Carboxypeptidase U (CPU, carboxypeptidase B2, activated thrombin-activatable fibrinolysis inhibitor) inhibition stimulates the fibrinolytic rate in different in vitro models.

Essentials AZD9684 is a potent inhibitor of carboxypeptidase U (CPU, TAFIa, CPB2). The effect of AZD9684 on fibrinolysis was investigated in four in vitro systems. The CPU system also attenuates fibrinolysis in more advanced hemostatic systems. The size of the observed effect on fibrinolysis is dependent on the exact experimental conditions. SUMMARY: Background Carboxypeptidase U (CPU, carboxypeptidase B2, activated thrombin-activatable fibrinolysis inhibitor) is a basic carboxypeptidase that attenuates fibrinolysis. This characteristic has raised interest in the scientific community and pharmaceutical industry for the development of inhibitors as profibrinolytic agents. Objectives Little is known about the contribution of CPU to clot resistance in more advanced hemostatic models, which include blood cells and shear stress. The aim of this study was to evaluate the effects of the CPU system in in vitro systems for fibrinolysis with different grades of complexity. Methods The contribution of the CPU system was evaluated in the following systems: (i) plasma clot lysis; (ii) rotational thromboelastometry (ROTEM) in whole blood; (iii) front lysis with confocal microscopy in platelet-free and platelet-rich plasma; and (iv) a microfluidic system with whole blood under arterial shear stress. Experiments were carried out in the presence or absence of AZD9684, a specific CPU inhibitor. Results During plasma clot lysis, addition of AZD9684 resulted in 33% faster lysis. In ROTEM, the lysis onset time was decreased by 38%. For both clot lysis and ROTEM, an AZD9684 dose-dependent response was observed. CPU inhibition in front lysis experiments resulted in 47% and 50% faster lysis for platelet-free plasma and platelet-rich plasma, respectively. Finally, a tendency for faster lysis was observed only in the microfluidic system when AZD9684 was added. Conclusions Overall, these experiments provide novel evidence that the CPU system can also modulate fibrinolysis in more advanced hemostatic systems. The extent of the effects appears to be dependent upon the exact experimental conditions.

The future of glycoprotein VI as an antithrombotic target.

The treatment of acute coronary syndromes has been considerably improved in recent years with the introduction of highly efficient antiplatelet drugs. However, there are still significant limitations: the recurrence of adverse vascular events remains a problem, and the improvement in efficacy is counterbalanced by an increased risk of bleeding, which is of particular importance in patients at risk of stroke. One of the most attractive targets for the development of new molecules with potential antithrombotic activity is platelet glycoprotein (GP)VI, because its blockade appears to ideally combine efficacy and safety. This review summarizes current knowledge on GPVI regarding its structure, its function, and its role in physiologic hemostasis and thrombosis. Strategies for inhibiting GPVI are presented, and evidence of the antithrombotic efficacy and safety of GPVI antagonists is provided.

An acquired inhibitor to the GPVI platelet collagen receptor in a patient with lupus nephritis.

BACKGROUND: GPVI is a major platelet collagen signaling receptor. In rare cases of immune thrombocytopenic purpura (ITP), autoantibodies to GPVI result in receptor shedding. OBJECTIVES: To investigate a possible pathogenic role of plasma anti-GPVI antibody located in a woman with lupus nephritis. METHODS: Measured were (i) platelet aggregation to collagen and convulxin, (ii) platelet GPVI expression (flow cytometry and western blotting), (iii) plasma soluble GPVI (sGPVI, dual antibody ELISA), and (iv) plasma anti-GPVI antibody (ELISA using recombinant sGPVI). RESULTS: In 2006 and early 2007, the patient had a normal platelet count but a virtual absence of platelet aggregation to collagen and convulxin. Her platelets responded normally to other agonists including cross-linking ITAM-dependent FcgammaRIIA by monoclonal antibody, IV.3. Flow cytometry and western blotting showed a platelet deficiency of GPVI. Plasma sGPVI levels were undetectable whereas ELISA confirmed the presence of anti-GPVI antibody. Sequencing revealed a normal GPVI cDNA structure. The patient's plasma and the isolated IgG3 fraction activated and induced GPVI shedding from normal platelets. A deteriorating clinical condition led to increasingly strict immunosuppressive therapy. This was globally associated with a fall in plasma anti-GPVI titres, the restoration of platelet GPVI and the convulxin response, and the loss of her nephrotic syndrome. CONCLUSIONS: Our results show that this patient acquired a potent anti-GPVI IgG3 antibody with loss of GPVI and collagen-related platelet function. Further studies are required to determine whether anti-GPVI antibodies occur in other lupus patients with nephritis.

