Interaction of beta 3 integrin-derived peptides 214-218 and 217-231 with alpha IIb beta 3 complex and with fibrinogen A alpha-chain.

beta 3 integrin-derived peptides 214-218 and 217-231 have been shown previously to inhibit platelet aggregation and fibrinogen binding to platelets and to purified receptor. In this paper we study the activity of both peptides in inhibition of binding of biotinylated fibrinogen to activated platelets and to immobilized alpha IIb beta 3 receptor. We found that the mechanism of this inhibition by both peptides is different 125I-labeled 214-218 peptide binds to alpha IIb beta 3 but in contrast, 125I-labeled 217-231 peptide binds to the A alpha-chain of native and gamma' fibrinogen, as judged by the cross-linking study. In solid phase assay both purified alpha IIb beta 3 and 217-231 peptide bound extensively to native and recombinant fibrinogen, and to fibrinogen with either D574E or D97E mutations in the A alpha-chain. Binding of purified alpha IIb beta 3 to gamma' fibrinogen was markedly impaired whereas binding of 217-231 was only slightly impaired in comparison with native fibrinogen. Binding of 217-231 to fibrinogen fragment X was also reduced suggesting that sequences other than RGDS and RGDF may represent binding sites for this peptide. We hypothesize that the close vicinity of fibrinogen binding site (217-231) and of the site participating in conformational changes of the alpha IIb beta 3 receptor (214-218) may facilitate fibrinogen interaction with its receptor.

Immunologic assessment of patients treated with bovine fibrin as a hemostatic agent.

Twenty-one cardiothoracic surgical patients have been treated with fibrin as a topical hemostatic/sealing agent, prepared from bovine fibrinogen clotted with bovine thrombin. Serum samples have been collected before treatment with fibrin and postoperatively between 1 and 9 days, 3 and 12 weeks, and 6 and 8 months. The titers of anti-bovine fibrinogen antibodies, measured by ELISA specific for immunoglobulins IgG or IgM, increased to maximal values after about 8 or 6 weeks, respectively. After 8 months, IgG titers were on average 20-fold lower than the mean maximal value, while IgM titers returned to the normal range. IgG was the predominant anti-bovine fibrinogen immunoglobulin as documented by ELISA, affinity chromatography and electrophoresis. Anti-bovine fibrinogen antibodies present in patients reacted readily with bovine fibrinogen, but did not cross-react with human fibrinogen as measured by ELISA or by immunoelectrophoresis. A significant amount of antibodies against bovine thrombin and factor V has been found, many cross-reacting with the human counterparts. No hemorrhagic or thrombotic complications, or clinically significant allergic reactions, occurred in any patient, in spite of antibody presence against some bovine and human coagulation factors. The treatment of patients with bovine fibrin, without induction of immunologic response against human fibrinogen, appeared to be an effective topical hemostatic/sealing measure.

Primary binding domain of bovine von Willebrand factor fragment expressed in E. coli.

Bovine vWF cDNA has been cloned from a bovine endothelial cell library. A fragment of this cDNA, corresponding to amino acid sequence Leu 469-Ser 723, called primary adhesion domain (PAD-1), and containing the binding sites for platelet glycoprotein Ib (GPIb), heparin and collagen, has been expressed in E. coli. The reduced and alkylated form of fragment PAD-1 inhibited native vWF binding to GPIb. Fragment PAD-1 bound to heparin and botrocetin in a specific and dose dependent manner as did the native vWF. In a solid-phase assay, fragment PAD-1 bound to calf skin collagen in contrast to a human vWF recombinant fragment (Ser 445-Val 733) which was inactive in the same assay. The studies presented in this paper demonstrated that the A1 domain of bovine vWF contained the GPIb, heparin, botrocetin as well as collagen binding sites and that integrity of the disulfide bond (Cys 509-Cys 695), did not seem to be essential for binding of bovine vWF fragment to GPIb.

Comparison of the kinetic fibrinogen assay with the von Clauss method and the clot recovery method in plasma of patients with conditions affecting fibrinogen coagulability.

