Fibrinogen Fragments Research Articles

Inhibition of coaggregation between Porphyromonas gingivalis and Streptococcus oralis by fibrinogen fragments.

The localization of regions of fibrinogen that inhibit coaggregation between Porphyromonas gingivalis and Streptococcus oralis was investigated. The coaggregation was inhibited by A alpha and gamma chains, but not by B beta chain. The inhibitory activity of fragment D was more potent than that of fragment E. Some cyanogen bromide-treated fragments isolated from A alpha and gamma chains including the NH2-terminal 148-207 amino acid residues of A alpha chain (A alpha 148-207) and gamma 1-78 showed inhibitory activities. A alpha 148-207 was further digested with lysyl endopeptidase. A alpha 158-176 and A alpha 192-206 which contained four and two arginine residues, respectively, retained the inhibitory activities. When the arginine residues of these two peptides were modified by phenylglyoxal, the inhibitory activities were much reduced. These findings suggest that the arginine residues of some specific regions of fibrinogen may play an important role in the inhibition of the coaggregation.

Extrahepatic expression of fibrinogen by granulosa cells: potential role in ovulation.

Granulosa cells from ovarian follicles were shown to express and secrete fibrinogen under the control of FSH. Conditioned medium was collected from granulosa cell cultures and found to contain FSH-dependent 50-kilodalton (kDa) and 93- to 95-kDa proteins. N-Terminal microsequence analysis identified these proteins as fibrinogen beta- and gamma-chains, respectively. Proteins migrating at 93 and 95 kDa contain identical gamma-chain sequences at the N-terminal, suggesting differential processing of fibrinogen. These fibrinogen chains were specifically detected with antifibrinogen antibodies in immunoblot and immuno-precipitation analysis. Fibrinogen gamma-chain mRNA was detected in granulosa cells by polymerase chain reaction analysis, confirming fibrinogen gene expression by these cells. Fibrinogen secretion by granulosa cells was measured by a competitive enzyme-linked immunosorbent assay. Granulosa cells treated with FSH (100 ng/ml) secreted 2-3 times more fibrinogen than untreated cells. These data show that fibrinogen, a major product of the liver, is also a secretory product of granulosa cells. This provides a novel extrahepatic site of fibrinogen expression. As hepatic parenchymal cells normally maintain high circulating levels of fibrinogen, the local production of fibrinogen in the ovary is anticipated to have specialized functions. Locally produced fibrinogen may be important in the clotting process following tissue rupture at ovulation. In addition, fibrinogen fragments may be involved in the mechanism of ovulation by increasing the activity of tissue-type plasminogen activator to control the proteolytic activity required for ovulation.

Extrahepatic expression of fibrinogen by granulosa cells: potential role in ovulation

Granulosa cells from ovarian follicles were shown to express and secrete fibrinogen under the control of FSH. Conditioned medium was collected from granulosa cell cultures and found to contain FSH-dependent 50-kilodalton (kDa) and 93- to 95-kDa proteins. N-Terminal microsequence analysis identified these proteins as fibrinogen beta- and gamma-chains, respectively. Proteins migrating at 93 and 95 kDa contain identical gamma-chain sequences at the N-terminal, suggesting differential processing of fibrinogen. These fibrinogen chains were specifically detected with antifibrinogen antibodies in immunoblot and immuno-precipitation analysis. Fibrinogen gamma-chain mRNA was detected in granulosa cells by polymerase chain reaction analysis, confirming fibrinogen gene expression by these cells. Fibrinogen secretion by granulosa cells was measured by a competitive enzyme-linked immunosorbent assay. Granulosa cells treated with FSH (100 ng/ml) secreted 2-3 times more fibrinogen than untreated cells. These data show that fibrinogen, a major product of the liver, is also a secretory product of granulosa cells. This provides a novel extrahepatic site of fibrinogen expression. As hepatic parenchymal cells normally maintain high circulating levels of fibrinogen, the local production of fibrinogen in the ovary is anticipated to have specialized functions. Locally produced fibrinogen may be important in the clotting process following tissue rupture at ovulation. In addition, fibrinogen fragments may be involved in the mechanism of ovulation by increasing the activity of tissue-type plasminogen activator to control the proteolytic activity required for ovulation.

