Fibrin-binding Domain Research Articles

Abstract 12419: Preclinical Safety and Efficacy of a Novel Thrombolytic Agent Administered by Rapid Bolus Injection: Clot Specific Streptokinase (CSSK/SMRX-11)

Introduction: Clot specific Streptokinase (CSSK), also known as SMRX-11, is a novel recombinant protein, produced and purified from a yeast system (Pichia pastoris). Novel fibrin binding domains attached to each end of streptokinase mask the ability of the streptokinase component of SMRX-11 to interact with blood plasminogen. As a result, SMRX-11 remains inactive in the blood circulation and does not convert blood plasminogen into plasmin until the fibrin binding domains are cleaved by clot-bound plasmin. This is in contrast to native Streptokinase (SK), which indiscriminately converts plasminogen into plasmin. The molecular weight of the glycosylated SMRX-11 protein is approximately 80kD, with a specific activity of 80,000 Units/mg of protein. Results. In vitro experiments comparing SMRX-11 to other fibrinolytics (SK and tPA), demonstrated that SMRX-11 lyses experimentally-preformed human blood clots without reducing residual plasma fibrinogen levels. Initial in vitro studies compared the activity of SMRX-11 to lyse human whole blood clots to native Streptokinase (SK) and tissue Plasminogen Activator (tPA). SMRX-11 had comparable clot lysis activity to SK, with tPA eliciting a slightly greater percent clot lysis. Additional in vitro studies determined the relative fibrinogenolytic effects of SMRX-11 versus tPA. SMRX-11 demonstrated superior fibrinogen sparing compared to tPA with minimal consumption of alpha-2 antiplasmin relative to native SK. In vivo, SMRX-11 (0.7-1.4 mg/kg as an iv bolus), lysed experimentally-induced femoral arterial thrombi in cynomolgus monkeys with relatively minor fluctuation in plasma fibrinogen, forearm bleeding time or hemodynamics. In GLP safety studies, treatment with SMRX-11 at doses of 1, 3 and 10 mg/kg/day for 5 consecutive days was generally well-tolerated in male and female cynomolgus monkeys. Thus, the low dose of 1 mg/kg was considered the No-Observed Adverse Effect Level (NOAEL) for daily repeat-dose administration of SMRX-11 administered via bolus i.v. injection in cynomolgus monkeys. Conclusion. SMRX-11 represents a novel pharmacophore with the appropriate preclinical safety and efficacy profile to warrant clinical investigation for acute treatment of myocardial infarction and stroke.

Engineering Interactions between Growth Factors and the Extracellular Matrix

In natural situations, growth factors such as VEGF and PDGF are present bound to the ECM, yet therapeutic use of such growth factors has focused on application in soluble forms. To explore ECM immobilization of growth factors, we have explored complexation with recombinant variants of EMC proteins such as fibronectin and engineering growth factors fused to a domain that binds strongly to ECM proteins. To explore noncovalent immobilization upon matrices, we have engineered a fibrin‐binding domain of fibronectin, containing the 12th‐14th type III repeat (which was known to bind VEGF‐A). In studies of the 12th‐14th type III repeat, we determined that the growth factor binding activity of this domain was promiscuous. Incorporation of this domain into fibrin, through engineered transglutaminase activity, provides a powerful and generalizable method to retain such growth factors into surgical matrices. Examination of promiscuous growth factor binding to ECM proteins revealed broad binding to not only fibronectin but also fibrinogen, vitronectin, osteopontin, and tenascin C, among others. We examined these affinities to determine that placenta growth factor‐2 (PlGF‐2) binds the mentioned ECM molecules very strongly. We identified the binding domain and engineered it into other growth factors to confer nM affinity to them, demonstrating enhanced efficacy in the context of angiogenesis, skin repair in chronic wound models, and bone repair. In this way, using variant growth factors that have super‐affinity for ECM proteins, either a fibrin matrix or a tissue itself may be turned into a reservoir for growth factor sequestration and presentation.