A Kinetic Fibrinogen Assay (KFA), a method based on the kinetic reaction of the developing fibrin clot, was used to determine fibrinogen concentration in plasma. Two other methods employing different quantification principles were used for comparison: the von Clauss method and the procedure measuring protein concentration in an isolated and washed plasma clot (World Health Organization [WHO] method). All three methods quantified functional thrombin-coagulable fibrinogen. Plasma specimens were obtained from three groups of patients: those with liver disease and those taking either coumarin derivative or heparin. In all of these conditions, there are deviations from the normal process of fibrin clot formation. The KFA method yielded results that were consistent and provided excellent precision and accuracy allowing quantification of plasma fibrinogen in the range of 70-800 mg/dL (2-23.5 microM). The determination of fibrinogen by the KFA method was not adversely affected using plasma from patients treated with heparin and those undergoing coumarin therapy. Statistical analysis of the results indicated that the KFA method compared very favorably with the von Clauss and WHO methods. In assessing the clinical utility of each method, the WHO method was found to be labor intensive and time consuming; therefore, not suitable for routine use in a clinical laboratory. The von Clauss method required a trained laboratory technician and some laboratory manipulations. The KFA method was not only reliable and accurate, but also fully automated, making it the easiest and the fastest to perform routinely.

The (DD)E complex is maintained by a composite fibrin polymerization site.

The (DD)E complex is the major cross-linked fibrin degradation fragment. Structural components required for maintenance of the (DD)E complex were examined in order to better understand clot structure and the contribution of specific polypeptide chain segments in the process of polymerization. First, the (DD)E complex was reversibly dissociated by peptides derived from the alpha-chain NH2-terminus of fibrin having a minimal sequence of GPR (alpha 17-19). In addition, the complex was partially dissociated by peptide beta 40-54, while beta 50-55 and peptides derived from the fibrin beta-chain NH2-terminus had no effect. Second, monoclonal antibody (mAb) 1B6, specific for the alpha-chain NH2-terminus of fibrin, reacted rapidly with fragment E1, but did not recognize the corresponding epitope on the (DD)E complex. On the other hand, mAb 59D8, specific for GHRPL at the beta-chain NH2-terminus of fibrin, reacted with the (DD)E complex in a dose-dependent manner. Third, the (DD)E complex was irreversibly dissociated by proteolytic cleavage of fragment E1 by either thrombin, which removed GPR from the alpha-chain NH2-terminus, or Crotalus atrox protease III, which released beta 15-42. It has been concluded that fragment E1 contains a composite polymerization site consisting at least of residues alpha 17-19 and beta 20-49, which together maintain the (DD)E complex. These results illustrate that the complex is kept together by complementary binding sites which form a nucleus of linear fibrin polymerization sites. The (DD)E complex can thus be considered as a soluble model of fibrin clot.(ABSTRACT TRUNCATED AT 250 WORDS)

ASP514 within the A1 domain of bovine von Willebrand factor is required for interaction with platelet glycoprotein Ib.

A mutant PAD-1 (D514-->Q) of the recombinant fragment PAD-1 comprising Leu469-Ser723 of the A1 domain of bovine von Willebrand factor (vWF) neither inhibited the binding of [125I]vWF to platelets nor the agglutination of human platelets induced by bovine vWF. PAD-1, on the other hand, inhibited human platelet agglutination induced by bovine vWF and [125I]vWF binding to human platelets. Collagen binding properties of the mutant, however, were indistinguishable from those of PAD-1. These results suggested that Asp514 within the A1 domain of vWF is required for interaction of bovine vWF with GPIb receptor on human platelets.

Localization of the cross-linking site of GPRVVERHK in the gamma-chain of human fibrinogen.

