Anti-fibrin Antibody Research Articles

Attachment of an antifibrin antibody to the amino terminus of tissue-type plasminogen activator impairs stimulation by Fibrin

On evidence that the potency and specificity of tissue-type plasminogen activator (t-PA) can be improved by chemical conjugation to an antifibrin antibody (59D8), we constructed a recombinant molecule, 59D8-t-PA(AB), containing a truncated 59D8 antibody attached through peptide linkage to the amino terminus of full-length t-PA. Initial analysis of recombinant 59D8-t-PA(AB) in the absence of fibrin revealed enzymatic characteristics indistinguishable from those of t-PA: for the S-2288 substrate, the respective Km values of t-PA and 59D8-t-PA(AB) were 0.480.02 mM and 0.520.02 mM; for plasminogen, the values were 82.35 ± 5.35 μM and 81.25 ± 2.03 μM. Recombinant 59D8-t-PA(AB) was up to 10-fold more potent than t-PA in an assay measuring the lysis of fibrin monomer. However, in contrast with the chemical conjugate, recombinant 59D8-t-PA(AB) was 2 to 3-fold less potent than t-PA in lysing clots in a plasma milieu. To determine whether this difference in plasma clot lysis was due to impaired fibrin stimulation of the t-PA portion of the recombinant molecule, we devised an assay that quantitates fibrin stimulation of t-PA's catalytic activity. Fibrin stimulation was indeed reduced by 15-fold for recombinant 59D8-t-PA(AB). This observation suggests that attaching a targeting antibody to the amino terminus of t-PA diminishes the ability of fibrin to stimulate the enzyme.

Design and evaluation of a thrombin-activable plasminogen activator.

A new chimeric plasminogen activator with high fibrin affinity was designed to bind fibrin and to initiate clot destruction, following activation by thrombin. The chimeric activator, 59D8-scuPA-T, was made from the Fab fragment of an anti-fibrin antibody (59D8) and a C-terminal portion of a thrombin-activable low molecular weight single-chain urokinase plasminogen activator, scuPA-T, obtained by deletion of Phe-157 and Lys-158 from low molecular weight single-chain urokinase-type plasminogen activator (scuPA) by site-directed mutagenesis. The chimeric molecule had a molecular mass of 91,000, a value consistent with one 59D8 light chain (M(r) = 27,000) and one 59D8 heavy-chain Fd fragment fused to low molecular weight scuPA (M(r) = 64,000). According to its design, 59D8-scuPA-T was activated by thrombin but not by plasmin, whereas the control chimeric molecule, 59D8-scuPA, was activated by plasmin but not by thrombin. When activated by thrombin, 59D8-scuPA-T converted plasminogen to plasmin. In vitro plasma clot lysis assays showed that 59D8-scuPA-T lysed clots performed by thrombin and that heparin and hirudin could prevent clot lysis. When incorporated as part of a thrombin-induced clot, only 59D8-scuPA-T was able to lyse the clot while 59D8-scuPA and high molecular weight scuPA were ineffective. Together these results demonstrate that 59D8-scuPA-T is a thrombin-activable plasminogen activator that offers selective thrombolysis of thrombin-rich clots over more established, aged clots, and may also act as an antithrombotic agent.

Correlation of beta-camera imaging and immunohistochemistry in radioimmunotherapy using90Y-labeled monoclonal antibodies in ovarian cancer animal models

Tumor stroma contains much fibrin and monoclonal antifibrin antibody targeting is possible in tumors. In this study, nude mouse human ovarian carcinoma xenograft specimens were investigated after treatment with 90Y-labeled monoclonal antifibrin antibody Fab fragment or with 90Y-labeled OC125-monoclonal antibody F(ab')2 fragments. The mice received the radioimmunotherapy activity either intratumorally, intraperitoneally, or intravenously. Beta-camera imaging (BCI) is a novel device for studying activity distribution in tissue specimens and, together with immunohistochemistry (IHC) with OC125, antifibrin, anticarcinoembryonic antigen, anti-cytokeratin, and anti-placental alkaline phosphatase antibodies, was used for correlation of activity distribution of tissue specimens. These results were in concordance: Antigen distribution measured with IHC and radioactivity distribution were similar with the same antibodies, antifibrin, and OC125: However, these antigens demonstrated rather different distribution. Tissue studies revealed that activity was concentrated also in the necrotic tumor tissue, indicating that cell death was also caused by radiation. Differences in the tumor cell morphology were observed using different routes of administration. With BCI, it is possible to quantitate activities in frozen sections (microdosimetry), and these results were in concordance with absolute activities as measured by tissue sampling and well-counting. Three-dimensional reconstruction of tissue slices combined with radioactivity distribution measured with BCI allows estimation of total absorbed radiation dose in tumor after an appropriate dose planning.