A Novel Molecular Mechanism In The Interplay Of Platelet GPIb-VWF-Fibrin In Thrombus Formation

It is established that the interplay between von Willebrand factor (VWF) and fibrinogen is relevant for the platelet-platelet interaction in thrombus formation. Although the concentration of fibrinogen is much higher than that of VWF in blood, normally, they do not interact. In fact, VWF in plasma has a poor binding activity for fibrin(ogen)-coated surface, which exposes fibrin-specific sequences when it is immobilized. This observation suggests that 1) the site recognized by fibrin is inaccessible in plasma VWF, and 2) a conformational change in the VWF protein is a pre-requisite to bind fibrin. The latter may be supported by a previous study indicating that VWF has to bind to its platelet receptor glycoprotein (GP)Ib first in order to bind fibrin. Another line of evidence suggests that the binding of VWF to the polymerizing fibrin facilitates the interaction of platelet GPIb with the fibrin-bound VWF during the growth of the thrombus, a mechanism apparently defective in Bernard-Soulier syndrome (BSS) and von Willebrand disease (VWD). Consistent with this, fibrin monomer, like ristocetin, can induce the binding of VWF to GPIb. The C1C2 domains have been described as the fibrin-binding domain in VWF, and it is unknown whether the structures of these domains change to bind to fibrin. In contrast, conformational changes within the A1A2 domains of VWF induced by ristocetin result in the binding to GPIb and the protease ADAMTS-13. Therefore, we investigated a potential contact site for fibrin among the A1A2 domains of VWF. We characterized the interaction of each plasma (p)VWF, purified pVWF, recombinant A1A2A3 domains protein, single A1, and single A2 domains with fibrinogen, fibrin, or D-dimer. We also examined the effect of ristocetin in the VWF-fibrin binding. Both pVWF and purified pVWF bound to immobilized fibrin(ogen) in a ristocetin dependent manner. An A1A2A3 mutant expressing increased GPIb-binding activity bound to fibrin(ogen) in the absence of ristocetin. Both A1 and A2 domains, but not the A3 domain had binding activity for immobilized fibrin(ogen). Both soluble fibrin monomer and D-dimer, unlike fibrinogen in solution, had a significant binding activity for the immobilized A2 domain. Moreover, the A2 domain was capable of delaying fibrin polymerization in vitro. The use of conformation-specific monoclonal antibodies demonstrated that, like collagen, fibrin(ogen) induces a change in the structure of the A1 domain, and platelets interacted effectively with the fibrin(ogen)-bound A1 domain under high shear stress. Interestingly, in contrast to A3 or A1domain, flowing platelets did not adhere to the fibrin(ogen)-coated surface incubated with the A2 domain. In conclusion, we have demonstrated a previously unknown fibrin(ogen)-binding activity for the A1 and A2 domains in VWF. These observations reveal new mechanisms that may help to understand the pathophysiology of the bleeding in VWD, in which a number of natural occurring mutations that cause the disease are found within the A1 and A2 domains, and the poor fibrin polymerization observed in BSS. Furthermore, these outcomes may have implications for a better understanding of the high risk for thrombosis when the levels of both VWF and fibrinogen are increased. Disclosures:No relevant conflicts of interest to declare.

Evaluation of 111In-Labeled EPep and FibPep as Tracers for Fibrin SPECT Imaging

Fibrin targeting is an attractive strategy for nuclear imaging of thrombosis, atherosclerosis and cancer. Recently, FibPep, an (111)In-labeled fibrin-binding peptide, was established as a tracer for fibrin SPECT imaging and was reported to allow sensitive detection of minute thrombi in mice using SPECT. In this study, we developed EPep, a novel (111)In-labeled fibrin-binding peptide containing the fibrin-binding domain of the clinically verified EP-2104R peptide, and sought to compare the potential of EPep and FibPep as tracers for fibrin SPECT imaging. In vitro, both EPep and FibPep showed high stability in serum, but were less stable in liver and kidney homogenate assays. Both peptide probes displayed comparable affinities toward human and mouse derived fibrin (Kd ≈ 1 μM), and similarly to FibPep, EPep showed fast blood clearance, low nontarget uptake and high thrombus uptake (6.8 ± 1.2% ID g(-1)) in a mouse carotid artery thrombosis model. Furthermore, EPep showed a similar affinity toward rat derived fibrin (Kd ≈ 1 μM), displayed high thrombus uptake in a rat carotid artery thrombosis model (0.74 ± 0.39% ID g(-1)), and allowed sensitive detection of thrombosis in rats using SPECT. In contrast, FibPep displayed a significantly lower affinity toward rat derived fibrin (Kd ≈ 14 μM) and low uptake in rat thrombi (0.06 ± 0.02% ID g(-1)) and did not allow clear visualization of carotid artery thrombosis in rats using SPECT. These results were confirmed ex vivo by autoradiography, which showed a 7-fold higher ratio of activity in the thrombus over the contralateral carotid artery for EPep in comparison to FibPep. These findings suggest that the FibPep binding fibrin epitope is not fully homologous between humans and rats, and that preclinical rat models of disease should not be employed to gauge the clinical potential of FibPep. In conclusion, both peptides showed approximately similar metabolic stability and affinity toward human and mouse derived fibrin, and displayed high thrombus uptake in a mouse carotid artery thrombosis model. Therefore, both EPep and FibPep are promising fibrin targeted tracers for translation into clinical settings to serve as novel tools for molecular imaging of fibrin.