The peptide alpha 17-24-Lys (GPRVVERHK) corresponding to the N-terminus of the alpha chain of fibrin was synthesized and used to localize its binding site in the fibrinogen molecule. The peptide was radioiodinated, incubated with fibrinogen, cross-linked with a bifunctional reagent disuccinimidyl suberate and the resulting product was analyzed in several ways, including plasmin digestion. The binding of the radioactive peptide was mainly to the gamma-chain and was inhibited by unlabelled GPRVVERHK and GPRP. After plasmin digestion, the radioactivity was present in fragment D1 and also in its gamma-chain remnant, but not in fragments D2, D3 or E3. Fragment D1 cross-linked with iodinated GPRVVERHK was purified by affinity chromatography on immobilized anti-fragment D IgG, further digested with plasmin in the presence of EGTA and the peptides were fractionated by reverse-phase HPLC. The amino acid sequence analysis of the radioactive peak revealed the presence of two peptides, gamma 357-373 and GPRVVERHK. It was concluded that the binding site for GPRVVERHK is in the sequence gamma 357-373 which is present in fragment D1 but absent in fragments D2 and D3.

Conformational changes in fibrinogen elicited by its interaction with platelet membrane glycoprotein GPIIb-IIIa.

The binding of fibrinogen to membrane glycoprotein GPIIb-IIIa on activated platelets leads to platelet aggregation. This interaction results in conformational changes in fibrinogen as evidenced by the expression of receptor-induced binding sites, RIBS, epitopes which are expressed by the bound but not the free ligand. In the present study, two RIBS epitopes have been localized. One sequence resides at gamma 112-119 and is recognized by mAb 9F9; the second is the RGDF sequence at A alpha 95-98 and is recognized by mAb 155B16. These epitopes are also exposed by adsorption of fibrinogen onto a plastic surface and digestion of the molecule by plasmin. Proteolytic exposure of the epitopes coincides with cleavage of the carboxyl-terminal aspects of the A alpha-chains to form fragment X2. The inaccessibility of the RGDF sequence at A alpha 95-98 in fibrinogen suggests that this sequence does not participate in the initial binding of the molecule to GPIIb-IIIa. The location of these RIBS epitopes suggests a model in which binding of fibrinogen to its receptor alters the conformation of the carboxyl-terminal aspects of the A alpha-chains, exposing the sequences which reside in the coiled-coil connector segments between the D and E domains of the molecule. These sequences may then serve as epitopes and may mediate unique functions of the receptor-bound molecule.

Beta 3 integrin derived peptide 217-230 inhibits fibrinogen binding and platelet aggregation: significance of RGD sequences and fibrinogen A alpha-chain.

beta 3 Integrin derived peptides 217-230 (DAPEGGFDAIMQAT) and 217-231 (Y) (DAPEGGFDAIMQATVY) at 100 microM inhibited 125I-fibrinogen binding to ADP-stimulated platelets and platelet aggregation. Peptide 217-231 (Y) (100 microM) significantly inhibited the binding of 125I-albolabrin (a disintegrin with a single RGD sequence) to ADP- and thrombin-activated platelets while it had only a slight effect on albolabrin binding to resting platelets. The 125I-beta 3 217-231 (Y) cross-linked selectively to the fibrinogen A alpha-chain. The interaction of the RGD sequence in the A alpha-chain of fibrinogen with beta 3 217-231 sequence appears to play a significant role in the events leading to platelet aggregation.

Interaction between complementary polymerization sites in the structural D and E domains of human fibrin.

