High Affinity For Fibrin Research Articles

Targeting platelet GPVI with glenzocimab: a novel mechanism for inhibition.

Platelet glycoprotein VI (GPVI) is attracting interest as a potential target for the development of new antiplatelet molecules with a low bleeding risk. GPVI binding to vascular collagen initiates thrombus formation and GPVI interactions with fibrin promote the growth and stability of the thrombus. In this study, we show that glenzocimab, a clinical stage humanized antibody fragment (Fab) with a high affinity for GPVI, blocks the binding of both ligands through a combination of steric hindrance and structural change. A cocrystal of glenzocimab with an extracellular domain of monomeric GPVI was obtained and its structure determined to a resolution of 1.9 Å. The data revealed that (1) glenzocimab binds to the D2 domain of GPVI, GPVI dimerization was not observed in the crystal structure because glenzocimab prevented D2 homotypic interactions and the formation of dimers that have a high affinity for collagen and fibrin; and (2) the light variable domain of the GPVI-bound Fab causes steric hindrance that is predicted to prevent the collagen-related peptide (CRP)/collagen fibers from extending out of their binding site and preclude GPVI clustering and downstream signaling. Glenzocimab did not bind to a truncated GPVI missing loop residues 129 to 136, thus validating the epitope identified in the crystal structure. Overall, these findings demonstrate that the binding of glenzocimab to the D2 domain of GPVI induces steric hindrance and structural modifications that drive the inhibition of GPVI interactions with its major ligands.

ПОКАЗНИКИ ДЕЯКИХ КОМПОНЕНТІВ СИСТЕМИ АКТИВАЦІЇ ПЛАЗМІНОГЕНУ ПРИ РАКУ СЕЧОВОГО МІХУРА

Bladder cancer (BC) continues to be a disease with a high mortality rate. Bladder cancer is the sixth for men and seventeenth for women in the incidence of malignancy worldwide. The invasion and metastasis of malignant tumors are caused by a sequence of processes, including loss of cell-cell and / or cell-matrix adhesion, proteolysis, and induction of angiogenesis. Different protease systems are involved in these processes, especially during the invasion and development of metastases. One such protease system is a plasminogen activation system or fibrinolysis system. Changes in the balance of plasminogen activation systems have been investigated in many types of malignancies, and these changes may not only indicate the functioning of this system but may also have prognostic significance. In malignancies, the components of this system are involved in the growth, invasion, and metastasis of tumors, affecting cell migration and angiogenesis. The main, but a well-studied component of the plasminogen activation system is serine proteinase – urokinase-type plasminogen activator (uPA). In contrast to uPA, tissue-type plasminogen activator (tPA) is characterized by a high affinity for fibrin and is involved in thrombolysis. Both types of plasminogen activators are synthesized in tumor tissues: tPA and uPA. The largest player among the inhibitors of fibrinolysis is the plasminogen activator inhibitor type 1 (PAI-1), involved in the pathogenesis of many cardiovascular diseases, as well as in cancer. The purpose of this study was to detect changes in the content of plasminogen activator tissue type tPA and PAI-1 in the blood plasma of patients with BC at different stages of the disease. The study involved 40 men who were verified with a diagnosis of BC. The content of tPA and PAI-1 in preoperative blood plasma was determined by enzyme immunoassay in ELISA modification. In our study, changes in the tPA and PAI-1 content of the blood plasma at different stages were identified, which can characterize tumor growth and invasion and can supplement existing disease information.

Multimodal Molecular Imaging Reveals High Target Uptake and Specificity of 111In- and 68Ga-Labeled Fibrin-Binding Probes for Thrombus Detection in Rats.