Introduction of lysine and clot binding properties in the kringle one domain of tissue-type plasminogen activator.

Despite the high overall similarity in primary structure between kringle one (K1) and kringle two (K2) of tissue-type plasminogen activator (t-PA) there exists an enormous functional difference. It is thought that, in contrast to K1, K2 mediates lysine binding and fibrin binding and is involved in stimulation of plasminogen activation by fibrin or derivatives as CNBr fragments of fibrinogen. Hypothesizing that sequence differences are responsible for differences in function, we compared the amino acid sequences of K1 and K2 with a consensus kringle sequence. Six consecutive amino acids unique to K2 of t-PA were found, i.e. from Asn248 to Trp253. To test whether these residues are involved in lysine binding, fibrin binding, and fibrin-dependent plasminogen activation, we constructed a set of t-PA mutant proteins containing only a kringle and the protease (P) domain: K2P, K1P, and k1P. In the latter molecule the original amino acid residues Ala160-Ser165 from K1 were substituted by Asn248-Trp253 from K2. As expected, K2P showed enhanced plasminogen activation in the presence of CNBr fragments of fibrinogen, bound to lysine-Sepharose and to a forming fibrin clot. K1P did not show any of these features. In contrast, k1P could be stimulated by CNBr fragments of fibrinogen and bound to lysine-Sepharose and a forming fibrin clot. These results indicate that at least a part of the functional differences between K1 and K2 of t-PA can be localized to a stretch of 6 amino acid residues from Asn248 to Trp253 present in K2.

Fibrinogen potentiates the effect of interleukin-3 on early human hematopoietic progenitors

The effect of human fibrinogen on the proliferation of purified SBA- CD34+ human bone marrow progenitors was investigated in clonal cultures. Fibrinogen alone or in combination with erythropoietin had no significant effect. However, in the presence of recombinant human interleukin-3 (IL-3), fibrinogen increased significantly in a dose- dependent manner the number of mixed and burst-forming unit-ethrocyte-- derived colonies, whereas the number of other colonies did not significantly change. In the presence of fibrinogen, low concentrations of IL-3 (0.17 U/mL) produced three times more mixed colonies than without fibrinogen, reaching the number of colonies obtained with optimal concentrations of IL-3 (1.67 U/mL). Fibrinogen fragment D had the same effect in the presence of IL-3 as intact fibrinogen, whereas fibrinogen fragment E and human collagen IV did not. This effect was not mediated by integrins, because peptides or monoclonal antibodies that block fibrinogen binding on integrins alpha IIb beta 3, alpha v beta 3 (RGD-peptides), alpha m beta 2 (OKM-1), and alpha x beta 2 (HC1/1) did not affect the observed mitogenic effect. The mitogenic effect of fibrinogen and its D fragment was not mediated by induction of IL-6 or granulocyte--colony-stimulating factor secretion, because it was not inhibited by blocking antisera against these two growth factors. Our results indicate that fibrinogen potentiates the effect of IL-3 on primitive hematopoietic progenitors and suggest that the mitogenic effect of fibrinogen could be mediated via a specific mitogenic receptor that does not belong to the integrin family.

Fibrinogen Potentiates the Effect of Interleukin-3 on Early Human Hematopoietic Progenitors

The effect of human fibrinogen on the proliferation of purified SBA- CD34+ human bone marrow progenitors was investigated in clonal cultures. Fibrinogen alone or in combination with erythropoietin had no significant effect. However, in the presence of recombinant human interleukin-3 (IL-3), fibrinogen increased significantly in a dose-dependent manner the number of mixed and burst-forming unit-ethrocyte--derived colonies, whereas the number of other colonies did not significantly change. In the presence of fibrinogen, low concentrations of IL-3 (0.17 U/mL) produced three times more mixed colonies than without fibrinogen, reaching the number of colonies obtained with optimal concentrations of IL-3 (1.67 U/mL). Fibrinogen fragment D had the same effect in the presence of IL-3 as intact fibrinogen, whereas fibrinogen fragment E and human collagen IV did not. This effect was not mediated by integrins, because peptides or monoclonal antibodies that block fibrinogen binding on integrins alpha IIb beta 3, alpha v beta 3 (RGD-peptides), alpha m beta 2 (OKM-1), and alpha x beta 2 (HC1/1) did not affect the observed mitogenic effect. The mitogenic effect of fibrinogen and its D fragment was not mediated by induction of IL-6 or granulocyte--colony-stimulating factor secretion, because it was not inhibited by blocking antisera against these two growth factors. Our results indicate that fibrinogen potentiates the effect of IL-3 on primitive hematopoietic progenitors and suggest that the mitogenic effect of fibrinogen could be mediated via a specific mitogenic receptor that does not belong to the integrin family.

