CHANGES IN THROMBIN GENERATION AND ANTITHROMBIN TITER FOLLOWING MASSIVE BLEEDING AND TRANSFUSION IN DOGS

1960 ◽  
Vol 38 (5) ◽  
pp. 475-480 ◽  
Author(s):  
F. C. Monkhouse ◽  
Susan Milojevic
Keyword(s):  
Plasma Protein ◽  
Thrombin Generation ◽  
Massive Bleeding ◽  
Clotting Factors ◽  
Protein Levels ◽  
Flow Fluctuations ◽  
Plasma Fraction ◽  
Dog Plasma ◽  
Total Plasma Protein

Thrombin generation, plasma antithrombin levels, and plasma protein levels were measured in dogs following rapid massive bleeding and transfusion. Three groups of four dogs per group were used. One group was transfused with saline, one with dextran, and one with a plasma fraction. The plasma fraction was prepared from mixed dog plasma by a procedure designed to remove most of the known clotting factors with a minimum of damage to other proteins. The in vivo dilution of blood by transfusion with two volumes of saline or one of plasma fraction increased the rate of thrombin generation. Dilution by transfusion with dextran decreased both rate and amount of thrombin generation. For the first few hours following transfusion with saline, plasma antithrombin increased more rapidly than total plasma protein. This was probably a result of increased lymph flow. Fluctuations in the coagulability of the blood were observed up to 72 hours following bleeding and transfusion. The fluctuations following saline transfusion were less prolonged than when dextran or plasma fraction was used.

CHANGES IN THROMBIN GENERATION AND ANTITHROMBIN TITER FOLLOWING MASSIVE BLEEDING AND TRANSFUSION IN DOGS

1960 ◽  
Vol 38 (1) ◽  
pp. 475-480
Author(s):  
F. C. Monkhouse ◽  
Susan Milojevic
Keyword(s):  
Plasma Protein ◽  
Thrombin Generation ◽  
Massive Bleeding ◽  
Clotting Factors ◽  
Protein Levels ◽  
Flow Fluctuations ◽  
Plasma Fraction ◽  
Dog Plasma ◽  
Total Plasma Protein

Thrombin generation, plasma antithrombin levels, and plasma protein levels were measured in dogs following rapid massive bleeding and transfusion. Three groups of four dogs per group were used. One group was transfused with saline, one with dextran, and one with a plasma fraction. The plasma fraction was prepared from mixed dog plasma by a procedure designed to remove most of the known clotting factors with a minimum of damage to other proteins. The in vivo dilution of blood by transfusion with two volumes of saline or one of plasma fraction increased the rate of thrombin generation. Dilution by transfusion with dextran decreased both rate and amount of thrombin generation. For the first few hours following transfusion with saline, plasma antithrombin increased more rapidly than total plasma protein. This was probably a result of increased lymph flow. Fluctuations in the coagulability of the blood were observed up to 72 hours following bleeding and transfusion. The fluctuations following saline transfusion were less prolonged than when dextran or plasma fraction was used.

scholarly journals THE BINDING OF MYOGLOBIN BY PLASMA PROTEIN

1960 ◽  
Vol 111 (1) ◽  
pp. 65-75 ◽  
Author(s):  
Willoughby Lathem
Keyword(s):  
Plasma Protein ◽  
Binding Sites ◽  
Binding Capacity ◽  
Plasma Hemoglobin ◽  
Renal Threshold ◽  
Dog Plasma ◽  
Beta Globulin ◽  
Maximal Binding