Ex vivo inhibition of thrombus formation by an anti-glycoprotein VI Fab fragment in non-human primates without modification of glycoprotein VI expression.

OBJECTIVES: Glycoprotein (GP)VI is an attractive target for the development of new antithrombotic drugs. Its deficiency protects animals in several models of thrombosis, arterial stenosis and ischemia--reperfusion while inducing no major bleeding tendency. The Fab fragment of one anti-GPVI monoclonal antibody (9O12.2) inhibits all GPVI functions in vitro. The aim of this study was to determine the ex vivo effects of 9O12.2 Fab on hemostasis, coagulation and thrombosis in non-human primates. METHODS AND RESULTS: Blood samples were collected from cynomolgus monkeys before and after (30, 90 and 150 min, 1 and 7 days) a bolus injection of 9O12.2 Fab (4 mg kg(-1)) or vehicle. Platelet counts and coagulation tests (prothrombin time, activated partial thromboplastin time) were not modified following Fab injection. The PFA-100 closure time increased during the first hours and returned to initial values on day + 1. Platelet-bound Fab was detected from 30 min to 24 h after Fab injection without GPVI depletion at any time. Collagen-induced platelet aggregation was selectively and fully inhibited at 30 min. Thrombus formation on collagen in flowing whole blood (1500 s(-1)) was delayed and decreased, and collagen-induced or tissue factor-induced thrombin generation in platelet-rich plasma was profoundly inhibited. CONCLUSION: The anti-GPVI 9O12.2 Fab inhibits thrombus formation ex vivo in non-human primates with a composite effect on platelet activation and thrombin generation in the absence of GPVI depletion.

Functional approach to investigate Lp(a) in ischaemic heart and cerebral diseases.

BACKGROUND: Lp(a), a major cardiovascular risk factor, contains a specific apolipoprotein, apo(a), which by virtue of structural homology with plasminogen inhibits the formation of plasmin, the fibrinolytic enzyme. A number of clinical reports support the role of Lp(a) as a cardiovascular or cerebral risk factor, and experimental data suggest that it may contribute to atherothrombosis by inhibiting fibrinolysis. DESIGN: A well-characterized model of a fibrin surface and an apo(a)-specific monoclonal antibody were used to develop a functional approach to detect pathogenic Lp(a). The assay is based on the competitive binding of Lp(a) and plasminogen for fibrin, and quantifies fibrin-bound Lp(a). High Lp(a) binding to fibrin is correlated with decreased plasmin formation. In a transversal case-control study we studied 248 individuals: 105 had a history of ischaemic cardiopathy (IC), 52 had cerebro-vascular disease (CVD) of thrombotic origin, and 91 were controls. RESULTS: The remarkably high apo(a) fibrin-binding in CVD (0.268 +/- 0.15 nmol L-1) compared with IC (0.155 +/- 0.12 nmol L-1) suggests the existence of peculiar and poorly understood differences in pro- or anti-thrombotic mechanisms in either cerebral and/or coronary arteries. CONCLUSIONS: Our results demonstrated that Lp(a) fibrin-binding and small Apo(a) isoforms are associated with athero-thrombotic disease.

Structure and properties of clots from fibrinogen Bicêtre II (gamma 308 Asn-->Lys). Increased permeability due to larger pores, thicker fibers, and decreased rigidity.

Fibrinogen Bicêtre II is a dysfibrinogenemia in which there is a substitution of Lys for Asn at gamma 308. We have studied the polymerization of this abnormal fibrinogen by measurement of turbidity and have characterized clot structure by scanning electron microscopy, permeation, and viscoelastic measurements. The results of these studies demonstrate that this amino acid substitution has substantial effects on the structure and properties of the clot, resulting in clots made up of thick fibers and large pores with greatly reduced stiffness and increased slippage of protofibrils.

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).

Kringles of the plasminogen--prothrombin gene family share conformational epitopes with recombinant apolipoprotein (a): specificity of the fibrin-binding site.

