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PubMed1980

Effects of fibrinopeptide cleavage on the plasmic degradation pathways of human cross-linked fibrin.

The presence of fibrinopeptide B in human fibrin has a significant effect on plasmic degradation pathways of cross-linked clots. Two types of fibrin were obtained from fibrinogen by incubation either with thrombin, to remove both fibrinopeptides A and B, or with batroxobin, to cleave fibrinopepitde A only. Fibrins obtained after various incubation times were characterized by the determination of the NH2-terminal amino acids, the content of fibrinopeptides, and the extent of cross-linking. The fibrins were digested by plasmin and were analyzed by polyacrylamide gel electrophoresis. The presence and concentration of the (DD)E complex, as well as fragments E1 and E2, in the digests were dependent upon the loss of fibrinopeptide B from cross-linked fibrin. These degradation products, and also fragment DD, appear to be useful molecular markers of fibrinolysis.

Amino Acids↗
PubMed2002

Analysis of engineered fibrinogen variants suggests that an additional site mediates platelet aggregation and that "B-b" interactions have a role in protofibril formation.

The C-terminal domain of the fibrinogen gamma-chain includes multiple functional sites that have been defined in high-resolution structures and biochemical assays. Calcium binds to this domain through the side chains of gammaD318 and gammaD320 and the backbone carbonyls of gammaF322 and gammaG324. We have examined variant fibrinogens with alanine at position gamma318 and/or gamma320 and found that calcium binding, fibrin polymerization, and fibrinogen-mediated platelet aggregation, but not FXIIIa-catalyzed cross-linking, were abnormal. When measured by turbidity, thrombin-catalyzed polymerization was severely reduced, and batroxobin-catalyzed polymerization was completely obliterated. Moreover, thrombin-catalyzed polymerization was abolished by the peptide GHRP, which binds to the polymerization site in the beta-chain but does not inhibit polymerization of normal fibrinogen. ADP-induced platelet aggregation was also severely impaired. In contrast, as measured by SDS-PAGE, FXIIIa introduced cross-links between gamma-chains for all three variants, as expected if the gamma-chain C-terminal sites were normal. In addition, binding of the monoclonal antibody 4A5, which recognizes the C-terminal residues, was not different from normal. These data suggest two specific conclusions: (1) a site in the gamma-module other than the C-terminus is critical for platelet aggregation and (2) "B-b" interactions have a role in protofibril formation.

Alanine↗
PubMed1986

Distinctive role of histidine-16 of the B beta chain of fibrinogen in the end-to-end association of fibrin.

Photooxidation of fibrinogen reduced the batroxobin-induced fibrin polymerization. The fibrin fragment des-AB N-DSK, which contains the binding sites termed A and B, lost the ability to bind to the site termed a in fibrinogen-Sepharose upon the oxidation of histidine-16 in the B beta chain of fibrinogen [Shimizu, A., Saito, Y., Matsushima, A. & Inada, Y. (1983) J. Biol. Chem. 258, 7915-7917]. Some of the fragments, which became unable to bind to fibrinogen-Sepharose due to the destruction of site A, however, retained the ability to bind to D-dimer-Sepharose, which contains both sites a and b. This shows that histidine-16 of the B beta chain of fibrinogen is essential for site A but may not be essential for site B. It is of interest that histidine-16 of the B beta chain, which is only one residue away from the thrombin-susceptible bond, makes a part of the site A for the end-to-end association created by the release of fibrinopeptide A.

Binding Sites↗
PubMed1995

A novel plasminogen activator from snake venom. Purification, characterization, and molecular cloning.