When added to dog plasma in vitro and in vivo, myoglobin was bound to plasma protein in a concentration which, maximally, averaged 21 ± 6 mg. per cent. Electrophoretically, bound myoglobin was separated from free myoglobin and migrated between alpha-2 and beta globulin. The electrophoretic characteristics of protein-bound myoglobin were similar to, although not identical with, those of protein-bound hemoglobin. The maximal binding capacity of plasma for myoglobin was less than for hemoglobin, which averaged 123 mg. per cent. At concentrations below the maximal binding capacity, from 15 to 50 per cent of the myoglobin was in the free, unbound state, differing from hemoglobin which was completely bound at all concentrations below the binding capacity. When myoglobin and hemoglobin were added together to plasma, hemoglobin appeared to interfere with the binding of myoglobin or to replace it at the binding sites. Myoglobin, however, did not appear to interfere with the binding of hemoglobin. These observations suggested that myoglobin and hemoglobin were bound at least in part by the same protein. When myoglobin was given intravenously, free myoglobin was excreted in the urine, whereas protein-bound myoglobin was not excreted. This suggests that protein-binding contributes to or determines the apparent renal threshold to myoglobin.

Total plasma protein level as an indicator of condition in wild American kestrels (Falco sparverius)

1997 ◽  
Vol 75 (5) ◽  
pp. 680-686 ◽  
Author(s):  
Russell D. Dawson ◽  
Gary R. Bortolotti
Keyword(s):  
Plasma Protein ◽  
Protein Level ◽  
Egg Laying ◽  
Falco Sparverius ◽  
Total Plasma ◽  
Protein Levels ◽  
American Kestrels ◽  
Total Plasma Protein ◽  
In The Wild ◽  
Plasma Protein Level

Total plasma protein levels were determined for 292 female and 228 male American kestrels (Falco sparverius) in the wild. Plasma protein levels were significantly higher in females than in males, and higher during prelaying than during incubation. For both sexes, plasma protein levels did not vary significantly with the number of days before or after egg laying on which the sample was taken, time of sampling, prey abundance, age, molt, or infection by the blood parasite Haemoproteus sp. Protein levels in females increased with date of sampling and body condition during prelaying, while the same pattern was seen in males during incubation. With the exception of those of prelaying females, plasma protein levels increased with ambient temperature. The results of this study suggest that at least some of the variation observed in total protein levels is attributable to physical condition. However, further investigation is required before the reliability of using total plasma protein level as a tool to assess the health and condition of kestrels is known.

scholarly journals PLASMA PROTEIN TURNOVER AND TISSUE EXCHANGE

1959 ◽  
Vol 109 (2) ◽  
pp. 173-186 ◽  
Author(s):  
C. L. Yuile ◽  
F. V. Lucas ◽  
J. P. Olson ◽  
A. B. Shapiro
Keyword(s):  
Plasma Protein ◽  
Dietary Protein ◽  
Protein Turnover ◽  
Nitrogen Retention ◽  
Total Plasma ◽  
Protein Breakdown ◽  
Protein Levels ◽  
Total Plasma Protein ◽  
Protein Feeding ◽  
The Difference

The rate of plasma protein turnover is more rapid in dogs receiving adequate dietary protein than when a diet devoid of protein is fed. Both albumin and combined globulins are involved in this change. The difference in turnover is reflected in a total protein half-life of 4.8 days with protein feeding versus 7.8 days without protein in the diet and in the metabolism of 1.0 and 0.65 gm. per kilogram of body weight per day on the respective diets. Additions of dietary protein from 10 to 30 per cent caused no further increase in the rate of plasma protein turnover. With protein depletion due to plasmapheresis and a very low protein diet there is evidence of reduced protein metabolism as indicated by nitrogen retention as well as a reduction in total plasma protein breakdown and interchange of isotope between plasma and tissue proteins. Following introduction of labeled plasma protein into the circulation the net amount of isotope transferred to tissues has been computed from the difference between total plasma protein breakdown and combined C14 excretion in urine and expired air. In animals receiving adequate dietary protein, tissue transfer amounts to 70 per cent of the total lost from the plasma proteins each day while the percentage rises to 85 in depleted dogs deprived of protein. In dogs with both plasma and tissue proteins labeled it can be estimated that, under conditions of protein feeding, an amount of C14 approximately equal to that lost from the plasma must recycle to account for the observed decrease in Apparent plasma protein turnover rate, (t½ of 15 versus 5 days). Without protein in the diet the isotope contribution of the tissues to the maintenance of plasma protein levels must be as great as or greater than that transferred in the opposite direction.