Plasmic degradation products of human fibrin, fragments DD, D, and E, bind to fibrin. It has been inferred from this observation that the binding occurs by attraction of complementary sites located in the NH2- and COOH-terminal domains of the fibrin molecule. The interaction between fragments D1 and E1 has been investigated in this work since it represents the first step in the process of fibrin clot formation. Fragment D1, that was initially as active as fragment DD, lost most of its anticoagulant activity after purification by cation-exchange chromatography. The lability of fragment D1 function explained the previous unsuccessful attempts to form a complex between fragments D1 and E1. The loss of fragment D1 anticoagulant activity was not associated with the cleavage of the gamma 63-85 chain segment, since fragments D1A and D1 identically inhibited the fibrin monomer polymerization rate. In order to demonstrate the formation of a complex between fragments D1 and E1, three lines of experiments were advanced. First, the anticoagulant activity of fragment D1 was neutralized by fragment E1 in a dose-dependent manner, demonstrating that the association between these fragments involved polymerization sites. Second, two products, D1.E1 and D1.E1.D1, were stabilized in a reaction with bifunctional cross-linking reagents, proving the formation of D.E complexes in aqueous solution. Third, immobilized fragment D1 bound fragments E1 and E2, but not fragment E3, showing that fragments E1 and E2 attached via a polymerization site to the complementary one in fragment D1, since this association was disrupted by fibrin polymerization inhibitory peptide GPRP. These results provided direct evidence for specific binding between the structural D and E domains of fibrin mediated through complementary polymerization sites. Thus, the initial formation of fibrin clot fibers appears to be driven by specific association of these sites.

Binding of fibrin fragments to one-chain and two-chain tissue-type plasminogen activator.

To explore whether fibrin fragments have binding affinity for the tissue-type plasminogen activator (t-PA) molecule, the interactions were studied of (DD)E complex and fragments DD, E1, and E3 with one-chain and two-chain t-PA. For this purpose, a solid-phase binding assay was developed using microtiter plates with nitrocellulose filters. It was found that (DD)E complex and fragments DD and E3 retained the t-PA binding function of the parent fibrin molecule, thus demonstrating that t-PA binds to both the D and E domains of fibrin. Unexpectedly, fragment E1 did not bind t-PA. Fibrin fragments had different binding properties for one-chain and two-chain t-PA. (DD)E complex had the highest and fragment E3 the lowest affinity for one-chain t-PA, both binding curves being consistent with one class of binding sites. However, binding of the fragments with two-chain t-PA was distinguished by more than one class of binding sites, with fragment E3 having the highest affinity for this form of the activator. epsilon-Aminocaproic acid, even at 50 mmol/L concentration, had only minimal effect on binding of (DD)E complex or fragment DD to either one-chain or two-chain t-PA. The potentiating effect of fibrin fragments on plasminogen activation by t-PA was measured by a chromogenic substrate assay. Fragment DD was the most effective stimulator of plasminogen activation by t-PA. In conclusion, (DD)E complex and fragment DD retained most of the regulatory functions of fibrin, which included t-PA binding and t-PA-mediated acceleration of plasminogen activation to plasmin.

Involvement of the alpha chain in fibrin clot formation. Effect of monoclonal antibodies.

Murine monoclonal antibodies 9C3, 7B1, and 9E9 have been obtained using native human fibrinogen as the antigen. The antibodies reacted with the epitopes in the COOH-terminal domain of the A alpha chain. Fragmentation of the A alpha chain with plasmin, and, as in the case of the 9E9 epitope, with V8 protease, followed by isolation of the smallest reacting peptides, allowed the localization of the epitopes for 9C3, 7B1, and 9E9 to the amino acid sequences of alpha 240-268, alpha 425-440, and alpha 541-574, respectively. All three monoclonal antibodies strongly inhibited the rate of fibrin polymer assembly from monomers, both in the purified system and in the human plasma. The mechanism of this strong inhibition implied a rapid formation of fibrin protofibrils, followed by capping with IgG molecules of protofibrils containing approximately ten monomers. These observations demonstrated that certain regions in the COOH terminus of the alpha chain may play an important role in the assembly of a fibrin clot, presumably being involved in lateral aggregation of protofibrils.

Interaction of hementin with fibrinogen and fibrin.