We recently showed the high target specificity and favorable imaging properties of 64Cu and Al18F PET probes for noninvasive imaging of thrombosis. Here, our aim was to evaluate new derivatives labeled with either with 68Ga, 111In, or 99mTc as thrombus imaging agents for PET and SPECT. In this study, the feasibility and potential of these probes for thrombus imaging was assessed in detail in 2 animal models of arterial thrombosis. The specificity of the probes was further evaluated using a triple-isotope approach with multimodal SPECT/PET/CT imaging. Radiotracers were synthesized using a known fibrin-binding peptide conjugated to 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid monoamide (DOTA-MA), or a diethylenetriamine ligand (DETA-propanoic acid [PA]), followed by labeling with 68Ga (FBP14, 68Ga-NODAGA), 111In (FBP15, 111In-DOTA-MA), or 99mTc (FBP16, 99mTc(CO)3-DETA-PA), respectively. PET or SPECT imaging, biodistribution, pharmacokinetics, and metabolic stability were evaluated in rat models of mural and occlusive carotid artery thrombosis. In vivo target specificity was evaluated by comparing the distribution of the SPECT and PET probes with preformed 125I-labeled thrombi and with a nonbinding control probe using SPECT/PET/CT imaging. All 3 radiotracers showed affinity similar to soluble fibrin fragment DD(E) (inhibition constant=0.53-0.83 μM). After the kidneys, the highest uptake of 68Ga-FBP14 and 111In-FBP15 was in the thrombus (1.0±0.2 percentage injected dose per gram), with low off-target accumulation. Both radiotracers underwent fast systemic elimination (half-life, 8-15 min) through the kidneys, which led to highly conspicuous thrombi on PET and SPECT images. 99mTc-FBP16 displayed low target uptake and distribution consistent with aggregation or degradation. Triple-isotope imaging experiments showed that both 68Ga-FBP14 and 111In-FBP15, but not the nonbinding derivative 64Cu-D-Cys-FBP8, detected the location of the 125I-labeled thrombus, confirming high target specificity. 68Ga-FBP14 and 111In-FBP15 have high fibrin affinity and thrombus specificity and represent useful PET and SPECT probes for thrombus detection.

Research Topic: A Study Of Fibrinolysis

According to present concepts, the enzyme responsible for the dissolution of fibrin both intra - and extra - vascularly is generated by the action of an “activator” upon the enzyme precursor plasminogen. Plasminogen is a B-globulin, of molecular weight of the order of 143,000, and a normal constituent of human plasma. It is known to possess a high affinity for both fibrin and fibrinogen, a property fundamental to the Sherry hypothesis of the physiological function of plasminogen where any fibrin clot is visualised as having an intrinsic plasminogen content sufficient to ensure its lysis when activated. The activation is achieved by activator either trapped within the clot at the moment of polymerisation, or diffusing subsequently into the clot from the surrounding liquid phase.

Lipoprotein(a) and Homocysteine Potentiate the Risk of Coronary Artery Disease in Male Subjects

Lipoprotein (Lp(a)) and homocysteine (Hcy) are independent risk factors for coronary artery disease (CAD). Hcy promotes the release of free apo(a) from Lp(a). The high fibrin affinity of free apo(a) inhibits plasminogen binding and plasmin generation. Hyperhomocysteinemia can result from a less active variant of methylene tetrahydrofolate reductase (variant C677T). Because the C677T genotype is estimated to be present in 32.2% of the Mexican population, we took advantage of this prevalence to determine the possible potentiating effect between high plasma Lp(a) and Hcy for increasing the risk of CAD in male patients. First, 222 male patients admitted for coronary angiography were recruited and classified as CAD+ or CAD-. Anthropometric measurements, traditional risk factors, and plasma total Hcy (tHcy) and Lp(a) levels were recorded in both groups. We performed a conditional logistic regression model adjusted for conventional risk factors of CAD and it became clear that Lp(a) ≥30mg/dl was a risk factor for CAD (odds ratio [OR] 5.06, 95% confidence interval [CI] 1.88-13.51, P=0.001), whereas Hcy was not related to CAD (OR 0.44, 95% CI 0.63-2.90, P=0.44). However, when both factors were considered together in an interaction model, high tHcy and high Lp(a) plasma concentrations showed a potentiated effect (OR 10.52, 95% CI 2.18-50.71, P=0.003). The combination of high Lp(a) and Hcy levels synergistically increases the likelihood of developing CAD in male patients.