Effect of Fibrin-binding Synthetic Oligopeptide on the Healing of Full-thickness Skin Wounds in Streptozotocin-induced Diabetic Rats

The aim of this study was to investigate whether topical application of fibrin-binding oligopeptides derived from FN promotes wound healing in streptozotocin (STZ)-induced diabetic rats. Oligopeptides including fibrin-binding sequences (FF3: CFDKYTGNTYRV, FF5 : CTSRNRCNDQ) of FN repeats were synthesized. Each peptide was loaded in 15 x 15 mm fibrous alginate dressings, and the release kinetics of the peptides was evaluated using trinitrobenzene sulfonic acid for 336 hours. Two full-thickness cutaneous wounds were prepared on the dorsal skin of each 75 diabetes induced rats. Each wound was divided into FF3-loaded alginate dressing group, FF5-loaded alginate dressing group, alginate dressing group and negative control group. Animals were sacrificed at day 0,3,7 and 14. The wound closure rate, inflammation degree, expression of TGF-β1 and hydroxyproline contents were evaluated. Both FF3 and FF5 peptides were released rapidly within the first 24 hours. FF3-loaded dressing treated wounds closed significantly faster than other wounds at day 3. And at day 14, FF3- & FF5- loaded dressing treated wounds demonstrated less inflammatory cells infiltration than alginate dressing treated and negative group wounds. TGF-β1 positive cells were more abundant in FF3-, FF5-treated alginate dressing treated wound at day 3 and 14. At last, the hyrdroxyproline contents in the FF3, FF5 group were higher at day 7 and day 14. Topical application of fibrin-binding domain synthetic oligopeptides from FN resulted in acceleration of full-thickness cutaneous wound healing in diabetic rats.

Effect of Fibrin-binding Synthetic Oligopeptide on the Healing of Full-thickness Skin Wounds in Streptozotocin-induced Diabetic Rats

The aim of this study was to investigate whether topical application of fibrin-binding oligopeptides derived from FN promotes wound healing in streptozotocin (STZ)-induced diabetic rats. Oligopeptides including fibrin-binding sequences (FF3: CFDKYTGNTYRV, FF5 : CTSRNRCNDQ) of FN repeats were synthesized. Each peptide was loaded in 15 x 15 mm fibrous alginate dressings, and the release kinetics of the peptides was evaluated using trinitrobenzene sulfonic acid for 336 hours. Two full-thickness cutaneous wounds were prepared on the dorsal skin of each 75 diabetes induced rats. Each wound was divided into FF3-loaded alginate dressing group, FF5-loaded alginate dressing group, alginate dressing group and negative control group. Animals were sacrificed at day 0,3,7 and 14. The wound closure rate, inflammation degree, expression of TGF-β1 and hydroxyproline contents were evaluated. Both FF3 and FF5 peptides were released rapidly within the first 24 hours. FF3-loaded dressing treated wounds closed significantly faster than other wounds at day 3. And at day 14, FF3- & FF5- loaded dressing treated wounds demonstrated less inflammatory cells infiltration than alginate dressing treated and negative group wounds. TGF-β1 positive cells were more abundant in FF3-, FF5-treated alginate dressing treated wound at day 3 and 14. At last, the hyrdroxyproline contents in the FF3, FF5 group were higher at day 7 and day 14. Topical application of fibrin-binding domain synthetic oligopeptides from FN resulted in acceleration of full-thickness cutaneous wound healing in diabetic rats.