The giant Amazon leech Haementeria ghilianii manufactures blood anticoagulant which is present in the posterior and anterior salivary glands. The mechanism of blood anticoagulation by Haementeria ghilianii is completely different from that used by Hirudo medicinalis. The anticoagulant is mostly associated with a fibrinogen-degrading proteinase, hementin. However, other inhibitors of blood coagulation are also present in the salivary glands. The salivary gland extract inhibits platelet aggregation that is mostly attributable to the degradation of fibrinogen. Hementin purified by various methods has a molecular weight in the range of 80,000-120,000 and appears to be a metalloproteinase that is regulated by calcium ions. The enzyme degrades both fibrinogen and fibrin. The Michaelis constant for human fibrinogen is 1 microM. The cleavage of the isolated chains of fibrinogen is inefficient implying that the native conformation of the substrate may play a role in the recognition mechanism. The pattern of fibrinogen degradation by hementin resembles that caused by plasmin since products analogous to fragments Y, D and E are generated. However, the unique action of hementin on fibrinogen is in the initial proteolytic attack in the coiled-coil connector region while proteolysis of the alpha-chain is very slow. In consequence, unique fibrinogen fragments are formed that contain the entire COOH-terminus of the alpha-chain. The mechanism of blood anticoagulation by hementin is very efficient since the cleavage of only three peptide bonds in the fibrinogen molecule disassembles its bivalent structure and renders it non-functional.

Polymerization site in the beta chain of fibrin: mapping of the B beta 1-55 sequence.

The formation of a fibrin clot occurs through binding of putative complementary sites, called fibrin polymerization sites, located in the NH2- and COOH-terminal domains of fibrin monomer molecules. In this study, we have investigated the structure of the NH2-terminal fibrin polymerization site by using fibrinogen-derived peptides and fragments. Fibrinogen was digested with Crotalus atrox protease III, to two major molecular species: a Mr 325,000 derivative (Fg325) and a peptide of Mr 5000. The peptide and its thrombin-cleavage product were purified by ion-exchange and reverse-phase HPLC; the authenticity of the B beta 1-42 and beta 15-42 peptides, respectively, was confirmed by amino acid sequencing. Since Fg325 had decreased thrombin coagulability, we addressed the question of whether the peptide B beta 1-42 contained a fibrin polymerization site. In order to identify and map the site, the peptides B beta 1-42 and beta 15-42 were tested for their ability to inhibit fibrin monomer polymerization. In addition the following peptides prepared by chemical synthesis were also tested: beta 15-18, beta 15-26, beta 24-42, beta 40-54, beta 50-55, and alpha 17-19-Pro. While B beta 1-42 had no inhibitory activity, the peptide devoid of fibrinopeptide B, beta 15-42, was a strong inhibitor. The peptides beta 15-18, beta 15-26, and beta 15-42 decreased the rate of fibrin polymerization by 50% at a molar excess of the peptide to fibrin monomer of 500, 430, and 50, respectively. The peptides beta 24-42, beta 40-54, and beta 50-55 were inactive.(ABSTRACT TRUNCATED AT 250 WORDS)

The polymerization and thrombin-binding properties of des-(B beta 1-42)-fibrin.