Plasmin Is Essential in Preventing Periodontitis in Mice

Periodontitis involves bacterial infection, inflammation of the periodontium, degradation of gum tissue, and alveolar bone resorption, which eventually leads to loss of teeth. To study the role of the broad-spectrum protease plasmin in periodontitis, we examined the oral health of plasminogen (Plg)-deficient mice. In wild-type mice, the periodontium was unaffected at all time points studied; in Plg-deficient mice, periodontitis progressed rapidly, within 20 weeks. Morphological study results of Plg-deficient mice revealed detachment of gingival tissue, resorption of the cementum layer, formation of necrotic tissue, and severe alveolar bone degradation. IHC staining showed massive infiltration of neutrophils in the periodontal tissues. Interestingly, doubly deficient mice, lacking both tissue- and urokinase-type plasminogen activators, developed periodontal disease similar to that in Plg-deficient mice; however, mice lacking only tissue- or urokinase-type plasminogen activator remained healthy. Supplementation by injection of Plg-deficient mice with human plasminogen for 10 days led to necrotic tissue absorption, inflammation subsidence, and full regeneration of gum tissues. Notably, there was also partial regrowth of degraded alveolar bone. Taken together, our results show that plasminogen is essential for the maintenance of a healthy periodontium and plays an important role in combating the spontaneous development of chronic periodontitis. Moreover, reversal to healthy status after supplementation of Plg-deficient mice with plasminogen suggests the possibility of using plasminogen for therapy of periodontal diseases.

Deep Vein Thrombosis of Lower Extremity: Direct Intraclot Injection of Alteplase Once Daily with Systemic Anticoagulation—Results of Pilot Study

Conclusion: Daily intraclot injection of a venous thrombus with alteplase can be an alternative to continuous infusion thrombolytic therapy for treatment of deep venous thrombosis (DVT). Summary: An alternative to continuous infusion of lytic agents is to intermittently lace the thrombus with a thrombolytic agent that binds to fibrin in the clot and is rapidly cleared from the general circulation. This can allow the thrombolytic agent to work at the site where it is needed but minimize the duration of systemic exposure to the thrombolytic agent. This study prospectively evaluated the outcome of patients with acute DVT in the lower extremity treated with daily lacing of the thrombus with alteplase. Alteplase has a high fibrin affinity and therefore continuous infusion of the thrombolytic agent is not required. Twenty patients with first-onset acute DVT were treated with direct daily lacing of the thrombus with alteplase with a maximum daily dosage of 50 mg/leg and a maximum of four treatments. Catheters were left in place between treatments. Patients were also systemically anticoagulated fully. Patients underwent ventilation perfusion (V/Q) scans, which were performed before treatment and during thrombolytic therapy. The study comprised 13 men and 7 women, aged 18 to 79 years. Antegrade blood flow was restored throughout the deep venous system in 16 patients (80%) during thrombolytic therapy. After 6 months of anticoagulation, 18 patients (90%) had complete resolution of symptoms. There was rapid clearance of circulating alteplase and recovery of plasminogen activator inhibitor-1 levels by pharmacokinetic studies ≤2 hours after termination of alteplase treatment. A 40% incidence of pulmonary embolism was documented by V/Q scanning before treatment and a 15% incidence of asymptomatic pulmonary embolism during thrombolytic therapy. No clinically important pulmonary embolism or serious bleeding occurred during thrombolytic therapy. During the mean follow-up of 3.4 years, no post-thrombotic syndrome or recurrent DVT developed in any patient. Comment: The most obvious practical advantage of this approach is that patients do not require intensive monitoring of a continuous infusion of a thrombolytic agent. However, intermittent administration of alteplase directly into the thrombus does require a substantial time commitment. Procedure times for the first treatment of an uncomplicated iliofemoral DVT were 1.5 hours. If there was extensive calf DVT, initial procedure times could be doubled or almost tripled. Procedure times for subsequent treatments are shorter because catheters are left in place after the initial treatment and can be exchanged using standard guidewire techniques. Overall, it appears if one has the catheter skills and time, intermittent administration of a thrombolytic agent directly into the clot may be a viable alternative to more standard continuous infusion techniques.

