300–15,900 TIU/mg protein. This is close to the upper limit of previously reported values of specific activity. We conclude that the precision of this determination compared with the wider range of values in the literature (8,000–16,000 thrombin inhibitory units [TIU]/mg) results from the use of good quality standard α-thrombin by all laboratories. This study has important implications for hirudin standardisation.

New multidetector cardiac computed tomography (MDCT) can image the heart within the span of a few beats, and as such, it is the favored noninvasive approach to assess coronary anatomy rapidly. However, MDCT has proven to be more useful for excluding coronary disease than for making positive diagnoses. The inability to detect unstable cardiac disease arises from the confounding attenuating effects of calcium deposits within atherosclerotic plaques, which obscure lumen anatomy, and from the insensitivity of CT X-rays to image low attenuating intraluminal thrombus adhered to a disrupted plaque cap, the absolute condition of ruptured plaque and unstable disease.[1–6] It is now well understood that the sensitive detection and quantification of small intravascular thrombus in coronary arteries with molecular imaging techniques could provide a direct metric to diagnose and risk stratify patients presenting with chest pain.[7,8]

Several production lots of Anistreplase (Eminase) were assayed for potency by either two fibrin plate assays or a clot lysis assay performed in 96-well microtiter plates. The 96-well plate assay yielded comparable data to the fibrin plate assays and had the advantage of greater efficiency with respect to both time and reagents. As a result the newer method appears to be a suitable alternative to the fibrin plate assays for lot release of Anistreplase.

SummarySince 1975 an International Standard for Thrombin of low purity has been used. While this standard was stable and of value for calibrating thrombins of unknown potency the need for a pure a-thrombin standard arose both for accurate calibration and for precise measurement of thrombin inhibitors, notably hirudin. An international collaborative study was undertaken to establish the potency and stability of an ampouled pure a-thrombin preparation. A potency of 97.5 international units (95% confidence limits 86.5-98.5) was established for the new a-thrombin standard (89/ 588) using a clotting-assay procedure. Stability data at various elevated temperatures indicated that the standard could be transported and stored with no significant loss of potency.Ampoules of lyophilised a-thrombin (coded 89/588) have been recommended as an International Standard for a-thrombin with an assigned potency of 100 international units per ampoule by the International Society for Thrombosis and Haemostasis (Thrombin and its Inhibitors Sub-Committee) in Barcelona, Spain in July 1990 while the Expert Committee on Biological Standardisation and Control of the World Health Organisation will consider its status at its next meeting in Geneva in 1991.

SummaryThere is increased interest in the relationship between plasma fibrinogen levels and the incidence of coronary artery disease. The National Institute for Biological Standards and Control (UK) has completed a study to establish an International Standard for plasma fibrinogen. This study was conducted using a recommended assay procedure to measure the clottable material present in the proposed lyophilised Standard (coded 89/644). Twenty-two laboratories from nine countries took part in the study and analysis of the data allowed the calibration of 89/644 at 2.4 mg/ml clottable protein. Agreement with this figure was established in two laboratories using three or more different assays for plasma fibrinogen. Degradation studies of the proposed plasma fibrinogen Standard suggested that no loss of clottable protein was observed when the lyophilised material was stored at 20° C for 1 year.The Fibrinogen Sub-Committee of the ISTH (Amsterdam, The Netherlands, June 1991) supported the establishment of 89/644 as an International Standard. This collaborative study will be presented to the Expert Committee on Biological Standardisation of the World Health Organisation at their 1992 session. In the meantime 89/644 will be distributed as the proposed International Standard for plasma fibrinogen measurement containing 2.4 mg/ ml clottable protein.

SummaryThe acquisition of monoclonal antibodies specific for human fibrin has been impaired by the similarity in chemical composition between fibrinogen and fibrin and the conformational difference between immobilised and soluble fibrinogen. Five monoclonal antibodies (mabs) with a known affinity for fibrin have been subjected to screening procedures which involved the presentation of different forms of both fibrinogen and fibrin to the test mabs. It was observed by scanning electron microscopy that dried fibrin (denoted fibrin D), immobilised on the wells of PVC plates was morphologically similar to the fibrin found in human clots whereas PVC-immobilised fibrin monolayers (fibrin M) and a homogenised form of fibrin (fibrin FF) presented two very different morphological appearances. It was shown that lack of cross reactivity of a mab with an antigen (e.g. fibrinogen) was validly demonstrated only when both mab and antigen were present in the soluble state. These findings have allowed the generation of a screening procedure which involves the use of fibrin D on PVC plates in conjunction with whole human plasma incubated with the test antibody. This screening procedure has been validated using two mabs, one of which has an exclusive fibrin affinity while the other has a broad spectrum crossreactivity with both fibrinogen and fibrin. This procedure would ensure the acquisition of all the five fibrin-specific mabs used in this study while other less reliable screening procedures might not.

Vascularly constrained, “soft” colloidal gold nanobeacons (GNB) demonstrate for the first time that GNBs can be characterized as exogenous photoacoustic contrast agents for targeted detection of fibrin, a major biochemical feature of thrombus. Fibrin-targeted GNBs provide a more than tenfold signal enhancement in photo acoustic tomography (PAT) in the NIR wavelength window, indicating their potential for diagnostic imaging with PAT.