Administration Of Thrombin Research Articles

Effects of Sclerosing Agents on the Local Blood Flow of the Mesenteric Vein in Rabbits

Abstract: To investigate the pathophysiology of scleroembolization as a treatment for esophageal varices, studies were made of the effects of the intravenous administration of Aethoxysklerol (AS), Ethanolamine Oleate (EO), ethanol and thrombin on the local blood flow of the mesenteric vein, the systemic hemodynamics and thrombus formation in blood vessels in rabbits.The dosages and concentrations of Aethoxysklerol used were from 0.5 to 2 ml and from 0.5% to 2%, respectively. This was done to ascertain the possible relationships of these variances of Aethoxysklerol on the local blood flow in vessels with a low‐flow and a high‐flow.99% ethanol, 50 U thrombin and 5% Ethanolamine Oleate were also administered into low‐flow and high‐flow vessels in doses of 0.5 ml and 1 ml, respectively.The increase in AS concentration from 0.5% to 2% caused no remarkable changes in the degree of blood flow inhibition in vessels with either a low‐or a high‐flow, but the time required for inhibition was 45–50 min in vessels with a high flow, which was longer than the 25 min necessary in vessels with a low‐flow. The increase in AS dosage from 0.5 to 2 ml was negligible for enhancing the inhibitory effect of the drug upon the blood flow, but the blood flow inhibition was significantly increased in the low‐flow vessels compared with the high‐flow vessels even when the AS was administered in the same volume and concentrations.The AS‐induced changes on the systemic blood pressure were minimal even with high dosages and high concentrations.99% ethanol and thrombin were less effective in blood flow inhibition in both the low‐and the high‐flow vessels. 5% Ethanolamine Oleate when administered in a volume of either 0.5 ml or 1 ml significantly inhibited the flow in the low‐flow vessels as compared to the flow in the high‐flow vessles.On the other hand, when AS was administered with thrombin, the inhibitory effects were accelerated to 88±26% in the low flow vessels and 44±16% in the high‐flow vessels as compared with administration of AS alone in both the low‐flow and the high‐flow vessels, respectively.We concluded that the embolizing effect of AS alone is rather weak, although it is a relatively safe drug. We consider it wise to combine it with thrombin.

The role of nitric oxide as an endogenous regulator of platelet and neutrophil activation within the pulmonary circulation of the rabbit.

1. Intravenous (i.v.) administration of adenosine diphosphate (ADP), platelet activating factor (PAF) and thrombin induced a dose-related accumulation of 111indium-labelled platelets within the thoracic region of anaesthetized rabbits. 2. I.v. administration of the inhibitor of NO biosynthesis, L-NG-nitro arginine methyl ester (L-NAME; 10 mg kg-1) significantly potentiated the peak platelet accumulation induced by ADP, PAF and thrombin. Additionally L-NAME prolonged the disaggregation of platelets in comparison to D-NAME (10 mg kg-1). Such changes were reversible by the administration of L-arginine (900 mg kg-1). 3. I.v. administration of PAF induced a small accumulation of 111indium-labelled neutrophils within the pulmonary circulation which could be greatly potentiated by pretreatment of the animals with L-NAME. In contrast, thrombin administration did not cause significant accumulation of 11indium-labelled erythrocytes in the pulmonary circulation of anaesthetized rabbits. 4. Intracarotid (i.c.) administration of thrombin induced a marked accumulation of radiolabelled platelets within the cranial vasculature which was not potentiated by the prior administration of L-NAME (at either 10 mg kg-1 or 100 mg kg-1). 5. These results suggest that endogenous NO may regulate platelet and polymorphonuclear leukocyte activation within the pulmonary but not the cerebral circulation of rabbits.

Dyspnea in aging rats due to disseminated intravascular coagulation (DIC).

During an 18-month oncogenicity study using rats, approximately 10% of the animals developed a form of respiratory distress very similar to that seen in the terminal stages of chronic respiratory disease, commonly associated with Mycoplasma pulmonis infection. Investigation of the lungs of the affected rats revealed not only that they did not have the consolidation usually associated with chronic respiratory disease, but they also appeared macroscopically normal. Further investigation of a number of cases revealed systemic intravascular thrombus formation of the type usually referred to as disseminated intravascular coagulation. Using an antiserum to fibrin we have demonstrated the presence of intravascular fibrin deposits in the lungs of the affected rats and have shown them to be the same as experimentally induced intravascular fibrin deposits induced in rat lungs by the administration of thrombin after blocking the fibrinolytic system. This is the first example of such a phenomenon being recorded in aging rats.