Deep Vein Thrombosis of Lower Extremity: Direct Intraclot Injection of Alteplase Once Daily with Systemic Anticoagulation—Results of Pilot Study

To prospectively evaluate the outcome of patients with acute deep vein thrombosis (DVT) of the lower extremity treated with "lacing" of the thrombus with alteplase (recombinant tissue plasminogen activator, or rTPA). This HIPAA-compliant study was approved by the Institutional Review Board of the National Heart, Lung, and Blood Institute and was funded by the National Institutes of Health. After giving written consent, 20 patients with first-onset acute DVT were treated with direct intraclot lacing of the thrombus with alteplase (maximum daily dose, 50 mg per leg per day; maximum of four treatments) and full systemic anticoagulation. Alteplase was chosen because its high fibrin affinity obviates continuous infusion of this thrombolytic agent. Ventilation-perfusion (V/Q) scans were performed for evaluation of embolic risks, and clinical and imaging examinations were supplemented with pharmacokinetic studies to enable further assessment of treatment outcomes. The 20 patients included 13 men and seven women aged 18-79 years. Antegrade blood flow was restored throughout the deep venous system in 16 patients (80%) during thrombolytic therapy, with complete resolution of symptoms in 18 patients (90%) after 6 months of anticoagulation. Pharmacokinetic studies showed rapid clearance of circulating alteplase and recovery of plasminogen activator inhibitor-1 levels within 2 hours after termination of alteplase treatment. V/Q scans revealed a 40% incidence of pulmonary embolism before treatment and a 15% incidence of asymptomatic pulmonary embolism during thrombolytic therapy. There were no cases of clinically important pulmonary embolism or serious bleeding during thrombolytic therapy. During a mean follow-up period of 3.4 years, no patient developed a postthrombotic syndrome or recurrent thromboembolism. Intraclot injection or lacing of the thrombus with a fibrin-binding thrombolytic agent such as alteplase is an alternative to continuous-infusion thrombolytic regimens and minimizes the duration of systemic exposure to thrombolytic agents.

Indocyanine Green as Useful Guide for Retinal Vein Cannulation and Injection of Tissue Plasminogen Activator in Rabbits

Central retinal vein occlusion (CRVO) is caused by a fibrin clot in central retinal vein and is one of the intractable diseases that deteriorate visual acuities. Tissue plasminogen activator (TPA) is a thrombolytic agent that has a high affinity for fibrin and that activates plasminogen into plasmin. Injection of TPA into the retinal vein was helpful to treat CRVO. But TPA injection into retinal vein was difficult for clinical use, because TPA solution was transparent and confirmation of its injection was not easy. Indocyanine green (ICG) has been used as an angiographic agent and a tissue-marking agent in ocular surgeries. We studied the effectiveness and safety of ICG assisted injection of TPA into the retinal vein in rabbits. The major retinal vein was punctured using a micropipette fabricated from a glass tube, then TPA/ICG was injected. Total of 12 eyes and 5 eyes were enucleated 7 days and 1 month after injection of TPA/ICG for histological observations including immunostaining of glial fibrillary acidic protein (GFAP) and TUNEL staining, respectively. GFAP is expressed in Müller cells under pathologic conditions and indicates retinal damages. TUNEL indicates apoptosis of sensory retina cells. Retinal vein cannulation was easily performed, as retinal vein became green following injection of TPA/ICG. Histologically, no retinal damages, due to the TPA/ICG solution, were observed. GFAP and TUNEL staining were negative. TPA/ICG causes no disturbance in retinal circulation when performed correctly. Because of its safety, ICG is a useful agent as a guide for retinal vein injection of TPA.