A novel plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) has been identified and purified to homogeneity. It is a single chain glycoprotein with an apparent molecular weight of 33,000 and an isoelectric point of pH 5.2. It specifically activates plasminogen through an enzymatic reaction. The activation of human native Glu-plasminogen by TSV-PA is due to a single cleavage of the molecule at the peptide bond Arg561-Val562. Purified TSV-PA, which catalyzes the hydrolysis of several tripeptide p-nitroanilide substrates, does not activate nor degrade prothrombin, factor X, or protein C and does not clot fibrinogen nor show fibrino(geno)lytic activity in the absence of plasminogen. The activity of TSV-PA was readily inhibited by phenylmethanesulfonyl fluoride and by p-nitrophenyl-p-guanidinobenzoate. Oligonucleotide primers designed on the basis of the N-terminal and the internal peptide sequences of TSV-PA were used for the amplification of cDNA fragments by polymerase chain reaction. This allowed the cloning of a full-length cDNA encoding TSV-PA from a cDNA library prepared from the venom glands. The deduced complete amino acid sequence of TSV-PA indicates that the mature TSV-PA protein is composed of 234 amino acids and contains a single potential N-glycosylation site at Asn161. The sequence of TSV-PA exhibits a high degree of sequence identity with other snake venom proteases: 66% with the protein C activator from Agkistrodon contortrix contortrix venom, 63% with batroxobin, and 60% with the factor V activator from Russell's viper venom. On the other hand, TSV-PA shows only 21-23% sequence similarity with the catalytic domains of u-PA and t-PA. Furthermore, TSV-PA lacks the sequence site that has been demonstrated to be responsible for the interaction of t-PA (KHRR) and u-PA (RRHR) with plasminogen activator inhibitor type 1.

Amides↗
PubMed2002

Crystallization of bothrombin, a fibrinogen-converting serine protease isolated from the venom of Bothrops jararaca.

Bothrombin, a snake-venom serine protease, specifically cleaves fibrinogen, releasing fibrinopeptide A to form non-crosslinked soft clots, aggregates platelets in the presence of exogenous fibrinogen and activates blood coagulation factor VIII. Bothrombin shares high sequence homology with other snake-venom proteases such as batroxobin (94% identity), but only 30 and 34% identity with human alpha-thrombin and trypsin, respectively. Single crystals of bothrombin have been obtained and X-ray diffraction data have been collected at the Laboratorio Nacional de Luz Sincrotron to a resolution of 2.8 A. The crystals belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 94.81, b = 115.68, c = 155.97 A.

Animals↗
PubMed1986

Soluble fibrin consists of fibrin oligomers of heterogeneous distribution.

Soluble fibrin is observed in patients with intravascular coagulation and represents an intermediary product of conversion of fibrin monomers into a fibrin clot whereby the presence of fibrinogen may suppress fibrin clot formation. The interactions between fibrin and fibrinogen and the occurrence of fibrin oligomers in soluble fibrin were studied by sucrose density ultracentrifugation. Different concentrations of soluble fibrin, prepared by mixing 125I-fibrin (24 nM - 1.5 microM) with a constant concentration of 131I-fibrinogen (6 microM) were analyzed at 37 degrees C in stable linear sucrose density gradients containing a uniform concentration of unlabelled fibrinogen (6 microM) and calcium ions in order to mimic the physiological situation. At any fibrin concentration, 125I-fibrin sedimented faster than 131I-fibrinogen through 5-30% (w/v) sucrose gradients. Sedimentation rates of fibrin increased from 9 S to 23 S depending on the initial fibrin concentration. The relative amount of residual fibrin monomer not incorporated into oligomers was calculated from the sedimentation profiles. At any fibrin concentration, the portion of free monomer was always more than twofold higher for batroxobin-generated (desAA-) fibrin than for thrombin-generated (desAABB-) fibrin. Apparent association constants for desAABB-fibrin were 3-10 times higher than those for desAA-fibrin indicating a stronger interaction between monomers of the former type of fibrin. In the presence of excess fibrinogen the predominant species in soluble desAA-fibrin were monomers and dimers, whereas dimers, trimers and higher-molecular-mass oligomers were present in soluble desAABB-fibrin. Strong interactions between both types of fibrin were demonstrated from their cosedimentation, whereby the size of these copolymers were shown to be governed by the oligomer size of the desAABB-fibrin type. These results provide evidence for the occurrence of differently sized oligomers of fibrin in soluble fibrin and for the concept of a cooperative polymerization process between both types of fibrin devoid of any stable complexes between fibrin and fibrinogen.

