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.

Fragment of human beta-fibrinogen induces a behavioral effect on mouse forced swimming.

We detected a peptide having a behavioral activity on mouse forced swimming from sera of healthy volunteers without affective and psychotic diseases. The amino acid sequence was GVNDNEEGF, which was found in the sequence of human beta-fibrinogen. The synthesized peptide also showed the behavioral activity dose-dependently, but human fibrinopeptide B (QGVDNEEGFFSAR) did not. The activity was decreased by dopamine 1 antagonist SCH-23390, but not by dopamine 2 antagonist sulpiride. These findings strongly suggest that metabolism of human beta-fibrinogen induces the fragment affecting the mouse behavior via the dopamine1 neuronal activity.

Fibrin monomer and fibrinopeptide B act additively to increase DNA synthesis in smooth muscle cells cultured from human saphenous vein.

PURPOSE: We investigated the hypothesis that fibrinogen increased DNA synthesis (and cell proliferation) of smooth muscle cells (SMCs) cultured from human saphenous vein and that the increased DNA synthesis was attenuated when cells were cultured on polymeric collagen. METHODS: SMCs were cultured from human saphenous vein on plastic, fibronectin, monomeric, and polymeric collagen. Fibrinogen products were prepared by proteolytic digestion. DNA synthesis was measured by bromodeoxyuridine incorporation into DNA, cell proliferation by cell counting, cyclic adenosine monophosphate by enzyme-linked immunosorbent assay, and fibrinopeptide B labeled with iodine 125 used for binding studies. RESULTS: Fibrin monomer (0.003-0.1 micromol/L) stimulated a concentration-dependent increase in DNA synthesis of up to 10-fold, which could be inhibited by the peptide Bbeta15-42. The stimulation of DNA synthesis was highest for cells cultured on plastic and lowest for cells cultured on type I collagen polymer. Much higher concentrations of fibrinogen (0.3-1 micromol/L) were required to effect similar increases in DNA synthesis. Fibrinogen had a particular effect to augment DNA synthesis, up to 14-fold, when cells were cultured on monomeric type I collagen. This augmented DNA synthesis was inhibited by a neutralizing antibody to urokinase-type plasminogen activator. Incubation of cells cultured on collagen monomer with fibrinogen resulted in production of fibrinopeptide B. Fibrinopeptide B (5 micromol/L) increased DNA synthesis by fourfold and had additive effects with fibrin monomer to increase DNA synthesis. Iodinated tyrosine fibrinopeptide B bound to SMCs (dissociation constant 0.6 micromol/L). CONCLUSION: Cultured human saphenous vein SMCs appear to have high-affinity receptors for fibrin monomer and fibrinopeptide B, the engagement of which stimulates DNA synthesis. These mechanisms may be pertinent to the association between fibrinogen and vein graft stenosis in vivo.

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.

Decreased levels of alpha 1(I) procollagen mRNA in dermal fibroblasts grown on fibrin gels and in response to fibrinopeptide B.

We have investigated human neonatal fibroblast synthetic activity in response to fibrin substrates and components of fibrin formation and degradation. Greater than threefold downregulation of procollagen mRNA levels was seen 24 hours after fibroblasts were grown on fibrin gels as compared to tissue culture plastic. This downregulation occurred in both reptilase-generated fibrin (retention of fibrinopeptide B) and thrombin-generated fibrin (loss of both fibrinopeptide A and B). However, fibroblasts grown on fibrin retained their capacity to respond to the stimulatory action of transforming growth factor (TGF)-beta 1. Fibroblasts seeded on reptilase-generated fibrin displayed an abnormal morphology manifested by dendritic appearance and cell rounding, while fibroblast attachment was enhanced by 30% on thrombin-generated fibrin substrate (P < 0.02). Fibrinopeptides A and B, which are generated during fibrin formation, increased and decreased procollagen mRNA levels, respectively. Tissue plasminogen activator (t-PA) increased procollagen mRNA and TGF-beta 1 levels as early as 6 hours after cells were grown on tissue culture plastic, but this stimulation did not occur in cells cultured on a fibrin substrate. We conclude that alpha 1(I) procollagen mRNA levels in cultures of human dermal fibroblasts are consistently down-regulated by a fibrin substrate and are directly and profoundly influenced by complex interactions between components involved in the formation and removal of fibrin.