Lipoproteína(a) e inibição da fibrinólise na doença arterial coronariana

O envolvimento da lipoproteína(a) nas doenças ateroscleróticas tem sido alvo de muitos estudos que têm demonstrado que esta lipoproteína é um fator de risco independente para a doença arterial coronariana (DAC). Devido à semelhança estrutural com o plasminogênio, a Lp(a) pode competir com os sítios de ligação deste, diminuindo a geração de plasmina e inibindo a fibrinólise. O trombo formado numa placa aterosclerótica rompida dispara a maioria dos eventos cardiovasculares isquêmicos. Como o trombo é dissolvido através do sistema fibrinolítico, surgiu a hipótese de que uma diminuição da atividade fibrinolítica poderia ser um fator de risco para eventos isquêmicos. Entretanto, alguns estudos demonstraram ausência de associação entre Lp(a) e DAC, enquanto alguns atribuem o real valor preditivo à uma sub-população de Lp(a) com alta afinidade por fibrina. Essa hipótese sugere que alguns fenótipos de Lp(a) não estão associados à aterotrombose. Sendo assim, as pesquisas envolvendo Lp(a) e DAC apresentam resultados controversos, altamente dependentes da população estudada. Na população brasileira, a qual apresenta uma heterogeneidade de etnias, são raros os estudos deste tipo. Diante das notáveis controvérsias, espera-se que a presente revisão possa contribuir para suscitar ânimos no sentido da realização de estudos adicionais envolvendo a dosagem da Lp(a) em pacientes com DAC na nossa população. O conhecimento acumulado ao longo do tempo sobre a associação entre Lp(a) e DAC foi obtido através de estudos conduzidos fora do nosso meio. Assim, estudos fundamentados dentro da nossa realidade poderão trazer respostas mais fidedignas e adequadas para a nossa população.

Soluble expression of a strong thrombolytic pro-urokinase mutant in Escherichia coli

A recombinant pro-urokinase mutant GZ5-sPA was successfully constructed by fusion of a high fibrin-affinity fragment GZ5 to the N-terminus of the serine protease domain of pro-urokinase (sPA). The fragment GZ5 contains a tetrapeptide GPRP and a tripeptide RGD, and was synthesized in bacterial preferred expressing codons. The mutant was then fused to the C-terminus of maltose binding protein (MBP) carried by pMAL-C2x vector, and expressed in Escherichia coli strain Origami (DE3). The produced fusion protein was highly soluble in the cytoplasm of the bacteria. After being cleaved with PreScission Protease to remove MBP tag, GZ5-sPA showed a molecular weight of 31 kDa on SDS–PAGE. GZ5-sPA maintained the same epitope as wild-type pro-urokinase and possessed a thrombolytic activity three times higher than standard urokinase did after being activated as two-chain form. The results could be a clue to other complicated heterogenous proteins similar to pro-urokinase.

Fibrin Affinity of Erythrocyte-Coupled Tissue-Type Plasminogen Activators Endures Hemodynamic Forces and Enhances Fibrinolysis in Vivo

Plasminogen activators (PAs; e.g., tissue-type, tPA) coupled to red blood cells (RBCs) display in vivo features useful for thromboprophylaxis: prolonged circulation, minimal extravasation, and preferential lysis of nascent versus preexisting clots. Yet, factors controlling the activity of RBC-bound PAs in vivo are not defined and may not mirror the profile of soluble PAs. We tested the role of RBC/PA binding to fibrin in fibrinolysis. RBC/tPA and RBC/tPA variant with low fibrin affinity (rPA) bound to and lysed plasminogen-containing fibrin clots in vitro comparably. In contrast, when coinjected in mice with fibrin emboli lodging in pulmonary vasculature, only RBC/tPA accumulated in lungs, which resulted in a more extensive fibrinolysis versus RBC/rPA (p < 0.01). Reconciling this apparent divergence between in vitro and in vivo behaviors, RBC/tPA, but not RBC/rPA perfused over fibrin in vitro at physiological shear stress bound to fibrin clots and caused greater fibrinolysis versus RBC/rPA (p < 0.001). These results indicate that because of high fibrin affinity, RBC/tPA binding to clots endures hemodynamic stress, which enhances fibrinolysis. Behavior of RBC/PAs under hemodynamic pressure is an important predictor of their performance in vivo.