Biopolymers↗
PubMedDate not supplied

Effect of platelet-rich plasma on migration and proliferation of SaOS-2 osteoblasts: role of platelet-derived growth factor and transforming growth factor-beta.

Platelet-enriched plasma (PRP) is used in therapy as a source of growth factors in bone fracture and wound healing; however, few data exist on its role in the different aspects of the healing process. The effect of PRP and of the two main growth factors present in this preparation (platelet-derived growth factor [PDGF] and transforming growth factor-beta [TGF-beta]) was evaluated in vitro using the human osteoblastic cell line SaOS-2, which was shown by reverse transcription-polymerase chain reaction to express both PDGF-alpha and -beta receptors. Batroxobine-activated PRP was added in different concentrations to SaOS-2 cells to assess cell migration (by a microchemotaxis assay) and cell proliferation (by [3H]-thymidine incorporation into the DNA). Immunoneutralization with anti-PDGF-beta or anti-TGF-beta antibodies allowed the assessment of the specific role of these growth factors. The overall results obtained indicate that PRP dose-dependently stimulates both chemotaxis and cell proliferation. PDGF and TGF-beta appear to exert distinct effects on the two parameters, the former involved in stimulating cell migration and the latter in inhibiting cell proliferation. It is concluded that the different growth factors present in activated PRP can specifically contribute to the main processes of tissue regeneration.

Analysis of Variance↗
PubMed1984

Xenopus fibrinogen: characterization of the mRNAs for the three subunits.

We purified and characterized the mRNAs coding for each of the three subunits of Xenopus fibrinogen. Purification was accomplished by electrophoretic separation of liver polyadenylated RNA in a fully denaturing gel, followed by recovery of the RNA from the gel via transfer to an ion-exchange membrane. This procedure yielded fractions which were highly enriched for the mRNAs for each of the fibrinogen chains. The fibrinogen mRNAs were identified by two methods: (i) in vitro translation followed by subunit-specific cleavage with the proteases thrombin and batroxobin; and (ii) cross-hybridization with cDNA clones for individual subunits of rat fibrinogen. The results demonstrate that the A alpha and gamma chains of frog fibrinogen are each coded by a single mRNA species. The A alpha mRNA is ca. 3,100 nucleotides in length, which is nearly twice the minimum size required to code for the A alpha precursor polypeptide. The gamma chain mRNA comprises about 1,600 bases and includes only a small untranslated region. In contrast, the B beta subunit is synthesized from two mRNAs, one of which is 2,500 and the other 1,800 nucleotides long. The 2,500-base mRNA includes a large noncoding region, whereas the smaller one is near the minimum required size. The larger B beta mRNA is ca, fivefold more abundant that the smaller species.

Animals↗
PubMed2005

The potential of Bothrops moojeni venom in the field of hemostasis. Established use and new insights.

Early studies in the 1930s on the venom of South American Lancehead snakesofthe Bothrops genuslead to the discovery of compounds active in blood coagulation such as batroxobin and botrocetin. The scope of our investigations is to have a deeper look at the crude venom of B. moojeni using state-of-the-art proteomics methods, as well as newly developed bioassays screening for activities in the different fields of application. The proteomics techniques used up to now have included different chromatography methods, mass spectrometry, and bio-computing. The bioassays are focussed on enzymatic and other activities in the field of hemostasis and fibrinolysis. Besides the known activities several new and interesting ones have been found. They still need to be studied and confirmed in more specific supplementary assays.

Animals↗
PubMed1983

Fibrinogen Seattle releases half the normal amount of fibrinopeptide B.

Fibrinogen Seattle, a clinically silent, slow-clotting dysfibrinogen, releases 50% of the normal amount of fibrinopeptide B as assessed by amino acid analysis. The reduced dysfibrin exhibited equal quantities of chains with B beta- and beta-charge mobility on polyacrylamide gel electrophoresis in 2 M urea at low pH. By these same techniques, the release of fibrinopeptide A was normal. Clots formed by repolymerizing the thrombin and batroxobin dysfibrin monomers showed a maximal turbidity that was lower than normal. Fibrinogen Seattle was indistinguishable from normal fibrinogen by radial immunodiffusion and immunoelectrophoresis. Degradation by plasmin and transamination by factor XIIIa were normal. The characteristics of fibrinopeptide release by fibrinogen Seattle distinguish it from other reported dysfibrinogens.