Endothelial desquamating activity of rat synthetic fibrinopeptide B and its analogues "in vivo": identification of responsible sequence.

The endothelial desquamating activity of the synthetic rat fibrinopeptide B (ATTDSDKVDLSIAR-OH), and its analogues was studied "in vivo" after intravenous administration to rats. Rat fibrinopeptide B (FPB) caused a significant increase in the count of circulating endothelial cell carcasses at the dose of 100 nmol/kg. Maximal effect reaching about 270% of the normal value was achieved with the dose of 600 nmol/kg in 30 min. after the injection. No significant thrombocytopenia, no hemolysis and no other life-threatening complications were observed. The same endothelial desquamating effect was achieved with N-terminal FPB(1-7) peptide ATTDSDK-OH, but very low activity exhibited the two FPB mutant peptides: ATDSDKVDLSIAR-OH and ATTNSNK-OH. Our results indicate that N-terminal sequence (1-7) consisting of N-terminal "pig tail" (ATT), acid region (DSD) and basic amino acid (K) is responsible for endothelial desquamating activity of rat FPB. Similar corresponding sequences may be recognized in FPB of different species. The conservation of this common "active site" sequence is less apparent in primates.

Interactions of plasminogen and fibrinogen with model silica glass surfaces: adsorption from plasma and enzymatic activity studies.

The adsorption of fibrinogen and plasminogen from plasma to silica glass, sulfonated silica glass, and lysine-derivatized silica glass has been investigated. The data indicate that the sulfonated material has a high affinity for both fibrinogen and plasminogen, but that the ratio of plasminogen to fibrinogen is greater on the lysine-derivatized surface. The adsorption data also suggest plasminogen as a possible contributor to the fibrinogen Vroman effect, whereby initially absorbed fibrinogen is displaced from the surface. The plasmin activity of plasminogen adsorbed to the lysine-derivatized silica glass and its sulfonated precursor was assessed by both a chromogenic substrate assay and a radioimmunoassay for the plasmin cleavage product of fibrinogen, the B beta 1-42 peptide. The data indicate that 1) the adsorbed plasminogen is not inherently plasmin-like; 2) the enzymatic activity associated with the bound plasminogen is significantly enhanced on both surfaces in the presence of activator; and 3) in the presence of activator, the plasmin activity per mole of bound plasminogen on the lysinized material is approximately a factor of two greater than on the sulfonated material based on the chromogenic substrate assay, and a factor of four greater based on the B beta 1-42 radioimmunoassay. The lysinized material thus exhibits several properties that are different from its sulfonated precursor. It adsorbs more plasminogen relative to fibrinogen after the Vroman peak, and this adsorbed plasminogen appears to be in a conformation that is more readily activated to plasmin. Once activated, the surface bound plasmin shows enhanced ability to cleave either a low molecular weight chromogenic substrate or a macromolecular substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

Effect of a peptide derived from fibrinogen degraded by leukocyte elastase on isolated bovine mesenteric arteries.

A peptide derived from fibrinogen degraded by leukocyte elastase, and corresponding to amino acids 30-43 in the B beta-chain of fibrinogen, was evaluated concerning its effects on isolated bovine mesenteric arteries. This peptide induced dilation of the arteries and an increase in both cyclic AMP and cyclic GMP in the vessels. In addition there was an increase in 6-keto-PGF1 alpha indicating an increased release of prostacyclin. The increase in cyclic nucleotides and 6-keto-PGF1 alpha was inhibited by indomethacin, as was the vasodilation. The increase in cyclic GMP was much larger than the increase in cyclic AMP. The effects of the studied peptide are similar to the effects of other vasoactive peptides with a similar structure, such as bradykinin, neurotensin and substance P. The increase in cyclic AMP is probably caused by prostacyclin, a probable mediator of vasodilation. In addition, in certain species vasodilation may be caused by an increase in cyclic GMP.

Human tissue-type plasminogen activator releases fibrinopeptides A and B from fibrinogen.

