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PubMed1975

Plasmin inhibition of thrombin-induced platelet aggregation.

The effects of plasmin treatment upon washed human platelets were studied in an attempt to elucidate the mechanisms underlying thrombin-induced platelet aggregation. At calcium concentrations of 10-20 muM, PLASMIN (0.2 CTA U/ml) inhibited thrombin-induced aggregation almost completely, but did not diminish the thrombin-induced release of adenine nucleotides, 5-hydroxytryptamine, or calcium. Increasing the calcium concentration partially antagonized plasmin's inhibition of aggregation. Studies utilizing calcium chelators and the Kunitz soybean trypsin inhibitor (SBTI) as a plasmin inhibitor indicated that in order to achieve maximal block of aggregation, plasmin must act upon a substrate made fully available only after an initial thrombin-platelet interaction has taken place. Moreover, the time course of this inhibition parallels the time course of the thrombin-induced release reaction. Plasmin inhibition of aggregation could not be mimicked by exposing the platelets to proteolytic digests of fibrinogen at concentrations as high as 17% total platelet protein. Nor could inhibitory activity be recovered from supernatants of plasmin-treated platelets, upon centrifugation and treatment with SBTI. With the use of a "cold initiation" technique, the release by thrombin of 46.7 plus or minus 6.7 (mean plus or minus SEM) mu-g of fibrinogen immunological equivalents per mg platelet protein could be demonstrated. Platelets in which thrombin-induced aggregation was abolished by plasmin treatment (and the plasmin subsequently inactivated by STBI) aggregated normally upon addition of as little as 10 mu-g human plasma fibrinogen per mg platelet protein. It is concluded that plasmin inhibition of aggregation most likely results from its attack upon a protein that is released or becomes fully available subsequent to interaction of thrombin with a platelet receptor mediating release. The results of this study are consistent with a cofactor role for fibrinogen in the aggregation of human platelets by thrombin.

Adenine Nucleotides↗
PubMed2003

Antithrombotic activity of LB30057, a newly synthesized direct thrombin inhibitor.

An amidrazonophenylalanine derivative, LB30057, inhibits the catalytic activity of thrombin potently by interaction with the active site of thrombin, and has high water solubility. In the present study, we evaluated the effect of LB30057 on the biological activities of thrombin at various tissues, and determined whether thrombin inhibition by LB30057 could reduce the incidence of occlusive thrombosis in an in vivo animal model. Treatment with LB30057 to human plasma prolonged clotting times in a concentration-dependent manner. LB30057 suppressed significantly thrombin-induced phosphatidylserine (PS) exposure in platelets, suggesting that LB30057 could inhibit blood coagulation accelerated by PS exposure. In human platelets, soluble thrombin- and clot-induced platelet aggregation was inhibited by LB30057 potently. Consistent with this finding, LB30057 showed concentration-dependent inhibitory effects on serotonin secretion and P-selectin expression induced by thrombin in platelets. In the blood vessel isolated from the guinea pig, treatment with LB30057 resulted in a concentration-dependent inhibition of thrombin-induced vascular contraction. In vivo study revealed that LB30057 following oral administration significantly increased the time to occlusion and improved carotid arterial patency using rat carotid artery thrombosis model. All these results suggest that LB30057 is a potent inhibitor of biological activities of thrombin at various target tissues and, therefore, might be developed as an antithrombotic agent for treatment and prevention of thrombotic diseases.

Administration, Oral↗
PubMedDate not supplied

[Human thrombin: enzymatic properties, stability and standardization of preparation].