Stimulation of tissue type plasminogen activator by leukaemic cell homogenates.

In patients with disseminated intravascular coagulation (DIC), hyperfibrinolysis was observed in patients with leukaemia, but hypofibrinolysis was seen in those with sepsis. Although the plasma tissue plasminogen activator (t-PA) level was higher in patients with DIC than in those without DIC, there was no significant difference in t-PA level between the patients with leukaemia and sepsis. Hyperfibrinolysis might not be caused by t-PA derived from leukaemic cells, although the PA antigen level in leukaemic cell homogenates was significantly higher in patients with DIC than in those without DIC. The activation of t-PA by leukaemic cell homogenates in the absence of bromocyan fibrinogen fragments suggested that leukaemic cell homogenates had t-PA stimulator activity. The t-PA stimulator activity was high in both acute myeloblastic leukaemia (AML) and acute lymphoblastic leukaemia (ALL), especially in DIC, but this activity was not detected in chronic myelocytic leukaemia (CML) or normal cells. Since fibrinogen and soluble fibrin monomer complex levels in leukaemic cells were also high in patients with DIC, fibrinogen degradation products might be the major t-PA stimulator in leukaemic cells. This might be one of the causes of hyperfibrinolysis in leukaemia.

Adhesive ligand binding to integrin alpha IIb beta 3 stimulates tyrosine phosphorylation of novel protein substrates before phosphorylation of pp125FAK.

Tyrosine phosphorylation of multiple platelet proteins is stimulated by thrombin and other agonists that cause platelet aggregation and secretion. The phosphorylation of a subset of these proteins, including a protein tyrosine kinase, pp125FAK, is dependent on the platelet aggregation that follows fibrinogen binding to integrin alpha IIb beta 3. In this report, we examined whether fibrinogen binding, per se, triggers a process of tyrosine phosphorylation in the absence of exogenous agonists. Binding of soluble fibrinogen was induced with Fab fragments of an anti-beta 3 antibody (anti-LIBS6) that directly exposes the fibrinogen binding site in alpha IIb beta3. Proteins of 50-68 KD and 140 kD became phosphorylated on tyrosine residues in a fibrinogen-dependent manner. This response did not require prostaglandin synthesis, an increase in cytosolic free calcium, platelet aggregation or granule secretion, nor was it associated with tyrosine phosphorylation of pp125FAK. Tyrosine phosphorylation of the 50-68-kD and 140-kD proteins was also observed when (a) fibrinogen binding was stimulated by agonists such as epinephrine, ADP, or thrombin instead of by anti-LIBS6; (b) fragment X, a dimeric plasmin-derived fragment of fibrinogen was used instead of fibrinogen; or (c) alpha IIb beta 3 complexes were cross-linked by antibodies, even in the absence of fibrinogen. In contrast, no tyrosine phosphorylation was observed when the ligand consisted of monomeric cell recognition peptides derived from fibrinogen (RGDS or gamma 400-411). Fibrinogen-dependent tyrosine phosphorylation was inhibited by cytochalasin D. These studies demonstrate that fibrinogen binding to alpha IIb beta 3 initiates a process of tyrosine phosphorylation that precedes platelet aggregation and the phosphorylation of pp125FAK. This reaction may depend on the oligomerization of integrin receptors and on the state of actin polymerization, organizational processes that may juxtapose tyrosine kinases with their substrates.

