Thrombin Generation in a Hemophilic Newborn

SummaryThrombin generation was studied in paediatric patients with congenital heart disease (CHD) undergoing cardiac surgery using the calibrated automated thrombography (CAT) in terms of the lag time until the onset of thrombin formation, time to thrombin peak maximum (TTP), endogenous thrombin potential (ETP), and thrombin peak height. The suitability to determine the coagulation status of these patients was investigated. Patients, material, methods: CAT data of 40 patients with CHD (age range from newborn to 18 years) were compared to data using standard coagulation parameters such as prothrombin (FII), antithrombin (AT), tissue factor pathway inhibitor (TFPI), prothrombin fragment 1.2 (F 1.2), thrombin-antithrombin (TAT), activated partial thromboplastin time (aPTT), and prothrombin time (PT). Results: A significant positive correlation was seen between ETP and FII (p < 0.01; r = 0.369), as well as between peak height and F II (p < 0.01; r = 0.483). A significant negative correlation was seen between ETP and TFPI values (p < 0.05; r = –0.225) while no significant correlation was seen between peak height and TFPI. A significant negative correlation was seen between F 1.2 generation and ETP (p < 0.05; r = –0.254) and between F 1.2 generation and peak height (p < 0.05; r = –0.236). No correlation was seen between AT and ETP or peak. Conclusions: CAT is a good global test reflecting procoagulatory and inhibitory factors of the haemostatic system in paediatric patients with CHD.

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

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615117 ◽

1998 ◽

Vol 79 (05) ◽

pp. 1041-1047 ◽

Cited By ~ 20

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.

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Fibrinogen and its Derivatives: Cofactors in the Intrinsic Generation of Thrombin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647923 ◽

1976 ◽

Vol 35 (02) ◽

pp. 305-313 ◽

Cited By ~ 1

Author(s):

D.C Triantaphyllopoulos ◽

L.T Ryan

Keyword(s):

Factor Viii ◽

Vitamin K ◽

Bovine Serum ◽

Thrombin Generation ◽

Factor V ◽

Simultaneous Addition ◽

Lower Effect

SummaryThe simultaneous addition of suboptimal concentrations of factor VIII and intact or plas-min-lysed fibrinogen into mixtures of the vitamin K dependent factors, phospholipids, adsorbed bovine serum (supplier of factor V) and calcium, increased the amount of thrombin which was generated three to twenty times over the sum of the amounts which were generated when factor VIII, or fibrinogen, or its derivatives were added separately into the thrombin generating mixture. When factor VIII was added together with both fibrinogen and its derivatives, the amount of thrombin generated was even greater, about 130% larger than the amount which was generated in the presence of equal concentrations of only intact fibrinogen plus factor VIII. Addition of albumin instead of fibrinogen or its derivatives has a similar but significantly lower effect on thrombin generation. It appears, therefore, that both intact fibrinogen and its plasminolytic derivatives, singly or in combination, and to a lesser extent albumin, act as cofactors in the reaction which is regulated by factor VIII.

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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

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651063 ◽

1987 ◽

Vol 57 (01) ◽

pp. 062-066 ◽

Cited By ~ 36

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.

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Experimental Studies on a Recombinant Hirudin, CGP 39393

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648151 ◽

1991 ◽

Vol 65 (04) ◽

pp. 355-359 ◽

Cited By ~ 12

Author(s):

E Gray ◽

J Watton ◽

S Cesmeli ◽

T W Barrowcliffe ◽

D P Thomas

Keyword(s):

Thrombin Generation ◽

Bleeding Time ◽

Thrombus Formation ◽

Experimental Studies ◽

Venous Stasis ◽

Antithrombotic Agent ◽

Recombinant Hirudin ◽

Excessive Bleeding ◽

Generation Test

SummaryThe in vitro anticoagulant activities of recombinant desulphatohirudin (r-hirudin) were studied in the activated partial thromboplastin time (APTT) and the thrombin generation test : systems. In the APTT at concentrations below 5 μg/ml, r-hirudin showed a dose-response curye. At concentrations above 5 μg/ml, the plasma became unclottable, but in the thrombin generation test , at least 10 μg/ml of r-hirudin was required for full inhibition of thrombin generation. The antithrombotic effect was assessed using a rabbit venous stasis model; 150 μg/ml r-hirudin completely prevented thrombus formation at 10 and 20 min stasis. At antithrombotic dose, the mean bleeding time ratio measured in a rabbit ear template model, was not prolonged over control values. At higher doses, the bleeding time ratios were higher than those observed for the same dosage of heparin. These data indicate that while r-hirudin is an effective antithrombotic agent, antithrombotic doses have to be carefully titrated to avoid excessive bleeding.

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

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651585 ◽

1993 ◽

Vol 69 (03) ◽

pp. 227-230 ◽

Cited By ~ 28

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.

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Plasma Factor V Activation Is Prevented by Activated Protein C in the Presence of Phospholipid Vesicles, Not Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651567 ◽

1993 ◽

Vol 69 (02) ◽

pp. 124-129 ◽

Cited By ~ 2

Author(s):

Susan Solymoss ◽

Kim Thi Phu Nguyen

Keyword(s):

Western Blotting ◽

Protein C ◽

Thrombin Generation ◽

Blood Platelets ◽

Activated Protein C ◽

Phospholipid Vesicles ◽

Factor V ◽

Two Stage ◽

One Stage ◽

Plasma Factor

SummaryActivated protein C (APC) is a vitamin K dependent anticoagulant which catalyzes the inactivation of factor Va and VIIIa, in a reaction modulated by phospholipid membrane surface, or blood platelets. APC prevents thrombin generation at a much lower concentration when added to recalcified plasma and phospholipid vesicles, than recalcified plasma and platelets. This observation was attributed to a platelet associated APC inhibitor. We have performed serial thrombin, factor V one stage and two stage assays and Western blotting of dilute recalcified plasma containing either phospholipid vesicles or platelets and APC. More thrombin was formed at a given APC concentration with platelets than phospholipid. One stage factor V values increased to higher levels with platelets and APC than phospholipid and APC. Two stage factor V values decreased substantially with platelets and 5 nM APC but remained unchanged with phospholipid and 5 nM APC. Western blotting of plasma factor V confirmed factor V activation in the presence of platelets and APC, but lack of factor V activation with phospholipid and APC. Inclusion of platelets or platelet membrane with phospholipid enhanced rather than inhibited APC catalyzed plasma factor V inactivation. Platelet activation further enhanced factor V activation and inactivation at any given APC concentration.Plasma thrombin generation in the presence of platelets and APC is related to ongoing factor V activation. No inhibition of APC inactivation of FVa occurs in the presence of platelets.

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