Amino Acids↗
PubMed1980

Cross-linking of alpha 2-plasmin inhibitor to fibrin by fibrin-stabilizing factor.

The concentration of alpha 2-plasmin inhibitor in blood plasma is higher than that in serum obtained from the blood clotted in the presence of calcium ions, but is the same as that in serum obtained in the absence of calcium ions. Radiolabeled alpha2-plasmin inhibitor was covalently bound to fibrin only when calcium ions were present at the time of clotting of plasma or fibrinogen. Whereas, when batroxobin, a snake venom enzyme that lacks the ability to activate fibrin-stabilizing factor, was used for clotting fibrinogen, the binding was not observed. When fibrin-stablizing, factor-deficient plasma was clotted, the specific binding of alpha 2-plasmin inhibitor to fibrin did not occur even in the presence of calcium ions and the concentration of alpha 2-plasmin inhibitor in serum was the same as that in plasma. Monodansyl cadaverine, a fluorescent substrate of the fibrin-stablizing factor, was incorporated into alpha 2-plasmin inhibitor by activated fibrin-stablizing factor. All these findings indicate that alpha 2-plasmin inhibitor is cross-linked to fibrin by activated fibrin-stabilizing factor when blood is clotted. Analysis of alpha 2-plasmin inhibitor-incorporated fibrin by sodium dodecyl sulfate gel electrophoresis showed that the inhibitor was mainly cross-linked to polymerized alpha-chains of cross-linked fibrin. Cross-linking of alpha 2-plasmin inhibitor to fibrin renders fibrin clot less susceptible to fibrinolysis by plasmin.

Calcium↗
PubMed2000

Two related thrombin-like enzymes present in Bothrops atrox venom.

This article describes the presence of two new forms of a thrombin-like enzyme, both with apparent molecular masses of 38 kDa, in Bothrops atrox venom. Both share the ability to cleave fibrinogen into fibrin and to digest casein. Both present identical K(m) on the substrate BApNA. Their N-terminal amino acid sequences are identical for 26 residues, sharing 80% homology with batroxobin and flavoxobin. Two groups of monoclonal antibodies (mAbs) raised against the purified enzyme forms recognized different epitopes of the putative corresponding enzymes present in B. atrox crude venom. On Western blotting analysis of B. atrox crude venom, mAbs 5DB2C8, 5AA10 and 5CF11, but not mAbs 6CC5 and 6AD2-G5, revealed two or more protein bands ranging from 25 to 38 kDa. By immunoprecipitation assays, the 6AD2-G5 mAb was able to precipitate protein bands of 36-38 kDa from B. atrox, B. leucurus, B. pradoi, B. moojeni, B. jararaca and B. neuwiedii crude venoms. Fibrinogen-clotting activity was inhibited when the same venom specimens were pre-incubated with mAb 6AD2-G5, except for B. jararaca and B. neuwiedii.

Animals↗
PubMed2000

[Effects of fibrinogen, fibrin and their degradation products on the behaviour of vascular smooth muscle cells].