Plasma calcium distribution in relation to parathyroid function

1961 ◽  
Vol 200 (2) ◽  
pp. 341-344 ◽  
Author(s):  
Moira Breen ◽  
Smith Freeman
Keyword(s):  
Plasma Protein ◽  
Plasma Calcium ◽  
Total Plasma ◽  
Calcium Distribution ◽  
Parathyroid Function ◽  
Dog Plasma ◽  
Parathyroid Extract ◽  
Simple Mass

The plasma calcium was fractionated into protein-bound and free fractions by means of an ultracentrifuge in normal, hypoparathyroid and hyperparathyroid dogs and normal and hypoparathyroid rats. The proportion of the total plasma calcium that was diffusible was greater in normal and hypercalcemic animals than in the hypoparathyroid state. Recalcification of hypoparathyroid dog plasma in vitro gave a similar calcium distribution to that obtained when parathyroid extract was injected into hypoparathyroid dogs. Although the binding of calcium per gram of plasma protein increased both in vivo and in vitro as the concentration rose, the results did not conform to the simple mass law relationship as usually expressed. The evidence presented indicates that parathyroid function only influences the distribution of plasma calcium indirectly by regulating its concentration.

Ancylostoma caninum Anticoagulant Peptide Blocks Metastasis In Vivo and Inhibits Factor Xa Binding to Melanoma Cells In Vitro

1998 ◽  
Vol 79 (05) ◽  
pp. 1041-1047 ◽  
Author(s):  
Kathleen M. Donnelly ◽  
Michael E. Bromberg ◽  
Aaron Milstone ◽  
Jennifer Madison McNiff ◽  
Gordon Terwilliger ◽  
...  
Keyword(s):  
Active Site ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Melanoma Cells ◽  
Factor V ◽  
Ancylostoma Caninum ◽  
Metastatic Activity

SummaryWe evaluated the in vivo anti-metastatic activity of recombinant Ancylostoma caninum Anticoagulant Peptide (rAcAP), a potent (Ki = 265 pM) and specific active site inhibitor of human coagulation factor Xa originally isolated from bloodfeeding hookworms. Subcutaneous injection of SCID mice with rAcAP (0.01-0.2 mg/mouse) prior to tail vein injection of LOX human melanoma cells resulted in a dose dependent reduction in pulmonary metastases. In order to elucidate potential mechanisms of rAcAP’s anti-metastatic activity, experiments were carried out to identify specific interactions between factor Xa and LOX. Binding of biotinylated factor Xa to LOX monolayers was both specific and saturable (Kd = 15 nM). Competition experiments using antibodies to previously identified factor Xa binding proteins, including factor V/Va, effector cell protease receptor-1, and tissue factor pathway inhibitor failed to implicate any of these molecules as significant binding sites for Factor Xa. Functional prothrombinase activity was also supported by LOX, with a half maximal rate of thrombin generation detected at a factor Xa concentration of 2.4 nM. Additional competition experiments using an excess of either rAcAP or active site blocked factor Xa (EGR-Xa) revealed that most of the total factor Xa binding to LOX is mediated via interaction with the enzyme’s active site, predicting that the vast majority of cell-associated factor Xa does not participate directly in thrombin generation. In addition to establishing two distinct mechanisms of factor Xa binding to melanoma, these data raise the possibility that rAcAP’s antimetastatic effect in vivo might involve novel non-coagulant pathways, perhaps via inhibition of active-site mediated interactions between factor Xa and tumor cells.