Immunodetection of human atherosclerotic plaque with 125I-labeled monoclonal antifibrin antibodies

To test the affinity of a new F(ab′) 2 monoclonal antibody (TRF1) against human fragment D dimer of cross-linked fibrin for atherosclerotic plaques free of detectable thrombi, 6 atherosclerotic segments of carotid and femoral artery, and as a control 5 segments of atherosclerosis-free internal mammary artery, were drawn from 11 male patients undergoing bypass surgery. All segments were carefully washed in order to remove possible endoluminal thrombi, and cut to obtain pairs of intimal fragments of similar weight, containing either plaques ( n = 16), or fatty streaks ( n = 12), or normal endothelium ( n = 20). Each fragment underwent a direct binding test to TRFI, or to a non-specific antibody, both labeled with 125I. The activity in each fragment was measured after 3 h of incubation at 37°C, and after washing the fragments every hour for 3 h. TRF1 binding (as percentage of initial activity) was significantly higher ( P < 0.001) in atherosclerotic than in normal fragments (26% ± 11.5%, vs. 9.2% ± 3.9% in fatty streaks, and 1.9% ± 0.6% in normal endothelium), and indirect immunofluorescence confirmed TRF1 uptake within the plaque wall. By contrast, the non-specific antibody did not show any significant binding. These preliminary results demonstrate the high specific affinity of TRF1 for atherosclerotic plaques, probably due to the hemorheologic phenomena that activate platelets and provoke the formation of fragment D dimers of cross-linked fibrin on the plaque surface.

Radioimmunotherapy with90Y-Labeled Monoclonal Antibodies in a Nude Mouse Ovarian Cancer Model

Tumor stroma contains much fibrin, and so monoclonal antifibrin antibody can accumulate in tumors. We treated nude mice bearing human ovarian carcinoma xenografts with 90Y-labeled monoclonal antifibrin antibody Fab fragments administered intratumorally. The survival time vs. a control group was significantly prolonged and tumor growth rate was decreased. Another group of animals was treated with 90Y-labeled OC 125-monoclonal antibody; these mice received the antibodies intratumorally, intraperitoneally or intravenously. The survival time was longest in the intratumorally treated group. There was no significant difference in survival between 90Y-labeled OC 125 and antifibrin in the intratumorally treated animal groups. The tissue activity distribution studies revealed that bone marrow is the critical organ. Intratumorally injected monoclonal 90Y-antifibrin antibodies were retained at least 36 h (up to 50% of injected activity per gram tumor tissue) in the xenograft after one treatment, causing cell death. Beta-camera imaging and immunohistochemistry were performed for studies of the correlation between 90Y activity and fibrin distribution in tumor specimens. These results were in concordance. In conclusion, intratumoral administration seems suitable for radioimmunotherapy, with an antibody that targets stromal structures. The accumulation can be successfully monitored by a beta-camera.

Thrombus Imaging with 99mTc-Hmpao-Labeled Platelets and 111In-Labeled Monoclonal Antifibrin Antibodies

Eighteen patients with suspicion of deep venous thrombosis (DVT) in the lower extremities were imaged both with autologous 99mTc-HMPAO-labeled platelets (Tc-PLT) and 111In-labeled monoclonal antifibrin antibodies (In-MoAbs) on the same day. Presence or absence of thrombosis was verified by venography. Tc-PLT was given i.v. followed after 30 min by In-MoAbs. Anterior and posterior projections of the lower extremities were obtained with a large field-of-view gamma camera at 5 to 25 min, 2 h, 4 to 6 h, and 20 h after administration of the marker. Both Tc-PLT and In-MoAbs detected DVT well but less frequently than venography. Thrombi were visualized at 2 to 4 h after injection. The quality of images was better with Tc-PLT than with In-MoAbs. In the patients treated during the study, heparin significantly (p < 0.01) inhibited the uptake of Tc-PLT but not of In-MoAbs. We conclude that both Tc-PLT and In-MoAbs are suitable agents for the detection of DVT especially in patients without anticoagulation.

Remaining perspectives of mutant and chimeric plasminogen activators.