The work deals with estimation of thrombin preparation having such features as: sedimentation activity 3000-3200 NIH un. per 1 mg of protein and 97% of active centres. The enzyme isolated has been estimated according to the amidolytic activity on synthetic substrates S-2160 and BAPNA being equal 5200 and 185 milli un/mg of protein, respectively. According to the electrophoresis in PAAG in the presence of Ds-Na the preparation is homogenous, its molecular mass is 36000. The fibrinogen sedimentation time dependence on the isolated thrombin concentration has been estimated as well as the comparative analysis with the thrombin of the firm "Sigma" with the previously calibrated activity using the international standartion (coded P4) has been conducted. The absence of proportionality between the substrate sedimentation time and the preparation concentration has been determined. It has been revealed, that if the experimental findings are presented in the units 1/t against the thrombin units NIH the right lines are received within the limits used. The defreezing and secondary freezing of the preparation preserved under -20 degrees C have been showed as rendering an essential effect on thrombin activity. In order of the enzyme stabilizing at preserving the thrombin isolated has been concentrated applying the amycon membranes (MWCo: 30,000). While applying the thrombin water-saline solution in the conditions selected the preparation has showed itself practically stable during a year without utilizing any admixtures. The essential effect on thrombin has been found from the side of 1% glycin, 0.5% PEG, 1% saccharose and so on. The thrombin isolated high functional homogeneity, its stability permit to recommend the preparation as an operative standard.

Binding Sites↗
PubMed1992

Synergistic induction of plasminogen activator inhibitor type-1 in HEP G2 cells by thrombin and transforming growth factor-beta.

Plasminogen activator inhibitor type-1 (PAI-1) is a physiologic modulator of the fibrinolytic system. Its activity in plasma increases in diverse thrombotic states. The large synthetic capacity of the liver make it a source of potentially large amounts of PAI-1. Because thrombin activity increases in association with thrombotic disorders and because specific binding sites for thrombin have been identified on hepatocytes, we characterized the effect of thrombin on hepatocyte PAI-1 production. Incubation of Hep G2 cells with human alpha-thrombin resulted in a dose- and time-dependent increase in the concentration of PAI-1 in conditioned media. This effect was inhibited completely by hirudin and by antithrombin III. Steady-state levels of both the 3.2-kb and 2.2-kb forms of PAI-1 mRNA increased after stimulation of the cells with thrombin, indicating that thrombin influences PAI-1 expression in Hep G2 cells at the pretranslational level. Incubation of Hep G2 cells with alpha-thrombin and either platelet lysates or purified transforming growth factor-beta (TGF-beta), both previously shown to augment hepatocyte PAI-1 expression, resulted in a synergistic increase in the concentration of PAI-1 in conditioned media. PAI-1 mRNA appeared to be synergistically increased as well. Thus, thrombin increases expression of both PAI-1 protein and mRNA in Hep G2 cells and exerts synergistic effects with TGF-beta. These results underscore the potential importance of inhibition of thrombin under conditions in which thrombolysis is induced pharmacologically.

Antithrombin III↗
PubMed1992

Alpha-thrombin stimulates nuclear diglyceride levels and differential nuclear localization of protein kinase C isozymes in IIC9 cells.

The mechanism by which an agonist, binding to a cell surface receptor, exerts an effect on events in the nucleus is not known. We have previously shown (Leach, K. L., Ruff, V. A., Wright, T. M., Pessin, M. S., and Raben, D. M. (1991) J. Biol. Chem. 266, 3215-3221) that alpha-thrombin treatment of IIC9 cells results in increased levels of cellular 1,2-diacylglycerol (DAG) and activation of protein kinase C (PKC). Here, we have examined whether changes in nuclear PKC and nuclear DAG also are induced following alpha-thrombin treatment. IIC9 cells were treated with 500 ng/ml alpha-thrombin, and nuclei were then isolated. Western blot analysis using isozyme-specific antibodies demonstrated the presence of PKC alpha, but not PKC epsilon or zeta in the nuclei of cells treated with either phorbol 12-myristate 13-acetate or alpha-thrombin. The increase in nuclear PKC alpha levels was accompanied by a 10-fold increase in nuclear PKC specific activity and stimulated phosphorylation of at least six nuclear proteins. The rise in nuclear PKC levels occurred rapidly and reached a maximum at 30-60 s, which was followed by a decline back to the control level over the next 15 min. In addition, alpha-thrombin treatment resulted in an immediate rise in DAG mass levels in the nuclear fractions. Kinetic analysis indicated that a maximum increase in DAG levels occurred 2.5-5 min after the addition of alpha-thrombin and remained elevated for at least 30 min. In cells labeled with [3H]myristic acid, alpha-thrombin treatment induced an increase in radiolabeled nuclear diglycerides, suggesting that the stimulated nuclear DAGs are derived, at least in part, from phosphatidylcholine. Our results suggest that increases in both nuclear DAG levels and PKC activity following alpha-thrombin treatment may play a role in mediating thrombin-induced nuclear responses such as changes in gene expression and cellular proliferation.