Anticoagulant Function of a 24-Kd Fragment Isolated From Human Fibrinogen Aα Chains

AbstractA fibrinogen fragment obtained by limited-plasmin proteolysis has been isolated and purified to apparent homogeneity by gel filtrations. This fragment, denoted as 24-Kd fragment, has an apparent Mr ≈24,000 and contains an N-terminal sequence of met-glu-leu-glu-arg-pro-gly-gly-asn-glu-ile. The fragment contains a large number of acidic amino acid residues, and its amino acid composition is similar to several fibrinogen Aα chains degradation fragments isolated previously. It corresponds to a peptide of the fibrinogen Aα chains, the N-terminal of which starts at α Met-240. This peptide delays thrombin plasma clotting time. It does not bind calcium ions and does not inhibit thrombin’s amidolytic activity. It binds to immobilized fibrin but not fibrinogen. It also inhibits the polymerization of desAA and desAABB fibrin monomers by simultaneously decreasing the maximum rate and the maximum level of the polymerization reaction. However, the initial lag period of this reaction is not affected by the fragment.

Anticoagulant function of a 24-Kd fragment isolated from human fibrinogen A alpha chains

A fibrinogen fragment obtained by limited-plasmin proteolysis has been isolated and purified to apparent homogeneity by gel filtrations. This fragment, denoted as 24-Kd fragment, has an apparent M(r) approximately 24,000 and contains an N-terminal sequence of met-glu-leu-glu-arg-pro- gly-gly-asn-glu-ile. The fragment contains a large number of acidic amino acid residues, and its amino acid composition is similar to several fibrinogen A alpha chains degradation fragments isolated previously. It corresponds to a peptide of the fibrinogen A alpha chains, the N-terminal of which starts at alpha Met-240. This peptide delays thrombin plasma clotting time. It does not bind calcium ions and does not inhibit thrombin's amidolytic activity. It binds to immobilized fibrin but not fibrinogen. It also inhibits the polymerization of desAA and desAABB fibrin monomers by simultaneously decreasing the maximum rate and the maximum level of the polymerization reaction. However, the initial lag period of this reaction is not affected by the fragment.

Comparison of the sequence of fibrinopeptide a cleavage from fibrinogen fragment e by thrombin, atroxin, or batroxobin

In order to investigate the sequence of fibrinopeptide release from the amino terminal end of a dimeric fibrinogen-derived substrate by thrombin or batroxobins, we studied their effects on plasmic fragment E 1, a core fragment from the central domain of fibrinogen containing both Aα chain fibrinopeptide A (FPA) sequences. Isoelectric focussing (IEF) was employed as a means of resolving des A-fragment E 1, from which one FPA had been cleaved, from des AA-fragment E 1 resulting from the loss of both FPA's. Using densitometric gel scanning for quantification of the levels of intact fragment E 1, des A-fragment E 1, and des AA-fragment E 1, in mixtures incubated with enzyme for various periods of time, we found similar catalytic rate constants (k 1, k 2) for release of the first fibrinopeptide A, (FPA 1) or the second, (FPA 2) from fragment E 1, with either thrombin or batroxobin (k 2 : k 1 ratios of 1.10 ± 0.42, 1.34 ± 0.26 respectively). Atroxin released FPA 2 more slowly than FPA 1 with a k 2 : k 1 ratio of 0.34 ± 0.1. Th finding that the cleavage of FPA 2 by Atroxin is three-fold slower than thrombin and almost four-fold slower than batroxobin, suggest that batroxobin and thrombin cleavage of FPA 2 may be cooperative in nature. However, the cooperativity in the cleavage sequence is insufficient to markedly suppress the evolution of intermediate des A fragment E species during early and intermediate phases of FPA cleavage from fragment E.

The Construction and Expression of Chimeric Urokinase-Type Plasminogen Activator Genes Containing Kringle Domains of Human Plasminogen