Monoclonal antibodies directed against recombinant apolipoprotein (a) (r-apo(a)) lacking plasminogen-like KIV-2 repeats were used to identify structurally related conformational epitopes in various members of the plasminogen-prothrombin gene family. A number of procedures including a fibrin-binding inhibition immunoassay and surface plasmon resonance studies were used. Two antibodies (A10.1 and A10.4) recognised common conformational structures in r-apo(a), prothrombin, factor XII, plasminogen and its tissue-type and urokinase-type activators. In contrast, two other antibodies recognised specifically an epitope comprising residues of the lysine-binding site (A10.2) or close to it (A10.5) and inhibited the fibrin-binding function of r-apo(a) (IC(50)=36 pmol/l and 9.76 nmol/l, respectively). Interestingly, these antibodies distinctly recognised the elastase-derived fragments of plasminogen K4 (A10.2) and K1+2+3 (A10.5) without affecting plasminogen binding to fibrin. These results suggest that highly conserved conformational regions are common to various proteins of the plasminogen-prothrombin gene family and are in agreement with the concept that these proteins constitute a monophyletic group derived from an ancestral gene.

Binding of recombinant apolipoprotein(a) to human platelets and effect on platelet aggregation.

The interaction of lipoprotein(a) [Lp(a)] with platelets is not well defined, particularly with regards to the individual contribution of the protein components of Lp(a), the apo B-100 and the apolipoprotein apo(a). This study investigated the binding of different recombinant apo(a) [r-apo(a)] isoforms, to human platelets and its effect on platelet aggregation. Scatchard analysis of saturation binding experiments demonstrated that human platelets display a single class of high affinity r-apo(a) binding sites (71 +/- 46 molec./platelet, Kd = 5.6 +/- 2.0 nmol/L). Platelet activation with strong agonists (thrombin, arachidonic acid) increased 2- to 10-fold the r-apo(a) binding, without affecting the affinity. Competition assays showed that the binding sites are highly specific for r-apo(a) and Lp(a). At high concentration t-PA could also bind to the r-apo(a) binding sites. By contrast, neither fibrinogen nor plasminogen inhibited to the r-apo(a) binding. The lysine analogue EACA inhibits the binding of r-apo(a) to platelets, thus suggesting the involvement of lysine residues in that interaction. Moreover, the r-apo(a) binding to platelets is unlikely mediated by GPIIb/IIIa-attached fibrin since it is not affected by platelet treatment with either LJ-CP8, a monoclonal antibody that specifically blocks fibrinogen binding to GPIIb/IIIa, nor GPRP, an inhibitor of fibrin polymerisation. Finally, we show that the distinct recombinant apo(a) proteins, as well as native Lp(a), promote an aggregation response of platelets to otherwise subaggregant doses of arachidonic acid. This proaggregant effect of r-apo(a) is dependent on its binding to platelets since it requires a minimum incubation time, and it is prevented by EACA at concentration inhibiting the r-apo(a)-platelet interaction. These results suggest that the prothrombotic action of Lp(a) may be in part mediated by modulating the platelet function through the interaction of its apo(a) subunit with a specific receptor at the platelet surface.

Effect of individual plasma lipoprotein(a) variations in vivo on its competition with plasminogen for fibrin and cell binding: An in vitro study using plasma from children with idiopathic nephrotic syndrome.