Ligation of the Fibrin-binding Domain by β-Strand Addition Is Sufficient for Expansion of Soluble Fibronectin

How fibronectin (FN) converts from a compact plasma protein to a fibrillar component of extracellular matrix is not understood. "Functional upstream domain" (FUD), a polypeptide based on F1 adhesin of Streptococcus pyogenes, binds by anti-parallel β-strand addition to discontinuous sets of N-terminal FN type I modules, (2-5)FNI of the fibrin-binding domain and (8-9)FNI of the gelatin-binding domain. Such binding blocks assembly of FN. To learn whether ligation of (2-5)FNI, (8-9)FNI, or the two sets in combination is important for inhibition, we tested "high affinity downstream domain" (HADD), which binds by β-strand addition to the continuous set of FNI modules, (1-5)FNI, comprising the fibrin-binding domain. HADD and FUD were similarly active in blocking fibronectin assembly. Binding of HADD or FUD to soluble plasma FN exposed the epitope to monoclonal antibody mAbIII-10 in the tenth FN type III module ((10)FNIII) and caused expansion of FN as assessed by dynamic light scattering. Soluble N-terminal constructs truncated after (9)FNI or (3)FNIII competed better than soluble FN for binding of FUD or HADD to adsorbed FN, indicating that interactions involving type III modules more C-terminal than (3)FNIII limit β-strand addition to (1-5)FNI within intact soluble FN. Preincubation of FN with mAbIII-10 or heparin modestly increased binding to HADD or FUD. Thus, ligation of FNIII modules involved in binding of integrins and glycosaminoglycans, (10)FNIII and (12-14)FNIII, increases accessibility of (1-5)FNI. Allosteric loss of constraining interactions among (1-5)FNI, (10)FNIII, and (12-14)FNIII likely enables assembly of FN into extracellular fibrils.

Interaction of fibrin with VE‐cadherin and anti‐inflammatory effect of fibrin‐derived fragments

The interaction of the fibrin βN-domain with VE-cadherin on endothelial cells is implicated in transendothelial migration of leukocytes, and the β15-42 fragment representing part of this domain has been shown to inhibit this process. However, our previous study revealed that only a dimeric (β15-66)(2) fragment, corresponding to the full-length βN-domain and mimicking its dimeric arrangement in fibrin, bound to VE-cadherin. To test our hypothesis that dimerization of β15-42-containing fragments increases their affinity for VE-cadherin and ability to inhibit transendothelial migration of leukocytes. Interaction of β15-42-containing fragments with VE-cadherin was characterized by ELISA and surface plasmon resonance. The inhibitory effect of such fragments was tested in vitro with a leukocyte transendothelial migration assay and in vivo with mouse models of peritonitis and myocardial ischemia-reperfusion injury. First, we prepared the monomeric β15-42 and β15-64 fragments and their dimeric forms, (β15-44)(2) and (β15-66)(2) , and studied their interaction with the fibrin-binding domain of VE-cadherin, VE-cad(3). The experiments revealed that both dimeric fragments bound to VE-cad(3) with high affinity, whereas the affinities of β15-42 and β15-64 were significantly lower. Next, we tested the ability of these fragments to inhibit leukocyte transmigration in vitro and infiltration into the inflamed peritoneum in vivo, and found that the inhibitory effects of the dimers on these processes were also superior. Furthermore, (β15-44)(2) significantly reduced myocardial injury induced by ischemia-reperfusion. The results confirm our hypotheses and indicate that (β15-66)(2) and (β15-44)(2) , which exhibited much higher affinity for VE-cadherin, are highly effective in suppressing inflammation by inhibiting leukocyte transmigration.

The promotion of neurological recovery in an intracerebral hemorrhage model using fibrin-binding brain derived neurotrophic factor

Brain derived neurotrophic factor (BDNF) has been shown to ameliorate recovery after intracerebral hemorrhage (ICH). The injured brain tissue after ICH is surrounded by hematoma formed from hemorrhage. Fibrin is abundant in hematoma, which could be a binding target for BDNF. In this work, we have fused a fibrin-binding domain (FBD) to BDNF (FBD-BDNF), and results demonstrate that FBD-BDNF has specific binding ability to fibrin and is retained in hematoma. Using the rat ICH model induced by bacterial collagenase, injected FBD-BDNF has been concentrated and retained at the hematoma. FBD has facilitated BDNF to exert targeting neuroprotective effect to the injured brain tissue around the hematoma after ICH. FBD-BDNF has significantly reduced the hemotoma volume, reduced tissue loss, promoted neural regeneration, and improved the rat behavioral performance.