In five patients with venous thromboembolic disease treated with recombinant tissue-type plasminogen activator (rt-PA), there was a marked increase in the mean concentrations of fibrinopeptide A (from 0.6 to 5.9 nM; P less than 0.0001) and desarginine fibrinopeptide B (from 5.6 nM to 24.1 nM; P less than 0.01) 30 min after a bolus of rt-PA (0.6 mg/kg). Thrombin was unlikely to be responsible because the levels of desarginine fibrinopeptide B exceeded those of fibrinopeptide A and the changes occurred despite concomitant heparin therapy. The purpose of this study therefore, was to determine whether rt-PA directly releases the fibrinopeptides from fibrinogen. Incubation of rt-PA with heparinized plasma or purified fibrinogen resulted in time and dose-dependent release of both fibrinopeptide A and B. Contaminating thrombin was not responsible for this activity by the following criteria: the rate of rt-PA mediated fibrinopeptide B release was considerably faster than that of fibrinopeptide A, and fibrinopeptide release was unaffected by heparin, hirudin, or a monospecific antithrombin IgG. Aprotinin also had no effect on fibrinopeptide release, indicating that this activity was not plasmin mediated. Fibrinopeptide release was shown to be due to rt-PA because this activity was completely blocked by a monoclonal antibody against the enzyme. Further, the specificity of rt-PA for the thrombin cleavage sites on fibrinogen was confirmed by the demonstration that rt-PA released fibrinopeptide A or fibrinopeptide B from fibrinopeptide A or B-containing substrates, respectively. These studies thus demonstrate that (a) rt-PA releases fibrinopeptides A and B from fibrinogen thereby indicating that this enzyme is not specific for plasminogen, and (b) plasma fibrinopeptide A and desarginine fibrinopeptide B levels are not specific markers of thrombin action on fibrinogen in patients receiving rt-PA.

Cardiovascular effects of fibrinopeptide B.

The effect of fibrinopeptide B (FpB) on isolated blood vessels and in the intact rat has been investigated. FpB contracted the rabbit superfused aorta preparation (EC50 = 7.5 nmol) and was some 20 times less potent than noradrenaline. Similarly, FpB (0.2-10 nmol) injected into the rat perfused kidney caused dose related, short-lived increases in perfusion pressure and potentiated the vasoconstrictor effect of injected noradrenaline. These effects were associated with increased efflux of PGE2 (but not TxB2) and were reduced but not abolished by indomethacin (10 microM). FpB injected intravenously into the urethane-anaesthetized rat exhibited vasoconstrictor activity (EC50 = 2.5 micrograms kg-1) but was less potent than noradrenaline (EC50 = 0.9 micrograms kg-1). The doses of FpB required to contract the rabbit isolated aorta and to constrict the vasculature of the rat kidney occur naturally in the bloodstream of patients with thrombotic disease. FpB released at the site of thrombus formation may play a part in regulating local blood vessel calibre.

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.

Fibrin(ogen) peptide B beta 15-42 inhibits platelet aggregation and fibrinogen binding to activated platelets.

The binding of fibrinogen to activated platelets leads to platelet aggregation. Fibrinogen has multiple binding sites to platelet membrane glycoprotein IIb-IIIa complex. At least two well-defined sequences in fibrinogen, Arg-Gly-Asp sequence of A alpha 95-97 and A alpha 572-574 and gamma 400-411, have been shown to interact with glycoprotein IIb-IIIa. A possible binding site on the amino-terminal end of fibrinogen to platelet glycoprotein IIb-IIIa has also been reported. In this paper the effect of synthetic peptides derived from the amino-terminal end of the B beta chain on platelet aggregation and fibrinogen binding has been examined. B beta 15-42 peptide inhibits platelet aggregation and 125I-fibrinogen binding to activated platelets in a dose-dependent manner. Since B beta 15-42 contains a previously identified fibrinogen binding site, B beta 15-18, exposed by thrombin cleavage of native fibrinogen, we also examined the effect of B beta 15-18, B beta 19-42, and B beta 1-14 (fibrinopeptide B) on platelet aggregation and fibrinogen binding. Synthetic fibrinopeptide B and B beta 15-18 had no effect on platelet aggregation and fibrinogen binding while B beta 19-42 retained the inhibitory effect. When fibrinogen is chromatographed on a column of agarose-bound B beta 15-42, a cation-dependent retention of fibrinogen on the peptide column was observed, and fibrinogen was eluted from the column by B beta 15-42 but not by B beta 1-14. Under the same conditions, platelet glycoprotein IIb-IIIa was not retained in the column. Thus, the observed inhibitory effect is due to its interaction with fibrinogen rather than to platelet glycoprotein IIb-IIIa.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

The release of small amounts of fibrinopeptide-B (FPB) is of critical importance for the thrombin clotting time.