Simultaneous natural changes in lipoprotein(a) [Lp(a)] and plasminogen occur in the nephrotic syndrome and offer a unique opportunity to investigate their effects on plasminogen activation under conditions fashioned in vivo. Plasminogen, Lp(a), and apolipoprotein(a) in plasma were characterized, and their competitive binding to carboxy-terminal lysine residues of fibrin and cell membrane proteins was determined in nephrotic children during a flare-up of the disease (61 cases) and after 6 weeks (33 cases) and 6 months (42 cases) of remission. Low plasminogen concentrations (median 1.34 micromol/L, range 0.39 to 1.96 micromol/L) and high Lp(a) levels (median 0.27 g/L, range 0.07 to 2. 57 g/L) were detected at flare-up. These changes were associated with an increased Lp(a) binding ratio onto fibrin (3.13+/-0.48) and cells (1.53+/-0.24) compared with binding ratios of control children (1.31+/-0.19 and 1.05+/-0.07, respectively) with normal plasminogen and low Lp(a) (median 0.071 g/L). After 6 weeks and 6 months of remission, the values for net decrease in Lp(a) binding to fibrin were 1.7+/-0.22 (after 6 weeks) and 1.88+/-0.38 (after 6 months) and were correlated with low Lp(a) concentrations (median 0.2 g/L, range 0.07 to 0.8 g/L; and median 0.12 g/L, range 0.07 to 1.34 g/L) and inversely associated with increased plasminogen levels (median 1.82 micromol/L, range 1.4 to 2.1 micromol/L; and median 1.58 micromol/L, range 1.1 to 2.1 micromol/L). These studies provide the first quantitative evidence that binding of Lp(a) to lysine residues of fibrin and cell surfaces is directly related to circulating levels of both plasminogen and Lp(a) and that these glycoproteins may interact as competitive ligands for these biological surfaces in vivo. This mechanism may be of relevance to the atherothrombotic role of Lp(a), particularly in nephrotic patients.

Evidence that modifications of Lp(a) in vivo inhibit plasmin formation on fibrin--a study with individual plasmas presenting natural variations of Lp(a).

In the present study we have investigated the effect of individual variations in the concentration of Lp(a) on plasmin formation at the surface of fibrin. The plasma Lp(a) concentrations from 20 nephrotic children were high at flare-up of the disease (0.43+/-0.45 g/l) and decreased progressively with remission at both 6 weeks (0.28+/-0.24 g/l) and 6 months (0.24+/-0.288 g/l). In contrast, the concentration of plasminogen showed an inverse variation, with low values at flare-up (1.27+/-0.34 microM) and normal values at remission (1.66+/-0.17 microM at 6 weeks and 1.99+/-0.21 microM at 6 months). An increase in plasmin formation (from 0.62+/-0.49 to 0.73+/-0.61, and 0.84+/-0.75 pmol/well) and a decrease in apo(a) binding (from 5.45+/-2.42 to 4.54+/-2.12, and 3.93+/-1.51 fmol/well) on the surface of fibrin, were concomitantly observed from flare-up to remission at 6 weeks and at 6 months, respectively. Values for plasmin formation parallel the amount of plasminogen bound. The low concentration of plasminogen found at flare-up may also have contributed to the increased binding of Lp(a) as indicated by a decrease in the maximal amount of Lp(a) bound (Bmax) to fibrin as a function of plasma plasminogen concentrations. Bmax was 1.51 fmol in the absence of plasminogen and decreased to 1.1 fmol and 0.93 fmol at respectively 1 and 2 microM of plasminogen. Altogether, these data constitute the first quantitative evidence indicating that plasmin formed at the surface of fibrin may vary with modifications of the concentration of Lp(a) in vivo.

A novel kringle-4 number-based recombinant apo[a] standard for human apo[a] phenotyping.

Apolipoprotein[a] phenotyping is a critically important method to explore the role of kringle-4 repeat number as a modulator of lipoprotein[a]-associated cardiovascular risk. The availability of a kringle-4 number-based reference standard is therefore necessary for a reliable and generally accepted classification of apo[a] phenotypes. We propose here a battery of recombinant apo[a] isoforms that may be used as the reference standard in various gel systems. Five plasmids encoding for r-apo[a] containing a known number (n = 9, 13, 17, 25, 33) of plasminogen-like kringle-4 copies were constructed, and transfected into the human embryonic kidney cell line 293. The electrophoretic mobility of the recombinant apo[a] isoforms expressed by these cells in a hollow-fiber bioreactor was determined after reduction by SDS-gel (agarose, acrylamide or a mixture of both) electrophoresis and immunoblotting using an antibody specific for human apo[a]. The equation of the linear relationship between log r-apo[a] kringle number and relative migration was used to determine the isoform size of apo[a] in normal human plasma. A very good correlation (r = 0.97) was found with the genotype (pulsed-field gel eletrophoresis of kpnI-digested restriction fragments of genomic DNA) and among electrophoretic methods. The proposed recombinant standard offers the possibility to identify apo[a] isoforms within a large range of molecular sizes, 9 to 33 kringle-4 copies, using simple electrophoretic techniques and a nomenclature based on its molecular structure, i.e., the number of kringle-4 repeats.-Anglés-Cano, E., S. Loyau, G. Cardoso-Saldaña, R. Couderc, and P. Gillery. A novel kringle-4 number-based recombinant apo[a] standard for human apo[a] phenotyping.