Extended Binding Site on Fibronectin for the Functional Upstream Domain of Protein F1 of Streptococcus pyogenes

The 49-residue functional upstream domain (FUD) of Streptococcus pyogenes F1 adhesin interacts with fibronectin (FN) in a heretofore unknown manner that prevents assembly of a FN matrix. Biotinylated FUD (b-FUD) bound to adsorbed FN or its recombinant N-terminal 70-kDa fibrin- and gelatin-binding fragment (70K). Binding was blocked by FN or 70K, but not by fibrin- or gelatin-binding subfragments of 70K. Isothermal titration calorimetry showed that FUD binds with Kd values of 5.2 and 59 nm to soluble 70K and FN, respectively. We tested sets of FUD mutants and epitope-mapped monoclonal antibodies (mAbs) for ability to compete with b-FUD for binding to FN or to block FN assembly by cultured fibroblasts. Deletions or alanine substitutions throughout FUD caused loss of both activities. mAb 4D1 to the 2FNI module had little effect, whereas mAb 7D5 to the 4FNI module in the fibrin-binding region, 5C3 to the 9FNI module in the gelatin-binding region, or L8 to the G-strand of 1FNIII module adjacent to 9FNI caused loss of binding of b-FUD to FN and decreased FN assembly. Conversely, FUD blocked binding of 7D5, 5C3, or L8, but not of 4D1, to FN. Circular dichroism indicated that FUD binds to 70K by β-strand addition, a possibility supported by modeling based on crystal structures of peptides bound to 2FNI-5FNI of the fibrin-binding domain and 8FNI-9FNI of the gelatin-binding domain. Thus, the interaction likely involves an extensive anti-parallel β-zipper in which FUD interacts with the E-strands of 2FNI-5FNI and 8FNI-9FNI.

A chimeric epidermal growth factor with fibrin affinity promotes repair of injured keratinocyte sheets

The aim of the present study is to create a novel chimeric protein of epidermal growth factor (EGF) with fibrin affinity and demonstrate its potential for repairing injured tissues by immobilization to fibrin. The chimeric protein (FBD–EGF) was produced by the fusion of the fibronectin fibrin-binding domain (FBD) to EGF. It showed dose-dependent binding to fibrin and its binding was stable for at least 7 days, while native EGF showed little affinity. FBD–EGF promoted the growth of fibroblasts and keratinocytes in the fibrin-bound state as well as in the soluble state. Its activity was further studied in a keratinocyte culture system in which fibrin was exposed upon injury of cell sheets. Fibrin-bound FBD–EGF promoted growth of the sheets over the injured area at a significantly faster rate (approximately eightfold) than native EGF ( p < 0.01). Wounds 2 mm wide were closed in 7–9 days. This repair process was inhibited by anti-EGF. Keratinocytes proliferated more extensively in the leading edges of sheets contacting fibrin with FBD–EGF, approximately 1.7-fold more than in the adjacent regions. These results imply that the stable binding of chimeric EGF to fibrin is effective for the repair of injured keratinocyte sheets, suggesting a potential use in tissue engineering.

The biologic effect of fibrin-binding synthetic oligopeptide on periodontal ligament cells

Purpose: Fibronectin(FN), one of the major components of ECM, mediates wide variety of cellular interactions including cell adhesion, migration, proliferation and differentiation. In this study, we used synthetic peptides based on fibrin binding sites of amino-terminal of FN and evaluated their biologic effects on periodontal ligament(PDL) cells. Materials and methods: PDL cells were cultured on synthetic oligopeptides coated dishes and examined for cell adhesion, proliferation via confocal microscope. For detection of ERK1/2, cells were plated and Western blot analysis was performed. Results: PDL cells on synthetic oligopeptide coated dishes showed enhanced cell adhesion and proliferation. Western blot analysis revealed increased level of ERK1/2 phosphorylation in cells plated on FN fragment containing fibrin-binding domain(FF1 and FF5) coated dishes. Conclusion: These results reveals that FN fragment containing fibrin-binding domain possess an enhanced biologic effect of PDL ligament cells. (J Korean Acad Periodontol 2009;39:45-52)

Connective tissue growth factor binds to fibronectin through the type I repeat modules and enhances the affinity of fibronectin to fibrin

