Fibrin-targeted perfluorocarbon nanoparticles for targeted thrombolysis.

BACKGROUND: Reperfusion of the ischemic brain is the most effective therapy for acute stroke, restoring blood flow to threatened tissues. Thrombolytics, such as recombinant tissue plasminogen activator, administered within 3 h of symptom onset can improve neurologic outcome, although the potential for adverse hemorrhagic events limits its use to less than 3% of acute ischemic stroke patients. Targeting of clot-dissolving therapeutics has the potential to decrease the frequency of complications while simultaneously increasing treatment effectiveness, by concentrating the available drug at the desired site and permitting a lower systemic dose. OBJECTIVES: We aimed to develop a fibrin-specific, liquid perfluorocarbon nanoparticle that is surface modified to deliver the plasminogen activator streptokinase. We also aimed to evaluate its effectiveness for targeted thrombolysis in vitro using quantitative acoustic microscopy. METHODS: Human plasma clots were formed in vitro and targeted with streptokinase-loaded nanoparticles, control nanoparticles or a mixture of both. Depending on the treatment group, clots were then exposed to either phosphate-buffered saline (PBS), PBS with plasminogen or PBS with plasminogen and free streptokinase. Spatially registered ultrasound scans were performed at 15-min intervals for 1 h to quantify changes in clot morphology and backscatter. RESULTS: Nanoparticles bound to the clot significantly increased the acoustic contrast of the targeted clot surface, permitting volumetric estimates. Profile plots of detected clot surfaces demonstrated that streptokinase-loaded, fibrin-targeted perfluoro-octylbromide nanoparticles in the presence of plasminogen induced rapid fibrinolysis (<60 min) without concurrent microbubble production and cavitation. Streptokinase-loaded or fibrin-targeted control nanoparticles insonified in PBS did not induce clot lysis. Morphologic changes in the treated group were accompanied by temporal and spatial changes in backscatter. Ultrasound exposure had no effect on the digestion process. Effective concentrations of targeted streptokinase were orders of magnitude lower than equivalently efficacious levels of free drug. Moreover, increasing competitive inhibition of fibrin-bound streptokinase nanoparticles reduced clot lysis in a monotonic fashion. As little as 1% surface targeting of streptokinase nanoparticles produced significant decreases in clot volumes (approximately 30%) in 1 h. CONCLUSION: This new nanoparticle-based thrombolytic agent provides specific and rapid fibrinolysis in vitro and may have a clinical role in early reperfusion during acute ischemic stroke.

An experimental clot model in sheep; generation of a heterologous clot and its detection in vivo using venography and (125)I labelled fibrinogen.

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.

A performance evaluation of commercial fibrinogen reference preparations and assays for Clauss and PT-derived fibrinogen.

The wide availability of fibrinogen estimations based on the prothrombin time (PT-Fg) has caused concern about the variability and clinical utility of fibrinogen assays. In a multi-centre study, we investigated fibrinogen assays using various reagents and analysers. Clauss assays generally gave good agreement, although one reagent gave 15-30% higher values in DIC and thrombolysis. Two commercial reference preparations had much lower potencies than the manufacturers declared, and plasma turbidity influenced parallelism in some Clauss assays. PT-Fg assays gave higher values than Clauss and showed calibrant dependent effects, the degree of disparity correlating with calibrant and test sample turbidity. Analyser and thromboplastin dependent differences were noted. The relationship between Clauss and PT-Fg assays was sigmoid, and the plateau of maximal PT-Fg differed by about 2 g/l between reagents. ELISA and immunonephelometric assays correlated well, but with a high degree of scatter. Antigen levels were higher than Clauss, but slightly lower than PT-Fg assays, which appeared to be influenced by degraded fibrinogen. Clauss assays are generally reproducible between centres, analysers and reagents, but PT-Fg assays are not reliable in clinical settings.

Differences between neonates and adults in tissue-type-plasminogen activator (t-PA)-catalyzed plasminogen activation with various effectors and in carbohydrate sequences of fibrinogen chains.

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.

Temperature dependence of ultrasonic enhancement with a site-targeted contrast agent.