When fibrinogen is clotted with thrombin, mainly fibrinopeptide-A (FPA) is released at gel point, the FPB-release being delayed. The present study present evidence that the small amounts of des-AABB fibrin found at visible gelation improves polymerization, thereby determining the thrombin clotting time. 1. Purified fibrinogen was clotted with thrombin or Reptilase, peptide release monitored radioimmunologically. With thrombin, significantly less fibrin (released FPA) was necessary for gel formation than when Reptilase was used. Furthermore, approximately 10% of the available FPB had been released by thrombin at gel point. 2. Polymerization of monomers prepared by incubation of fibrinogen with Reptilase (des-AA) and thrombin (des-AABB) in urea, were examined by light scattering. Des-AA fibrin monomers polymerised after a lag-phase of 4 minutes. When 20% of these monomers were substituted with des-AABB monomers, the lag was reduced to 1 1/2 minutes. 3. Finally, polymerization of preformed des-AA monomers was studied with and without the addition of small amounts of thrombin, and the FPB-release monitored. The presence of 0.04 NIH U/ml of thrombin reduced the lag from 10 minutes to 3 minutes. At this time approximately 10% of FPB had been released. It is concluded that even small amounts of des-AABB fibrin substantially improves polymerization of des-AA fibrin.

Participation of prostaglandins E2 and F2 alpha in the action of fibrinopeptides A and B on the central dopaminergic system.

The participation of prostaglandins (PGs) E2 and F2 alpha administered icv in the action of fibrinopeptides A and B (FAB) on the central dopamine neurons was investigated. PGE2 and PGF2 alpha (0.5 microgram) attenuated the intensity of apomorphine (APO)-induced stereotypy and completely abolished the stimulatory action of FAB. PGE2 given together with FAB attenuated, while PGF2 alpha did not affect the amphetamine-induced stereotypy and haloperidol-induced catalepsy. The investigated PGs did not affect or normalized the level of dopamine depressed by peptides in some parts of the brain. The results indicate that PGE2 alpha and PGF2 alpha do not participate in the mechanism of the central action of FAB.

Action of fibrinopeptides A and B on the central dopaminergic system of rats pretreated with indomethacin.

Fibrinopeptides A and B (FAB) stimulate central dopamine neurons. Inhibition of prostaglandin synthesis by administration of indomethacin potentiated the stimulatory action of FAB on apomorphine stereotypy, while induced inhibitory action of FAB on amphetamine stereotypy. Results of studies on the level and turnover of dopamine do not explain the changes in the stereotyped behavior. The data suggest that prostaglandins may to some extent participate in the mechanism of the central action of FAB.

Fibrin(ogen)-derived peptide B beta 30-43 increases coronary blood flow in the anesthetized dog.

The hemodynamic effects of intracoronary administration of a fibrin (ogen)-derived peptide B beta 30-43 (Arg-Pro-Ala-Pro-Pro-Pro-Ile-Ser-Gly-Gly-Gly-Tyr-Arg-Ala) were evaluated in open-chest anesthetized dogs. Coronary blood flow (CBF) increased and coronary vascular resistance (CVR) decreased with intracoronary administration of peptide B beta 30-43. These changes were dependent on the amount of the peptide B beta 30-43 administered. There were no significant effects of peptide B beta 30-43 on aortic & left ventricular end-diastolic pressures. Plasma 6-keto-PGF1 alpha (stable hydrolysis product of PGI2) concentrations increased in coronary sinus blood samples in conjunction with increase in CBF. Intravenous administration of indomethacin (5 mg/kg) inhibited the release of PGI2 and almost completely abolished the effects of the fibrin(ogen)-derived peptide on CBF. This study suggests that this fibrin (ogen)-derived peptide has potent effects on the coronary vascular bed of the dog, and that these effects are in large part mediated through PGI2 release. These coronary hemodynamic effects of fibrin(ogen)-derived products may have important autoregulatory effects in atheromatous coronary circulation, wherein thrombi may form spontaneously.