Connective tissue growth factor (CTGF) is a member of the CCN family of the cysteine-rich proteins and involved in wound healing and fibrosis. We have previously shown a biochemical interaction between the CTGF and fibronectin (FN) using the yeast two-hybrid system. In this study, we confirmed the interaction between the CTGF and FN using the surface plasmon resonance (SPR) and solid-phase binding analysis. Our results show that the regions containing the FN type I repeat modules (the N-terminal fibrin, the gelatin-collagen and the C-terminal fibrin binding domains) of FN and the C-terminal domain of CTGF are required for the interaction. We also demonstrated that CTGF enhances the affinity of FN to fibrin. It appears that CTGF contributes to the extracellular matrix accumulation in wound healing and tissue fibrosis by enhancing the affinity of FN to fibrin. Because CTGF is up-regulated during the tissue repair and in coagulation cascade-associated fibrotic disorders, the new function of CTGF found in this study is consistent with its physiological role.

Homocysteine targeting of plasma proteins in hemodialysis patients

Hyperhomocysteinemia, an independent, modifiable risk factor for cardiovascular disease, is found in most patients with end-stage renal disease. In this issue, Perna et al. examine the extent of protein-S-linked and protein-N-linked homocysteinylation in uremic patients on hemodialysis and the effect of folate treatment on protein homocysteinylation. Their findings show that protein-N-linked homocysteinylation, but not S-linked homocysteinylation, can be normalized by folate therapy.

Homocysteine binds to human plasma fibronectin and inhibits its interaction with fibrin.

More than 70% of circulating homocysteine is disulfide-bonded to protein, but little is known about the specific proteins that bind homocysteine and their function as a consequence of homocysteine binding. When human plasma was incubated with [(35)S]L-homocysteine, most of the homocysteine bound to albumin. However, additional homocysteine-binding proteins were detected, and 1 of them comigrated with fibronectin. Treatment with 2-mercaptoethanol removed the bound homocysteine, demonstrating the involvement of disulfide bonding. In contrast, [35S]L-cysteine did not bind to fibronectin. Purified fibronectin bound approximately 5 homocysteine molecules per fibronectin dimer. SDS-PAGE of a limited trypsin digestion of homocysteinylated fibronectin showed that several tryptic fragments contained [35S]homocysteine. Sequence analysis demonstrated that the fragments containing bound homocysteine had localized mainly to the C-terminal region, within and adjacent to the fibrin-binding domain. Homocysteinylation of fibronectin significantly inhibited its capacity to bind fibrin by 62% (P<0.005). In contrast, neither the binding of fibronectin to gelatin nor its capacity to serve as an attachment factor for aortic smooth muscle cells was affected. These results suggest that homocysteine may alter normal thrombosis and delay or interfere with wound healing by impairing the interaction of fibronectin with fibrin.

Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) binds to fibronectin (FN): demonstration of IGF-I/IGFBP-3/fn ternary complexes in human plasma.

We used a yeast two-hybrid system to identify binding partners for insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3). A partial complementary DNA encoding the carboxyl-terminal of fibronectin (FN), including the cell binding site, the heparin-binding domain, and the fibrin-binding domain, was identified in a screen of a human placental complementary DNA library. The interaction of IGFBP-3 with FN and the 40-kDa heparin-binding carboxyl-terminal fragment of FN was confirmed using Western ligand blotting. Both glycosylated and nonglycosylated IGFBP-3 bound to FN with a K(d) of approximately 0.3 nmol/L. IGF-I and IGFBP-1 had no effect on IGFBP-3 binding to FN. Competitive inhibition of IGFBP-3 binding to FN was observed in the presence of IGFBP-5 and heparin. The binding affinity of the immobilized IGFBP-3/FN complex for [(125)I]IGF-I (K(d) = 0.8 nmol/L) was similar to that of IGFBP-3 alone. The presence of IGF-I/IGFBP-3/FN ternary complexes in human plasma was demonstrated by coimmunoprecipitation of IGFBP-3 and [(125)I]IGF-I with anti-FN monoclonal antibody. These data indicate that FN may have a role in the transportation of IGFBP-3 and IGF-I in the circulation and the sequestration of these proteins in tissues.