Animals↗
PubMed1992

[Activation of Na/H exchange by thrombin in peritoneal mast cells].

Using the fluorescent probe, BCECF, the changes in intracellular pH (pHi) in rat peritoneal mast cells were studied. alpha-Thrombin (0.1 nM) induced biphasic changes in pHi which consisted in a temporary decrease in pH with its subsequent steady increase due to the Na/H exchange activation which was inhibited by EIPA and controlled by extracellular Na+. The biphasic changes in pHi induced by DIP-alpha-thrombin (0.1 pM-1 nM), a catalytically inactive form with an intact recognition site, were similar to those of alpha-thrombin, whereas beta/gamma-thrombin (10-1000 pM), a catalytically active form characterized by structural disturbances in the recognition site, was able to induce only the initial phase of acidification. The thrombin recognition site modulators, alpha 1-thymosin and heparin, blocked the ability of the enzyme to induce the alkalinization of pHi. Nigericin stimulated the Na/H-exchange in mast cells. The rate of the Na/H-exchange activation determined with nigericin, decreased with an increase in the alpha-thrombin dose from 0.1 pM up to 10 nM. Activation of protein kinase C (PKC) in mast cells by PMA used at 1 nM and 10 nM led to the alkalinization of the cytoplasm as a result of the Na/H-exchange activation blocked by EIPA. The PKC inhibitor, H-7, suppressed the pHi increase induced by both PMA and alpha-thrombin. The alpha-thrombin-induced acidification of the cytoplasm was completely blocked by SITS in Ca(2+)-free media, whereas in media with Ca2+ SITS inhibited the pHi decline. Acidification of the cytoplasm by thrombin seems to be due to both Ca2+ influx and activation of Cl- fluxes. It is concluded that the observed activation of the Na/H-exchange by thrombin is induced by a cascade of intracellular reactions involving PKC.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Thrombin induction of plasminogen activator inhibitor mRNA in human umbilical vein endothelial cells in culture.

We have previously reported that incubation of human umbilical vein endothelial cells (HUVECs) with human alpha-thrombin causes a time- and concentration-dependent increase in secreted plasminogen activator inhibitor type 1 (PAI-1) activity (Gelehrter TD, Sznycer-Laszuk R. J Clin Invest 1986;77:165-9). Here we report that the regulation of PAI-1 activity by thrombin is secondary to the thrombin-induced increase in PAI-1 mRNA accumulation. Incubation of HUVECs for 6 to 24 hours with 0.3 to 1.0 U/ml thrombin causes a 1.8-fold to 10-fold increase in PAI-1 activity and a 1.5-fold to threefold increase in accumulation of both the 3.2 and the 2.2 kilobase PAI-1 mRNAs. These effects are prevented by the prior addition of hirudin, a specific thrombin inhibitor. Inhibition of RNA synthesis with actinomycin D blocks the thrombin induction of PAI-1 mRNA accumulation. The protein synthesis inhibitor, cycloheximide, which itself markedly stimulates the accumulation of PAI-1, appears to prevent the induction by thrombin, suggesting that thrombin may act by inducing another effector such as interleukin-1. Consistent with this hypothesis is our observation that simultaneous addition of antibodies to interleukin-1-alpha prevents the thrombin induction of PAI-1 activity and mRNA.

