RESUMEN
In the human circulation, factor VII is present in relatively low plasma concentration (0.01 microM) and has been reported to have a short half-life (t(1/2); 6 h). In contrast, prothrombin is present in a relatively high plasma concentration (2 microM) and has a relatively long catabolic half-life (t(1/2) = approximately 2-3 days). This report examines the metabolic characteristics of purified rabbit plasma factor VII and prothrombin, radiolabeled with (125)I and (131)I, respectively, in healthy young rabbits. From the plasma clearance curves of protein-bound radioactivities, fractional catabolic rates and compartmental distributions were calculated using a three-compartment model. Turnover of factor VII within the intravascular space (2.95 days) exceeded that of prothrombin (1.9 days). However, the whole body fractional catabolic rate of factor VII (0.34 days(-1); catabolic t(1/2) = 2.04 days) was significantly slower than that of prothrombin (0.53 days(-1); t(1/2) = 1.31 days). Furthermore, the fractional distributions of factor VII in the intravascular (0.14) and extravascular compartments (0.76) differed from those of prothrombin (0.29 and 0.53). Absolute quantities of factor VII and prothrombin catabolized by a 3-kg rabbit amounted to 0.18 and 24.0 mg/day, respectively (molar ratio of prothrombin to factor VII = 100). The molar ratio of catabolism was compared with the release rates of factor VII and prothrombin from rabbit livers perfused ex vivo. After correction for uptake of factor VII and prothrombin by the liver, the molar ratio of released prothrombin to factor VII in the perfusate was approximately 293:1 over a 0.25- to 3-h interval. These results indicate that, compared with prothrombin, factor VII in the healthy rabbit circulates as a relatively long-lived protein. This behavior does not reflect that reported for factor VII in the human circulation.
Asunto(s)
Factor VII/metabolismo , Protrombina/metabolismo , Animales , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Semivida , Radioisótopos de Yodo , Cinética , Hígado/metabolismo , Tasa de Depuración Metabólica , ConejosRESUMEN
Plasminogen (PLG) exists in the circulation as two glycoforms, I and II. Angiostatin (AST) is a polypeptide that has been cleaved from the kringle region of PLG and has strong anti-angiogenic properties. AST-I and AST-II, which consisted only of kringles 1 through 3, were prepared by the action of urokinase on purified rabbit PLG-I and PLG-II, respectively, in the presence of N-acetyl cysteine, followed by affinity chromatography on lysine-Sepharose. Purified AST-I and AST-II were tested for functional activity with a chick chorioallantoic membrane (CAM) model; when similar amounts were applied to a 6-day CAM, AST-I was substantially more effective than AST-II in decreasing vascular supply to the CAM over a 72-hour period; this activity correlated with a loss of capillaries, probably through apoptosis of endothelial cells. Radiolabeled AST-I and AST-II (iodine 125 and iodine 131) were co-injected intravenously into healthy rabbits to determine their clearances from plasma measured over 3 days. Over a dose range of 0.08 to 2.7 microg/kg, the fractional catabolic rate within the intravascular space (j(3)) indicated that AST-I was cleared 3-fold to 4-fold more rapidly than AST-II (P < .001). The catabolic half-life of AST-I (2.01 +/- 0.19 days) was significantly less than that of AST-II (2.62 +/- 0.20 days). The faster clearance of AST-I from the intravascular space was matched by its more rapid passage than AST-II to the extravascular space of various organs over 60 minutes in vivo. This property of AST-I as compared with AST-II may partially explain its greater anti-angiogenic potential. From the plasma concentrations of PLG-I and PLG-II and their relative behaviors toward rabbit VX-2 lung tumors in vivo, we predict that substantially greater quantities of AST-II than AST-I may be released into the extravascular space of tumors.