Potential approaches to improve thrombolytic agents comprise the construction of mutants and variants of tissue-type plasminogen activator (tPA) or of single chain urokinase-type plasminogen activator (scuPA, pro-urokinase), of chimeric plasminogen activators and of conjugates of plasminogen activators with monoclonal antibodies. tPA mutants have been constructed with altered pharmacokinetic properties or altered functional properties, including binding to and stimulation by fibrin, resistance to plasmin and to protease inhibitors. Mutants of tPA described to date, obtained by deletion/substitution of functional domains or of single amino acids, have markedly reduced clearances, but usually also reduced specific thrombolytic potencies. Mutants of scuPA with improved thrombolytic potencies have thus far not been reported. Chimeric molecules containing functional domains of both tPA and scuPA have intact enzymatic properties of uPA and some fibrin affinity of tPA. Surprisingly, chimeras endowed with fibrin affinity usually have unaltered or reduced thrombolytic potencies. However, a chimera consisting of amino acids 87-274 of tPA and amino acids 138-411 of scuPA, with negligible fibrin affinity, has a 10-fold higher thrombolytic potency than scuPA in animal models of venous thrombosis, as a result of a delayed in vivo clearance and a relatively maintained specific thrombolytic activity. Plasminogen activators conjugated with antifibrin or antiplatelet monoclonal antibodies, either chemically or by recombinant DNA technology, are targeted to blood clots, resulting in a 5- to 10-fold increased thrombolytic potency. Thus, it is possible to develop plasminogen activators with improved thrombolytic potency. Whether such agents will be clinically useful remains to be established.

Evaluation of Indium-111 Antifibrin Monoclonal Antibody Imaging in Deep Venous Thrombosis Diagnosis

Objective: To evaluate Indium-111 antifibrin monoclonal antibody in the diagnosis of deep vein thrombosis. Design: Prospective study in twelve patients undergoing major surgical treatment, including hip surgery, who would be considered at high risk of developing subsequent deep vein thrombosis. Setting: Departments of Angiology and Vascular Surgery, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain. Patients: Twelve patients receiving surgical treatment. Interventions: Bilateral contrast phlebography was performed in all patients, combined with an ***immunoscintigraphic test of an Indium-111 labelled monoclonal ***antibody to fibrin. Patients with deep vein thrombosis were treated with full anticoagulation using heparin. Main outcome measures: The presence of thrombosis on phlebography and the presence of isotopic labelling of suspected deep vein thrombosis on immunoscintigraphic scanning. Results: Phlebography demonstrated venous thrombosis at 23 sites in veins, of which 22 were also detected by immunoscintigraphic scanning. Conclusions: Indium-111 labelled monoclonal antibodies to fibrin may provide an alternative method of investigation for suspected deep vein thrombosis as an alternative to other non-invasive diagnosis methodologies.

Availability of the Bβ(15-21) Epitope on Cross-Linked Human Fibrin and Its Plasmic Degradation Products

The binding of radiolabeled monoclonal antifibrin antibody 59D8 (specific for fibrin but not fibrinogen) to a series of degraded fibrin clots showed that the availability of the B beta(15-21) epitope (against which 59D8 had been raised) was inversely proportional to the extent of clot lysis. Examination of digest supernatants revealed that the B beta(15-21) epitope was released from clots as a high molecular weight degradation product in the presence of calcium ions but that the generation of low molecular weight peptides occurred in the absence of calcium ions. To address the question of epitope accessibility, we compared levels of fibrin clot binding among four radioactively labeled antibodies: antifibrin monoclonal antibody 59D8, two antifibrinogen monoclonal antibodies that cross-reacted with fibrin, and an affinity-purified polyclonal antifibrinogen antibody. We expected that the antifibrinogen antibodies would show enhanced binding to clots in comparison with the antifibrin antibody. However, the epitope accessibility experiments showed that all four antibody preparations bound fibrin clots at comparable levels. Taken together, these studies demonstrated that one fibrin-specific epitope, B beta(15-21), remains available on clots as they undergo degradation by plasmin and, importantly, that the epitope is not solubilized at a rate faster than the rate at which the clot is itself solubilized. The availability of the B beta(15-21) epitope during the course of plasminolysis assures the potential utility of antifibrin antibodies such as 59D8 for detecting thrombi and targeting plasminogen activators.

Echo-doppler veineux : futur examen de rétérence dans le diagnostic des thromboses des membres inférieurs ?