Cells, Cultured↗
PubMed1992

Thrombin, but not thromboxane, stimulates megakaryocytic differentiation in human megakaryoblastic leukemia cells.

Physiologic stimuli for differentiation of bone marrow megakaryoblasts, a critical event leading to formation of platelets, have not been identified. Therefore, we examined the effects of two potent platelet agonists, thrombin and the thromboxane A2 mimetic U46619 [(15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z, 13E-dienoic acid], on the developmental regulation of cultured CHRF-288-11 megakaryoblast cells. Both thrombin and U46619 increased intracellular free calcium in fura-2 loaded, serum-deprived CHRF-288-11 cells with EC50 values of 0.07 +/- 0.01 U/ml and 194 +/- 27 nM, respectively (n = 4 each). Thrombin, but not U46619, increased intracellular pH from 7.44 +/- 0.03 to 7.55 +/- 0.06 (n = 4, P = .012) in 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein-loaded cells. Because increases in cytosolic free calcium and/or cytosolic pH are associated with mitogenic responses in many cell systems, we determined if thrombin or U46619 stimulated CHRF-288-11 proliferation or differentiation. U46619 (1 microM) inhibited cell growth but had no effect on cell size or morphology. In contrast, thrombin (0.2 U/ml) inhibited cell division and increased overall cell size to 116 +/- 2% of control (P less than .05). Microscopically, thrombin-treated cells had increased cytoplasmic content, nuclear lobulation and nucleolar content--hallmarks of megakaryocytic differentiation. Thrombin, but not U46619, increased levels of CHRF-288-11 c-fos and c-myc mRNA transcripts (protooncogene expression). We conclude that thrombin, but not thromboxane A2, induces terminal megakaryocytic differentiation of CHRF-288-11 cells. This suggests that thrombin may act as a physiologic regulator of platelet formation in vivo.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed2005

[Effect of thrombin on survival of hippocampal neurons].

The effect of thrombin on the rat hippocampal neurons death in model of neurotoxicity induced by hemoglobin or glutamate, was studied. Thrombin (10 nM) was shown to inhibit 100-mkM glutamate--or 10-mkM hemoglobin-induced apoptosis of the rat hippocampal neurons. With the aid of PAR1 (protease-activated receptor1) agonist peptide and PAR1 antagonist, the PAR1 was found to be necessary for protective action of thrombin in hippocampal neurons in models of neurotoxicity induced by hemoglobin or glutamate. Because the prolonged elevation [Ca2+] ib neurons is a critical part of neurodestructive processes in CNS, the effect of thrombin on Ca2+-homeostatis of neurons after its injury by the inducer of neuronal apoptosis: a synthetic agonist of the NMDA receptors N-methyl-D-aspartate (NMDA), was studied. We hypothesized that thrombin via receptors PAR may prove to be neuroprotective for the hippocampus. Thrombin was shown to stimulate via PAR1 a transient increase in [Ca2+] in neurons in a concentration-dependent manner. Thrombin (1 nM) decreased the [Ca2+] signal induced by activation of the NMDA-subtype of glutamate receptors. This thrombin effect may be one of the reasons of the protective action of thrombin in hippocampal neurons.

Animals↗
PubMed2005

[Expression of intercellular adhesion molecule-1 and neutrophil infiltration induced by injection of thrombin in rats].

OBJECTIVE: To investigate the role of the thrombin in inducing inflammatory response in brain tissue adjacent to hematoma in the brain. METHODS: Seventy-two male Wistar rats were randomly divided into two groups: sham-operated control group and thrombin injection group. Immunohistochemical technique and myeloperoxidase (MPO) determination were respectively used to evaluate intercellular adhesion molecule-1 (ICAM-1) expression and neutrophil infiltration in adjacent brain tissue of the thrombin injection zone at 6, 12, 24, 48, 72 and 128 hours after thrombin injection. RESULTS: The expression of ICAM-1 in tissues adjacent to thrombin injection started to increase 6 hours after the injection, peaking at 72 hours, and lasting for 120 hours. MPO activity of tissues in the area increased 6 hours, reached a high level at 24 hours, then decreased slightly, and it peaked at 72 hours after thrombin injection, correlating closely with the increase in ICAM-1 expression except the changes at 24 hours. CONCLUSION: The increased expression of ICAM-1 after thrombin injection is correlated with the degree of the neutrophil infiltration in the brain tissue around the thrombin injection zone, indicating thrombin is important to produce secondary brain damage after cerebral hemorrhage.