The transition of fibrinogen to fibrin and to their degradation products within the arterial wall has been reported to be accompanied by atherosclerotic progression. A major step in the pathogenesis of atherosclerosis is the vectorial migration of vascular smooth muscle cells (SMCs) from the arterial media through the internal elastic lamina into the intima and their subsequent proliferation in the intima. I have been studying the effects of fibrinogen, fibrin and their degradation products on the behaviour, particularly migration, of SMCs. Fibrinogen/fibrin stimulates the adhesion and migration of SMCs and their effects are mediated by both the RGD-containing region of the alpha chain of fibrinogen/fibrin and integrin alpha v beta 3 on the cell surface. SMCs migrate into fibrin gel even with no other chemotactic stimuli. SMCs displayed two-fold increase in migration into crosslinked fibrin gels compared to non-crosslinked gels, suggesting the importance of fibrin crosslinking by factor XIIIa on its three-dimensional structure for the migration of SMCs. Fibrin gels prepared with batroxobin, which cleaves only fibrinopeptide A, with ACTE, which cleaves only fibrinopeptide B, and with protamine sulfate, which cleaves nothing, but forms a fibrin-like gel, induce migration of SMCs in a manner similar to the gel prepared with thrombin, suggesting that the cleavage of fibrinopeptides is not involved in the migration of SMCs. Both anti-fibrinogen fragment D and E antibodies inhibit the migration of SMCs into fibrin gel, suggesting that both D and E regions of fibrin are involved in the migration of SMCs into fibrin gel. The migration of SMCs into fibrin gel also depends on the RGD-containing region and integrin alpha v beta 3. Both fibrinogen fragments D and E inhibit the migration of SMCs into fibrin gels, suggesting that these fragments may be involved in the regulation of SMC migration into fibrin gel as the result of fibrinolysis. Although subcultured SMCs usually show a synthetic phenotype, the behaviour of contractile SMCs may be crucial for the subsequent migration of the cells. We employed an in vitro assay system to evaluate the effects of fibrin gels on the migration of SMCs from explants taken from rabbit aorta. alpha v beta 3 integrin and the RGD-containing region are involved in the migration of SMCs into the fibrin gels. SMCs which migrated from the explants showed positive staining with monoclonal antibodies against SMC myosin heavy chain isoforms, SMemb, SM1 and SM2, suggesting that they are in an intermediate state changing from a contractile to synthetic state. These findings show that fibrin (ogen) itself induces adhesion and migration of SMCs without other chemotactic or chemokinetic substances, suggesting a crucial role for fibrin (ogen) in the development and progression of such vascular diseases as atherosclerosis, thrombosis and restenosis following balloon angioplasty.

Animals↗
PubMed2001

Fibrinogen Longmont. A heterozygous abnormal fibrinogen with B beta Arg-166 to Cys substitution associated with defective fibrin polymerization.

B beta Arg166 to Cys substitution was identified in an abnormal fibrinogen named fibrinogen Longmont. The proband, a young woman, and her mother were heterozygous; both experienced episodes of severe hemorrhage at childbirth. The neo-Cys residues were found to be disulfide-bridged to either an isolated Cys amino acid or to the corresponding Cys residue of another abnormal fibrinogen molecule, forming dimers. Thrombin and batroxobin induced fibrin polymerization were impaired, despite normal release of fibrinopeptides A and B. Moreover, the polymerization defect was not corrected by removing the dimeric species or adding calcium. Fibrinogen Longmont had normal polymerization site a, as evidenced by normal GPRP-peptide binding. Thus, the sites A and a can interact to form protofibrils, as evidenced by dynamic light scattering measurements. These protofibrils, however, do not associate laterally in a normal manner, leading to an abnormal clot formation.

Biopolymers↗
PubMedDate not supplied

[Local therapy with platelet-derived growth factors for chronic diabetic ulcers in haemodialysis patients].

BACKGROUND: The increasing number of diabetic patients who need dialysis treatment is the cause for the growing interest of nephrologists in ulcers of the lower limbs. It has been reported that platelet-derived growth factors, such as PDGF and TGF beta 1, are able to heal skin lesions due to diabetes. Therefore, a platelet gel (PG) has been developed to locally treat foot ulcers with a limited number of applications (3+/-1.7). METHOLOGY AND PATIENTS: The PG is a mix of concentrated platelets and cryoprecipitate activated by batroxobin in the presence of calcium chloride (or gluconate) that must be applied to the wound for three days. The surgeon and the dermatologist should then plan successive treatments because the necrotic tissue needs to be removed. We treated a 62-year-old diabetic man, who has been under treatment with insulin since the age of 36 and has been dialysed for three years. His right lower leg was amputated and he developed a chronic left foot ulcer that resulted in many septic episodes. His ulcer recovered in two months after 8 PG applications. CONCLUSIONS: Conventional therapy is not successful in 15-20% of diabetic feet. Uraemia and malnutrition may represent important risk factors for foot ulcers in diabetic patients who need advanced technology to stimulate tissue repair. The use of platelet growth factor may ameliorate ulcer healing thus reducing disease-associated morbidity and lowering health care costs.