Duplex ultrasound imaging provides both an ultrasound picture of the tissue being explored and the sound of the flow speed in a particular area. Colour Doppler imaging gives colour codes for the different flow rates. Venous pathology is always investigated, with ultrasound, on both sides, at the iliac and caval, femoral, and foreleg levels. For the latter, excellent equipment and patient position are required. Ultrasound criteria of deep venous thrombosis (DVT) are the incompressibility of the vein being investigated, and detection of the thrombus with the ultrasound probe. Doppler criteria are asymmetric spontaneous flows, the loss of their respiratory rhythm, and the lack of flow induced by contraction of muscle. The colour Doppler draws provides the contours of a partial or floating thrombus. Compared with phlebography, ultrasound imaging has a sensitivity and specificity of 96 %. Colour Doppler imaging is even more accurate : both sensitivity and specificity increase to 100 %. The limitations of ultrasound are : its dependence on the investigator, the difficulty of making the diagnosis of recurring DVT when there already are previous sequellae, obesity, intra-abdominal gas, skin lesions or inflammatory oedema. Phlebography remains necessary in 5 % of cases. The advantages of this technique are its non invasiveness, excellent diagnostic performance, low cost, and the possibility of diagnosing another cause, whether responsible for the clinical signs or not. It is indicated for the diagnosis of DVT, routine screening in patients at risk, and the early and late follow-up of patients. Today, ultrasound imaging should always be carried out before other investigations. A multicenter trial comparing phlebography, ultrasound imaging and a third investigation, acting as a reference (scintigraphy with anti-fibrin antibodies), is now required to redefine the standard investigation for venous thrombosis.

Noninvasive arterial thrombus imaging with 99mTc monoclonal antifibrin antibody.

The T2G1s monoclonal antifibrin antibody binds specifically to fibrin but not to fibrinogen. In a canine model of acute arterial thrombosis, we determined the feasibility of imaging thrombi using a 99mTc-labeled Fab' fragment. In 14 dogs, 10 carotid and 13 femoral artery thrombi were produced using 2-hour temporary occlusion, crush injury, and local thrombin injection methods. A sham-operated carotid artery served as control. Antifibrin antibody was injected intravenously at the end of temporary occlusion. Serial planar radionuclide images were obtained immediately and at 1 and 2 hours. Following killing the dogs at 2 hours, we measured antibody uptake ex vivo in 5-mm-long segments of thrombus, the adjacent injured artery, and a control artery. Antibody was cleared from the blood with a mean +/- SD t1/2 of 121 +/- 23 minutes. The thrombi weighed 218 +/- 140 mg. Antibody uptake in the thrombi was patchy, and the thrombi were closely adherent to the injured arterial wall. In the segment with maximal ex vivo antibody uptake, the ratio of control artery to blood counts/g/sec was 0.65 +/- 0.46, the injured artery-to-blood ratio was 2.35 +/- 1.01 (p less than 0.0001 versus control), and the thrombus-to-blood ratio was 4.24 +/- 2.58 (p less than 0.0001 versus control). In three dogs, an isotype-matched ovarian tumor antibody labeled with 111In was injected with T2G1s but was not taken up in the thrombus or the adjacent arterial wall. Visual analysis of the in vivo carotid radionuclide images showed uptake by 2 hours in all 10 carotid thrombi. Quantitative image analysis, measured as the thrombus-to-opposite carotid artery ratio, showed increasing uptake over time with ratios of 1.1 +/- 0.3, 1.6 +/- 2.0, and 2.2 +/- 1.3 on the immediate, 1-hour, and 2-hour images, respectively. All quantitative ratios of 1.3 or greater were visually identified. 99mTc-labeled Fab' fragments of the T2G1s antibody are taken up specifically by acute arterial thrombi after intravenous injection. Uptake is progressive over a 2-hour period, and all thrombi are detected by radionuclide imaging at 2 hours. These results show that it is feasible to noninvasively detect arterial thrombi within 2 hours of formation.

Preclinical evaluation of 99mtechnetium-labeled DD-3B6/22 FAB' for thrombus detection

Injection of 99mTc-labeled Fab' fragments of the anti-fibrin antibody DD3B6/22, in the baboon, resulted in clear visualisation of both fresh and aged autologous thrombi by gamma scintigraphy. Whole body scintigraphy, pharmacokinetics and urine analysis showed rapid renal excretion of the conjugate with little accumulation of label in other organs. 99mTc-DD-3B6/22 Fab' appears a suitable candidate for further investigation as a radioimaging agent for thrombi in humans.