Investigation of the Interaction of Blood Platelets with the Coagulation System at the Site of Plug Formation In Vivo in Man - Effect of Low-Dose Aspirin

1987 ◽  
Vol 57 (01) ◽  
pp. 062-066 ◽  
Author(s):  
P A Kyrle ◽  
J Westwick ◽  
M F Scully ◽  
V V Kakkar ◽  
G P Lewis
Keyword(s):  
Platelet Activation ◽  
Thrombin Generation ◽  
Low Dose ◽  
Bleeding Time ◽  
Blood Platelets ◽  
Dose Aspirin ◽  
Low Dose Aspirin ◽  
Plug Formation ◽  
Granule Release

SummaryIn 7 healthy volunteers, formation of thrombin (represented by fibrinopeptide A (FPA) generation, α-granule release (represented by β-thromboglobulin [βTG] release) and the generation of thromboxane B2 (TxB2) were measured in vivo in blood emerging from a template bleeding time incision. At the site of plug formation, considerable platelet activation and thrombin generation were seen within the first minute, as indicated by a 110-fold, 50-fold and 30-fold increase of FPA, TxB2 and PTG over the corresponding plasma values. After a further increase of the markers in the subsequent 3 minutes, they reached a plateau during the fourth and fifth minute. A low-dose aspirin regimen (0.42 mg.kg-1.day-1 for 7 days) caused >90% inhibition of TxB2formation in both bleeding time blood and clotted blood. At the site of plug formation, a-granule release was substantially reduced within the first three minutes and thrombin generation was similarly inhibited. We conclude that (a) marked platelet activation and considerable thrombin generation occur in the early stages.of haemostasis, (b) α-granule release in vivo is partially dependent upon cyclo-oxygenase-controlled mechanisms and (c) thrombin generation at the site of plug formation is promoted by the activation of platelets.

The Effects of Heparin and Annexin V on Fibrin Accretion after Injury in the Jugular Veins of Rabbits

1993 ◽  
Vol 69 (03) ◽  
pp. 227-230 ◽  
Author(s):  
J Van Ryn-McKenna ◽  
H Merk ◽  
T H Müller ◽  
M R Buchanan ◽  
W G Eisert
Keyword(s):  
Vessel Wall ◽  
Thrombin Generation ◽  
Antithrombin Iii ◽  
Specific Effect ◽  
Annexin V ◽  
Inhibitory Effect ◽  
Jugular Veins ◽  
Prothrombinase Complex ◽  
Wall Injury

SummaryWe compared the relative abilities of unfractionated heparin and annexin V to prevent fibrin accretion onto injured jugular veins in vivo. Heparin was used to accelerate the inhibition of thrombin by antithrombin III, and annexin V was used to inhibit the assembly of the prothrombinase complex on phospholipid surfaces, thereby blocking thrombin generation. Rabbit jugular veins were isolated in situ, a 2 cm segment was injured by perfusing it with air, and then blood flow was re-established. Five minutes later, each rabbit was injected with heparin (20 U/kg) or annexin V (0.3 mg/kg) and then with 125I-fibrinogen. The amount of 125I-fibrin accumulation onto each injured vessel wall segment was measured 4 h later. Each injured vessel was completely deendothelialized as a result of the air perfusion as demonstrated by electron microscopy. 125I-fibrin accretion onto the injured jugular veins was enhanced 2.4-fold as compared to the uninjured veins in sham-operated animals. Heparin treatment did not reduce fibrin accretion, whereas, annexin V treatment decreased fibrin accretion by 60%, p <0.05. This latter effect was achieved without sustained circulating anticoagulation. Additional experiments confirmed that the inhibitory effect of annexin V on fibrin accretion was associated with a surface specific effect, since more annexin V bound to the injured jugular vein segments as compared to the non-injured jugular veins. We conclude that, i) mild vessel wall injury (selective de-endothelialization) in veins results in a thrombogenic vessel wall; ii) the thrombogenecity of which is not inhibited by prophylactic doses of heparin; but iii) is inhibited by annexin V, which binds to injured vessel wall surface, and inhibits thrombin generation independently of antithrombin III.