Radiolabeled peptides in the detection of deep venous thrombosis

Venous thrombosis and pulmonary embolism are major clinical problems that result in significant morbidity and mortality. It is estimated that 600,000 cases of pulmonary embolism occur each year in the United States, resulting in the death of approximately 100,000 patients. Most of these pulmonary emboli arise from deep venous thrombosis (DVT). The clinical diagnosis of DVT is unreliable. Only a third of patients with a clinical suspicion of DVT have objective evidence of the disease, and half of patients with proven DVT do not have any clinical symptoms. Although ascending contrast venography is the present standard for the diagnosis of DVT, duplex ultrasonography, which is increasingly used in combination with color Doppler flow imaging, is accepted as a useful clinical afternative to contrast venography. Both contrast venography and ultrasonography are imaging procedures that detect changes in venous anatomy that are caused by the presence of an intraluminal thrombus that is sufficiently formed either to reduce vascular filling with contrast medium or to resist compression. However, these imaging procedures do not reflect the metabolic activity of the clot, and therefore, they may overestimate the presence of active clots. The sensitivity of ultrasonography is also limited by various disease-related and technical factors. An alternative approach to the diagnosis of acute DVT is to detect a molecular marker of acute DVT that is not present in old, organized DVT. Recent advances in biotechnology permit the use of highly specific synthetic peptide or small molecular markers, which are involved in the acute stages of DVT formation and can be labeled efficiently with 99mTc. 99mTc-apcitide, a glycoprotein (GP IIb/IIIa) receptor antagonist previously known as 99mTc-P280, has been approved recently by the Food and Drug Administration for the clinical detection of acute DVT. Two other agents are currently under clinical investigation: 99mTc-DMP 444, which is another GP IIb/IIIa receptor antagonist, and 99mTc-Fibrin-Binding Domain (FBD), a radio-labeled fibrin-binding domain of fibronectin. Different clinical studies have shown a high diagnostic accuracy with these synthetic 99mTc-labeled peptides in the detection of acute DVT. Although further studies are needed to fully appreciate all of the diagnostic potential of these radiopharmaceuticals, the clinical introduction of 99mTcapcitide scintigraphy will certainly be helpful in expanding the use of nuclear medicine in a specific field in which it used to play a relatively marginal role.

Recombinant Polypeptides Derived from the Fibrin Binding Domain of Fibronectin Are Potential Agents for the Imaging of Blood Clots

Thrombus formation in the circulation is accompanied by covalent linkage of fibronectin (FN) through transglutamination of glutamine no. 3 in the fibrin binding amino terminal domain (FBD) of FN. We have exploited this phenomenon for thrombus detection by the employment of radioactively-labelled recombinant polypeptide molecules derived from the 5-finger FBD of human FN. Three recombinant FBD polypeptides, 12 kDa ("2 fingers"), 18.5 kDa ("3 fingers") and 31 kDa FBD ("5 fingers"), were prepared and compared to native FN-derived 31 kDa-FBD with respect to their ability to attach to fibrin clots in vitro and in vivo. The accessibility of Gln-3 in these molecules was demonstrated by the incorporation of stoichiometric amounts of 14C-putrescine in the presence of plasma transglutaminase. Competitive binding experiments to fibrin have indicated that, although the binding affinities of the FBD molecules are lower than that of FN, substantial covalent linkage was obtained in the presence of transglutaminase, and even in the presence of excess FN or heparin. The biological clearance rates of radioactively labelled FBD molecules in rats and rabbits were much higher than those of FN and fibrinogen, thus indicating their potential advantage for use as a diagnostic imaging tool. Of the three molecules, the 12 kDa FBD exhibited the highest rate of clearance. The potential of the 12 kDa and 31 kDa FBDs as imaging agents was examined in a stainless steel coil-induced thrombus model in rats and in a jugular vein thrombus model in rabbits, using either [125I] or [111In]-labelled materials. At 24 h, clot-to-blood ratios ranged between 10 and 22 for [125I]-12 kDa FBD and 40 and 60 for [111In]-12 kDa FBD. In the rat model, heparin did not inhibit the uptake of FBD. Taken together, the results indicate that recombinant 12 kDa FBD is a good candidate for the diagnosis of venous thrombosis.

