Fibrinopeptide A induces C-reactive protein expression through the ROS-ERK1/2/p38-NF-κB signal pathway in the human umbilical vascular endothelial cells.

Atherosclerosis is a chronic inflammatory disease of the arterial wall. Inflammation causes endothelial injury and dysfunction, which is an initial step of atherosclerosis. Fibrinopeptide A (FPA) is a biomarker of the activation of the coagulation system, and a high concentration of FPA in the blood occurs in patients with ischemic cardiocerebrovascular diseases. The present research observed that FPA stimulated the generation of C-reactive protein (CRP), IL-1ß, and IL-6 in human umbilical vascular endothelial cells (HUVECs); and anti-IL-1 ß and anti-IL-6 neutralizing antibodies did not alter FPA-induced CRP expression in HUVECs. The subchronic administration of FPA into rats increased the plasma FPA and CRP levels. Further studies showed that FPA stimulated superoxide anion generation, activated ERK1/2 and p38, promoted nuclear factor κB (NF-κB) nuclear translocation, and raised the NF-κB level in the nuclei of HUVECs. Antioxidant N-acetylcysteine (NAC), complex II inhibitor thenoyltrifluoroacetone (TTFA), and NADPH oxidase inhibitor diphenyleneiodonium (DPI) inhibited FPA-stimulated generation of superoxide anion, and NAC reduced FPA-induced expressions of the phosphorylated ERK1/2 and p38. NAC, TTFA, DPI, inhibitors of ERK1/2, p38, and NF-κB all downregulated FPA-induced CRP expression. These results indicate that FPA induces CRP expression in HUVECs via the ROS-ERK1/2/p38-NF-κB signal pathway. Moreover, this is the first report that FPA produces a proinflammatory effect on the vascular endothelial cells.

D-Amino Acids in Peptides from Animals, Including Human: Occurrence, Structure, Bioactivity and Pharmacology.

All life forms typically possess homochirality, with rare exceptions. In the case of peptides and proteins, only L-amino acids are known to be encoded by genes. Nevertheless, D-amino acids have been identified in a variety of peptides, synthesized by animal cells. They include neuroexcitatory and neuroprotective peptides, cardioexcitatory peptides, hyperglycemic hormones, opioid peptides, antimicrobial peptides, natriuretic and defensin-like peptides, and fibrinopeptides. This article is a review of their occurrence, structure and bioactivity. It further explores the pharmacology and potential medical applications of some of the peptides.

Infectivity of Lactobacillus rhamnosus and Lactobacillus paracasei isolates in a rat model of experimental endocarditis.

The potential pathogenicity of selected (potentially) probiotic and clinical isolates of Lactobacillus rhamnosus and Lactobacillus paracasei was investigated in a rat model of experimental endocarditis. In addition, adhesion properties of the lactobacilli for fibrinogen, fibronectin, collagen and laminin, as well as the killing activity of the platelet-microbicidal proteins fibrinopeptide A (FP-A) and connective tissue activating peptide 3 (CTAP-3), were assessed. The 90 % infective dose (ID(90)) of the L. rhamnosus endocarditis isolates varied between 10(6) and 10(7) c.f.u., whereas four of the six (potentially) probiotic L. rhamnosus isolates showed an ID(90) that was at least 10-fold higher (10(8) c.f.u.) (P<0.001). In contrast, the two other probiotic L. rhamnosus isolates exhibited an ID(90) (10(6) and 10(7) c.f.u.) comparable to the ID(90) of the clinical isolates of this species investigated (P>0.05). Importantly, these two probiotic isolates shared the same fluorescent amplified fragment length polymorphism cluster type as the clinical isolate showing the lowest ID(90) (10(6) c.f.u.). L. paracasei tended to have a lower infectivity than L. rhamnosus (ID(90) of 10(7) to > or =10(8) c.f.u.). All isolates had comparable bacterial counts in cardiac vegetations (P>0.05). Except for one L. paracasei strain adhering to all substrates, all tested lactobacilli adhered only weakly or not at all. The platelet peptide FP-A did not show any microbicidal activity against the tested lactobacilli, whereas CTAP-3 killed the majority of the isolates. In general, these results indicate that probiotic lactobacilli display a lower infectivity in experimental endocarditis compared with true endocarditis pathogens. However, the difference in infectivity between L. rhamnosus endocarditis and (potentially) probiotic isolates could not be explained by differences in adherence or platelet microbicidal protein susceptibility. Other disease-promoting factors may exist in these organisms and warrant further investigation.