Phospholipid-Protein Interactions in the Formation of Prothrombin Activator

1965 ◽  
Vol 14 (03/04) ◽  
pp. 431-444 ◽  
Author(s):  
E. R Cole ◽  
J. L Koppel ◽  
J. H Olwin
Keyword(s):  
Complex Formation ◽  
Protein Interactions ◽  
Calcium Ions ◽  
Fixed Number ◽  
Factor V ◽  
Clotting Factors ◽  
Number Of Binding Sites ◽  
C Complex ◽  
Autoprothrombin C

SummarySince Ac-globulin (factor V) is involved in the formation of prothrombin activator, its ability to complex with phospholipids was studied. Purified bovine Ac-globulin was complexed to asolectin, there being presumably a fixed number of binding sites on the phospholipid micelle for Ac-globulin. In contrast to the requirement for calcium ions in the formation of complexes between asolectin and autoprothrombin C, calcium ions were not required for complex formation between asolectin and Ac-globulin to occur ; in fact, the presence of calcium prevented complex formation occurring, the degree of inhibition being dependent on the calcium concentration. By treating isolated, pre-formed aso- lectin-Ac-globulin complexes with calcium chloride solutions, Ac-globulin could be recovered in a much higher state of purity and essentially free of asolectin.Complete activators were formed by first preparing the asolectin-calcium- autoprothrombin C complex and then reacting the complex with Ac-globulin. A small amount of this product was very effective as an activator of purified prothrombin without further addition of calcium or any other cofactor. If the autoprothrombin C preparation used to prepare the complex was free of traces of prothrombin, the complete activator was stable for several hours at room temperature. Stable preparations of the complete activator were centrifuged, resulting in the sedimentation of most of the activity. Experimental evidence also indicated that activator activity was highest when autoprothrombin C and Ac-globulin were complexed to the same phospholipid micelle, rather than when the two clotting factors were complexed to separate micelles. These data suggested that the in vivo prothrombin activator may be a sedimentable complex composed of a thromboplastic enzyme, calcium, Ac-globulin and phospholipid.

Cellular Localization of Enzymatically Active Thrombin in Intact Human Tissues by Hirudin Binding

1995 ◽  
Vol 73 (05) ◽  
pp. 793-797 ◽  
Author(s):  
Leo R Zacharski ◽  
Vincent A Memoli ◽  
William D Morain ◽  
Jean-Marc Schlaeppi ◽  
Sandra M Rousseau
Keyword(s):  
Tumor Cell ◽  
Cell Carcinoma ◽  
Cellular Localization ◽  
Thrombin Generation ◽  
Normal Lung ◽  
Cancer Tissue ◽  
Immunohistochemical Techniques ◽  
Tumor Types ◽  
Negative Controls

SummaryCellular sites of coagulation activation within complex, intact tissues have been studied by immunohistochemical techniques. Hirudin, a specific and high affinity inihibitor of the active site of thrombin, together with antibody to hirudin were applied to sections of AMeX-fixed specimens of normal lung, kidney, placenta, freshly incised skin and unperturbed skin obtained at fresh autopsy; to rheumatoid synovial tissue; and to malignant tissue from a variety of tumor types. Staining for thrombin was observed selectively on pulmonary alveolar, rheumatoid synovial, and placental macrophages that express an intact extrinsic coagulation pathway. Staining was also observed restricted to the endothelium of capillaries in freshly incised skin but not in either unperturbed skin or in aged incisions. Staining of tumor cell bodies was observed in small cell carcinoma of the lung, renal cell carcinoma, and malignant melanoma tissues that we found previously to show tumor cell-associated procoagulant activity. This staining occurred commonly on cells within the tumor mass that were distant from stromal fibrinogen/fibrin. By contrast, tumor-associated macrophage but not tumor cell staining was seen in adenocarcinoma and squamous cell carcinoma of the lung, and little or no staining was seen in colon cancer tissue. Negative controls in which either the hirudin probe or its antibody were omitted failed to show staining. These results are in accord with previous findings and suggest that such techniques may be useful for studying the cellular sites of thrombin generation in intact tissues. We postulate that administration of potent and specific thrombin antagonists, such as hirudin, to patients with relevant tumor types might be followed by homing of hirudin to tumor cells in vivo so that effects of local thrombin generation on malignant progression can be determined.

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