A series of chimeric urokinase-type plasminogen activator (uPA) genes, which contain combinations of kringle domains of human plasminogen (HPg) in place of the uPA kringle (KuPA), has been constructed and expressed. Some of the resulting recombinant (r) variant uPA chimeras contain modules that potentially mediate the macroscopic binding of HPg to its activation effectors, fibrin(ogen) and 6-aminohexanoic acid (EACA). Such binding sites are not possessed by KuPA, but are present in certain of the HPg kringles, viz., kringle 1 (K1HPg), kringle 4 (K4HPg), and kringle 5 (K5HPg). The recombinant (r) chimeras constructed included molecules with replacements of KuPA with K1HPg (r-[KuPA → K1HPg]uPA), and with KuPA replaced by double kringle combinations of K1HPgK4HPg (r-[KuPA → K1HPgK4HPg]uPA), K2HPgK3HPg (r-[KuPA → K2HPgK3HPg]uPA), and K4HPgK5HPg (r[KuPA → K4HPgK5HPg]uPA). All of these variant genes, along with their wild-type (wt) r-uPA counterparts, were expressed in human kidney 293 cells. In cases wherein EACA-binding kringles from HPg have been placed in uPA, this property has been retained in the chimeric molecule and employed as an essential part of the purification procedures for the variants. The steady state amidolytic activity of two-chain (tc) wtr-uPA toward the chromogenic substrate, H-D-pyroglutamyl-Gly-L-Arg-p-nitroanilide (S2444), is characterized by a kcat/KM (pH 7.4, 37°C) of 120 s−1 mM−1. This value ranges from 92 s−1 mM−1 (tcr-[KuPA → K1HPg]uPA) to 166 s−1 mM−1 (tcr[KuPA → K1HPgK4HPg]uPA) for each of the variants, demonstrating that the catalytic efficiency of the active site is altered only in a small way by changes in the noncatalytic domain of uPA. Small differences are also observed in the abilities of these tcr variants to interact with the fast-acting plasma inhibitor of uPA, viz., plasminogen activator inhibitor-1 (PAI-1). The second-order rate constant for the interaction of PAI-1 with tcr-uPA, 0.46 × 107 M−1s−1 (pH 7.4, 10°C), ranges from 0.29 × 107 M−1s−1 (tcr-[KuPA → K1HPgK4HPg]uPA) to 1.08 × 107 M−1s−1 (tcr-[KuPA → K4HPgK5HPg]uPA), for the tcr-chimeric variants. Neither wtr-uPA nor any of its chimeric r-variants interacted macroscopically with a fibrin clot under conditions that allowed binding of 74% of single-chain r-tissue-type plasminogen activator. However, the tcr-chimeric uPA variants provided HPg-enriched clot lysis times between 0.2 (r-[KuPA → K1HPgK4HPg]uPA) and 2.4 (r-[KuPA → K2HPgK3HPg]uPA) relative to that of wtr-uPA. These results demonstrate that no perceptible relationship exists between macroscopic fibrin binding and the clot lytic properties of the variants. In addition, under conditions wherein the HPg activation by tissue-type plasminogen activator was significantly stimulated by fibrinogen, fibrin, and CNBr fragments of fibrinogen, this same activation was not similarly stimulated by these effectors when wtr-uPA or any of the chimeric variants were employed as the activators. These results demonstrate that despite the retention of the EACA binding properties of K1HPg, K1HPgK4HPg, and K4HPgK5HPg, a factor that supports the independent domainal character of HPg kringle domains, neither fibrin-binding capabilities nor fibrin-selectivity for HPg activation was engineered into uPA when the domains were inserted into uPA in place of KuPA.

Localization of a fibrin polymerization site complementary to Gly-His-Arg sequence

Dansyl-labeled tetrapeptide Gly-His-Arg-Pro which mimics the central fibrin polymerization site was used to investigate its binding to a number of fibrinogen fragments containing different numbers of domains. The tetrapeptide was found to bind to fragments D H(95 kDa), D L(82 kDa) and D Y(63 kDa) but not to the TSD(28 kDa) fragment. The D Y fragment differs from the TSD by the presence of β and βC domains. Therefore these domains, which are formed by the C-terminal part of the β chain, possess a polymerization site complementary to the Gly-His-Arg containing counterpart.

