fibrinopeptide-a and Disease-Models--Animal

The pharmacokinetics of heparin differ markedly from those of its fractions both in man and in experimental animal models. The route of administration determines the relative availability of different molecular species that exert the anti-Xa, anti-IIa, fibrinopeptide A generation inhibiting actions and the release of tissue plasminogen activator-like activity from the endothelial cell lining. The bioavailability of heparin fractions has proved to be much greater than heparin after subcutaneous or intraperitoneal administration. Most of the low molecular weight heparin fractions exhibit sustained antiprotease and antithrombotic actions. The pharmacokinetics of the specific anti-IIa and anti-Xa actions of heparin and its fractions is dependent on the molecular composition of these agents. Even if the fractions are standardized for identical potencies by the in vitro assays, the elimination rate of anti-Xa and anti-IIa actions are significantly different for each fraction. The antithrombotic actions of heparin and its fractions also vary widely in the rabbit stasis thrombosis model. Different fractions show variable antithrombotic actions against defined thrombogenic challenges. Moreover, selection and potency of a thrombogenic agent is of crucial importance in these studies. The primate (Macaca mulatta) model offers a useful preclinical model for the pharmacologic evaluation of the low molecular heparin fractions. Since the coagulation system and heparinizability index of this model approximate a human response, the data may be used to reflect therapeutic and prophylactic responses, as well as to assess toxic effects, such as bleeding.

Topics: Animals; Biological Availability; Bleeding Time; Chemical Phenomena; Chemistry; Disease Models, Animal; Factor X; Factor Xa; Fibrinopeptide A; Heparin; Humans; Kinetics; Macaca mulatta; Molecular Weight; Oligosaccharides; Partial Thromboplastin Time; Polymers; Prothrombin; Rabbits; Structure-Activity Relationship; Thrombosis

Topics: Animals; Antithrombin III; Biological Availability; Biotransformation; Blood Coagulation Tests; Chemical Phenomena; Chemistry; Disease Models, Animal; Drug Evaluation; Drug Stability; Factor X; Factor Xa; Fibrinolysis; Fibrinopeptide A; Hemorrhage; Heparin; Humans; Kinetics; Prothrombin; Pulmonary Embolism; Structure-Activity Relationship; Thrombophlebitis

Engineered recombinant factor X (FX) variants represent a promising strategy to bypass the tenase complex and restore hemostasis in hemophilia patients. Previously, a thrombin-activatable FX variant with fibrinopeptide-A replacing the activation peptide (FX-delAP/FpA) has been described in this regard. Here we show that FX-delAP/FpA is characterized by a sixfold shorter circulatory half-life compared with wild-type FX, limiting its therapeutical applicability. We therefore designed a variant in which the FpA sequence is inserted C-terminal to the FX activation peptide (FX/FpA). FX/FpA displayed a similar survival to wt-FX in clearance experiments and could be converted into FX by thrombin and other activating agents. In in vitro assays, FX/FpA efficiently restored thrombin generation in hemophilia A and hemophilia B plasmas, even in the presence of inhibitory antibodies. Expression following hydrodynamic gene transfer of FX/FpA restored thrombus formation in FVIII-deficient mice in a laser-induced injury model as well as hemostasis in a tail-clip bleeding model. Hemostasis after tail transection in FVIII-deficient mice was also corrected at 5 and 90 minutes after injection of purified FX/FpA. Our data indicate that FX/FpA represents a potential tenase-bypassing agent for the treatment of hemophilia patients with or without inhibitors.

Topics: Animals; Antibodies; Disease Models, Animal; Factor X; Female; Fibrinopeptide A; Genetic Variation; Hemophilia A; Hemostasis; Hepatocytes; Kinetics; Male; Mice; Mice, Inbred C57BL; Microcirculation; Peptides; Protein Domains; Recombinant Proteins; Thrombin

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.