Mechanism for the homocysteine-enhanced antifibrinolytic potential of lipoprotein(a) in human plasma

Lipoprotein(a) and total plasma homocysteine levels are now established as independent atherothrombogenic risk factors. A distinctive pathophysiological feature of lipoprotein(a) is its antifibrinolytic activity, an effect dependent on plasma concentration and high affinity for fibrin of its small size apo(a) component. A stimulating effect of homocysteine on purified lipoprotein(a) has been proposed. However, little is known about their specific interactions in human plasma. We demonstrate by immunochemical, ligand-binding and plasminogen activation studies, that homocysteine modifies the structure and function of lipoprotein(a) in human plasma; it reduces the apo(a)/apoB disulfide bond causing the appearance of free apo(a) with high affinity for fibrin that inhibits plasminogen binding and plasmin formation (r= -0.995, p =0.002). These effects were evident particularly in plasma samples containing lipoprotein(a) with low affinity for fibrin and more than 22 kringles apo(a) isoforms. In contrast, for plasmas containing high fibrin affinity lipoprotein(a) (less than 22 kringles apo[a] isoforms) no significant change neither in fibrin binding nor in plasmin formation was observed. Furthermore, isolated apo(a) recombinants (10 to 34 kringles) that have been shown to display size-independent high affinity for fibrin were not affected by homocysteine, thus confirming lipoprotein(a) as its main target. These results suggest that the pro-atherogenic role already conferred to lipoprotein(a) by small apo(a) isoforms may be extended to large apo(a) isoforms if released in plasma by homocysteine, as this mechanism reveals their high fibrin affinity. Lipoprotein(a) and homocysteine may therefore constitute, if acting in concert, a new risk factor for athero-thrombotic vascular disease.

Inhibition of fibrinolysis by lipoprotein(a).

A high plasma concentration of lipoprotein Lp(a) is now considered to be a major and independent risk factor for cerebro- and cardiovascular atherothrombosis. The mechanism by which Lp(a) may favour this pathological state may be related to its particular structure, a plasminogen-like glycoprotein, apo(a), that is disulfide linked to the apo B100 of an atherogenic LDL-like particle. Apo(a) exists in several isoforms defined by a variable number of copies of plasminogen-like kringle 4 and single copies of kringle 5 and the catalytic region. At least one of the plasminogen-like kringle 4 copies present in apo(a) (kringle IV type 10) contains a lysine binding site (LBS) that is similar to that of plasminogen. This structure allows binding of these proteins to fibrin and cell membranes. Plasminogen thus bound is cleaved at Arg561-Val562 by plasminogen activators and transformed into plasmin. This mechanism ensures fibrinolysis and pericellular proteolysis. In apo(a) a Ser-Ile substitution at the Arg-Val plasminogen activation cleavage site prevents its transformation into a plasmin-like enzyme. Because of this structural/functional homology and enzymatic difference, Lp(a) may compete with plasminogen for binding to lysine residues and impair, thereby, fibrinolysis and pericellular proteolysis. High concentrations of Lp(a) in plasma may, therefore, represent a potential source of antifibrinolytic activity. Indeed, we have recently shown that during the course of the nephrotic syndrome the amount of plasminogen bound and plasmin formed at the surface of fibrin are directly related to in vivo variations in the circulating concentration of Lp(a) (Arterioscler. Thromb. Vasc. Biol., 2000, 20: 575-584; Thromb. Haemost., 1999, 82: 121-127). This antifibrinolytic effect is primarily defined by the size of the apo(a) polymorphs, which show heterogeneity in their fibrin-binding activity--only small size isoforms display high affinity binding to fibrin (Biochemistry, 1995, 34: 13353-13358). Thus, in heterozygous subjects the amount of Lp(a) or plasminogen bound to fibrin is a function of the affinity of each of the apo(a) isoforms and of their concentration relative to each other and to plasminogen. The real risk factor is, therefore, the Lp(a) subpopulation with high affinity for fibrin. According to this concept, some Lp(a) phenotypes may not be related to atherothrombosis and, therefore, high Lp(a) in some individuals might not represent a risk factor for cardiovascular disease. In agreement with these data, it has been recently reported that Lp(a) particles containing low molecular mass apo(a) emerged as one of the leading risk conditions in advanced stenotic atherosclerosis (Circulation, 1999, 100: 1154-1160). The predictive value of high Lp(a) as a risk factor, therefore, depends on the relative concentration of Lp(a) particles containing small apo(a) isoforms with the highest affinity for fibrin. Within this context, the development of agents able to selectively neutralise the antifibrinolytic activity of Lp(a), offers new perspectives in the prevention and treatment of the cardiovascular risk associated with high concentrations of thrombogenic Lp(a).