Animals↗
PubMed1992

A collaborative study to establish an international standard for alpha-thrombin.

Since 1975 an International Standard for Thrombin of low purity has been used. While this standard was stable and of value for calibrating thrombins of unknown potency the need for a pure alpha-thrombin standard arose both for accurate calibration and for precise measurement of thrombin inhibitors, notably hirudin. An international collaborative study was undertaken to establish the potency and stability of an ampouled pure alpha-thrombin preparation. A potency of 97.5 international units (95% confidence limits 86.5-98.5) was established for the new alpha-thrombin standard (89/588) using a clotting-assay procedure. Stability data at various elevated temperatures indicated that the standard could be transported and stored with no significant loss of potency. Ampoules of lyophilised alpha-thrombin (coded 89/588) have been recommended as an International Standard for alpha-thrombin with an assigned potency of 100 international units per ampoule by the International Society for Thrombosis and Haemostasis (Thrombin and its Inhibitors Sub-Committee) in Barcelona, Spain in July 1990 while the Expert Committee on Biological Standardisation and Control of the World Health Organisation will consider its status at its next meeting in Geneva in 1991.

Drug Stability↗
PubMedDate not supplied

[Interrelation between thrombin structure and its stability].

Temperature inactivation of human thrombin has been studied when finding out the mechanism of this enzyme stabilization by amino acids. Effect of a number of amino acids on thrombin in the conditions (pH) of the highest activity of proteinase has been investigated. It is established that most amino acids are characterized to more or less extent by the protective action, when hampering the temperature inactivation of the enzyme. The correspondence was mainly found between the stabilizing effect of amino acids and thrombin specificity. Thrombin is stabilized by L-arginine and DL-lysine more intensively than by other amino acids. A stabilizing effect of L-glutamic acid was shown in contrast to the action of the latter on trypsin that was obviously connected with the original structure of the active centre of thrombin, that is the availability of anionic binding centre which includes Lys68, Arg72, Arg77. High thrombin stabilization by such amino acids as phenylalanine, DL-serine, DL-methonine was an exception. It was established that amino acids stabilize thrombin with formation of a compound with the reactive centre of its molecule, like the compounds enzyme-substrate. The macrostructure stability probably depends, to a considerable extent, on the state of the enzyme reactive centre: thrombin molecules, which contain a free reactive centre, are more labile than those which reactive centre is bound to the reagent of more or less specific character. The inhibition of the autolysis process may be another manifestation of thrombin stabilization by amino acids.

Amino Acids↗
PubMed1991

Function of glycoprotein Ib alpha in platelet activation induced by alpha-thrombin.

We have obtained evidence that selective inhibition of high affinity thrombin-binding sites located in the amino-terminal domain of the membrane glycoprotein (GP) Ib alpha results in impaired platelet activation, as shown by abrogation or reduction of the following responses induced in normal platelets by exposure to less than 1 nM alpha-thrombin: (i) increase in intracellular ionized calcium concentration ([Ca2+]i), (ii) release of dense granule content, (iii) binding of fibrinogen, (iv) aggregation. An anti-GP Ib monoclonal antibody, LJ-Ib 10, which does not inhibit von Willebrand factor binding to platelets, obliterated the high affinity alpha-thrombin-binding sites on normal platelets. Isotherms of alpha-thrombin binding to normal platelets treated with saturating amounts of the antibody were virtually identical to those obtained with platelets from a patient with classical Bernard-Soulier syndrome. In parallel with decreased binding of the agonist, this antibody caused 50% inhibition of the maximal extent of platelet aggregation and 90% inhibition of ATP release induced by 0.3 nM alpha-thrombin. By inhibiting alpha-thrombin binding to GP Ib, the antibody prevented the activation of platelets exposed to low concentrations of the agonist, as demonstrated by abrogation of the increase in intraplatelet ionized calcium concentration induced in control platelets by 0.18 nM alpha-thrombin; under these conditions, fibrinogen binding was inhibited by 84%. Therefore, there is a correlation between occupancy of the high affinity sites for alpha-thrombin on GP Ib alpha and platelet activation, secretion, and aggregation, suggesting that GP Ib alpha is part of an alpha-thrombin receptor relevant for platelet function.