Characterization and Targeting of the Murine α2-Antiplasmin Gene

Alpha2-Antiplasmin (alpha2-AP) is the main physiological plasmin inhibitor in mammalian plasma. As a first step toward the generation of alpha2-AP deficient mice, the murine alpha2-AP gene was characterized and a targeting vector for homologous recombination in embryonic stem (ES) cells constructed. Alignment of nucleotide sequences obtained from genomic subclones allowed location of exons 2 through 10 of the alpha2-AP gene, but failed to identify the 5' boundary of exon 1. Compared to the human gene, exons 2 through 9 in the murine gene have identical size and intron-exon boundaries obeying the GT/AG rule. The 5' boundary of exon 10 is identical in both genes while the 3' non-coding region is 64 bp longer in the human gene. Introns 2, 3, 6 and 8 have similar sizes in the mouse and human genes; intron 1 is 6-fold smaller, introns 5, 7 and 9 are 2- to 3-fold smaller, whereas intron 4 is about 2-fold larger in the mouse gene. Compared to the human 5' flanking sequence, an insertion of a simple repeat region with sequence (TGG)n has occurred. The open reading frame of the mouse alpha2-AP gene encodes a 491-amino-acid protein comprising the experimentally determined NH2-terminus of the mature protein Val-Asp-Leu-Pro-Gly-. A targeting vector, pPNT.alpha2-AP, was constructed by introducing a homologous sequence of 8.3 kb in total in the parental pPNT vector. In pPNT.alpha2-AP, the neomycin resistance expression cassette replaces a 7 kb genomic fragment comprising exon 2 through part of exon 10 (including the stop codon), which represents the entire sequence encoding the mature protein, including the fibrin-binding domain, the reactive site peptide bond and the plasmin(ogen)-binding region. Electroporation of 129R1 embryonic stem (ES) cells with the linearized vector pPNT.alpha2-AP yielded three targeted clones with correct homologous recombination at the 5'- and 3'-ends, as confirmed by Southern blot analysis of purified genomic DNA with appropriate restriction enzymes and probes. These targeted clones will be used to generate alpha2-AP deficient mice.

Differentiation of the roles of the apolipoprotein(a) and LDL moiety in the binding of lipoprotein(a) to limited plasmin digested des AA fibrin

Elevated plasma levels of lipoprotein (a) (Lp(a)) in humans represent a major inherited risk factor for atherosclerosis. Lp(a) consists of a LDL-like particle with an additional glycoprotein, apolipoprotein(a) (apo(a)), linked to apolipoprotein B100. The apo(a) moiety is highly homologous to plasminogen as it contains multiple repeats resembling plasminogen kringle IV, a lysine and fibrin binding domain. In the present study, the individual contribution of the two constituents of Lp(a), namely LDL and apo(a), in the binding of Lp(a) to limited plasmin digested des AA fibrin (Desafib-X) is examined. Lp(a) was isolated by sequential ultracentrifugation followed by gel filtration chromatography. Lysine binding (Lp(a)lys+) and non-lysine binding (Lp(a)lys−) Lp(a) were obtained by affinity chromatography using lysine-Sepharose. Lp(a)-free LDL was isolated by ultracentrifugation followed by Lp(a)-free LDL was isolated by ultracentrifugation followed by chromatofocusing. 125I-labelled Lp(a), LDL, Lp(a)lys−, and Lp(a)lys+ preparations were incubated with Desafib-X coated wells in the presence or absence of ɛ-aminocaproic acid (ɛACA, a lysine-analogue) and/or autologous LDL. Total Lp(a) contained 86±8% Lp(a)lys+. The mean apparent dissociation constant (Kd in nM) for Lp(a), LDL, Lp(a)lys−, and Lp(a)lys+ binding to Desafib-X was 48±11, 28±4, 7±4, and 43±23, respectively. The binding of Lp(a) and Lp(a)lys+ to Desafib-X could be inhibited to a similar degree by 0.2m ɛACA (for 31±14% and 37±15% respectively), indicating lysine specific binding of these preparations. The binding of Lp(a)lys− and LDL could not be inhibited by ɛACA. A ten times molar excess of LDL could inhibit the binding of Lp(a), Lp(a)lys−, and Lp(a)lys+ to Desafib-X by 60 to 80%. For Lp(a) and Lp(a)lys+, an additional binding-inhibition can be observed when adding 0.2M ɛACA. In conclusion, the overall findings indicate that the binding of Lp(a) to fibrin(-ogen) is more complex than previously thought and imposes an influence of the LDL moiety and LDL, additional to the apo(a) moiety, in the binding of Lp(a) to lysine-containing proteins like Desafib-X.