Cardiovascular adjustments to pulmonary vascular injury in dogs.

The hemodynamic effects of blood volume augmentation and mechanical ventilation (MV) with positive end-expiratory pressure (PEEP) were studied in nine Beagles anesthetized with halothane before and after thrombin-induced pulmonary hypertension. The effect of therapy with dopamine, norepinephrine with and without nitroglycerin (NTG), and intraaortic balloon pumping (IABP) were studied in a second series of six Beagles. Before thrombin, dextran (35 ml.kg-1) caused a significant increase in right and left ventricular end-diastolic and end-systolic volumes (RV and LVEDV, and RV and LVESV). However, RV and LV performance, as estimated by ejection fraction, was unchanged during volume loading and MV with PEEP when the pulmonary vasculature was intact. The response to volume loading and MV with PEEP was altered significantly once PVR had been increased with the administration of thrombin. Stroke volumes were decreased, and remained so, despite volume loading and MV with PEEP. LVEDV decreased without a decrease in LVEDP, indicating a decreased LV compliance. Dopamine and norepinephrine with and without NTG increased stroke volumes and RV ejection fraction in contrast to IABP. Assessment of LV performance, according to the Frank-Starling mechanism, requires a measure of end-diastolic volume when diffuse pulmonary vasoconstriction leads to RV distension and LV hypovolemia secondary to septal shift. Measurement of LV filling pressures can provide misleading values to estimate changes in LV volume in this setting. Measurement of ventricular volumes is required for optimal management of patients with severe acute respiratory failure and pulmonary hypertension.

Effect to coagulation-fibrinolysis and platelet function on local administration of thrombin to tonsillar bed

Effect to coagulation-fibrinolysis and platelet function on local administration of thrombin to tonsillar bed

Prostaglandin I 2 (PG I 2) fails to inhibit ADP induced platelet aggregation in vivo in rats

There is a simple method of inducing cardiopulmonary dyfunction in rats through platelet aggregation. after rapid i.v. administration of ADP and thrombin. Infusion of 25 ng/min/kg PGI 2 completely inhibited the decrease of both breathing and heart rate induced by 30 IU/kg thrombin. The same PGI infusion failed to influence the cardiopulmonary effects of the different ADP doses (between 75 and 0.1 mg/kg). Some-possible reasons for the differential effectiveness of PGI 2 are discussed on the basis of the modes of action of ADP, thrombin, and PGI 2. The results stress the necessity of testing antiaggregatory substances in in vivo models.

Plasminogen activator release from the perfused isolated dog leg by thrombin

Vascular endotherial cells release plasminogen activator (plg-act) which is thought to play a key role in maintaining the fluidity of circulatory blood. It was reported that endotherial cells had thrombin receptors, so the present study was performed to evaluate the effect of purified thrombin on plg-act release from the vascular wall of the perfused hind leg of dog with physiological solution. Plg-act activity was measured using a standard fibrin plate. When 5units/ml thrombin (5ml) was administered to the perfused hind leg, plg-act was released within 5 sec. and reached to the peak activity at 15-30 sec. A dose response of plasminogen activator activity to thrombin was also detected to a certain extent (1units/ml-50units/ml). The peak activity of released plg-act after the administration of thrombin (5units/ml) was in the range of 0.38 CTA units/ml-0.44 CTA units/ml. The effects of DFP-thrombin and TLCK-thrombin, which are inactive in fibrinogen-fibrin conversion, on plg-act release were examined. The two sorts of chemically modified thrombin did not release plg-act. Furthermore, the effect of acetylated thrombin was also examined. The acetylated thrombin did not cause plg-act release. These results obtained indicate that thrombin releases plg-act from the vascular wall so long as thrombin maintains the intact active center of fibrinogen-fibrin conversion.

Effect of fibrin degradation products and thrombin on fibrinogen synthesis.

The effect of intravenous administration of homologous fibrin degradation products and thrombin on fibrinogen synthesis was assessed in rabbits. The relative fibrinogen synthesis rate was calculated as a ratio of the amount of radiolabelled lysine incorporated into fibrinogen to the amount incorporated into albumin during the same measurement period. An increase in this ratio above control would indicate a relatively specific stimulation of fibrinogen synthesis as compared with albumin, which is not an acute-phase reactant. Injection of 45 mg of 'early' or 'late' fibrin degradation products failed to produce a significant increase in the relative fibrinogen synthesis rate, suggesting that fibrin degradation products play no feedback role in controlling fibrinogen synthesis. Infusion of small amounts of homologous thrombin (15--25 NIH u) was followed by a small but statistically significant elevation of the relative fibrinogen synthesis rate. This was not accompanied by any increase in the levels of fibrinogen degradation products in plasma, or by any decrease in plasma fibrinogen concentration, possibly suggesting that thrombin can stimulate fibrinogen synthesis by a mechanism independent of significant fibrinogenolysis or intravascular coagulation.