Antibodies, Monoclonal↗
PubMed1991

Comparison of the effects of heparin and hirudin on thrombin binding to the normal and the de-endothelialized rabbit aorta in vitro.

The properties of heparin and hirudin to inhibit thrombin from binding to the freshly-excised rabbit aorta wall were compared in vitro. When aorta segments were incubated with 125I-thrombin (4.4 +/- 0.4 nM) in the presence of heparin or hirudin, both anticoagulants inhibited 125I-thrombin binding to the endothelium in a concentration-dependent manner (IC50: 0.1 USP U heparin/ml; 0.1 ATU hirudin/ml). Endothelium-bound 125I-thrombin was displaced by either heparin (50% liberated at 4.1 U/ml) or hirudin (0.4 U/ml). Using de-endothelialized aortas, heparin inhibited thrombin binding by the exposed subendothelium (IC50: 1.8 U/ml) whereas hirudin was without effect. Neither heparin nor hirudin was able to significantly liberate thrombin bound to the exposed subendothelium. These observations suggest that both heparin and hirudin mask the binding site on thrombin to the endothelial cell membrane. A separate site on thrombin must bind to the subendothelium because only heparin inhibits binding. Thrombin, although bound reversibly to the endothelium, is bound irreversibly to the exposed subendothelium due, probably, to reaction with endogenous extracellular antithrombin activities (e.g. antithrombin-III, protease nexin-1).

Adsorption↗
PubMed1991

Thrombin attenuates the stimulatory effect of histamine on Ca2+ entry in confluent human umbilical vein endothelial cell cultures.

Human umbilical vein endothelial cells were grown to confluence, as first passage cells, on coverslips. Treatment with ionomycin or histamine caused a sustained rise in intracellular Ca2+ (measured by Fura-2 fluorescence), but after treatment with thrombin, only a transient rise in Ca2+ was observed. Furthermore, the addition of thrombin after ionomycin or histamine lowered the raised Ca2+ back to near control levels. This effect of thrombin was concentration dependent, with increasing concentrations producing increases in both the rate and extent of the lowering of Ca2+. A similar effect of thrombin was seen on video imaging of Fura-2-loaded cell monolayers. The Ca2(+)-lowering effect of thrombin was not mimicked by phorbol 12-myristate 13-acetate nor blocked by staurosporine, indicating a lack of involvement of protein kinase C; intracellular pH also does not appear to be involved. The mechanism by which thrombin lowers cytoplasmic Ca2+ is due mainly to inhibition of Ca2+ entry since thrombin prevented the stimulated influx of Mn2+ caused by histamine or ionomycin. It may therefore be that in vivo under certain physiological or pathological conditions, thrombin's effects on intracellular Ca2+ are more transient than those of histamine, and thrombin also may induce transience in histamine's actions.

Calcium↗
PubMed1991

Fibrin moderates the catalytic action of heparin but not that of dermatan sulfate on thrombin inhibition in human plasma.