Asunto(s)
Neovascularización Fisiológica/efectos de los fármacos , Fragmentos de Péptidos/farmacocinética , Plasminógeno/farmacocinética , Angiostatinas , Animales , Capilares/metabolismo , Embrión de Pollo , Endotelio Vascular/metabolismo , Radioisótopos de Yodo , Isomerismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Plasminógeno/química , Plasminógeno/aislamiento & purificación , Conejos , Especificidad de la Especie , Articulaciones Tarsianas/metabolismoRESUMEN
SERP-1 is a secreted myxoma virus-encoded 55-kd protein of the serine proteinase inhibitor ("serpin") family that strongly inhibits the mitosis of medial arterial smooth muscle cells, thus preventing stenosis in injured rabbit and rat arteries. We have measured the fractional catabolic rate (FCR) and compartmental distribution of 1251-SERP-1 after injection of various doses into the circulation of healthy rabbits. The FCR within the intravascular space decreased from 2.99 d(-1) to 2.39 d(-1) and the whole-body FCR decreased from 0.66 d(-1) to 0.51 d(-1) as the dose was increased 35-fold from 0.11 microg/kg to 3.8 microg/kg. The fractional distribution of SERP-1 between the intravascular (0.21), noncirculating vascular wall (0.09), and extravascular compartments (0.70) at equilibrium did not change significantly over this dose range. SERP-1 did not appear to selectively accumulate in any organ in any of 11 rabbits studied over a 6-day interval. In comparison to other rabbit plasma serpins, the behavior of SERP-1 in vivo most closely resembled that of heparin cofactor II.
Asunto(s)
Myxoma virus/genética , Inhibidores de Serina Proteinasa/farmacocinética , Animales , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Semivida , Radioisótopos de Yodo , Conejos , Inhibidores de Serina Proteinasa/sangre , Inhibidores de Serina Proteinasa/genética , Distribución TisularRESUMEN
During their growth, many malignant solid tumors elicit a hemostatic response in the host. In this report, the fluxes of various rabbit plasma hemostatic proteins into pulmonary VX-2 tumors have been measured in vivo. VX-2 cells were contained within a small rabbit plasma clot which was injected intravenously into rabbits. At 28 d, each rabbit was injected intravenously with two radiolabeled (131I, 125I) proteins selected from fibrinogen, prothrombin, antithrombin-alpha, heparin cofactor II, or plasminogen-I or -II. After allowing the labeled proteins to circulate for 10-200 min, each rabbit was perfused with Krebs-Henseleit solution and the lungs excised. Solid tumors were isolated, weighed and measured for radioactivity content. A mean of 14 tumors/pair of lungs with a mean tumor weight of 0.3 g was obtained. Radioactivity per g of tumor was related to radioactivity/ml of blood at exsanguination for each protein at each time interval. Maximum flux rates were calculated (as pmol/g of tumor/min): Fibrinogen, 65.0; prothrombin, 53.0; antithrombin-alpha, 24.1; HCII, 17.2; plasminogen-II, 5.0; and plasminogen-I, 3.2. Using immunocytochemical staining, fibrin(ogen) was distributed heterogeneously within the VX-2 tumor, appearing largely in the perinodular region and in the necrotic core. From the net fluxes of these proteins, the profile of chronic hemostasis associated with the VX-2 tumor was shown to differ markedly from the hemostatic response that occurs after an acute vascular injury.