Characterization of the Interactions of Plasminogen and Tissue and Vampire Bat Plasminogen Activators with Fibrinogen, Fibrin, and the Complex of d-Dimer Noncovalently Linked to Fragment E

Vampire bat plasminogen activator (b-PA) causes less fibrinogen (Fg) consumption than tissue-type plasminogen activator (t-PA). Herein, we demonstrate that this occurs because the complex of D-dimer noncovalently linked to fragment E ((DD)E), the most abundant degradation product of cross-linked fibrin, as well as Fg, stimulate plasminogen (Pg) activation by t-PA more than b-PA. To explain these findings, we characterized the interactions of t-PA, b-PA, Lys-Pg, and Glu-Pg with Fg and (DD)E using right angle light scattering spectroscopy. In addition, interactions with fibrin were determined by clotting Fg in the presence of various amounts of t-PA, b-PA, Lys-Pg, or Glu-Pg and quantifying unbound material in the supernatant after centrifugation. Glu-Pg and Lys-Pg bind fibrin with Kd values of 13 and 0.13 microM, respectively. t-PA binds fibrin through two classes of sites with Kd values of 0.05 and 2.6 microM, respectively. The second kringle (K2) of t-PA mediates the low affinity binding that is eliminated with epsilon-amino-n-caproic acid. In contrast, b-PA binds fibrin through a single kringle-independent site with a Kd of 0.15 microM. t-PA competes with b-PA for fibrin binding, indicating that both activators share the same finger-dependent site on fibrin. Glu-Pg binds (DD)E with a Kd of 5.4 microM. Lys-Pg binds to (DD)E and Fg with Kd values of 0.03 and 0.23 microM, respectively. t-PA binds to (DD)E and Fg with Kd values of 0.02 and 0.76 microM, respectively; interactions were eliminated with epsilon-amino-n-caproic acid, consistent with K2-dependent binding. Because it lacks a K2-domain, b-PA does not bind to either (DD)E or Fg, thereby explaining why b-PA is more fibrin-specific than t-PA.

Factors that control extravascular fibrinolysis.

The physiological and pathophysiological state of tissues determines the exudation of plasma proteins, hemostasis, and fibrinolysis, i.e., inflammation, injury, and malignancy. The physiological controls of extravascular fibrinolysis ultimately rest on a balance between generation of the fibrinolytic enzyme(s), i.e., plasmin, elastase, cathepsins, etc., and inhibitors of the fibrinolytic enzyme(s), i.e., plasminogen activator inhibitors, alpha-2 plasmin inhibitor, alpha 1-protease inhibitors, etc. Moreover, it is the structural modification of fibrin that determines its stability toward proteolytic enzymes and physical duress. The structural modification of fibrin involves factor XIIIa-mediated cross-linking of interfibrin chains and alpha 2-plasmin inhibitor to fibrin. In turn, there are a number of agents that influence factor XIIIa catalytic activity (e.g., sulfhydryl agents, albumin, erythrocytes). The two key proenzymes, factor XIII and plasminogen, are tightly bound with the circulating fibrinogen molecules. Such high selective affinity for fibrin(ogen) provides the reaction specificity in a complex tissue fluid milieu and governs the kinetics of fibrinolysis. Any agents that interfere with such binding reactions, e.g., autoantibodies, may also affect the fibrinolytic reactions. Understanding these unique biochemical controls of factors involved in fibrinolysis may provide an insight into the complex regulatory process of extravascular fibrinolysis.