Molecular imaging contrast agents specifically detect the biochemical "signatures" of disease before anatomical manifestations are apparent. Sensitive and specific localization of fibrin both in vivo and in vitro has been demonstrated with the use of a ligand-directed liquid perfluorocarbon nanoparticle. Since the acoustic properties of perfluorocarbons are known to vary with temperature, it was hypothesized that temperature could be used to augment the magnitude of enhancement imparted by targeted nanoparticles. Accordingly, the acoustic backscatter of two different substrates, nitrocellulose membrane and human plasma clot, targeted by the nanoparticles was measured at temperatures ranging from 27 degrees to 47 degrees C in 5 degrees C increments. Classic avidin-biotin interactions were utilized to couple biotinylated nanoparticles to avidin-conjugated nitrocellulose membranes. Ultrasonic contrast enhancement of the nitrocellulose membrane at 25 MHz, measured by acoustic microscopy, increased from 2.0+/-0.3 dB at 27 degrees C to 3.7+/-0.4 at 47 degrees C. In a similar experiment, antifibrin nanoparticles bound to human plasma clots also exhibited temperature-dependent ultrasonic signal enhancement ranging from 13.9+/-1.5 dB at 27 degrees C to 18.1+/-1.5 dB at 47 degrees C. The increase in ultrasonic contrast enhancement measured was well described by a simple, acoustic transmission line model with temperature-dependent impedance. These results suggest that temperature-dependent changes in acoustic backscatter may be used to further differentiate tissues targeted with site-specific nanoparticles from surrounding normal soft tissues.

Novel MRI contrast agent for molecular imaging of fibrin: implications for detecting vulnerable plaques.

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 approximately 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.

Pesticide resistance: can we make it a renewable resource?

Negative cross-resistance (NCR) occurs when a mutant allele confers (i) resistance to one toxic chemical and (ii) hyper-susceptibility to another. Sequential deployment of NCR toxins is useful for insect control in few situations (Pittendrigh et al., 2000). Using Monte Carlo simulations, we investigated the concurrent use of a pair of NCR toxins to control a hypothetical insect pest population. When the toxins killed more heterozygotes than homozygotes, the resistance allele became either extremely common or rare depending on starting allelic frequency. If the NCR toxins did not kill the two homozygous groups equally, then the toxin with lesser toxicity eventually played a greater role in the control of the pest population. Based on our results, we present an approach for the systematic development of an NCR toxin after the commercial release of the first toxin. First, large-scale screens are performed to find chemicals that kill the resistant homozygous insects, but not the susceptible ones. Chemicals that preferentially kill resistant insects are then tested for toxicity to the heterozygotes. Those highly toxic to both homo- and heterozygotes are given the highest priority for development. This screen can be adapted to identify compounds useful in controlling antibiotic-, herbicide- or fungicide-resistant organisms.

Fibrin degradation products. A review of structures found in vitro and in vivo.

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.

Differences between neonates and adults in the urokinase-plasminogen activator (u-PA) pathway of the fibrinolytic system.

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.

High-resolution MRI characterization of human thrombus using a novel fibrin-targeted paramagnetic nanoparticle contrast agent.

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 T(1)-weighted MRI, while targeted contrast agent dramatically improved the detectability of all clots. Decreases in T(1) and T(2) 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.

A collaborative study to establish the 2nd International Standard for Fibrinogen, Plasma.

An International Collaborative Study involving 12 laboratories in 7 different countries was undertaken in order to replace the 1st International Standard (IS) for Fibrinogen, Plasma (89/644). The candidate replacement standard was the ampouled and freeze-dried residue of solvent/detergent treated plasma and was calibrated as coded duplicates (A and B) versus the 1st IS Fibrinogen, Plasma by automated Clauss assay and by a recommended clot collection (gravimetric) assay. This latter method had been used to calibrate the 1st IS Fibrinogen, Plasma. Comparing the ratios of the potency estimates of sample A to sample B (the coded duplicates), all of the laboratories obtained a ratio within 5% of the expected value of 1.0 by automated Clauss assay, which suggests that the laboratories were able to perform this assay well. Scrutiny of the data obtained from the gravimetric assays revealed that in almost all cases the results were invalid. The results of these assays are included in this report but clearly should be treated with caution and indeed produced significantly lower mean estimates of potency than the other assay methods. The overall geometric mean of all estimates of potency of the proposed 2nd IS Fibrinogen, Plasma (98/612) is 2.19 mg/ampoule by the automated Clauss assay. These data have been presented to the Fibrinogen Sub-Committee of the Standardisation and Scientific Committee (SSC) of the International Society on Thrombosis and Haemostasis (ISTH) (Washington, DC, August 1999), which recommended the establishment of 98/612 as the 2nd IS Fibrinogen, Plasma. This report has been presented to the Expert Committee on Biological Standardisation of the World Health Organisation (ECBS-WHO) at their 1999 session and 98/612 was established as the 2nd IS Fibrinogen, Plasma with a potency of 2.2 mg/ampoule.

In vivo penetration of experimentally produced clots by monoclonal antibodies.