Topics: Animals; Bacterial Adhesion; Disease Models, Animal; Dose-Response Relationship, Drug; Endocarditis, Bacterial; Female; Fibrinopeptide A; Humans; Lacticaseibacillus rhamnosus; Lactobacillus; Peptides; Probiotics; Rats; Rats, Wistar; Species Specificity; Virulence

Forced swimming is considered a depression model. Repeated electroconvulsive shock treatment shows an anti-depressive effect in mice forced swimming. In serum of the mice treated with repeated electroconvulsive shock, the humoral anti-depressive factors were detected. The factors were the glycolipid having GalNAc alpha1-3GalNAc and mouse fibrinopeptide A having amino acid sequence TDTEKDGEFLSGGGV. The behavioral anti-depressive activity of the glycolipid was decreased by the low doses (100 to approximately 10 microg/kg) of dopamine 2 receptor antagonist sulpiride. Behavioral activity of the peptide was also decreased by the low doses (100 to approximately 1 microg/kg) of dopamine 1 receptor antagonist SCH-23390. The present findings clearly indicate that repeated electroconvulsive shock treatment induces the humoral anti-depressive factors affecting the dopaminergic neuronal system in mice.

Topics: Amino Acid Sequence; Animals; Antidepressive Agents; Depression; Disease Models, Animal; Electroconvulsive Therapy; Electroshock; Fibrinopeptide A; Glycolipids; Male; Mice; Molecular Sequence Data

Topics: Animals; Anticoagulants; Artifacts; Blood Coagulation Factors; Blood Specimen Collection; Disease Models, Animal; Fibrinopeptide A; Rats; Records; Reproducibility of Results; Research Design; Thrombosis

Disseminated intravascular coagulation (DIC) may lead to severe thrombotic or hemorrhagic complications. The present work was undertaken to study the effect of interleukin 6 (IL-6) on variations of key coagulation and fibrinolytic parameters in plasma in a baboon model of experimental DIC induced by injection of factor Xa and phospholipids at dosages leading to partial (48%) or complete fibrinogen depletion. Transient increases of D-dimer, fibrinopeptide A, thrombin-antithrombin and the activated partial thromboplastin time were observed. Each parameter had a particular (time and Xa/phospholipid dose dependent) pattern of changes. The principal effect of IL-6 was a more rapid restoration of fibrinogen concentrations and of overall coagulation tests. Injection of factor Xa/phospholipids led also to a rapid increase of tissue-type plasminogen activator (t-PA) the extent of which was dependent on Xa/phospholipid dose. Pretreatment with IL-6 induced a threefold increase of basal t-PA and a corresponding increase of the t-PA response. Plasminogen activator inhibitor type 1 (PAI-1) concentrations did not change after low dose Xa/phospholipids, but increased eightfold after high dose Xa/phospholipids, IL-6 pretreatment induced within 8 h a twentyfold increase of PAI-1 but no further increase was observed after injection of factor Xa/phospholipids. Thus, in vivo thrombin generation leads to dynamic modifications of the coagulation and fibrinolytic systems. The principal effect of IL-6 is a more rapid normalization of overall coagulation tests, due to normalization of fibrinogen, and an increased t-PA release response which is partially counteracted by increased PAI-1 concentrations.

Topics: Animals; Antithrombin III; Blood Coagulation; Disease Models, Animal; Disseminated Intravascular Coagulation; Factor Xa; Fibrin Fibrinogen Degradation Products; Fibrinolysis; Fibrinopeptide A; Interleukin-6; Papio; Partial Thromboplastin Time; Peptide Hydrolases; Phospholipids; Plasminogen Activator Inhibitor 1; Recombinant Proteins; Thrombin; Time Factors; Tissue Plasminogen Activator

Rethrombosis limits the efficacy of coronary thrombolysis and may result from surface-associated thrombin, de-novo prothrombin activation, or both. This study was designed to determine the relative roles of thrombin, factor Xa, and the complex of tissue factor and factor VIIa in the procoagulant activity on injured arteries with evolving thrombi.. Extensive vascular injury and platelet-rich thrombi were induced in the abdominal aorta of 25 anesthetized rabbits by applying anodal current through a transluminal electrode for 3 h. Injured vessel segments were excised and placed in a chamber permitting perfusion over the luminal surface and associated thrombus.. Vessel segments perfused with recalcified, citrated human plasma induced marked increases in the concentration of fibrinopeptide A, a marker of thrombin-induced fibrin formation, in the effluent plasma after 10 min (4636 +/- 1894% of fibrinopeptide A in the nonperfused plasma, n = 5). Perfusion with plasma depleted of vitamin K-dependent coagulation factors prevented the increase in fibrinopeptide A (122 +/- 30%, n = 4), indicating the lack of preformed functional thrombin. Furthermore, appearance of fibrinopeptide A was attenuated by perfusion with plasma containing 0.1 mumol/l recombinant tick anticoagulant peptide, a specific inhibitor of factor Xa (594 +/- 320%, n = 3), and by preincubation of vessel segments with a monoclonal antibody to rabbit tissue factor (438 +/- 220%, n = 3).. Procoagulant activity on injured vessels and associated thrombi is mediated by factor Xa, a product of the functional initiation of coagulation by factor VIIa associated with tissue factor. Accordingly, inhibition of tissue factor-mediated coagulation may be effective for attenuation of active thrombogenesis on injured vessels and during thrombolysis.