The cleavage sequence of fibrinopeptide A from fibrinogen fragment E by thrombin, atroxin or batroxobin

The cleavage sequence of fibrinopeptide A from fibrinogen fragment E by thrombin, atroxin or batroxobin

The cleavage sequence of fibrinopeptide A from fibrinogen fragment E by thrombin, atroxin or batroxobin

Calculations of data from fibrin polymerization and cross-linking experiments infer that thrombin-catalysed release of the second of the two fibrinopeptides A (FpA2) from fibrinogen is concerted, although other data suggest that FpA2 release is random. In the concerted pattern of FpA release, divalent monomer (des AA-fibrin) formation predominates throughout the enzymatic conversion of fibrinogen to fibrin, an effect leading to relatively rapid fibril assembly. Alternatively, random FpA2 release would result in a substantial population of monovalent monomer (des A-fibrin) intermediates during early and intermediate phases of the enzymatic conversion to fibrin. Their formation would cause a delay in fibrin fibril assembly. In order to address the question of the pattern of FpA release directly, we purified plasmic fibrinogen fragment E1 isoforms containing both FpA sequences and studied the sequence of FpA release by thrombin or batroxobin. Des A-fragment E1 intermediates formed by loss of one FpA (FpA1), and des AA-fragment E1 products (lacking both FpA1 and FpA2) were identified by analytical isoelectric focusing and quantified by densitometry. The catalytic rate of release of FpA1 (k1) and FpA2 (k2) by thrombin or batroxobin was similar. The ratio of these rates, k2:k1, was 1.10 +/- 0.42 for thrombin and 1.34 +/- 0.26 for batroxobin. These findings indicate that these enzymes cleave FpA2 randomly from fragment E1.

Binding of a new monoclonal antibody against N-terminal heptapeptide of fibrin α-chain to fibrin polymerization site ‘A’

A novel murine monoclonal antibody against the fibrin alpha-chain N-terminus is presented, which reacts with desAA- and desAABB-fibrin. In immunoblot procedures, the antibody reacted with fibrin degradation products X and Y of non-crosslinked fibrin, and fragment E. No binding was observed to the fibrin fragment D-dimer, and fibrinogen fragments D and E. Minor binding to fibrinogen fragments X, and Y, and desBB-fibrin were presumably due to minor contamination with (desAA)-fibrin. A prerequisite for binding was release of fibrinopeptides A (FpA), the binding site being a fibrin-specific neo-epitope. No binding was observed to fibrinogen or to thrombin-treated dysfibrinogen MANNHEIM III (A alpha 16 Arg-->Cys) molecules, which do not release FpA. The antibody bound to abnormal fibrin molecules prepared from dysfibrinogen MANNHEIM I (A alpha 19 Arg-->Gly), albeit to a lesser extent than to normal fibrin. Binding of the antibody to the fibrin epitope was greatly enhanced by denaturation, e.g. by heat, or by treatment with chaotropic ions. Soluble fibrin in clinical samples is generally found as a complex with fibrinogen, since polymerization sites 'A' exposed by release of FpA react with complementary binding sites 'a' on the D-domains of other fibrin and fibrinogen molecules. Treatment of samples with NaSCN caused dissociation of fibrin monomer complexes. Reassociation was prevented by denaturation of both polymerization sites 'A' and 'a'. The antibody in combination with NaSCN-treatment of samples was useful for specific detection of fibrin monomer in plasma samples. Measurement was not influenced by fibrinogen degradation products, whereas fibrin degradation products at very high concentration caused some underestimation of fibrin monomer concentration.

Characterization of fibronectin and fibrin(ogen) fragments in gingival crevicular fluid.

A total of 49 crevicular fluid (CF) samples were collected with paper strips from 12 healthy adults. Each sample was eluted into sterile saline and two aliquots were drawn for SDS-PAGE, one for fibronectin and one for fibrin analysis. Peptides were transferred to nitrocellulose membranes, and fibronectin and fibrin were detected using specific antibodies. The relative amounts of different molecular forms of fibronectin and fibrin were analyzed using a laser densitometer. After the sample collection, Plaque Index, Papilla Bleeding Index and pocket depth were measured. Bone loss was estimated from the orthopanthomograms. Fibronectin fragments were seen in all CF samples. Intact fibronectin was seen in 21 samples, of which 76% were collected from periodontitis-affected sites. There was a positive correlation between the proportion of intact fibronectin and the clinical parameters. Intact fibrin and fibrin fragments were seen in all samples. Fibrin-positive material with larger molecular weight than intact fibrin was also seen in all samples. A negative correlation was found between the proportion of intact fibrin and the clinical parameters. There was no correlation between total amounts and concentrations of fibronectin and fibrin. Molecular forms of fibronectin and fibrin may affect the pathogenesis and healing of periodontal diseases, since the biologic effects of the fragments of these molecules differ from those of the intact molecules.