Asunto(s)
Hemostasis , Neoplasias Pulmonares/sangre , Neoplasias Experimentales/sangre , Animales , Antitrombinas/metabolismo , Fibrinógeno/metabolismo , Cofactor II de Heparina/metabolismo , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Experimentales/irrigación sanguínea , Neovascularización Patológica , Plasminógeno/metabolismo , Protrombina/metabolismo , ConejosRESUMEN
In the rabbit blood stream, plasminogen circulates as two glycoforms, plasminogen I (PLG-I) and plasminogen II (PLG-II), in a molar ratio of 1:2.2. To compare their relative behaviors toward a site of vascular injury, radiolabeled samples of PLG-I and PLG-II were coinjected intravenously into NZW rabbits before inducing a de-endothelializing (balloon catheter) injury to the thoracic aorta. At various times (5 to 60 minutes) after injury, each rabbit was anesthetized and exsanguinated, the aorta was excised, and the radioactivity per centimeters squared of aortic intima-media (IM) was measured relative to that of blood at exsanguination. The uptake of iodine 125-labeled PLG-I and iodine 131-labeled PLG-II showed that the IM was essentially saturated by both glycoforms by 30 to 40 minutes after injury. Extrapolation of the flux rates to 1 minute after injury indicated that the uptake of PLG-II (2.4 pmol/min/cm2) exceeded PLG-I (0.5 pmol/min/cm2) almost five-fold. This result is consistent with an earlier report (Metabolism 1994;43:1430-7) that PLG-II is released by the liver and catabolized in vivo approximately five times faster than PLG-I. By molar comparison, the flux of total plasminogen (ie, PLG-I plus PLG-II) into the injured aorta wall in vivo was 2.4 times greater than that for prothrombin. Assuming both zymogens are converted to their respective proteases within the wound site, then approximately 2 to 3 molecules of plasmin are released for each molecule of thrombin in vivo. The possible significance of this plasmin:thrombin ratio is discussed in respect to the turnover of fibrin(ogen) within the site of vascular injury.
Asunto(s)
Aorta Torácica/lesiones , Aorta Torácica/metabolismo , Endotelio Vascular/lesiones , Endotelio Vascular/metabolismo , Plasminógeno/metabolismo , Animales , Antitrombinas/metabolismo , Cateterismo/efectos adversos , Modelos Animales de Enfermedad , Fibrinógeno/metabolismo , Glicosilación , Hemostasis , Cofactor II de Heparina/metabolismo , Cinética , Masculino , Plasminógeno/aislamiento & purificación , Isoformas de Proteínas/aislamiento & purificación , Isoformas de Proteínas/metabolismo , Protrombina/metabolismo , ConejosRESUMEN
The initiation of a denuding injury to the vascular endothelium rapidly leads to a deposition of platelets and fibrin at the site of injury. We have measured previously the responses of rabbit fibrinogen, prothrombin, and antithrombin to a deendothelializing balloon-catheter injury to the rabbit aorta in vivo. In this study, rabbit iodine 125-labeled HCII and iodine 125-labeled AT were coinjected intravenously into anesthetized rabbits 5 minutes before deendothelialization of the thoracic aorta. The rabbit was exsanguinated at 5 to 60 minutes after injury, the aorta was excised, and the accumulation of each radiolabeled protein in each layer of aorta wall was determined relative to the concentration of the respective native protein in circulating blood at exsanguination. The maximum flux rates into the aorta wall (i.e., platelet layer and intima-media) in the first minute after injury were calculated from the uptake data; approximately 2.8 molecules of AT accumulated for each HCII molecule. By comparison with previous measurements, the maximum flux rate of AT was similar to that of prothrombin. Further, the molar ratio of accumulated prothrombin/AT + HCII) in the aorta wall was 0.75. Detergent extracts of the injured aorta intima-media contained unreacted HCII and HCII complexes; the uninjured aorta contained only unreacted HCII. By contrast, high molecular weight AT complexes and unreacted AT were extracted from the uninjured, and in greater quantity from the injured, aorta wall. We conclude that, of the plasma antithrombins, AT accumulated more rapidly than HCII in vivo and appeared to be the more active inhibitor at the site of vascular injury. HCII may play a relatively minor role as an antithrombin and possibly only after injury.