Administration, Topical↗
PubMed2002

[Significens of blood platelet CD62p in treating sudden deafness].

OBJECTIVE: To compare different treatment methods for sudden deafness of different blood platelet CD62p levels. METHODS: Blood platelet was measured with flow cytometer. Patients with platelet CD62p over or less than 2% were treated with routine treatment (low molecular weight dextran, ATP, CoA and dexamethasone) or routine treatment plus batroxobin (thrombolysis) separately. RESULTS: In the group with platelet CD62p > or = 2%, the effective rates of routine treatment was 60.5%, and that of routine treatment plus thrombolysis was 84.6%. There was significant difference between them. In the group with platelet CD62p < 2%, the effective rates of two methods were 65.0% and 71.8% respectively(no significant difference between them). CONCLUSIONS: Detecting CD62p in sudden deafness patients makes sense for deciding treatment project.

Adolescent↗
PubMed2002

[Vasodilators for sudden sensorineural hearing loss: a systematic review of randomized controlled trials].

OBJECTIVE: To assess the effects and safety of vasodilators on sudden sensorineural hearing loss(SSHL). SEARCH STRATEGY: Electronic databases: MEDLINE from 1966, EMBASE from 1974, the Cochrane Controlled Trails Register, Chinese Bio-medicine Database from 1989. Hand search: Five kinds of Chinese otolaryngology journals were selected. Literature references were checked intensively. SELECTION CRITERIA: Randomized controlled trials comparing vasodilators with placebo or other drugs in patients with SSHL. DATA COLLECTION AND ANALYSIS: Three reviewers independently accessed the quality of trials and extracted the data. RESULTS: Thirteen trials with 1,155 patients were eligible and covered in the systematic review. Ten of the trials were conducted in developed countries, and three in China. None of the four trials showed the effects of vasodilators on SSHL were better than that of placebo. None of the seven trials comparing different drugs showed the effects of one kind of vasodilators were better than that of the other vasodilators. Two trials showed that some other drugs, such as batroxobin and hypaque were probably better than the vasodilators (dextran, papaverine, 654-2, danshen). Eight trials showed side effects of vasodilators, such as pruritus, allergy, etc. CONCLUSIONS: Based on the systematic review of current eligible randomized controlled clinical trials, there is no evidence to prove that vasodilator therapy is more effective than placebo or other therapies for SSHL, or the effects of one kind of vasodilators to be better than that of the other vasodilators. So far we can't draw a reliable conclusion about the effects of vasodilators for SSHL. In addition, we must pay attention to their potential adverse reactions.

Databases, Bibliographic↗
PubMed2003

Patient-derived fibrin sealant: clinical, preclinical, and biophysical aspects.

Today, there is an enormous interest in surgical sealant, not only for hemostasis, but also for binding of tissues together during surgery, and to improve wound healing. Man has imitated nature in developing fibrin sealant that is biodegradable. However, the risk of transmission of both known and unknown infectious agents can generally not be ruled out completely for plasma products from donors. In addition there is a considerable immunologic risk of using biological products of animal origin. Preclinical and clinical data has demonstrated that a safe and useable surgical fibrin sealant can be prepared from the patient's own blood using the enzyme batroxobin. Experimental data showed that patient-derived fibrin sealant provided enhanced instant adhesion strength and elasticity compared with conventional fibrin sealant due to its faster polymerisation rate. Test methods for fibrin sealant were found to be inaccurate, and we constructed and validated a new computer assisted test method to get information about elasticity and other dynamic properties of biological sealant. The method is highly reproducible and is the first validated using vital human tissue as the adhesion substrate. It takes about one half hour to prepared patient-derived fibrin sealant, which may turn a good operation into a perfect operation without any known risk.

Adhesiveness↗