Fibrinogen-induced erythrocyte aggregation: erythrocyte-binding site in the fibrinogen molecule.

The effect of fibrinogen and fibrinogen-derived products on the velocity of rouleau formation of human erythrocytes was quantitatively examined with a rheoscope combined with a video-camera, an image analyzer and a computer. (i) The velocity of rouleau formation by naturally occurring low-molecular-weight fibrinogen of 305 kDa and by desialylated fibrinogen was the same as that by native fibrinogen of 340 kDa. (ii) Concerning fibrinogen degradation products by plasmin, the velocity of rouleau formation decreased upon going from fibrinogen greater than fragment X greater than fragment Y (the ratio of molar concentration of fibrinogen, fragment X and fragment Y for giving a certain velocity of rouleau formation was approx. 1:2:5). The effect of fragments X and Y on the fibrinogen-induced rouleau formation was additive. (iii) Fragments D and E could not induce rouleau formation and did not affect the fibrinogen-, fragment X- and fragment Y-induced rouleau formation. (iv) Fibrinopeptides A and B and artificial tetrapeptides (Gly-Pro-Arg-Pro and Gly-His-Arg-Pro) did not affect the fibrinogen-induced rouleau formation. (v) The possible erythrocyte-binding site in fibrinogen molecule for leading to rouleaux was proposed to be in A alpha-chain (probably, around residues No. 207-303) near the terminal domain of the trinodular structure of fibrinogen.

Inhibition of human thrombin assessed with different substrates and inhibitors. Characterization of fibrinopeptide binding interaction.

The activity of human thrombin has been assessed with fibrinogen, N-alpha-benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide, N-alpha-benzoyl-arginine-p-nitroanilide, N-alpha-carbobenzoxy-tyrosine-p-nitrophenyl ester and p-nitrophenylacetate: increased rates of hydrolysis were found for N-alpha-carbobenzoxy-tyrosine-p-nitrophenyl ester and N-alpha-benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide compared to N-alpha-benzoyl-arginine-p-nitroanilide and p-nitrophenylacetate. Phenylmethyl sulfonyl fluoride and N-alpha-tosyl-L-lysine chloromethyl ketone inhibited, to the same degree, the activity toward each substrate. Inclusion of N-alpha-tosyl-arginine methyl ester in the phenylmethyl sulfonyl fluoride reaction mixtures protected the enzyme from inhibition as shown with N-alpha-benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide and N-alpha-carbobenzoxy-tyrosine-p-nitrophenyl ester. N-Acetylimidazole inhibited the activity towards fibrinogen, N-alphrosine-p-nitrophenyl ester to varying degrees. Inhibition of N-alpha-benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide was completely reversible with neutral hydroxylamine, whereas coagulant activity towards fibrinogen was only partially regained. Human fibrinopeptide A inhibited activity toward N-alpha-benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide and N-alpha-carbobenzoxy-tyrosine-p-nitrophenyl ester. The mode of inhibition of N-alpha-benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide by fibrinopeptide A was non=competitive (K1 = 3.02.10(-5) M), whereas N-alpha-toysyl-arginine methyl ester was a competitive inhibitor of this substrate (K1 = 2.6.10(-5) M). These studies demonstrate more than one binding domain for fibrinogen on human thrombin.

Human fibrinopeptide A mediates allergic reaction in mice in the acute phase.

We detected a human humoral peptide that deglycosylates mouse antibody IgE, and found that this peptide had the amino acid sequence ADSGEGDFLAEGGGV. The peptide synthesized according to the sequence also deglycosylated the antibody IgE. The deglycosylated IgE did not induce mouse systemic anaphylaxis. Human fibrinopeptide A has the amino acid sequence indicated above, and a polypeptide extracted from human urine showed an antiallergic effect on humans and mice, which strongly suggests that fibrinopeptide A mediates allergic reaction via antibody IgE-deglycosylation, and is excreted as a polypeptide in urine.