Asunto(s)
Antitrombinas/metabolismo , Aorta Torácica/metabolismo , Endotelio Vascular/metabolismo , Fibrinógeno/metabolismo , Cofactor II de Heparina/metabolismo , Protrombina/metabolismo , Animales , Aorta Torácica/lesiones , Cateterismo/métodos , Electroforesis en Gel de Poliacrilamida , Endotelio Vascular/lesiones , Radioisótopos de Yodo , Masculino , ConejosRESUMEN
Fibrinogen and platelets rapidly saturate the exposed subendothelium of a freshly deendothelialized aorta in vivo. As thrombin generated within the site of injury is largely responsible for fibrin(ogen) deposition, we questioned whether various anticoagulant treatments would inhibit uptake of both fibrinogen and platelets in vivo. Rabbits were anticoagulated by pretreatment with either Warfarin, Ancrod, or recombinant hirudin. Each anesthetized, anticoagulated (or saline-injected control) rabbit was injected i.v. with rabbit 51Cr-platelets and 125I-fibrinogen before a balloon-catheter deendothelializing (or sham) injury of the thoracic aorta. At 10 minutes after injury, the rabbit was exsanguinated and the aorta excised. Platelet adsorption by the deendothelialized aorta surface was substantially reduced in anticoagulated rabbits (controls, 2.2x10(5)/mm2; Warfarin-treated, 1.2x10(5)/mm2; Ancrod-treated, 5.3x10(4)/mm2; r-hirudin-treated [5 mg/kg], 5.3x10(4)/mm2), and a significant reduction of fibrinogen associated with the platelet layer (from 5.3 to 1 to 2 pmol/cm2) and within the underlying intima-media layer (from 16.9 to 5 to 6 pmol/cm2) was observed in the r-hirudin-and Warfarin-treated rabbits. The pattern of aorta-deposited 51Cr-platelets and 125I-fibrin in the anticoagulated rabbits corresponded well with an assessment by transmission electron microscopy of aortic tissue samples. We conclude that approximately 70% of fibrinogen uptake is thrombin dependent and that approximately 80% of platelet adsorption depends on codeposited fibrin(ogen) during the 10-minute interval after balloon injury. Pretreatment with an agent that interferes with either thrombin or fibrin production will inhibit the immediate interaction of fibrinogen and platelets with the freshly exposed subendothelium.
Asunto(s)
Ancrod/farmacología , Anticoagulantes/farmacología , Aorta/metabolismo , Cateterismo/métodos , Fibrinógeno/farmacocinética , Hirudinas/farmacología , Warfarina/farmacología , Animales , Aorta/efectos de los fármacos , Aorta/lesiones , Aorta/patología , Plaquetas/metabolismo , Radioisótopos de Cromo , Fibrinógeno/metabolismo , Radioisótopos de Yodo , Marcaje Isotópico , Conejos , Proteínas Recombinantes/farmacologíaRESUMEN
The metabolic characteristics of two rabbit plasma thrombin inhibitors, heparin cofactor II (HCII) and antithrombin (AT), have been compared in healthy young rabbits. Purified HCII and AT-alpha were differentially radiolabeled (125I, 131I) and injected intravenously; blood samples were taken at prescribed intervals over 7 days. From the plasma clearance curves of protein-bound radioactivities, fractional catabolic rates and compartmental distributions were calculated using a three-compartment model. The whole body fractional catabolic rate for HCII (jt, 0.43/day, equivalent to t1/2 = 1.61 days) was significantly faster than for AT (jt, 0.37/day; t1/2 = 1.89 days; P < 0.005). The fractional distribution of HCII in the intravascular compartment (Ap, 0.20) and in the extravascular compartment (Ac, 0.63) differed significantly from AT (Ap, 0.30; Ac, 0.56). From the catabolic data and blood concentrations, absolute quantities of HCII and AT catabolized by a 3-kg rabbit amounted to 12.8 and 19.9 mg/day, respectively, equivalent to a molar ratio, AT/HCII, of 1.7. The catabolic molar ratio was compared with the relative release rates of HCII and AT from perfused rabbit livers. Both proteins were released from the liver, the molar ratio in the perfusate rising to approximately 1.4 at 2.5 h. This report increases our understanding of the in vivo dynamics of these two proteins.