Fibrino(geno)lytic properties of purified hementerin, a metalloproteinase from the leech Haementeria depressa.

The fibrino(geno)lytic protein designated hementerin contained in crude extracts of the salivary complex of Haementeria depressa leeches was purified to apparent homogeneity by gel filtration, ion exchange chromatography and preparative SDS-PAGE. It is a single-chain 80 kDa, PhMeSO2F-resistant, calcium-dependent, metalloproteinase, which specifically degrades fibrin(ogen) through a plasminogen-independent pathway. The amino terminal sequence of 8 residues shows 80% similarity with hementin, another fibrino(geno)lytic protein purified from Haementeria ghilianii leeches. However, their activities differ somewhat in terms of kinetics and with regard to the structure of the fibrin(ogen) fragments they may produce. Cleavage by hementerin of fibrinogen Aalpha, gamma and Bbeta chains, in that order, produces 270 kDa to 67 kDa fragments which differ from those produced by plasmin. Hementerin was also able to degrade cross-linked fibrin although at a lower rate as compared to fibrinogen. In conclusion, hementerin is a plasminogen-independent fibrino(geno)lytic metalloproteinase that degrades fibrinogen faster than fibrin, prevents blood coagulation and destroys fibrin clots in vitro.

Antibodies to fibrin-bound tissue-type plasminogen activator in systemic lupus erythematosus are associated with Raynaud's phenomenon and thrombosis.

Fibrinolysis triggered by t-PA bound to fibrin is one of the main antithrombotic mechanisms. Defects in the fibrinolytic system-decreased tissue-type plasminogen activator (t-PA) activity and elevated levels of plasminogen activator inhibitor (PAI-1), in patients with SLE have been associated with an increased tendency to thrombosis. In the present study, 43 patients with SLE fulfilling the ACR criteria for the disease, were studied for the presence of autoantibodies to fibrin-bound t-PA, i.e. the physiological active form of this plasminogen activator. A solution of 200 IU/ml of t-PA was incubated with solid-phase fibrin prepared as previously described (Anal Biochem 1986; 153; 201-210). Sera diluted 1:50 were incubated with fibrin-bound t-PA, the plates were then washed, and bound immunoglobulins were detected using a polyvalent peroxidase-labeled goat anti-human Ig. Plates coated with fibrin alone were used as controls. Sera were considered positive when A490/630 obtained with normal human sera in two independent test was greater than the mean plus 2 SD. Eleven of 43 (26%) SLE sera demonstrated antibody reactivity against fibrin-bound t-PA. Within the anti-t-PA positive group there was a higher proportion of SLE patients with severe Raynaud's phenomenon and thrombotic events when compared to the anti-t-PA negative group: 36% vs 6% and 18% vs 6% respectively. These results suggest that autoantibodies to fibrin-bound t-PA could play a role in the pathogenesis of vascular disease in some SLE patients.

Antibodies to fibrin bound tissue type plasminogen activator in systemic sclerosis.

OBJECTIVE: Abnormalities of tissue type plasminogen activator (tPA) and plasminogen activator inhibitor have been described in some patients with systemic sclerosis (SSc). We studied 128 unselected SSc sera for the presence of autoantibodies to fibrin bound tPA. METHODS: A solid phase fibrin-tPA immunoassay utilized 500 IU/ml tPA bound to solid phase fibrin. Sera diluted 1/50 were incubated with the fibrin bound tPA, the plates were washed, and bound immunoglobulins were detected using polyvalent peroxidase labelled goat antihuman immunoglobulins. Controls included plates coated with fibrin alone or tPA passively adsorbed to the plastic. Sera were considered positive when the A490/630 was above the mean + 2 SD (> 0.055) obtained with normal human serum in 2 independent tests. RESULTS: 25/128 (20%) SSc sera demonstrated antibody reactivity with fibrin bound tPA (mean A490/630 = 0.112). Detailed clinical data were available on 117/128 patients with SSc and on 21/25 anti-tPA positive patients. The mean age of the anti-tPA positive group was 51 yrs and of the anti-tPA negative group 49.6 yrs. Within the anti-tPA positive group there was a significantly higher proportion (p > 0.05) of patients with the CREST (calcinosis, Raynaud's esophageal dysmotility, sclerodactyly, telangiectasias) variant of SSc (7/25 = 28% vs 11/103 = 11%) and pulmonary hypertension (5/21 = 24% vs 6/96 = 6%). CONCLUSION: Our study demonstrates that 20% of unselected patients with SSc have anti-tPA antibodies and that there is a higher representation of patients with CREST syndrome in this subgroup. The high frequency of pulmonary hypertension in the anti-tPA positive group suggests that these autoantibodies may play a pathogenic role in certain patients with SSc.