Multiple factors affect the thrombin-catalyzed conversion of fibrinogen to fibrin, including: fibrinopeptide (FPA and FPB) release leading to exposure of two types of polymerization domains ("A" and "B," respectively) in the central portion of the molecule, and exposure of a noncatalytic "secondary" thrombin-binding site in fibrin. Fibrinogen containing the FPA sequence but lacking the B beta 1-42 sequence ("des-(B beta 1-42)-fibrinogen"), was compared to native fibrinogen (containing both FPA and FPB) to investigate the role played by B beta 1-42 in the polymerization of alpha-fibrin (i.e. fibrin lacking FPA), to compare reptilase and thrombin cleavage of FPA from fibrinogen, and to explore the location and function of the secondary thrombin-binding site. Electron microscopy of evolving polymer structures (mu, 0.14; pH 7.4) plus turbidity measurements, showed that early thin fibril formation as well as subsequent lateral fibril associations were impaired in des-(B beta 1-42)-alpha-fibrin, thus indicating that the B beta 1-42 sequence contributes to the A polymerization site. Reptilase-activated des-(B beta 1-42)-alpha-fibrin polymerized even more slowly than thrombin-activated des-(B beta 1-42)-alpha-fibrin, differences that disappeared when repolymerization of preformed fibrin monomers was carried out. Since existing data indicate that thrombin releases FPA in a concerted manner, resulting in relatively rapid evolution of fully functional divalent alpha-fibrin monomers, it can be inferred that delayed fibrin assembly of reptilase fibrin is due to slower formation of divalent alpha-fibrin monomers. Thrombin-activated des-(B beta 1-42)-alpha-fibrin polymerized more rapidly at low ionic strength (mu, 0.04) than did native alpha,beta-fibrin, a reversal of their behavior at physiological ionic strength (mu, 0.14). Concomitant measurement of FPA release revealed modest slowing of release at low ionic strength from des-(B beta 1-42)-fibrinogen (t1/2, 36.5 versus 21.5 min) and marked slowing from native fibrinogen (t1/2, 138 versus 22.2 min). This behavior correlated with increased thrombin binding to native alpha,beta-fibrin at low ionic strength, coupled with weak thrombin binding to des-(B beta 1-42)-alpha-fibrin, and indicates that secondary thrombin binding plays an important role in regulating thrombin diffusion and catalytic activity. Des-(B beta 1-42)-fibrinogen lacks or has a markedly defective secondary thrombin-binding site, from which we conclude that the B beta 15-42 sequence in fibrin plays a major role in forming or providing this site.

A unique proteolytic fragment of human fibrinogen containing the A alpha COOH-terminal domain of the native molecule.

The COOH-terminal portion of the A alpha chain of human fibrinogen is highly susceptible to proteolytic degradation. This property has prevented isolation of the COOH-terminal domain of fibrinogen for the direct investigation of its functional characteristics. Human fibrinogen was degraded with hementin, a fibrinogen-olytic protease from the posterior salivary glands of the leech, Haementeria ghilianii. Two initial fragments, Yhem1 and Dhem1, produced by cleavage through the three polypeptide chains in the connector region, were characterized and shown to retain the entire A alpha COOH-terminal domain. Late cleavages by hementin occurred in the A alpha chain COOH-terminal region to produce fragments Yhem and Dhem with shorter A alpha chain remnants. Fragments Dhem were isolated from an intermediate hementin digest of fibrinogen using anion-exchange chromatography. Fragment Dhem1 was separated further from Dhem fragments with shorter alpha chain remnants by affinity chromatography on immobilized plasma fibronectin. Fragment Dhem1 represents a unique proteolytic fragment of fibrinogen containing an intact A alpha chain COOH-terminal region. NH2-terminal sequence analysis of isolated chains from fragment Dhem1 located hementin cleavage sites in the connector region to A alpha Asn102-Asn103, B beta Lys130-Gln131, and gamma Pro76-Asn77. The specific interaction of fragment Dhem1 with immobilized fibronectin indicated that the binding site probably was located within the COOH-terminal 111 amino acids of the A alpha chain. The overall pattern of fibrinogen cleavage by hementin is similar to that of plasmin, yet hementin cleaves preferably in the coiled-coil connector, sparing the A alpha COOH-terminal domain.

Purification and characterization of hementin, a fibrinogenolytic protease from the leech Haementeria ghilianii.

The fibrinogenolytic enzyme hementin, present in extracts of the posterior salivary glands of the giant leech Haementeria ghilianii, was isolated by ultrafiltration, high-performance ion-exchange chromatography and subsequent reversed-phase liquid chromatography. Approximately 100 micrograms (1 nmol) of hementin, present at less than 0.5% in the crude leech salivary extract, was brought to about 90% purity in three steps. Hementin migrated at an Mr of about 73,000 on non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and at 82,000 on reducing SDS-PAGE. The amino terminal sequence was determined to be TTLTE-PEPDL. The amino terminal sequences of two inactive proteins that partially coeluted with hementin in the first chromatographic step were also determined.

Direct detection of activated platelets and platelet-derived microparticles in humans.