Three recent studies have reported that fibrin in solution significantly inhibits the ability of heparin to catalyze the inhibition of thrombin by antithrombin III. In addition, heparin inhibits the release of fibrinopeptide A by clot-bound thrombin less effectively than it inhibits the release of fibrinopeptide A by thrombin in solution. We have also reported that dermatan sulfate, which catalyzes thrombin inhibition by heparin cofactor II, inhibits thrombus growth in rabbits more effectively than heparin. Because the results of these studies suggest that fibrin inhibits the reactivity of thrombin with antithrombin III-heparin but not with heparin cofactor II-dermatan sulfate, we compared the relative catalytic effects of heparin and dermatan sulfate on thrombin inhibition in plasma, both in the presence and absence of fibrin. We quantitated the rates of thrombin inhibition by antithrombin III and heparin cofactor II by specific enzyme-linked immunosorbent assays. When it was generated, fibrin was kept in solution by adding 2 mmol/L Gly-Pro-Arg-Pro to plasma. Fibrinogen-fibrin reduced the reactivity of thrombin with plasma antithrombin III, both in the presence of and in the absence of heparin. In contrast, the catalytic action of dermatan sulfate on thrombin inhibition by plasma heparin cofactor II was unimpaired by fibrinogen-fibrin. Based on the ability of dermatan sulfate to inhibit thrombus growth in rabbits, failure of fibrinogen-fibrin to moderate the catalytic action of dermatan sulfate may account for its greater antithrombotic effectiveness relative to that of heparin.

Antithrombin III↗
PubMed1990

A study of the lethal effects of intravenous injection of thrombin in rabbits.

Thrombin can stop bleeding during ophthalmic surgery by directly converting fibrinogen to fibrin. Little attention has been given to the potential risk of inadvertent intravenous administration of thrombin causing massive intravascular coagulation. We injected rabbits with a dose of thrombin, adjusted for body weight, equivalent to the amount used in ocular surgery. We also studied the effects of treating the rabbits with heparin. The rabbit injected with thrombin alone died within 30 seconds due to total intravascular coagulation. Three additional rabbits injected with thrombin but treated with heparin (intraperitoneally 40 minutes before thrombin, intravenously 30 minutes before thrombin, and intravenously immediately after thrombin injection) survived without any ill effects noted over the next month. For extraocular cases we recommend color coding the thrombin with some methylene blue. Using an eye dropper, rather than a syringe, reduces the chance of intravascular injection.

Animals↗
PubMed1990

Enhanced binding of fibrinogen by the subendothelium after treatment of the rabbit aorta with thrombin.

Damage to the endothelial surface of an artery invokes a hemostatic response that causes platelet deposition and activation of the coagulation and fibrinolytic pathways on the exposed subendothelial surface. Plasma fibrinogen is rapidly adsorbed at the site of injury. To gain insight into fibrinogen uptake, undamaged and de-endothelialized rabbit thoracic aortas were pretreated with various concentrations of thrombin and then incubated with fibrinogen labeled with iodine 125 in vitro. Uptake of fibrinogen by the subendothelium was not affected by low thrombin concentrations (less than 10 nmol/L), probably because of the antithrombin capacity of the vessel wall to inactivate any thrombin adsorbed. Over the thrombin concentration range of 10 to 90 nmol/L. fibrinogen binding increased linearly as binding of thrombin labeled with iodine 131 increased. In contrast, treatment of the subendothelium with enzymatically inactive thrombin did not enhance fibrinogen binding. Fibrinogen binding was inhibited by exposing the thrombin-treated subendothelium to hirudin or phenylalanyl-prolyl-arginyl-chloromethyl ketone. High thrombin concentrations (greater than 100 nmol/L) caused either a steadily decreasing uptake of fibrinogen with low fibrinogen concentrations or fibrin coagulation on the subendothelial surface from a high fibrinogen concentration. Glycyl-prolyl-arginyl-proline (0.1 mg/ml), a selective inhibitor of fibrin polymerization, inhibited 72% to 78% of fibrinogen uptake by the thrombin-treated subendothelium. Fibrinogen uptake was Ca2(+)-dependent, but ethylenediaminetetraacetic acid (10 mmol/L) did not displace subendothelium-bound fibrinogen. Plasmin effectively removed at least 75% of bound fibrinogen, indicating an extracellular location for the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