Detection of deep venous thrombosis with indium 111-labelled monoclonal antibody against tissue plasminogen activator

The administration of a radiolabelled monoclonal antibody against tissue plasminogen activator allows detection of areas with increased fibrinolytic activity, i.e. those with an active thrombotic lesion. Eight patients with phlebographically verified deep venous thrombosis were examined. At the time of immunoscintigraphy study they were examined receiving anticoagulant therapy. Some 75-85 MBq indium 111-labelled antibody were injected, and scintigrams were obtained after 30 min and after 24 h. The precise site of the thrombus could not be visualized after 30 min due to high background activity, whereas after 24 h it was detectable in all patients. The thrombus/background ratios achieved are twice as high as those observed in a human antifibrin antibody study. These preliminary data suggest a high sensitivity of our t-PA-specific antibody for the detection of active deep venous thrombosis in man, and our antibody seems to offer theoretical advantages over both platelet and fibrin-specific antibodies.

Anti-fibrin monoclonal antibody for immunoscintigraphic detection of thrombi

Anti-fibrin monoclonal antibody for immunoscintigraphic detection of thrombi

Radiolabeled antifibrin antibody in the detection of venous thrombosis: preliminary results.

The recent development of monoclonal antibodies against components of coagulated blood may provide new approaches to the diagnosis of venous thrombosis. Scanning with an indium-111-labeled Fab fragment of a murine monoclonal antifibrin antibody (59D8) and ascending contrast venography were performed in 33 patients. Images of the calves, popliteal fossae, thighs, and pelvis were obtained immediately, 4-6 hours, and 24 hours after injection of 2 mCi (74 MBq) of the antibody. All images were read in a blinded manner. Findings in both studies were positive in 28 patients and negative in three. In 19 patients not undergoing heparin therapy, 19 specific anatomic sites were positive on venograms and 29 were positive on antibody images (19 sites matched). In 14 patients undergoing heparin therapy, 34 sites were positive on venograms and 27 were positive on antibody images (22 sites matched). In most patients, positive results were noted within 1 hour of antibody injection. No adverse effects were noted with the antibody preparation. Preliminary data suggest that antifibrin antibody imaging is sensitive in detecting clots, is safe to use, and may have a role in diagnosing and managing venous thrombosis.

Bispecific Monoclonal Antibodies Produced by Somatic Cell Fusion Increase the Potency of Tissue Plasminogen Activator

Bispecific monoclonal antibodies that bind simultaneously to human fibrin and tissue plasminogen activator (tPA) enhance the fibrinolytic potency of tPA. Two bispecific antibodies (F36.23 and F32.1) were generated by somatic cell fusion. Antibody F36.23 derives its tPA binding from monoclonal anti-tPA antibody TCL8 and its fibrin binding from monoclonal antifibrin antibody 59D8. After purification from cell supernatants and ascites by two steps of affinity chromatography, hybrid-hybridoma bispecific antibody F36.23 simultaneously bound tPA and fibrin in solution and in solid-phase assays. In an assay for the lysis of human fibrin monomer, F36.23 increased the fibrinolytic potency of tPA by 5 to 10 fold, regardless of whether the bispecific antibody had been combined with the tPA before or during the assay. Bispecific F36.23 F(ab')2 also bound tPA and fibrin simultaneously, and the enhancement in fibrinolysis in the presence of F36.23 F(ab')2 was identical to that in the presence of intact F36.23. The second bispecific antibody, F32.1, was produced by an alternative strategy that has a wider potential for application in other systems. Hybridoma bispecific antibody F32.1 was derived from the fusion of immune splenocytes (in mice immunized with a synthetic oligopeptide representing the amino terminus of the alpha-chain of human fibrin) with the anti-tPA cell line TCL8. The properties of hybridoma bispecific antibody F32.1 and its F(ab')2 were indistinguishable from those of hybrid-hybridoma bispecific antibody F36.23 in solid-phase binding assays and in assays of fibrinolysis. Bispecific antibodies produced by somatic cell fusion, particularly in the form of F(ab')2, may have potential for use in clinical thrombolysis.

Deep vein thrombosis: scintigraphic diagnosis with In-111-labeled monoclonal antifibrin antibodies.