Molecular assembly of plasminogen and tissue-type plasminogen activator on an evolving fibrin surface.

A well characterized model of an intact and a degraded surface of fibrin that represents the states of fibrin during the initiation and the progression of fibrinolysis was used to quantitatively characterize the molecular interplay between tissue-type plasminogen activator (t-PA), plasminogen and fibrin. The molecular assembly of t-PA and plasminogen on these surfaces was investigated using combinations of proteins that preclude complications due to side reactions caused by generated plasmin: native plasminogen with di-isopropylphosphofluoridate-inactivated t-PA, and a recombinant human plasminogen with the active-site Ser741 mutagenized to Ala which renders the catalytic site inactive. Under these conditions, neither the affinity nor the maximal number of binding sites for plasminogen were modified by the presence of t-PA, indicating that binding sites for plasminogen pre-exist in intact fibrin and are not dependent on the presence of t-PA. In contrast, when plasminogen activation is allowed, increasing binding of plasminogen to the progressively degraded fibrin surface is directly correlated (r = 0.98) to the appearance of the fibrin E-fragment as shown using a monoclonal antibody (FDP-14) that has its epitope in the E domain of fibrin. t-PA was shown to bind with a high affinity to both the intact (Kd = 3.3 +/- 0.6 nM) and the degraded surface of fibrin (Kd = 1.2 +/- 0.4 nM). Binding of t-PA to carboxy-terminal lysine residues of degraded fibrin was shown to be efficiently competed by physiological concentrations of plasminogen (2 microM), indicating that the affinity of t-PA for these residues was lower than that of plasminogen (Kd = 0.66 +/- 0.22 microM) and unrelated to the high affinity of t-PA for specific binding sites on intact fibrin. These data confirm and establish that the generation of carboxy-terminal lysine residues on fibrin during ongoing fibrinolysis, and the binding of plasminogen to these sites, is an important pathway in the acceleration of clot dissolution.

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.

Familial association of high levels of histidine-rich glycoprotein and plasminogen activator inhibitor-1 with venous thromboembolism.

High levels of histidine-rich glycoprotein (HRGP) and plasminogen activator inhibitor-1 (PAI-1) have been claimed to contribute to the hypofibrinolytic state observed in patients with venous thrombosis. These abnormalities were detected, respectively, in eight and 10 members of a family from which four of seven members with both abnormalities had venous thromboembolism. Binding of tissue plasminogen activator (t-PA) by PAI-1 may induce hypofibrinolysis. To determine whether plasminogen binding by HRGP may influence plasminogen activation, we studied the fibrinolytic activity of members of this family cohort with a system that detects modifications in plasmin generation by proteins interfering with the binding of plasminogen to fibrin. Plasminogen activation was performed by adding plasma to fibrin surfaces to which t-PA had been previously bound in the presence of 40 mg/ml bovine serum albumin and 20 mumol/L of the lysine analog trans-4-(aminomethyl)-cyclohexane carboxylic acid to prevent nonspecific binding and thereby the inhibitory effect of elevated PAI-1 levels. The generation of plasmin as a function of time was detected (1) by photometric analysis with a chromogenic substrate highly selective for plasmin and (2) by measuring the binding and activation of plasminogen at the fibrin surface with radiolabeled plasminogen. The amount of plasmin generated by plasma from patients having high levels of HRGP (160% to 280%) was similar to that of a control group having normal levels of HRGP (100% +/- 22%). Similar results were obtained with a plasma artificially depleted in HRGP and supplemented with various amounts of this protein. No correlation between HRGP level and t-PA-mediated plasminogen activation was observed.(ABSTRACT TRUNCATED AT 250 WORDS)