Flow cytometry was used to determine whether activated platelets and platelet-derived microparticles can be detected directly in whole blood after a hemostatic insult. Two different in vivo models of platelet activation were examined: (1) a standardized bleeding time, and (2) cardiopulmonary bypass. Platelets and microplatelets were identified with a biotinylated anti-glycoprotein (GP)lb antibody and a fluorophore, phycoerythrin-streptavidin. Microparticles were distinguished from platelets by light scatter. Activated platelets were detected with three fluorescein-labeled monoclonal antibodies (MoAbs): (1) PAC1, which binds to the activated form of GPIIb-IIIa; (2) 9F9, a newly developed antibody that is specific for fibrinogen bound to the surface of activated platelets; and (3) S12, which binds to an alpha-granule membrane protein expressed on the platelet surface after granule secretion. In nine normal subjects, bleeding times ranged from 4.5 to 7.5 minutes. Over this time, there was a progressive increase in the amount of PAC1, 9F9, and S12 bound to platelets in blood emerging from the bleeding time wound. With all three antibodies, platelet activation was apparent as early as 30 seconds after the incision (P less than .03). Activation was accompanied by a progressive decrease in the concentration of platelets in blood from the wound, while the concentration of microparticles increased slightly. In nine patients undergoing open heart surgery, 1 hour of cardiopulmonary bypass caused a 2.2-fold increase in the relative proportion of microparticles in circulating blood (P less than .001). Moreover, bypass caused platelet activation as evidenced by a mean two- to threefold increase in PAC1 binding to platelets. Although this increase was significant (P less than .02), PAC1 binding exceeded the normal range for unstimulated control platelets in only 5 of 9 patients, and 9F9 and S12 binding exceeded the normal range in only two patients. Taken together, these studies demonstrate that it is now feasible using flow cytometry to evaluate the extent of platelet activation and the presence of platelet-derived microparticles in the circulation of humans.

Differences between binding of one-chain and two-chain tissue plasminogen activators to non-cross-linked and cross-linked fibrin clots.

Interaction of tissue plasminogen activator (t-PA) with fibrin plays a key role in regulation of plasminogen activation and clot dissolution. Previous investigations of t-PA-fibrin interaction, using incorporation of t-PA into polymerizing fibrin clots, have suggested that no significant differences exist in the binding of one-chain or two-chain t-PA to non-cross-linked or cross-linked fibrin. In the present study, binding of 125I-labeled and affinity-purified one-chain and two-chain forms of t-PA to preformed non-cross-linked or cross-linked, sonicated suspension of fibrin was investigated. Interaction of one-chain t-PA with cross-linked fibrin involved a single type of binding site with dissociation constant (kd) of 0.58 mumol/L and a stoichiometry (n) of 1.5. Interaction of one-chain t-PA with non-cross-linked fibrin, however, involved two classes of binding sites with dissociation constants of 0.32 and 1.5 mumol/L and corresponding number of binding sites equal to 0.57 and 2.0, respectively. In contrast to the binding of one-chain t-PA to cross-linked fibrin by a limited number of sites, two-chain t-PA appeared to involve a considerably greater number of sites (minimum six) whose dissociation constant was 3.2 mumol/L. Interaction of two-chain t-PA with non-cross-linked fibrin also showed the presence of many binding sites (minimum seven) with approximate dissociation constant of 6.4 mumol/L, as well as a few (n = 0.012) high-affinity sites with a kd of 0.011 mumol/L epsilon-Aminocaproic acid did not completely reverse the binding of either one-chain t-PA or two-chain t-PA to fibrin. The present findings suggest that the fibrin-binding properties of t-PA undergo considerable changes on proteolytic conversion from one-chain to two-chain t-PA, catalyzed under physiologic conditions by plasmin. The cleavage of one-chain t-PA to two-chain t-PA allows to bind to a large number of low-affinity binding sites on fibrin. Cross-linking of fibrin by factor XIIIa results in masking of high-affinity binding sites that are present in non-cross-linked fibrin. We propose that both plasmin and factor XIIIa play an important regulatory role in dissolution of blood clots by modulating t-PA-fibrin interaction.