Effect of fibrinopeptides A and B on human and rat platelet aggregation in vitro.

The effect of fibrinopeptides on platelet aggregation is reported. Fibrinopeptide A (minimal effective concentration, 0.65 microM) aggregated human (but not rat) platelets suspended in plasma and at lower concentrations (0.01-0.1 microM) potentiated platelet aggregation due to ADP and collagen in both species. Fibrinogen mimicked these effects of fibrinopeptide A. P-bromophenacyl bromide (100 microM), mepacrine (10 microM), indomethacin (10 microM) and dazoxiben (10 microM) inhibited human platelet aggregation induced by fibrinopeptide A and fibrinogen. In both species, fibrinopeptide B (0.65-6.5 microM) antagonised the platelet inhibitory effect of PGI2 and PGD2 but not adenosine. Antagonism was non-competitive in nature. The concentration of fibrinopeptide A required to potentiate platelet aggregation occurs naturally in the plasma of patients with thrombotic disease suggesting this effect may be of physiological significance during the formation of a thrombus. The novel action of fibrinopeptide B to reduce the platelet inhibitory effect of PGI2 and PGD2 may also contribute to the control of thrombus formation.

Role of D and E domains in the migration of vascular smooth muscle cells into fibrin gels.

The structure of fibrin plays an important role in the organization of thrombi, the development of atherosclerosis, and restenosis after PTCA. In this study, we examined the mechanisms of the migration of vascular smooth muscle cells (SMCs) into fibrin gels, using an in vitro assay system. Cultured SMCs from bovine fetal aortic media migrated into fibrin gels prepared with thrombin, which cleaves both fibrinopeptides A and B from fibrinogen, without other chemotactic stimuli. Both desA fibrin gels prepared with batroxobin, which cleaves only fibrinopeptide A, and desB fibrin gels prepared with Agkistrodon contortrix thrombin-like enzyme (ACTE), which cleaves only fibrinopeptide B, similarly induced the migration of SMCs compared to fibrin gels prepared with thrombin. These results suggest that the cleavage of fibrinopeptides is not necessary, but rather that the three-dimensional structure of the gel may be important for the migration of SMCs. Furthermore, gels prepared with protamine sulfate, which forms fibrin-like gels non-enzymatically, similarly induced the migration of SMCs compared to the gels prepared with thrombin. Both anti-fibrin(ogen) fragment D and anti-fibrin(ogen) E antibodies inhibited the migration of SMCs into fibrin gels, suggesting that both the D and E domains of fibrin(ogen) are involved in the migration of SMCs into fibrin gels. The addition of GRGDS, a synthetic RGD-containing peptide, but not that of GRGES, a control peptide, partially inhibited the migration of SMCs into fibrin gels, suggesting that the migration of SMCs into fibrin gels is at least in part dependent on the RGD-containing region of the alpha chain. The migration of SMCs into fibrin gels was also inhibited by a monoclonal antibody for integrin alpha v beta 3 and alpha 5 beta 1, indicating that migration is dependent on these integrins. Furthermore, both fibrin(ogen) fragments D and E inhibited the migration of SMCs into fibrin gels, suggesting that these fragments, generated during fibrino(geno)lysis, may be relevant in the regulation of SMC migration into fibrin gels.

Haemostatic derangements associated with arenavirus infection in the guinea-pig: radioimmunoassay of fibrinopeptide A to assess thrombin action in infected animals.

Pichinde virus infection of inbred guinea-pigs is a model for arenaviral infections in humans. Infected animals experience reduced levels of multiple coagulation factors caused by either consumption coagulopathy or impaired factor synthesis. A radioimmunoassay (RIA) of guinea-pig fibrinopeptide A (gFPA) has been developed to measure the degree of thrombin action in vivo. gFPA was synthesized via the solid-phase method and conjugated to bovine serum albumin (BSA). A double antibody RIA was established employing goat anti-rabbit IgG to precipitate the primary complex composed of either 125I-5-Tyr-gFPA or 125I-12-Tyr-gFPA and rabbit anti-gFPA-BSA. The cross-reaching material was removed by mixing the plasma with 3 vol of ethanol. The supernatant was filtered through a hollow fibre apparatus by centrifugation. Plasma gFPA immunoreactivities of outbred guinea-pigs averaged 6.56 ng/ml. The gFPA-RIA was validated by determining the quantity of gFPA released from thrombin-degraded fibrinogen. A transient elevation of gFPA levels was detected in Pichinde-infected animals by the gFPA-RIA using 125I-12-Tyr-gFPA as a tracer. The pathogenic mechanism by which the increased gFPA levels may lead to the lethality of Pichinde virus infection remains to be elucidated. It is possible that the coagulopathy triggers changes in immune and inflammatory pathways that induces high cytokine concentrations, with deleterious effects on organs such as the heart and lungs.