Studies on Antithrombin III (AT III)

Changes in canine AT III metabolism after i. v. administration of bovine thrombin were studied using I-125-AT III. I-125-AT III was prepared by the method of McFarlane.(1) On the second day after intravenous injection of I-125-AT III, bovine thrombin (30u/kg/hr) was infused continuously for 3hrs. Plasma fibrinogen level was decreased to 60% of initial level, while plasma AT III concentration and AT III metabolism did not change markedly. AT III. metabolism in the dogs infused larger amount of thrombin was studied. Total amount of 5, 000-15, 000u of thrombin was administered intravenously with simultaneous injection of the same units of heparin. Plasma concentration of AT III and the radioactivity of I-125-AT III decreased to 70-80% of the initial levels, although plasma concentration of fibrinogen did not change significantly.(2) When, 50μg of I-125-AT III, 1, 000u of bovine thrombin and 1, 000u of heparin were injected simultaneously, the half life of 1st phase of the plasma disappearance curve of I-125-AT III was shortend to 1.6 hours compared to the normal value of 5.5 hours.From these data, it is suggested that AT III forms a complex with thrombin and thereafter the AT III-thrombin complex is cleared out rapidly from blood stream.

Influence of dihydroergotoxine on platelet aggregation in rabbits

Adrenaline potentiates the thrombin-induced aggregation of rabbit blood platelets in platelet-rich citrated plasma in vitro. In rabbits, simultaneous administration of adrenaline and thrombin results in a decrease in platelet count and the appearance of fibrin monomer. The adrenaline-potentiated effect of thrombin is prevented in vitro and in vivo by previous administration of dihydroergotoxine.

Fibrinogen synthesis in rabbits: effects of altered levels of circulating fibrinogen.

The effects of altered fibrinogen concentrations on fibrinogen synthesis in rabbits were evaluated by determining the rate of appearance of [75Se]selenomethionine (75SeM) in circulating fibrinogen. Fibrinogen levels were maintained at twice normal by infusion of homologous fibrinogen for either 1 or 6 days before the intravenous injection of 20 micronCi of 75SeM, and the rate of appearance of labeled fibrinogen was measured during the subsequent 24 h. In both groups, synthesis was unchanged. Five hours after induction of partial defibrinogenation by the infusion of bovine thrombin, fibrinogen synthesis was increased threefold. Stimulation was not attributable to decreased fibrinogen concentrations; synthesis was increased equally when levels were maintained above normal by infusion of fibrinogen before administration of thrombin. Heat-inactivated thrombin and diisopropylfluorophosphate-inactivated thrombin did not stimulate fibrinogen synthesis. Thrombin produced elevated titers of fibrinogen-fibrin degradation products (FDP-fdp). However, fibrinogen synthesis was not increased in rabbits that had received FDP-fdp from thrombin-treated donors. These data suggest that neither the fibrinogen concentration nor FDP-fdp influenced fibrinogen synthesis.

Semiquantitative Determination of Soluble Fibrin Monomer Complexes by Chromatography and Serial-dilution Protamine Sulfate Test

Plasmas were prepared from blood obtained from rabbits and from healthy individuals and patients with thrombotic disorders. Gel filtration (Sepharose-4B) chromatography and the serial-dilution protamine sulfate (SDPS) test were performed on each sample. For healthy individuals and rabbits, a narrow fibrinogen peak was obtained on chromatogram and was associted with a negative SDPS test. After administration of thrombin or endotoxin to rabbits and in plasmas from patients with intravascular coagulation, major shifts to earlier effluent volumes occurred. These higher-molecular-weight derivatives corresponded to soluble fibrin monomer complexes (FM). Positive SDPS tests were found in these plasmas. There was a highly significant correlation between percentage area occupied by FM on chromatogram and SDPS test titer. It is concluded that the SDPS test is a reliable semiquantitative measure of FM that can be used clinically as well as in experimental animal models.

The role of fibrin monomer and an in vivo thrombin-induced-anticoagulant in experimental venous thrombosis.