Topics: Animals; Aorta, Abdominal; Blood Coagulation; Coronary Thrombosis; Disease Models, Animal; Factor VIIa; Factor Xa; Fibrinopeptide A; Humans; Microscopy, Electron, Scanning; Prothrombin; Rabbits; Thrombin; Thrombolytic Therapy; Thromboplastin; Thrombosis

Modification of hemoglobin (Hb) by crosslinking and polymerization results in an improved oxygen release capacity and a prolonged vascular retention time. Modification improves the efficacy and prevents certain side effects. It eliminates leakage of Hb through the kidneys and accumulation in the tubuli. Another important issue is the degree of purification of Hb solutions. Traces of membrane fragments may cause immunogenic and thrombogenic side effects. To determine the contamination with erythrocyte membrane fragments, we developed assays for glycophorin-alpha and phospholipids. Special models were evaluated for testing the maximum allowable level of membrane contamination. As an in vitro model for thrombogenicity we used confluent monolayers of human umbilical vein endothelial cells. These cells were incubated with Hb solutions and subsequently tested on tissue factor (TF) procoagulant activity. TF was tested by the factor VII-catalyzed activation of factor X. The lower detection limit of this assay for endotoxin was 0.5 ng/ml. Hb did not cause any tissue factor expression even after prolonged incubation. No cooperation was found within endotoxin. As an in vivo test on thrombogenicity we developed a guinea pig model in which we can follow the generation of fibrinopeptide A (FPA). This is one of the most sensitive markers for thrombin activation in vivo. When slightly contaminated Hb solutions (phospholipid content 2 nmol/ml) were infused in the presence of factor Xa at a dose (9 micrograms/kg) which in itself did not induce FPA generation, we observed an increase in FPA levels in the plasma from 1.2 +/- 0.4 ng/ml to 5.2 +/- 0.7 ng/ml. Factor Xa is used to mimic a stressed clinical condition with activated coagulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Blood Substitutes; Cells, Cultured; Disease Models, Animal; Endothelium, Vascular; Exchange Transfusion, Whole Blood; Fibrinopeptide A; Guinea Pigs; Hemoglobins; Humans; Models, Biological; Solutions; Thrombosis

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.

Topics: Amino Acid Sequence; Animals; Arenaviridae Infections; Disease Models, Animal; Fibrin Fibrinogen Degradation Products; Fibrinopeptide A; Guinea Pigs; Hemorrhagic Fevers, Viral; Hemostasis; Molecular Sequence Data; Radioimmunoassay; Thrombin

A non-stasis rodent model of thrombogenicity has been used for dose-ranging studies with a conventional prothrombin complex concentrate (PCC) and to evaluate high purity factor IX concentrates from different manufacturers. Fibrin monomer (soluble fibrin) and fibrinopeptide A (FPA) were monitored before and after infusion of test solution. FPA was found to be the more sensitive and reproducible indicator of thrombogenicity and exhibited a dose-related elevation after infusion of the PCC at doses of between 100-300 IU/kg. In contrast the amounts of FPA generated after 300 IU/kg of the high purity factor IX products were similar to control infusions of albumin.

Topics: Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Factor IX; Female; Fibrin; Fibrinopeptide A; Prothrombin; Rats; Rats, Sprague-Dawley; Thrombosis

Artificial insemination with frozen semen was used to breed beagles homozygous for a deficiency in clotting factor VII, with plasma concentrations of 2-3% of normal adult beagle pooled plasma. The factor VII-deficient beagles were used to determine if factor VII activity has a role in the thrombogenic activity of prothrombin complex concentrates (PCCs) containing factors II, IX and X by evaluating changes in selected indicators of thrombogenicity. No differences were seen between the responsiveness of the factor VII-deficient and normal beagles, indicating that the thrombogenic activities of PCCs are mediated via effects on the intrinsic clotting system.