17] Lipoprotein (a): Purification and kinetic analysis

Publisher Summary This chapter discusses the purification and kinetic analysis of lipoprotein. Lipoprotein (a) levels in excess of 30 mg/dl are associated with a two-to fivefold increased risk of atherosclerosis. Elevated Lp(a) levels also are linked to stenosis of carotid and cerebral arteries and rethrombosis of venous grafts. Lp(a) downregulates fibrinolysis by competing with Pg for various vascular binding sites, including cellular binding sites, fibrinogen fragments, and heparin. Pg cellular binding sites on endothelial cells, macrophages, and other cells locally concentrate Pg, which is then activated by adjacently bound Pg activators. Lp(a) binds to these sites with an affinity comparable to Pg as a result of the apo(a) kringle-4 domains. Lp(a) displacement of Pg from these cellular binding sites may prevent Pg from interacting with Pg activators, such as tissue-type Pg activator (t-PA) and urinary-type Pg activator (u-PA) and may, thereby depress fibrinolysis. Thus in vitro Lp(a) competes with Pg for various cellular binding sites, inhibits plasmin generation by Pg activators, and promotes plasmin inhibition, but protects Pg activators from inhibition.

Purification and Characterization of Hepatic Plasminogen Activator (h-PA) in the Conditioned Medium of Rat Hepatocytes in Primary Culture

Plasminogen activator (PA) produced by rat hepatocytes (hepatic PA, h-PA) was purified from 5.2 liters of the conditioned medium of primary cultures. The three-step procedure, consisting of a carboxymethyl-Sepharose gel, arginine-Sepharose column, and benzamidine-Sepharose column chromatographies yielded 35 µg of h-PA with a specific activity of 42,800 IU/mg. The molecular mass of the purified h-PA was 70kD, which is similar to that of rat bilokinase (BK), a biliary PA, 70 kD; and to that of human melanoma tissue-type PA (t-PA), 72kD. However, it is quite different from that of high (54 kD) or low (31 kD) molecular weight urinary PA, urokinase (UK). The h-PA had a much higher affinity for fibrin than UK. Unlike UK activity, the h-PA activity was augmented by CNBr-digested fibrinogen fragments (a stimulator). Thus h-PA should be categorized as a tissue-type PA, and may have high efficacy for recanalization of vessels occluded by thrombi. The characteristics of h-PA are almost identical to those of BK, whic...

Identification of proteins absorbed to hemodialyser membranes from heparinized plasma.

The protein layers formed during contact of plasma with hemodialysis membranes were studied. Dialysers having membranes of cellulose acetate (CA), saponified cellulose ester (SCE), cuprophane (CUP), polymethylmethacrylate (PMMA), and polyacrylonitrile (PAN) were used. Heparinized human plasma was recirculated through the dialysers for four hours. They were then rinsed and the proteins adsorbed to the membranes were eluted with 2% SDS. The yields of protein from the different membranes increased in the order: PMMA < CA < SCE < CUP < PAN. This is the probable order of increasing hydrophilicity. SDS-PAGE and Western blots were performed on the dialyser eluates. The blots were positive for most of the twenty proteins tested for. There were some interesting differences in adsorption patterns among the different membrane materials, notably for high molecular weight kininogen (HMWK), plasminogen and the C3 component of complement. HMWK was intact in the eluates from CA, CUP and SCE, whereas on PMMA and PAN there was evidence of cleavage, suggesting that activation of the contact phase of coagulation was more extensive on the latter two materials. Intact plasminogen was visible on all the blots. However, low molecular weight fragments were visible in the PAN eluates, suggesting activation of the fibrinolytic pathway. Low molecular weight fibrinogen fragments eluted from PAN membranes support this conclusion. C3 was visible in the blots obtained for all membrane materials, and the data suggest that complement is activated by all the membranes. A C3 fragment at about 30 kD (possibly C3d) was seen in the blots for the cellulosic membranes but not for PMMA or PAN.