Antifibrin 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 (131)I 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 1H10 and examining the clot excised after 1, 6 and 24 h using immunofluorescence. In a further series of similar experiments (125)I 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.

Textilinins from Pseudonaja textilis textilis. Characterization of two plasmin inhibitors that reduce bleeding in an animal model.

The incidence of vein-graft occlusion associated with myocardial infarction and thrombosis following the use of the plasmin inhibitor, aprotinin, to reduce blood loss during vascular surgery has prompted the isolation of an alternative kinetically distinct inhibitor of plasmin from the venom of Pseudonaja textilis. This inhibitor has been called textilinin (Txln) and two distinct forms have been isolated from the Brown-snake venom (molecular weight, 6688 and 6692). A comparison of plasmin inhibitor constants for aprotinin and the Txlns 1 and 2 indicated that the former bound very tightly (inhibitor constant, Ki approximately 10(-11) mol/l), while both of the latter bound less tightly (Ki approximately 10(-9) mol/l). Homogeneity of Txlns 1 and 2 was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and mass spectrometry. A sequence difference of six amino acids was observed between the two forms of Txln. Txln 1 and 2 showed, respectively, 45 and 43% homology with aprotinin, while there was 58 and 55% homology, respectively, with a plasmin inhibitor from the venom of eastern Taipan, Oxyuranus scutellatus. Both Txlns have six cysteines, like other inhibitors of this group, and homology was determined by alignment of these cysteines. Both have been shown to reduce blood loss by about 60% in a murine tail vein bleeding model. It is proposed that the kinetic profiles of Txln 1 and 2 for plasmin allow the arrest of haemorrhage without the possible threat of thrombosis.

Time evolution of enhanced ultrasonic reflection using a fibrin-targeted nanoparticulate contrast agent.

Complex molecular signaling heralds the early stages of pathologies such as angiogenesis, inflammation, unstable atherosclerotic plaques, and areas of remote thrombi. In previous studies, acoustic enhancement of blood clot morphology was demonstrated with the use of a nongaseous, fibrin-targeted acoustic nanoparticle emulsion delivered to areas of thrombosis both in vitro and in vivo. In this study, a system was designed and constructed that allows visualization of the evolution of acoustic contrast enhancement. To evaluate the system, two targets were examined: avidin-complexed nitrocellulose membrane and human plasma clots. The time evolution of enhancement was visualized in 10-min increments for 1 h. A monotonic increase was observed in ultrasonic reflection enhancement from specially treated nitrocellulose membranes for targeted emulsions containing perfluorooctylbromide (1.30+/-0.3 dB) and for perfluorooctane (2.64+/-0.5 dB) within the first 60 min of imaging. In comparison, the inherently nonechogenic plasma clots showed a substantial increase of 12.0+/-0.9 dB when targeted with a perfluoro-octane emulsion. This study demonstrates the concept of molecular imaging and provides the first quantifiable time-evolution report of the binding of a site-targeted ultrasonic contrast agent. Moreover, with the incorporation of specific drug treatments into the nanoparticulate contrast agent, ultrasonic molecular imaging may yield reliable detection and quantification of nascent pathologies and facilitate targeted drug therapy.

Development of an ELISA for the quantification of fibrin in canine tumours.

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 microg 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 intraassay 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 microg/ml. Using this assay, we reported the levels of fibrin in three different types of malignant canine tissue.

The haemostatic balance -- Astrup revisited.

Prof. Tage Astrup first elaborated the notion that blood fluidity involved a balance between the tendency of blood to clot and for such clots to lyse. It would seem that, at that time, this haemostatic balance involved the notion that forming fibrin orchestrated its own destruction by stimulating fibrinolytic activity. In this review, we have clarified the detail of this balance and developed the thesis that Astrup's far-sighted balance notions involve a variety of control mechanisms. These involve the notion that thrombin, being at first sight a procoagulant, can also, in conjunction with thrombomodulin, act as a stimulus of anticoagulant activity by the generation of activated protein C. The thrombin-activatable fibrinolytic inhibitor (TAFI) is also involved in this balance since the generation of thrombin provokes the neutralisation of fibrinolysis by the TAFI pathway. The kallikrein/factor XII/urokinase pathway is discussed indicating yet another aspect of balance between the generation of coagulation and fibrinolysis. The overall theme of this review, apart from an insight into various aspects of the haemostatic balance, is that blood has a strong tendency to clot when tissue is damaged, and the intact vasculature requires major anticoagulant systems to prevent clots adhering to and stabilising in the vasculature.

In vitro characterization of a novel, tissue-targeted ultrasonic contrast system with acoustic microscopy.

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.

The role of the monocyte in the generation and dissolution of arterial and venous thrombi.

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.