Topics: Animals; Blood Coagulation Factors; Disease Models, Animal; Dogs; Dose-Response Relationship, Drug; Factor VII Deficiency; Fibrin Fibrinogen Degradation Products; Fibrinopeptide A; Partial Thromboplastin Time; Thrombosis

The development of atherosclerotic lesions involves many cell types, including macrophages. Fibrinopeptide B (FPB) has been shown to be a potent chemotactic agent for macrophages, which are abundant as intimal foam cells in atherosclerotic lesions, especially in cholesterol-fed rabbits. We hypothesize that intimal low-density lipoproteins also cause fibrinogen in the intima to release FPB and that FPB attracts macrophages in response to the high lipid levels associated with lesion development. To test our hypothesis, we used an atherosclerotic model. Silk sutures containing either FPB, fibrinopeptide A (FPA), lipopolysaccharide (LPS), or saline control were prepared. One suture of each type was placed in the adventitia of the femoral artery of a rabbit. Animals were killed at 1 or 2 weeks. Only vessels exposed to either FPB or LPS showed significant intimal thickening in the region adjacent to the suture site. Semi-thin electron microscopic sections indicated that the intimal wall was highly cellular and that many cells contained lipid vacuoles after 2 weeks. These sections also showed that the endothelium remained intact and that no injury to the media of the artery had occurred. Electron microscopy of the tissue samples showed the proliferation of smooth muscle cells and deposition of extracellular matrix in the 2-week animals, whereas foam cells were present in the 1-week animals. We conclude that FPB does indeed attract macrophages to the intima and that these macrophages may become foam cells. The model we have developed can be used to study possible mechanisms for the entry of macrophages into the intima during early lesion development and to further understand the complex interactions of FPB, fibrinogen, and lipids in atherosclerotic lesion development.

Topics: Animals; Arteriosclerosis; Disease Models, Animal; Endothelium, Vascular; Fibrinogen; Fibrinopeptide A; Fibrinopeptide B; Foam Cells; Lipopolysaccharides; Rabbits

Coagulation abnormalities associated with severe pancreatitis were studied in 24 dogs. Group I consists of six control subjects who had duodenotomy alone. Group II consists of six dogs with pancreatitis induced by bile injection ( lcm3 /kg) into the pancreatic duct. The six dogs in Group III and the six in Group IV were given aprotinin (trasylol) 1.0 mg/kg and S-2441 (10mg/kg), a new synthetic protease inhibitor, respectively. These were given over 10 minutes by intravenous infusion, 20 minutes after bile induced pancreatitis. Blood was drawn for amylase, prothrombin time (PT), partial thromboplastin time (PTT), fibrinogen, and platelets, in addition to markers for hypercoagulation, fibrinopeptide A, antithrombin III, and markers for fibrinolysis, B beta 15-42 immunoreactive peptides and alpha 2 antiplasmin at baseline, 30 minutes, 1 hour, 3 hours, 6 hours, and daily for 3 days after injection of bile or duodenotomy. There was no significant difference in PT, platelets, antithrombin III, and fibrinopeptide A among the four groups. Fibrinogen levels and PTT were minimally elevated in animals with bile induced pancreatitis, but these changes reached significance only at 24 hours and 48 hours, respectively (P less than 0.05). Immunoreactive B beta 15-42 became elevated at 30 minutes indicating fibrinolysis in animals with pancreatitis, and these changes were significant compared with Group I control subjects (P less than 0.05) throughout the study. Levels of alpha 2 antiplasmin were decreased in Group II animals with pancreatitis, which also suggests fibrinolysis. Amylase was elevated in Group II animals with pancreatitis (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acute Disease; alpha-2-Antiplasmin; Amylases; Animals; Antithrombin III; Aprotinin; Blood Coagulation; Disease Models, Animal; Dogs; Fibrin Fibrinogen Degradation Products; Fibrinogen; Fibrinolysis; Fibrinopeptide A; Oligopeptides; Pancreatitis; Peptide Fragments; Platelet Count; Protease Inhibitors; Prothrombin Time

Topics: Animals; Blood Coagulation; Cross Reactions; Disease Models, Animal; Fibrinogen; Fibrinopeptide A; Half-Life; Hematoma; Humans; Models, Biological; Radioimmunoassay; Rats; Species Specificity