Demonstration of antiinflammatory activity of fibrinogen and fibrinopeptides in rats.

Carrageenin-induced inflammatory rat paw swelling was significantly inhibited by intraperitoneal injection of rat fibrinogen. Whole-body radioscanning of the rat after intraperitoneal administration of 131I-labeled fibrinogen revealed the accumulation of radiolabeled material in the inflammed rat paw. Resorption studies showed that not more than 4% of the intraperitoneally administered [125I] fibrinogen could be demonstrated in the peripheral blood. Furthermore only 1/3 of this circulating radiolabeled protein was able to take part in clot formation, suggesting that inhibition of edema formation is mediated by fibrinogen-derived split products. This is further supported by the finding that rat fibrinopeptides, released by the action of thrombin, also diminish edema formation both after intracardial and intraperitoneal injection into carrageenin-stimulated rats.

Dynamic changes of fibrinopeptides A and B in sera of burn patients and their effects on vascular endothelial cells.

In this study, dynamic changes of fibrinopeptides A and B (FPA, FPB) in sera of burns patients are determined with an HPLC method. On this basis detrimental effects of FPA and FPB on vascular endothelial cells (VEC) are observed in vitro. The main results are as follows: (1) FPA and FPB in 18 burn patients' sera increase at day 2 postburn, reach peaks at days 5-7 postburn, then decline after day 15 postburn, and return to the control levels at day 25 postburn. The fluctuations in FPA and FPB levels in burn patients' sera are parallel to the progress of burn illness. The measurements of FPA and FPB levels in burn patients' sera may be useful in evaluating patients' condition. (2) FPA and FPB are detrimental to the cultured VEC in vitro. Characteristics of their injurious effects are: specific, irreversible and dose dependent. FPA and FPB may play an important role in endothelial injury.

The effect of bovine fibrinopeptides on the central action of chlorpromazine and amphetamine in rats.

A mixture of fibrinopeptides A and B did not evoke any significant central effects when given by intraperitoneal injection, whereas it increased psychomotor activity when injected into a cerebral ventricle. The fibrinopeptides when given by intraperitoneal injection interacted with amphetamine to increase locomotor activity and with chlorpromazine to decrease both locomotor activity and body temperature. It is suggested that the release of fibrinopeptides in various clinical conditions where there is increased fibrinogen-fibrin conversion may lead to an altered sensitivity to centrally acting drugs.

Amino acids and peptides. XVI. Synthesis of N-terminal tetrapeptide analogs of fibrin alpha-chain and their inhibitory effects on fibrinogen/thrombin clotting.

N-Terminal tetrapeptide analogs of fibrin alpha-chain were synthesized by the solution method using a new active ester, the ester of the oxime of p-nitroacetophenone, and by the solid-phase method. Their inhibitory effects on fibrinogen/thrombin clotting were examined. Of the synthetic peptides, amide analogs of Gly-Pro-Arg-Pro exhibited a more potent inhibitory effect.

A alpha and B beta chains of fibrinogen stimulate proliferation of human fibroblasts.

During blood coagulation and wound healing, fibrinogen polymerises to form a fibrin matrix, providing a substratum over which connective tissue cells migrate and proliferate. Although a number of growth factors have been implicated in this process, a possible role for the fibrin(ogen) molecules themselves has not been considered. In this study we have investigated the ability of the constituent chains of fibrin(ogen) to induce fibroblast replication. Fibrinogen chains (A alpha 1, A alpha 2, B beta and gamma) were separated by cation exchange chromatography and their mitogenic activity was assessed before and after treatment with thrombin. The A alpha 1, A alpha 2 and B beta chains where all found to stimulate fibroblast replication (23 +/- 2.9%, 29.2 +/- 5.3% and 31.4 +/- 5% stimulation above control, respectively) and on the addition of thrombin this activity was enhanced. No activity was observed in the gamma chain before or after treatment with thrombin. These results indicate that growth promoting activity is inherent in fibrin(ogen) structure, suggesting a novel mechanism for fibroblast proliferation during wound healing.