Fifty-two patients suspected of having deep vein thrombosis under-went scintigraphy with an indium-111-labeled monoclonal antifibrin antibody. Venography disclosed deep vein thrombosis in 31 patients. With the whole limb considered an anatomic entity, antifibrin antibody scintigrams obtained 2 hours after injection had a specificity and sensitivity of 81% and 84%, respectively. A higher sensitivity (92%) was found for a subgroup of patients (n = 44) with symptoms for less than 10 days. Regional sensitivities for all patients and for the subgroup, respectively, were 92% and 100% in the calf, 82% and 94% in the popliteal region, 63% and 71% in the thigh, and only 18% and 13% in the pelvis. Additional imaging performed 6 hours and 21 hours after injection in 12 patients and quantitative analysis done from scintigrams with and without blood-pool (technetium-99m human serum albumin) correction did not improve sensitivity. In-111-antifibrin antibody scintigraphy is an accurate method for diagnosis of acute established deep vein thrombosis of the calf and popliteal region; its sensitivity in the thigh is lower, and it is not feasible for diagnosis in the pelvic area.

Current status of immunoscintigraphy in the detection of thrombosis and thromboembolism

This review describes the use of monoclonal antibodies (MoAbs) for immunoscintigraphic detection of thrombosis and thromboembolism. Two major groups of MoAbs have been tested: Antibodies directed against platelets and antibodies directed against fibrin. Several antiplatelet antibodies have been developed that are directed against either the platelet membrane glycoprotein complex, IIb/IIIa, which binds fibrinogen, or against two different proteins, alpha granule membrane protein GMP-140 and thrombospondin (TSP) that are expressed during platelet activation. Platelets labeled with radioactive antibody or antibody fragments have characteristics similar to platelets labeled in vitro with lipophilic complexes. Studies in animal models indicate that antiplatelet antibody and antiactivated platelet factor antibody imaging have a high sensitivity for clots less than 24 hours old and that images can be positive as early as one to two hours after antibody administration. A limited number of antiplatelet antibody studies have been performed in patients. This technique appears to yield accurate results provided that active platelet deposition is occurring at the time of the study. Several antifibrin MoAbs, specific for fibrin monomers, have been developed. Compared with antiplatelet antibodies, antifibrin antibodies and antibody fragments appear to be capable of detecting older experimental clots (up to five days old). Images in experimental thrombosis models are routinely positive within two hours of antibody administration. Recently reported clinical trials indicate a high sensitivity in all anatomic locations within the extremities whether or not patients were receiving heparin.

Diagnosis of Deep Venous Thrombosis of the Leg Using Immunoscintigraphy with111In-Labelled Monoclonal Antifibrin Antibody Fragments

Fourteen unselected consecutive patients with clinically suggested deep venous thrombosis were studied using 111In-labelled monoclonal antibody Fab-fragments (59D8) in order to evaluate the accuracy of antifibrin imaging. Scintigrams of the legs were obtained at 1 to 4 h and 20 to 28 h in all patients and in seven dynamic studies were also performed. Contrast medium phlebography used as diagnostic standard revealed thrombosis in six patients who also had increased uptake of the tracer at immunoscintigraphy. Three patients had a positive finding at immunoscintigraphy without thrombosis. Because of the high sensitivity of this method, normal antifibrin images may be used to exclude thrombosis. Phlebography may then be restricted to positive cases to confirm the presence of thrombosis.

活性ペプチドのＤＤＳ 血栓溶解剤のＤＤＳ

Recently, a remarkable progress has been done in thrombolytic therapy. The progress mainly depends on the newly develped route of administration (for example, intracoronary), and the development of more fibrin-specific fibrinolytic agents. The newly developed thrombolytic drugs, called as the “third generation” agents are t-PA(tissue-type plasminogen activator), which is fibrin-specific, scu-PA(single-chain urokinase-type plasminogen activator), which is activated on fibrin surface, and APSAC(acylated plasminogen-streptokinase activator complex), which has rather long in vivo half-life. The physiological half-lives of these agents in the circulation are short except a kind of acylated plasminogen-streptokinase activator complex (RBL 33575). Plasmin(“first generation” agent)is not used because of its extremely short half-life(0.1 sec)and its lack of fibrin-specificity. However, the “second” and the “third” generation drugs are useful notwithstanding their relatively short half-lives, because fibrinolytic agents must be administered as quick as possible before the development of organ failure due to disturbances of circulation. Quite recently, the drugs with more increasing fibrin selectivity have begun to be designed. These are mutants of the scu-PA, kimeras of these drugs with plasminogen, and hybrids of fibrinolytic agents and anti-fibrin antibody. Moreover, fibrotide, which inhibits activity of PAI, which neutralizes t-PA and urokinase.