The functional role of soluble fibrin monomer (FM) complexes in vivo is not known and the clinical significance of its laboratory measurement unclear. The effect of FM on the development of experimental venous thrombosis was evaluated. Laboratory determination of FM by ethanol gelation and protamine sulfate paracoagulation in the presence of serum‐induced thrombosis was performed. FM in vivo was formed by the administration of thrombin and by the infusion of soluble fibrin. FM was found not to significantly potentiate thrombus formation in this experimental model. Furthermore, extensive thrombosis occurred without demonstrable circulating FM. The results suggest that the presence of FM in the blood is not a reliable measure of incipient venous thrombosis. The laboratory demonstration of FM in association with deep vein thrombosis probably reflects the result and not the cause of the thrombus. During this study, a potent anticoagulant and antithrombotic activity induced by thrombin in vivo was demonstrated. Some of the experimental results were related to this activity. Preliminary studies of this activity are presented and its pathophysiologic significance considered.

FORMATION OF HYALINE MICROTFIROMBI IN THE MOUSE

Small, globular, eosinophilic bodies, similar in appearance to the hyaline microthrombi (HMT) previously reported in man, were found regularly in the mouse liver. The occurrence of HMT in the mouse liver was subjected to quantitative analysis. In man, HMT are a frequent finding in bronchopneumonic patients. Intranasal inoculation of pneumococci gave bronchopneumonia in mice, but HMT formation was not influenced. Inhibition of plasminogen activation by epsilon‐amino‐caproic acid reduced significantly the frequency of HMT. Activation of plasminogen by urokinase more than doubled the frequency of HMT. The difference from findings in the controls was highly significant. Heparinization did not significantly affect HMT formation. Furthermore, intravenous thrombin injections showed no significant effect on HMT formation, but more than 50 per cent of the injected animals showed platelet‐fibrin thrombi in pulmonary vessels. Administration of thrombin after urokinase reversed the increase in HMT found after urokinase alone. Possibly, HMT or their precursors combine with newly formed fibrin and are removed from the circulation. It is concluded that HMT are formed as a result of fibrinolytic activity, possibly through spontaneous aggregation of fibrinogen split products.

The Effect of Ellagic Acid on the Thrombin-Induced Hemorrhagic Syndrome in the Rat

Abstract The intraperitoneal administration of thrombin increased the bleeding due to the amputation of the tail in the rat (average blood loss 4.6 ml.). The intravenous administration of ellagic acid in thrombin-injected rats reduced the average blood loss to 0.6 ml. This value was slightly more than the average blood loss noted in animals given only ellagic acid (0.2 ml.). Both these figures were much less than the average blood loss observed in control animals (3.2 ml.).

Hemorrhagic Syndrome in Dogs Induced by Intravenous Thrombin

SummaryThe intravenous administration of thrombin (70 N. I. H. u/kg) caused in the dog a decrease in platelets, factor V, factor VIII and fibrinogen, a prolongation of glass and silicone clotting times, prothrombin and partial thromboplastin times, decreased prothrombin consumption and defective thromboplastin generation. This resulted in a generalized bleeding syndrome. Increased fibrinolysis occurred after an initial phase of inhibition of fibrinolysis. It is suggested that this hemorrhagic syndrome is similar to some conditions observed clinically.

The behaviour of platelets in vivo.

Observations have been made on platelet behaviour in non‐inflamed and growing blood vessels in the rabbit ear chamber. Platelets showed little tendency to aggregate or to adhere to endothelium. Most commonly a few adhered to leucocytes which were attached to or rolled along the endothelium.Platelets adhered to leucocytes in larger numbers and aggregated to form microthrombi and emboli following mild trauma and intravascular injection of adenosine diphosphate, Pelikan ink and methyl cellulose. Following intravascular administration of thrombin and bacterial lipopolysaccharides (S. typhosa and E. coli) platelet aggregation was observed and later hyaline thrombi. In addition, aggregation of erythrocytes occurred with methyl cellulose.

Effect on Coagulation Time of Oral Administration of Rabbit Thrombin

Parfentjev1 recently described a method for the preparation of pseudoglobulin of rabbit plasma having a high clot-promoting activity. Parfentjev's preparation has been repeated and the activity associated with this pseudoglobulin was found to be thrombic in nature.2 This was indicated by the ability of the pseudoglobulin fraction to coagulate pure fibrinogen solution in the absence of calcium and to coagulate citrated and oxalated plasma from which pro-thrombin had been removed. Evidence indicating that this material may have clinical applications as a powerful hemostatic when applied locally to small wounds has also been presented.3The present communication reports the effect of the oral administration of relatively large amounts of pseudoglobulin from rabbit plasma, hereafter referred to as “rabbit thrombin”, to dogs, normal subjects and patients suffering from hemophilia.Methods. Coagulation times were determined on venous blood by the standard procedure formerly described.4 In certain instances the bl...