Recombinant variants of tissue-type plasminogen activator containing amino acid substitutions between positions 37 and 42

The finger domain deletion mutant of tissue type plasminogen activator (t-PA) has a prolonged half-life in vivo but tends to be accompanied by a decrease in affinity for fibrin. We considered that further refinements in this mutant would reduce the clearance rate and maintain high affinity for fibrin. Two variants, t-PA N37S.S38V G39V R40E.A41S.Q42S and t-PA N37S.S38V G39V R40E.A41F Q42S had a 5-fold increased half-life, but the amount of these variants that bound fibrin was lower than that of wild-type t-PA. To further investigate the effects of these modifications and create a t-PA variant with higher affinity for fibrin, we produced six variants in which residues 37–42 were substituted by more hydophobic amino acids. These variants had various degrees of decreased fibrin binding. Two of them, t-PA N37S.S38VG39VR40I.A41VQ42S and t-PA N37S.S38V.G39V R40I.A41V.Q42L, bound to fibrin stronger than the variants, t-PA N37S.S38VG39VR40E.A41S.Q42S and t-PA N37S.S38VG39VR40E.A41FQ42S, accompanied by a prolonged half-life in vivo. The other objective of this study was to estimate the effects of the amino acid substitution at position 42. Two variants, t-PA N37S.S38VG39VR40E.A41S and t-PA N37S.S38VG39VR40E.A41F had about a 6-fold longer half life in vivo. These data suggest that changes in the pharmacokinetic characteristics of the 37–42 variants are mainly due to modifications at amino acid residues 37–41.

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.

Does apolipoprotein(a) heterogeneity influence lipoprotein(a) effects on fibrinolysis?

AbstractHigh plasma levels of lipoprotein(a) [Lp(a)] are considered to be an independent risk factor for premature cardiovascular disease and are inversely associated with apolipoprotein(a) [apo(a)] isoform sizes. The contribution of apo(a) polymorphism to the inhibition of fibrinolysis, a mechanism that may favor thrombus development, was therefore evaluated by measuring the ability of Lp(a) particles of distinct apo(a) isoform content to compete with plasminogen for fibrin binding during plasminogen activation by fibrin-bound tissue-type plasminogen activator. The rate of plasmin generation was most efficiently inhibited by an isoform with a molecular weight (M(r)) of approximately 540 Kd. An isoform with M(r) approximately 590 Kd produced a less pronounced effect, whereas the isoform with M(r) approximately 610 Kd failed to inhibit plasminogen activation. These effects were directly proportional to the amount of Lp(a) bound to the carboxy-terminal lysine residues of degraded fibrin. The relative affinity of the binding (kd range, 16 to 180 nmol/L) reflected the ability of individual Lp(a) isoforms to inhibit the binding of plasminogen (kd, 600 nmol/L). The question of the influence of kringle sequence variability on the binding to fibrin was not addressed by the present work. These data suggest that apo(a) isoform types with high affinity for fibrin may influence the ability of Lp(a) to interfere with fibrinolysis and contribute thereby to the association of elevated levels of Lp(a) with atherosclerotic and thrombotic risks.© 1993 by The American Society of Hematology.

Does apolipoprotein(a) heterogeneity influence lipoprotein(a) effects on fibrinolysis?

High plasma levels of lipoprotein(a) [Lp(a)] are considered to be an independent risk factor for premature cardiovascular disease and are inversely associated with apolipoprotein(a) [apo(a)] isoform sizes. The contribution of apo(a) polymorphism to the inhibition of fibrinolysis, a mechanism that may favor thrombus development, was therefore evaluated by measuring the ability of Lp(a) particles of distinct apo(a) isoform content to compete with plasminogen for fibrin binding during plasminogen activation by fibrin-bound tissue-type plasminogen activator. The rate of plasmin generation was most efficiently inhibited by an isoform with a molecular weight (M(r)) of approximately 540 Kd. An isoform with M(r) approximately 590 Kd produced a less pronounced effect, whereas the isoform with M(r) approximately 610 Kd failed to inhibit plasminogen activation. These effects were directly proportional to the amount of Lp(a) bound to the carboxy-terminal lysine residues of degraded fibrin. The relative affinity of the binding (kd range, 16 to 180 nmol/L) reflected the ability of individual Lp(a) isoforms to inhibit the binding of plasminogen (kd, 600 nmol/L). The question of the influence of kringle sequence variability on the binding to fibrin was not addressed by the present work. These data suggest that apo(a) isoform types with high affinity for fibrin may influence the ability of Lp(a) to interfere with fibrinolysis and contribute thereby to the association of elevated levels of Lp(a) with atherosclerotic and thrombotic risks.