The effect of products of degradation of fibrinogen A and B on the arterial blood pressure in the rat.

Fibrinopeptides A and B (FAB) and the products of their degradation with chymotrypsin and trypsin elevate the aortal blood pressure in the rat. The products of FAB digestion do not affect the hypertensive action of dopamine. It seems that the action of these peptides on blood pressure and the effect of dopamine are dependent on at least two unrelated mechanisms.

Regulation of fibrinogen biosynthesis: effect of fibrin degradation products, low-molecular-weight peptides of fibrinogenolysis, and fibrinopeptides A and B.

Fibrinogen synthesis in rabbits was evaluated following intravenous infusions of stage 3 degradation products of homologous fibrinogen or fibrin, prepared in vitro. Fibrinogen production was measured by determining the rate of appearance of 75SeM into circulating fibrinogen. Fibrinogen synthesis increased threefold after the administration of stage 3 FDP (D and E), dialyzed to remove LMW digestion fragments, In contrast, the fdp obtained by plasminolysis of crosslinked thrombin clots or of noncrosslinked ancrod or thrombin clots failed to enhance basal fibrinogen production. Accelerated fibrinogen production was not accompanied by alterations in haptoglobin concentration or by increased incorporation of 75SeM into haptoglobin. Fibrinogen synthesis was not increased after infusions of FPA and FPB.

The effect of fibrinopeptides A and B on the circulatory action of dopamine.

Fibrinopeptides A and B potentiate the hypertensive action of dopamine in the rat and potentiate its hypotensive effects after alpha- and beta-adrenergic blockade. Dopamine receptor blockade with haloperidol completely abolished the effects of FAB. The possible mechanism of the observed effects is discussed.

Some pharmacological and biochemical effects of fibrinopeptides A and B in the circulatory system of rats.

Human fibrinopeptides A and B (FAB) increased the arterial blood pressure, accelerated the heart rate and elevated permeability of capillaries, produced a vasodilatory effect, evoked positive chronotropic and inotropic action on the isolated heart and did not affect the coronary flow. They lowered the content of glycogen in the heart muscle. They did not affect the concentration of glucose and elevated the content of lactic acid and free fatty acids (FFA) in the blood. As FAB are present in large quantities in the blood during disseminated intravascular coagulation, they may play an essential role in pathology of the circulatory system of mammals.

Cell proliferation on fibrin: modulation by fibrinopeptide cleavage.

Fibrin forms the cohesive network of hemostatic plugs and thrombi, and it also provides the temporary matrix for initial support of healing and revascularization. Because cell proliferation is needed for revascularization after vessel injury, we have characterized structural requirements of fibrin needed to support cell proliferation on fibrin in vitro. Proliferation of cultured human endothelial cells and fibroblasts was measured by 3H-thymidine incorporation on fibrin surfaces varying in structure. Fibrin prepared with thrombin and lacking both fibrinopeptides A and B (desAB fibrin) supported proliferation of both endothelial cells and fibroblasts. In contrast, fibrin prepared with reptilase, which cleaves only fibrinopeptide A, supported significantly less proliferation. Also, fibrin prepared by thrombin treatment of fibrinogen lacking residues beta 1-42 supported only a low level of proliferation. Therefore, fibrinopeptide B cleavage and exposure of beta 15-42 enhanced proliferation of cells on fibrin. Specific proteolytic inhibitors were used to eliminate the potential mitogenic effects of residual fibrin-bound thrombin. Additional controls showed that neither catalytically inactive thrombin nor addition of the thrombin receptor-activating peptide (SFLLRNPNDKYEPF [SFLL]) stimulated proliferation on desA fibrin. The results indicate that cell proliferation on fibrin is enhanced by fibrinopeptide B cleavage and exposure of the amino terminus of the fibrin beta chain. They also show that specific structural features of the temporary fibrin matrix formed at sites of injury may modulate the proliferative response of vascular cells.