Asunto(s)
Antitrombina III/metabolismo , Cofactor II de Heparina/metabolismo , Animales , Antitrombina III/farmacocinética , Sangre/metabolismo , Cofactor II de Heparina/farmacocinética , Técnicas In Vitro , Hígado/metabolismo , Perfusión , Conejos , Trombina/antagonistas & inhibidores , Distribución TisularRESUMEN
Previous studies have shown that alloxan-induced diabetes in rabbits effects a slower release of plasma proteins from the liver, a slower synthesis of 35S-glycosaminoglycan in the extracellular matrix of the arterial wall, and a concurrent reduction in the fractional catabolic rates of several plasma proteins. In the present study, the catabolism of two hemostatic proteins, prothrombin and antithrombin, are compared in alloxan-induced diabetic rabbits (of 6 months' duration) and age-matched control rabbits. Differentially radiolabeled prothrombin and antithrombin were injected intravenously, and arterial blood was sampled over a 7-day period to measure the clearance from plasma. A three-compartment model was used to determine the fractional catabolic rate and compartmental distribution of the two proteins. As observed for other plasma proteins, the whole-body fractional catabolic rates (jt) for prothrombin and antithrombin were significantly less in diabetic rabbits (prothrombin, 0.33 d-1; antithrombin, 0.27 d-1) than in control rabbits (prothrombin, 0.37 d-1; antithrombin, 0.30 d-1; P < .001 and P < .005, respectively). In absolute terms, the catabolism of antithrombin and prothrombin in diabetic rabbits was 5.1 and 6.2 mg.kg-1.d-1, respectively, equivalent to a molar ratio for antithrombin to prothrombin of 0.94. For the control rabbits, catabolism accounted for 6.3 mg.kg-1.d-1 of antithrombin and 7.3 mg.kg-1.d-1 of prothrombin, equivalent to a molar ratio of 1.01. The fractional distribution of these proteins was not significantly different within the intravascular and extravascular spaces in diabetic and control rabbits. The decreased catabolic rates observed for prothrombin and antithrombin in diabetic rabbits conform with results obtained previously for other plasma proteins, and probably reflect a generally decreased rate of plasma protein production by diabetic rabbit liver compared with control liver.
Asunto(s)
Antitrombina III/metabolismo , Diabetes Mellitus Experimental/metabolismo , Protrombina/metabolismo , Aloxano , Animales , Antitrombina III/administración & dosificación , Antitrombina III/análisis , Autorradiografía , Diabetes Mellitus Experimental/sangre , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Inyecciones Intravenosas , Radioisótopos de Yodo , Hígado/metabolismo , Protrombina/administración & dosificación , Protrombina/análisis , ConejosRESUMEN
The ability of the rabbit aorta intima-media (IM) layer to adsorb certain plasma proteins was measured for up to 20 months after a deendothelializing injury in vivo. Purified radioiodinated rabbit fibrinogen, antithrombin, or prothrombin was injected intravenously into either uninjured or sham-injured rabbits (controls) or rabbits at various times (5 minutes to 20 months) after a balloon-catheter injury to the aorta. After a 10-minute circulation time, a blood sample was taken, and the rabbit was exsanguinated rapidly (via a carotid cannula) and the aorta excised. Uptake of each radiolabeled protein was measured as bound radioactivity per square centimeter of platelet- or endothelium-free aorta IM and was compared with the radioactivity (ergo concentration) in blood at exsanguination. Fibrinogen adsorption by the IM was maximal at 5 minutes after injury (10.9 +/- 2.3 pmol/cm2 IM) and declined slowly to 4 to 6 pmol/cm2 at 12 months (controls: 0.8 +/- 0.1 pmol/cm2). Uptake of prothrombin (3.7 +/- 0.5 pmol/cm2 at 5 minutes) decreased to approximately 2 pmol/cm2 at 12 months (controls: 0.3 pmol/cm2). Antithrombin adsorption by the IM (3.3 +/- 0.4 pmol/cm2 at 5 minutes) paralleled that of prothrombin over 12 months (controls: 0.3 to 0.4 pmol/cm2), the molar ratio ranging from 0.8 to 1.2. At 20 months, the ballooned aorta had a significantly thickened intima and was approximately 90% reendothelialized. Injection of horseradish peroxidase (HRP) into rabbits at 1 or 12 months after balloon injury showed clearly that HRP activity was present throughout the entire depth of the deendothelialized, but not the reendothelialized, thickened intima. These results may indicate that an elevated turnover of hemostatic proteins continues within the deendothelialized intima after injury, conceivably until reendothelialization is complete.
Asunto(s)
Antitrombina III/metabolismo , Aorta/metabolismo , Fibrinógeno/metabolismo , Protrombina/metabolismo , Animales , Aorta/patología , Transporte Biológico , Cateterismo , Colorantes , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Peroxidasa de Rábano Silvestre , Masculino , Conejos , Túnica Íntima/metabolismo , Túnica Íntima/patologíaRESUMEN
After an injury to the vascular endothelium, certain blood proteins collect rapidly at the site of damage to prevent blood loss and maintain blood flow. The uptake of fibrinogen, plasminogen, and antithrombin--but not prothrombin--have been measured previously at the rabbit aorta wall after injury in vivo. This report describes the clearance of rabbit iodine 131-labeled prothrombin from the rabbit circulation to measure the distribution and fractional catabolic rate and compares the behavior of 131I-labeled prothrombin with either iodine 125-labeled fibrinogen or 125I-labeled antithrombin at the balloon catheter-injured aorta wall. When injected into young rabbits, 131I-labeled prothrombin was cleared from the intravascular space to yield a plasma curve that was best described by three exponentials. Fractional plasma and whole body catabolic rates were 2.0 day-1 and 0.41 day-1, respectively, equivalent to a catabolic half-life of 1.7 days. Fractional distribution of prothrombin amounted to 0.21, 0.24, and 0.55 within the intravascular, vascular endothelial, and extravascular compartments, respectively. Samples of 131I-labeled prothrombin and either 125I-labeled fibrinogen or 125I-labeled antithrombin were injected into anesthetized rabbits before balloon de-endothelialization of the thoracic aorta. The uptake of each radiolabeled protein by the aorta intima-media was measured at various times (5 to 60 minutes) after injury. Whereas uptake of plasma fibrinogen by the balloon-injured intima-media was maximal (20 pmol/cm2) in less than 5 minutes after injury, maximum uptake of prothrombin (5 to 6 pmol/cm2) took approximately 15 minutes. Uptake of prothrombin was initially faster than that of antithrombin, although approximately equimolar amounts of prothrombin and antithrombin were bound by the intimamedia by 60 minutes. The results are discussed in relation to thrombin production and the demand for antithrombin by the damaged aorta wall in vivo.
Asunto(s)
Aorta/metabolismo , Endotelio Vascular/lesiones , Protrombina/metabolismo , Túnica Íntima/patología , Albúminas/metabolismo , Animales , Antitrombina III/metabolismo , Aorta/citología , Transporte Biológico/fisiología , Cateterismo , Endotelio Vascular/metabolismo , Fibrinógeno/metabolismo , Radioisótopos de Yodo , Protrombina/análisis , Conejos , Túnica Íntima/metabolismo , Cicatrización de Heridas/fisiologíaRESUMEN
The metabolism of plasminogen glycoforms I and II was measured in alloxan-induced diabetic and in age-matched control rabbits. Radiolabeled plasminogen I and II were degraded significantly more slowly in diabetic compared with control rabbits; plasminogen II [half-time (T1/2), 1.31 days] was degraded faster than plasminogen I (T1/2), 1.86 days) in diabetic rabbits and in control rabbits (T1/2, 1.18 and 1.58 days, respectively). From the catabolic rates and relative quantities in plasma, we calculated that approximately four molecules of plasminogen II were degraded for one molecule of plasminogen I in the diabetic and control rabbits. To verify this later observation, plasminogen I and II production by diabetic rabbit livers was compared with that by the control livers in vitro. During perfusion with [3H]leucine, 3H-labeled protein was released more slowly from diabetic than from control livers, but no quantitative difference in total plasminogen yield between diabetic and control livers was found. Nevertheless, plasminogen II was produced 0.7 +/- 0.4 and 4.3 +/- 0.3 times faster than plasminogen I by diabetic and control livers, respectively. Plasminogen metabolism in the diabetic rabbit did not differ qualitatively from that in the control rabbit except that catabolism was slowed.